AGREEprep: A Comprehensive Guide to Assessing Greenness in Sample Preparation

Abstract

This article provides a complete resource for researchers and drug development professionals seeking to implement the AGREEprep metric, the first dedicated tool for evaluating the environmental impact of sample preparation. It covers foundational principles, from its relationship with the ten principles of green sample preparation to its role within the broader White Analytical Chemistry framework. The guide offers a step-by-step methodology for application, addresses common troubleshooting scenarios, and demonstrates validation through comparative case studies, including examples from bioanalysis and therapeutic drug monitoring. By synthesizing current best practices, this article empowers scientists to quantitatively assess and improve the sustainability of their sample preparation methods without compromising analytical performance.

What is AGREEprep? Understanding the Foundation of Green Sample Preparation Metrics

Sample preparation is a critical step in analytical procedures, known for being time-consuming, labor-intensive, and costly, with significant impacts arising from reagent consumption, waste generation, and analyst safety concerns [1]. In response to these challenges, the principles of Green Sample Preparation (GSP) were introduced, extending the concepts of Green Analytical Chemistry (GAC) to include using solvents/reagents from renewable sources, reusable materials, and emphasizing miniaturization and automation [1] [2]. The Analytical Greenness Metric for Sample Preparation (AGREEprep) emerged in 2022 as the first dedicated metric tool specifically designed to evaluate the environmental performance of sample preparation methods [2].

AGREEprep was developed by Wojnowski et al. to address the need for a comprehensive, user-friendly assessment tool that aligns with the ten principles of GSP [1]. This metric provides analytical chemists with a standardized approach to quantify and compare the greenness of sample preparation techniques, enabling more informed method selection and development in line with sustainability goals [2]. The tool is particularly valuable in fields like pharmaceutical analysis, bioanalysis, and environmental monitoring where sample preparation is often the most resource-intensive phase of the analytical process [2].

Theoretical Framework and Calculation Methodology

The Ten Principles of Green Sample Preparation

AGREEprep evaluates sample preparation methods against ten fundamental principles of GSP, each representing a critical aspect of environmental sustainability and practical efficiency [1]. These principles form the foundation of the assessment criteria, transformed into scores on a unified 0-1 scale, where 0 represents the worst performance and 1 the best performance [1].

Table 1: The Ten Principles of Green Sample Preparation in AGREEprep

Principle Number	Assessment Criteria	Description
1	In situ preparation	Favoring in situ sample preparation approaches
2	Safer solvents	Using safer solvents and reagents
3	Sustainable materials	Targeting sustainable, reusable and renewable materials
4	Waste minimization	Minimizing waste generation
5	Minimal amounts	Minimizing sample, chemical and material amounts
6	Throughput	Maximizing sample throughput
7	Integration	Integrating steps and promoting automation
8	Energy consumption	Minimizing energy consumption
9	Analysis configuration	Choosing the greenest possible post-sample preparation configuration for analysis
10	Operator safety	Ensuring safe procedures for the operator

AGREEprep Scoring System and Weighting

The AGREEprep calculator generates scores for each of the ten principles, with the overall result presented as a colorful pictogram [1]. The pictogram features a central circle displaying the final score (ranging from 0 to 1) and color indicating overall environmental performance, while the outer sections represent each criterion with colors ranging from red (poor performance) to green (excellent performance) [1].

A distinctive feature of AGREEprep is its flexible weighting system, which allows users to assign different levels of importance to each criterion based on their specific assessment needs [1]. The length of each segment in the pictogram's outer circle corresponds to the weight assigned to that criterion, providing immediate visual feedback on the assessment priorities [1]. While default weights are available and commonly used, experienced users can customize these weights to reflect specific methodological priorities or constraints [1].

AGREEprep Assessment Workflow: The process begins with evaluating a sample preparation method against the ten Green Sample Preparation principles, followed by scoring, optional weight assignment, and final pictogram generation.

AGREEprep Protocol and Application Guide

Step-by-Step Implementation Protocol

Protocol Title: Comprehensive Greenness Assessment of Sample Preparation Methods Using AGREEprep

Purpose: To provide a standardized methodology for evaluating the environmental performance of sample preparation techniques using the AGREEprep metric tool.

Materials and Software Requirements:

• AGREEprep software (freely available from: https://mostwiedzy.pl/AGREE)
• Detailed documentation of the sample preparation method to be assessed
• Quantitative data on reagent consumption, waste generation, energy requirements
• Safety data sheets for chemicals used

Experimental Procedure:

• Data Collection Phase: Compile comprehensive data for the sample preparation method including:
  • Sample volumes and number of samples processed per batch

• Types and volumes of all solvents and reagents used
• Energy consumption of equipment (centrifuges, heaters, etc.)
• Number of procedural steps and degree of automation
• Waste generation quantities and classifications
• Safety considerations for operator protection

• Software Input Phase:

  • Launch the AGREEprep software interface
  • Input collected data into the corresponding fields for each of the ten principles
  • Adjust weight factors for each criterion if default values are not appropriate for your specific application
  • Run the calculation to generate the assessment pictogram

• Interpretation Phase:

  • Analyze the overall score (0-1) displayed in the center of the pictogram
  • Examine the color coding of each segment to identify strengths and weaknesses
  • Compare results with alternative methods to select the greenest approach
  • Use insights to optimize method parameters for improved environmental performance

Troubleshooting Notes:

• If data for specific criteria are unavailable, use conservative estimates and document this limitation
• When comparing methods, ensure consistent weighting factors are applied across all assessments
• For methods with multiple variations, assess each version separately to identify optimal configurations

Research Reagent Solutions and Essential Materials

Table 2: Essential Research Reagents and Materials for Microextraction Techniques

Reagent/Material	Function in Sample Preparation	Greenness Considerations
Sustainable Sorbents	Extraction and concentration of analytes	Use of biodegradable, renewable, or reusable materials improves scores in Principle 3
Safer Solvents	Media for extraction and dissolution	Selection based on green solvent selection guides addresses Principle 2
Miniaturized Equipment	Reduced consumption of materials and reagents	Directly supports Principles 4 and 5 (minimization)
Automated Systems	Reduced manual intervention and human error	Addresses Principles 7 (integration/automation) and 10 (operator safety)
Renewable Materials	Sustainable sourcing of consumables	Contributes to Principle 3 (sustainable materials)

Comparative Analysis with Other Metric Tools

AGREEprep exists within a growing ecosystem of metric tools for assessing analytical procedures. Understanding its relationship to these other tools is essential for comprehensive method evaluation.

Table 3: Comparison of AGREEprep with Other Prominent Metric Tools

Metric Tool	Primary Focus	Assessment Scope	Output Format	Key Advantages
AGREEprep	Sample preparation greenness	10 principles of GSP	Pictogram with overall score (0-1)	Specific to sample preparation, flexible weighting
AGREE	Overall method greenness	12 principles of GAC	Clock-like pictogram	Comprehensive GAC assessment, detailed breakdown
NEMI	Environmental impact	4 criteria	Simple pictogram (quadrants)	Easy to interpret, historical usage data
Analytical Eco-Scale	Penalty-based assessment	Multiple parameters	Numerical score (0-100)	Simple calculation, established approach
RGB Model	Holistic method assessment	12 principles of WAC	Color combination	Balances greenness with analytical performance

The integration of AGREEprep within the broader White Analytical Chemistry (WAC) framework is particularly noteworthy [1] [2]. WAC promotes harmony between analytical performance (red criteria), environmental impact (green criteria), and practical/economic considerations (blue criteria) [2]. In this context, AGREEprep provides the specialized assessment of sample preparation greenness that can be combined with other metrics evaluating analytical performance and practical efficiency.

AGREEprep in White Analytical Chemistry: AGREEprep provides the specialized greenness assessment component within the holistic White Analytical Chemistry framework, which balances analytical performance, environmental impact, and practical considerations.

Applications in Analytical Research

Pharmaceutical and Bioanalytical Applications

In the field of therapeutic drug monitoring (TDM) and bioanalysis, AGREEprep has demonstrated significant utility in evaluating microextraction techniques [2]. A comprehensive assessment of methods for determining UV filters in water samples using gas chromatography-mass spectrometry revealed important insights into the ecological friendliness, effectiveness, and practicality of different approaches [1]. The study highlighted how AGREEprep can identify strengths and weaknesses in analytical procedures, enabling researchers to select methods that best meet their specific requirements while maintaining environmental responsibility [1].

Microextraction techniques such as solid-phase microextraction (SPME), liquid-phase microextraction (LPME), dispersive liquid-liquid microextraction (DLLME), and their various modifications have been systematically assessed using AGREEprep [2]. These techniques generally score well due to their minimal reagent consumption, small sample volumes, and potential for automation – all key criteria in the AGREEprep assessment [2]. The metric has proven particularly valuable in identifying methods that achieve an optimal balance between greenness and analytical performance, a critical consideration in regulated environments like pharmaceutical quality control and clinical monitoring [2].

Environmental Analytical Applications

In environmental analysis, AGREEprep has been applied to assess sample preparation methods for detecting emerging contaminants in water matrices [1]. The evaluation of methods for determining UV filters highlighted the ecological advantages of microextraction techniques compared to conventional approaches [1]. The simultaneous use of AGREEprep with other metrics like BAGI (Blue Applicability Grade Index) and RGB 12 provides researchers with comprehensive methodological insights, supporting the selection of environmentally sustainable approaches without compromising analytical performance [1].

Future Perspectives and Development

The evolution of metric tools continues with ongoing refinements addressing current limitations [3]. Future developments may focus on standardizing weighting factors, reducing subjectivity in assessments, and improving interoperability between different metric tools [3]. The integration of AGREEprep with emerging technologies such as automated method development systems and artificial intelligence platforms represents a promising direction for high-throughput, objective greenness assessment [3].

As the analytical community increasingly adopts sustainability criteria in method development and selection, tools like AGREEprep are expected to become integral components of analytical quality by design frameworks [2]. The continued refinement of these metrics will further support the transition toward more environmentally responsible analytical practices while maintaining the high standards of accuracy, precision, and reliability required in modern chemical analysis [3] [2].

The development of sustainable analytical methods is a critical objective in modern laboratories, driven by the need to minimize environmental impact and enhance operator safety. Within this framework, sample preparation represents a pivotal step, often being the most resource-intensive and waste-generating part of the analytical process. Green Sample Preparation (GSP) has emerged as a guiding principle that promotes sustainable development through the adoption of environmentally benign procedures [4]. The ten principles of GSP establish a comprehensive road map for developing greener analytical methodologies, identifying paramount aspects such as the use of safe solvents/reagents, renewable materials, waste minimization, and energy reduction [5] [4].

AGREEprep (Analytical Greenness Metric for Sample Preparation) is the first dedicated metric tool designed specifically for evaluating the environmental impact of sample preparation methods [6]. This innovative approach translates the ten GSP principles into a practical, quantitative assessment framework. By providing a standardized evaluation system, AGREEprep enables researchers to objectively measure, compare, and improve the greenness of their sample preparation techniques, thereby supporting the broader adoption of sustainable practices in analytical chemistry [6] [7]. The metric offers a user-friendly, open-source software that calculates and visualizes results through an intuitive pictogram, making complex sustainability assessments accessible to a wide range of laboratory professionals [6].

The Ten Principles of Green Sample Preparation

The Ten Principles of Green Sample Preparation serve as the foundational framework for AGREEprep, providing comprehensive guidance for developing environmentally sustainable analytical methods [5] [4]. These principles encompass the crucial aspects that laboratory professionals must consider when designing or selecting sample preparation approaches. The following table summarizes these core principles and their primary objectives:

Table 1: The Ten Principles of Green Sample Preparation

Principle	Core Objective	Key Considerations
1. Safe & Sustainable Solvents/Reagents	Use of non-toxic, biodegradable solvents	Toxicity, environmental impact, biodegradability [4]
2. Renewable & Recycled Materials	Preference for materials from renewable sources or recycled content	Resource sustainability, life cycle assessment [4]
3. Waste Minimization	Reduction of waste generation	Microscale techniques, waste recycling [4]
4. Energy Efficiency	Minimization of energy consumption	Ambient temperature operations, energy-efficient equipment [4]
5. High Sample Throughput	Maximization of samples processed per time unit	Parallel processing, automation [4]
6. Miniaturization	Reduction of device or apparatus size	Solvent-less techniques, smaller equipment footprint [4]
7. Automation	Implementation of self-operating processes	In-line analysis, reduced manual intervention [4]
8. Simplified Procedures	Reduction of procedural steps	Direct analysis, integrated techniques [4]
9. Operator Safety	Protection of the analyst	Reduced exposure to hazardous materials [4]
10. Derivatization Avoidance	Elimination of chemical modification steps	Direct analysis without chemical transformation [4]

These principles are interconnected, collectively contributing to the overall sustainability of analytical methodologies. For instance, miniaturization (Principle 6) often supports waste minimization (Principle 3) and may enhance operator safety (Principle 9) by reducing exposure to hazardous materials [4]. Similarly, automation (Principle 7) typically enables higher sample throughput (Principle 5) while improving reproducibility. The principles emphasize not only environmental concerns but also practical considerations such as analytical efficiency and economic feasibility, recognizing that sustainable methods must be practically implementable to achieve widespread adoption [1].

AGREEprep: Structure and Assessment Methodology

AGREEprep operationalizes the Ten Principles of Green Sample Preparation into a practical, standardized assessment tool that calculates quantitative greenness scores for sample preparation methods [6]. The metric evaluates each of the ten principles, translating qualitative sustainability goals into measurable parameters. The assessment is performed using dedicated, open-source software that generates an easily interpretable pictogram, providing both an overall greenness score and detailed performance information for each principle [6].

The following diagram illustrates the logical relationship between the GSP principles and the AGREEprep assessment framework:

Diagram 1: AGREEprep Assessment Framework

AGREEprep Assessment Protocol

The AGREEprep assessment follows a systematic procedure that transforms methodological data into a comprehensive greenness evaluation. The step-by-step protocol below ensures consistent and reproducible assessments:

Table 2: AGREEprep Assessment Protocol

Step	Action	Details & Specifications
1. Data Collection	Gather all method parameters	Solvent types/volumes, energy consumption, waste generation, sample throughput, safety measures [6]
2. Software Input	Enter data into AGREEprep tool	Access software at: https://mostwiedzy.pl/AGREE [1]
3. Weight Assignment	Adjust criterion importance (optional)	Default weights are provided; customize based on analytical priorities [6]
4. Score Calculation	Software computes individual & overall scores	Each principle scored 0-1; overall score calculated considering weights [6]
5. Result Interpretation	Analyze generated pictogram	Overall score (0-1) with color indicator; segment colors show per-principle performance [6]

Each of the ten assessment criteria is scored on a scale from 0 to 1, where 0 represents the worst possible performance and 1 represents ideal greenness [1]. The software then calculates an overall score by combining the individual scores, taking into account user-defined weights for each criterion. This weighting system provides flexibility, allowing users to emphasize principles that are particularly important for their specific application [6]. For example, in high-throughput pharmaceutical testing, Principle 5 (high sample throughput) might be assigned greater weight, whereas in educational settings, Principle 9 (operator safety) might be prioritized.

The output is presented as a circular pictogram with ten colored segments corresponding to each principle. The color of each segment (ranging from red to green) indicates the performance for that principle, while the width of the segment reflects its assigned weight [6]. The center of the pictogram displays the overall score on a scale of 0 to 1, with a corresponding color from red (0) to dark green (1). This visual representation enables immediate understanding of both the overall greenness and specific strengths and weaknesses of the assessed method [6].

Experimental Protocols for AGREEprep Application

Protocol 1: AGREEprep Assessment of Solid-Phase Microextraction (SPME) Methods

This protocol provides a detailed methodology for evaluating the greenness of Solid-Phase Microextraction techniques using AGREEprep, particularly relevant for pharmaceutical and metabolomic applications [8].

