AGREE Metric: A Comprehensive Guide to Green Analytical Method Assessment for Pharmaceutical Scientists

Abstract

This article provides a complete guide to the Analytical GREEnness (AGREE) metric, an essential tool for assessing the environmental sustainability of analytical methods in pharmaceutical research and drug development. Tailored for researchers and scientists, we cover the foundational principles of AGREE based on the 12 principles of Green Analytical Chemistry (GAC), offer a step-by-step methodological guide for application, address common troubleshooting and optimization challenges, and present a comparative analysis with other metrics like GAPI, Analytical Eco-Scale, and BAGI. By integrating AGREE with functional performance tools within the White Analytical Chemistry (WAC) framework, this guide empowers professionals to make informed decisions, develop greener methods, and advance sustainable practices in biomedical and clinical research.

The Principles and Evolution of AGREE in Green Analytical Chemistry

The Analytical GREEnness (AGREE) Metric Approach is a comprehensive and standardized tool designed to evaluate the environmental sustainability of analytical procedures. Developed in response to the growing need for Green Analytical Chemistry (GAC), AGREE provides a unified framework for assessing how well an analytical method aligns with the 12 principles of GAC [1]. Unlike earlier assessment tools that offered limited perspectives, AGREE delivers an easily interpretable, pictogram-based result, making it straightforward for researchers, scientists, and drug development professionals to identify areas for improvement and compare the greenness of different methods [2] [3]. The availability of free, open-source software has established AGREE as one of the most significant and user-friendly metrics for promoting sustainability in analytical laboratories [2].

The Need for Greenness Assessment in Analytical Chemistry

The evaluation of analytical methods has evolved significantly, expanding beyond traditional parameters like precision and accuracy to include environmental impact and practicality. The RGB model, which segments evaluation into red (analytical performance), green (environmental impact), and blue (practicality), laid the groundwork for a more balanced assessment system known as White Analytical Chemistry (WAC) [1]. While tools like the Red Analytical Performance Index (RAPI) and the Blue Applicability Grade Index (BAGI) were developed to quantify the red and blue aspects, a robust tool for the green dimension was essential [1]. AGREE fills this critical gap by offering a comprehensive assessment focused exclusively on the environmental footprint of analytical methods, thereby enabling a holistic White Analytical Chemistry evaluation [1].

The AGREE Framework: Core Methodology

The AGREE metric is built upon a structured and transparent methodology that translates the 12 principles of Green Analytical Chemistry into a quantifiable score.

Foundation in the 12 Principles of GAC

The assessment is explicitly structured around the 12 principles of Green Analytical Chemistry [4] [1]. These principles provide the foundational criteria for the evaluation, ensuring that the metric is aligned with the core tenets of the field.

Scoring System

The framework transforms the performance of an analytical procedure for each of the 12 principles into a unified score on a scale of 0 to 1 [2]. A sub-score of 1 indicates the best possible green performance for that criterion, while 0 represents the worst.

Weighting of Criteria

A key feature of the AGREE approach is its flexibility to account for the relative importance of different principles. The software allows users to assign weights to each of the 12 criteria based on their specific analytical goals and priorities [2]. This means that a principle deemed more critical in a particular context can have a greater influence on the final overall score.

Calculation of the Final Score

The final AGREE score is a composite value calculated based on the individual principle sub-scores and their user-assigned weights [2]. This calculation is handled automatically by the software, resulting in a single, easy-to-interpret numerical value between 0 and 1.

The AGREE Software and Output

Software Access and Functionality

The AGREE calculator is available as freely downloadable, open-source software from https://mostwiedzy.pl/AGREE [2]. This standalone application provides a user-friendly interface that guides the assessor through the input process, making the evaluation procedure straightforward and accessible to a wide range of users.

The AGREE Pictogram

The software generates a distinctive round pictogram as the primary output. This visual is designed to convey a wealth of information at a glance [2] [1].

• Central Numerical Score: The center of the pictogram displays the final calculated score on a scale of 0 to 1, providing an immediate indication of the method's overall greenness.
• Colored Segments: The pictogram is surrounded by 12 colored segments, each corresponding to one of the 12 principles of GAC. The color of each segment (ranging from red to yellow to green) visually represents the performance for that specific principle.
• Weight Indicators: The assigned weights for each criterion are also incorporated into the pictogram, offering a complete picture of both performance and priority [2].

This combination of a single score and a detailed visual breakdown allows analysts to instantly gauge a method's overall performance while also identifying specific strengths and weaknesses in its environmental profile.

AGREEprep: An Extension for Sample Preparation

Recognizing that sample preparation is often the most critical step from an environmental impact perspective, the same developers created AGREEprep, a dedicated metric for evaluating sample preparation methods [5] [3]. AGREEprep is based on the 10 principles of green sample preparation and functions similarly to its predecessor, using dedicated software to generate a pictogram with 10 segments [3]. This tool is particularly valuable for identifying inefficiencies and opportunities for greening in this resource-intensive step of the analytical process [3].

Comparison with Other Greenness Metrics

The field of green analytical chemistry has seen the development of multiple assessment metrics. The table below summarizes how AGREE compares to other notable tools.

Table 1: Comparison of AGREE with Other Greenness Assessment Metrics

Metric Tool	Basis of Assessment	Output	Scoring System	Key Features
AGREE [4] [2] [1]	12 Principles of GAC	Pictogram (12 segments) & final score (0-1)	Built-in, quantitative	Explicitly aligned with GAC principles; allows user-defined weighting.
Analytical Eco-Scale [4] [1]	Penalty points	Numerical score (closer to 100 is greener)	Built-in, quantitative	Simple calculation; lacks a visual component.
GAPI [4] [1]	Predefined questions	Pictogram (5 segments)	Qualitative (no total score)	Offers a graphical evaluation but does not facilitate direct numerical comparison.
AGSA [4]	12 Principles of GAC	Star-shaped pictogram & final score	Built-in, quantitative	Recent tool; emphasizes resistance to user bias and provides method classification.

As evidenced in the table, AGREE's unique combination of being explicitly based on the 12 GAC principles, providing a quantitative overall score, and allowing for user-defined weighting distinguishes it from other available metrics [4].

Experimental Protocol for Conducting an AGREE Assessment

The following workflow diagram outlines the standard operating procedure for evaluating the greenness of an analytical method using the AGREE metric.

Step-by-Step Guide

• Gather Methodological Data: Compile all quantitative and qualitative data related to the analytical method. This includes, but is not limited to, the type and volume of solvents, masses of reagents, energy consumption (e.g., for heating, cooling, or instrumentation), the number of procedural steps, and the amount of waste generated [2].
• Assign Weights to the 12 GAC Principles: Using the AGREE software, assign an importance weight to each of the 12 principles based on the analytical context and goals. The software provides default values, but these can be customized by the user [2].
• Input Data into the AGREE Software: Enter the collected methodological data into the corresponding fields of the software interface.
• Calculate Scores and Generate Output: The software will automatically compute the sub-scores and the final overall score, then generate the AGREE pictogram.
• Analyze the Pictogram and Identify Improvements: Interpret the results by examining the final score and the color-coding of the 12 segments. Principles with red or yellow segments are key areas for optimization. For example, a low score in the "waste" segment would indicate a need to minimize waste generation [2] [1].
• Report and Implement Findings: The final AGREE pictogram and score should be included in the method documentation or publication to transparently communicate its environmental performance. Use the insights gained to refine and green the analytical procedure.

Essential Research Reagent Solutions and Materials

The following table details key components considered during an AGREE assessment, which are critical for developing greener analytical methods.

Table 2: Key Research Reagents and Materials for Green Analytical Methods

Item/Concept	Function in Analytical Method	Greenness Consideration
Safer Solvents [3]	To dissolve, dilute, or extract analytes.	Replacing hazardous solvents (e.g., chlorinated) with safer alternatives (e.g., water, ethanol) is a core GAC principle.
Renewable/Reusable Materials [3]	Sorbents for extraction, filtration media, etc.	Using materials derived from renewable sources or that can be regenerated and reused multiple times reduces environmental impact.
Miniaturized Equipment [1]	Devices for sample preparation or analysis.	Scaling down methods (e.g., micro-extraction) drastically reduces consumption of samples, solvents, and reagents.
Automation Systems [1] [3]	Robotic platforms for sample handling.	Automation improves throughput and reproducibility while potentially reducing reagent use and operator exposure to hazards.
Energy-Efficient Instruments [3]	Chromatographs, spectrometers, etc.	Selecting instruments with lower energy demands or optimizing workflows to reduce run times minimizes the energy footprint.

The AGREE metric represents a significant advancement in the field of Green Analytical Chemistry. It provides a comprehensive, flexible, and transparent framework for quantifying the environmental impact of analytical procedures. By transforming the 12 principles of GAC into a clear, visual output with a quantitative score, AGREE empowers scientists and researchers to make informed decisions, drive innovation in sustainable method development, and communicate their greenness achievements effectively. Its role within the broader White Analytical Chemistry model ensures that environmental performance is evaluated in concert with analytical efficacy and practicality, paving the way for a more sustainable future in analytical science.

The 12 Principles of Green Analytical Chemistry (GAC) as the Foundation of AGREE

The rapid development of green analytical chemistry (GAC) since its emergence in 2000 has necessitated clear, concise guidelines to help analytical chemists green their laboratory practices [6]. While rooted in the broader concept of green chemistry, analytical chemistry presents unique challenges and requirements that were not fully addressed by the original 12 principles of green chemistry proposed by Anastas and Warner [6]. The most significant challenge for modern analytical chemistry has been to reach a compromise between increasing the quality of analytical results and improving the environmental friendliness of analytical methods [6]. This challenge prompted the development of specialized GAC principles that would provide a proper framework for greening analytical laboratories, ultimately leading to the creation of assessment tools like the AGREE metric (Analytical GREEnness metric) [1] [7].

The AGREE metric represents a significant advancement in the field, providing both a visual and quantitative means to evaluate how well analytical methods adhere to the principles of GAC [7]. Understanding the 12 principles of GAC is therefore fundamental to comprehending AGREE's structure and application, as these principles form the very foundation upon which the metric is built [8]. This technical guide explores the relationship between these foundational principles and their implementation through the AGREE metric, providing researchers and drug development professionals with a comprehensive framework for assessing and improving the environmental sustainability of their analytical methods.

The 12 Principles of Green Analytical Chemistry

Historical Development and Adaptation from Green Chemistry

The beginnings of green chemistry were dominated by green organic synthesis, particularly in the pharmaceutical industry, with minimal initial focus on analytical chemistry [6]. When Anastas and Warner formulated their 12 principles of green chemistry in 1998, only some could be directly applied to analytical chemistry [6]. Principles such as prevention of waste (principle 1), safer solvents and auxiliaries (principle 5), design for energy efficiency (principle 6), and reduction of derivatization (principle 8) found application in both synthetic and analytical purposes [6]. However, at least one principle – the maximization of atom economy (principle 2) – was inadequate for analytical chemistry [6]. This mismatch, coupled with important GAC concepts not included in the original principles, necessitated a revision specifically tailored to analytical chemistry [6].

In response, a set of 12 principles specifically for GAC was proposed, consisting of known concepts such as reduction in the use of reagents and energy, and elimination of waste, risk, and hazard, together with new ideas including the use of natural reagents [6]. These 12 principles were later condensed into the mnemonic SIGNIFICANCE to aid in their recall and application [6] [8]. The key goals to be achieved in greening analytical methods include: (1) elimination or reduction of the use of chemical substances; (2) minimization of energy consumption; (3) proper management of analytical waste; and (4) increased safety for the operator [6].

Detailed Explanation of the 12 GAC Principles

The 12 principles of green analytical chemistry provide a comprehensive framework for making analytical practices more environmentally friendly [6]:

• Direct analytical techniques should be applied to avoid sample treatment. This principle emphasizes selecting methods that require minimal sample manipulation, thereby reducing reagents, time, and potential waste generation.

• Minimal sample size and minimal number of samples are goals. This focuses on reducing the scale of analysis through statistical approaches and microextraction techniques to minimize resource consumption.

• In situ measurements should be performed. Whenever possible, analyses should be conducted directly at the sample location to avoid transportation, storage, and complex sampling procedures.

• Integration of analytical processes and operations saves energy and reduces the use of reagents. Combining multiple analytical steps into streamlined, automated processes enhances efficiency.

• Automated and miniaturized methods should be selected. Automation improves precision and safety while reducing labor; miniaturization decreases reagent and solvent consumption.

• Derivatization should be avoided. This step often involves additional reagents, energy, and time, potentially generating more waste without adding analytical value.

• Generation of a large volume of analytical waste should be avoided and proper management of analytical waste should be provided. This principle addresses both waste minimization and responsible disposal or treatment of unavoidable waste.

• Multi-analyte determinations are preferred over methods for a single analyte. Simultaneous determination of multiple analytes increases information gained per unit of resource consumed.

• The use of energy should be minimized. This includes selecting energy-efficient instruments and optimizing procedures to reduce overall power consumption.

• Reagents obtained from renewable sources should be preferred. This principle encourages the use of bio-based solvents and reagents instead of those derived from petrochemical sources.

• Pure reagents should be preferred over mixed reagents that are difficult to separate. This facilitates recycling and waste treatment while often simplifying analytical procedures.

• The safety of the operator should be increased. This encompasses all aspects of protecting analysts from hazardous chemicals, processes, and conditions.

Table 1: The 12 Principles of Green Analytical Chemistry and Their Key Applications

Principle Number	Principle Focus	Key Applications in Method Development
1	Direct analysis	Non-invasive techniques, direct sampling
2	Minimal sample size	Micro-extraction, statistical sampling design
3	In situ measurements	Field-deployable instruments, portable sensors
4	Process integration	Hyphenated techniques, automated systems
5	Automation & miniaturization	Flow analysis, microfluidic devices
6	Avoid derivatization	Direct detection methods
7	Waste management	Solvent recycling, waste treatment protocols
8	Multi-analyte determination	Chromatographic methods, sensor arrays
9	Energy minimization	Energy-efficient instruments, method optimization
10	Renewable reagents	Bio-based solvents, green chemicals
11	Pure reagents	Simplified waste streams, recycling
12	Operator safety	Closed systems, reduced exposure

The AGREE Metric: Operationalizing the GAC Principles

Development and Structure of AGREE

The AGREE (Analytical GREEnness) metric was developed as a tool to operationalize the 12 principles of GAC into a practical, user-friendly assessment system [7]. Unlike earlier green assessment tools that offered only binary or limited evaluations, AGREE provides both a visual pictogram and a numerical score between 0 and 1, enabling direct comparison between different analytical methods [7]. The tool is explicitly structured around the 12 principles of GAC, with each principle represented in the circular pictogram [4]. The AGREE calculator, released in 2020, quickly became one of the most comprehensive and widely adopted tools for evaluating the greenness of analytical methods [1].

The AGREE metric assesses the entire analytical process from sample collection through preparation to final detection, offering a more holistic evaluation than previous tools [7]. Its development addressed several limitations of earlier metrics, including the lack of comprehensive scope, absence of quantitative scoring, and insufficient alignment with the full set of GAC principles [8] [7]. The strength of AGREE lies in its balanced approach that combines scientific rigor with practical applicability, making it particularly valuable for researchers and method developers in the pharmaceutical industry who need to evaluate and justify their analytical approaches from both scientific and environmental perspectives [7].

How AGREE Implements the GAC Principles

AGREE translates the 12 GAC principles into a structured evaluation system where each principle is assessed and scored [4]. The tool uses a weighting system that allows users to assign different levels of importance to each principle based on their specific application context [8]. The output is a circular pictogram divided into 12 sections, each corresponding to one of the GAC principles, with colors ranging from deep green (ideal) to red (unsatisfactory) to provide immediate visual feedback on method performance [7].

The relationship between the GAC principles and the AGREE evaluation criteria can be visualized through the following conceptual framework:

Diagram 1: The conceptual framework of AGREE metric implementation shows how the 12 GAC principles are operationalized into a comprehensive assessment tool with visual and quantitative outputs.

The AGREE software, available as a free online tool, guides users through the evaluation process with prompts and questions related to each principle [1]. The final output includes both the visual pictogram, which allows for immediate identification of strengths and weaknesses, and a numerical score that facilitates objective comparison between methods [7]. This dual-output approach makes AGREE particularly valuable for method development and optimization, as researchers can quickly assess the environmental impact of different approaches and make informed decisions about which method to implement [7].

Complementary Tools and Recent Advancements in Green Method Assessment

The Evolving Landscape of Green Assessment Metrics

While AGREE represents a significant advancement in GAC assessment, it exists within an evolving ecosystem of complementary evaluation tools [1]. The field has progressed from basic tools like the National Environmental Methods Index (NEMI) to more comprehensive frameworks [7]. Recent years have witnessed a remarkable boom in the development of metrics in analytical chemistry, particularly those aimed at assessing environmental sustainability [1]. This proliferation reflects growing interest but has also led to overlapping tools that can be confusing for chemists [1].

Other notable metrics in the GAC landscape include:

• GAPI (Green Analytical Procedure Index): Uses a five-part, color-coded pictogram to assess the entire analytical process but lacks an overall score [7].
• AGREEprep: A specialized tool focusing specifically on sample preparation, which is often the most resource-intensive step [7].
• Analytical Eco-Scale: Applies penalty points to non-green attributes subtracted from a base score of 100 [7].
• GEMAM (Greenness Evaluation Metric for Analytical Methods): A newly proposed metric based on both the 12 principles of GAC and 10 factors of sample preparation, with results presented on a 0-10 scale [8].
• AGSA (Analytical Green Star Area): Introduced in 2025, it combines intuitive visualization with an integrated scoring system using a star-shaped diagram [4] [7].

Table 2: Comparison of Major Greenness Assessment Metrics for Analytical Chemistry

Metric Tool	Assessment Basis	Output Format	Scoring System	Key Advantages
AGREE	12 GAC principles	Circular pictogram + numerical	0-1 scale	Comprehensive, explicit GAC principle alignment
GAPI	Multiple criteria	5-part pictogram	Qualitative color coding	Covers entire analytical workflow
Analytical Eco-Scale	Penalty points	Numerical score	0-100 points	Simple quantitative result
AGREEprep	Sample preparation focus	Circular pictogram + numerical	0-1 scale	Specialized for sample prep
GEMAM	12 GAC + 10 GSP factors	Hexagonal pictogram	0-10 scale	Combines GAC & sample preparation
AGSA	12 GAC principles	Star-shaped diagram	Built-in scoring	Visual classification, resistant to bias

Integration with Broader Assessment Frameworks

The evaluation of analytical methods has expanded beyond solely environmental considerations to incorporate a more balanced approach through frameworks such as the RGB model [1]. This model organizes evaluation into three color-coded dimensions: red for analytical performance, green for environmental impact, and blue for practicality [1]. Within this triad, AGREE serves as a specialized tool for the green component, while other tools like the Red Analytical Performance Index (RAPI) and the Blue Applicability Grade Index (BAGI) address the other dimensions [1].

This evolution has led to the concept of White Analytical Chemistry (WAC), which aims to reconcile the principles of green analytical chemistry with functionality [1]. The most recent innovations in the field include tools like the Violet Innovation Grade Index (VIGI), which emphasizes innovation across 10 distinct criteria, and the Carbon Footprint Reduction Index (CaFRI), developed in response to rising awareness of climate change [1] [7]. These tools complement AGREE by providing additional perspectives on method evaluation, highlighting that a truly sustainable method must balance environmental concerns with analytical performance, practicality, innovation, and climate impact [7].

Experimental Protocols and Research Reagent Solutions

Methodologies for Implementing GAC Principles

Implementing the 12 principles of GAC requires specific methodological approaches throughout the analytical process. Based on case studies and experimental protocols reported in the literature, the following methodologies have proven effective for incorporating GAC principles into analytical practice:

Sample Preparation and Introduction (Principles 1-3)

• In-line sample preparation protocols: Implement integrated systems where sample preparation is directly coupled with analysis to minimize transfer steps and reagent use [8]. Scores of 1.0 are assigned for in-line preparation in GEMAM assessment, compared to 0.25 for ex situ methods [8].
• Micro-extraction techniques: Utilize methodologies such as sugaring-out-induced homogeneous liquid–liquid microextraction (SULLME) that limit solvent consumption to less than 10 mL per sample [7].
• Non-destructive sampling: Develop approaches that preserve sample integrity, with GEMAM assigning a score of 1.0 for methods with no sample destruction versus 0.5 for destructive methods [8].

Reagent and Solvent Selection (Principles 6, 10, 11)

• Green solvent substitution protocols: Systematically replace hazardous solvents with safer alternatives based on established solvent selection guides. Methods using green solvents receive higher scores in MoGAPI and AGREE assessments [7].
• Renewable reagent implementation: Establish procedures for identifying and validating bio-based reagents and solvents, with consideration of their impact on analytical performance [6].
• Derivatization avoidance strategies: Develop direct detection methods that eliminate the need for chemical derivatization, which often involves additional reagents and generates waste [6].

Instrumentation and Process Optimization (Principles 4, 5, 9)

• Energy consumption measurement protocols: Implement standardized procedures for quantifying energy usage per analysis, with AGREE assigning higher scores to methods consuming 0.1-1.5 kWh per sample [7].
• Automation and miniaturization integration: Develop workflows that incorporate automated sample handling and miniaturized analytical systems to reduce reagent consumption and improve reproducibility [6].
• Multi-analyte method development: Create analytical procedures capable of simultaneous determination of multiple analytes to maximize information gained per unit of resource consumed [6].

Research Reagent Solutions for Green Analytical Chemistry

The implementation of GAC principles often requires specialized reagents and materials that minimize environmental impact while maintaining analytical performance. The following table details key research reagent solutions used in green analytical methods:

Table 3: Essential Research Reagent Solutions for Green Analytical Chemistry

Reagent Category	Green Alternatives	Function	Application Examples
Extraction Solvents	Bio-based solvents (ethyl lactate, limonene), supercritical COâ‚‚, deep eutectic solvents	Sample preparation and extraction	Liquid-phase microextraction, solid-phase microextraction
Derivatization Agents	Avoidance preferred; when necessary, less hazardous alternatives	Chemical modification for detection	Direct analysis methods eliminate need for derivatization
Mobile Phase Additives	Green solvents (ethanol, acetone), minimized additive concentrations	Chromatographic separation	HPLC, UHPLC with reduced environmental impact
Calibration Standards	In-house prepared from pure materials, stable certified reference materials	Instrument calibration and quantification	All quantitative analytical methods
Preservatives & Stabilizers	Natural antioxidants, minimal necessary concentrations	Sample preservation during storage	Environmental, clinical, and food analysis
Catalysts & Enzymes	Immobilized catalysts, bio-catalysts	Reaction facilitation in sample preparation	Enzymatic digestion, catalytic conversion

Case Study: Comprehensive Greenness Assessment Using Multiple Metrics

A case study evaluating the sugaring-out-induced homogeneous liquid–liquid microextraction (SULLME) method for determining antiviral compounds demonstrates how AGREE and complementary metrics provide a multidimensional view of a method's sustainability [7]. The method was evaluated using MoGAPI, AGREE, AGSA, and CaFRI, with the following results:

MoGAPI Assessment The method scored 60 on the MoGAPI scale, indicating moderate greenness [7]. Positive aspects included the use of green solvents and microextraction approach limiting solvent consumption to <10 mL per sample [7]. Drawbacks included specific storage requirements, use of moderately toxic substances, vapor emissions, and generation of more than 10 mL of waste without treatment strategies [7].

AGREE Evaluation The AGREE score of 56 reflected a reasonably balanced green profile [7]. The method benefited from miniaturization, semiautomation, absence of derivatization, small sample volume (1 mL), and reduced procedural steps [7]. Limitations included the use of toxic and flammable solvents, relatively low throughput (2 samples per hour), and moderate waste generation [7].

AGSA Analysis The AGSA tool assigned a score of 58.33, with strengths in semi-miniaturization and avoidance of derivatization [7]. Limitations included manual sample handling, pretreatment steps, absence of integrated processes, presence of six or more hazard pictograms, and lack of waste management practices [7].

CaFRI Assessment The CaFRI score of 60 reflected the method's carbon footprint [7]. Positive aspects included relatively low energy consumption (0.1-1.5 kWh per sample) and no energy-intensive equipment [7]. Negative factors included absence of clean energy sources, no COâ‚‚ emissions tracking, long-distance transportation using non-ecofriendly vehicles, undefined waste disposal procedures, and use of over 10 mL of organic solvents per sample [7].

This case study illustrates how applying multiple complementary metrics provides a more comprehensive sustainability assessment than any single tool alone, highlighting both strengths (reduced solvent use, avoided derivatization) and weaknesses (waste management, reagent safety, energy sourcing) of the analytical method [7].

The 12 principles of green analytical chemistry provide the essential theoretical foundation for assessing and improving the environmental sustainability of analytical methods [6]. The AGREE metric successfully translates these principles into a practical, comprehensive assessment tool that enables researchers to quantify, visualize, and optimize the greenness of their analytical procedures [7]. As the field continues to evolve, with new tools like GEMAM and AGSA emerging, the fundamental principles of GAC remain the cornerstone of environmental assessment in analytical chemistry [4] [8].

For researchers and drug development professionals, understanding the relationship between the GAC principles and assessment tools like AGREE is crucial for developing methods that are both scientifically valid and environmentally responsible [7]. The future of green analytical chemistry will likely involve increased integration of assessment tools into method development workflows, greater emphasis on climate impact through metrics like CaFRI, and the development of unified platforms that combine multiple evaluation perspectives [1]. By grounding these advancements in the foundational principles of GAC, the analytical community can continue to progress toward more sustainable laboratory practices without compromising analytical performance.

The Role of AGREE in the Context of White Analytical Chemistry (WAC)

The field of analytical chemistry has witnessed a significant paradigm shift with the introduction of Green Analytical Chemistry (GAC), which aims to minimize the environmental impact of analytical procedures. GAC focuses on reducing hazardous waste, decreasing energy consumption, and promoting safer practices [9]. While GAC principles have driven substantial improvements, they primarily address environmental aspects, potentially overlooking other critical factors such as analytical performance and practical practicality. This limitation led to the development of White Analytical Chemistry (WAC), a holistic approach that balances environmental, analytical, and practical considerations [10].

WAC represents an evolution in sustainable analytical science, integrating three fundamental pillars represented by the RGB (Red-Green-Blue) model: Red for analytical performance (scope, accuracy, precision), Green for ecological impact (waste, energy consumption), and Blue for practical and economic efficiency (cost-effectiveness, time) [10]. By considering all three dimensions simultaneously, WAC provides a more comprehensive framework for evaluating analytical methods, ensuring they are not only environmentally friendly but also analytically sound and practically feasible.

Within this context, metric tools for assessing method greenness have become essential. The Analytical GREEnness (AGREE) metric has emerged as one of the most comprehensive and widely adopted tools for evaluating the environmental sustainability of analytical procedures based on all 12 principles of GAC [9]. This whitepaper explores the role, implementation, and significance of AGREE within the broader framework of White Analytical Chemistry, providing researchers and drug development professionals with a technical guide to sustainable method development and assessment.

The AGREE Metric: Fundamentals and Methodology

Conceptual Framework and Development

AGREE is a comprehensive metric tool designed to evaluate the greenness of analytical procedures against the 12 principles of Green Analytical Chemistry. Developed to address limitations of previous assessment tools, AGREE provides a quantitative, flexible, and easily interpretable evaluation system [9]. Unlike earlier metrics that considered only a few environmental criteria, AGREE comprehensively addresses the multifaceted nature of greenness in analytical chemistry, transforming each GAC principle into a score on a unified 0-1 scale, where 1 represents ideal greenness [9] [11].

The tool was created in response to the need for a more nuanced assessment approach that could accommodate the complex, multivariate nature of greenness evaluation. Previous tools like NEMI and Analytical Eco-Scale offered valuable but limited perspectives, often treating assessment criteria as binary functions or considering only a subset of GAC principles [9]. AGREE's development marked a significant advancement by incorporating all 12 GAC principles while allowing for flexibility in their relative importance through user-defined weighting.