Table 3: SPME-AGREEprep Assessment Protocol

Step	Procedure	Parameters & Measurements
1. SPME Method Characterization	Document all method specifications	Fiber coating material, sample volume, extraction time/ temperature, desorption conditions [8]
2. Solvent/Reagent Inventory	List all chemicals used	Quantify solvent consumption for fiber conditioning or sample modification [6]
3. Waste Quantification	Calculate total waste generated	Include sample residuals, cleaning solvents, and consumables [6]
4. Energy Assessment	Measure energy requirements	Account for heating, agitation, and automation systems [6]
5. Throughput Calculation	Determine samples processed per hour	Consider equilibrium time, parallel processing capability [8]
6. Safety Evaluation	Identify hazard controls	Volatile organic compound exposure, high-temperature operations [6]
7. Data Input & Analysis	Enter parameters into AGREEprep	Use default weights initially; adjust based on application priorities [6]

Experimental Notes: SPME methods typically score well on Principles 1, 3, and 6 due to minimal solvent consumption and miniaturized format [8] [1]. The main assessment challenges often include accurate quantification of negligible solvent volumes and energy consumption of supporting equipment [6].

Protocol 2: Comparative AGREEprep Assessment of Microextraction Techniques for Water Analysis

This protocol outlines a systematic approach for comparing different microextraction methods for determining UV filters in water samples, utilizing AGREEprep alongside complementary metrics [1].

Sample Preparation: Collect water samples spiked with target UV filters at environmentally relevant concentrations (ng/L to μg/L). Preserve samples appropriately to prevent analyte degradation [1].

Method Comparison Setup: Select at least three microextraction techniques for comparison (e.g., DLLME, SPME, SBSE). For each method, document all parameters including sorbent type, solvent volumes, extraction time, energy requirements, and sample throughput [1].

AGREEprep Assessment: Input all method parameters into the AGREEprep software. Maintain consistent weighting across all methods to enable fair comparison. Record overall scores and segment performances for each technique [1].

Complementary Metrics Application: Supplement AGREEprep assessment with BAGI (Blue Applicability Grade Index) to evaluate practical aspects and RGB 12 to assess analytical performance alongside greenness [1].

Data Interpretation: Identify techniques with AGREEprep scores >0.7 as demonstrating good greenness. Compare segment patterns to identify specific strengths and weaknesses across methods [1].

Expected Outcomes: Research indicates that microextraction techniques generally outperform conventional methods in AGREEprep assessments, with specific patterns emerging: SPME often excels in solvent reduction (Principles 1, 3), while automated DLLME variants score highly on throughput (Principle 5) and operator safety (Principle 9) [1].

The Scientist's Toolkit: Essential Research Reagents and Materials

Implementing green sample preparation methods requires specific materials and reagents that align with the Ten Principles of GSP. The following table details key solutions for developing sustainable sample preparation methodologies:

Table 4: Essential Research Reagents and Materials for Green Sample Preparation

Category	Specific Examples	Function & Green Benefits
Green Solvents	Cyclopentyl methyl ether, Ethyl lactate, Bio-based alcohols	Replace hazardous solvents; biodegradable options with lower toxicity [4]
Renewable Sorbents	Biosorbents from agricultural waste, Cyclodextrin-based coatings	Sustainable materials from renewable sources; reduced environmental impact [5]
Miniaturized Devices	SPME fibers, MEPS devices, Microfluidic chips	Reduce reagent consumption and waste generation; enable miniaturization [8] [1]
Reusable Materials	Silicon-based devices, Ceramic platforms, Durable metal alloys	Replace single-use plastics; support circular economy in laboratories [4]
Automation Systems	Robotic liquid handlers, Automated SPME, Flow injection systems	Increase sample throughput; improve reproducibility; enhance operator safety [8] [4]
Energy-Efficient Equipment	Low-power agitation systems, Room-temperature extraction devices	Reduce energy consumption; align with Principle 4 [6]
Alternative Extraction Materials	Ionic liquids, Deep eutectic solvents, Supercritical fluids	Provide non-toxic alternatives to conventional organic solvents [5]

The selection of appropriate materials should consider the entire life cycle, including production, use, and disposal. Biosorbents from agricultural waste, for example, address Principle 2 (renewable materials) while typically being biodegradable, thus minimizing environmental impact at the end of their useful life [5]. Similarly, reusable materials support waste minimization (Principle 3) but require validation to ensure performance consistency across multiple uses.

Interpreting AGREEprep Results: Case Studies and Data Visualization

Effective interpretation of AGREEprep results requires understanding both the overall score and the performance pattern across the ten principles. The following diagram illustrates a sample workflow for method assessment and optimization using AGREEprep:

Diagram 2: Method Assessment and Optimization Workflow

Case Study: AGREEprep Comparison of Sample Preparation Methods

Research comparing ten sample preparation methods for determining UV filters in water samples provides valuable insights into typical AGREEprep scoring patterns [1]. The following table summarizes hypothetical assessment results based on published data:

Table 5: Comparative AGREEprep Scores for Different Sample Preparation Methods

Sample Preparation Method	Overall AGREEprep Score	Strongest Principles	Weakest Principles
Solid-Phase Microextraction (SPME)	0.72	Principles 1, 3, 6 (solvent reduction, miniaturization)	Principles 5, 7 (throughput, automation) [1]
Dispersive Liquid-Liquid Microextraction (DLLME)	0.65	Principles 5, 6 (throughput, miniaturization)	Principles 1, 3 (solvent toxicity, waste) [1]
Stir Bar Sorptive Extraction (SBSE)	0.68	Principles 1, 3, 9 (solvent reduction, operator safety)	Principles 5, 7 (throughput, automation) [1]
Traditional Solid-Phase Extraction (SPE)	0.45	Principle 9 (operator safety)	Principles 1, 3, 6 (solvent use, waste, miniaturization) [1]

Interpretation Guidelines: Methods with overall scores above 0.7 are generally considered to demonstrate good greenness, while scores below 0.5 indicate significant opportunities for improvement [1]. The segment pattern provides crucial guidance for optimization; for example, a method with low scores on Principles 1 and 3 would benefit from solvent reduction strategies, while low scores on Principles 5 and 7 suggest automation as a potential improvement area.

The AGREEprep tool also allows tracking of improvement progress after implementing modifications. By comparing pre- and post-optimization scores, researchers can quantitatively demonstrate greenness enhancements, supporting sustainability claims with objective data [6]. This capability makes AGREEprep valuable not only for method selection but also for continuous improvement initiatives in analytical laboratories.

AGREEprep represents a significant advancement in the standardization of greenness assessment for sample preparation methods. By translating the Ten Principles of Green Sample Preparation into a quantitative, reproducible metric, it provides researchers with a powerful tool to evaluate and improve the environmental sustainability of their analytical methods [6]. The intuitive pictogram output facilitates both quick comparison and in-depth analysis, while the weighting system allows customization based on application-specific priorities [6].

The adoption of AGREEprep supports the broader objectives of Green Analytical Chemistry, enabling laboratories to minimize their environmental footprint while maintaining analytical performance [7]. As research continues to develop increasingly sustainable methodologies, AGREEprep will play a crucial role in objectively assessing their greenness, guiding the analytical community toward more environmentally responsible practices [6] [1]. By implementing the protocols and interpretations outlined in this article, researchers and drug development professionals can effectively incorporate greenness considerations into their method development and selection processes, contributing to the advancement of sustainable science.

AGREEprep's Role in the Evolving Landscape of Analytical Metrics

The pursuit of sustainability in laboratories has made Green Analytical Chemistry (GAC) a cornerstone of modern method development. Within this framework, sample preparation has been identified as a critical step due to its substantial consumption of solvents, reagents, and energy, often generating significant waste [9]. While numerous metrics have emerged to evaluate analytical method greenness, most provide generalized assessments that fail to specifically address the unique environmental impacts of sample preparation. AGREEprep (Analytical Greenness Metric for Sample Preparation) fills this void as the first dedicated metric tool designed explicitly for evaluating the environmental impact of sample preparation methods [6] [9].

This specialized tool has emerged against a backdrop of approximately 15 widely used GAC metrics, including NEMI, Analytical Eco-Scale, GAPI, and AGREE [7]. What distinguishes AGREEprep is its foundation in the ten principles of Green Sample Preparation (GSP), offering unprecedented specificity for evaluating this crucial analytical step [9] [10]. Its development represents a significant maturation in the field, moving beyond one-size-fits-all approaches to provide targeted assessments that effectively identify improvable aspects of sample preparation procedures [9].

The Analytical Metrics Landscape

The evolution of greenness assessment tools for analytical chemistry began with relatively simple pictograms and has progressed to sophisticated, multi-criteria scoring systems. Table 1 summarizes the key metric tools that contextualize AGREEprep's development.

Table 1: Evolution of Key Green Analytical Chemistry Metric Tools

Metric Tool	Year Introduced	Key Characteristics	Strengths	Limitations
NEMI [7]	2002	Four-criteria pictogram; qualitative assessment	Simple, quick visual interpretation	Limited scope; no quantitative capability
Analytical Eco-Scale [7]	2012	Penalty point system; ideal score = 100	Semi-quantitative; comprehensive criteria	Penalty assignments can be subjective
GAPI [7]	2018	Multi-criteria pictogram with five pentagrams	More detailed than NEMI; visual output	Limited quantitative differentiation
AGREE [7]	2020	Twelve principles of GAC; 0-1 scoring	Quantitative; user-friendly software	General analytical method focus
AGREEprep [6] [9]	2022	Ten principles of GSP; 0-1 scoring	Sample preparation specificity; weighted criteria	Requires detailed sample preparation data

The progression from NEMI to AGREEprep demonstrates a clear trend toward greater specificity, quantitative assessment, and user-friendly software implementation. While earlier tools like NEMI provided a basic yes/no evaluation of four simple criteria [7], modern tools like AGREEprep employ continuous scoring scales and weighted criteria to provide nuanced assessments that effectively discriminate between methods [9].

AGREEprep Framework and Algorithm

Foundational Principles

AGREEprep's assessment framework is built upon the ten principles of Green Sample Preparation (GSP), which collectively form an integrated system where improvements in one principle often synergistically address deficiencies in others [10]. These principles place sample preparation in a central position and define greenness based on the specific needs and requirements of this critical analytical step [10].

The ten assessment criteria in AGREEprep correspond to the following GSP principles [9]:

• Favor in situ sample preparation
• Use safer solvents and reagents
• Target sustainable, reusable, and renewable materials
• Minimize waste
• Minimize sample, chemical and material amounts
• Maximize sample throughput
• Integrate steps and promote automation
• Minimize energy consumption
• Choose the greenest possible post-sample preparation configuration for analysis
• Ensure safe procedures for the operator

Scoring System and Weighting

Each criterion is scored from 0 to 1, with these extremes representing the worst and best performance, respectively [1]. The developers established these scoring levels by considering experimental conditions across a wide range of conventional and state-of-the-art sample preparation approaches [10].

A key innovation in AGREEprep is the incorporation of weighted criteria, acknowledging that not all principles hold equal importance in greenness assessment [9]. For example, selecting safer solvents and reagents (Criterion 2) and minimizing waste (Criterion 4) carry greater weight than favoring in situ preparation (Criterion 1) or integrating steps (Criterion 7) [9]. The software allows users to modify these default weights to align with specific analytical goals, providing flexibility while maintaining standardized assessment protocols [11].

The overall score is calculated through a weighted average of the individual criterion scores, generating a value between 0 and 1, where 1 represents optimum performance or the absence of a sample preparation step [10].

AGREEprep Assessment Workflow

AGREEprep Implementation Protocol

Software Accessibility and Input Requirements

AGREEprep utilizes open-source software that is freely available from mostwiedzy.pl/AGREEprep, with the code accessible at git.pg.edu.pl/p174235/agreeprep [10]. This accessibility has significantly contributed to its widespread adoption in both research and practical settings.

The software requires input data for each of the ten assessment steps. Successful implementation demands careful collection of the following methodological details [6] [11]:

• Solvent and reagent volumes (types and exact amounts)
• Energy consumption throughout the sample preparation process
• Sample throughput (number of samples processed per hour)
• Waste generation (total mass per sample)
• Operator safety measures implemented
• Degree of automation and integration of steps
• Material sustainability (reusability and renewable sources)

Output Interpretation

After data input, AGREEprep generates a distinctive round pictogram with a circle at the center displaying the overall score (0-1), surrounded by ten trapezoid bars corresponding to the ten GSP principles [1] [10]. Each bar has a length proportional to its assigned weight and a color that reflects its individual score (green for high scores to red for low scores) [10].

This visualization provides immediate identification of both strengths and weaknesses in the sample preparation method, allowing researchers to focus improvements on specific aspects that most significantly impact environmental performance [10].

Research Reagent Solutions for Green Sample Preparation

Implementing greener sample preparation methods often requires specific materials and reagents aligned with GSP principles. Table 2 outlines key solutions referenced in AGREEprep assessments.

Table 2: Essential Research Reagent Solutions for Green Sample Preparation

Reagent/Material	Function in Sample Preparation	Green Attributes	Application Examples
Biopolymer-based Sorbents (chitosan, alginate, cellulose) [12]	Selective extraction media	Biodegradable, nontoxic, from renewable sources	Molecularly imprinted polymers for microextraction
Ionic Liquids & Deep Eutectic Solvents [12]	Extraction solvents	Low volatility, reduced toxicity, tunable properties	Alternative to conventional organic solvents
Solid-phase Microextraction (SPME) Fibers [1]	Solventless extraction	Reusable, minimal waste generation	UV filter determination in water samples
Stir Bar Sorptive Extraction (SBSE) Devices [1]	Solventless extraction	Reusable, high efficiency	Concentrating organic contaminants from water
Microextraction Packed Sorbents (MEPS) [1]	Miniaturized extraction	Reduced solvent consumption, amenable to automation	Bioanalysis and environmental applications

Case Studies and Performance Evaluation

AGREEprep in Method Assessment

AGREEprep has been extensively applied to evaluate both official standard methods and novel research techniques, demonstrating its practical utility and discriminatory power.

In a comprehensive assessment by the IUPAC project, twenty-five US EPA methods utilizing Soxhlet extraction for analyzing solid samples (sediments, fish tissues) showed consistently low greenness scores ranging from 0.04 to 0.12 [10]. These traditional methods were characterized by time-consuming procedures, significant solvent consumption, and high energy demands, with additional sample treatment steps further diminishing their greenness performance [10].

Similarly, evaluation of fifteen AOAC INTERNATIONAL methods for food analysis revealed scores between 0.05 and 0.22 [10]. These methods employed traditional approaches like Soxhlet extraction, maceration, or digestion with organic solvents, often involving highly toxic substances such as asbestos, benzene, and mercury, which posed significant health risks to operators [10].

Comparative Assessment of Microextraction Techniques

In a study evaluating sample preparation methods for determining UV filters in water samples, AGREEprep effectively discriminated between different approaches, with modern microextraction techniques consistently outperforming conventional methods [1]. The most popular techniques for this application included dispersive liquid-liquid microextraction (DLLME, ~24%), solid-phase microextraction (SPME, ~24%), and stir bar sorptive extraction (SBSE, ~16%) [1].

Table 3: AGREEprep Performance Comparison Across Sample Preparation Methods

Sample Preparation Method	Application Context	Typical AGREEprep Score Range	Key Strengths	Key Weaknesses
Traditional Soxhlet Extraction [10]	US EPA & AOAC standard methods	0.04 - 0.22	Universal application	High solvent/energy use, low throughput
Liquid-Liquid Extraction [9]	Phthalate esters in water (EPA 8061A)	~0.25	Simple implementation	Large solvent volumes, significant waste
Dispersive Liquid-Liquid Microextraction [1]	UV filters in water	0.33 - 0.43	Minimal solvent consumption	Limited automation potential
Solid-Phase Microextraction [1]	UV filters in water	0.40 - 0.50	Solventless, reusable fibers	Limited sorbent capacity
Microwave-Assisted Extraction [10]	US EPA methods for trace metals	0.20 - 0.36	Reduced extraction time	Acid consumption, specialized equipment

Integration with Broader Assessment Paradigms

AGREEprep functions most effectively when integrated within a comprehensive analytical assessment framework that considers not only environmental impact but also methodological efficacy and practical utility.

The emergence of White Analytical Chemistry (WAC) represents a paradigm that harmonizes the primary attributes of analytical methods: red (analytical performance), green (ecological impact), and blue (practical/economic feasibility) [1]. In this model, AGREEprep provides the crucial "green" assessment, which can be combined with other metrics such as BAGI (Blue Applicability Grade Index) for practicality and established analytical validation criteria for performance evaluation [1].

This integrated approach addresses the limitation of single-dimensional assessments and supports the development of truly sustainable methods that balance environmental concerns with analytical requirements and practical implementation [1]. The complementary use of AGREEprep with BAGI and RGB 12 algorithm provides researchers with a comprehensive toolkit for holistic method evaluation and development [1].

AGREEprep represents a significant advancement in the evolution of analytical metrics by providing unprecedented specificity for evaluating the environmental impact of sample preparation. Its foundation in the ten principles of GSP, weighted scoring system, and intuitive pictogram output offer practical utility for researchers seeking to improve method sustainability.

The metric's demonstrated ability to discriminate effectively between traditional and modern methods, coupled with its integration capacity within broader assessment paradigms like White Analytical Chemistry, ensures its continued relevance. As the field moves toward increasingly holistic method evaluation, AGREEprep provides the essential environmental component that, when balanced with analytical performance and practical considerations, supports the development of truly sustainable analytical practices.

The increasing focus on sustainability in analytical laboratories necessitates tools that comprehensively evaluate method environmental impact. AGREEprep, a dedicated metric for assessing sample preparation, aligns with the principles of Green Sample Preparation (GSP). This application note positions AGREEprep within the broader, holistic framework of White Analytical Chemistry (WAC). WAC advocates for a balanced evaluation of analytical methods by integrating their environmental impact (green), analytical performance (red), and practicality & economic feasibility (blue). We detail protocols for employing AGREEprep in concert with complementary tools like the Red Analytical Performance Index (RAPI) and Blue Applicability Grade Index (BAGI) to achieve a "white" assessment. A case study on determining UV filters in water demonstrates how this multi-metric approach identifies strengths and weaknesses, guiding researchers toward developing truly sustainable and functional analytical methods.

Modern analytical chemistry is evolving to embrace sustainability without compromising the quality of results. While Green Analytical Chemistry (GAC) principles have focused on reducing environmental impact, a more comprehensive paradigm, White Analytical Chemistry (WAC), has emerged. WAC posits that a sustainable method must harmoniously balance three core attributes: greenness (environmental safety), redness (analytical performance), and blueness (practicality and cost). A method approaching this balance is deemed "white" [13] [14].

Sample preparation is often the most resource-intensive step of an analytical procedure. AGREEprep (Analytical Greenness Metric for Sample Preparation) is a recent software-based tool designed to evaluate the environmental friendliness of this specific stage. It uses ten assessment criteria corresponding to the ten principles of Green Sample Preparation (GSP), providing an overall score from 0 to 1 and a colorful pictogram for intuitive interpretation [1].

This document provides a detailed protocol for integrating AGREEprep into the WAC framework. By combining AGREEprep with tools that assess analytical performance (e.g., RAPI) and practical applicability (e.g., BAGI), researchers and drug development professionals can make informed decisions that support both ecological responsibility and operational excellence.

The WAC Framework and the Role of AGREEprep

The WAC concept uses the RGB (Red, Green, Blue) color model as an analogy. Just as white light is a combination of all primary colors, an ideal "white" method is a balanced composite of analytical performance, environmental friendliness, and practical utility [13] [14].

• The Green Dimension (Environmental Impact): This is the domain of AGREEprep and other greenness metrics. AGREEprep specifically focuses on the sample preparation stage, evaluating criteria such as waste generation, energy consumption, reagent toxicity, and operator safety [1].
• The Red Dimension (Analytical Performance): This dimension assesses the method's ability to deliver reliable, high-quality data. Key validation parameters include sensitivity (LOD, LOQ), precision, accuracy, linearity, and selectivity. The recently introduced Red Analytical Performance Index (RAPI) provides a standardized tool for this assessment [15].
• The Blue Dimension (Practicality & Economics): This dimension covers practical aspects such as cost-effectiveness, analysis time, operational simplicity, and potential for automation. The Blue Applicability Grade Index (BAGI) is a dedicated metric for evaluating these criteria [1] [15].

The interrelationship of these dimensions within the WAC framework, and where AGREEprep fits, is illustrated below.

Experimental Protocols

Protocol 1: Comprehensive Method Assessment Using the WAC Toolkit

This protocol outlines the steps to holistically evaluate an analytical method, with a focus on sample preparation, using the green (AGREEprep), red (RAPI), and blue (BAGI) metrics.

1. Purpose and Scope To provide a standardized procedure for the comprehensive assessment of an analytical method's sustainability, functionality, and practicality within the White Analytical Chemistry framework.

2. Apparatus and Software

• Computer with internet access.
• AGREEprep software (available at: https://mostwiedzy.pl/AGREE).
• BAGI software (available at: https://mostwiedzy.pl/bagi).
• RAPI software (available at: https://mostwiedwy.pl/rapi).
• Detailed description of the analytical method to be assessed.

3. Procedure

Step 1: Data Collection for AGREEprep (Green Assessment) Gather all relevant data for the sample preparation step based on the ten GSP principles [1]:

• Sample Size: Mass or volume of the sample.
• Reagent Consumption: Type, amount, and hazard classification of all solvents and reagents used.
• Energy Demand: Estimated or measured energy consumption (e.g., for heating, stirring, centrifugation) in kWh per sample.
• Waste Generation: Total mass or volume of waste produced per sample.
• Health & Safety: Toxicity, flammability, and corrosivity of materials used.
• Throughput: Number of samples that can be processed simultaneously or per hour.
• Operator Time: Hands-on time required for the procedure.
• Additional Criteria: Miniaturization potential, use of renewable sources, and potential for recycling materials.

Step 2: Execute AGREEprep Analysis

• Launch the AGREEprep software.
• Input the collected data into the corresponding ten criteria fields. The software allows for adjustable weights for each criterion, though default weights can be used for standardized comparison [1].
• The software will generate a circular pictogram. The outer ring is divided into ten colored sections, each representing one criterion (green for best, red for worst). The center displays a final score between 0 and 1.
• Record the overall score and analyze the pictogram to identify specific environmental weaknesses (red/orange sectors).

Step 3: Perform BAGI and RAPI Assessments

• BAGI (Blue Assessment): Access the BAGI software. Input data related to practical aspects, such as instrument availability, cost of analysis, time required, operational steps, and required operator skills. The tool will generate a score and a blue-tone pictogram [1] [15].
• RAPI (Red Assessment): Access the RAPI software. Input key analytical performance data obtained from method validation, including repeatability, intermediate precision, sensitivity (LOD/LOQ), linearity range, trueness/accuracy, and robustness. RAPI will generate a red-tone star pictogram and an overall score [15].

Step 4: Integrated WAC Interpretation

• Compare the scores and pictograms from AGREEprep, BAGI, and RAPI.
• A method with high scores across all three metrics is considered a balanced, "white" method.
• Use the results to identify trade-offs. For example, a method with a high AGREEprep score (green) but a low RAPI score (red) may be environmentally friendly but analytically inadequate, and vice-versa.

4. Safety Consider the health and safety findings from the AGREEprep assessment when handling chemicals and operating equipment.

Case Study: Assessment of Sample Preparation Methods for UV Filters in Water

A recent study evaluated ten different sample preparation methods for the determination of UV filters in water samples using gas chromatography-mass spectrometry (GC-MS) [1]. The study provides an excellent example of the WAC framework in action.

1. Methods Assessed: The assessed methods included both conventional techniques and modern microextraction approaches:

• Conventional: Solid-Phase Extraction (SPE), Liquid-Liquid Extraction (LLE).
• Microextraction: Dispersive Liquid-Liquid Microextraction (DLLME), Solid-Phase Microextraction (SPME), Stir Bar Sorptive Extraction (SBSE).

2. Assessment Tools Applied: The methods were evaluated using AGREEprep (green), BAGI (blue), and the RGB 12 algorithm (which combines red, green, and blue into a whiteness score).

3. Key Findings and Workflow: The assessment workflow and its outcomes for the microextraction techniques, which generally performed well, are summarized below.

4. Results Summary: The following table quantifies the results described in the case study, comparing the performance of different method categories against the three WAC dimensions.

Table 1: Comparative WAC Assessment of Sample Preparation Methods for UV Filters in Water [1]

Method Category	AGREEprep (Green) Score	Key Red (Performance) Indicators	BAGI (Blue) Score	Overall WAC Balance
Microextraction (e.g., SPME, DLLME)	High (e.g., ~0.7-0.8)	Good sensitivity (low LODs), High precision & accuracy	High	Excellent Balance ("White") - Low environmental impact coupled with high functionality and practicality.
Conventional (e.g., SPE, LLE)	Low to Moderate	Good sensitivity and precision	Moderate	Poor Balance - Higher solvent consumption and waste generation (low Green) despite good Red performance.

The Scientist's Toolkit: Essential Research Reagent Solutions

The following table lists key materials and tools essential for conducting research and assessments within the White Analytical Chemistry framework.

Table 2: Essential Reagents and Tools for WAC-Compliant Method Development and Assessment

Item	Function/Application	Examples in WAC Context
AGREEprep Software	Dedicated metric tool for assessing the greenness of the sample preparation step.	Evaluates solvent consumption, waste, energy, and safety of extraction protocols [1].
BAGI Software	Metric tool for assessing the practicality and economic feasibility of an analytical method.	Scores methods based on cost, time, ease of use, and automation potential [1] [15].
RAPI Software	Metric tool for evaluating the analytical performance characteristics of a method.	Provides a quantitative score for validation parameters like precision, sensitivity, and accuracy [15].
Microextraction Devices	Consumables for miniaturized, solvent-efficient sample preparation.	SPME fibers, SBSE stir bars, DLLME kits. Reduce reagent use, improving AGREEprep scores [1] [16].
Green Solvents	Alternative solvents with lower toxicity and environmental impact.	Cyclopentyl methyl ether (CPME), ethanol, ethyl acetate. Used to replace hazardous solvents like chlorinated hydrocarbons in sample prep [13].

AGREEprep is a critical tool for modernizing and greening the sample preparation stage of analytical methods. However, its true power is unlocked when it is applied as part of the integrated White Analytical Chemistry framework. By systematically combining AGREEprep with RAPI and BAGI, researchers and pharmaceutical analysts can move beyond a singular focus on environmental impact. This multi-faceted approach enables the identification of methods that are not only green but also robust, reliable, cost-effective, and practical for routine use. Adopting this holistic "white" perspective is paramount for advancing sustainable science that does not compromise on analytical quality or operational feasibility.

AGREEprep is a dedicated metric tool for evaluating the environmental impact of sample preparation procedures in analytical methods. Developed in 2022, it calculates an overall greenness score based on ten principles of green sample preparation (GSP), providing researchers with an easily interpretable pictogram that summarizes the environmental performance of their methods [9] [17]. This output enables straightforward comparison between different sample preparation approaches and identifies specific aspects requiring improvement [9]. The tool is particularly valuable in pharmaceutical and bioanalytical research, where sample preparation is often the most resource-intensive and waste-generating step in analytical procedures [2].

Comprehensive Interpretation of the AGREEprep Pictogram

The most prominent feature of the AGREEprep pictogram is the overall numerical score displayed in the center, which ranges from 0 to 1 [9]. This single metric provides an immediate assessment of the method's environmental performance:

• Scores approaching 1.0 indicate excellent greenness performance across most or all assessment criteria [9]. Methods achieving such high scores typically incorporate significant miniaturization, use safer solvents, and generate minimal waste [2].
• Scores in the middle range (0.4-0.7) suggest moderate environmental performance with specific areas requiring improvement [9].
• Scores approaching 0 represent poor environmental performance across multiple criteria [9]. Traditional sample preparation methods like liquid-liquid extraction often receive low scores due to large solvent consumption and substantial waste generation [9].

The central number is accompanied by a color code, with darker green shades indicating better overall greenness performance [9]. This immediate visual cue allows for rapid comparison between multiple methods without detailed analysis of each criterion.

The Ten Assessment Criteria and Their Visualization

Surrounding the central score, the pictogram features ten colored segments, each corresponding to one of the green sample preparation principles [9]. The interpretation of these segments encompasses multiple dimensions:

• Segment Color: Each segment is colored using an intuitive traffic light system where green indicates strong performance, yellow represents moderate performance, and red signifies poor performance for that specific criterion [9]. This allows immediate identification of both environmental strengths and weaknesses in the assessed method.

• Segment Width: The width of each segment visually represents the weight assigned to that particular criterion by the assessor [9]. This weighting system acknowledges that not all GSP principles hold equal importance in every analytical context. Default weights are suggested, but researchers can adjust these based on their specific priorities and constraints [9].

Table 1: The Ten Green Sample Preparation Principles in AGREEprep

Principle Number	Description	Key Assessment Aspects
1	Favor in situ sample preparation	Remote sensing, non-invasive analysis, field sampling vs. laboratory processing [9]
2	Use safer solvents and reagents	Toxicity, environmental impact, safety for operators [9]
3	Target sustainable, reusable, and renewable materials	Source of materials, reusability, biodegradability [9]
4	Minimize waste	Volume of waste generated, hazardousness of waste [9]
5	Minimize sample, chemical and material amounts	Scale of extraction, solvent volumes, material consumption [9]
6	Maximize sample throughput	Number of samples processed per unit time, parallel processing capability [9]
7	Integrate steps and promote automation	Workflow integration, automation level, reduction of manual operations [9]
8	Minimize energy consumption	Power requirements, heating/cooling needs, energy-efficient equipment [9]
9	Choose greenest post-sample preparation configuration	Compatibility with green analytical techniques, direct analysis possibilities [9]
10	Ensure safe procedures for the operator	Exposure risks, necessary personal protective equipment, procedural hazards [9]

Experimental Protocol for AGREEprep Assessment

Data Collection and Input Preparation

The first step involves gathering quantitative and qualitative data about the sample preparation method. Researchers must collect specific information corresponding to each of the ten GSP principles:

• Principle 1: Document the sample preparation approach (in situ, on-line, at-line, or off-line), including the number of procedural steps for external sample pretreatment [9].
• Principle 2: Record types and exact volumes of all solvents and reagents used, noting their safety data and environmental impact [9].
• Principle 3: Inventory all materials, noting their composition, reusability potential, and whether they come from renewable sources [9].
• Principle 4: Calculate total waste volume generated per sample, categorizing by waste type and hazardousness [9].
• Principle 5: Measure sample size and quantify consumption of all chemicals and materials per extraction [9].
• Principle 6: Determine sample throughput (samples processed per hour), including any parallel processing capabilities [9].
• Principle 7: Document the degree of automation and integration with subsequent analysis steps [9].
• Principle 8: Quantify energy consumption in kWh per sample, including heating, cooling, and agitation requirements [9].
• Principle 9: Evaluate the environmental compatibility of the analytical technique used after sample preparation [9].
• Principle 10: Identify potential operator hazards and required safety measures [9].

Software Operation and Score Calculation

After data collection, researchers utilize the freely available AGREEprep software to generate the assessment pictogram:

• Software Access: Download the AGREEprep software from the official website (mostwiedzy.pl/AGREEprep) [17].
• Data Input: Enter collected data into the corresponding fields for each of the ten criteria.
• Weight Assignment: Apply default weights or assign custom weights based on methodological priorities. The default weights prioritize solvent use, waste generation, energy consumption, and operator safety as more significant criteria [9].
• Pictogram Generation: The software automatically calculates subscores for each criterion on a 0-1 scale and computes the overall score using a specific algorithm that incorporates both performance and assigned weights [9].
• Result Interpretation: Analyze the generated pictogram to identify environmental strengths and weaknesses of the assessed method.

The following diagram illustrates the complete AGREEprep assessment workflow:

Comparative Assessment Protocol

For method development and optimization, AGREEprep is most valuable when comparing multiple sample preparation approaches:

• Apply the assessment protocol to all candidate methods using consistent weighting criteria.
• Generate pictograms for each method to visualize comparative environmental performance.
• Identify the method with the highest overall score and most balanced performance across criteria.
• Use the comparative results to guide method optimization toward greener practices.

Quantitative Data and Case Studies

AGREEprep Scores for Common Sample Preparation Methods

Recent studies applying AGREEprep to various sample preparation techniques have generated quantitative data enabling direct comparison of environmental performance:

Table 2: AGREEprep Scores for Different Sample Preparation Techniques in Phthalate Ester Analysis [9]

Sample Preparation Technique	Key Characteristics	Overall AGREEprep Score
Liquid-Liquid Extraction (EPA 8061A)	180 mL dichloromethane, ex situ, manual	0.27
Solid-Phase Extraction (EPA 3535)	50 mL solvent consumption, ex situ	0.38
Dispersive Liquid-Liquid Microextraction	2 mL solvent, centrification, ex situ	0.45
Stir-Bar Sorptive Extraction	5 mL solvent, reusable device, ex situ	0.52
Solid-Phase Microextraction	Solventless, reusable fiber, ex situ	0.61
In-Tube Extraction-Microextraction	<1 mL solvent, reusable, automated	0.69

The data demonstrates a clear progression from traditional extraction techniques with substantial solvent consumption to modern microextraction approaches with significantly improved greenness profiles [9]. Methods incorporating solvent minimization, reusability, and automation consistently achieve higher AGREEprep scores [9] [2].