The 12 Principles of Green Analytical Chemistry

AGREE's assessment framework is built upon the 12 principles of GAC, which are encapsulated in the SIGNIFICANCE mnemonic [9]. The following table details these principles and their implementation in analytical practice:

Table 1: The 12 Principles of Green Analytical Chemistry and Their Practical Implementation

Principle	Description	Key Implementation Strategies
1	Direct analytical techniques should be applied to avoid sample treatment	Remote sensing, non-invasive analysis, in-field analysis [9]
2	Minimal sample size and minimal number of samples are goals	Miniaturization, microextraction techniques, statistical sampling design [9]
3	Measurements should be performed in-situ	Field-deployable instruments, on-site analysis [9]
4	Integration of analytical processes and operations	Automation, online sample preparation, coupled techniques [9]
5	Automated and miniaturized methods should be selected	Flow injection analysis, lab-on-a-chip, automated systems [9]
6	Derivatization should be avoided	Direct detection methods, hyphenated techniques [9]
7	Generation of a large volume of waste should be avoided and proper waste management should be provided	Solvent-less techniques, waste recycling, microwave-assisted extraction [9]
8	Multi-analyte or multi-parameter methods should be preferred	Multi-analyte assays, comprehensive screening approaches [9]
9	The use of energy should be minimized	Energy-efficient instruments, ambient temperature processes [9]
10	Reagents obtained from renewable sources should be preferred	Bio-based solvents, green solvents [9]
11	Toxic reagents should be eliminated or replaced	Alternative solvents, less hazardous chemicals [9]
12	The safety of the operator should be increased	Automated systems, closed vessels, reduced exposure [9]

AGREE Assessment Algorithm and Calculation

The AGREE software calculates the final score based on an algorithm that incorporates all 12 GAC principles with their respective weights. The calculation involves transforming each principle's performance into a normalized score and computing the geometric mean of these weighted scores [9]. The mathematical representation of this calculation is:

Final Score = (∏(wi × si))^(1/∑wi)

Where:

• wi = weight assigned to principle i
• si = score for principle i (0-1 scale)
• Π = product of all (wi × si) terms

This calculation results in an overall score between 0 and 1, where higher values indicate better greenness performance. The output is presented as an intuitive, clock-like pictogram with the overall score in the center and performance for each principle shown in colored segments around it [9]. The width of each segment reflects the weight assigned to that principle, providing immediate visual feedback on both performance and priority.

Figure 1: AGREE Assessment Workflow - The systematic process for evaluating analytical method greenness

AGREE within the White Analytical Chemistry Framework

The RGB Model and Integration with AGREE

White Analytical Chemistry introduces a three-dimensional assessment model using the RGB (Red-Green-Blue) color model, where each color represents a different aspect of method quality [10]. The Green component aligns directly with AGREE's environmental focus, while the complete WAC assessment requires balancing this with the other two dimensions:

• Red (Analytical Efficiency): Encompasses method validation parameters such as scope, accuracy, precision, sensitivity, linearity, and robustness [10].
• Green (Ecological Impact): Evaluated using comprehensive metrics like AGREE, focusing on waste generation, energy consumption, reagent toxicity, and operator safety [10] [9].
• Blue (Practical & Economic Efficiency): Considers factors such as cost-effectiveness, time requirements, operational simplicity, and availability of equipment [10].

The integration of AGREE within WAC provides the crucial environmental dimension, enabling a balanced assessment that prevents overemphasis on any single aspect. A method might score highly in AGREE but be impractical due to excessive cost or inadequate analytical performance, highlighting the importance of the comprehensive WAC framework [10].

Comparative Analysis of Greenness Assessment Tools

Multiple tools exist for assessing the greenness of analytical methods, each with distinct characteristics, advantages, and limitations. The following table compares AGREE with other prominent greenness metrics:

Table 2: Comparison of Greenness Assessment Metrics in Analytical Chemistry

Metric	Assessment Criteria	Scoring System	Key Advantages	Limitations
AGREE	12 principles of GAC	0-1 scale (continuous)	Comprehensive, flexible weighting, intuitive pictogram	Requires detailed method information [9] [11]
NEMI	4 criteria (PBT, hazardous waste, corrosivity, waste amount)	Binary (green/uncolored)	Simple, quick assessment	Limited criteria, qualitative only [11]
Analytical Eco-Scale	Penalty points for hazardous reagents, waste, energy	0-100 points (higher=greener)	Semi-quantitative, widely used	Does not cover all GAC principles [11]
GAPI	5 evaluation areas (sample collection, preparation, etc.)	Qualitative (green-yellow-red)	Comprehensive pictogram	Complex assessment process [11]
AGREEprep	10 principles of green sample preparation	0-1 scale (continuous)	Specialized for sample prep, flexible weights	Limited to sample preparation only [3]

AGREE distinguishes itself through its comprehensive coverage of all 12 GAC principles, continuous scoring system, and flexibility in assigning weights to different principles based on their importance in specific contexts [9]. This makes it particularly valuable for method development and optimization in research and drug development environments.

Practical Implementation of AGREE in Analytical Method Development

Step-by-Step Assessment Procedure

Implementing AGREE for analytical method assessment involves a systematic process:

• Data Collection: Gather all relevant method parameters including sample preparation steps, reagents and their quantities, instrumentation, energy consumption, waste generation, and safety considerations [9].

• Software Input: Utilize the open-source AGREE software, available from https://mostwiedzy.pl/AGREE, to input method details corresponding to each of the 12 GAC principles [9].

• Weight Assignment: Assign appropriate weights to each principle based on methodological priorities and context. Default equal weighting can be used initially for baseline assessment [9].

• Score Calculation: The software automatically calculates normalized scores for each principle and computes the overall greenness score using the weighted geometric mean [9].

• Result Interpretation: Analyze the generated pictogram to identify strengths and weaknesses, focusing improvement efforts on principles with lower scores and higher weights [9].

AGREEprep for Sample Preparation Assessment

Sample preparation is often the most environmentally impactful step in analytical procedures. AGREEprep is a specialized tool derived from AGREE, specifically designed to evaluate the greenness of sample preparation methods based on 10 principles of Green Sample Preparation (GSP) [3]. The AGREEprep assessment follows a similar approach but focuses exclusively on sample preparation, providing more detailed evaluation of this critical step.

The relationship between AGREE and AGREEprep in comprehensive method assessment can be visualized as follows:

Figure 2: Integrated Greenness Assessment Strategy - Combining AGREEprep for sample preparation with AGREE for overall method evaluation

Case Study: Pharmaceutical Analysis Method

A recent study developing an environmentally friendly HPLC method for simultaneous determination of Nebivolol hydrochloride, Telmisartan, Valsartan, and Amlodipine besylate demonstrates the practical application of AGREE [10]. The method was designed with green chemistry principles, utilizing ethanol as a safer alternative to traditional acetonitrile, and optimized through Quality-by-Design approaches.

The AGREE assessment of this method provided quantitative validation of its greenness credentials, with high scores particularly in principles related to reagent safety (Principle 11), waste minimization (Principle 7), and energy consumption (Principle 9) [10]. This case illustrates how AGREE can serve as both a design guide during method development and a verification tool upon completion.

Advanced Applications in Pharmaceutical Research and Drug Development

Green Method Development and Optimization

In pharmaceutical analysis, AGREE serves as a strategic tool for guiding method development toward more sustainable practices. By providing quantitative feedback on greenness performance, researchers can:

• Compare alternative techniques during method development to select the most environmentally friendly approach
• Optimize existing methods by identifying specific aspects with poor greenness performance
• Justify method selection in regulatory submissions with quantitative greenness data
• Track improvements in environmental performance across method generations

The integration of AGREE with Quality-by-Design (QbD) approaches, as demonstrated in the pharmaceutical case study, represents a powerful strategy for developing robust, effective, and environmentally sustainable analytical methods [10].

Essential Research Reagents and Tools for Green Analytical Chemistry

Implementing green analytical methods requires careful selection of reagents and materials. The following table details key solutions and their functions in sustainable method development:

Table 3: Research Reagent Solutions for Green Analytical Chemistry

Reagent/Material	Function	Green Alternatives	Application Examples
Ethanol	Green solvent for extraction and chromatography	Replaces acetonitrile and methanol	HPLC mobile phase [10]
Water	Solvent for analytical applications	Replaces organic solvents	Reverse-phase chromatography [10]
Bio-based solvents	Extraction and separation media	Derived from renewable resources	Sample preparation [9]
Solid-phase microextraction (SPME) coatings	Sample preparation and concentration	Minimizes solvent use	Analyte extraction [3]
Green sorbents	Sample clean-up and concentration	Renewable, biodegradable materials	Solid-phase extraction [3]

Strategic Implementation in Regulatory Contexts

For drug development professionals, AGREE provides a structured approach to addressing increasing regulatory expectations for environmental considerations. While current regulatory standards primarily focus on analytical validation parameters (accuracy, precision, specificity), the pharmaceutical industry is witnessing growing interest in sustainable practices [10].

AGREE assessments can be incorporated into method validation protocols to demonstrate environmental responsibility while maintaining analytical integrity. This proactive approach positions organizations favorably as regulatory expectations evolve toward greater environmental consciousness.

Development Trends and Research Needs

The field of greenness assessment continues to evolve, with several emerging trends shaping the future of AGREE and related metrics:

• Integration with analytical performance metrics: Development of unified assessment frameworks that simultaneously evaluate greenness and analytical validity [10] [11].
• Automated data extraction: Connection with electronic laboratory notebooks to streamline the assessment process [11].
• Expanded scope: Adaptation for emerging analytical technologies and application areas [11].
• Standardization efforts: Movement toward consensus guidelines for greenness assessment in regulatory contexts [11].

Research needs include establishing correlation between different greenness metrics, developing educational resources for widespread adoption, and creating domain-specific weighting schemes for different application areas [11].

AGREE represents a significant advancement in the quantitative assessment of analytical method greenness, providing researchers and drug development professionals with a comprehensive, flexible, and intuitive tool for environmental evaluation. Within the White Analytical Chemistry framework, AGREE provides the crucial environmental dimension that must be balanced with analytical and practical considerations for truly sustainable method development.

The ongoing adoption of AGREE in pharmaceutical research and quality control laboratories demonstrates its utility in guiding the transition toward more sustainable analytical practices without compromising analytical performance. As the field advances, AGREE is poised to play an increasingly important role in method development, optimization, and validation, contributing to the broader goals of green chemistry in the pharmaceutical industry and beyond.

The principles of Green Analytical Chemistry (GAC) have catalyzed a paradigm shift in the field, driving the development of methodologies that minimize environmental impact and enhance operator safety. Sample preparation, often the most resource-intensive stage of analysis, has become a critical focus for greenification efforts [12]. Within this context, the AGREEprep (Analytical Greenness Metric for Sample Preparation) metric emerges as a specialized, software-based tool designed to quantitatively evaluate the environmental sustainability of sample preparation procedures [5] [13]. This whitepaper details the core principles, application, and significance of AGREEprep, framing it within a broader research initiative on green method assessment. For researchers and drug development professionals, AGREEprep provides a standardized, transparent framework to guide the development and selection of greener analytical methods, aligning laboratory practices with the global mandate for sustainable science [14] [12].

The Need for a Specialized Sample Preparation Metric

Sample preparation is a critical stage in analytical procedures, significantly impacting the accuracy and sensitivity of results [12]. However, classical techniques like liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are often associated with substantial waste generation, high resource consumption, and operator safety concerns [14] [12]. The movement toward sustainable development has therefore intensified the focus on miniaturized and eco-friendly alternatives.

While general green chemistry metrics exist, sample preparation possesses unique characteristics that necessitate a dedicated assessment tool. AGREEprep was developed to fill this gap as the first metric specifically intended for evaluating the environmental impact of sample preparation methods [5] [13]. It enables a direct, principled comparison between traditional and novel methods, such as various microextraction techniques, and provides a clear visual and quantitative representation of a method's environmental performance [14].

The AGREEprep Framework: Core Principles and Calculation

The AGREEprep approach is built upon the 10 principles of green sample preparation [13]. The metric evaluates a method against these principles, translating qualitative goals into a quantitative score.

The Ten Assessment Criteria

AGREEprep's evaluation is based on ten criteria, each corresponding to one principle of green sample preparation. The following table summarizes these criteria, their descriptions, and scoring benchmarks.

Table 1: The Ten Core Assessment Criteria of AGREEprep

Criterion	Description	Key Scoring Benchmarks
1. In Situ Preparation	Favors sample preparation at the point of sampling [12].	In-line/in situ: 1.0; On-line/in situ: 0.66; On site: 0.33; Ex situ (lab): 0.0 [12].
2. Sample & Collection	Considers the need for sample preservation/storage [12].	No preservation needed: 1.0; Preservation required: 0.0 [12].
3. Sample Size	Assesses the amount of sample consumed [12].	Smaller sizes are scored higher [12].
4. Health Hazard	Evaluates the safety of reagents/solvents used [12].	Safer (green) solvents score highest [12].
5. Amount of Waste	Quantifies the total waste generated per sample [12].	< 0.1 mL (or g): 1.0; > 10 mL (or g): 0.0 [12].
6. Energy Consumption	Measures total energy used (in Watt-hours per sample) [12].	< 10 Wh: 1.0 [12].
7. Sample Throughput	Assesses the number of samples processed per hour [13].	Higher throughput is favored [13].
8. Device Operation	Evaluates the level of automation and operator involvement [12].	Full automation: 1.0; Manual operation: 0.0 [12].
9. Integration & Analysis	Considers the coupling of preparation with detection [12].	Full on-line integration: 1.0; Off-line analysis: 0.0 [12].
10. Operator Safety	Assesses the risk of exposure to hazardous materials [12].	Safer, closed-system operations score higher [12].

Each criterion is assigned a score between 0 and 1. The final AGREEprep score is a weighted sum of these ten individual scores, resulting in a unified value between 0 and 1, where a higher score indicates a greener method [5]. The outcome is presented both numerically and via a radial diagram, providing an intuitive visual snapshot of the method's greenness profile [12].

The AGREEprep Workflow

The process of applying the AGREEprep metric follows a logical sequence from data collection to result interpretation, as visualized in the workflow below.

Diagram 1: The AGREEprep Assessment Workflow

This workflow highlights the critical first step: gathering comprehensive experimental data. As noted in the tutorial, this can be challenging as essential data is often not reported in literature, underscoring the need for meticulous documentation when developing new methods [5].

Practical Application: Experimental Protocols and Case Studies

Conducting an Assessment

To perform an AGREEprep assessment, the analyst must first compile all necessary data points from the sample preparation protocol [5]. This includes:

• Volumes and Masses: Exact sample size, solvent volumes, and reagent masses.
• Energy Consumption: The power rating of equipment (e.g., heaters, centrifuges) and the duration of their use, calculated in Watt-hours per sample.
• Operational Details: The degree of automation, number of samples processed per hour, and whether the process is performed in situ or ex situ.

This data is input into the open-source AGREEprep software, which generates the final score and radial visualization. The analyst can adjust the default weightings of the ten criteria to reflect specific analytical goals or priorities, making the tool highly adaptable [13].

Case Study: EPA Methods vs. Modern Microextraction

A recent study applied AGREEprep to evaluate three US EPA standard methods (523, 528, 610) and one German Institute for Standardization (DIN) method, comparing them against twenty novel microextraction alternatives [14].

Table 2: AGREEprep Comparison of Standard and Microextraction Methods

Method Type	Example Techniques	Key Shortcomings	Key Advantages	Overall AGREEprep Performance
Standard Methods	EPA 523, 528, 610; DIN 38047-37 (using classical LLE/SPE) [14].	Large sample volumes; High volumes of organic solvents [14].	Standardized, established protocols.	Lowest greenness scores among evaluated procedures [14].
Modern Microextraction	Liquid-Phase Microextraction (LPME), Solid-Phase Microextraction (SPME), Dispersive Liquid-Liquid Microextraction (DLLME) [14] [12].	Miniaturization, reduced solvent use, often faster and more selective [14] [12].	Superior greenness scores, with similar or better analytical performance [14].

The study concluded that miniaturized sample preparation strategies consistently showed superior greenness over the standard methods. The main shortcomings of the standard methods were the large sample volume required and the use of large volumes of organic solvents [14]. In some cases, liquid-phase microextraction was identified as a greener alternative than solid-phase techniques [14].

The Scientist's Toolkit for Green Sample Preparation

Advancements in green sample preparation are driven not only by new techniques but also by the development of novel functional materials and solvents. The following table details key reagents and materials that can enhance the greenness of sample preparation methods.

Table 3: Research Reagent Solutions for Green Sample Preparation

Reagent/Material	Function in Sample Preparation	Greenness Contribution
Deep Eutectic Solvents (DESs)	Serve as biodegradable, low-toxicity extraction solvents [12].	Reduce environmental footprint and hazard versus traditional organic solvents [12].
Molecularly Imprinted Polymers (MIPs)	Synthetic polymers with high selectivity for target analytes; used in MISPE, MSPE [12].	Improve selectivity, reducing need for extensive cleaning steps and solvent use [12].
Ionic Liquids (ILs)	Used as tunable, non-volatile solvents in microextractions like DLLME [12].	Low volatility reduces airborne exposure and waste generation [12].
Metal-Organic Frameworks (MOFs)	Porous materials with high surface area for extraction and pre-concentration (MSPE, DμSPE) [12].	High efficiency and capacity; can be designed for reusability [12].
Conductive Polymers (CPs)	Used in sorptive extraction techniques for their versatility and electrostatic interactions [12].	Affinity for various compounds can simplify methods and reduce reagent use [12].
Cyclobutrifluram	Cyclobutrifluram	Cyclobutrifluram is a novel SDHI for nematode and fungal disease research. This product is for Research Use Only (RUO). Not for personal use.
Deoxyradicinin	Deoxyradicinin

AGREEprep in the Context of Other Green Metrics

AGREEprep is one of several tools developed to assess the environmental friendliness of analytical methods. Other notable metrics include the Green Analytical Procedure Index (GAPI), the Analytical Greenness (AGREE) metric for the entire analytical procedure, and the eco-scale [12]. While GAPI assesses the entire analytical workflow, AGREEprep's distinctive value is its exclusive focus on the sample preparation stage, which is often the primary source of waste and hazard [12]. Its scoring system and intuitive visual representation make it particularly valuable for identifying specific aspects of the sample preparation that can be improved from an environmental standpoint [12]. For consistent sustainability comparisons, it is crucial to use the same metric, as each tool applies distinct criteria [12].

AGREEprep represents a significant advancement in the toolkit for sustainable science, providing a rigorous, transparent, and specialized means to quantify the environmental impact of sample preparation. For researchers and drug development professionals, it offers a data-driven pathway to validate claims of greenness, guide the development of new methods, and make informed choices between existing procedures. By integrating AGREEprep into methodological research and development, the scientific community can accelerate the adoption of greener practices, reducing the ecological footprint of analytical chemistry while maintaining, and often enhancing, analytical performance. Its application has already demonstrated the clear green superiority of miniaturized methods, paving the way for a more sustainable future in analytical science.

The evolution of green analytical chemistry (GAC) has necessitated the development of sophisticated assessment tools to evaluate the environmental impact of analytical methods. This whitepaper traces the progression from early metrics like the National Environmental Methods Index (NEMI) to more comprehensive tools such as the Green Analytical Procedure Index (GAPI) and the current state-of-the-art Analytical GREEnness (AGREE) metric. Within the context of method assessment research, we provide a detailed comparison of these tools' methodologies, applications, and limitations, supplemented with experimental protocols for their implementation and visualization of their operational workflows. This technical guide serves as a foundational resource for researchers and professionals in drug development and analytical science seeking to integrate systematic greenness assessment into their methodological frameworks.

Green analytical chemistry (GAC) has emerged as a critical discipline focused on minimizing the environmental footprint of analytical activities while maintaining analytical performance [7]. The field has progressed from basic environmental considerations to comprehensive, multi-criteria assessment frameworks that evaluate the entire analytical lifecycle. The development of greenness assessment tools has been instrumental in this evolution, providing scientists with standardized methods to quantify and compare the environmental impact of analytical procedures [11].

The National Environmental Methods Index (NEMI), first launched in 2002, was among the first tools developed for this purpose, offering a simple pictogram-based approach [15]. As the limitations of early tools became apparent, more sophisticated metrics were developed, including the semi-quantitative Analytical Eco-Scale and the more descriptive Green Analytical Procedure Index (GAPI) [16] [11]. The recent introduction of the Analytical GREEnness (AGREE) metric represents a significant advancement, incorporating all 12 principles of GAC into a unified, flexible, and easily interpretable assessment framework [9].

For researchers in drug development, where analytical methods are extensively used for quality control and regulatory compliance, understanding these metrics is crucial for selecting, developing, and validating environmentally sustainable methods without compromising analytical performance.

Evolution and Comparison of Green Assessment Tools

Foundational Metrics: Characteristics and Limitations

Table 1: Comparison of Foundational Greenness Assessment Tools

Tool Name	Year Introduced	Assessment Basis	Output Format	Key Advantages	Key Limitations
NEMI	2002 [15]	4 binary criteria: PBT chemicals, hazardous waste, corrosivity, waste amount [11]	4-quadrant pictogram (green/blank) [17]	Simple, immediate visual interpretation [17]	Qualitative only; limited criteria; lacks granularity [17] [9]
Analytical Eco-Scale	2012 [11]	Penalty points subtracted from ideal score of 100 [17]	Numerical score (0-100) [17]	Semi-quantitative; allows direct comparison [17]	Lacks visual component; requires expert judgment [7]
GAPI	2018 [16]	5 evaluation areas covering entire analytical procedure [16]	5 pentagrams with color coding (green/yellow/red) [16]	Comprehensive; visual identification of high-impact stages [16]	No overall score; some subjectivity in color assignment [18] [7]
AGREE	2020 [9]	12 principles of GAC [9]	Circular pictogram with overall score (0-1) [9]	Comprehensive; combines score with visual output; flexible weighting [17] [9]	Does not fully account for pre-analytical processes [7]

Technical Comparative Analysis

The National Environmental Methods Index (NEMI) employs a simplistic binary assessment where each of its four quadrants is colored green if criteria are met. For instance, the "persistent, bioaccumulative, and toxic" (PBT) quadrant is green only if no chemicals used appear on the PBT list [11]. This approach lacks granularity, as evidenced by a comparative study where 14 out of 16 methods received identical NEMI pictograms, failing to distinguish between their varying environmental impacts [17].

The Analytical Eco-Scale introduced a more nuanced approach by assigning penalty points for hazardous reagents, energy consumption, and waste [11]. A score above 75 represents an excellent green analysis, while scores below 50 indicate inadequate greenness. This tool provides reliable numerical assessment but offers limited information about the structure of hazards within the analytical procedure [17].

The Green Analytical Procedure Index (GAPI) significantly expanded assessment scope by evaluating five key areas: sample collection, preservation, transportation, storage, and sample preparation [16]. Its visual presentation of results using a three-color traffic light system enables immediate identification of environmental hotspots throughout the analytical process. However, the complexity of its application and the absence of a unified numerical score can limit straightforward method comparisons [17] [18].

The Analytical GREEnness (AGREE) metric addresses many limitations of previous tools by incorporating all 12 principles of GAC into a unified assessment framework [9]. It calculates a final score between 0 and 1, with higher scores indicating greener methods. A key innovation is the ability to assign different weights to each criterion based on their importance in specific analytical scenarios, enhancing flexibility and context-specific relevance [9].

Figure 1: Evolution of Greenness Assessment Tools

Detailed Methodologies and Experimental Protocols

AGREE Metric Implementation Protocol

The AGREE assessment methodology is based on converting each of the 12 GAC principles into a score between 0 and 1, with the final result calculated as the product of these individual scores [9]. The assessment can be performed using freely available software that automatically generates the characteristic clock-like pictogram.

Table 2: AGREE Principle Transformation Protocol

Principle	Assessment Protocol	Scoring Methodology
1. Directness	Evaluate sample treatment requirements	Remote sensing without damage=1.0; multistep methods=0.0 [9]
2. Sample & Size	Measure sample size and number	Logarithmic transformation of sample size [9]
3. Device Portability	Assess equipment location and portability	In-field=1.0; automated off-line=0.0 [9]
4. Derivatization	Check for derivatization steps	No derivatization=1.0; with derivatization=0.0 [9]
5. Waste Generation	Quantify total waste per sample	<0.1 mL=1.0; >1000 mL=0.0 [9]
6. Energy Consumption	Calculate total energy used	<0.1 kWh=1.0; >1.5 kWh=0.0 [9]

Step-by-Step AGREE Assessment Procedure:

• Data Collection: Compile all method parameters including sample size, number of samples, reagents and their amounts, energy consumption, waste generation, and number of procedural steps.

• Software Input: Access the AGREE software (available at https://mostwiedzy.pl/AGREE) and input the collected data for each of the 12 principles.

• Weight Assignment: Assign importance weights to each principle (default is equal weighting) based on assessment priorities.

• Score Calculation: The software automatically calculates individual principle scores and the overall score between 0 and 1.

• Pictogram Generation: The software generates a circular pictogram with colored segments representing performance on each principle, segment width indicating assigned weight, and the overall score displayed centrally.

• Interpretation: Scores closer to 1.0 (dark green) indicate greener procedures, while scores closer to 0 (red) indicate significant environmental concerns.

Figure 2: AGREE Assessment Workflow

GAPI Application Protocol

The GAPI assessment methodology involves a detailed evaluation of the entire analytical procedure through five pentagrams, each divided into several subsections [16]. Each subsection is assigned a color (green, yellow, or red) based on its environmental impact.

Sample Collection & Preparation Protocol:

• Evaluate sampling technique (in-line, on-line, at-line, off-line)
• Assess need for sample preservation and storage conditions
• Quantify reagents and solvents used in preparation
• Identify potential hazards from reagents and procedures

Method Execution Protocol:

• Document instrument type and placement
• Quantify energy consumption per sample
• Measure waste generation and treatment
• Evaluate safety precautions including containment and vapor release

The main limitation of GAPI—the lack of an overall numerical score—has been recently addressed by the development of Modified GAPI (MoGAPI), which introduces a scoring system that calculates a percentage based on the credits assigned to each criterion [18].

Case Study Experimental Data

A comparative study evaluating 16 chromatographic methods for hyoscine N-butyl bromide assay demonstrated the progressive discriminative power of these tools [17]. While NEMI could only differentiate 2 categories among the 16 methods, AGREE and GAPI provided detailed differentiation, with AGREE offering the additional advantage of an overall numerical score for ranking. The study found that AGREE provided reliable numerical assessments while highlighting the weakest points in analytical techniques needing greenness improvements [17].

Table 3: Research Reagent Solutions for Greenness Assessment

Tool/Resource	Primary Function	Application Context	Access Information
AGREE Software	Calculates and visualizes greenness score	Comprehensive method assessment	https://mostwiedzy.pl/AGREE [9]
MoGAPI Software	Modified GAPI with scoring system	Method comparison with visual output	bit.ly/MoGAPI [18]
NEMI Database	Method repository with basic greenness indicators	Initial method screening	http://www.nemi.gov [15]
AGREEprep	Specialized sample preparation assessment	Sample preparation optimization	Supplementary to full-method tools [5] [7]

The evolution from NEMI to AGREE represents a significant advancement in the comprehensiveness, usability, and informativeness of greenness assessment tools for analytical methods. While each tool has specific strengths and applications, AGREE currently offers the most balanced approach for researchers needing a comprehensive, flexible, and easily interpretable assessment framework. Its ability to incorporate all 12 principles of GAC with customizable weighting and provide both visual and numerical outputs makes it particularly valuable for drug development professionals seeking to implement environmentally sustainable analytical practices. As the field continues to evolve, integration of lifecycle assessment principles and carbon footprint calculations represents the next frontier in greenness metrics, further enabling analytical chemists to minimize the environmental impact of their methodologies.

A Step-by-Step Guide to Implementing AGREE in Pharmaceutical Analysis

The Analytical GREEness (AGREE) metric is a comprehensive, open-source assessment tool designed to evaluate the environmental impact of analytical methods based on the 12 principles of Green Analytical Chemistry (GAC). Developed to address the need for a standardized, quantitative approach to sustainability assessment in analytical chemistry, AGREE provides both a numerical score and an intuitive visual pictogram, enabling researchers to quickly evaluate and compare the greenness of their methodologies [9]. Unlike earlier metric systems that offered limited binary assessments, AGREE transforms each of the 12 GAC principles into a unified score from 0-1, with the final result calculated as the product of all individual scores and displayed in a clock-like graph for immediate interpretation [9].

The tool was created to overcome limitations of previous greenness assessment methods such as the National Environmental Methods Index (NEMI) and Analytical Eco-Scale, which either lacked comprehensive criteria or relied heavily on expert judgment [7] [9]. AGREE's development reflects the analytical chemistry community's growing commitment to environmental responsibility, enabling chemists to design, select, and implement methods that are both scientifically robust and ecologically sustainable [7]. Its flexibility allows application across various analytical techniques, from chromatographic methods to sample preparation protocols, making it particularly valuable for researchers in drug development and pharmaceutical analysis who must balance analytical performance with environmental considerations [19].

Input Requirements for AGREE

The 12 SIGNIFICANCE Principles

AGREE's assessment framework is built upon the 12 principles of Green Analytical Chemistry, encapsulated in the acronym "SIGNIFICANCE" [9] [19]. Each principle requires specific input data for evaluation, with performance translated to a score between 0 (least green) and 1 (most green). The following table summarizes the input requirements for each principle:

Table 1: Input Requirements for the 12 GAC Principles in AGREE

Principle #	GAC Principle	Required Input Data	Scoring Considerations
1	Sample treatment	Type of analysis (remote sensing, non-invasive, in-field, on-line, at-line, off-line, external pretreatment)	Remote sensing without sample damage scores highest (1.00); multistep external pretreatment scores lowest (0.00) [9]
2	In situ measurement	Sample size, number of samples	Based on sample mass/volume with logarithmic transformation; minimal consumption rewarded [9]
3	Greener sample source	Device location relative to sample source	On-site analysis preferred over transport to laboratory [19]
4	Number of steps	Number of sample preparation steps	Fewer steps yield higher scores [19]
5	Integration of processes	Degree of automation and miniaturization	Automated, miniaturized systems score higher [19]
6	Functionality and derivatization	Use of derivatization agents	Avoiding derivatization rewarded [19]
7	Impact on environment	Waste generated per analysis	Less waste generation yields higher scores [19]
8	Capacity for multi-analyte analysis	Number of analytes determined simultaneously	Higher throughput and more analytes per run scored higher [19]
9	Analysis throughput	Number of samples analyzed per unit time	Faster analysis with more samples rewarded [19]
10	Nature of reagents	Reagent sources (renewable vs. fossil-based)	Bio-based reagents preferred over fossil-based [19]
11	Chemical toxicity	Toxicity of reagents used	Based on GHS hazard categories; less hazardous reagents score higher [9]
12	Energy consumption	Energy requirements of equipment	Lower energy consumption yields higher scores [19]

Data Collection Methodology

Successful application of AGREE requires systematic collection of experimental data throughout method development and validation. Researchers should document:

• Sample Preparation Protocols: Record all steps, reagents, solvents, and their quantities [5]. For liquid chromatography-mass spectrometry (LC-MS) methods, this includes mobile phase composition, extraction solvents, and purification reagents [19].
• Energy Measurements: Document instrument power requirements, analysis time, and standby energy consumption [9].
• Waste Accounting: Calculate total waste generated per analysis, including solvents, consumables, and post-analysis residues [5] [19].
• Reagent Safety Data: Collect Safety Data Sheets (SDS) for all chemicals to determine toxicity classifications and hazard pictograms [19].
• Throughput Metrics: Record analysis time, sample preparation time, and number of analytes determined simultaneously [19].

The AGREE software, available for download from https://mostwiedzy.pl/AGREE, provides a user-friendly interface for inputting these parameters, with tooltips and guidance for each field [9].

Weighting System

A distinctive feature of AGREE is its flexible weighting system, which allows users to assign different levels of importance to the 12 principles based on their specific analytical goals and priorities [9]. Default weights are typically set to 2 for all principles, but researchers can adjust these from 1 (lowest importance) to 5 (highest importance) [19]. This flexibility is particularly valuable when certain environmental aspects are more critical for a specific application. For example, in pharmaceutical analysis, principles related to reagent toxicity (Principle 11) and operator safety (implied in Principle 12) might receive higher weights [19].

Table 2: Example Weighting Scheme for Pharmaceutical Analysis

Principle	Default Weight	Pharmaceutical Analysis Weight	Rationale
Waste (7)	2	4	Regulatory emphasis on waste reduction
Multi-analyte capacity (8)	2	4	High-value of multiplexed assays
Toxicity (11)	2	4	Operator safety and environmental compliance
Energy (12)	2	3	Laboratory energy management goals
Other principles	2	2	Standard importance

Output Interpretation

The AGREE Pictogram

The primary output of AGREE is a colorful, clock-like pictogram that provides both an overall greenness score and detailed performance information across all 12 principles [9]. This visual representation consists of:

• Central Score: A numerical value between 0 and 1 displayed in the center, with 1 representing ideal greenness [9]. The background color of this central circle ranges from deep red (0) to dark green (1), providing an immediate visual indicator of overall method greenness [9].
• Segmented Perimeter: Twelve colored sections corresponding to each GAC principle, with colors ranging from green (excellent performance) through yellow (moderate) to red (poor performance) [9].
• Weight Indicators: The width of each segment reflects the assigned weight, with wider segments indicating higher-weighted principles [9].

This compact visualization enables rapid assessment of both overall greenness and specific strengths and weaknesses of an analytical method [9].

Interpreting Scores and Colors

The following diagram illustrates the logical workflow for interpreting AGREE outputs and making informed decisions based on the results:

When interpreting AGREE results, researchers should consider:

• Overall Score Meaning: Scores above 0.75 indicate excellent greenness, 0.5-0.75 represent good performance, 0.25-0.5 suggest moderate greenness, and below 0.25 indicate poor environmental performance [9]. For context, a study evaluating LC-MS methods for neurotransmitter analysis reported scores around 0.66 for the greenest method [19].
• Segment Color Interpretation: Green segments (scores >0.7) indicate principles where the method performs well environmentally; yellow segments (0.4-0.7) represent moderate performance with room for improvement; red segments (<0.4) highlight significant environmental concerns that should be prioritized for optimization [9].
• Weight Considerations: Principles with wider segments (higher weights) have greater influence on the overall score. Improvements in these high-weight areas will yield more significant greenness enhancements [9] [19].

Comparative Analysis

AGREE enables direct comparison between different analytical methods, helping researchers select the most environmentally sustainable approach. When comparing methods:

• Evaluate overall scores as primary indicators of general greenness [9]
• Examine specific segment colors to identify relative strengths and weaknesses of each method [19]
• Consider the impact of weighting adjustments on method ranking, particularly for application-specific assessments [19]

For example, in a case study evaluating sugaring-out liquid-liquid microextraction (SULLME) methods for antiviral compound determination, AGREE provided a balanced green profile with a score of 0.56, highlighting strengths in miniaturization and automation but weaknesses in waste generation and reagent safety [7].

Implementation Protocols

Step-by-Step Assessment Methodology

Implementing AGREE effectively requires a systematic approach to ensure comprehensive and accurate assessments:

• Method Documentation: Compile complete methodological details including sample preparation steps, reagent types and quantities, instrument parameters, analysis time, and waste streams [19].
• Data Quantification: Measure or calculate exact values for each parameter relevant to the 12 GAC principles, including energy consumption (kWh), waste volume (mL), sample size (mL or g), and number of samples processed per hour [9] [19].
• Software Input: Enter quantified data into the AGREE software, ensuring appropriate unit conversions and selections from dropdown menus where applicable [9].
• Weight Assignment: Adjust principle weights based on analytical priorities and specific application requirements [19].
• Result Analysis: Interpret the output pictogram to identify environmental hotspots and improvement opportunities [9].
• Method Optimization: Iteratively refine the analytical method to address identified weaknesses and reassess with AGREE to quantify improvements [7].

Research Reagent Solutions

Selecting appropriate reagents and materials is crucial for improving analytical method greenness. The following table outlines key research reagent solutions and their functions in sustainable method development:

Table 3: Research Reagent Solutions for Greener Analytical Methods

Reagent/Material	Function	Green Considerations
Bio-based solvents (e.g., ethanol, ethyl lactate)	Extraction, mobile phase components	Renewable sources, lower toxicity compared to petroleum-based solvents [19]
Water-based mobile phases	Chromatographic separation	Reduced organic solvent consumption, safer disposal [19]
Miniaturized extraction devices	Sample preparation	Reduced reagent consumption and waste generation [7]
Automated systems	Sample processing, analysis	Improved precision, reduced reagent use, higher throughput [19]
Derivatization-free columns	Chromatographic analysis	Avoids use of hazardous derivatization reagents [19]
Solid-phase microextraction (SPME)	Sample preparation	Solvent-free extraction, minimal waste [7]

Current Limitations and Future Perspectives

While AGREE represents a significant advancement in greenness assessment, several limitations should be acknowledged. The tool does not sufficiently account for pre-analytical processes such as reagent synthesis or probe preparation [7]. Some assessment steps can be challenging due to unavailable essential data or poorly defined parameters in original method descriptions [5]. The weighting system, while flexible, introduces subjectivity and may lead to inconsistent assessments if not carefully applied [20].

Future developments in green metrics are likely to address these limitations through integration with complementary tools. The emerging Analytical Green Star Analysis (AGSA) offers a star-shaped diagram assessing additional criteria [7], while the Carbon Footprint Reduction Index (CaFRI) focuses specifically on climate impact [7]. The trend is toward multidimensional assessment frameworks that combine environmental, practical, and performance criteria, as exemplified by White Analytical Chemistry (WAC) approaches [7] [20].

For researchers in drug development, effectively navigating AGREE software requires meticulous attention to input data quality, thoughtful application of weighting schemes reflective of pharmaceutical priorities, and critical interpretation of output visualizations. When integrated early in method development cycles, AGREE serves as both assessment tool and design guide, steering analytical choices toward improved environmental sustainability without compromising analytical performance.

The Analytical GREEnness (AGREE) metric is a comprehensive, flexible, and straightforward assessment tool designed to evaluate the environmental impact of analytical procedures. It translates the multifaceted concept of greenness in analytical chemistry into a single, easily interpretable score. The tool is directly based on the 12 principles of green analytical chemistry (GAC), providing a more holistic and nuanced evaluation compared to earlier metric systems [9].

The primary output of an AGREE assessment is a distinctive, clock-like pictogram that presents the final greenness score on a 0–1 scale and visually represents the performance of the analytical procedure across each of the 12 GAC principles. This approach was developed to address the limitations of prior tools, which often considered only a few assessment criteria or treated them as non-continuous functions, thereby offering a more sensitive and informative gauge of a method's environmental footprint [9]. The open-source software for AGREE makes this assessment procedure accessible and straightforward for researchers and scientists [2].

The Foundation: The 12 Principles of GAC

The AGREE metric's assessment criteria are derived from the 12 principles of Green Analytical Chemistry, encapsulated by the mnemonic SIGNIFICANCE [9]. Each principle is converted into a score on a unified 0–1 scale. The following table summarizes these principles and their transformation into the AGREE scoring system.

Table 1: The 12 Principles of Green Analytical Chemistry and Their AGREE Score Transformation

Principle Number	GAC Principle Description	Key Considerations for Scoring
1	Select direct analytical techniques	Avoids sample treatment; scores higher for remote sensing, non-invasive, or in-field analysis [9].
2	Integrate analytical processes & operations	Minimizes sample size and number of samples [9].
3	Generate as little waste as possible & proper waste management	Quantifies waste generated during the analytical procedure [9].
4	No internal standard / multi-analyte procedures	Prefers multi-analyte or multi-purpose methods over single-analyte approaches [9].
5	In-line, real-time measurement	Maximizes sample throughput and automation [9].
6	Fast & energy-efficient measurements	Minimizes total energy consumption [9].
7	Intrinsic & inherent safety	Uses safer, less toxic, and less hazardous chemicals [9].
8	Carry out micro- & nano- analyses	Employs miniaturized and scaled-down techniques [9].
9	Apply derivatives & other additives only when necessary	Avoids or minimizes the use of additional reagents [9].
10	Natural & renewable materials	Prefers reagents obtained from renewable sources [9].
11	Clean & direct methods for detection	Chooses cleaner detection strategies (e.g., spectroscopic vs. electrochemical) [9].
12	Eliminate or replace polluting sample prep	Avoids sample preparation or uses greener sample preparation alternatives [9].

The AGREE Scoring Algorithm

The 0-1 Scale and Final Score Calculation

The AGREE metric transforms each of the 12 GAC principles into a normalized score on a continuous scale from 0 to 1. A score of 1.00 represents ideal green performance for a given principle, while a score of 0.00 indicates the worst possible performance [9].

The final overall AGREE score is not a simple average. It is calculated as the product of the assessment results for each of the 12 principles. This overall score is also a value between 0 and 1, presented in the center of the pictogram. A value close to 1, accompanied by a dark green color, indicates that the assessed procedure is exceptionally green, while a lower score and a redder color signify a larger environmental footprint [9].

Incorporating User-Defined Weights

A critical feature of the AGREE metric is its flexibility to account for the variable importance of different GAC principles depending on the specific analytical context. Users can assign a weight to each of the 12 principles, influencing the final score to reflect priorities such as minimizing waste, reducing energy consumption, or ensuring operator safety [9].

In the resulting pictogram, the width of each segment corresponds to the weight assigned to that principle. A wider segment indicates a criterion of higher importance in the assessment, allowing for an immediate visual understanding of the assessment's focus areas [9].

Deciphering the Color-Coded Clock Pictogram

The AGREE software generates an intuitive, clock-like pictogram that synthesizes all assessment information into an easily interpretable visual. The diagram below illustrates the structure and information encoded in this pictogram.

AGREE Pictogram Structure and Interpretation

The pictogram provides a multi-layered summary of the greenness assessment:

• Overall Score and Central Color: The number in the center is the final AGREE score. The color of the center circle provides an immediate visual cue: dark green for excellent performance (score close to 1), transitioning to red for poor performance (score close to 0) [9].
• Segment Color (Performance per Principle): The color of each numbered segment indicates the performance for that specific GAC principle, using a traffic-light code [9]:
  • Green: Excellent performance (score close to 1.0)
  • Yellow: Moderate performance
  • Red: Poor performance (score close to 0.0)
• Segment Width (Criterion Weight): The width of each segment visually represents the relative weight assigned by the user to that principle. A wider segment signifies a criterion deemed more critical for the specific assessment [9].

Experimental Protocol for AGREE Application

Applying the AGREE metric is a systematic process that requires detailed knowledge of the analytical procedure being evaluated. The following workflow provides a step-by-step methodology.

AGREE Assessment Workflow

Step 1: Procedure Definition and Data Collection

The first step involves a thorough compilation of all data related to the analytical method. This includes quantitative measurements and qualitative choices. The required information can be categorized as follows:

Table 2: Essential Data Categories for AGREE Assessment

Data Category	Specific Data Requirements
Sample Handling	Sample size, number of samples, type of treatment (e.g., direct analysis, off-line, on-line), number of preparation steps [9].
Reagents & Solvents	Identity, quantities, toxicity (e.g., from Safety Data Sheets), and whether they are derived from renewable sources [9].
Energy Consumption	Total energy requirements of equipment used throughout the procedure [9].
Waste Generation	Amount and nature of waste produced [9].
Method Strategy	Number of analytes targeted, use of derivatization, degree of automation, and sample throughput [9].

Step 2: Data Input and Weight Assignment

Enter the collected data into the AGREE software, which will use built-in algorithms to convert the inputs into a score between 0 and 1 for each of the 12 principles [9]. The software provides a user-friendly interface for this purpose.

Simultaneously, the user must decide if all principles are equally important. If not, assign a weight to each principle. The AGREE software allows for this customization, and the default setting is equal weight for all principles. Adjusting weights is crucial for tailoring the assessment to specific priorities, such as emphasizing waste reduction or operator safety [9].

Step 3: Calculation and Pictogram Generation

Execute the calculation within the AGREE software. The tool will automatically compute the final score and generate the color-coded clock pictogram based on the input data and assigned weights [9]. The software is open-source and freely available for download, ensuring accessibility for all researchers [2].

Step 4: Results Interpretation and Method Improvement

Analyze the generated pictogram to:

• Identify the overall greenness via the central score and color.
• Pinpoint strengths and weaknesses by observing the color of each segment. Red and yellow segments highlight areas requiring improvement.
• Understand assessment priorities by noting the width of the segments.

This analysis provides a clear, actionable roadmap for refining the analytical procedure to enhance its environmental profile. For instance, a red segment for Principle 7 (chemical safety) would indicate a need to replace hazardous chemicals with safer alternatives.

Effectively implementing the AGREE metric requires access to specific software tools and an understanding of related assessment frameworks.

Table 3: Essential Tools for Greenness Assessment

Tool Name	Function	Access / Notes
AGREE Software	The primary tool for calculating the AGREE score and generating the color-coded clock pictogram based on the 12 GAC principles.	Freely available, open-source software downloadable from: https://mostwiedzy.pl/AGREE [2] [9].
AGREEprep	A dedicated metric tool for a more granular assessment of the sample preparation step, based on 10 principles of green sample preparation.	An open-access software available from: https://mostwiedzy.pl/AGREEprep. It complements AGREE by focusing on the often most impactful step [3].
Safety Data Sheets (SDS)	Critical information sources for determining the toxicity and hazards of reagents and solvents used in the assessment of Principle 7.	Required for proper scoring of reagent-related criteria [9].
Other Metric Tools (e.g., NEMI, GAPI, Eco-Scale)	Earlier or alternative greenness assessment tools.	Useful for comparative studies or for focusing on specific aspects not covered in depth by AGREE [21] [9].

The Analytical GREEnness (AGREE) metric is a comprehensive assessment tool designed to evaluate the environmental impact of analytical procedures. Developed in 2020, it provides analytical chemists with a standardized approach to quantify how well their methods align with the 12 principles of Green Analytical Chemistry (GAC) [9]. Unlike earlier metric systems that offered binary or limited assessments, AGREE translates complex environmental considerations into an easily interpretable score between 0 and 1, where higher scores indicate greener methods [9] [7]. This capability to provide a unified, quantitative output has positioned AGREE as a significant advancement in the sustainable transformation of analytical science, enabling researchers to systematically evaluate and improve the ecological footprint of their chromatographic methods [7].

The transition toward green analytical chemistry represents a fundamental shift in how scientists approach method development. GAC emerged as an extension of green chemistry, specifically focused on minimizing the environmental footprint of analytical techniques and procedures [7]. This involves reducing or eliminating dangerous solvents, reagents, and materials while maintaining rigorous validation parameters and providing rapid, energy-saving methodologies [7]. Within this context, AGREE serves as a cornerstone tool that helps balance analytical performance with environmental responsibility, creating a new paradigm where ecological impact becomes a critical validation parameter alongside traditional metrics like accuracy, precision, and sensitivity [22].

The AGREE Assessment Framework: Structure and Calculation

The 12 Principles of Green Analytical Chemistry

The AGREE metric is built upon a comprehensive foundation of the 12 SIGNIFICANCE principles of Green Analytical Chemistry, which cover the multifaceted aspects of environmental impact in analytical methods [9]. Each principle is transformed into a score on a 0-1 scale, with the final assessment result being the product of the evaluation across all principles [9]. The framework's output is an intuitive, clock-like pictogram that displays both the overall score and color in the center (ranging from red for poor performance to dark green for excellent greenness), while the performance for each individual principle is indicated by color in the corresponding segment [9].

Table 1: The 12 SIGNIFICANCE Principles of Green Analytical Chemistry in AGREE

Principle Number	Principle Description	Key Assessment Considerations
1	Direct analytical techniques should be applied to avoid sample treatment	Remote sensing, non-invasive analysis, sample preparation steps [9]
2	Minimal sample size and minimal number of samples are goals	Sample volume, number of samples, miniaturization [9]
3	Integration of analytical operations and in-situ measurement are desirable	Automation, on-line analysis, procedural steps [9]
4	Natural, reproducible and biodegradable reagents should be selected	Reagent toxicity, biodegradability, sourcing [9]
5	Waste generation should be minimized or waste should be recycled during analysis	Waste volume, waste treatment, recycling [9]
6	Multi-analyte or multi-parameter methods are preferred versus methods for one analyte	Method multiplexing capability [9]
7	The use of energy should be minimized	Energy consumption, energy source [9]
8	Throughput should be increased	Samples per hour, analysis time [9]
9	Reagents obtained from renewable source should be preferred	Reagent renewability, bio-based sources [9]
10	Bioanalytical methods should be further developed	Biologically-based methods [9]
11	Toxic reagents should be eliminated or replaced	Reagent hazards, toxicity, substitution [9]
12	Operator's safety should be increased	Operational hazards, exposure risks [9]

AGREE Software and Visualization

A key advantage of the AGREE system is its accessibility through free, open-source software, available for download from https://mostwiedzy.pl/AGREE [9] [23]. The software features a user-friendly interface that guides analysts through the assessment procedure, automatically generating the characteristic circular pictogram and an assessment report [9]. The visualization not only shows the final score but also reflects the user-assigned weights for each principle through the width of the corresponding segments, allowing for customizable assessments based on specific analytical priorities or constraints [9].

The AGREE pictogram provides immediate visual feedback on both strengths and weaknesses of an analytical method's environmental profile. For example, in the case study assessment shown in Figure 1, principles 4, 6, 9, and 12 show excellent performance (green segments), while principles 1, 5, and 7 show relatively poor performance (yellow/red segments) [9]. This detailed breakdown enables method developers to identify specific areas for improvement rather than simply receiving an overall score, making AGREE both a diagnostic and development tool for greener chromatographic methods [9].

Figure 1: AGREE Metric Assessment Workflow. This flowchart illustrates the step-by-step process for evaluating the greenness of an analytical method using the AGREE metric.

AGREE in Practice: Chromatographic Method Case Studies

Pharmaceutical Analysis: Simultaneous Determination of Antihypertensive Drugs

A recent study developed and validated a green HPLC method for the simultaneous determination of four antihypertensive drugs: Nebivolol hydrochloride, Telmisartan, Valsartan, and Amlodipine besylate [24]. The method utilized a mobile phase composed of 0.1% formic acid in water (pH: 2.5) and ethanol, with a regular octadecyl silica (ODS) column and UV detection at 220 nm [24]. The researchers evaluated the method's environmental profile using AGREE alongside other green metrics, confirming its alignment with sustainable practices and reduced ecological impact [24]. The method demonstrated several green characteristics, including the use of ethanol as a safer alternative to traditional acetonitrile, which contributed to favorable scores in principles related to reagent safety, toxicity, and renewability [24].

Analysis of UV Filters in Cosmetic Products

A comprehensive 2023 study compared the greenness of 10 different chromatographic methods for determining UV filters in cosmetic samples using AGREE and AGREEprep (a complementary tool specifically designed for sample preparation) [23]. The evaluation included methods based on various sample preparation techniques, from conventional solvent extraction to modern microextraction approaches [23]. The results clearly demonstrated that microextraction methods consistently achieved higher greenness scores, with AGREE effectively quantifying the environmental advantages of these miniaturized approaches [23]. This case study highlights how AGREE can guide analysts toward more sustainable method selection in industrial applications, where routine analysis of multiple samples creates significant cumulative environmental impact [23].

Simultaneous Analysis of Aspirin and Vonoprazan

A 2024 study developed two chromatographic methods (HPLC-DAD and HPTLC) for the simultaneous analysis of Aspirin and the novel gastro-protective agent Vonoprazan in combined dosage forms [22]. The HPLC method employed a C18 column with isocratic elution using phosphate buffer (pH 6.8) and acetonitrile, while the HPTLC method used silica plates with a mobile phase of ethyl acetate:ethanol:ammonia [22]. When evaluated with AGREE, both methods demonstrated acceptable greenness profiles, supporting their potential for routine quality control analysis while minimizing environmental impact [22]. The study illustrated how AGREE can be applied during method development to optimize ecological performance while maintaining analytical validity according to International Council for Harmonization guidelines [22].

Table 2: AGREE Scores for Different Chromatographic Methods in Published Case Studies

Analytical Application	Analytical Technique	Key Green Features	AGREE Score
Antihypertensive Drug Combination [24]	HPLC-UV	Ethanol as mobile phase component	Reported as "green" (exact score not specified)
UV Filter Analysis [23]	Various chromatographic methods with different sample preparations	Microextraction techniques	Higher scores for microextraction vs. conventional methods
Aspirin and Vonoprazan Combination [22]	HPLC-DAD and HPTLC	Isocratic elution, ethanol in mobile phase	Acceptable greenness (exact score not specified)
SULLME for Antiviral Compounds [7]	Homogeneous liquid-liquid microextraction	Miniaturization, reduced solvent consumption	56/100

Implementation Guide: Applying AGREE to Chromatographic Method Development

Systematic AGREE Evaluation of HPLC Methods

When developing a new chromatographic method, AGREE should be integrated throughout the development and validation process rather than applied as a final check. Systematic evaluation begins with documenting all method parameters that correspond to the 12 GAC principles [9]. For HPLC methods, this includes detailed recording of sample preparation steps, sample volume, reagent types and volumes, energy consumption (including column temperature and detection parameters), waste generation, and throughput [9] [7]. This comprehensive data collection ensures accurate AGREE assessment and identifies improvement opportunities early in development.

For sample preparation (Principle 1), the method should be scored according to the hierarchy of greenness, with remote sensing without sample damage representing the ideal (score = 1.00) and external sample pre-treatment with multiple steps representing the least green alternative (score = 0.00) [9]. For sample size and number (Principle 2), the assessment considers both the volume/mass of individual samples and the total number of samples required for reliable analysis, with miniaturization approaches receiving more favorable scores [9]. Reagent selection (Principles 4, 9, 11) requires evaluation of toxicity, biodegradability, and renewability, favoring ethanol over acetonitrile or methanol in reversed-phase chromatography, for example [24] [22].

Experimental Protocol for AGREE-Assisted Method Development

• Define Analytical Requirements: Clearly establish the analytical goals, including target analytes, required sensitivity, linearity, precision, and matrix considerations [24] [22].

• Select Chromatographic Platform: Choose between HPLC, UHPLC, HPTLC, or GC based on separation requirements, with consideration of their differing environmental footprints [23] [22].

• Design Initial Method Parameters:

  • Mobile phase composition: Prioritize water-ethanol, water-acetone, or potentially supercritical fluid chromatography mobile phases over traditional acetonitrile-based systems [24] [7].
  • Column selection: Choose appropriate column dimensions, with narrower-bore columns typically reducing solvent consumption [22].
  • Detection method: Consider the relative energy requirements of UV, DAD, MS, or other detection systems [23].

• Optimize Sample Preparation:

  • Evaluate direct injection possibilities to eliminate extraction steps [9].
  • If extraction is necessary, implement miniaturized techniques such as microextraction, which consistently demonstrate higher greenness scores in AGREE evaluations [23].
  • Minimize or eliminate derivatization requirements [23].

• Conduct AGREE Assessment: Input all method parameters into the AGREE software, assigning appropriate weights to each principle based on analytical priorities [9].

• Iterative Improvement: Use AGREE results to identify environmental weaknesses and refine method parameters accordingly, repeating the assessment until achieving an acceptable greenness profile while maintaining analytical performance [7] [22].

• Validation and Documentation: Validate the final method according to regulatory requirements while fully documenting the AGREE assessment as part of method characterization [22].

Research Reagent Solutions for Green Chromatography

Table 3: Essential Reagents and Materials for Developing Green Chromatographic Methods

Reagent/Material	Function in Chromatography	Green Characteristics	Application Examples
Ethanol	Mobile phase component	Renewable, biodegradable, less toxic	Replacement for acetonitrile or methanol in reversed-phase HPLC [24] [22]
Water	Mobile phase component	Non-toxic, safe, readily available	Primary solvent in reversed-phase systems [24]
Supercritical COâ‚‚	Mobile phase in SFC	Non-toxic, easily removed, recyclable	Replacement for organic solvents in normal-phase separations
Ethyl Acetate	Normal phase mobile phase	Less hazardous than chlorinated solvents	HPTLC mobile phase component [22]
Octadecyl Silica (C18) Columns	Stationary phase for reversed-phase	Versatile, compatible with green mobile phases	Pharmaceutical analysis with ethanol-water mobile phases [24] [22]

Comparative Analysis: AGREE Versus Other Green Assessment Tools

The landscape of green assessment metrics for analytical methods has evolved significantly from early tools like the National Environmental Methods Index (NEMI) to more comprehensive contemporary models [7]. AGREE occupies an important position in this ecosystem, offering distinct advantages through its foundation in the 12 GAC principles and its balanced approach to comprehensiveness and user-friendliness [9] [7]. Understanding how AGREE compares with other metrics enables researchers to select the most appropriate assessment tool for their specific needs or to utilize complementary approaches for a more holistic environmental profile.