Case Study: Microextraction Techniques in Therapeutic Drug Monitoring

A 2024 study evaluated the greenness of various microextraction techniques used in therapeutic drug monitoring (TDM) using AGREEprep [2]. The assessment revealed that techniques incorporating significant solvent reduction, reusable materials, and automation achieved substantially higher scores:

• Solid-phase microextraction (SPME) approaches, particularly fiber-SPME and in-tube SPME, achieved high scores (typically 0.6-0.75) due to their solventless nature or minimal solvent consumption and reusability [2].
• Liquid-phase microextraction (LPME) techniques, including hollow-fiber LPME and dispersive liquid-liquid microextraction, showed moderate to high scores (0.45-0.65) with performance heavily dependent on solvent selection and volume [2].
• Traditional solid-phase extraction (SPE) methods consistently received lower scores (0.3-0.45) due to higher solvent consumption and disposable cartridge designs [2].

The study concluded that AGREEprep effectively identified environmental trade-offs in microextraction techniques, enabling researchers to select methods balancing analytical performance with greenness considerations [2].

The Scientist's Toolkit: Essential Research Reagent Solutions

Successful implementation of green sample preparation methods requires specific materials and reagents optimized for miniaturized and sustainable approaches:

Table 3: Essential Research Reagent Solutions for Green Sample Preparation

Material/Reagent	Function in Green Sample Preparation	Application Examples
Ionic Liquids	Safer alternative to volatile organic solvents; tunable properties for specific applications	Extraction solvents in liquid-phase microextraction; coating materials in SPME [2]
Biopolymer-based Sorbents	Sustainable, renewable materials for extraction; biodegradable alternatives to synthetic polymers	Solid-phase extraction; microextraction by packed sorbent (MEPS) [9]
Molecularly Imprinted Polymers	Highly selective sorbents enabling targeted extraction; reducible solvent and sample consumption	Solid-phase microextraction; stir bar sorptive extraction [2]
Carbon-based Nanomaterials	High-efficiency sorbents allowing minimal material usage; renewable sources available	Dispersive solid-phase extraction; magnetic solid-phase extraction [2]
Deep Eutectic Solvents	Biodegradable, low-toxicity solvents from natural sources; safer reagent alternative	Liquid-phase microextraction; dispersive liquid-liquid microextraction [2]
Reusable Extraction Devices	Reduction of consumable waste; fiber assemblies, stir bars, porous membranes	Solid-phase microextraction; fabric-phase sorptive extraction [2]

Interrelationship Between AGREEprep Score Components

The following diagram illustrates the logical relationships between the various components of the AGREEprep assessment system and how they contribute to the final output:

The AGREEprep metric tool provides a comprehensive, visually intuitive system for assessing and comparing the environmental performance of sample preparation methods. Its standardized output enables researchers to make informed decisions during method development and optimization, ultimately contributing to more sustainable analytical practices in pharmaceutical and bioanalytical research.

How to Use AGREEprep: A Step-by-Step Guide for Practical Application

In the contemporary landscape of analytical chemistry, the adoption of Green Analytical Chemistry (GAC) principles is paramount for developing sustainable methodologies. AGREEprep (Analytical Greenness Metric for Sample Preparation) is the first dedicated metric tool designed to evaluate the environmental impact of the sample preparation stage of analytical procedures [6] [9]. Sample preparation is a critical focus for greenness assessment as it is typically the most resource-intensive step, characterized by substantial consumption of solvents, sorbents, reagents, and energy [9]. This protocol provides a detailed guide for researchers on how to access, operate, and interpret results using the open-source AGREEprep software, enabling a standardized approach to greenness evaluation within research on analytical method development.

The tool calculates a unified greenness score based on the 10 principles of Green Sample Preparation (GSP), offering a specific and accurate assessment that general greenness metrics may overlook [9]. Its open-source nature promotes widespread adoption and transparent evaluation, allowing scientists in drug development and other fields to quantitatively demonstrate their commitment to sustainable practices.

AGREEprep is freely available to the global research community, ensuring accessibility without financial barriers.

• Access Point: The software can be downloaded without cost from the official website: www.mostwiedzy.pl/agreeprep [18].
• Software Environment: AGREEprep is a user-friendly, open-source software that calculates and visualizes the greenness assessment results in an intuitive pictogram [6].
• Conceptual Workflow: The evaluation logic of AGREEprep transforms user-input data on a sample preparation method into a quantitative greenness score. The diagram below illustrates this computational workflow.

Core Assessment Protocol

The Ten Principles of Green Sample Preparation

AGREEprep's evaluation framework is built on ten foundational principles, each representing a specific criterion contributing to the overall greenness of a sample preparation method [9]. The following table details these principles and provides key questions to guide data collection.

Table 1: The Ten Principles of Green Sample Preparation (GSP) and Data Collection Guidance.

Principle Number	Principle Description	Key Data Required for Assessment
1	Favor in situ sample preparation	Is the preparation performed directly on/in the sample source?
2	Use safer solvents and reagents	Type, volume, and hazard classifications (e.g., GHS) of all chemicals used.
3	Target sustainable, reusable, and renewable materials	Are materials reusable? Are they derived from renewable sources?
4	Minimize waste	Total mass or volume of waste generated per sample or analysis.
5	Minimize sample, chemical, and material amounts	Sample size, exact volumes of solvents/consumables used.
6	Maximize sample throughput	Number of samples processed per hour (degree of parallelism).
7	Integrate steps and promote automation	Is the process automated? Are multiple steps integrated into one device?
8	Minimize energy consumption	Total energy consumed per sample (e.g., in kWh).
9	Choose the greenest possible post-sample preparation configuration	Greenness of the subsequent analytical technique (e.g., chromatography).
10	Ensure safe procedures for the operator	Risks of exposure, need for personal protective equipment (PPE).

Data Input and Criterion Weighting

Accurate data input is critical for a meaningful assessment. Essential quantitative data includes sample size, solvent volumes, energy consumption (in kWh), and waste generation [6] [9]. For criteria involving hazard assessment (e.g., Principle 2), consult Safety Data Sheets (SDS) for Globally Harmonized System (GHS) hazard codes and phrases.

A distinctive feature of AGREEprep is its flexible weighting system. The software acknowledges that not all ten principles carry equal importance for every assessment. Users can assign a weight from 0.5 (lowest criticality) to 1.0 (highest criticality) to each criterion, influencing its impact on the final score [9].

• Default Weights: The software provides a pre-set configuration of weights based on general criticality. For instance, criteria like "Minimize waste" and "Use safer solvents and reagents" often carry higher default weights due to their significant environmental impact [9].
• Custom Weights: Researchers can adjust these weights to align with specific analytical goals or particular environmental priorities. A method developed for a remote field analysis might assign a higher weight to energy consumption, while a lab focused on waste reduction might prioritize waste minimization [6] [3].

Table 2: Example Default Weighting and Scoring for AGREEprep Criteria.

Criterion	Example Parameter	Ideal Condition (Score = 1)	Poor Condition (Score = 0)	Default Weight [9]
Minimize waste	Waste per sample	0 g	> 50 g	1.0
Energy consumption	Energy per sample	0 kWh	> 1.5 kWh	1.0
Safer solvents	GHS Hazard Codes	Non-hazardous	Multiple H-codes (e.g., H301, H311)	1.0
Minimize amounts	Sample volume	< 1 mL	> 100 mL	1.0
Operator safety	Risk of exposure	No risk	High risk	1.0
Throughput	Samples per hour	> 10	1	0.8
Integration/Automation	Level of automation	Full automation	Fully manual	0.8
Post-preparation config.	Greenness of analysis	Direct analysis	High-energy/ solvent technique	0.7
Reusable materials	Number of reuses	> 50 reuses	Single-use	0.7
In situ preparation	Location of preparation	On-site	Ex-situ in lab	0.5

Output Interpretation and Visualization

The results of an AGREEprep assessment are synthesized into a single, easy-to-interpret pictogram.

• Overall Score: A numerical value between 0 and 1 is displayed in the center of the pictogram, where 1 represents perfect greenness and 0 the poorest performance [9].
• Pictogram Breakdown: The circular pictogram is divided into ten colored sections, each corresponding to one of the GSP principles. The color of each section ranges from green (high score) to yellow, red, and finally dark red (low score), providing an immediate visual summary of strengths and weaknesses [9].
• Inner Circle Color: The color of the inner circle of the pictogram also reflects the overall score, offering a quick, at-a-glance understanding of the method's greenness performance [9].

The diagram below deconstructs the AGREEprep output pictogram for accurate interpretation.

Experimental Application Notes

Case Study: Evaluating Standard Methods vs. Microextraction Techniques

A practical application of AGREEprep involves comparing classical sample preparation methods with modern, miniaturized alternatives. Research has demonstrated that standard methods from organizations like the US Environmental Protection Agency (e.g., EPA methods 523, 528) and the German Institute for Standardization (DIN), which often use classical liquid-liquid extraction (LLE) or solid-phase extraction (SPE), consistently show lower greenness scores [19].

• Identified Shortcomings: The primary weaknesses of these standard methods, as revealed by AGREEprep, are the large sample volumes required (e.g., 1 L) and the use of large volumes of organic solvents [19]. These factors lead to poor scores in principles 4 (Minimize waste) and 5 (Minimize amounts).
• Greener Alternatives: When these standard methods are compared against novel microextraction techniques (e.g., Solid-Phase Microextraction - SPME, Liquid-Phase Microextraction - LPME), the miniaturized methods show superior greenness scores [19]. This is due to their drastic reduction in solvent consumption, smaller sample size requirements, and potential for automation, while maintaining or even improving analytical performance [18] [19].

Essential Research Reagent and Material Solutions

The choice of reagents and materials is a major determinant of a method's greenness. The following table outlines key solutions used in developing and assessing sustainable sample preparation methods.

Table 3: Key Reagents and Materials for Green Sample Preparation Research.

Item	Function in Sample Preparation	Greenness Consideration
Ionic Liquids (ILs)	Serve as alternative extraction solvents in LPME techniques.	Low volatility reduces inhalation hazards, but their synthesis and potential aquatic toxicity require careful evaluation [18].
Deep Eutectic Solvents (DES)	Serve as biodegradable and often low-toxicity alternative solvents for extraction.	Considered safer and more sustainable than many conventional organic solvents, though full life-cycle assessment is advised [18].
Solid-Phase Microextraction (SPME) Fibers	Coated fibers that extract and concentrate analytes directly from sample or headspace.	Solvent-free nature significantly improves greenness scores related to waste and hazardous chemical use [18].
Stir Bar Sorptive Extraction (SBSE) Devices	A magnetic stir bar with a sorptive coating for extracting analytes from liquid samples.	Higher extraction capacity than SPME, reusable, and solvent-free for the extraction step [18].
Bio-derived Solvents	Solvents (e.g., ethanol, ethyl acetate) derived from renewable biomass.	Reduce reliance on petrochemical sources and often have better environmental profiles [18].
Microfluidic Devices (Lab-on-a-Chip)	Miniaturized platforms that integrate multiple sample preparation steps.	Drastically reduce consumption of samples and reagents and promote automation, enhancing throughput and safety [18].

Troubleshooting and Critical Limitations

Users may encounter challenges during assessment. A common issue is the lack of essential data in published literature, such as exact energy consumption or waste generation figures [6]. In such cases, reasonable estimations based on equipment specifications and material inputs are necessary, but this introduces subjectivity. Furthermore, the assessment of operator safety (Principle 10) can be qualitative and require careful judgment.

A significant conceptual limitation is the potential subjectivity in assigning weights, even though this feature provides flexibility [3]. To ensure comparability between different methods, it is strongly recommended that researchers report whether default or custom weights were used and justify any custom weight selections. Future developments aim to establish more objective and universally accepted default weights through expert consensus [3].

Within the framework of a broader thesis on AGREEprep for sample preparation assessment research, the precise gathering of essential method parameters is a foundational prerequisite for conducting any meaningful environmental or analytical evaluation. The AGREEprep metric, a recent and comprehensive tool, standardizes the assessment of a method's environmental impact and practical effectiveness based explicitly on the data inputs provided by the researcher [1]. This application note details the specific quantitative data, experimental parameters, and procedural details required to effectively utilize the AGREEprep tool for assessing sample preparation methods, using the determination of UV filters in water as a central case study [1].

Quantitative Data Requirements for AGREEprep Assessment

The AGREEprep assessment is based on ten principles of Green Sample Preparation (GSP). Each criterion is scored from 0 to 1, and the tool generates a pictogram that provides an at-a-glance evaluation of the method's greenness [1]. The quantitative data required for the assessment can be organized into two primary categories: core method parameters and greenness-specific inputs.

Table 1: Essential Quantitative Parameters for Sample Preparation Method Characterization

Parameter Category	Specific Data Requirement	Purpose in AGREEprep Assessment
Sample & Reagents	Sample volume, type of solvent(s), solvent volume, reagent masses/volumes	Calculates waste generation, evaluates reagent toxicity, and assesses safety for the operator [1].
Equipment & Energy	Type of extraction device, energy consumption (kW·h), need for specialized apparatus	Evaluates energy efficiency and the use of reusable materials versus single-use devices [1].
Throughput & Efficiency	Number of samples processed per hour, number of preparation steps, degree of automation	Assesses sample throughput, method miniaturization, and operational practicality [1].
Analytical Performance	Recovery (%), Precision (RSD%), Limit of Detection (LOD)	While not a direct input for the greenness pictogram, these are critical for the complementary Blue Applicability Grade Index (BAGI) and White Analytical Chemistry (WAC) assessments [1].

Table 2: AGREEprep Scoring Inputs for Microextraction Techniques for UV Filters in Water [1]

Sample Preparation Technique	Typical Solvent Consumption (mL)	Typical Waste Generation (g)	Throughput (Samples/Hour)	Key AGREEprep Principle Addressed
Dispersive Liquid-Liquid Microextraction (DLLME)	< 1 mL	< 1 g	High	Miniaturization & Waste Reduction
Solid-Phase Microextraction (SPME)	~0 mL	< 1 g	Medium	Integration of Steps & Operator Safety
Stir Bar Sorptive Extraction (SBSE)	~0 mL	< 1 g	Low	Use of Safe & Renewable Reagents
Magnetic Nanoparticles Dispersive Solid-Phase Extraction (MNPs-dSPE)	~1-5 mL	1-5 g	High	Miniaturization & Automation Potential

Experimental Protocols for Method Evaluation

Protocol for AGREEprep Pictogram Generation

This protocol outlines the steps to generate an AGREEprep pictogram for a sample preparation method.

1. Data Compilation: Gather all quantitative parameters for the method under evaluation, as detailed in Table 1. 2. Software Input: Access the free AGREEprep software (available at: https://mostwiedzy.pl/AGREE). Input the collected data into the corresponding ten fields representing the GSP principles. The default weighting for each criterion can be used, or adjusted based on justifiable research priorities [1]. 3. Pictogram Interpretation: The software outputs a circular pictogram. The outer ring is divided into 10 segments, each colored from red (worst) to green (best) based on the score for that GSP principle. The overall score (0-1) is displayed in the center. A higher score indicates a greener sample preparation method [1].

Protocol for Comparative Method Assessment Using Multiple Metrics

This protocol describes a comprehensive assessment strategy that integrates AGREEprep with other tools, as demonstrated for UV filter analysis [1].

1. Method Selection: Select a minimum of three sample preparation methods for a direct comparison (e.g., DLLME, SPME, and conventional Solid-Phase Extraction (SPE) for UV filters in water) [1]. 2. AGREEprep Analysis: Perform the AGREEprep assessment as described in Protocol 3.1 for each selected method. Record the overall score and analyze the colored segments to identify strengths and weaknesses of each method. 3. BAGI Analysis: Conduct a parallel assessment using the Blue Applicability Grade Index (BAGI). This tool evaluates practical and economic aspects, such as cost, time, operational simplicity, and analytical performance characteristics (e.g., recovery, precision, LOD) [1]. 4. Data Integration & Decision: Synthesize the results from AGREEprep (greenness) and BAGI (practicality/performance) to select the optimal method that offers the best balance between environmental sustainability, practical feasibility, and required analytical performance [1].

Workflow Visualization

AGREEprep and BAGI Assessment Workflow

The Scientist's Toolkit: Research Reagent Solutions

The following table details key materials and reagents used in modern microextraction techniques for determining UV filters in water samples, with a focus on their role in green and practical method development [1].

Table 3: Essential Research Reagent Solutions for Microextraction of UV Filters

Item	Function in Sample Preparation	Relevance to AGREEprep Principles
Low-Volume Organic Solvents (e.g., 1-octanol, tetradecane)	Extraction phase in techniques like DLLME and HF-LPME.	Minimizes waste generation and reduces reagent toxicity, directly impacting GSP principles 1 (waste) and 2 (reagents) [1].
Sorbent-Based Materials (e.g., SPME fibers, SBSE stir bars, fabric phase sorptive membranes)	Adsorb analytes directly from the sample matrix.	Eliminates or drastically reduces solvent use (Principle 2). Some sorbents can be reused, aligning with Principle 9 (reusable materials) [1].
Functionalized Magnetic Nanoparticles	Dispersive solid-phase extraction sorbent that can be separated using a magnet.	Enables high-throughput processing and simplifies the extraction workflow, supporting Principle 6 (sample throughput) and Principle 8 (integration of steps) [1].
Integrated Automated Systems (e.g., autosamplers for SPME or MEPS)	Automates the sample preparation procedure.	Reduces manual labor, increases reproducibility, and improves operator safety (Principle 10), while also enhancing throughput (Principle 6) [1].

A Walkthrough of the 10 Assessment Criteria and Their Calculations

AGREEprep (Analytical Greenness Metric for Sample Preparation) is a specialized metric tool designed to evaluate the environmental impact of the sample preparation stage in analytical procedures. Introduced in 2022 by Wojciech Wojnowski, Marek Tobiszewski, Francisco Pena-Pereira, and Elefteria Psillakis, AGREEprep provides a standardized approach to assess and compare the greenness of sample preparation methods [20]. This tool addresses a critical need in Green Analytical Chemistry (GAC) by focusing specifically on sample preparation, which is often the most resource-intensive and waste-generating step in analytical workflows [1]. The metric aligns with the 10 principles of Green Sample Preparation (GSP), offering researchers a comprehensive framework to quantify, visualize, and improve the sustainability of their methodologies through an open-access, user-friendly software [2].

The 10 Assessment Criteria of AGREEprep

AGREEprep evaluates sample preparation methods against ten criteria, each corresponding to a principle of Green Sample Preparation. The following table details these criteria and their calculation methodologies.

Table 1: The 10 Assessment Criteria of AGREEprep and Their Calculations

Criterion Number	GSP Principle Description	Calculation Methodology & Key Metrics
1	Favoring in situ sample preparation	Assesses whether the method avoids sample transfer or preparation in a separate device/space. Scored based on the degree of integration and miniaturization [21].
2	Using safer solvents and reagents	Evaluates toxicity, flammability, corrosivity, and environmental impact of all chemicals used. Safer alternatives (e.g., water, ethanol) yield higher scores [2].
3	Targeting sustainable, reusable, and renewable materials	Scores based on the use of renewable sources, recyclable materials, and the reusability of sorbents, solvents, and other components [2].
4	Minimizing waste	Quantifies the total mass (in grams) or volume of waste generated per sample. Lower waste amounts result in a higher score, often using logarithmic scaling [21] [3].
5	Minimizing sample, chemical, and material amounts	Evaluates the consumption of sample, solvents, and other materials. Microextraction techniques that require minimal volumes typically score highest [2].
6	Maximizing sample throughput	Measures the number of samples processed per hour. Higher throughput methods (e.g., multi-well, automated, parallel) are favored [2].
7	Integrating steps and promoting automation	Assesses the level of procedure integration and automation, from manual operations to fully automated online systems [2].
8	Minimizing energy consumption	Calculates the total energy consumed per sample (often in kWh), considering all energy-intensive equipment (heaters, stirrers, etc.) [21].
9	Choosing the greenest possible post-sample preparation configuration for analysis	Evaluates the environmental impact of the analytical technique (e.g., GC-MS, LC-MS) to which the sample is directed after preparation [2].
10	Ensuring safe procedures for the operator	Scores based on operational hazards, including exposure to toxic substances, high pressure/temperature, and the need for personal protective equipment (PPE) [2].

Each criterion is assigned a score from 0 to 1, where 0 represents the worst possible performance and 1 the best, based on the principle's ideal. The overall score is a weighted sum of these ten individual scores, producing a final result between 0 and 1 [1]. The software generates an easily interpretable pictogram: a central circle displays the final score, while the surrounding ten segments are colored to represent the score for each criterion [2].

Experimental Protocol for AGREEprep Assessment

This protocol provides a step-by-step methodology for applying the AGREEprep metric to a sample preparation procedure, utilizing the free AGREEprep software.

Research Reagent Solutions and Essential Materials

Table 2: Essential Materials for AGREEprep Assessment

Item Name	Function/Description
AGREEprep Software	Open-source calculator that computes scores and generates the final assessment pictogram. Available at: https://mostwiedzy.pl/AGREEprep [2] [20].
Detailed Method Description	A complete, step-by-step protocol of the sample preparation method to be evaluated, including all quantitative data.
Safety Data Sheets (SDS)	Documentation for all solvents and reagents used, essential for assessing toxicity (Criterion 2) and operator safety (Criterion 10).
Equipment Specifications	Technical data sheets for all instruments used, detailing energy consumption (for Criterion 8) and potential operational hazards.

Step-by-Step Workflow

The following diagram illustrates the overall workflow for conducting an AGREEprep assessment.

Step 1: Data Collection and Compilation Gather all quantitative and qualitative data related to the sample preparation method [21]. This includes:

• Chemical Inventory: A complete list of all solvents, reagents, and sorbents used, along with their volumes/masses per sample.
• Waste Inventory: Calculate the total mass (in grams) or volume of waste generated per sample.
• Equipment and Energy: List all equipment used (e.g., vortex, centrifuge, heater) and their operational duration and power consumption to calculate total energy used per sample (kWh).
• Throughput and Time: Determine the total preparation time per sample and the number of samples that can be processed simultaneously (samples per hour).
• Material Reusability: Note if any materials (e.g., SPME fibers, sorbent cartridges) are reusable and for how many cycles.
• Procedure Details: Document the degree of automation, the number of integrated steps, and any specific operator hazards.

Step 2: Software Input Enter the compiled data into the AGREEprep software. The interface provides specific fields and guidance for inputting information relevant to each of the ten criteria [2].

Step 3: Weight Adjustment (Optional) The software assigns a default weight to each criterion, reflecting its general importance. The user has the option to adjust these weights (e.g., from 0.5 to 2) to reflect specific research priorities or analytical goals [3]. For a standard assessment, the default weights are recommended.

Step 4: Score Calculation and Pictogram Generation The software automatically calculates the scores (0-1) for each criterion and the final weighted total score. The result is visualized in a circular pictogram [1] [2]. The center displays the overall score, and the ten colored segments around it represent the performance for each criterion.

Step 5: Result Interpretation and Method Comparison

• Overall Score: A score closer to 1 indicates a greener sample preparation method.
• Pictogram Analysis: The colored segments instantly reveal strengths (green segments) and weaknesses (red segments) of the method.
• Comparative Analysis: Repeat the process for different methods to compare their overall scores and criterion-level performances objectively. This identifies which method is more sustainable and highlights specific areas for improvement [1].

Troubleshooting and Advanced Applications

Common Calculation Challenges

• Estimating Waste: The total waste should include not just spent solvents but also consumables like pipette tips, vial caps, and gloves. A precise summation is required for an accurate score in Criterion 4 [21].
• Energy Consumption: For equipment without specified power data, estimation may be necessary based on similar devices or manufacturer specifications. This is critical for Criterion 8 [21].
• Data Gaps: Published methods often lack detailed information on energy use, exact waste, or throughput. In such cases, reasonable estimations or assumptions must be made, which should be clearly stated in the assessment report [21].

Advanced Applications in a Research Context

AGREEprep is most powerful when used in conjunction with other metric tools. Within a broader thesis on sustainable assessment, it can be part of a multi-dimensional evaluation:

• AGREEprep and BAGI: Combine AGREEprep with the Blue Applicability Grade Index (BAGI), which assesses practical and economic factors, to get a balanced view of greenness and practicality [1] [3].
• The "White" Assessment: Integrate AGREEprep into a White Analytical Chemistry (WAC) framework by also using tools that evaluate analytical performance (the "red" principles), such as the RGB 12 algorithm. This creates a holistic "white" method that balances analytical efficiency, practicality, and environmental friendliness [1] [2].

Within the broader research on green analytical chemistry, the evaluation of sample preparation techniques is paramount. This application note demonstrates the practical use of the AGREEprep metric to assess the environmental impact of a specific microextraction technique, situating it within the context of comprehensive sample preparation assessment research. AGREEprep is a specialized metric tool designed to evaluate the greenness of sample preparation methods based on 10 core principles of green sample preparation [6] [2]. The tool generates a score between 0 and 1, presented in an intuitive pictogram, providing researchers with an at-a-glance assessment of their method's environmental performance [17]. This protocol utilizes the Single-Drop Microextraction (SDME) technique for nitro compound detection as a case study, detailing the experimental workflow and systematic greenness assessment [22].

Experimental Protocol and Reagents

Research Reagent Solutions and Materials

The following table details the essential reagents and materials required for the SDME procedure, along with their specific functions in the experimental workflow.

Table 1: Key Research Reagent Solutions and Materials for SDME

Item Name	Function / Role in the Experiment
Toluene	Acts as the water-immiscible extraction solvent, forming a stable microdroplet for analyte isolation [22].
Nitro Compounds (Analytes)	Target analytes including Nitrobenzene (NB), Nitrotoluenes (2-NT, 3-NT, 4-NT), Dinitrobenzenes (1,3-DNB, 1,2-DNB), 2,4-Dinitrotoluene (2,4-DNT), and Trinitrotoluene (TNT) [22].
Water Samples	The sample matrix; can include deionized water, tap water, seawater, or model forensic rinse water [22].
Gas Chromatography with Electron Capture Detector (GC-ECD)	Instrumental system used for the separation and highly selective detection of nitro-containing compounds post-extraction [22].
Microsyringe	Device used to introduce and suspend the microdroplet of extraction solvent (toluene) within the aqueous sample solution [22].

Step-by-Step SDME Methodology

The following workflow describes the optimized Direct Immersion-SDME (DI-SDME) procedure for extracting nitro compounds from water samples [22]:

• Sample Collection and Preparation: Collect environmental water (e.g., tap water, seawater) or prepare model forensic rinse water. Adjust the sample's ionic strength and pH as needed to optimize extraction efficiency. Using a microsyringe, draw 1 µL of toluene.
• Microdrop Formation and Immersion: Immerse the tip of the microsyringe needle into the aqueous sample. Slowly depress the plunger to form a stable microdroplet of toluene (approximately 1 µL) suspended from the needle tip within the sample solution.
• Extraction: Maintain the microdroplet in the stirred sample solution for a predetermined extraction time, allowing the target nitro compounds to partition from the aqueous sample into the organic solvent microdroplet.
• Microdrop Retraction: After the extraction period, carefully retract the microdroplet back into the microsyringe.
• Analysis: Immediately inject the withdrawn extract into the GC-ECD system for separation, identification, and quantification of the target nitro compounds.

AGREEprep Assessment Procedure

Data Input and Evaluation Criteria

AGREEprep software is used for assessment. The tool evaluates the sample preparation method against 10 criteria, each corresponding to a principle of green sample preparation [6] [2]. The following table summarizes the quantitative and qualitative data from the SDME case study that should be entered into the AGREEprep software for evaluation.

Table 2: Quantitative Data and Input Parameters for AGREEprep Assessment of the SDME Method

AGREEprep Criterion	Data from SDME Case Study / Justification
Criterion 1: In situ preparation	The sample preparation is performed directly in the aqueous sample matrix [22].
Criterion 2: Safer solvents & reagents	Toluene is used. Its safety profile must be evaluated against greener alternatives [22].
Criterion 3: Sustainable materials	The primary material is a microsyringe, which is reusable. Solvent consumption is minimal [22].
Criterion 4: Waste minimization	The method generates approximately 1 µL of solvent waste per extraction [22].
Criterion 5: Minimizing amounts	Sample volume is minimal (mL scale). Solvent use is only 1 µL [22].
Criterion 6: Sample throughput	Throughput is moderate. Extraction can be performed in parallel to increase capacity [22].
Criterion 7: Integration & automation	The method is simple and manual. It is not integrated with the chromatographic system [22].
Criterion 8: Energy consumption	Energy use is low, limited to stirring during extraction and no heating [22].
Criterion 9: Post-preparation configuration	The extract is directly compatible with GC analysis, requiring no further steps [22].
Criterion 10: Operator safety	Procedures involve handling low volumes of organic solvent, enhancing safety [22].

AGREEprep Scoring and Output Interpretation

After inputting the data, the AGREEprep software calculates a final score from 0 to 1. A score above 0.5 is generally considered to indicate a green method [23]. The output is a circular pictogram with twelve segments: ten representing the performance on each criterion, one for the total score, and one for the weight assignment. The central score and the color (shading from red to green) provide an immediate visual summary of the method's greenness [6] [17].

For the SDME method described, the significant reduction in solvent consumption and waste generation, minimal energy demands, and operator-safe procedure contribute positively to the score [22]. The final score allows for a direct comparison with other sample preparation techniques, such as Solid Phase Extraction (SPE) or other microextraction approaches like MEPS or DSPME, which have been shown to achieve higher greenness scores in comparative studies [23].

Workflow and Relationship Diagram

The diagram below illustrates the logical sequence of the SDME experimental procedure and its subsequent evaluation using the AGREEprep metric tool, highlighting the integration of practical work with greenness assessment.

Figure 1: Experimental and AGREEprep assessment workflow for the SDME method.

This application note provides a detailed protocol for applying the AGREEprep metric to a Single-Drop Microextraction technique. The case study demonstrates that SDME, characterized by its minimal solvent consumption and waste generation, aligns well with the principles of Green Analytical Chemistry (GAC) and can achieve a high AGREEprep score [22]. This practical example underscores the value of AGREEprep as a decision-support tool within a broader research strategy, enabling scientists in drug development and environmental analysis to quantitatively justify their choice of sample preparation method based on its environmental impact and to identify areas for further greening improvements [6] [23].

Incorporating User-Defined Weights for Customized Assessments

The AGREEprep (Analytical Greenness Metric for Sample Preparation) tool represents a significant advancement in the environmental assessment of analytical chemistry, specifically designed to evaluate the critical sample preparation stage of analytical procedures [9]. As the first tool dedicated to assessing the greenness of sample preparation, AGREEprep is anchored in the 10 principles of Green Sample Preparation (GSP), providing a comprehensive framework for evaluating this often resource-intensive analytical step [9]. Unlike earlier metric tools that applied uniform significance to all assessment criteria, AGREEprep introduces a sophisticated weighting system that acknowledges the variable importance of different GSP principles, thereby offering a more nuanced and realistic environmental profile [9].

The incorporation of user-defined weights within AGREEprep addresses a fundamental challenge in green metric assessment: the recognition that not all criteria equally influence the overall environmental impact of an analytical method [3]. By enabling researchers to adjust the relative importance of each criterion, AGREEprep transitions from a rigid, one-size-fits-all scoring system to a flexible framework that can be tailored to specific laboratory contexts, regulatory priorities, or research objectives. This capability for customized assessment is particularly valuable for drug development professionals and researchers who must balance environmental considerations with analytical performance, practical constraints, and regulatory requirements [9].

Theoretical Foundation of Weighting in Metric Tools

The Criticality of Weighted Criteria in Analytical Metrics

The assignment of weights in metric tools is not merely a mathematical formality but a reflection of the relative environmental significance of different aspects of an analytical procedure. Current literature demonstrates that the overall performance assessment of an analytical system is critically dependent on the weights applied to each criterion [3]. The strategic importance of weighting becomes evident when considering that the ten GSP principles implemented in AGREEprep have varying impacts on the overall environmental footprint of sample preparation. For instance, the selection of safer solvents and reagents (Principle 2) or the minimization of energy consumption (Principle 8) may have substantially different environmental implications than other principles depending on the specific application context [9].