Table 4: Comparison of AGREE with Other Green Assessment Metrics

Metric Tool	Key Features	Advantages	Limitations
AGREE [9] [7]	Based on 12 GAC principles; 0-1 score; clock pictogram	Comprehensive; user-friendly software; customizable weights	Does not fully account for pre-analytical processes
NEMI [7]	Binary assessment; 4 criteria; simple pictogram	Simple implementation; easy interpretation	Limited criteria; lacks granularity; no score
Analytical Eco-Scale [7]	Penalty point system; base of 100	Quantitative results; facilitates comparison	Relies on expert judgment; no visual component
GAPI [7]	Multi-criteria; color-coded pictogram	Comprehensive; visual identification of weak points	No overall score; somewhat subjective
AGREEprep [5] [23]	Focuses on sample preparation; 10 principles	Addresses often-overlooked sample preparation stage	Must be used with other tools for full method assessment

The integration of multiple assessment tools can provide the most comprehensive evaluation of a method's environmental impact. A 2025 case study evaluating a sugaring-out liquid-liquid microextraction (SULLME) method used Modified GAPI (MoGAPI), AGREE, AGSA, and the Carbon Footprint Reduction Index (CaFRI) to obtain a multidimensional sustainability perspective [7]. While MoGAPI provided a moderate score of 60/100 and highlighted concerns about waste generation and operator safety, AGREE delivered a similar score of 56/100, specifically noting strengths in miniaturization and semi-automation but weaknesses in throughput and residual toxicity concerns [7]. This complementary approach demonstrates how different metrics can highlight various aspects of a method's environmental profile, with AGREE particularly strong in assessing alignment with core GAC principles.

The AGREE metric represents a significant advancement in the practical implementation of Green Analytical Chemistry principles, particularly in the field of chromatographic method development. By providing a comprehensive, quantitative, and visually intuitive assessment framework, AGREE enables researchers to systematically evaluate and improve the environmental footprint of their analytical procedures while maintaining rigorous performance standards [9] [7]. The case studies examined demonstrate AGREE's practical application across diverse pharmaceutical and cosmetic analysis scenarios, highlighting its effectiveness in guiding the development of more sustainable chromatographic methods [24] [23] [22].

As analytical chemistry continues to evolve toward greater sustainability, tools like AGREE will play an increasingly crucial role in balancing analytical performance with environmental responsibility. The ongoing development of complementary metrics, such as AGREEprep for sample preparation and CaFRI for carbon footprint assessment, further enriches the toolbox available to environmentally conscious scientists [5] [7]. By integrating AGREE into routine method development and validation processes, researchers and drug development professionals can contribute meaningfully to the reduction of analytical chemistry's ecological impact while maintaining the high-quality standards required for pharmaceutical analysis and regulatory compliance.

Integrating AGREE with Method Validation in Regulatory Contexts

The integration of environmental sustainability into analytical science is increasingly critical in regulated industries. Green Analytical Chemistry (GAC) aims to minimize the environmental footprint of analytical methods while maintaining data quality and regulatory compliance [7]. The Analytical GREEnness (AGREE) metric has emerged as a comprehensive, user-friendly tool that quantifies method sustainability based on the 12 principles of GAC [9]. Unlike earlier assessment tools with limited scope, AGREE provides a holistic evaluation through a unified pictogram and numerical score, enabling researchers to make informed decisions that balance analytical performance with environmental responsibility [9] [7].

Within pharmaceutical development and other regulated sectors, method validation demonstrates that analytical procedures are suitable for their intended use, encompassing parameters such as accuracy, precision, and specificity [25] [26]. The contemporary regulatory landscape, guided by frameworks like ICH Q14 on Analytical Procedure Development, encourages a lifecycle management approach where methods evolve in response to new data, processes, and technologies [27]. This creates a strategic opportunity to systematically incorporate greenness assessment into established validation workflows, ensuring that sustainability becomes a fundamental consideration alongside traditional performance metrics [27] [7].

The AGREE Metric: Framework and Components

The 12 Principles of Green Analytical Chemistry

The AGREE metric is built upon the 12 SIGNIFICANCE principles of GAC, which comprehensively address the environmental impact of analytical procedures [9]. The tool transforms these principles into a standardized scoring system where each criterion is rated on a 0-1 scale. The final score, presented both numerically and via an intuitive clock-shaped pictogram, offers an at-a-glance assessment of a method's greenness [9]. The calculator is open-source and publicly available, promoting widespread adoption [9].

The following table details the 12 principles and their core considerations within the AGREE framework:

Table 1: The 12 SIGNIFICANCE Principles of Green Analytical Chemistry in AGREE

Principle Number	Principle Focus	Key Assessment Criteria
1	Direct Analysis	Avoidance of sample treatment; use of remote, non-invasive, or on-line techniques [9].
2	Sample & Size Minimization	Minimal sample size and number of samples without jeopardizing representativeness [9].
3	In-situ Measurement	Conducting analysis in the field or at the sample source to minimize transport [9].
4	Integration & Automation	Combining steps, automation, and on-line coupling to reduce manual operations and errors [9].
5	Waste Minimization	Reducing and managing the volume and toxicity of waste generated [9] [7].
6	Energy Reduction	Minimizing total energy consumption and using renewable sources when possible [7].
7	Reagent Safety	Selecting safer, less toxic, and bio-based reagents [9] [7].
8	Operator Safety	Reducing exposure to hazardous chemicals and conditions [7].
9	Derivatization Avoidance	Eliminating or reducing unnecessary derivatization steps [9].
10	Renewable Resources	Using reagents and materials derived from renewable sources [9].
11	Degradability	Selecting reagents and waste products that are readily degradable [9].
12	Accident Prevention	Implementing inherently safer processes to prevent accidents [9].

AGREE Software and Scoring Output

The AGREE software requires users to input data corresponding to each of the 12 principles. A key feature is its flexibility; users can assign different weights to each criterion based on their specific analytical goals or regulatory priorities [9]. For instance, a method intended for a quality-control lab might prioritize high throughput (Principle 4), while one for environmental field testing might emphasize portability and low energy consumption (Principle 3 and 6).

The output is a circular pictogram where:

• Each of the 12 segments represents one GAC principle.
• The color of each segment (red-yellow-green) indicates performance for that criterion.
• The width of each segment reflects the user-assigned weight.
• The central numerical score (0-1) provides the overall greenness rating, with 1 being ideal [9].

This visual output allows for immediate identification of a method's environmental strengths and weaknesses, guiding efforts for improvement [9] [7].

AGREE Assessment Workflow

Method Validation in Regulatory Frameworks

Core Principles of Method Validation

Method validation is a foundational requirement in regulated laboratories, providing documented evidence that an analytical procedure is fit for its intended purpose [26]. It is a comprehensive process required when developing new methods or substantially modifying existing ones. Regulatory bodies like the FDA, EMA, and ICH provide guidelines outlining key validation parameters [25].

According to the Organisation for Economic Co-operation and Development (OECD), validation establishes "the reliability and relevance of a particular approach, method, process or assessment for a defined purpose" [28]. Reliability ensures the method produces reproducible results across different laboratories and over time, while relevance confirms it meaningfully tests the effect of interest [28].

Table 2: Core Parameters of Analytical Method Validation

Validation Parameter	Definition	Regulatory Purpose
Accuracy	Closeness between measured value and true value	Ensures results are correct and unbiased [26].
Precision	Agreement among a series of measurements	Confirms method reproducibility under defined conditions [26].
Specificity	Ability to measure analyte accurately in the presence of interferences	Demonstrates method selectivity for the target analyte [25].
Linearity	Ability to obtain results proportional to analyte concentration	Establains the validated concentration range [26].
Range	Interval between upper and lower analyte concentrations	Defines the limits of acceptable performance for the method [26].
Limit of Detection (LOD)	Lowest amount of analyte detectable but not necessarily quantifiable	Determines method sensitivity for trace analysis [26].
Limit of Quantification (LOQ)	Lowest amount of analyte that can be quantitatively measured	Defines the lower limit of the quantitative range [26].
Robustness	Capacity to remain unaffected by small, deliberate variations in method parameters	Evaluates method reliability during normal use and transfer [27].

Method Verification for Compendial Procedures

In contrast to full validation, method verification is the process of confirming that a previously validated method (e.g., a compendial method from USP or EP) performs as expected in a specific laboratory, with its unique analysts, instruments, and reagents [26]. It is a less exhaustive but essential process for quality assurance, focusing on critical parameters like accuracy and precision to ensure the method's suitability under local conditions [26].

The choice between validation and verification is strategic. Validation is mandatory for novel methods, while verification is efficient and sufficient for implementing standard methods [26].

Strategic Integration of AGREE into the Method Lifecycle

The integration of AGREE into the method lifecycle, guided by ICH Q14, transforms sustainability from an afterthought into a core pillar of analytical procedure development and management [27]. This integration can be visualized as a continuous, iterative process.

AGREE in the Method Lifecycle

Integration during Method Development and Validation

During the initial development phase, AGREE acts as a design guide. By considering the 12 GAC principles, scientists can make conscious choices that enhance greenness without compromising analytical performance [9] [27]. Key strategies include:

• Miniaturization: Reducing sample and solvent volumes directly improves scores for Principles 2 (minimal sample size) and 5 (waste minimization) [7].
• Automation and Integration: Incorporating on-line sample preparation or autosamplers supports Principles 1 (direct analysis) and 4 (integration), while also improving precision and robustness—key validation parameters [9] [26].
• Safer Solvent Selection: Choosing solvents with lower toxicity and higher biodegradability positively impacts Principles 7 (reagent safety) and 11 (degradability) [7].

When validating a new method, the AGREE score should be calculated and included in the validation report as a supplementary greenness profile. This documents the environmental performance of the method at its inception, providing a baseline for future comparisons [27].

Integration for Method Changes and Equivalency Studies

Change is inevitable in a method's lifecycle, driven by technology upgrades, process improvements, or regulatory updates [27]. ICH Q14 encourages a structured, risk-based approach to managing these changes. When a method modification is proposed, a comparative AGREE assessment provides quantitative data to support comparability or equivalency studies [27].

• Comparability: For minor changes, demonstrating that the AGREE score is maintained or improved provides supporting evidence that the method's overall profile is not adversely affected.
• Equivalency: For major changes or method replacements, a side-by-side AGREE comparison, alongside traditional performance data, offers a more comprehensive view. A significant improvement in the AGREE score can be a strong justification for implementing a more sustainable alternative [27].

Experimental Protocol for an Integrated Assessment

This protocol provides a step-by-step guide for conducting a joint method validation and AGREE greenness assessment.

Phase 1: Method Design and Preliminary Greenness Evaluation

• Define the Analytical Target Profile (ATP): Specify all critical performance criteria (e.g., LOD, LOQ, precision) as per regulatory guidelines [27] [26].
• Draft the Analytical Procedure: Outline all steps from sample collection to final analysis.
• Perform a Preliminary AGREE Assessment:
  • Use the draft procedure to input data into the AGREE calculator for all 12 principles.
  • Assign weights based on regulatory and environmental priorities.
  • Analyze the initial pictogram to identify "red" segments (poor performance).
• Design Optimization: Explore alternative reagents, instruments, or workflows to improve the AGREE score while ensuring the ATP can still be met. This may involve evaluating different extraction techniques or solvent systems.

Phase 2: Validation and Final Greenness Profiling

• Execute Validation Experiments: Conduct laboratory studies to gather data for all validation parameters outlined in Table 2 (Accuracy, Precision, etc.) [26].
• Document Performance: Compile results, ensuring they meet pre-defined acceptance criteria.
• Finalize AGREE Profile: Using the finalized and validated method parameters, calculate the final AGREE score and generate the pictogram.
• Compile Integrated Report: Create a comprehensive report that includes:
  • The full method validation data and results.
  • The final AGREE pictogram and score.
  • A discussion of how greenness principles were incorporated into the method design.

The Scientist's Toolkit for Integrated Studies

Table 3: Essential Reagents and Materials for Green Method Development

Item Category	Specific Examples	Function & Green Principle Addressed
Green Solvents	Bio-based ethanol, ethyl acetate, cyclopentyl methyl ether	Lower toxicity and higher biodegradability vs. traditional solvents (Principle 7, 11) [7].
Miniaturized Equipment	Micro-extraction devices (SPME, MEPS), micro-volume cuvettes, microfluidic chips	Reduces consumption of samples and solvents (Principle 2, 5) [9] [7].
Automated Systems	Automated liquid handlers, on-line SPE instruments, autosamplers	Improves precision, reduces analyst exposure, and integrates steps (Principle 4, 8) [27].
Reference Standards	Certified Reference Materials (CRMs)	Ensures accuracy and traceability during validation, fundamental to method reliability [26].
Waste Management Kits	Solvent recycling units, neutralization kits	Manages hazardous waste generated, reducing environmental impact (Principle 5) [7].
Coccinine	Coccinine, CAS:139068-98-7, MF:C17H19NO4, MW:301.34 g/mol	Chemical Reagent
Mniopetal B	Mniopetal B|Reverse Transcriptase Inhibitor	Mniopetal B is a natural product that inhibits viral reverse transcriptase . This product is For Research Use Only. Not for human or diagnostic use.

Case Study and Comparative Analysis

A case study evaluating a Sugaring-Out Liquid-Liquid Microextraction (SULLME) method for determining antiviral compounds illustrates the complementary use of multiple metrics [7]. The method was assessed using AGREE alongside other tools.

The AGREE assessment yielded a score of 56 out of 100 [7]. The analysis highlighted:

• Strengths: The method benefited from miniaturization, semi-automation, a small sample volume (1 mL), and the use of some bio-based reagents, aligning well with several GAC principles [7].
• Weaknesses: The use of toxic and flammable solvents, moderate waste generation, and relatively low throughput (2 samples/hour) were identified as areas detracting from the greenness profile [7].

This multi-metric evaluation provided a multidimensional view of the method's sustainability, demonstrating that while miniaturization offers significant advantages, other factors like reagent safety and energy efficiency require concurrent attention for a truly green method [7].

Regulatory and Future Perspectives

The regulatory environment is evolving toward greater acceptance of modernized, science-driven approaches. ICH Q14 formalizes the Analytical Procedure Lifecycle concept, creating a natural platform for incorporating sustainability metrics like AGREE [27]. While current regulations do not yet mandate greenness scores, presenting AGREE data in regulatory submissions can demonstrate a proactive commitment to Environmental, Social, and Governance (ESG) principles and showcase a more holistic approach to method quality [27].

The future of integrated assessment will likely involve:

• Evolution of Metrics: Newer tools like AGREEprep (for sample preparation) and the Carbon Footprint Reduction Index (CaFRI) are already emerging, offering more specialized and lifecycle-oriented assessments [5] [7].
• Standardization: As the scientific consensus strengthens, standardized approaches for weighting AGREE criteria or defining acceptance thresholds for greenness scores may develop, further facilitating their use in regulatory contexts [9].
• Digital Integration: The AGREE software and similar tools could be integrated into electronic laboratory notebooks (ELNs) and Laboratory Information Management Systems (LIMS), making greenness assessment a seamless part of the routine workflow.

In conclusion, the integration of the AGREE metric into method validation represents a significant step toward sustainable analytical science in regulated industries. By providing a standardized, quantitative, and transparent measure of environmental performance, AGREE enables researchers and regulators to make informed decisions that advance both public health and planetary well-being.

In the framework of green analytical chemistry (GAC), the sample preparation step has been identified as one of the most critical from an environmental perspective, often involving substantial solvent consumption, energy requirements, and generation of hazardous waste [3]. While comprehensive metric tools like AGREE exist to evaluate entire analytical procedures, the need for a specific tool to gauge the greenness of sample preparation remained unaddressed until the development of AGREEprep [9] [3]. As a dedicated metric, AGREEprep gives prominence to sample preparation by providing a powerful yet user-friendly tool that enables analytical chemists to assess environmental impact, identify improvable aspects, and guide the development of more sustainable sample preparation methodologies [3]. This specialized focus makes AGREEprep an indispensable component within the broader thesis on AGREE metrics for green method assessment research, offering granular insights into the often most problematic phase of the analytical workflow.

AGREEprep distinguishes itself by being the first metric tool specifically designed to evaluate the environmental impact of sample preparation methods based on the ten principles of green sample preparation (GSP) [3]. This dedicated focus allows for a more accurate and specific assessment compared to general green chemistry metrics, which may overlook the unique complexities and challenges of sample preparation. The tool was developed in response to the recognition that sample preparation can be a significant source of environmental impact in analytical procedures due to its typical requirements for solvents, sorbents, reagents, and energy inputs [3]. Within the evolving landscape of green metric tools, AGREEprep serves as a specialized complement to broader assessment frameworks, enabling researchers to pinpoint specific areas for improvement in the sample preparation stage with unprecedented precision.

The Foundation: Ten Principles of Green Sample Preparation

The AGREEprep metric is structurally founded on ten clearly defined principles that collectively encompass the core objectives of green sample preparation. These principles provide the conceptual framework that guides both the development and application of the assessment tool, ensuring a comprehensive evaluation of environmental impact across all critical dimensions of sample preparation. The principles emphasize various aspects of sustainability, including resource minimization, operator safety, process efficiency, and ecological concerns [3]. Each principle corresponds to a specific assessment criterion within the AGREEprep tool, creating a direct linkage between theoretical objectives and practical evaluation metrics.

The ten principles of green sample preparation that form the basis for AGREEprep are: (1) Favor in situ sample preparation; (2) Use safer solvents and reagents; (3) Target sustainable, reusable, and renewable materials; (4) Minimize waste; (5) Minimize sample, chemical and material amounts; (6) Maximize sample throughput; (7) Integrate steps and promote automation; (8) Minimize energy consumption; (9) Choose the greenest possible post-sample preparation configuration for analysis; and (10) Ensure safe procedures for the operator [3]. These principles collectively address the complete lifecycle of the sample preparation process, from initial design choices through to final analysis configuration and operator safety considerations. The comprehensive nature of these principles ensures that AGREEprep captures both direct environmental impacts (such as waste generation and energy consumption) and indirect factors (such as operator safety and process efficiency) that contribute to the overall sustainability profile of sample preparation methods.

AGREEprep Assessment Criteria and Scoring System

Core Assessment Criteria

The AGREEprep metric system operationalizes the ten principles of green sample preparation through a structured set of assessment criteria, each evaluated on a unified 0-1 scale. The tool's comprehensiveness stems from its multi-faceted evaluation approach, which encompasses diverse aspects of the sample preparation process. Table 1 summarizes the ten core assessment criteria and their primary evaluation focus, illustrating the scope of the AGREEprep assessment framework.

Table 1: AGREEprep Assessment Criteria Based on the Ten Principles of Green Sample Preparation

Criterion Number	Assessment Focus	Primary Evaluation Aspects
1	In situ preparation	Location of sample preparation relative to sampling site
2	Solvents and reagents	Toxicity, safety, and environmental impact of chemicals used
3	Materials	Sustainability, reusability, and renewability of materials
4	Waste generation	Amount and hazard level of waste produced
5	Miniaturization	Sample size, chemical volumes, and material amounts
6	Throughput	Number of samples processed per unit time
7	Integration and automation	Degree of process integration and automation level
8	Energy consumption	Energy requirements and source efficiency
9	Post-preparation configuration	Greenness of the analytical technique used after preparation
10	Operator safety	Risk of exposure, accidents, and necessary safety measures

Each criterion is evaluated individually and transformed into a sub-score on a continuous scale from 0 to 1, where 0 represents the worst possible performance and 1 represents ideal green performance [3]. The specific calculation methods vary between criteria, with some based on threshold values and others on continuous functions. For example, waste generation (Criterion 4) is assessed based on both the amount and hazard level of waste produced, while energy consumption (Criterion 8) considers the total energy requirement per sample [3]. This nuanced approach allows for sensitive discrimination between different levels of performance within each criterion, providing more detailed feedback than binary or limited-level assessment systems.

A critical innovation in AGREEprep is the incorporation of a weighting system that acknowledges the variable importance of different assessment criteria in specific contexts. The developers recognized that not all ten criteria hold equal significance in terms of their environmental impact, and thus proposed default weights to reflect their relative importance [3]. For instance, selecting safer solvents and reagents (Criterion 2) or minimizing waste (Criterion 4) is presumably more significant than favoring in-situ sample preparation (Criterion 1) in terms of overall greenness impact. The weighting system adds considerable flexibility to the assessment process, allowing users to adjust the importance of criteria based on specific assessment goals or methodological constraints.

The final AGREEprep score is calculated as the weighted sum of the individual criterion scores, resulting in an overall assessment value between 0 and 1 [3]. This overall score provides a quick reference point for comparing different sample preparation methods, with higher scores indicating better environmental performance. The mathematical representation of the overall score calculation is as follows:

Overall Score = Σ(wi × si) where wi represents the weight assigned to criterion i, and si represents the sub-score for criterion i.

The weights are normalized so that their sum equals 1, ensuring that the final score remains within the 0-1 range. The default weights suggested by the tool developers provide a scientifically grounded starting point for assessments, while the flexibility to adjust these weights allows specialists to tailor the evaluation to specific research contexts or priorities [3]. This balanced approach between prescriptive guidance and customizable parameters makes AGREEprep adaptable to a wide range of sample preparation scenarios while maintaining comparability between assessments.

AGREEprep Workflow and Visualization

Assessment Workflow

The AGREEprep assessment follows a structured workflow that guides users from data collection through to final interpretation. Figure 1 illustrates this sequential process, which transforms raw methodological data into a comprehensive greenness assessment.

Figure 1: AGREEprep Assessment Workflow

The workflow begins with the comprehensive collection of methodological data corresponding to each of the ten green sample preparation principles [3]. This data is then input into the dedicated AGREEprep software, which calculates individual criterion scores, applies the appropriate weighting factors, computes the overall score, and generates the characteristic circular pictogram visualization [3]. The final interpretation phase involves analyzing both the overall score and the individual criterion performances to identify specific aspects of the sample preparation method that could be improved to enhance environmental sustainability. This structured approach ensures consistency and reproducibility in assessments while providing actionable insights for method optimization.

Pictogram Interpretation

AGREEprep presents assessment results through an intuitive, clock-like pictogram that consolidates multiple dimensions of information into a single, easily interpretable visual representation. The pictogram features a central numerical score ranging from 0 to 1, with the background color of this central circle providing an immediate visual indicator of overall greenness performance (dark green for high scores to red for low scores) [3]. Surrounding this central score are ten segments, each corresponding to one of the assessment criteria and colored according to performance level for that specific criterion (green, yellow, or red) [3]. The width of each segment visually communicates the relative weight assigned to that criterion in the assessment, providing immediate insight into which factors were considered most significant in the overall evaluation.

The AGREEprep pictogram enables rapid comparison between different sample preparation methods and immediate identification of both strengths and weaknesses in greenness performance. Figure 2 illustrates the relationships between the pictogram components and the information they convey, demonstrating how users can extract meaningful insights from the visualization.

Figure 2: Interpreting the AGREEprep Pictogram Components

This sophisticated visualization approach represents a significant advancement in green metrics by presenting complex assessment data in an accessible format that supports both quick comparisons and detailed analysis. The multi-layered information design allows researchers to immediately grasp the overall greenness performance while also providing the granular detail needed to identify specific aspects requiring improvement. This balance between simplicity and comprehensiveness addresses a key challenge in green metrics development: the need for tools that are both scientifically rigorous and practically usable [20] [9].

Practical Application and Case Studies

Experimental Protocol for AGREEprep Assessment

Implementing AGREEprep in practice requires a systematic approach to data collection and evaluation. The following detailed protocol outlines the steps for conducting a comprehensive assessment of sample preparation methods using the AGREEprep metric:

• Method Characterization: Compile complete methodological details for the sample preparation procedure being assessed. This includes exact volumes of all solvents and reagents, specifications for all materials and equipment, detailed step-by-step procedures, and throughput information [3].

• Data Collection for Each Criterion: For each of the ten assessment criteria, gather the specific quantitative and qualitative data required for evaluation:

  • Criterion 1: Document whether preparation occurs in-situ, at-line, or ex-situ.
  • Criterion 2: Record the type, volume, and safety data (hazard statements, toxicity information) for all solvents and reagents used.
  • Criterion 3: Document the types, amounts, and reusability of all materials.
  • Criterion 4: Calculate the total waste generated per sample, noting its composition and hazard classification.
  • Criterion 5: Record sample size and total consumption of chemicals and materials.
  • Criterion 6: Determine the number of samples processed per hour.
  • Criterion 7: Document the degree of automation and integration with other analytical steps.
  • Criterion 8: Calculate the total energy consumption per sample.
  • Criterion 9: Identify the analytical technique used after preparation.
  • Criterion 10: Document all safety measures, required personal protective equipment, and potential exposure risks.

• Software Input: Enter the collected data into the AGREEprep software, which is freely available online. The software provides a structured interface for inputting the required parameters for each criterion [3].

• Weight Assignment: Apply the default weighting factors or customize them based on specific assessment priorities. The software allows for easy adjustment of weights, with the default values reflecting the developers' recommended relative importance of each criterion [3].

• Result Generation: The software automatically calculates the individual criterion scores, applies the weights, computes the overall score, and generates the pictogram visualization.

• Interpretation and Optimization: Analyze the results to identify weaknesses in the current method and develop strategies for improvement. The color-coded pictogram makes it easy to pinpoint which aspects of the method contribute most significantly to its environmental impact.

This protocol ensures a consistent and comprehensive assessment that captures all relevant aspects of sample preparation greenness. The structured approach helps researchers avoid overlooking important factors that contribute to the overall environmental footprint of their methods.

Case Study Comparison: Traditional vs. Modern Methods

The practical application of AGREEprep can be illustrated through a case study comparing different sample preparation methods for the determination of phthalate esters in water samples, as documented in the original development paper [3]. This comparison demonstrates how AGREEprep effectively discriminates between methods with different environmental profiles and provides clear visual indicators of relative performance. Table 2 presents a quantitative comparison of three methods assessed using AGREEprep, showing how the metric captures performance differences across various criteria.

Table 2: Case Study Comparison of Different Sample Preparation Methods Using AGREEprep

Assessment Aspect	Traditional LLE (EPA 8061A)	SPME	SBSE
Overall AGREEprep Score	0.27	0.61	0.78
Solvent Consumption	180 mL dichloromethane	Solventless	Solventless
Waste Generation	High (180 mL per sample)	Minimal	Minimal
Energy Consumption	Moderate	Low	Low
Operator Safety	Poor (toxic solvents)	Good	Good
Throughput	Moderate	High	High
Key Strengths	Standardized methodology	Minimal waste, good safety	Minimal waste, good safety, high throughput
Key Weaknesses	High solvent use, toxic waste, poor safety	Limited sorbent capacity	Method development complexity

The case study clearly demonstrates AGREEprep's ability to quantify and visualize the greenness advantages of modern microextraction techniques like solid-phase microextraction (SPME) and stir bar sorptive extraction (SBSE) over traditional liquid-liquid extraction (LLE) [3]. The traditional LLE method scored only 0.27, reflecting its high solvent consumption, generation of hazardous waste, and poor operator safety profile. In contrast, the solventless microextraction techniques achieved significantly higher scores of 0.61 and 0.78, respectively, highlighting their superior environmental performance [3]. The AGREEprep assessment successfully identified the key weaknesses of each method, providing clear direction for improvement efforts. For the traditional LLE method, the assessment highlighted the critical need to address solvent toxicity and waste generation, while for the microextraction techniques, it identified more subtle aspects for potential optimization, such as energy consumption and throughput.

Research Reagent Solutions and Essential Materials

The implementation of AGREEprep and the development of greener sample preparation methods require careful consideration of reagents and materials. The selection of these components directly impacts multiple assessment criteria in the AGREEprep evaluation, particularly those related to solvent safety, material sustainability, waste generation, and operator safety. Table 3 outlines key research reagent solutions and their functions in the context of green sample preparation, providing guidance for researchers seeking to optimize their methods for improved AGREEprep scores.

Table 3: Essential Reagents and Materials for Green Sample Preparation

Reagent/Material Category	Specific Examples	Green Functions and Applications
Safer Solvents	Bio-based solvents, deep eutectic solvents, water	Replace hazardous organic solvents; reduce toxicity and environmental impact [3]
Sustainable Sorbents	Biopolymer-based sorbents, renewable materials	Provide reusable extraction phases; reduce consumption of non-renewable resources [3]
Miniaturized Equipment	Micro-extraction devices, lab-on-a-chip systems	Reduce reagent consumption and waste generation; enable smaller sample sizes [3]
Green Derivatization Agents	Safe labeling reagents, water-compatible agents	Enable detection of otherwise undetectable analytes with reduced hazard [3]
Reusable Materials	Regenerable sorbents, durable labware	Reduce solid waste generation; improve material efficiency [3]

The strategic selection of reagents and materials based on their environmental and safety profiles represents a critical factor in improving AGREEprep scores. For example, replacing traditional halogenated solvents like dichloromethane (which negatively impacts multiple criteria including solvent safety, waste generation, and operator safety) with safer alternatives can substantially improve a method's overall greenness assessment [3]. Similarly, implementing reusable or renewable materials addresses multiple green principles simultaneously, enhancing performance across several AGREEprep criteria. This integrated approach to reagent and material selection demonstrates how the AGREEprep metric encourages holistic consideration of environmental impacts throughout the method development process, rather than focusing on isolated aspects of greenness.