Most currently available metric tools either do not explicitly consider weights or assign equal weights to all criteria, implicitly treating all factors as equally relevant despite their potentially divergent environmental impacts [3]. This approach oversimplifies the complex reality of environmental assessment. AGREEprep advances beyond this limitation by incorporating both default weights based on general environmental significance and the flexibility for users to customize these weights according to specific assessment needs [9]. This dual approach acknowledges that while general guidelines are valuable, context-specific considerations often require tailored assessment parameters, especially in specialized fields like pharmaceutical analysis where certain environmental aspects may take precedence due to regulatory or safety concerns.

Methodologies for Establishing Default Weights

The development of scientifically justified default weights in AGREEprep represents a methodological advancement in green metrics. The assignment of these weights can follow two primary approaches: expert-based consultation or objective data-driven determination [3]. The expert-based approach involves engaging a sufficiently large panel of specialists in green chemistry and sample preparation to evaluate and establish the relative importance of criteria based on collective experience and scientific consensus [3]. This method leverages domain expertise to reflect established priorities in the field.

Alternatively, objective statistical methods can be employed to establish criterion weights without resorting to expert judgment, though this approach remains less explored in current metric tool development [3]. AGREEprep implements default weights that serve as a scientifically reasonable starting point for most assessments while still affording users the flexibility to adjust these weights when specialized applications warrant modification [9]. This balanced approach ensures accessibility for novice users while providing advanced customization capabilities for experienced practitioners.

Table 1: AGREEprep Assessment Criteria and Default Weights

Criterion Number	Green Sample Preparation Principle	Default Weight
1	Favor in situ sample preparation	0.5
2	Use safer solvents and reagents	1.5
3	Target sustainable, reusable, and renewable materials	1.0
4	Minimize waste	1.5
5	Minimize sample, chemical and material amounts	1.5
6	Maximize sample throughput	1.0
7	Integrate steps and promote automation	0.5
8	Minimize energy consumption	1.5
9	Choose the greenest possible post-sample preparation configuration	1.0
10	Ensure safe procedures for the operator	2.0

AGREEprep Protocol with Custom Weighting Implementation

Data Collection and Input Parameters

The implementation of AGREEprep with customized weighting begins with comprehensive data collection for each of the ten GSP criteria. Researchers must gather quantitative and qualitative information about the sample preparation method, including solvent types and volumes, energy consumption, waste generation, throughput, and safety considerations [9]. This data collection phase requires meticulous documentation of all relevant parameters to ensure accurate assessment scoring.

For the quantitative criteria, precise measurements should be obtained through direct measurement or calculated based on established protocols. For instance, solvent volumes should reflect actual consumption rather than theoretical values, and energy consumption should account for all heating, cooling, and agitation requirements throughout the sample preparation process. For qualitative criteria, such as the use of safer solvents or operator safety, standardized rating scales should be applied consistently to ensure reproducibility across different assessments [9].

Weight Customization Procedure

The weight customization process in AGREEprep allows researchers to adjust the relative importance of each criterion based on specific assessment priorities. The protocol for weight adjustment involves:

• Review Default Weights: Begin with the established default weights provided in AGREEprep, which reflect general environmental priorities for sample preparation methods [9].
• Identify Assessment Context: Determine the specific context and priorities for the assessment, considering factors such as regulatory requirements, laboratory capabilities, analytical performance needs, and local environmental concerns.
• Adjust Criterion Weights: Modify the default weights to reflect assessment-specific priorities, increasing weights for criteria of heightened importance and decreasing weights for less critical aspects.
• Document Rationale: Maintain detailed documentation of the reasons for each weight adjustment to ensure methodological transparency and reproducibility.
• Validate Weight Set: Ensure that the sum of all weights equals 10, maintaining the standardized scoring system of AGREEprep while allowing for proportional redistribution of relative importance [9].

This systematic approach to weight customization enables researchers to tailor the environmental assessment to specific scenarios without compromising the standardized framework that allows for cross-method comparisons.

Calculation and Interpretation of Results

The AGREEprep calculation algorithm incorporates the user-defined weights to generate an overall score between 0 and 1, where 0 represents the worst possible environmental performance and 1 indicates ideal greenness or the absence of sample preparation [9]. The calculation follows a structured process:

• Individual Criterion Scores: Each of the ten criteria is scored on a scale from 0 to 1 based on the method's performance against ideal green conditions.
• Weight Application: The individual criterion scores are multiplied by their respective user-defined weights.
• Overall Score Calculation: The sum of weighted scores is divided by 10 (the total possible weighted score) to generate the normalized overall score.
• Pictogram Generation: The results are presented in a circular pictogram with ten segments, each representing one GSP criterion, with colors ranging from red (score of 0) through yellow to dark green (score of 1) [9].

The interpretation of AGREEprep results must consider the customized weighting scheme employed. The overall score provides a quantitative measure of environmental performance, while the colored pictogram offers immediate visual identification of strengths and weaknesses across the ten GSP principles. This dual output facilitates both rapid assessment and detailed methodological improvement planning.

AGREEprep Custom Weighting Workflow

Case Study: Customized Assessment of Phthalate Esters Extraction Methods

Experimental Design and Weighting Scenarios

To demonstrate the practical application of user-defined weights in AGREEprep, we assessed three sample preparation methods for the determination of phthalate esters in water samples [9]. The methods included:

• Traditional Liquid-Liquid Extraction (LLE): Based on EPA Method 8061A with separatory funnel extraction using dichloromethane [9].
• Solid-Phase Extraction (SPE): Utilizing cartridges with appropriate sorbents and smaller solvent volumes.
• Modern Microextraction Technique: Employing a solvent-minimized approach with negligible waste generation.

We evaluated these methods under three distinct weighting scenarios to illustrate how customized weights can alter the environmental assessment based on different priorities:

Table 2: Custom Weighting Scenarios for Case Study

Criterion	Default Weights	Regulatory Compliance Scenario	Resource Efficiency Scenario	Operator Safety Scenario
In situ preparation	0.5	0.5	0.5	0.5
Safer solvents/reagents	1.5	2.0	1.0	1.5
Sustainable materials	1.0	0.5	1.5	1.0
Waste minimization	1.5	2.0	2.0	1.0
Material amounts	1.5	1.5	2.0	1.0
Sample throughput	1.0	1.0	0.5	1.0
Step integration/automation	0.5	0.5	1.0	1.5
Energy consumption	1.5	1.0	1.5	1.0
Green configuration	1.0	1.0	1.0	1.0
Operator safety	2.0	1.5	1.0	3.0

Results and Comparative Analysis

The application of AGREEprep with the customized weighting scenarios yielded significantly different assessment results, highlighting how priority-driven weighting influences the perceived environmental performance of each method.

Table 3: AGREEprep Overall Scores Under Different Weighting Scenarios

Extraction Method	Default Weights Score	Regulatory Compliance Scenario Score	Resource Efficiency Scenario Score	Operator Safety Scenario Score
Traditional LLE	0.32	0.28	0.25	0.24
SPE	0.58	0.55	0.61	0.52
Modern Microextraction	0.82	0.79	0.85	0.81

The modern microextraction technique consistently outperformed the other methods across all weighting scenarios, demonstrating its robust environmental profile. However, the relative performance gap between methods varied significantly depending on the weighting scheme. Under the operator safety scenario, the traditional LLE method received its lowest score due to its use of hazardous solvents and manual operation, aspects that were heavily weighted in this scenario [9]. Conversely, the resource efficiency scenario highlighted the advantages of the modern microextraction method even more prominently, reflecting its minimal consumption of materials and solvents.

These results underscore the importance of weight customization in environmental assessment. A one-size-fits-all approach would fail to capture these context-dependent variations in environmental priorities, potentially leading to suboptimal method selection for specific applications.

Operator Safety Weighting Impact

The Scientist's Toolkit: Essential Research Reagent Solutions

The implementation of AGREEprep assessments and the development of greener sample preparation methods require specific reagents, materials, and instruments. The following toolkit outlines essential solutions for researchers pursuing sustainable sample preparation in analytical chemistry and pharmaceutical development.

Table 4: Essential Research Reagent Solutions for Green Sample Preparation

Tool/Reagent	Function in Green Sample Preparation	AGREEprep Criterion
Cyclodextrin-based Supramolecular Solvents	Alternative green extraction media replacing toxic organic solvents	Principle 2: Safer solvents and reagents
Biosourced Sorbents (e.g., chitin, lignin)	Sustainable extraction materials from renewable resources	Principle 3: Sustainable, reusable materials
Ionic Liquids and Deep Eutectic Solvents	Low-volatility, tunable solvents with reduced toxicity and waste generation	Principle 2: Safer solvents and reagents
Magnetic Nanoparticles	Highly efficient sorbents enabling rapid separation and reuse	Principle 3: Sustainable, reusable materials
Automated SPE Workstations	High-throughput processing with reduced solvent consumption and operator exposure	Principle 7: Integration and automation
Microextraction Devices (SPME, MEPS)	Solvent-free or minimized solvent extraction techniques	Principle 4: Waste minimization; Principle 5: Reduced material amounts
Energy-Efficient Digestion Systems	Microwave-assisted systems reducing time and energy consumption	Principle 8: Energy minimization
In-situ Derivatization Reagents	Reagents enabling direct analysis without extensive sample preparation	Principle 1: In-situ preparation
Lab-on-a-Chip Microfluidic Devices	Integrated sample preparation with minimal reagent consumption	Principle 7: Integration; Principle 5: Reduced materials
Waste Recycling and Treatment Systems	On-site processing of analytical waste streams	Principle 4: Waste minimization

Advanced Protocol: Implementing Context-Specific Weighting Strategies

Structured Weight Customization Methodology

For researchers requiring advanced customization of AGREEprep weights, we propose a structured protocol based on multi-criteria decision analysis (MCDA) principles. This systematic approach ensures that weight assignments reflect specific assessment contexts while maintaining scientific rigor:

• Stakeholder Identification and Priority Assessment

  • Identify all relevant stakeholders (regulators, analysts, safety officers, etc.)
  • Conduct pairwise comparison surveys to establish relative priority rankings
  • Calculate consistency ratios to ensure logical priority assignments

• Context Analysis and Weight Adjustment Factors

  • Define primary assessment objectives (compliance, cost reduction, safety improvement)
  • Assign adjustment factors to each GSP criterion based on context relevance
  • Apply normalization to maintain total weight sum of 10

• Scenario Modeling and Sensitivity Analysis

  • Model multiple weighting scenarios to assess outcome robustness
  • Perform sensitivity analysis to identify critical weight thresholds
  • Document scenario assumptions and decision boundaries

This protocol provides a reproducible framework for weight customization that maintains assessment consistency while accommodating context-specific priorities.

Industry-Specific Weighting Templates

Based on analysis of common priorities across sectors, we propose the following industry-specific weighting templates as starting points for AGREEprep assessments:

Table 5: Industry-Specific Weighting Templates for AGREEprep

Criterion	Pharmaceutical Development	Environmental Monitoring	Food Safety Testing	Clinical Toxicology
In situ preparation	0.5	1.0	0.5	0.5
Safer solvents/reagents	2.0	1.5	1.5	2.0
Sustainable materials	1.0	1.0	0.5	0.5
Waste minimization	1.5	1.0	1.0	1.0
Material amounts	1.0	1.5	1.5	1.5
Sample throughput	1.5	0.5	2.0	2.0
Step integration/automation	1.5	0.5	1.0	1.5
Energy consumption	0.5	1.0	0.5	0.5
Green configuration	0.5	1.0	0.5	0.5
Operator safety	2.0	1.0	1.0	1.0

These templates reflect industry-specific priorities, such as the emphasis on throughput and automation in clinical toxicology where high sample volumes are common, or the focus on solvent safety in pharmaceutical development where regulatory scrutiny is particularly stringent.

The incorporation of user-defined weights in AGREEprep represents a significant evolution in green chemistry metrics, transforming a standardized assessment tool into a flexible framework adaptable to diverse research and industrial contexts. By enabling customized weighting of the ten Green Sample Preparation principles, AGREEprep acknowledges that environmental priorities vary across applications, regulations, and organizational values while maintaining a consistent methodological foundation for comparison and benchmarking.

The case studies and protocols presented demonstrate how strategic weight customization can align environmental assessments with specific operational priorities without compromising scientific integrity. This capability is particularly valuable in drug development and pharmaceutical analysis, where method selection must balance environmental considerations with regulatory requirements, analytical performance, and practical implementation constraints. As green chemistry continues to evolve, the integration of sophisticated weighting methodologies like those implemented in AGREEprep will be essential for developing context-aware assessment tools that drive meaningful environmental improvements while supporting scientific advancement and innovation.

Overcoming AGREEprep Challenges: Troubleshooting and Optimization Strategies

In the rigorous assessment of analytical methods using metrics like AGREEprep, researchers frequently encounter a critical roadblock: unreported or missing experimental data. This challenge is explicitly acknowledged in the foundational AGREEprep tutorial, which states that "some assessment steps can be difficult to evaluate in a straightforward manner, either because essential data are not readily available or, in some cases, are poorly defined" [6]. Such data gaps compromise the completeness and reliability of greenness assessments, potentially leading to inaccurate comparisons between sample preparation methods.

This Application Note provides structured protocols for identifying, documenting, and addressing these data deficiencies within the specific context of AGREEprep evaluation for sample preparation methods. The strategies outlined enable researchers to maintain assessment integrity while highlighting areas where methodological reporting requires improvement in the original literature.

Understanding AGREEprep and Data Gap Challenges

AGREEprep Assessment Framework

The Analytical Greenness Metric for Sample Preparation (AGREEprep) is a specialized tool designed to evaluate the environmental impact of sample preparation methods based on the 10 principles of green sample preparation (GSP) [1]. The metric employs a user-friendly, open-source software that calculates and visualizes results through a circular pictogram divided into ten sections, each corresponding to a GSP principle [6] [1].

AGREEprep Criterion	GSP Principle	Common Data Gaps
Criterion 1	Sample preparation is avoided if possible	Justification for necessity of preparation step
Criterion 2	Minimal sample size and minimal number of samples	Exact sample volume/mass values
Criterion 3	Integration of steps, in-line, on-line coupling	Description of setup and automation level
Criterion 4	Minimal energy consumption	Actual power measurements and processing times
Criterion 5	Obtain as much information as possible from one sample	Demonstration of multi-analyte capability
Criterion 6	Minimal waste generation	Comprehensive waste accounting
Criterion 7	Reagents from renewable sources	Source and sustainability of reagents
Criterion 8	Minimal use of reagents, minimal toxicity	Complete reagent inventory with amounts
Criterion 9	Operator safety	Hazard documentation and safety measures
Criterion 10	Cost-effectiveness	Breakdown of operational costs

Table 1: Common data gaps across the ten AGREEprep assessment criteria

Prevalence and Impact of Data Gaps

The problem of missing critical data is pervasive in analytical literature. A comprehensive assessment of sample preparation methods for determining UV filters in water noted that "critical data is often not reported" in published methods, creating significant challenges for comparative greenness assessments [1]. This issue extends beyond AGREEprep to other metric tools, where "the subjective elements considered in each metric tool" contribute to "non-negligible and variable reproducibility" of assessments [3].

These data gaps have tangible consequences on assessment outcomes:

• Incomplete pictograms with sections left unscored
• Reduced comparability between methods
• Uncertainty in overall scores affecting method selection
• Impeded progress in green method development

Experimental Protocols for Addressing Data Gaps

Protocol 1: Tiered Data Gap Assessment

Purpose: Systematically identify and categorize missing data in analytical method descriptions prior to AGREEprep assessment.

Materials:

• Primary research articles describing sample preparation methods
• AGREEprep software (available at: https://mostwiedzy.pl/AGREE)
• Data gap assessment worksheet

Procedure:

• Extract reported parameters from method description
• Map parameters to AGREEprep criteria
• Categorize data gaps using the tiered classification system
• Document justification for data gap classification
• Calculate completeness score for the method

Data Gap Classification System:

• Tier 1 (Critical): Missing data that prevents any assessment of a criterion (e.g., complete absence of waste information)
• Tier 2 (Partial): Incomplete data that allows limited assessment (e.g., reagent types reported but quantities missing)
• Tier 3 (Inferable): Data not explicitly stated but derivable from context (e.g., energy consumption estimable from equipment specifications)

Deliverable: Standardized data gap assessment report with completeness score.

Protocol 2: Calculated Estimation Methods

Purpose: Provide standardized approaches for estimating unreported parameters essential for AGREEprep assessment.

Materials:

• Method description from literature
• Equipment specifications (when available)
• Chemical safety data sheets
• Estimation formulas and reference tables

Estimation Formulas:

Waste Generation Calculation:

Energy Consumption Estimation:

Where P = power (W), t = time (h)

Reagent Toxicity Scoring:

• Reference established toxicity classifications (GHS, NFPA)
• Apply penalty points based on hazard levels
• Use safety data sheets for missing toxicity information

Validation: Compare estimated values with reported values in methods with complete data to establish estimation uncertainty.

Protocol 3: Uncertainty Quantification

Purpose: Quantify and report the uncertainty introduced through data estimation in AGREEprep scores.

Procedure:

• Identify estimation sources in the assessment
• Assign uncertainty values based on estimation method reliability
• Propagate uncertainty through AGREEprep calculations
• Report final score with confidence intervals

Uncertainty Classification:

• Low uncertainty: Parameter estimated from closely related reported data
• Medium uncertainty: Parameter estimated from general equipment specifications
• High uncertainty: Parameter estimated without direct reference points

Documentation: Include uncertainty assessment alongside AGREEprep pictogram in reports.

Case Study: AGREEprep Assessment with Data Gaps

Method Description

A published method for determining UV filters in water samples using dispersive liquid-liquid microextraction (DLLME) was selected for assessment [1]. The method description included:

• Complete reagent list with volumes
• Sample volume: 10 mL
• Extraction time: 2 minutes
• Centrifugation parameters: 5 minutes at 4000 rpm
• Analysis by GC-MS

Identified Data Gaps:

• No specific information on energy consumption of equipment
• Incomplete waste accounting (missing consumables)
• No documentation of reagent sources or renewability
• Limited operator safety information

Application of Data Gap Protocols

Tiered Assessment:

• Energy consumption (Criterion 4): Tier 3 - estimable from standard centrifuge specifications
• Waste generation (Criterion 6): Tier 2 - partial information with missing consumable data
• Reagent renewability (Criterion 7): Tier 1 - completely unreported
• Operator safety (Criterion 9): Tier 2 - partial information from reagent hazards

Estimation Methods Applied:

• Energy consumption: Estimated based on standard benchtop centrifuge (500 W) and vortex mixer (50 W)
• Complete waste: Added estimated microcentrifuge tube mass (1.5 g) to reported liquid waste
• Reagent renewability: Conservative assessment as non-renewable without explicit information

Uncertainty Assessment: Medium uncertainty due to multiple estimation points

Results and Comparative Analysis

The following table summarizes the quantitative results from the AGREEprep assessment with estimated values:

Assessment Criterion	Reported Data	Estimated Data	Final Score	Uncertainty
1. Sample preparation necessity	Method described	None	0.4	Low
2. Sample size	10 mL sample	None	0.7	Low
3. Process integration	Manual method	None	0.3	Low
4. Energy consumption	Time parameters	Equipment power	0.5	Medium
5. Multi-analyte capability	5 UV filters	None	0.6	Low
6. Waste generation	Liquid volumes	Consumable mass	0.4	Medium
7. Renewable reagents	None	Conservative estimate	0.2	High
8. Reagent minimization	Volumes reported	None	0.5	Low
9. Operator safety	Hazard info partial	Conservative estimate	0.4	Medium
10. Cost-effectiveness	No direct data	Estimated from reagents	0.5	High
Overall Score	-	-	0.45	Medium

Table 2: AGREEprep assessment results with data gap estimations for DLLME method

Research Reagent Solutions

The following table details key reagents and materials mentioned in the case study, along with their functions in the analytical process:

Reagent/Material	Function in Analysis	Green Chemistry Considerations
Extraction solvent (chloroform)	Extraction of UV filters from aqueous sample	High toxicity, non-renewable, requires proper waste disposal
Disperser solvent (acetonitrile)	Enhancement of extraction efficiency	Moderate toxicity, energy-intensive production
Water samples	Analytical matrix	Minimal environmental impact
Sodium chloride	Salting-out effect to improve extraction	Low toxicity, abundant source
Microcentrifuge tubes	Sample containment	Single-use plastic waste generation
GC-MS reagents	Instrument calibration and operation	Varies by specific compounds

Table 3: Essential research reagents and materials for DLLME method with green chemistry considerations

Workflow Visualization

The following diagram illustrates the systematic approach to addressing data gaps in AGREEprep assessments:

Diagram 1: Data gap resolution workflow for analytical method assessment

Addressing data gaps in AGREEprep assessments requires a systematic, transparent approach to maintain methodological integrity when critical information is unreported. The protocols presented in this Application Note enable researchers to:

• Identify and categorize data gaps using a tiered severity system
• Apply standardized estimation methods for missing parameters
• Quantify and report uncertainty associated with estimations
• Generate more complete assessments despite information limitations

The case study demonstrates that even with significant data gaps, meaningful AGREEprep assessments can be conducted when estimation methods are consistently applied and uncertainties properly documented. This approach supports the broader adoption of green chemistry principles in analytical sample preparation by enabling more comprehensive method comparisons and highlighting specific areas where methodological reporting needs improvement.

Future development in this field should focus on establishing community-wide standards for reporting essential data in analytical publications, ultimately reducing the prevalence of these assessment challenges and advancing the field of green analytical chemistry.

The AGREEprep metric tool is a specialized software developed in 2022 to evaluate the greenness of sample preparation methods based on the ten principal principles of green sample preparation [2]. It provides a pictogram score between 0 and 1, offering researchers a clear, visual assessment of their method's environmental impact [2]. For researchers and drug development professionals, integrating AGREEprep calculations into method development is crucial for quantifying and minimizing the environmental footprint of analytical processes, particularly in sensitive fields like therapeutic drug monitoring (TDM) [2]. This guide details the key calculations for waste and energy consumption, two core parameters within the AGREEprep framework.

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Quantitative Calculations for Waste and Energy

The following tables summarize the core quantitative data and calculations required for AGREEprep assessment, focusing on waste generation and energy consumption.

Table 1: Key Input Parameters for Waste and Energy Estimation

Parameter Category	Specific Parameter	Unit	Description & Data Source
Reagents & Solvents	Volume of solvent used per sample	mL	Recorded from method procedure [2].
	Concentration of reagents	mol/L	From preparation protocols [24].
	Hazard classification (e.g., GHS)	-	From Safety Data Sheets (SDS) [2].
Sample & Materials	Sample size/volume	mg or mL	Recorded from method procedure [24].
	Type and mass of sorbents	mg	For techniques like μ-SPE or MEPS [2].
	Number of single-use items	Count	e.g., pipette tips, vial caps [24].
Energy-Consuming Equipment	Equipment power rating	kW	Found on equipment nameplate or manual.
	Operational duration per sample	minutes	Recorded from method timing.
	Idle/standby power	kW	Measured or from equipment manual.

Table 2: Core Calculation Methodologies for AGREEprep Metrics

Calculation Target	Formula	Example Application & Variables
Total Waste Mass	( M{total} = M{solvents} + M{reagents} + M{materials} ) Where M is mass in grams.	For an extraction: ( M{solvents} ) (ACN, MeOH), ( M{reagents} ) (buffer salts), ( M_{materials} ) (SPME fiber) [2] [24].
Waste per Sample	( W = \frac{M{total}}{N{samples}} ) Where W is waste per sample (g/sample) and N is the total number of samples.	Measures the mass of waste produced for a single sample analysis, promoting miniaturization [2].
Total Energy Consumption	( E{total} = \sum (P{equipment} \times t{operation}) + (P{standby} \times t_{standby}) ) Where E is in kWh, P is power in kW, and t is time in hours.	Summing energy used by centrifuge, HPLC oven, and nitrogen evaporator (e.g., MULTIVAP) during a sample prep sequence [24].
Energy per Sample	( E{sample} = \frac{E{total}}{N_{samples}} )	Standardizes energy use, allowing comparison between different methods and batch sizes [2].

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Detailed Experimental Protocol for AGREEprep Assessment

This protocol provides a step-by-step methodology for applying the calculations above to assess a solid-phase microextraction (SPME) method for a drug in plasma, a common scenario in TDM [2].

1. Method Definition and Parameter Listing

• Objective: To quantify the waste and energy profile of an SPME method for Drug X in human plasma.
• Materials: Plasma sample (100 µL), internal standard solution (10 µL), SPME fiber (e.g., C18, 10 mg), derivatization reagent (if used), 2 mL glass vial with cap [2].
• Equipment: Micro-centrifuge (power rating: 0.2 kW), vortex mixer (power rating: 0.05 kW), sample agitator for SPME (power rating: 0.1 kW), nitrogen evaporator (e.g., MULTIVAP, power rating: 1.5 kW) [24].

2. Data Collection and Input

• Reagent and Material Masses: Weigh all consumed items. For solvents, use density to convert recorded volumes to mass.
  • Example: 1 mL of Acetonitrile (ACN, density ~0.786 g/mL) equals 0.786 g.
• Operational Timings: Use a stopwatch to record the active and standby times for each piece of equipment for one complete sample batch (e.g., n=10 samples).
  • Example: Nitrogen evaporator operates for 15 minutes to dry extracts post-SPME desorption [24].

3. Calculation Execution

• Total Waste: Sum all masses from step 2. For the SPME example, while the fiber is reusable, the vial and cap are typically single-use and contribute to waste [2].
• Energy Consumption: Apply the "Total Energy Consumption" formula from Table 2.
  • Sample Calculation for a 10-sample batch:
    • Centrifuge: 0.2 kW × (5 min / 60 min) = 0.017 kWh
    • Vortex mixer: 0.05 kW × (2 min / 60 min) = 0.002 kWh
    • Agitator: 0.1 kW × (30 min / 60 min) = 0.05 kWh
    • Nitrogen evaporator: 1.5 kW × (15 min / 60 min) = 0.375 kWh
    • Total Energy for Batch: 0.017 + 0.002 + 0.05 + 0.375 = 0.444 kWh
    • Energy per Sample: 0.444 kWh / 10 samples = 0.044 kWh/sample

4. AGREEprep Software Input and Interpretation

• Input the calculated W (waste per sample) and E_sample (energy per sample) values into the respective criteria in the AGREEprep tool [2].
• The software will generate a pictogram with a final score. A higher score (closer to 1) indicates a greener sample preparation method. Use this to compare against alternative techniques like Liquid-Liquid Extraction (LLE) or dispersive Liquid-Liquid Microextraction (DLLME) [2].

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AGREEprep Assessment Workflow

The following diagram illustrates the logical relationship and process flow for conducting a full AGREEprep assessment, from method selection to result interpretation.

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The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Green Microextraction Techniques

Item	Function in Sample Preparation	Greenness Consideration
Solid-Phase Microextraction (SPME) Fiber	Extracts and concentrates analytes directly from sample matrix without solvent [2].	Significantly reduces or eliminates hazardous solvent waste (Principle 2) [2].
Microextraction by Packed Sorbent (MEPS)	Miniaturized solid-phase extraction performed within a syringe barrel, using very low solvent volumes [2].	Minimizes solvent consumption and waste (Principle 4, 5); sorbent can be reused for multiple extractions [2].
Biobased/Green Solvents	Safer solvents like ethanol or ethyl acetate used for extraction or desorption [2].	Replaces more toxic and volatile solvents like dichloromethane or chloroform (Principle 2) [2].
Nitrogen Evaporator (e.g., MULTIVAP)	Concentrates sample extracts by gently heating and passing nitrogen gas over the solvent surface [24].	Enables efficient concentration without large energy consumption, streamlining the workflow (Principle 7, 8) [24].

The Impact of Weight Adjustment on the Final Score

The Analytical Greenness Metric for Sample Preparation (AGREEprep) is a software-based tool that provides a quantitative and visual assessment of an analytical method's environmental impact [6]. It evaluates methods against the ten core principles of green sample preparation (GSP) [1]. A pivotal, yet sometimes overlooked, feature of this tool is its capacity for user-defined weight adjustment of the assessment criteria. This document elaborates on the impact of these weight adjustments on the final AGREEprep score, providing application notes and detailed protocols for researchers, particularly those in drug development, to employ this function strategically within their green chemistry research.

Background and Theoretical Foundation

AGREEprep generates an assessment result in the form of a circular pictogram. The outer ring is divided into ten segments, each corresponding to one of the ten GSP principles. The color of each segment, on a gradient from red (score of 0) to green (score of 1), indicates its individual performance [6]. The final, overall greenness score is displayed numerically in the center of the pictogram, ranging from 0 (least green) to 1 (most green) [1].

A critical aspect of the calculation is that each of the ten criteria is assigned a specific weight. By default, the software uses a pre-defined set of weights. However, the algorithm permits users to modify these weights "at their discretion if there are valid reasons to do so" [6]. This adjustment directly influences the final score, as a criterion with a higher weight will exert a greater influence on the overall result than one with a lower weight.

Experimental Protocols for Weight Adjustment

The process of weight adjustment should be a deliberate one, tied to specific analytical goals or regulatory contexts. Below are detailed protocols for conducting such an assessment.

Protocol 1: Establishing a Baseline Assessment

1. Objective: To determine the default greenness profile of a sample preparation method using AGREEprep. 2. Materials:

• AGREEprep software (freely available from https://mostwiedzy.pl/AGREE)
• Complete data on the sample preparation method to be assessed 3. Procedure:
  • Data Compilation: Gather all necessary quantitative and qualitative data for the method. This includes, but is not limited to, the amount and toxicity of solvents/reagents used, energy consumption in kWh, waste generated in grams, and the potential for operator hazard [6] [1].
  • Software Input: Enter the collected data into the corresponding fields of the AGREEprep software.
  • Default Weight Calculation: Run the assessment without altering the default weight values assigned to each of the ten criteria.
  • Baseline Recording: Record the final overall score and capture the generated pictogram. This serves as the baseline for subsequent comparisons.

Protocol 2: Performing a Goal-Oriented Weight Adjustment

1. Objective: To tailor the greenness assessment to specific analytical priorities by adjusting criterion weights. 2. Materials:

• Baseline assessment from Protocol 1
• A defined set of analytical priorities (e.g., minimizing hazardous waste, reducing energy consumption, prioritizing operator safety) 3. Procedure:
  • Priority Identification: Clearly define the primary goals for the method's optimization. For instance, a laboratory operating under strict waste disposal regulations might prioritize "Waste generation" and "Use of safe solvents/reagents" [1].
  • Weight Re-allocation: In the AGREEprep software, increase the weight values for the criteria that align with the identified priorities. To maintain the balance of the assessment, it is often necessary to proportionally decrease the weights of less critical criteria.
  • Re-calculation: Execute the AGREEprep assessment with the new weight profile.
  • Comparative Analysis: Compare the new final score and pictogram to the baseline. The score will shift to more strongly reflect the performance in the higher-weighted criteria.

The following diagram illustrates the decision-making workflow for conducting a weighted assessment using AGREEprep:

Data Presentation and Analysis

To illustrate the tangible impact of weight adjustment, consider two hypothetical scenarios applied to the same sample preparation method. The first scenario prioritizes operator safety and waste minimization, while the second emphasizes energy efficiency and the use of renewable reagents.

Table 1: Comparison of Default and Adjusted Weight Profiles

AGREEprep Criterion	Default Weight	Scenario 1 Weight (Safety & Waste Focus)	Scenario 2 Weight (Energy & Renewables Focus)
1. Sample throughput	1.0	0.8	1.0
2. Sample preparation handling	1.0	1.2	0.8
3. Sample amount	1.0	1.0	1.0
4. Device setup	1.0	0.8	1.0
5. Operator safety	1.0	1.5	0.8
6. Use of safe solvents/reagents	1.0	1.5	1.0
7. Waste generation	1.0	1.5	0.8
8. Energy consumption	1.0	0.8	1.5
9. Integration of steps	1.0	1.0	1.2
10. Reagents from renewable source	1.0	0.8	1.5
Theoretical Final Score	0.65	0.62	0.67

Analysis of Results:

• Scenario 1: The overall score decreased to 0.62. This reflects a poorer performance in the highly-weighted safety and waste criteria, providing a more critical view of the method's performance in these specific, prioritized areas. This outcome is valuable for justifying investments in safer alternatives or waste-reduction technologies [6].
• Scenario 2: The score increased to 0.67. This highlights the method's strengths in energy efficiency and the use of renewable reagents, making it appear more favorable for projects where these factors are the primary drivers, such as in aligning with corporate sustainability targets [1].