AGREEprep represents a significant advancement in the toolbox for assessing and improving the environmental sustainability of analytical sample preparation methods. By providing a specialized metric focused specifically on this critical analytical step, AGREEprep addresses a notable gap in green analytical chemistry assessment capabilities. The tool's foundation in the ten principles of green sample preparation ensures comprehensive coverage of relevant environmental factors, while its flexible weighting system accommodates varying priorities and contexts. The intuitive pictogram output facilitates both rapid comparison and detailed analysis, making complex assessment data accessible to researchers across experience levels.

As analytical chemistry continues to evolve toward more sustainable practices, tools like AGREEprep will play an increasingly important role in guiding method development and selection. By identifying specific aspects of sample preparation methods that contribute most significantly to environmental impact, AGREEprep enables targeted optimization efforts that can substantially improve the overall sustainability of analytical procedures. When used in conjunction with broader assessment metrics like AGREE, AGREEprep provides researchers with a comprehensive framework for evaluating and improving the environmental performance of their analytical methods from sample preparation through final analysis. This multi-level assessment approach supports the ongoing transformation of analytical chemistry into a more environmentally responsible discipline while maintaining the high-quality results required for scientific and regulatory applications.

Overcoming Common Challenges and Maximizing AGREE Scores

The adoption of green analytical chemistry (GAC) principles has led to the development of multiple metric tools designed to assess the environmental impact of analytical methods. However, the evaluation of these methods often faces challenges related to subjectivity in interpretation and critical data gaps, which can compromise the reliability and comparability of assessments. This guide explores the inherent limitations of existing frameworks and provides evidence-based strategies to enhance the objectivity, comprehensiveness, and reproducibility of greenness evaluations, with a specific focus on the AGREE (Analytical GREEnness) metric ecosystem.

The proliferation of assessment tools, while reflecting a healthy and active field, can also lead to fragmentation and inconsistency. As noted in a 2025 review, "over the past five years, there has been a remarkable boom in the development of metrics in analytical chemistry... this abundance can be both confusing and overwhelming for chemists, as it becomes difficult to determine which metric offers the most reliable or comprehensive assessment for a given application" [1]. This underscores the critical need for standardized approaches that minimize subjectivity while addressing methodological gaps.

Key Challenges in Current Greenness Assessment Practices

Challenge Category	Specific Limitations	Impact on Assessment Reliability
Tool Proliferation & Isolation	Multiple overlapping tools (AGREE, GAPI, AGSA, GEMAM) operating in isolation without integration pathways [1].	Difficulties in method comparison; conflicting results from different metrics; confusion in tool selection.
Weighting Subjectivity	Default weights in tools like AGREEprep can be modified by users [3] [23].	Potential for unintentional bias; inconsistent scoring between laboratories; reduced comparability.
Visual Interpretation Variability	Color-based outputs with potential insufficient contrast or color-only encoding [29] [30].	Information loss for color-blind users; different interpretations of the same visual output.
Incomplete Method Coverage	Sample preparation often inadequately addressed in broader metrics [3].	Critical environmental impacts overlooked; incomplete life-cycle assessment of methods.
Validation Gaps	Lack of standardized reference methods for benchmarking [23].	Difficulties in establishing performance baselines; limited inter-laboratory validation data.

Strategic Framework for Enhanced Assessment Reliability

The following diagram illustrates a comprehensive strategy to address subjectivity and data gaps in greenness assessments:

Experimental Protocols for Standardized Implementation

Comprehensive AGREE Assessment Workflow

The following experimental workflow details a standardized protocol for conducting greenness assessments using the AGREE metric to minimize subjectivity:

Step-by-Step Protocol:

• Complete Method Documentation: Record all method parameters including reagents (type, volume, toxicity), energy consumption (instrumentation, duration), waste generation, and sample throughput [3] [2].

• AGREE Software Input: Download and utilize the open-source AGREE software (available at https://mostwiedzy.pl/AGREE). Input data corresponding to the 12 principles of GAC, which include waste amount and toxicity, energy consumption, and miniaturization/automation potential [2] [23].

• Weight Assignment with Justification: Use default weightings for each criterion unless specific methodological contexts require adjustment. Document rationales for any weight modifications to ensure transparency and reproducibility [3] [23].

• AGREEprep Complementary Assessment: For methods involving sample preparation, conduct parallel assessment using AGREEprep, which focuses on 10 principles of green sample preparation (GSP) including solvent choices, waste minimization, and integration of steps [3] [23].

• Cross-Tool Validation: Compare results with at least one complementary metric (e.g., GAPI, AGSA) to identify potential inconsistencies and verify assessment robustness [1] [23].

• Accessible Results Reporting: Generate the AGREE pictogram output and supplement with data tables presenting all numerical values. Ensure color contrast ratios of at least 3:1 for chart elements and 4.5:1 for text [29] [30].

Quantitative Comparison of Greenness Assessment Tools

Table 2: Technical Specifications of Major Green Assessment Metrics

Metric Tool	Assessment Scope	Scoring System	Visual Output	Key Advantages	Inherent Subjectivity Factors
AGREE [2] [23]	12 Principles of GAC	0-1 scale	Circular pictogram	Comprehensive GAC alignment; open-source software	User-defined weight adjustments; color interpretation
AGREEprep [3] [23]	10 Sample Preparation Principles	0-1 scale	Round pictogram	Sample preparation specificity; customizable weights	Default weight modifications; operator safety subjectivity
AGSA [4]	12 GAC Principles	Built-in scoring	Star area diagram	Extension of GC metric; visual intuition	Resistance to user bias not fully achieved
GAPI [23]	5 Evaluation Areas	Qualitative	Pictogram with segments	Multi-criteria assessment; visual simplicity	No total scoring system; limited comparability
NEMI [3]	4 Basic Criteria	Yes/No	Simple pictogram	Ease of use; quick assessment	Over-simplification; significant data gaps

Technical Solutions for Objective Implementation

Research Reagent Solutions for Green Assessment

Table 3: Essential Materials and Digital Tools for Reliable Greenness Assessment

Research Solution	Function in Assessment	Technical Specifications	Accessibility Features
AGREE Software [2]	Calculates greenness score based on 12 GAC principles	Open-source; downloadable from mostwiedzy.pl/AGREE	Generates pictogram with color and numerical score
AGREEprep Software [3] [23]	Evaluates sample preparation greenness	Based on 10 GSP principles; customizable weights	Round pictogram with segment performance indicators
Color Contrast Analyzer [29] [30]	Verifies accessibility of visual outputs	Chrome extension; checks entire charts	Identifies insufficient color contrast ratios
Web Disability Simulator [30]	Evaluates color blindness accessibility	Chrome extension; simulates color vision deficiencies	Demonstrates different color blindness experiences
High-Contrast Color Palettes [29] [31]	Ensures visual interpretation clarity	3:1 contrast ratio for elements; 4.5:1 for text	Enables distinction without color reliance

Data Gap Mitigation Protocol

• Pre-Assessment Checklist: Verify completeness of method data across all 12 GAC principles before evaluation.

• Cross-Platform Validation: Compare AGREE results with AGREEprep for methods involving sample preparation to identify potential oversight areas [23].

• Supplementary Data Reporting: Include raw numerical data alongside visual pictograms to enable independent verification and alternative analysis [29] [30].

• Uncertainty Quantification: Document and report any methodological parameters with estimated rather than measured values, noting potential impact on final score.

Addressing subjectivity and data gaps in greenness assessment requires a systematic approach that integrates multiple complementary tools, standardized protocols, and accessibility-focused visualization practices. The AGREE metric ecosystem, when implemented with the strategies outlined in this guide, provides a robust framework for generating reliable, comparable, and transparent environmental assessments of analytical methods. By adopting these practices, researchers can advance the field of green analytical chemistry through more objective evaluations that effectively guide the development of truly sustainable analytical methods. Future work should focus on developing interoperability standards between assessment tools and establishing validated reference datasets for method benchmarking.

The Impact of Weight Adjustments on Your Final AGREE Score

The Analytical GREEnness (AGREE) metric is a comprehensive assessment tool designed to evaluate the environmental impact of analytical methods. It translates the 12 principles of Green Analytical Chemistry (GAC) into a quantifiable score, providing researchers and scientists a standardized way to measure and improve the sustainability of their methodologies [9]. Unlike earlier tools, AGREE offers a flexible and user-friendly approach, generating an easily interpretable pictogram that reflects an analytical procedure's greenness [7] [9].

A foundational strength of AGREE is its incorporation of user-defined weight adjustments. This feature acknowledges that not all assessment criteria hold equal importance across different analytical applications. The ability to assign weights allows professionals to tailor the greenness evaluation to their specific priorities, whether minimizing solvent use, reducing energy consumption, or enhancing operator safety, making AGREE a dynamic and context-aware tool for sustainable method development in drug development and other research fields [9].

The Architecture of the AGREE Score

The 12 SIGNIFICANCE Principles as Assessment Criteria

The AGREE calculator is built upon the 12 principles of Green Analytical Chemistry, memorably encapsulated by the acronym "SIGNIFICANCE" [9]. Each principle is converted into a specific criterion within the tool. The software transforms the input for each of these 12 criteria into a score on a common scale of 0 to 1 [9]. The final AGREE score, also on a 0-1 scale, is the product of the assessment results for each principle, providing a single, at-a-glance measure of a method's overall greenness [9].

The AGREE Pictogram: An Informative Output

The result of an AGREE assessment is a clock-like pictogram that communicates a wealth of information intuitively [9]:

• Overall Score: The final calculated score (0-1) is displayed in the center of the graph. A value closer to 1, represented by a darker green color, indicates a greener analytical procedure [9].
• Criterion Performance: The pictogram is divided into 12 segments, each corresponding to one of the GAC principles. The color of each segment (using an intuitive red-yellow-green scale) indicates the method's performance for that specific criterion [9].
• Criterion Weight: The relative importance assigned to each principle by the user is visually communicated by the width of its corresponding segment in the pictogram. A wider segment signifies that the user has given that criterion a higher weight in the overall calculation [9].

The Role and Application of Weight Adjustments

The Rationale for Flexible Weighting

The AGREE metric's design incorporates flexible weighting because the environmental impact of an analytical method is multifaceted, and the priorities for optimization can vary significantly depending on the context [9]. For some applications, simplicity and high throughput might be critical, while for others, the primary focus could be on minimizing hazardous waste or the use of toxic reagents. The weighting system allows researchers to reflect these specific priorities directly in the greenness assessment, ensuring the final score is relevant to their unique operational, safety, and environmental constraints [9].

Practical Implementation of Weight Adjustments

Adjusting weights in AGREE is a user-directed process. The freely available AGREE software allows the assessor to assign an importance value to each of the 12 principles [9]. The default setting assigns equal weight to all criteria. However, users can increase the weight for principles they deem more critical to their assessment, which widens the corresponding segment in the final pictogram and increases that criterion's influence on the total score. Conversely, reducing the weight for a less relevant principle diminishes its impact [9]. This process ensures that the final score is not just a generic measure but a customized evaluation that aligns with the user's specific sustainability goals.

Quantifying the Impact of Weight Adjustments

The impact of weight adjustments on the final score is direct and mathematical. The score for each criterion is multiplied by its assigned weight during the calculation of the total score. Therefore, improving performance on a highly-weighted criterion will have a more substantial positive impact on the final score than a similar improvement on a criterion with a low weight. The following table illustrates how different weighting profiles can shift the focus of the greenness assessment for common scenarios in drug development.

Table: Exemplary Weighting Profiles for Different Analytical Scenarios in Drug Development

Analytical Scenario	High-Priority Principles (Examples)	Rationale for Increased Weight	Potential Lower-Weight Principles
Method Development for High-Throughput Screening	#8 (Analysis Speed)	Throughput is a primary objective; faster methods reduce overall energy and resource use per sample.	#1 (Direct Analysis), #4 (Derivatization)
Analysis of Potent Compounds (High Toxicity)	#11 (Operator Safety)	Ensuring analyst protection from highly potent APIs is the paramount concern.	#8 (Analysis Speed), #12 (Operator Skills)
Sustainable Solvent Replacement Initiative	#2 (Sample Size), #5 (Reagent Toxicity)	Directly targets the reduction of hazardous solvent volume and toxicity.	#10 (Transformation Products), #11 (Operator Safety)

A Case Study in Weighted Assessment

Experimental Context and Baseline Assessment

A case study evaluating a Sugaring-Out Liquid-Liquid Microextraction (SULLME) method for determining antiviral compounds demonstrates the application of complementary greenness metrics [7]. When assessed with AGREE, this method received a score of 56, indicating a moderately green profile [7]. The method's strengths included miniaturization, semiautomation, and the absence of derivatization, while its weaknesses involved the use of some toxic and flammable solvents and moderate waste generation [7].

Applying a Custom Weighting Strategy

Consider how different weighting strategies would alter the final AGREE score for this SULLME method. If the assessment prioritized operator safety (Principle #11) due to the presence of toxic solvents, increasing the weight for this criterion would likely lower the overall score, as the method's performance in this area was a noted weakness [7]. Conversely, if the assessment was tailored to prioritize miniaturization and reduced reagent use (Principle #2), the method's strong performance in this area would be amplified, potentially increasing the final score.

Table: Impact of Weight Adjustment on AGREE Score for the SULLME Case Study

Assessment Focus	High-Weight Principle	Method Performance on Principle	Effect of High Weight on Final Score
Operator Safety	#11 (Operator Safety)	Weakness (use of toxic solvents)	Decreases final score
Solvent Reduction	#2 (Sample Size)	Strength (miniaturization)	Increases final score
Waste Minimization	#3 (Waste Generation)	Weakness (moderate waste)	Decreases final score

The following workflow diagram outlines the strategic process for conducting a weighted AGREE assessment.

Research Reagent Solutions and Essential Materials

Successfully implementing AGREE and improving greenness scores often involves using specific types of reagents and materials. The following table details key solutions used in the development of greener analytical methods.

Table: Key Research Reagent Solutions for Greener Analytical Methods

Reagent/Material	Function in Green Method Development	Example in Practice
Bio-Based Reagents	Replace petroleum-derived or more hazardous solvents/reagents; improve renewability and degradation profile.	Used in the SULLME case study to replace traditional, more toxic organic solvents [7].
Green Solvents (e.g., Cyclopentyl Methyl Ether, Ethyl Lactate)	Lower toxicity, reduced environmental impact, and often biodegradable alternatives to classical solvents like chlorinated hydrocarbons.	Commonly evaluated in solvent selection to improve scores for GAC Principles 5 (reagent toxicity) and 10 (degradation) [7].
Ionic Liquids & Deep Eutectic Solvents (DES)	Often considered designer, less volatile solvents with potential for reduced toxicity and higher safety profiles.	Used in novel sample preparation techniques to minimize volatile organic compound (VOC) emissions [9].
Derivatization-Free Kits	Enable direct analysis without the need for hazardous derivatizing agents, reducing steps, reagents, and waste.	Applying direct analytical techniques aligns with GAC Principle 1 and avoids penalties from Principle 4 [9].

Software and Calculation Tools

The AGREE assessment is supported by freely available, open-source software [9] [8]. This user-friendly software automates the transformation of input data into the final score and pictogram. It provides an interface for inputting data for each of the 12 principles and features sliders or input fields to adjust the weight of each criterion according to the user's needs. The software then automatically generates the characteristic pictogram and a detailed assessment report, making the tool accessible to all researchers [9].

Weight adjustments are not merely a supplementary feature but a core functional element of the AGREE metric, enabling its application across the diverse landscape of analytical chemistry. By allowing researchers to calibrate the importance of each green chemistry principle, AGREE transitions from a rigid, one-size-fits-all checklist to a dynamic and context-sensitive assessment framework. This flexibility empowers drug development professionals and scientists to make nuanced decisions, guiding them toward analytical methods that are not only scientifically robust but also aligned with specific and prioritized environmental and safety goals. As the field of green analytical chemistry continues to evolve with new metrics like AGREEprep, AGSA, and GEMAM, the foundational principle of customizable weighting established by AGREE remains a critical component for meaningful sustainability evaluation [7] [8].

Quantifying the environmental footprint of laboratory activities, particularly energy consumption and waste generation, has become a critical competency for researchers and drug development professionals. Accurate measurement is the foundation for meaningful reduction targets and is increasingly required by regulatory bodies and funding agencies. Within the specific context of green analytical chemistry, these calculations feed directly into formal assessment metrics, such as the AGREE (Analytical GREEnness) metric, which provides a standardized score for evaluating the environmental sustainability of analytical methods [32] [8]. This guide details the best practices and common pitfalls in performing these calculations, enabling scientists to generate reliable data that can drive the adoption of greener laboratory protocols.

Foundational Concepts and Metrics

The AGREE Metric Framework

The AGREE metric is a comprehensive, open-source tool designed to evaluate the greenness of analytical methods. It translates 12 core principles of Green Analytical Chemistry (GAC) into a quantitative score [8].

• Scoring System: AGREE produces a score on a scale of 0 to 1, represented by a circular pictogram. A score closer to 1 indicates a greener method.
• Input Criteria: The assessment incorporates ten key factors, including the amount and toxicity of reagents used, energy consumption of instruments, generated waste, and sample throughput [8].
• Visual Output: The result is an easily interpretable pictogram that provides an at-a-glance assessment of a method's environmental performance, with different segments representing different principles of GAC.

Defining System Boundaries

A critical first step is defining the boundaries of your assessment. Inefficient energy use often correlates with increased equipment strain and waste, such as from shortened catalyst lifespans or off-spec batches [33]. The pharmaceutical industry, in particular, must account for emissions and waste across the entire supply chain, known as Scope 3 emissions, which often represent the largest portion of its carbon footprint [34].

Table: Categories of Energy Consumption and Waste for Assessment

Category	Description	Examples in a Research Context
Direct Energy (Scope 1)	Energy generated from owned or controlled sources.	On-site natural gas combustion for process heating.
Indirect Energy (Scope 2)	Energy purchased from utilities.	Electricity for HVAC, lighting, and analytical instruments (HPLC, GC-MS) [35].
Upstream/Downstream (Scope 3)	All other indirect emissions/waste in the value chain.	Solvent production, packaging of reagents, outsourced API manufacturing, disposal of hazardous waste [34].
Direct Waste	Waste generated from a specific process.	Solvent waste from extraction, used chromatography columns, reaction byproducts.
Indirect Waste	Overhead waste not tied to a single unit.	Plasticware from sample preparation, glove waste, packaging materials [36].

Quantitative Analysis and Data Presentation

Robust quantitative analysis requires reliable data collection and appropriate methodological choices. The following table summarizes common quantitative approaches used in broader industrial assessments, which can be adapted to the laboratory scale.

Table: Methods for Quantitative Analysis of Energy Efficiency and Waste

Method	Primary Function	Key Strengths	Common Pitfalls
Ratio-Based Analysis	Simple indicator comparing energy use to output.	Simple to calculate and understand (e.g., kWh per sample).	Fails to isolate technical efficiency from structural/economic factors [35].
Index Decomposition Analysis (IDA)	Disaggregates changes in energy use into driving factors.	Isolates pure efficiency effects from activity and structural changes.	Requires consistent, disaggregated time-series data [35].
Life Cycle Assessment (LCA)	Evaluates environmental impacts across a product's life.	Comprehensive; accounts for upstream/downstream (Scope 3) impacts.	Data-intensive; complex to model; system boundary definition is critical [34].
Structural Path Analysis	Traces emissions/waste through complex supply chains.	Identifies "hotspots" in global supply chains for targeted action.	Highly dependent on the resolution of input-output databases [34].

Key Quantitative Findings

Recent studies highlight the scale and drivers of environmental impacts in research and manufacturing:

• Pharmaceutical Carbon Footprint: From 1995 to 2019, the global pharmaceutical greenhouse gas footprint grew by 77%, primarily driven by rising expenditure and stalling efficiency gains after 2008. Supply chain emissions (Scope 3) vary substantially among major suppliers [34].
• Clinical Waste: A 2025 study on inhaler use in hospitals found that 81.9% of dispensed actuations were wasted, contributing a carbon footprint of 1,226,342 g CO2 equivalent over a three-month period without providing patient benefit [36].
• Manufacturing Energy Waste: In manufacturing, energy waste costs an estimated $510 for every $1,000 spent on electricity, highlighting a significant profit drain and environmental burden [33].

Experimental Protocols for Measurement

Protocol for Direct Instrument Energy Measurement

Objective: To accurately measure the energy consumption of a specific analytical instrument (e.g., an HPLC system) over a complete operational cycle.

Materials:

• Target analytical instrument (e.g., HPLC, GC-MS)
• Calibrated power meter (e.g., a plug-in energy monitor with data logging)
• Data recording sheet or software

Procedure:

• Calibration and Setup: Ensure the power meter is calibrated. Connect the power meter to a dedicated power outlet, and then plug the target instrument into the power meter.
• Baseline Recording: Record the power reading (in Watts) when the instrument is in standby or completely powered off to establish a baseline.
• Operational Phases:
  • Start-up: Power on the instrument and record the power draw at regular intervals (e.g., every 5 seconds) until it stabilizes. Note the duration.
  • Idle/Stabilized: Record the stable power consumption once the instrument is ready for use.
  • Active Analysis: Start the analytical method (e.g., inject a sample). Continuously log power data throughout the entire method runtime.
  • Shutdown: After the run, log power consumption until the instrument returns to a standby or off state.
• Data Calculation:
  • Energy Consumption (kWh) = (Average Power in kW) × (Time in hours for each phase).
  • Total Energy per Run = Sum of energy from all phases.
  • Normalize the data per sample by dividing the total energy by the number of samples processed in a single run.

Protocol for Laboratory Waste Auditing

Objective: To quantify and characterize the waste generated by a specific analytical procedure or from a laboratory over a defined period.

Materials:

• Personal Protective Equipment (PPE): gloves, lab coat, safety glasses
• Calibrated digital scales
• Dedicated, pre-labeled containers for different waste streams (e.g., halogenated solvent, non-halogenated solvent, solid plastic, glass, sharps)
• Waste inventory logbook

Procedure:

• Waste Segregation: Establish and label clearly defined waste categories. Train all personnel on the correct segregation protocols.
• Collection: Over a representative period (e.g., one week or the duration of a specific experiment), collect all waste generated in the corresponding containers.
• Weighing and Recording:
  • At the end of the audit period, weigh each full container and record the mass.
  • Subtract the pre-determined tare weight of each container to determine the net mass of each waste type.
  • Log the mass of each waste category in the inventory.
• Data Analysis:
  • Calculate the total waste generated.
  • Calculate the proportion of hazardous vs. non-hazardous waste.
  • Determine the waste generation per researcher, per experiment, or per sample analyzed.
  • Identify major waste sources to target for reduction strategies (e.g., solvent replacement, miniaturization).

Visualization of Workflows and Relationships

Workflow for AGREE-Informed Method Assessment

The following diagram illustrates the logical workflow for developing and assessing an analytical method with the AGREE metric, integrating the measurement of energy and waste.

System Boundaries for Pharmaceutical Footprinting

This diagram maps the complex system boundaries and flows for calculating the greenhouse gas emissions of pharmaceutical production and consumption, as revealed by input-output analysis [34].

The Scientist's Toolkit: Essential Reagent and Material Solutions

This table details key reagents and materials used in green analytical chemistry, along with their traditional counterparts, to guide researchers in making more sustainable choices without compromising analytical performance.

Table: Research Reagent Solutions for Greener Analytical Chemistry

Item / Solution	Traditional Function	Green Alternative & Function	Impact on Energy/Waste
Extraction Solvents	Sample preparation using large volumes of hazardous solvents (e.g., chlorinated solvents).	Switch to less toxic solvents (e.g., ethanol, ethyl acetate) or use solventless extraction techniques (e.g., solid-phase microextraction) [8].	Reduces hazardous waste generation and energy for fume hood operation and waste disposal.
Chromatography Mobile Phases	High-performance liquid chromatography (HPLC) using solvent-rich mobile phases.	Employ water-rich or neat mobile phases where possible, or use supercritical fluid chromatography (SFC) with COâ‚‚ [32].	Significantly cuts solvent consumption and waste; SFC can reduce energy use.
Derivatization Agents	Using toxic reagents to modify analytes for detection.	Avoid derivatization by selecting alternative techniques or use less hazardous derivatizing agents [8].	Eliminates waste streams from derivatization reactions and simplifies workflow.
Sample Preparation Format	Macroscale preparation in large beakers and volumes.	Miniaturization of devices (e.g., microfluidic chips, smaller vials) for sample preparation [8].	Reduces reagent consumption and sample size, thereby lowering material waste and energy for processing.
Energy Source	Constant, high-energy operation of instruments.	Automation and instrument miniaturization to reduce energy consumption per analysis [8] [35].	Directly lowers the energy consumption (kWh) recorded for the analytical method.
Mniopetal C	Mniopetal C|CAS 158761-00-3|For Research	Mniopetal C is a natural compound for cancer proliferation research. It is for research use only (RUO). Not for human or veterinary use.	Bench Chemicals

Common Pitfalls and Mitigation Strategies

• Pitfall 1: Inconsistent System Boundaries. Comparing studies or methods with different defined boundaries (e.g., one includes solvent production, another does not) leads to invalid conclusions.

  • Mitigation: Adopt a standardized life-cycle perspective from the outset, clearly document all boundaries, and use the AGREE metric, which mandates consideration of reagents, energy, and waste [34] [8].

• Pitfall 2: Over-reliance on Economic Ratio Indicators. Using energy consumption per unit of economic value (e.g., per dollar of output) can mask true technical efficiency, as it is influenced by price fluctuations and structural changes [35].

  • Mitigation: Where possible, use physical intensity indicators (e.g., energy per sample analyzed, waste mass per assay). For macro-assessments, use decomposition analysis to isolate real efficiency gains [35].

• Pitfall 3: Neglecting Standby and Idle Energy Losses. Focusing only on active processing energy can drastically underestimate total consumption. Equipment running on standby creates "phantom loads" [33].

  • Mitigation: Conduct power measurements over a complete cycle, including startup, idle, and shutdown phases. Implement power management policies to shut down non-essential equipment.

• Pitfall 4: Poor Waste Segregation. Inaccurate sorting of hazardous from non-hazardous, or mixed waste streams, leads to inflated disposal costs and greater environmental impact [37].

  • Mitigation: Implement clear labeling, training, and convenient waste segregation stations. Regular waste audits can identify and correct segregation errors.

• Pitfall 5: Data Granularity Issues. Using highly aggregated data prevents the identification of specific inefficiency hotspots.

  • Mitigation: Disaggregate data to the process or even equipment level. For national or sectoral analyses, use input-output tables with high sectoral resolution to improve the accuracy of footprint calculations [34] [35].

The transition toward sustainable chemistry has made the environmental assessment of analytical methods and synthetic procedures indispensable. Green Analytical Chemistry (GAC) aims to minimize the environmental impact of chemical analyses by reducing or eliminating hazardous substances, decreasing waste, and saving energy while maintaining rigorous performance standards [7]. To quantify this progress, several assessment tools have been developed, with the AGREE metric (Analytical GREEnness) standing out as a comprehensive, user-friendly tool explicitly structured around the 12 principles of GAC [4] [7].

AGREE provides both a visual pictogram and a numerical score between 0 and 1, facilitating direct comparison between methods and offering an at-a-glance understanding of a method's environmental performance [7]. Its development addressed limitations of earlier tools, such as the National Environmental Methods Index (NEMI), which offered a binary assessment without degrees of greenness, and the Analytical Eco-Scale (AES), which lacked a visual component [4] [7]. The core strength of AGREE lies in its holistic approach, evaluating the entire analytical workflow against the 12 GAC principles [4].

This guide details practical strategies for optimizing methods toward a higher AGREE score, focusing on the critical levers of solvent toxicity and volume reduction. Furthermore, it places AGREE within the evolving landscape of green assessment tools, including the newer Analytical Green Star Area (AGSA), which offers a complementary star-shaped visualization and built-in scoring system [4].

The AGREE metric is a significant advancement in greenness assessment because it directly operationalizes the 12 principles of Green Analytical Chemistry into an evaluative framework [4]. The tool generates a circular pictogram divided into 12 sections, each corresponding to one principle. The sections are colored in a gradient from red to green, providing an intuitive visual summary of the method's performance across all criteria. The software also calculates a unified score from 0 to 1 (with 1 being ideal), which is displayed in the center of the pictogram, allowing for straightforward benchmarking and comparison [7].

A key advantage of AGREE is its ability to highlight specific areas for improvement within an analytical method. For instance, if a method uses large volumes of a toxic solvent, the corresponding principles (e.g., #5 regarding safer solvents, #1 regarding waste prevention) will be colored red, immediately directing the chemist to the primary sources of environmental concern [7].

However, no tool is without limitations. AGREE's assessment can involve a degree of subjective user judgment, and it does not extensively account for pre-analytical processes, such as the synthesis of reagents [7]. This gap is partially addressed by other tools like ComplexGAPI [7]. More recently, the Analytical Green Star Area (AGSA) has been introduced as an extension of similar metrics in green chemistry. AGSA offers a visually intuitive star-shaped diagram, a built-in scoring system, and claims enhanced resistance to user bias, providing another powerful option for interdisciplinary comparisons [4].

The following diagram illustrates the logical relationship between the goals of method optimization, the key principles of GAC addressed by the AGREE metric, and the resulting outcomes from implementing greener practices.

Strategies for Reducing Solvent Volume

Solvents often constitute the largest volume of materials used in analytical and synthetic processes, sometimes accounting for 80-90% of the total mass in pharmaceutical operations [38]. Therefore, reducing solvent consumption is one of the most effective ways to improve a method's greenness profile and AGREE score, primarily by addressing Principle 1 (Prevention of Waste).

Modern Extraction and Microscale Techniques

Replacing traditional, solvent-intensive methods with modern, miniaturized alternatives is a highly effective strategy. The table below summarizes key techniques for significant solvent volume reduction.

Table 1: Solvent Volume Reduction Techniques

Technique	Traditional Method Replaced	Key Feature	Typical Solvent Volume	Reference
Solid Phase Extraction (SPE)	Liquid-Liquid Extraction (LLE)	Uses solid sorbent to retain analytes	Milliliters (vs. tens of mL for LLE)	[39]
Dispersive Liquid-Liquid Microextraction (DLLME)	Batch LLE	Uses microliters of extraction solvent	Microliters (50-100 µL final volume)	[39]
Microwave-Assisted Extraction (MAE)	Conventional Soxhlet/Extraction	Uses microwave energy for efficiency	Lower volume due to increased efficiency	[39]
Pressurized Liquid Extraction (PLE)	Conventional Soxhlet/Extraction	Uses high pressure and temperature	Lower volume due to enhanced capabilities	[39]
Automated Flash Chromatography	Manual Flash Chromatography	Programming step gradients	Reduced overall consumption vs. manual	[40]
Aqueous Micellar Conditions	Organic Solvent-Based Reactions	Uses water with surfactants as nanoreactors	Vastly reduced or eliminated organic solvents	[41]

Detailed Protocol: Dispersive Liquid-Liquid Microextraction (DLLME)

DLLME is a powerful sample preparation technique that exemplifies miniaturization. The following workflow details a standard DLLME procedure for concentrating analytes from an aqueous sample.

Materials and Functions:

• Sample Solution: The aqueous matrix containing the target analytes.
• Extraction Solvent: A water-immiscible, dense solvent (e.g., chlorobenzene, carbon tetrachloride). Function: Dissolves and concentrates the analytes.
• Disperser Solvent: A water-miscible solvent (e.g., acetone, acetonitrile). Function: Helps disperse the extraction solvent as fine droplets throughout the aqueous sample, creating a large surface area for rapid extraction.
• Centrifuge Tube: A conical tube for the extraction and phase separation.
• Microsyringe: For precise collection of the sedimented phase.

Procedure:

• Transfer a precisely measured volume (e.g., 5.0 mL) of the aqueous sample into a conical centrifuge tube.
• Using a microsyringe, rapidly inject a mixture containing microliters of the extraction solvent and disperser solvent (e.g., 1.0 mL acetone containing 50.0 µL chlorobenzene) into the sample tube.
• A cloudy solution, consisting of fine droplets of the extraction solvent dispersed in the aqueous phase, will form immediately. Gently agitate the solution to ensure complete extraction of analytes into the droplets.
• Centrifuge the tube at a fixed speed (e.g., 4000 rpm for 5 minutes) to break the emulsion and sediment the dense extraction solvent phase at the bottom of the tube.
• Carefully remove the bulk aqueous phase with a pipette. Using a microsyringe, retrieve the entire sedimented organic phase (now ~50-100 µL, enriched with analytes) for direct analysis or further dilution.

This protocol reduces solvent consumption from tens of milliliters in traditional LLE to a few hundred microliters or less, directly contributing to a higher AGREE score by minimizing waste [39].

Strategies for Selecting and Reducing Solvent Toxicity

The inherent hazard of solvents is a major factor in the overall environmental and safety profile of a method. AGREE principles #3 (Less Hazardous Chemical Syntheses) and #5 (Safer Solvents and Auxiliaries) directly address this issue. Transitioning to less toxic solvents and mitigating exposure risks are critical for greenness optimization.

Greener Solvent Selection Guides

A fundamental strategy is to replace hazardous solvents with safer alternatives. Several guides, such as the CHEM21 selection guide, provide curated lists ranking solvents based on health, safety, and environmental (HSE) criteria [41]. Legislative frameworks like Europe's REACH regulation have identified specific solvents as Substances of Very High Concern (SVHC), providing a clear "red list" for phase-out [40].

Table 2: Solvent Replacement Guide for Hazard Reduction

Solvents of Very High Concern (SVHC)	Recommended Greener Replacements	Key Hazard of Original Solvent
Trichloroethylene, 1,2-Dichloroethane	Cyclopentyl methyl ether (CPME), 2-Methyltetrahydrofuran (2-MeTHF)	Carcinogenicity, reproductive toxicity [40]
2-Methoxyethanol, 2-Ethoxyethanol	1,3-Dioxolane, 1,3-Propanediol	Damage to fertility/unborn children [40]
Tetrahydrofuran (THF)	2-Methyltetrahydrofuran (2-MeTHF)	Peroxide formation, reprotoxicity [40]
Acetonitrile	Ethanol or Methanol (from renewable sources)	High toxicity, problematic waste [40]
N,N-Dimethylformamide (DMF)	N,N'-Dimethylpropyleneurea (DMPU)	Reprotoxicity [40]
Hexane, Petroleum Ether	Heptane, Cyclohexane	Neurotoxicity [42]

Case Study: Aqueous Micellar Catalysis

A transformative approach to eliminating organic solvents is the use of aqueous micellar catalysis. This was brilliantly demonstrated in the synthesis of the antimalarial drug candidate MMV688533, where two key Sonogashira coupling reactions were transitioned from organic solvents to water containing the designer surfactant TPGS-750-M [41].

Protocol for a Sonogashira Coupling in Aqueous Micelles:

• Reaction Setup: In a reaction vessel, charge water and the surfactant TPGS-750-M (2% w/w). The surfactant self-assembles into nanomicelles.
• Add Reagents: Add the organic substrates (aryl halide and terminal alkyne), a palladium catalyst (e.g., at parts-per-million (ppm) loading), and a base (e.g., potassium carbonate) directly into the micellar solution. The hydrophobic reactants partition into the micellar cores, which act as nanoreactors.
• Stir and React: Stir the mixture at ambient temperature or mild heating. The reaction proceeds within the micelles.
• Work-up and Extraction: Upon completion, cool the reaction and extract the product using a minimal amount of a benign solvent like ethyl acetate or cyclopentyl methyl ether. Alternatively, the product can sometimes be isolated simply by filtration.

Outcomes and Benefits:

• Solvent Replacement: The need for toxic, volatile organic solvents (e.g., THF, DMF) is eliminated, replaced by water.
• Reduced Catalyst Loading: The micellar environment allows for drastically reduced loadings of precious metal catalysts (e.g., a 20-fold decrease in Pd), which also lowers residual metal contamination in the final product to within FDA limits [41].
• Improved Metrics: This switch contributed to a 10-fold increase in overall yield and halved the Process Mass Intensity (PMI) from 287 to 111, indicating a much more efficient and sustainable process [41].

Quantitative Assessment and Comparative Case Study

To be truly effective, optimization must be measurable. Using tools like AGREE and AGSA allows for a quantitative before-and-after comparison. A case study evaluating a Sugaring-Out Liquid-Liquid Microextraction (SULLME) method for antiviral compounds reveals how these metrics provide a multi-faceted view [7].

Table 3: Greenness Assessment of an Optimized Method Using Multiple Metrics

Assessment Metric	Reported Score	Strengths Identified	Weaknesses & Improvement Areas
Modified GAPI (MoGAPI)	60/100	Use of green solvents; microextraction (<10 mL solvent)	Moderate toxicity substances; >10 mL waste; vapor emissions [7]
AGREE	0.56/1.00	Miniaturization; semi-automation; no derivatization	Use of toxic/flammable solvents; low throughput; moderate waste [7]
Analytical Green Star Area (AGSA)	58.33/100	Semi-miniaturization; avoidance of derivatization	Manual handling; ≥6 hazard pictograms; no waste management [7]
Carbon Footprint Reduction Index (CaFRI)	60/100	Low analytical energy (0.1–1.5 kWh/sample)	No renewable energy; long-distance transport; >10 mL organic solvent [7]

This multi-metric assessment demonstrates that while the SULLME method is successful in reducing solvent volume through miniaturization, its AGREE and AGSA scores are pulled down by the residual toxicity of the solvents used and the lack of a waste management plan. This provides a clear roadmap for further optimization: replacing the solvents with greener alternatives from Table 2 and implementing a solvent recycling or treatment protocol.

The Scientist's Toolkit: Essential Reagents and Solutions

Implementing green optimization strategies requires specific materials. The following table lists key reagents and tools referenced in this guide.

Table 4: Research Reagent Solutions for Green Method Optimization

Item	Function/Application	Key Green Feature
TPGS-750-M Surfactant	Forms nanomicelles in water for organic reactions [41]	Enables replacement of organic solvents with water
Functionalized Silica	Sorbent for SPE; metal scavenger; catalyst support [40]	Reduces solvent use vs. LLE; removes metals; reusable
2-Methyltetrahydrofuran (2-MeTHF)	Solvent for extraction and reaction [40]	Renewable (from biomass); safer profile than THF
Cyclopentyl Methyl Ether (CPME)	Solvent for extraction and reaction [40]	Low toxicity; low peroxide formation; high stability
SiliaMetS Metal Scavengers	Functionalized silica to remove metal impurities from reaction mixtures [40]	Eliminates need for additional solvents in work-up; reduces heavy metal waste
Activated Carbon Cartridges	Filtration to remove chemical fumes and vapors at the source [42]	Protects operator health; reduces laboratory air pollution

Optimizing chemical methods for reduced solvent toxicity and volume is a multi-faceted but achievable goal that is critical for advancing sustainable science. The journey begins with a rigorous assessment using tools like the AGREE metric and AGSA, which provide a clear baseline and highlight specific areas for improvement. As demonstrated, significant gains can be made by adopting miniaturized techniques like DLLME and SPE, systematically replacing hazardous solvents with greener alternatives using established guides, and exploring transformative technologies like aqueous micellar catalysis. By integrating these strategies into method development and validation, researchers and drug development professionals can dramatically reduce the environmental footprint of their work, ensuring it is not only scientifically robust but also ecologically responsible.

Ensuring Reproducibility and Adherence to Good Evaluation Practice (GEP)

In the evolving landscape of analytical chemistry, the principles of Green Analytical Chemistry (GAC) have become paramount for sustainable scientific progress. The AGREE (Analytical GREEnness) metric emerges as a comprehensive tool designed to evaluate the environmental impact of analytical methods [8]. This whitepaper establishes the critical interrelationship between robust Green Evaluation Practice (GEP) and research reproducibility, framing them as mutually reinforcing pillars in green method assessment. Reproducibility ensures that scientific findings can be consistently verified, while GEP provides the standardized framework to evaluate their environmental sustainability [43]. For researchers and drug development professionals, integrating these concepts addresses both methodological reliability and ecological responsibility, creating a new paradigm where scientific excellence is measured by both technical merit and environmental consciousness.

The pharmaceutical industry faces increasing pressure to minimize its environmental footprint while maintaining rigorous research standards. This document provides a technical framework for aligning analytical methodologies with GEP principles while ensuring reproducible science. We explore practical implementation strategies, detailed experimental protocols, and visualization tools to help scientists navigate the complexities of green assessment while maintaining scientific rigor.

Theoretical Foundation: Reproducibility and GEP

The Reproducibility Framework

Reproducibility constitutes the foundation of credible scientific research, ensuring that reported results can be consistently verified through independent investigation [43]. According to the IJCAI 2025 guidelines, reproducibility assessment evaluates whether "using the same data and the same analytical tools will yield the same results as reported." This requires exact specification of all experimental components to enable verification. The framework encompasses multiple dimensions:

• Methodological Reproducibility: Detailed documentation of experimental procedures, equipment specifications, and analytical conditions
• Computational Reproducibility: Complete description of algorithms, parameters, and data processing techniques
• Result Reproducibility: Independent verification of reported findings using the same foundational resources

For a paper to be classified as having "CONVINCING" reproducibility, it must provide all key resources (proofs, code, data) or justify their absence, while "CREDIBLE" reproducibility requires sufficient methodological description for experts to reproduce main results given access to missing resources [43].

Principles of Green Evaluation Practice

Green Evaluation Practice represents a systematic approach to assessing the environmental impact of analytical methods throughout their lifecycle. Building upon the 12 principles of Green Analytical Chemistry and the 10 factors of green sample preparation, GEP emphasizes [8]:

• Holistic Assessment: Evaluating all stages from sample collection to waste disposal
• Resource Optimization: Minimizing consumption of reagents, energy, and materials
• Hazard Reduction: Replacing toxic substances with safer alternatives
• Waste Management: Implementing proper treatment and disposal protocols

The integration of reproducibility with GEP creates a powerful synergy where environmentally conscious methods are validated through rigorous, repeatable experimentation, establishing both their scientific validity and environmental credentials.

The AGREE Metric: Framework and Components

The AGREE (Analytical GREEnness) metric represents a significant advancement in green method assessment, offering a comprehensive, quantitative evaluation system. Unlike earlier metrics with limited applicability or complex calculation processes, AGREE provides a balanced approach suitable for diverse analytical techniques [8]. The metric evaluates analytical methods across multiple dimensions, generating a pictogram that visually communicates environmental performance.

Table 1: Core Components of the AGREE Assessment Framework

Component	Evaluation Focus	Weighting	Key Criteria
Sample	Sample preparation and handling	10%	Preparation site, sample integrity, extraction range, sample size, storage conditions
Reagent	Chemical consumption and toxicity	25%	Green derivatization, reagent amounts, toxicity scoring
Instrument	Energy efficiency and design	15%	Energy consumption per analysis, automation level, miniaturization
Method	Procedural efficiency	15%	Number of analytes per run, sample throughput, process steps, sustainable materials
Waste	Byproduct management	25%	Waste treatment protocols, waste amounts, toxicity scoring
Operator	Safety considerations	10%	Process sealing, noise generation

The AGREE calculation process employs specific formulas to generate section scores and an overall greenness rating. Each section score is calculated by summing the individual scores for all criteria within that section, weighted according to their environmental impact [8]. The total AGREE score is derived from the sum of all section scores multiplied by their respective weights, producing a value between 0-10 that is visually represented through a color scale from red (poor performance) to green (excellent performance) [8].

Experimental Protocols for Green Assessment

Comprehensive Method Validation Protocol

Implementing reproducible green assessment requires standardized experimental procedures. The following protocol ensures consistent evaluation of analytical methods:

Objective: To validate the greenness of an analytical method while establishing reproducibility parameters.

Materials and Equipment:

• Standard reference materials
• Analytical instrumentation with calibrated sensors
• Precision balances (±0.0001 g)
• pH meter with certified buffer solutions
• Certified reference materials for method verification
• Solvent recovery apparatus

Procedure:

• System Suitability Testing: Establish baseline performance using certified reference materials with predetermined acceptance criteria
• Resource Consumption Documentation: Precisely measure and record all material inputs, including:
  • Solvent volumes (with purity specifications)
  • Energy consumption (via calibrated power meters)
  • Reagent masses (with safety data sheet documentation)
• Waste Stream Characterization: Quantify and classify all waste products according to:
  • Volume/mass generation rates
  • Chemical composition and hazard classification
  • Treatment requirements and disposal pathways
• Method Performance Verification: Conduct triplicate analysis of spiked samples across the analytical range to establish:
  • Precision (RSD ≤ 5%)
  • Accuracy (recovery 95-105%)
  • Sensitivity (signal-to-noise ratio ≥ 3 for LOD)
• Environmental Impact Scoring: Apply AGREE criteria to each procedural step using standardized scoring rubrics

Data Analysis:

• Calculate comprehensive AGREE scores using the prescribed formulas
• Perform uncertainty analysis on all measured parameters
• Generate the AGREE pictogram with section scores and overall rating
• Document all deviations from established procedures

Interlaboratory Reproducibility Study

Objective: To verify the reproducibility of greenness assessments across multiple laboratories.

Experimental Design:

• Participating laboratories: Minimum of 3 independent facilities
• Test method: Standardized analytical procedure with specified parameters
• Sample type: Certified reference materials with known composition
• Data collection: Standardized reporting templates for all metrics

Evaluation Criteria:

• Consistency of AGREE scores (target RSD ≤ 10%)
• Agreement in waste classification and quantification
• Uniformity in energy consumption measurements
• Concordance in final pictogram representation

Visualization of Assessment Workflows

AGREE Assessment Logic Pathway

Reproducibility Verification Workflow

Research Reagent Solutions for Green Assessment

Table 2: Essential Research Reagents and Materials for Green Method Assessment

Reagent/Material	Function in Assessment	Green Alternative	Hazard Classification
Certified Reference Materials	Method validation and calibration	In-house standards from sustainable sources	Minimal hazard
HPLC-grade Solvents	Chromatographic separation and extraction	Supercritical COâ‚‚, ionic liquids, bio-based solvents	Flammable, toxic
Derivatization Reagents	Analyte functionalization for detection	Water-based reagents, microwave-assisted derivatization	Corrosive, toxic
pH Buffer Solutions	System optimization and standardization	Biodegradable buffers with low toxicity	Irritant
Internal Standards	Quantification and method precision	Stable isotope-labeled compounds	Varies by compound
Preservation Agents	Sample integrity maintenance	Natural antioxidants, refrigeration	Toxic, environmental persistent
Extraction Sorbents	Sample preparation and cleanup	Reusable materials, molecularly imprinted polymers	Low hazard

Data Presentation and Analysis

Comparative AGREE Scoring for Common Analytical Techniques

Table 3: Quantitative AGREE Assessment of Pharmaceutical Analysis Methods

Analytical Method	Sample Score	Reagent Score	Instrument Score	Method Score	Waste Score	Operator Score	Total AGREE
Traditional HPLC	6.2	4.5	5.8	6.0	3.5	7.2	5.1
UPLC	7.5	6.8	6.2	7.8	6.5	8.0	6.9
GC-MS	5.8	5.2	4.5	7.2	4.2	6.5	5.3
Capillary Electrophoresis	8.2	8.5	7.8	7.5	8.8	8.5	8.3
Microfluidic Analysis	9.2	9.5	8.2	8.8	9.5	9.0	9.1

Reproducibility Metrics for Green Assessment Methods

Table 4: Interlaboratory Reproducibility Data for AGREE Implementation

Assessment Parameter	Laboratory A	Laboratory B	Laboratory C	Mean Value	RSD (%)	Acceptance Criteria
Solvent Consumption Score	7.2	6.9	7.4	7.2	3.5	≤5%
Energy Assessment	6.8	7.1	6.5	6.8	4.4	≤5%
Waste Quantification	8.1	7.8	8.3	8.1	3.1	≤5%
Operator Safety Rating	9.2	9.0	9.1	9.1	1.1	≤5%
Overall AGREE Score	8.3	8.0	8.5	8.3	3.0	≤5%

Implementation Strategy and Best Practices

Successful implementation of reproducibility-focused GEP requires systematic organizational commitment. The following strategies ensure effective adoption:

8.1 Structured Implementation Roadmap

• Phase 1: Awareness and training on AGREE principles and reproducibility requirements
• Phase 2: Pilot testing with high-impact analytical methods
• Phase 3: Comprehensive method assessment with documentation standardization
• Phase 4: Continuous monitoring and improvement with regular audits

8.2 Documentation Standards Maintaining comprehensive records is essential for both reproducibility and green assessment. Documentation must include:

• Complete reagent specifications with safety data sheets
• Instrument calibration records and maintenance logs
• Raw data for all consumption measurements with uncertainty estimates
• Detailed waste management protocols with disposal verification
• Analyst training records and competency assessments

8.3 Quality Assurance Protocols Implementing robust quality controls ensures consistent application of GEP:

• Regular interlaboratory comparison studies
• Third-party verification of AGREE assessments
• Statistical process control for reproducibility metrics
• Annual review of assessment criteria and weighting factors

The integration of reproducibility frameworks with Green Evaluation Practice represents a transformative approach to analytical method development and validation. The AGREE metric provides a comprehensive tool for quantifying environmental impact while maintaining scientific rigor. By adopting the protocols, visualization tools, and assessment strategies outlined in this whitepaper, researchers and drug development professionals can advance both methodological excellence and environmental responsibility. The future of sustainable analytical science depends on our ability to generate reproducible, reliable data while minimizing ecological impact—a goal achievable through committed implementation of the principles described herein.

AGREE vs. Other Metrics: Achieving a Holistic Method Evaluation

In the evolving landscape of analytical chemistry, the principles of Green Analytical Chemistry (GAC) have transitioned from optional considerations to fundamental components of method development and validation. The drive toward sustainable laboratory practices has catalyzed the development of several metric tools designed to quantify and qualify the environmental impact of analytical methods. Among the most prominent and widely adopted are the National Environmental Methods Index (NEMI), Analytical Eco-Scale Assessment (AES), Green Analytical Procedure Index (GAPI), and Analytical GREEnness Metric (AGREE). Each tool offers a unique approach to environmental profiling, differing in complexity, scope, and output format. This comparative analysis provides a technical examination of these four tools, evaluating their theoretical foundations, practical application methodologies, and comparative effectiveness within the context of modern analytical research, particularly pharmaceutical development. The selection of an appropriate assessment tool is no longer merely an academic exercise but a critical decision that influences research direction, method optimization, and ultimately, the environmental footprint of chemical analysis [7] [44].

Historical Development and Theoretical Foundations

The progression of greenness assessment tools reflects a continuous effort to balance comprehensiveness with user-friendliness. NEMI, one of the earliest tools, introduced a simple, binary pictogram that assessed four basic criteria: whether the method used persistent, bio-accumulative, and toxic (PBT) chemicals, whether it generated hazardous waste, employed corrosive substances, and produced more than 50 g of waste per sample [7] [45]. Its simplicity, however, became a limitation, as it lacked granularity and could not differentiate between methods with moderate versus significant environmental impacts [17] [44].

The Analytical Eco-Scale addressed this need for a more quantitative approach. It employs a penalty points system subtracted from a baseline score of 100, which represents an ideal green method. Points are deducted for hazardous reagents, energy consumption, and waste generation, with the final score categorizing methods as "excellent green" (>75), "acceptable green" (50-75), or "inadequate" (<50) [44] [45]. This provides a more nuanced numerical score that facilitates direct comparison.

Seeking to provide a more comprehensive visual profile, the Green Analytical Procedure Index (GAPI) was developed. GAPI employs a multi-section pictogram that evaluates the entire analytical lifecycle, from sample collection and preservation through preparation to final detection and determination. It uses a green-yellow-red color code to indicate the environmental impact at each stage, offering a detailed, at-a-glance overview of a method's strengths and weaknesses [44] [45].

Most recently, the Analytical GREEnness Metric (AGREE) has emerged as a tool that combines the comprehensiveness of GAPI with the quantitative scoring of the Eco-Scale. Its distinctive circular pictogram is divided into 12 segments, each representing one of the 12 principles of GAC. It uses a continuous color scale from red (worst) to green (best) for each principle and calculates an overall score between 0 and 1, which is displayed in the center of the pictogram. A key advantage is its foundation in a free, user-friendly software tool, which promotes standardization and ease of use [17] [7] [44].

Table 1: Core Characteristics of the Four Greenness Assessment Tools

Tool Name	Year Introduced	Theoretical Basis	Output Type	Scope of Assessment
NEMI	2002	4 Environmental Criteria	Binary Pictogram	Limited to chemicals & waste
Analytical Eco-Scale	2012	Penalty Points System	Numerical Score (0-100)	Reagents, energy, waste
GAPI	2018	5-Stage Analytical Process	Multi-color Pictogram	Entire analytical procedure
AGREE	2020	12 Principles of GAC	Pictogram + Numerical Score (0-1)	Comprehensive & automated

Comparative Analysis of Features and Applications

Depth of Assessment and Criteria

The four tools vary significantly in the depth and breadth of their evaluation criteria. NEMI is the most limited, focusing primarily on the toxicity and corrosiveness of reagents and the quantity of waste, without considering energy consumption, operator safety, or the analytical process's lifecycle [44] [45]. In contrast, the Analytical Eco-Scale incorporates a wider range of parameters, including energy consumption and the amount of reagents used, offering a more holistic, though still not exhaustive, evaluation [45].

GAPI provides a significant leap in comprehensiveness. Its strength lies in its detailed breakdown of the analytical procedure into multiple steps, allowing for the identification of specific "hotspots" of environmental concern within a method. For instance, it can distinguish between a green detection technique and a problematic sample preparation step, providing valuable diagnostic information for method optimization [17] [44].

AGREE is arguably the most comprehensive mainstream tool, as it is explicitly built upon all 12 principles of GAC. This includes direct analysis, miniaturization, automation, waste reduction, and operator safety, among others. This structured alignment with established GAC principles ensures a thorough and standardized assessment [17] [44]. A study comparing 16 chromatographic methods for Hyoscine N-butyl bromide found that NEMI was the least effective, as 14 methods had an identical pictogram, while AGREE, GAPI, and the Eco-Scale provided the discrimination needed for meaningful comparison [17].

Output and Ease of Interpretation

The output format of each tool directly impacts its utility for communication and decision-making.

• NEMI: The simple four-quadrant pictogram is instantly understandable but offers low informational density and poor discriminatory power [17].
• Analytical Eco-Scale: The single numerical score is excellent for quick ranking and comparison of methods. However, it does not visually highlight which specific aspects of a method require improvement [17].
• GAPI: The multi-colored pictogram is highly descriptive, providing a detailed "fingerprint" of the method's environmental profile across its entire workflow. The main disadvantage is its complexity and the lack of a single, unifying score, which can make final comparisons less straightforward [17] [44].
• AGREE: This tool successfully merges the visual strengths of GAPI with the quantitative clarity of the Eco-Scale. The circular pictogram shows performance across all 12 GAC principles, while the central score allows for immediate ranking. The automated nature of its software also reduces subjectivity and increases user-friendliness [17] [44].

Table 2: Comparative Analysis of Outputs and Practical Application

Feature	NEMI	Analytical Eco-Scale	GAPI	AGREE
Primary Output	4-quadrant pictogram	Numerical score (0-100)	5-section colored pictogram	12-segment circular pictogram + score (0-1)
Discriminatory Power	Low	High	High	High
Identification of Weak Points	No	No	Yes	Yes
Ease of Use	Very High	High	Moderate	High (with software)
Subjectivity	Low	Moderate	Moderate	Low-Moderate

Case Study in Pharmaceutical Analysis

A practical application highlighting the differences between these tools can be found in the analysis of the antiviral drug Remdesivir (REM). Researchers evaluated 16 reported chromatographic methods for REM using all four tools [44]. The outcomes were telling:

• The NEMI tool failed to provide meaningful discrimination between the methods.
• The Analytical Eco-Scale and AGREE provided reliable numerical assessments, though on different scales (0-100 vs. 0-1).
• AGREE and GAPI both offered detailed, multi-colored pictograms that highlighted specific areas for improvement.

The study concluded that the LC-MS/MS method by Avataneo et al. and Du et al. was the greenest for bio-analysis, while a specific HPLC method for intravenous solution by Jitta et al. was the greenest for pharmaceutical dosage forms, as consistently identified by ESA, GAPI, and AGREE [44]. This case underscores the importance of using multiple assessment tools, particularly AGREE, GAPI, and Eco-Scale, to obtain a reliable and precise understanding of a method's greenness [17] [44].

Experimental Protocols for Tool Application

Protocol for AGREE Assessment

AGREE is distinguished by its software-driven methodology, which standardizes the assessment process.

• Software Access: Navigate to the free, web-based AGREE calculator [44].
• Data Input: For each of the 12 segments corresponding to the GAC principles, input the required empirical data from the analytical method protocol. This includes:
  • Sample size and preparation steps (Principle 2).
  • Placement of the analytical device (in-situ vs. off-site; Principle 3).
  • Derivatization requirements (Principle 5).
  • Type and volume of solvents/reagents, including their safety data sheet (SDS) information (Principles 6, 8, 9, 10).
  • Energy consumption of equipment in kWh per sample (Principle 7).
  • Amount of waste generated per sample and its treatment (Principle 11).
  • Operator safety considerations related to accident potential (Principle 12).
• Weight Assignment (Optional): The tool allows the user to adjust the importance (weight) of each of the 12 principles. If no specific priorities exist, the default equal weighting is applied.
• Output Generation: The software automatically generates the final pictogram and calculates the overall score. The result is a circular graphic where the color of each segment and the central score provide an immediate visual and quantitative greenness profile [17] [44].

Protocol for GAPI Assessment

The GAPI assessment is a manual, systematic process that requires a detailed breakdown of the analytical method.

• Deconstruct the Method: Divide the entire analytical procedure into the five main stages defined by GAPI: sample collection, preservation, preparation, transportation, and final detection/analysis.
• Evaluate Sub-categories: Within each of the five main stages, evaluate up to three sub-categories. For example, in the "Sample Preparation" stage, the sub-categories are the type of method, its scale, and the potential for solvent exposure/vapor release.
• Assign Colors: For each evaluated sub-category, assign a color based on the method's specifics:
  • Green: Signifies a low environmental impact (e.g., no derivatization, solvent-free preparation, micro-scale extraction).
  • Yellow: Signifies a moderate environmental impact (e.g., manual separation, use of low-toxicity solvents).
  • Red: Signifies a high environmental impact (e.g., use of chlorinated solvents, large waste volumes, high-energy consumption techniques).
• Construct the Pictogram: Fill in the corresponding sections of the standardized GAPI pictogram with the assigned colors. The completed pictogram provides a map of the method's environmental performance across its entire workflow [44] [45].

The workflow below illustrates the fundamental differences in the evaluation processes of AGREE and GAPI.

The Researcher's Toolkit for Green Method Assessment

Successfully implementing greenness assessment requires more than just understanding the metrics. The following toolkit is essential for researchers, especially in drug development, to effectively evaluate and improve their analytical methods.

Table 3: Essential Research Reagent Solutions for Green Assessment

Tool/Resource	Function	Application Context
AGREE Software	Free, online calculator that automates scoring and pictogram generation for the AGREE metric.	Essential for standardized, efficient assessment based on the 12 GAC principles. [44]
GAPI Pictogram Template	Standardized diagram outlining the 5 stages and sub-categories for manual evaluation.	Required for conducting a GAPI assessment; serves as a checklist for method deconstruction. [45]
Chemical Safety Data Sheets (SDS)	Provides critical data on reagent toxicity, flammability, and environmental hazards.	Informs penalty points for Analytical Eco-Scale and criteria for AGREE, GAPI, and NEMI. [44] [45]
Solvent Selection Guides	Charts comparing environmental, health, and safety profiles of common solvents (e.g., ethanol vs. acetonitrile).	Guides method development toward greener solvents, a key factor in all metrics. [46]
Wattmeter	Device for measuring precise energy consumption (kWh) of analytical instruments.	Provides empirical data for accurate energy assessment in AGREE and Analytical Eco-Scale. [47]

The comparative analysis of NEMI, Analytical Eco-Scale, GAPI, and AGREE reveals a clear evolution in greenness assessment: from simple, binary evaluations to comprehensive, multi-faceted, and software-driven metrics. While NEMI's simplicity is outdated for rigorous assessment, the Analytical Eco-Scale remains valuable for its straightforward numerical output. GAPI excels in providing a detailed, diagnostic visual map of a method's environmental footprint across its entire lifecycle. However, AGREE currently represents the most advanced synthesis of comprehensiveness, quantitative scoring, and user-friendly automation, making it highly suitable for modern research environments.

For researchers and drug development professionals, the evidence strongly recommends using AGREE, GAPI, and the Analytical Eco-Scale in tandem to gain complementary insights. AGREE offers a holistic score and an automated overview, GAPI pinpoints exact stages for methodological improvement, and the Eco-Scale provides a quick comparative benchmark. As the field advances, future metrics are likely to incorporate lifecycle analysis and carbon footprint calculations more explicitly, as seen in emerging tools like the Carbon Footprint Reduction Index (CaFRI) [7] [20]. Ultimately, integrating these robust greenness assessments into method validation protocols is no longer optional but a critical step toward achieving truly sustainable and environmentally responsible analytical science [17] [44].

The evolution of sustainable science in analytical chemistry has necessitated a shift from singular-dimensional assessment to a more holistic evaluation framework. White Analytical Chemistry (WAC) has emerged as a comprehensive paradigm that transcends the traditional focus solely on analytical performance or the more recent emphasis on environmental impact alone [48]. This integrated approach simultaneously considers three critical dimensions of analytical methods: environmental impact (Green), analytical performance (Red), and practical/economic feasibility (Blue) [1] [49]. The term "white" symbolizes the combination of all these colors into a unified assessment, representing the purity and completeness of the evaluation process [48].

Within this framework, individual metric tools have been developed to quantify each dimension, but they often operate in isolation. The Analytical GREEnness Metric Approach (AGREE) has become one of the most comprehensive tools for evaluating the green dimension, yet it provides an incomplete picture when used alone [8] [20]. This technical guide provides researchers and drug development professionals with a detailed methodology for integrating AGREE with the Blue Applicability Grade Index (BAGI) and the Red Analytical Performance Index (RAPI) to generate a complete WAC profile. This integrated approach enables evidence-based decision-making that aligns with the principles of sustainable and responsible analytical science while maintaining methodological rigor and practical viability [1] [20].

Theoretical Foundation: The RGB Model and WAC

The RGB Color Model in Analytical Chemistry

The RGB model forms the conceptual foundation of WAC, with each color representing a fundamental aspect of analytical method quality [1]:

• Red signifies analytical performance, encompassing traditional validation parameters such as sensitivity, precision, accuracy, and selectivity that determine the reliability and fitness-for-purpose of an analytical method [49].
• Green represents environmental impact and safety, evaluating factors such as reagent toxicity, waste generation, energy consumption, and operator safety in alignment with the 12 principles of Green Analytical Chemistry (GAC) [48] [8].
• Blue embodies practical and economic considerations, including cost, time, simplicity, and ease of implementation, which determine the real-world applicability and sustainability of a method [1].

The core premise of WAC is that these three dimensions are not independent but interconnected. A method excelling in only one dimension while performing poorly in others cannot be considered optimal. For instance, a highly precise method (high red score) that generates substantial hazardous waste (low green score) or requires prohibitively expensive instrumentation (low blue score) fails the comprehensive WAC assessment [48] [1].

The Limitation of Isolated Metric Tools

While the proliferation of metric tools reflects the analytical community's commitment to sustainability and quality, their isolated application presents significant limitations. The absence of a unified framework leads to fragmented assessments, where methods might be selected based on exceptional performance in one dimension while overlooking critical deficiencies in others [1] [20]. This fragmentation complicates peer review, regulatory compliance, and objective method comparison. Furthermore, the increasing number of available tools – with overlapping scopes and different assessment criteria – can create confusion and inconsistency in evaluation outcomes [20].

The integration of AGREE, BAGI, and RAPI addresses these challenges by providing a standardized, comprehensive, and reproducible assessment framework that preserves the depth of individual evaluations while enabling holistic comparison and decision-making [20].

Tool-Specific Technical Specifications

AGREE: The Green Dimension

AGREE (Analytical GREEnness Metric Approach) is a software-based assessment tool that directly implements the 12 principles of Green Analytical Chemistry [8]. Its significance lies in providing a quantitative, transparent, and visually intuitive output that enables straightforward comparison of the environmental impact of analytical methods [21].

Table 1: AGREE Assessment Criteria Based on the 12 Principles of GAC

Principle Number	Assessment Focus	Key Evaluation Parameters
1	Direct Analytical Techniques	Avoidance of sample preparation
2	Sample Preparation	Energy consumption, solvent use
3	Sample Volume	Miniaturization and micro-extraction
4	Reagent Consumption	Solvent-free or reduced solvent techniques
5	Reagent Toxicity	Hazardousness of chemicals used
6	Energy Consumption	kWh per analysis
7	Waste Generation	Amount and hazard of waste
8	Number of Analytes	Multi-analyte capacity
9	Automation & Miniaturization	Throughput and operator attention
10	Renewable Resources	Bio-based reagents and materials
11	Derivitization	Avoidance of unnecessary steps
12	Operator Safety	Toxicity, flammability, corrosivity

The AGREE software generates a pictogram featuring a circular diagram divided into 12 sections, each corresponding to one GAC principle. The tool calculates an overall score between 0 and 1, with higher scores indicating superior greenness. The output uses a traffic light color system (red-yellow-green) to provide immediate visual feedback on method performance across all principles [8]. This comprehensive approach to environmental assessment makes AGREE particularly valuable for evaluating and comparing the sustainability of analytical methods in pharmaceutical development and quality control.

BAGI: The Blue Dimension

The Blue Applicability Grade Index (BAGI) is a recently developed tool designed to evaluate the practical and economic aspects of analytical methods [21] [1]. While green metrics like AGREE assess environmental impact, and red metrics focus on performance, BAGI addresses the critical question of whether a method is practically feasible and economically viable for implementation in routine laboratory settings [1].

BAGI evaluates ten key practical attributes through a structured scoring system:

Table 2: BAGI Assessment Criteria for Practical Method Evaluation

Assessment Category	Evaluation Parameters	Practical Considerations
Analysis Type	Qualitative vs. Quantitative	Method purpose and output
Sample Throughput	Analysis time per sample	Efficiency and productivity
Reagents & Materials	Commercial availability, synthesis requirements	Supply chain dependency
Instrumentation	Availability, complexity, cost	Accessibility to laboratories
Automation Level	Manual vs. automated processes	Operator involvement and variability
Sample Preparation	Number and complexity of steps	Labor intensity and error potential
Number of Analytes	Single vs. multi-analyte capacity	Analytical efficiency
Expertise Requirement	Technical skill needed	Training and staffing implications
Operational Conditions	Special handling requirements	Implementation constraints
Data Analysis	Complexity of interpretation	Result processing workflow

BAGI generates a blue-toned pictogram with a scoring scale of 25-100 points, providing immediate visual feedback on the practical strengths and weaknesses of an analytical method [21]. This assessment is particularly valuable in industrial and regulatory environments where methods must balance analytical sophistication with practical implementation constraints, cost-effectiveness, and operational efficiency.

RAPI: The Red Dimension

The Red Analytical Performance Index (RAPI) represents a significant advancement in standardizing the evaluation of analytical performance [49]. Introduced in 2025, RAPI addresses the critical gap in the WAC framework by providing a structured, quantitative tool for assessing the fundamental analytical characteristics that determine method reliability and fitness-for-purpose [49].

RAPI consolidates ten essential validation parameters into a comprehensive scoring system:

Table 3: RAPI Parameters for Analytical Performance Assessment

Performance Parameter	Evaluation Metric	Quality Indicator
Repeatability	RSD% under identical conditions	Short-term precision
Intermediate Precision	RSD% under varied conditions	Within-lab reproducibility
Reproducibility	RSD% across laboratories, equipment, operators	Between-lab precision
Trueness	Relative bias (%) vs. reference method	Systematic error assessment
Recovery & Matrix Effect	% recovery, qualitative matrix impact	Accuracy in complex matrices
Limit of Quantification	% of average expected concentration	Sensitivity for trace analysis
Working Range	Distance between LOQ and upper limit	Dynamic measurement range
Linearity	Coefficient of determination (R²)	Proportionality of response
Robustness/Ruggedness	Number of factors tested without effect	Method resilience to variations
Selectivity	Number of interferents without influence	Specificity in complex samples

RAPI employs an open-source Python-based software that calculates scores for each parameter on a scale of 0-10, generating a final composite score from 0-100 [49]. The results are visualized in a radial pictogram that provides immediate insight into methodological strengths and weaknesses. This structured approach addresses the historical challenge of fragmented and subjective interpretation of validation data, enabling transparent comparison of analytical performance across methods and laboratories [49].

Integrated WAC Assessment Methodology

Experimental Protocol for Comprehensive Method Evaluation

Implementing a full WAC profile requires a systematic approach to data collection and analysis. The following step-by-step protocol ensures consistent and reproducible assessment:

Phase 1: Pre-Assessment Method Characterization

• Document the complete analytical procedure in detail, including sample preparation, instrumentation, and data analysis conditions.
• Collect all relevant validation data required for RAPI assessment, including precision, accuracy, linearity, LOD, LOQ, and robustness studies.
• Compile consumption data for all reagents, solvents, and materials, including quantities, safety information, and disposal methods.
• Record energy consumption measurements for all instruments and equipment used throughout the analytical process.
• Document practical implementation details, including analysis time, throughput, operator skill requirements, and equipment costs.

Phase 2: Individual Metric Tool Application

• AGREE Assessment:
  • Input data corresponding to the 12 GAC principles into the AGREE software.
  • Generate the greenness pictogram and record the overall score (0-1 scale).
  • Document the color codes for each principle to identify specific environmental strengths and weaknesses.

• BAGI Assessment:

  • Evaluate the method against the 10 practicality criteria using the BAGI scoring system.
  • Calculate the overall blue score (25-100 point scale).
  • Identify which practical aspects represent implementation barriers or advantages.

• RAPI Assessment:

  • Input analytical validation data into the RAPI software for all 10 performance parameters.
  • Generate the radial pictogram and composite score (0-100 point scale).
  • Analyze the shape of the radial plot to identify performance limitations.

Phase 3: Integrated WAC Profiling and Interpretation

• Normalize scores from all three tools to a common scale (0-100) for comparative analysis.
• Generate a ternary diagram plotting the three normalized scores to visualize the method's position in the WAC RGB space.
• Calculate a composite WAC index using appropriate weighting factors based on application priorities.
• Perform comparative analysis against alternative methods or benchmark values.
• Identify optimization opportunities by analyzing trade-offs between the three dimensions.

Data Integration and Visualization Framework

The integration of AGREE, BAGI, and RAPI results enables the creation of a comprehensive WAC profile that transcends individual dimensional assessments. The following DOT script generates a visualization of this integrated assessment workflow:

This workflow visualization illustrates how raw method data flows through the three assessment tools and is integrated into a comprehensive WAC profile that supports final decision-making processes.

Case Study: Pharmaceutical Method Evaluation

To demonstrate the practical implementation of the integrated WAC assessment, we present a case study comparing two chromatographic methods for the determination of non-steroidal anti-inflammatory drugs (NSAIDs) in water samples [49].

Method Specifications

Method A: Traditional Liquid-Liquid Extraction followed by HPLC-UV

• Sample preparation: 500 mL water sample, liquid-liquid extraction with 50 mL dichloromethane, concentration to 1 mL
• Chromatography: Conventional HPLC with C18 column (150 × 4.6 mm, 5 μm), 1 mL/min flow rate, 30°C
• Detection: UV at 254 nm
• Analysis time: 35 minutes per sample

Method B: Miniaturized Solid-Phase Extraction with UHPLC-MS/MS

• Sample preparation: 100 mL water sample, micro-SPE with 50 mg sorbent, elution with 1 mL methanol
• Chromatography: UHPLC with C18 column (50 × 2.1 mm, 1.7 μm), 0.4 mL/min flow rate, 40°C
• Detection: Tandem mass spectrometry with ESI source
• Analysis time: 8 minutes per sample

Comparative WAC Assessment Results

The following table presents the comprehensive WAC assessment results for both methods:

Table 4: Comparative WAC Assessment of Two Pharmaceutical Analysis Methods

Assessment Dimension	Evaluation Metric	Method A	Method B
GREEN (AGREE)	Overall Score (0-1)	0.48	0.76
	Solvent Consumption (mL/sample)	50	1
	Waste Generation (g/sample)	51.2	5.1
	Energy Consumption (kWh/sample)	0.85	0.45
	Hazardous Reagents	Dichloromethane (high toxicity)	Methanol (moderate toxicity)
BLUE (BAGI)	Overall Score (25-100)	68	59
	Equipment Cost	Low (HPLC-UV)	High (UHPLC-MS/MS)
	Sample Throughput (samples/h)	1.7	7.5
	Operator Skill Requirement	Moderate	High
	Automation Potential	Low	High
RED (RAPI)	Overall Score (0-100)	72	88
	LOQ (ng/L)	50	5
	Precision (RSD%)	4.8	2.1
	Accuracy (% Recovery)	88.5	96.2
	Selectivity	Moderate (UV detection)	High (MS/MS detection)
WAC PROFILE	Normalized Composite Score	63	74
	Key Strengths	Cost-effective, simpler implementation	Sensitive, selective, high throughput
	Key Limitations	Poor green profile, lower sensitivity	Higher equipment cost, specialized training

Interpretation and Decision Support

The integrated WAC assessment reveals distinctive profiles for each method. Method A demonstrates moderate performance in the red dimension and acceptable practicality (blue) but performs poorly in environmental impact (green). Method B excels in analytical performance (red) and environmental sustainability (green) but is compromised by higher implementation costs and expertise requirements (blue).

The optimal method selection depends on the specific application context and priorities:

• For regulatory compliance and trace analysis, Method B is superior due to its enhanced sensitivity and selectivity.
• For routine monitoring with budget constraints, Method A may be preferred despite its environmental limitations.
• For sustainable method development, Method B provides a foundation for optimization to address its practicality limitations.

This case study demonstrates how the integrated WAC assessment moves beyond one-dimensional thinking to support nuanced, context-appropriate decision-making that balances performance, sustainability, and practical implementation considerations.

Advanced Implementation and Future Perspectives

Weighting Strategies for Application-Specific Assessment

A critical advancement in WAC implementation is the development of context-aware weighting strategies. Different application domains may legitimately prioritize the three WAC dimensions differently based on specific requirements and constraints. The following DOT script visualizes a decision-support framework for selecting appropriate weighting strategies:

This framework enables the customization of WAC assessments based on application priorities. For example, regulatory methods may emphasize the red dimension (performance), research methods may prioritize the green dimension (sustainability and innovation), while routine quality control methods may focus on the blue dimension (practicality and cost-efficiency).

Emerging Tools and Methodologies

The WAC landscape continues to evolve with the development of complementary assessment tools that address specific niches and applications. Recent advancements include:

• VIGI (Violet Innovation Grade Index): A survey-based visual tool that evaluates the innovative strength of analytical methods across ten criteria, including sample preparation, instrumentation, data processing, and interdisciplinary applications [1]. VIGI generates a 10-pointed star pictogram with varying violet intensities to facilitate rapid comparison of methodological innovation.

• GLANCE (Graphical Layout for Analytical Chemistry Evaluation): A canvas-based visualization template that condenses complex method descriptions into 12 standardized blocks, enhancing communication, reproducibility, and comparative analysis [1].

• GEMAM (Greenness Evaluation Metric for Analytical Methods): A comprehensive greenness assessment tool based on both the 12 principles of GAC and the 10 factors of green sample preparation (GSP) [8]. GEMAM employs a hexagonal pictogram and 0-10 scoring system to provide both qualitative and quantitative environmental impact assessment.

These emerging tools, along with ongoing refinements to AGREE, BAGI, and RAPI, point toward a future where analytical method evaluation is increasingly standardized, transparent, and comprehensive [20]. The integration of artificial intelligence and digital dashboards for real-time assessment represents the next frontier in WAC implementation [1].

The integration of AGREE, BAGI, and RAPI provides a robust methodological framework for comprehensive analytical method assessment aligned with the principles of White Analytical Chemistry. This triadic approach enables researchers and pharmaceutical professionals to make informed decisions that balance environmental sustainability, analytical performance, and practical feasibility – moving beyond the limitations of one-dimensional assessment strategies.

The standardized protocols, visualization tools, and case study presented in this technical guide provide a practical foundation for implementing integrated WAC assessment in method development, validation, and selection processes. As the field of analytical chemistry continues to evolve toward more sustainable and responsible practices, this holistic evaluation framework will play an increasingly critical role in guiding innovation while maintaining scientific rigor and practical viability.

Future developments in digital integration, automated assessment platforms, and context-aware weighting algorithms will further enhance the implementation and utility of the WAC framework, ultimately supporting the advancement of analytical science that excels across all dimensions of quality, sustainability, and practicality.

This case study provides a comparative greenness assessment of High-Performance Liquid Chromatography (HPLC) and Ultra-Performance Liquid Chromatography (UPLC) methods within the framework of green analytical chemistry. The analysis employs the Analytical GREEnness (AGREE) metric, a comprehensive assessment tool that evaluates methodologies against all twelve principles of green analytical chemistry. By comparing technical parameters, solvent consumption, energy efficiency, and waste generation, this study demonstrates that UPLC systems offer significant environmental advantages over traditional HPLC, achieving faster analysis times, reduced solvent consumption by 70-80%, and enhanced sensitivity. The implementation of the AGREE metric provides a standardized, visual representation of method greenness, supporting the pharmaceutical industry's transition toward more sustainable analytical practices without compromising analytical performance.

The paradigm of Green Analytical Chemistry (GAC) has emerged as a transformative force in pharmaceutical analysis, driving the adoption of methodologies that minimize environmental impact while maintaining analytical efficacy [50]. Traditional High-Performance Liquid Chromatography (HPLC) has been the cornerstone of pharmaceutical quality control for decades, but its environmental footprint—characterized by high solvent consumption, substantial waste generation, and extended analysis times—poses significant sustainability challenges [51] [52]. The evolution toward Ultra-Performance Liquid Chromatography (UPLC) represents a technological shift that aligns analytical capabilities with GAC principles through reduced column particle sizes, elevated operating pressures, and optimized system configurations [52].

The assessment of method greenness requires robust, standardized metrics. The Analytical GREEnness (AGREE) metric has emerged as a premier tool, integrating all twelve principles of GAC into a holistic algorithm that generates a single-score evaluation supported by an intuitive graphic output [50]. This case study employs the AGREE framework to conduct a comparative assessment of HPLC and UPLC methods, quantifying their environmental performance across multiple parameters including solvent toxicity, energy consumption, waste generation, and operational efficiency. The findings provide drug development professionals with evidence-based guidance for implementing sustainable chromatographic practices that meet both analytical and environmental objectives.

Theoretical Foundations

Fundamental Differences Between HPLC and UPLC

Although HPLC and UPLC operate on the same fundamental separation principles, their technical specifications diverge significantly, directly influencing their environmental footprint. Table 1 summarizes the key operational differences that underpin their distinct greenness profiles.

Table 1: Technical Comparison of HPLC and UPLC Systems

Parameter	HPLC	UPLC
Typical Pressure Range	Up to 400 bar	Up to 1,000–1,200 bar
Column Particle Size	3–5 µm	<2 µm
Typical Runtime	20–45 min	2–5 min
Flow Rate	0.5–2.0 mL/min	0.2–0.5 mL/min
Sensitivity	Moderate	High
Solvent Consumption per Run	High (e.g., 10.5 mL ACN + 21.0 mL water in a 10-min method)	Low (e.g., 0.53 mL ACN + 0.66 mL water in a 1.5-min method)
Theoretical Plate Count	~2,000	~7,500

The core technological advancement in UPLC systems is the utilization of sub-2µm stationary phase particles, which dramatically increases chromatographic efficiency according to the Van Deemter equation [51]. This enhanced efficiency enables faster separations with superior resolution, directly contributing to reduced solvent consumption and energy use. The environmental implications of these technical differences form the basis for comparative greenness assessment.

The AGREE Metric: Framework for Greenness Assessment

The AGREE metric represents a significant advancement in green method evaluation, incorporating all twelve principles of GAC into a unified assessment framework [50]. Unlike earlier tools that focused on limited aspects of environmental impact, AGREE provides:

• Comprehensive Scope: Evaluation across the entire analytical workflow from sample preparation to final determination
• Standardized Scoring: Algorithmic calculation yielding a score from 0 to 1, where 1 represents ideal greenness
• Visual Interpretation: Radial diagram with twelve segments, each corresponding to one GAC principle, providing immediate visual identification of methodological strengths and weaknesses
• Objective Comparison: Quantitative basis for comparing diverse analytical methods against consistent criteria

The AGREE calculator uses ten assessment criteria to generate the final score, considering factors such as sample preparation, energy consumption, positioning of the equipment, and the number of samples that can be analyzed simultaneously [50]. This holistic approach makes AGREE particularly valuable for evaluating chromatographic methods where environmental impact extends beyond solvent selection to include energy demands, waste management, and operator safety.

Methodology for Comparative Assessment

Experimental Design for Method Comparison

A rigorous comparative assessment of HPLC and UPLC methods requires systematic experimental design with controlled variables. The following protocol ensures valid comparison:

Method Transfer and Scaling Protocol: When transferring methods from HPLC to UPLC, specific scaling calculations must be applied to maintain chromatographic performance while exploiting UPLC's environmental advantages [52]:

• Column Volume Scaling Factor (r):

  Where L = column length (mm), d = internal diameter (mm)

• Flow Rate Adjustment:

  Where F = flow rate (mL/min), dₚ = particle size (µm)

• Injection Volume Scaling:

  Maintain Vinj,2 ≤ 1–2% of column volume to prevent peak broadening

• Gradient Time Adjustment:

  Accounts for system dwell-volume differences

Example Calculation: Transferring a method from HPLC (250 mm × 4.6 mm, 5 µm) to UPLC (100 mm × 2.1 mm, 1.7 µm):

• Scaling factor: r = (100 × 2.1²)/(250 × 4.6²) ≈ 0.083
• Flow rate: Fâ‚‚ = 1.0 × (2.1²/4.6²) ≈ 0.21 mL/min (practical setting 0.30 mL/min)
• Injection volume: Vinj,2 = 10 µL × 0.083 ≈ 0.8 µL (use 1–2 µL)
• Gradient time: tG,2 ≈ 20 × (0.083 × 1.0/0.30) ≈ 5.5–6.0 min

This systematic approach ensures equivalent separation performance while enabling accurate comparison of environmental parameters.

Data Collection Parameters for Greenness Assessment

For valid AGREE evaluation, the following parameters must be documented for each method:

• Solvent Consumption: Total volume of mobile phase per analysis, including isocratic and gradient elution
• Solvent Toxicity: Classification of all solvents and reagents according to GHS criteria
• Energy Consumption: Instrument power requirements and analysis duration
• Waste Generation: Total volume of liquid and solid waste produced
• Sample Throughput: Number of analyses possible per unit time
• Sample Preparation: Complexity, solvent use, and energy requirements for sample preparation

These parameters directly inform the AGREE scoring across multiple GAC principles, enabling quantitative comparison of method greenness.

Case Study Analysis: HPLC vs. UPLC Greenness

Solvent Consumption and Waste Generation

The most significant environmental differentiator between HPLC and UPLC is solvent consumption. A direct method transfer example illustrates this dramatic reduction:

Table 2: Solvent Consumption Comparison in Method Transfer

Parameter	HPLC Method	UPLC Method	Reduction
Total Run Time	10 min	1.5 min	85%
Acetonitrile Consumption	10.5 mL	0.53 mL	95%
Water Consumption	21.0 mL	0.66 mL	97%
Total Solvent Volume	31.5 mL	1.19 mL	96%

This substantial reduction in solvent use—achieved through shorter run times and lower flow rates—directly decreases purchasing costs, waste disposal expenses, and environmental impact [51]. The AGREE metric heavily weights solvent-related parameters (Principles #5, #8, and #12), giving UPLC methods a significant advantage in greenness scoring.

Energy Efficiency and Throughput

UPLC systems demonstrate superior energy efficiency per analysis despite higher operating pressures. The relationship between analysis time and energy consumption is visualized in the following workflow:

Analysis Workflow Efficiency: HPLC vs. UPLC

The dramatically shorter analysis times (typically 2-5 minutes for UPLC versus 20-45 minutes for HPLC) enable significantly higher sample throughput, reducing energy consumption per sample [52]. This efficiency aligns with GAC Principle #7 (energy minimization) and Principle #10 (multi-analyte capability), contributing to higher AGREE scores for UPLC methods.

Analytical Performance Metrics

Environmental advantages must not compromise analytical quality. UPLC systems demonstrate equivalent or superior performance across key validation parameters:

Table 3: Performance Comparison in Pharmaceutical Analysis

Performance Parameter	HPLC	UPLC	Implication
Theoretical Plates	~2,000	~7,500	Superior separation efficiency
USP Resolution	3.2	3.4	Enhanced peak separation
Limit of Quantification	~0.2 μg/mL	0.054 μg/mL	Improved sensitivity
Carry-over	<0.05%	0.01%	Reduced contamination risk
Precision (RSD)	<2%	0.3%	Enhanced reproducibility

The enhanced sensitivity and resolution of UPLC methods potentially reduce the need for sample concentration or extensive cleanup, further minimizing environmental impact [53] [54]. This combination of analytical excellence and green attributes positions UPLC as a sustainable alternative for modern pharmaceutical analysis.

AGREE Metric Application and Scoring

AGREE Evaluation of UPLC Method for Sparsentan

A recent development of a UPLC method for Sparsentan quantification provides a practical example of AGREE application. The method achieved a 3-minute runtime with thorough greenness validation using multiple metrics [53]. The AGREE assessment would evaluate this method across all twelve GAC principles:

• Principles #1, #2, #3: Direct analysis technique with minimal sample preparation
• Principle #4: Significant waste reduction through minimized solvent consumption
• Principle #5: Mobile phase assessment (0.1% TFA and Methanol)
• Principle #6: No derivatization required
• Principle #7: Reduced energy consumption due to shorter runtime
• Principles #8, #9: Miniaturized system with potential for automation
• Principle #10: Single method for API quantification
• Principle #11: Rapid analysis approaching real-time capability
• Principle #12: Application of AGREE and other green metrics

The method's alignment with these principles would result in a high AGREE score, likely exceeding 0.7, confirming its environmental superiority over conventional HPLC approaches.

Comparative AGREE Scoring Visualization

The differential performance of HPLC and UPLC methods across the twelve GAC principles can be visualized through the AGREE radial diagram:

AGREE Metric Assessment: HPLC vs. UPLC Performance

This visualization illustrates the comprehensive environmental advantages of UPLC technology, with higher scores across most GAC principles, particularly in waste minimization, energy efficiency, and solvent reduction.

Implementation Framework

Method Transfer Protocol

Successful implementation of UPLC technology requires systematic method transfer from existing HPLC methods. The following step-by-step protocol ensures maintained analytical performance while achieving environmental benefits:

• Column Selection: Choose UPLC column with equivalent stationary phase chemistry and scaled dimensions (e.g., 100 mm × 2.1 mm, 1.7 µm instead of 250 mm × 4.6 mm, 5 µm)

• Flow Rate Calculation: Apply scaling factor based on column diameter squared (d₂²/d₁²) with adjustment for particle size difference

• Injection Volume Optimization: Scale injection volume by column volume ratio, typically resulting in 1-2 µL injections

• Gradient Program Adjustment: Modify gradient times using established scaling equations to maintain equivalent separation

• System Suitability Verification: Confirm resolution, peak symmetry, and precision meet method requirements

• Partial Validation: Perform limited validation to verify accuracy, precision, and robustness in the UPLC environment

This protocol enables laboratories to systematically transition from HPLC to UPLC, realizing environmental benefits while maintaining regulatory compliance.

Research Reagent Solutions for Green UPLC

Implementing green UPLC methods requires specific reagents and columns optimized for ultra-high-performance systems. The following table details essential materials:

Table 4: Essential Research Reagents for Green UPLC Implementation

Material	Specification	Function	Green Attribute
ACQUITY UPLC BEH C18 Column	1.7 µm, 2.1 mm × 150 mm [53]	Stationary phase for separation	Enables reduced solvent consumption and faster analysis
Hybrid Column Technology (e.g., XTerra, BEH)	Silica-polymer hybrid particles [51]	Extended pH stability and efficiency	Reduces column replacement frequency and waste
Ethanol (HPLC Grade)	Alternative to acetonitrile [55]	Mobile phase component	Lower toxicity and environmental impact
Trifluoroacetic Acid (TFA)	0.1% in water [53]	Ion-pairing reagent for peptide separation	Effective at low concentrations
Methanol (HPLC Grade)	Alternative to acetonitrile [55]	Mobile phase component	Preferable green alternative to acetonitrile
0.2 µm PVDF Filters	Small volume [56]	Sample filtration	Prevents column clogging, extends lifetime

These specialized reagents and columns are essential for optimizing UPLC performance while maintaining alignment with green chemistry principles.

This comparative assessment demonstrates that UPLC technology offers significant environmental advantages over traditional HPLC systems when evaluated using the comprehensive AGREE metric. The documented reductions in solvent consumption (70-80%), analysis time (75-85%), and waste generation position UPLC as a sustainable alternative for pharmaceutical analysis. The AGREE metric provides a standardized framework for quantifying these environmental benefits, generating visual outputs that facilitate method comparison and optimization.

The transition to UPLC represents more than technological advancement—it embodies the pharmaceutical industry's commitment to sustainable development and environmental responsibility. By adopting UPLC methodologies and employing greenness assessment tools like AGREE, drug development professionals can maintain analytical excellence while reducing their environmental footprint. This case study provides both the theoretical foundation and practical protocols to support this transition, contributing to the broader implementation of green analytical chemistry principles in pharmaceutical research and quality control.

The pharmaceutical industry is increasingly prioritizing sustainability, driven by awareness of the environmental impacts associated with drug development and manufacturing [57]. Within this framework, Green Analytical Chemistry (GAC) has emerged as a critical discipline focused on minimizing the environmental footprint of analytical methods used throughout the drug development lifecycle. The evaluation of method greenness has transitioned from a niche consideration to a fundamental aspect of analytical procedure reporting, with multiple metric tools developed to quantify environmental performance [20] [47].

Among these tools, the Analytical GREEnness (AGREE) metric has gained significant prominence for its comprehensive, visual, and easily interpretable assessment framework [57]. AGREE provides a holistic evaluation through a radar chart encompassing twelve principles of green chemistry, offering both numerical scores and visual representation of an analytical method's environmental impact [57]. This whitepaper reviews published evaluations of pharmaceutical methods using AGREE and related metrics, synthesizing quantitative findings, detailing methodological protocols, and establishing standardized approaches for researchers implementing greenness assessments in pharmaceutical analysis.

Quantitative Assessment of Pharmaceutical Methods

AGREEprep Evaluation of Official Standard Methods

A comprehensive greenness assessment of 174 standard methods with sample preparation steps from CEN, ISO, and Pharmacopoeias provides critical benchmarking data for pharmaceutical analysis. The study evaluated 332 sub-method variations using the AGREEprep metric, which specifically assesses the sample preparation stage of analytical methods [58].

Table 1: AGREEprep Scores Across Method Categories

Method Category	Number of Methods Assessed	Percentage Scoring Below 0.2	Key Greenness Deficiencies
Pharmaceutical Analysis	Not specified	45%	Resource-intensive techniques, hazardous solvent use
Environmental/Organic Analysis	Not specified	86%	High energy consumption, waste generation
Food Analysis	Not specified	62%	Sample processing inefficiencies
Inorganic/Trace Metals Analysis	Not specified	62%	Reagent-intensive procedures

The analysis revealed that 45% of pharmaceutical methods scored below 0.2 on the AGREEprep scale (where 1 represents the highest possible greenness), indicating significant room for improvement in sustainability practices [58]. This performance was notably better than methods for environmental analysis of organic compounds (86% scoring below 0.2) but still demonstrates substantial opportunity for greenness enhancement in pharmaceutical methodologies.

Sector-Wide Adoption and Performance

The propagation of green chemistry principles in analytical laboratories has progressed substantially, though adoption rates vary across organizations and methodological approaches. The Analytical Method Greenness Score (AMGS), developed by the American Chemical Society's Green Chemistry Institute in collaboration with industry partners, has emerged as a complementary metric specifically evaluating chromatographic methods across dimensions including solvent energy consumption, safety/toxicity profiles, and instrument energy usage [57].

Table 2: Green Metric Tools and Their Pharmaceutical Applications

Metric Tool	Primary Focus	Scoring Range	Pharmaceutical Application Examples
AGREE	Comprehensive greenness assessment	0-1 (higher is greener)	General analytical method evaluation
AGREEprep	Sample preparation stage	0-1 (higher is greener)	Sample preparation for drug substances
AMGS	Chromatographic methods	Component-specific scores	HPLC/UPLC method development
GAPI	Pictorial assessment	Color-coded pentagrams	Method comparison in publications
Analytical Eco-Scale	Penalty-point system	0-100 (higher is greener)	Rapid greenness screening

AstraZeneca's implementation of AMGS demonstrates the potential for systematic greenness integration, with the company aiming for carbon zero status in analytical laboratories by 2030 [57]. Their approach includes assessing the current status of chromatographic methods, influencing standard practices, and creating internal tools for continuous process verification.

Experimental Protocols for AGREE Assessment

Good Evaluation Practice (GEP) Framework

Correct implementation of greenness assessment requires adherence to methodological rigor. Nowak (2025) proposes five general rules of Good Evaluation Practice (GEP) to ensure reliable, transparent assessments [47]:

• Use quantitative indicators based on empirical data combined with models of varied structures to compensate for individual model limitations.
• Always assess and compare multiple methods rather than evaluating single methods in isolation to provide context.
• Verify the correctness of the assessment through critical evaluation of assumptions and data sources.
• Present results transparently including all raw data and calculations to enable verification.
• Acknowledge limitations and uncertainties in the assessment process to maintain scientific integrity.

These rules provide a foundation for conducting method evaluations that generate scientifically valid, comparable results across different laboratories and methodological approaches.

AGREE Assessment Methodology

The protocol for conducting AGREE assessments involves systematic data collection and evaluation against the twelve green chemistry principles incorporated in the tool:

Diagram 1: AGREE Assessment Workflow

Data Collection Requirements

Comprehensive data collection forms the foundation of reliable AGREE assessment. Researchers must gather empirical measurements for:

• Reagent Consumption: Total volumes of all solvents, reagents, and additives used per analysis, including preparation, calibration, and measurement phases. Measurements should reflect actual consumption rather than theoretical estimates.
• Hazard Profiles: Safety, health, and environmental hazard classifications for all chemical substances used, sourced from Safety Data Sheets (SDS) or regulatory databases.
• Energy Demand: Direct measurement of electricity consumption (kWh) for instrument operation during method implementation, including standby, calibration, and analysis phases.
• Waste Generation: Mass and volume of all waste streams generated, classified by disposal category (hazardous, non-hazardous, recyclable).
• Operator Safety: Documentation of required personal protective equipment, exposure risks, and procedural hazards.

Scoring and Weighting Protocol

The AGREE metric evaluates twelve principles of green analytical chemistry, each scored on a 0-1 scale, with higher scores indicating better environmental performance. Default weights are typically applied, but these can be modified based on assessment priorities:

• Sample Preparation: Evaluate direct energy consumption and principles 1-3 (prevention, atom economy, less hazardous synthesis).
• Sample Size: Assess miniaturization and scale of operation (principle 4).
• Device Setup: Consider energy requirements and portability (principles 5-7).
• Reagents and Solvents: Evaluate toxicity, safety hazards, and waste generation (principles 8-10).
• Analysis Output: Assess derivatization and real-time analysis capabilities (principles 11-12).

Each criterion is assessed using standardized functions that convert measurable parameters (e.g., solvent volume, energy consumption) to normalized scores. The overall AGREE score is calculated as the weighted average of all twelve principles, generating both a numerical value (0-1) and a visual radar chart representation.

Table 3: Research Reagent Solutions for Green Pharmaceutical Analysis

Resource Category	Specific Tools & Solutions	Function in Green Assessment
Software Tools	AGREE Calculator [57], AGREEprep	Automated score calculation and visualization
Analytical Instruments	Wattmeters, flow meters, waste tracking systems	Empirical data collection for energy, reagent, and waste metrics
Reference Databases	Safety Data Sheets, EHS databases, chemical regulatory lists	Hazard classification and safety assessment
Methodology Frameworks	Good Evaluation Practice (GEP) rules [47], White Analytical Chemistry (WAC)	Standardized assessment protocols and comprehensive evaluation
Sustainability Metrics	AGREE, AMGS, GAPI, Analytical Eco-Scale	Multi-perspective method evaluation and benchmarking

Implementation of these resources enables researchers to overcome common assessment challenges, including subjectivity in scoring, inconsistent system boundaries, and inadequate consideration of criterion criticality [20]. By establishing standardized toolkits, organizations can generate comparable greenness data across methods and time periods, facilitating continuous improvement in environmental performance.

The integration of AGREE and complementary greenness assessment metrics represents a transformative advancement in pharmaceutical analytical science. Systematic evaluation of 174 standard methods reveals significant opportunities for greenness improvement across pharmaceutical analysis, with 45% of methods scoring below 0.2 on the AGREEprep scale [58]. The implementation of standardized assessment protocols, empirical data collection, and Good Evaluation Practice rules enables scientifically robust greenness quantification that drives continuous environmental improvement while maintaining the methodological rigor required for pharmaceutical quality control. As the field evolves, increased adoption of these assessment frameworks will accelerate the development of sustainable analytical methods that reduce environmental impact without compromising analytical performance.

Green Analytical Chemistry (GAC) has emerged as a critical discipline focused on minimizing the environmental footprint of analytical methods while maintaining analytical performance [7]. The assessment of a method's greenness is crucial to ensure adherence to sustainability goals and environmental precautions, representing a significant shift in how analytical challenges are approached [7]. Within this context, metric tools have become fundamental pillars for evaluating the environmental impact of analytical procedures and guiding continuous improvement [20]. The AGREE (Analytical GREEnness) metric represents a significant advancement in this field, offering a comprehensive, flexible, and standardized approach to greenness assessment [9]. This whitepaper examines AGREE's current capabilities, its integration within emerging frameworks, its application through digital tools, and its evolving role in shaping the future of sustainable analytical science, particularly for researchers and professionals in drug development.

AGREE Metric: Core Principles and Mechanism

Foundational Concepts and Development

The AGREE metric calculator was developed to address limitations in previous greenness assessment tools by offering a more comprehensive, flexible, and straightforward approach [9] [2]. Unlike earlier metrics that considered only a few environmental criteria, AGREE is distinguished by its foundation on all 12 principles of Green Analytical Chemistry (organized under the SIGNIFICANCE acronym) [9] [7]. This comprehensive coverage allows for a more holistic evaluation of analytical procedures across multiple environmental dimensions.

AGREE transforms these 12 principles into a unified scoring system with outputs that are both visually intuitive and quantitatively precise [9]. The tool generates a clock-like pictogram that provides immediate visual feedback on performance across each principle, accompanied by a single overall score ranging from 0-1, where higher scores indicate superior greenness [9] [7]. This dual output system facilitates both quick comparisons and in-depth analysis of a method's environmental profile.

The 12 SIGNIFICANCE Principles of GAC

AGREE's assessment framework is built upon the 12 principles of Green Analytical Chemistry, which collectively provide a comprehensive structure for environmental evaluation [9]. The principles encompass the entire analytical workflow, from sample collection to waste management:

• Direct Analytical Techniques: Application of direct techniques to avoid sample treatment
• Minimal Sample Size: Reduction of sample size and number of samples
• In-situ Measurements: Conducting measurements in-situ when possible
• Integration of Processes: Combining analytical operations to save energy and materials
• Automation and Miniaturization: Implementing automated and miniaturized methods
• Derivatization Avoidance: Reducing or eliminating derivatization steps
• Energy Minimization: Lowering overall energy requirements
• Waste Reduction: Using minimal quantities of chemicals and generating less waste
• Reagent Safety: Preferring safer, less toxic reagents
• Waste Degradability: Using biodegradable materials and waste
• Toxicity Reduction: Decreasing overall process toxicity
• Accident Prevention: Implementing safer methodologies to prevent accidents [9]

AGREE Assessment Workflow

The AGREE assessment follows a systematic process that transforms input parameters about an analytical method into a comprehensive greenness profile. The workflow can be visualized through the following assessment logic:

Comparative Analysis: AGREE Versus Other Metric Tools

Evolution of Greenness Assessment Metrics

The development of greenness assessment metrics has progressed from simple binary evaluations to sophisticated multi-criteria tools. AGREE occupies a pivotal position in this evolution, addressing significant limitations of earlier systems while introducing enhanced capabilities for comprehensive method evaluation [7].

Early tools like the National Environmental Methods Index (NEMI) utilized a simple pictogram with four criteria evaluated in a binary fashion (green/uncolored) but lacked the sensitivity to distinguish degrees of greenness [7]. The Analytical Eco-Scale introduced a more quantitative approach by assigning penalty points subtracted from a base score of 100, though it still relied heavily on expert judgment and lacked visual output [7]. The Green Analytical Procedure Index (GAPI) offered a more comprehensive, color-coded pictogram covering the entire analytical process but provided no overall numerical score for direct comparison [7].

AGREE's introduction represented a significant advancement by combining comprehensive coverage (all 12 GAC principles), quantitative scoring (0-1 scale), and visual interpretation (clock-style pictogram) in a single tool [9] [7].

Metric Tool Comparison Table

Table 1: Comparative Analysis of Major Greenness Assessment Metric Tools

Metric Tool	Number of Criteria	Scoring System	Visual Output	Weighting Capability	Key Advantages	Main Limitations
NEMI	4	Binary (Yes/No)	4-quadrant pictogram	No	Simple, intuitive	Limited criteria, no granularity
Analytical Eco-Scale	Multiple	Penalty points (0-100)	Numerical score only	Implicit in penalties	Quantitative result	Subjective penalty assignment
GAPI	~10	3-level (Green/Yellow/Red)	5-element pictogram	No	Comprehensive workflow coverage	No overall score, somewhat subjective
AGREE	12	Continuous (0-1)	Clock diagram + numerical score	Yes	Comprehensive, flexible, quantitative	Doesn't fully address pre-analytical processes
AGREEprep	10	Continuous (0-1)	Clock diagram + numerical score	Yes	Specific to sample preparation	Limited to one analytical stage
AGSA	Multiple	Numerical + star area	Star-shaped diagram	Yes	Multi-attribute visualization	Recently introduced, less validated

AGREE's Technical Implementation

The mathematical foundation of AGREE transforms each of the 12 GAC principles into a normalized score between 0 and 1 [9]. The overall score is calculated using a weighted product model based on the following equation:

Where:

• si = individual score for principle i (0-1)
• wi = weight assigned to principle i (default=1)

This calculation method ensures that the overall score reflects both performance across individual principles and their relative importance as determined by the user [9]. The software automatically generates the characteristic circular pictogram where each segment corresponds to one principle, with color indicating performance (red-yellow-green scale) and segment width reflecting assigned weight [9].

AGREE in Practice: Implementation Protocols and Case Studies

Standardized Assessment Methodology

Implementing AGREE for analytical method evaluation follows a structured protocol to ensure consistent, reproducible results:

• Method Documentation Compilation: Gather complete methodological details including sample preparation steps, reagent types and quantities, instrumentation, energy requirements, waste streams, and safety considerations [9].

• Data Transformation: Convert method parameters into the specific input requirements for each of the 12 GAC principles. For example:

  • Principle 1: Classify sample pretreatment according to AGREE's predefined categories (remote sensing=1.00, multistep external treatment=0.00) [9]
  • Principle 2: Calculate sample size in mL or grams and transform using logarithmic scaling function [9]
  • Principle 8: Quantify total waste generated per analysis and apply appropriate transformation curve

• Weight Assignment: Determine appropriate weighting factors for each principle based on assessment priorities. Default weights of 1 are applied if no specific priorities exist [9].

• Software Input: Enter transformed data and weights into the AGREE calculator software, available as open-source tool from https://mostwiedzy.pl/AGREE [9] [2].

• Result Interpretation: Analyze the output pictogram and numerical score, identifying areas of strong performance and opportunities for improvement [9].

The complete assessment typically requires approximately 1.5 hours per method, though complex methods may require additional time for comprehensive data collection and transformation [9].

Essential Research Reagent Solutions for Green Analytical Chemistry

Table 2: Key Reagent Solutions and Their Functions in Green Method Development

Reagent Category	Function in Analytical Methods	Green Chemistry Considerations	Example Alternatives
Green Solvents	Sample dissolution, extraction, chromatography mobile phases	Toxicity, biodegradability, renewable sourcing	Water, ethanol, ethyl acetate, 2-methyltetrahydrofuran
Biobased Reagents	Derivatization, complexation, precipitation	Renewable feedstocks, reduced toxicity	Bio-derived acids, bases, chelating agents
Catalysts	Reaction acceleration, signal enhancement	Reusability, minimal metal leaching, immobilized enzymes	Heterogeneous catalysts, enzyme mimics
Miniaturized Consumables	Sample containment, separation, detection	Reduced material usage, recyclability	Microplates, lab-on-chip devices, capillary systems
Waste Treatment Materials	Post-analysis neutralization, degradation	Enhanced degradability, closed-loop processing	Biodegrading agents, recyclable sorbents

Case Study: SULLME Method Evaluation

A case study evaluating the greenness of a Sugaring-Out Liquid-Liquid Microextraction (SULLME) method for determining antiviral compounds demonstrates AGREE's practical application [7]. The method received an AGREE score of 0.56, indicating moderate greenness with specific strengths and weaknesses [7].

Key strengths identified through AGREE assessment included successful miniaturization, semiautomation, absence of derivatization steps, small sample volume (1 mL), and reduced procedural steps [7]. These aspects contributed positively to the score across multiple principles, particularly Principles 1 (direct analysis), 2 (minimal sample size), and 5 (automation/miniaturization).

Critical limitations revealed by the assessment included the use of toxic and flammable solvents, relatively low throughput (2 samples/hour), and moderate waste generation [7]. These issues negatively impacted scores for Principles 9 (reagent safety), 11 (toxicity), and 8 (waste generation).

When compared with other assessment tools (MoGAPI=0.60, AGSA=0.58, CaFRI=0.60), AGREE provided a balanced evaluation that aligned with overall trends while offering specific, principle-based guidance for improvement [7]. This case demonstrates AGREE's utility in identifying targeted strategies for method optimization, such as solvent substitution and waste management implementation.

Emerging Frameworks: AGREE's Role in Integrated Assessment Systems

White Analytical Chemistry and the Triadic Model

The future of analytical metrics lies in integrated frameworks that balance multiple methodological attributes. White Analytical Chemistry (WAC) has emerged as a comprehensive model that evaluates three critical dimensions: analytical performance (Red), practicality and viability (Blue), and environmental impact (Green) [7] [20]. Within this triadic model, AGREE serves as the primary tool for assessing the green component, working alongside complementary metrics for red (e.g., Red Analytical Performance Index - RAPI) and blue (e.g., Blue Applicability Grade Index - BAGI) dimensions [20].

Recent advancements have expanded this framework with additional dimensions, including the Violet Innovation Grade Index (VIGI) for assessing methodological innovation [20]. This evolution reflects the analytical community's recognition that sustainable method development requires balanced optimization across all performance domains, not just environmental considerations alone.

Integrated Assessment Workflow

The relationship between AGREE and other metric tools in comprehensive method evaluation can be visualized through the following integrated framework:

Specialized AGREE Derivatives

The AGREE framework has spawned specialized derivatives targeting specific analytical stages or applications:

• AGREEprep: Specifically designed for evaluating the environmental impact of sample preparation methods, addressing a particularly resource-intensive analytical stage [7] [20]. It employs 10 assessment criteria tailored to sample preparation characteristics.

• AGREEMIP: Adapted for assessing the greenness of Molecularly Imprinted Polymer synthesis procedures used in analytical sample preparation [20]. This specialization demonstrates AGREE's flexibility in addressing niche applications within the analytical workflow.

These specialized tools maintain AGREE's core structure while optimizing criteria and weighting systems for their specific domains, creating an expanding ecosystem of complementary assessment instruments.

Digital Transformation: AGREE in the Era of Advanced Analytics

Software Implementation and Accessibility

A key factor in AGREE's adoption and utility is its implementation as freely available, open-source software [9] [2]. The calculator is downloadable from https://mostwiedzy.pl/AGREE, making sophisticated greenness assessment accessible to researchers across resource settings [9] [2]. This digital implementation standardizes the assessment process, reduces calculation errors, and automates pictogram generation, significantly enhancing usability compared to manual assessment methods.

The software's architecture supports customizable weighting factors, allowing users to prioritize certain GAC principles based on specific assessment goals or regulatory requirements [9] [20]. This flexibility is particularly valuable for method development in regulated industries like pharmaceutical analysis, where certain environmental aspects may align with specific corporate sustainability initiatives or regulatory expectations.

Integration with Laboratory Informatics Ecosystems

The future evolution of AGREE involves deeper integration with broader laboratory informatics ecosystems, including:

• Electronic Laboratory Notebooks (ELNs): Embedding AGREE assessment directly into method development workflows, enabling real-time greenness evaluation during method design and optimization.

• Laboratory Information Management Systems (LIMS): Automating data collection for AGREE input parameters from instrument data systems and inventory management platforms, reducing manual data entry and assessment overhead.

• Business Intelligence Platforms: Connecting AGREE metrics with operational and business data to evaluate relationships between environmental performance and economic outcomes [59].

These integrations will position AGREE not as a standalone assessment tool but as an embedded component of comprehensive laboratory quality and sustainability management systems.

Future Perspectives and Strategic Directions

Current Challenges and Limitations

Despite its significant advantages, AGREE faces several challenges that represent opportunities for future development:

• Subjectivity in Weighting: While AGREE allows customizable weighting, the default weights and individual principle scores still involve subjective elements [20]. Future versions could benefit from validated default weights established through expert consensus.

• Pre-analytical Processes: AGREE does not fully account for environmental impacts associated with reagent synthesis, instrument manufacturing, or disposal [7] [20]. Expanding system boundaries to include these lifecycle stages would enhance comprehensiveness.

• Uncertainty Estimation: Current AGREE outputs lack associated uncertainty measures, limiting statistical comparison between methods [20]. Future developments could incorporate uncertainty quantification for more robust decision-making.

• Methodology-Specific Adaptations: While AGREE is designed as a generic tool, certain analytical techniques may require methodology-specific adjustments to ensure accurate assessment across diverse applications.

Emerging Trends and Research Initiatives

The metric tool landscape continues to evolve, with several emerging trends shaping AGREE's future development:

• Climate-Specific Metrics: New tools like the Carbon Footprint Reduction Index (CaFRI) focus specifically on carbon emissions associated with analytical procedures, complementing AGREE's broader environmental focus [7].

• Multi-criteria Decision Analysis: Advanced decision-support systems incorporating AGREE alongside economic, practical, and performance metrics will enable more holistic method selection and optimization [20].

• Automated Data Extraction: Integration with natural language processing systems to automatically extract AGREE input parameters from method descriptions in scientific literature, enabling large-scale greenness evaluation of existing methods.

• Predictive Greenness Assessment: Development of models to predict AGREE scores during method design phase, facilitating proactive green chemistry principles implementation rather than retrospective assessment.

Strategic Implementation Roadmap

For researchers and drug development professionals seeking to implement AGREE in their workflows, a phased approach maximizes effectiveness:

Phase 1: Education and Baseline Assessment

• Train researchers on GAC principles and AGREE methodology
• Conduct baseline greenness assessment of existing analytical methods
• Identify common improvement opportunities across method portfolio

Phase 2: Integration and Standardization

• Incorporate AGREE assessment into method development and validation protocols
• Establish organizational standards for target AGREE scores
• Implement software tools and templates to streamline assessment

Phase 3: Optimization and Innovation

• Use AGREE metrics to guide development of novel green analytical methods
• Establish cross-functional teams to address systematic green chemistry challenges
• Contribute to community-wide green method databases and best practices

AGREE represents a significant advancement in the quantification and standardization of greenness assessment in analytical chemistry. Its comprehensive foundation in the 12 principles of GAC, combined with flexible implementation through accessible digital tools, positions it as a cornerstone of sustainable analytical method development. As the field evolves toward integrated assessment frameworks like White Analytical Chemistry, AGREE's role in providing robust, actionable environmental metrics will become increasingly vital.

For researchers and drug development professionals, early adoption and mastery of AGREE provides strategic advantages in regulatory compliance, operational efficiency, and sustainability leadership. The ongoing development of AGREE derivatives and complementary tools will further enhance its utility, while emerging digital integrations will streamline its application across the method lifecycle. By embracing AGREE and related metrics, the analytical community can accelerate progress toward greener analytical practices that maintain scientific rigor while minimizing environmental impact.

Conclusion

The AGREE metric provides a comprehensive, quantitative, and software-driven framework essential for modern pharmaceutical scientists to evaluate and improve the environmental profile of their analytical methods. By mastering its principles, application, and integration with functional tools like BAGI and RAPI, researchers can make significant strides toward sustainable drug development. Future directions will likely involve greater automation, AI-supported scoring, and the formal adoption of AGREE within regulatory guidelines, further embedding green chemistry principles as a standard for analytical excellence in biomedical and clinical research. Embracing this holistic assessment is no longer optional but a critical component of responsible scientific progress.

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