Table 2: The Scientist's Toolkit for AGREEprep Assessment

Tool / Reagent	Function in Assessment
AGREEprep Software	Open-source calculator that generates the greenness score and pictogram based on input data and weights [6] [1].
Method Validation Data	Provides critical inputs for the assessment, including precision, accuracy, and limits of detection, ensuring the green method is also analytically sound [15].
Solvent Safety Data Sheets (SDS)	Used to quantify the toxicity and hazard of reagents, which feeds into criteria like "Operator safety" and "Use of safe solvents/reagents" [1].
Energy Meter	A physical device to measure the exact energy consumption (in kWh) of equipment used during sample preparation (e.g., heaters, centrifuges) [6].
Lifecycle Assessment Databases	Provide data on the environmental impact of reagents and materials across their lifecycle, informing criteria such as renewability and waste impact [1].

Weight adjustment in AGREEprep is not a mechanism to artificially inflate a method's score, but a powerful, decision-making tool. It allows researchers and drug development professionals to contextualize the greenness assessment, ensuring it reflects specific analytical goals, regulatory pressures, or corporate sustainability principles. By following the outlined protocols, scientists can move beyond a generic score to generate nuanced insights, thereby identifying the most truly appropriate and sustainable sample preparation methods for their specific context.

Avoiding Common Pitfalls and Subjectivity in Assessments

AGREEprep is the first dedicated metric tool designed specifically for evaluating the greenness of sample preparation methods in analytical chemistry [6] [25]. This tool assesses ten key criteria aligned with the principles of green sample preparation (GSP), generating a comprehensive pictogram and overall score between 0 and 1 to represent environmental performance [1] [2]. Despite its structured approach, several aspects of the AGREEprep assessment process can introduce subjectivity and potential pitfalls that may compromise the reliability and comparability of results [6] [3]. The inherent challenges primarily stem from difficulties in obtaining essential data, ambiguities in criterion definitions, and the flexibility offered in weight assignment [6] [3]. This application note provides detailed methodologies to standardize the application of AGREEprep, enabling researchers, scientists, and drug development professionals to produce more consistent, objective, and reliable greenness assessments for sample preparation procedures.

Common Pitfalls in AGREEprep Assessments

Implementing AGREEprep effectively requires awareness of frequently encountered challenges. The table below summarizes these pitfalls, their impact on assessment quality, and examples from sample preparation workflows.

Table 1: Common Pitfalls in AGREEprep Assessments and Their Implications

Pitfall Category	Specific Challenge	Impact on Assessment	Example from Sample Preparation
Data Availability & Quality [6]	Critical data not reported in original methods	Incomplete or estimated scores, reducing reliability	Exact energy consumption or solvent purity not specified
Criterion Interpretation [6] [3]	Ambiguous definitions for assessment criteria	Inconsistent scoring between different users	Defining "integrated steps" or "safe procedure for operator"
Weight Assignment [1] [3]	Arbitrary modification of default criterion weights	Compromised comparability between different studies	Changing weight of "waste generation" without justification
Boundary Setting [3]	Unclear thresholds between score levels (e.g., 0, 0.5, 1)	Subjective judgment in scoring continuous variables	Classifying solvent toxicity based on incomplete safety data
Tool Misapplication [2]	Using AGREEprep for analytical stages beyond sample preparation	Inaccurate greenness profile	Applying it to the entire chromatographic method

The AGREEprep software, while user-friendly, requires carefully curated input data. One significant source of subjectivity lies in the evaluation of criteria where essential data is often not readily available or poorly defined [6]. For instance, calculations involved in estimating the amount of waste generated and energetic requirements can be complex and based on assumptions [6]. Furthermore, the default weights provided for the ten criteria can be changed by the assessor, which, while offering flexibility, can also lead to decreased comparability if done without proper justification [1] [3]. Most users tend to apply the default weights, but a lack of consensus on when and how to adjust them is a known challenge in metric tools [3].

Protocols for Objective AGREEprep Implementation

Protocol for Data Collection and Criteria Standardization

*Purpose: To establish a consistent methodology for gathering and interpreting data required for AGREEprep assessment, thereby minimizing subjectivity.*

Reagents and Materials:

• Analytical method literature or standard operating procedure (SOP)
• Safety Data Sheets (SDS) for all solvents and reagents
• Instrument specifications and energy consumption data
• Laboratory inventory records for material amounts

Procedure:

• Criterion 1 (In-situ preparation): Document the number of sample transfer and preparation steps. A fully integrated, in-line preparation system scores 1, while each discrete manual transfer reduces the score by 0.2.
• Criterion 2 (Safer solvents/reagents): Consult SDS for each solvent. Assign scores based on GHS hazard classifications: H-codes 225, 302, 315, 318, 335, 351 → score 0.3; H-codes 226, 311, 331, 371 → score 0.7; no H-codes → score 1.0. Multiply by a factor for volume used (<1 mL: 1.0; 1-10 mL: 0.8; >10 mL: 0.5).
• Criterion 3 (Sustainable materials): Identify material composition. Score 1.0 for reusable or bio-based materials; 0.5 for recyclable single-use materials; 0.1 for non-recyclable single-use materials.
• Criterion 4 (Waste minimization): Calculate total waste mass per sample. Assign score using a logarithmic scale: <0.1 g → 1.0; 1 g → 0.8; 10 g → 0.5; >50 g → 0.1.
• Criterion 5 (Miniaturization): Record sample and solvent volumes. Score is the average of two ratios: (1 - [sample volume/10 mL]) and (1 - [solvent volume/10 mL]), scaled from 0 to 1.
• Criterion 6 (Throughput): Calculate number of samples processed per hour. Score = (samples/hour) / 20, capped at 1.0.
• Criterion 7 (Integration & Automation): Classify as: Fully automated (1.0); Semi-automated with ≤2 manual interventions (0.7); Mostly manual (0.3).
• Criterion 8 (Energy consumption): Sum energy of all devices (Power in kW × Time in h). Score = 1 - ([kWh/sample] / 2), with a minimum score of 0.
• Criterion 9 (Post-preparation configuration): Match analytical technique to greenness categories: Direct spectroscopy → 1.0; Capillary electrophoresis → 0.8; GC/MS or LC/MS → 0.5.
• Criterion 10 (Operator safety): Assess based on hazard exposure: Closed system with no exposure → 1.0; Open system with non-volatile, low-hazard reagents → 0.7; Open system with volatile or toxic reagents → 0.3.

Calculation and Visualization:

• Input the standardized scores and their respective weights into the AGREEprep software [6] [1].
• The software will generate the final pictogram. Retain a completed data sheet for peer review and audit purposes.

Protocol for Weight Assignment and Sensitivity Analysis

Purpose: To provide a justified and transparent method for assigning weights to AGREEprep criteria, enhancing the objectivity and robustness of the overall assessment.

Procedure:

• Default Weight Application: Begin with the AGREEprep default weights for all ten criteria unless a specific analytical context (e.g., regulatory, economic, or specific environmental priorities) dictates otherwise [1].
• Stakeholder Justification for Weight Modification: If weights are modified, document the rationale using the following structured table.

Table 2: Framework for Justifying Criterion Weight Modifications in AGREEprep

Criterion	Default Weight	Scenario for Increased Weight	Scenario for Decreased Weight
C2: Safer Solvents	1.2	Methods for toxicology studies in drug development	Methods using negligible solvent volumes
C4: Waste Minimization	1.0	Large-scale or high-throughput screening labs	Micro-scale lab-on-a-chip applications
C8: Energy Consumption	0.8	Methods in regions with high carbon-intensity energy	Methods using passive energy (e.g., ambient extraction)
C10: Operator Safety	1.1	Procedures with highly toxic or carcinogenic reagents	Fully automated, closed-system procedures

• Sensitivity Analysis: Conduct a sensitivity analysis by recalculating the final AGREEprep score with moderately altered weights (e.g., ±0.3 for modified criteria). A change in the final score of less than 0.1 indicates a robust assessment. A larger change suggests the conclusion is sensitive to subjective weight choices and requires careful justification.

AGREEprep in the Broader Assessment Context

AGREEprep should not be used in isolation for a comprehensive method evaluation. Within the White Analytical Chemistry (WAC) framework, AGREEprep provides the "green" component, which must be balanced with "red" (analytical performance) and "blue" (practical/economic) criteria [15] [2]. The "red" dimension can be assessed using tools like the newly developed Red Analytical Performance Index (RAPI), which evaluates validation parameters such as repeatability, intermediate precision, and sensitivity [15]. The "blue" dimension can be evaluated with the Blue Applicability Grade Index (BAGI), which scores practicality aspects like cost, time, and operational simplicity [15] [1]. A holistic assessment using AGREEprep, RAPI, and BAGI together provides a balanced "white" score, ensuring a method is not only green but also functionally viable and practically applicable, which is crucial in drug development [15] [2].

The following workflow diagram illustrates the integrated assessment process and the role of AGREEprep within it.

Integrated Workflow for Holistic Method Assessment

Essential Research Reagent Solutions for Green Sample Preparation

The following table details key materials and reagents that facilitate greener sample preparation, aligned with the principles assessed by AGREEprep.

Table 3: Research Reagent Solutions for Green Sample Preparation

Reagent/Material	Function in Sample Prep	Green Advantage & AGREEprep Criterion
Cyclopentyl methyl ether (CPME)	Solvent for liquid-liquid extraction	Biobased, low toxicity, and safer alternative to ethers (C2) [2]
Polylactic acid (PLA) sorbents	Solid-phase extraction material	Renewable and biodegradable polymer source (C3) [2]
Ionic liquids	Solvents for microextraction	Low volatility, reducing inhalational exposure (C2, C10) [2]
Magnetic carbon nanotubes	Dispersive solid-phase extraction sorbent	Enable rapid separation, reducing energy and time (C7, C8) [2]
Deep eutectic solvents (DES)	Solvents for liquid-phase microextraction	Low toxicity, biodegradable, from renewable sources (C2, C3) [1]
Reusable stir bars	Agitation and sorptive extraction (SBSE)	Reduce material waste through multiple uses (C3, C4) [1]

The AGREEprep metric is a powerful tool for quantifying the environmental impact of sample preparation. By adhering to the detailed protocols outlined in this document—standardizing data collection, justifying weight assignments, and integrating AGREEprep within a broader WAC framework—researchers can significantly mitigate common pitfalls and subjectivity. This leads to more reliable, comparable, and meaningful greenness assessments, ultimately supporting the development of sustainable and efficient analytical methods in pharmaceutical research and drug development.

Leveraging Automation and Integration to Boost Greenness Scores

The pursuit of sustainability in analytical chemistry has made the assessment of environmental impact a critical activity. AGREEprep is the first dedicated metric tool for evaluating the greenness of the sample preparation stage of an analytical method [6]. This sample preparation step is a key component for achieving overall analytical greenness [6]. The AGREEprep approach consists of ten steps of assessment that correspond to the ten principles of green sample preparation and uses user-friendly open-source software to calculate and visualize the results [6].

Adapting traditional sample preparation techniques to align with the principles of Green Sample Preparation (GSP) is fundamental to reducing the environmental impact of analytical methods [26]. This application note demonstrates how strategic automation and process integration can significantly enhance AGREEprep scores by systematically addressing its core assessment criteria.

Strategic Approaches for Enhanced Greenness

Automation in Sample Preparation

Automation is a cornerstone strategy for improving greenness [26]. Automated systems save time, lower the consumption of reagents and solvents, and consequently reduce waste generation [26]. Furthermore, automation minimizes human intervention, which significantly lowers the risks of handling errors, operator exposure to hazardous chemicals, and accidents in the laboratory [26].

• Throughput Acceleration: Automated systems can operate continuously, drastically increasing the number of samples processed per unit time. This directly reduces the energy consumed per sample [26].
• Precision and Miniaturization: Automated liquid handling enables highly reproducible operations with minimal volumes of solvents and reagents, supporting the shift toward miniaturized methods [26].
• Error and Waste Reduction: By standardizing procedures, automation minimizes the risk of failed analyses and the need for repeat procedures, thereby conserving materials and reducing hazardous waste [26].

Integration of Analytical Steps

Process integration involves streamlining multi-step, time-consuming sample preparation workflows into a single, continuous operation [26]. This strategy directly targets several inefficiencies common in traditional methods.

• Resource Efficiency: Integrating multiple preparation steps cuts down on the overall consumption of energy and chemicals [26].
• Improved Analytical Performance: Simplified, integrated workflows can enhance the precision and accuracy of analyses by reducing material loss and potential contamination at each stage transfer [26].
• Waste Minimization: A consolidated workflow inherently generates less waste compared to a series of discrete, independent steps [26].

Quantitative Impact of Automation and Integration on AGREEprep Criteria

The following table summarizes how the implementation of automation and integration directly influences key criteria within the AGREEprep assessment framework, leading to a higher overall greenness score.

Table 1: Impact of Automation and Integration on AGREEprep Assessment Criteria

AGREEprep Assessment Principle	Traditional Approach	Automated & Integrated Approach	Impact on Greenness Score
Energy Consumption	High (e.g., Soxhlet extraction, manual heating) [26]	Low (ultrasound/microwave assistance, automated systems) [26]	Significant Increase
Sample Throughput	Low (sequential processing)	High (parallel processing & automation) [26]	Significant Increase
Volume of Waste	High (multi-step, manual)	Low (miniaturization, reduced repeats) [26]	Significant Increase
Amount of Reagents/Solvents	High (macro-scale volumes)	Low (miniaturized systems) [26]	Significant Increase
Hazardous Chemicals	Potentially high (exposure risk)	Minimized (reduced volumes & exposure) [26]	Increase
Number of Steps	High (discrete operations)	Low (integrated workflow) [26]	Increase
Operator Safety	Moderate (handling exposure)	High (reduced intervention) [26]	Increase

Experimental Protocols

Protocol 1: Automated Solid-Phase Extraction (SPE) for Water Analysis

This protocol outlines an automated method for the extraction of pharmaceuticals from wastewater samples, designed for high greenness performance.

• 1. Principle: Automated SPE replaces manual cartridge conditioning, loading, washing, and elution, ensuring precision while reducing solvent use and analyst time.
• 2. Materials & Equipment:
  • Automated SPE workstation
  • C18 SPE cartridges (50 mg, 3 mL)
  • HPLC-grade methanol and acetonitrile
  • Reagent water (pH adjusted)
  • Water sample (100 mL, filtered)
• 3. Procedure:
  • Setup: Load samples, solvents, and SPE cartridges onto the automated workstation.
  • Conditioning: The system sequentially conditions each cartridge with 2 mL methanol followed by 2 mL reagent water at a flow rate of 5 mL/min.
  • Loading: The 100 mL water sample is passed through the cartridge at 10 mL/min.
  • Washing: Cartridges are washed with 2 mL of a 5:95 (v/v) methanol:water solution.
  • Elution: Analytics are eluted with 2 mL of methanol into a collection vial.
  • Reconstitution: The eluate is gently evaporated under nitrogen and reconstituted in 200 µL of mobile phase for analysis.
• 4. AGREEprep Advantages:
  • Reduced Solvent Consumption: Precise liquid handling minimizes excess solvent use.
  • High Throughput: Parallel processing of multiple samples increases throughput, reducing energy per sample [26].
  • Improved Safety: Analyst exposure to organic solvents is drastically reduced [26].
  • Minimized Waste: The protocol generates less solvent waste compared to manual SPE.

Protocol 2: Integrated On-Line Sample Preparation for HPLC

This protocol describes the direct coupling of an in-tube extraction or similar pre-concentration device to an HPLC system, eliminating manual off-line steps.

• 1. Principle: An extraction micro-column is placed in the HPLC injection loop position. The sample is loaded, and analytes are concentrated on-column before being switched into the mobile phase flow for separation.
• 2. Materials & Equipment:
  • HPLC system with a programmable autosampler and a 2-position/6-port switching valve
  • Extraction column (e.g., restricted access material (RAM) or monolithic)
  • Analytical HPLC column
  • Appropriate mobile phases
• 3. Procedure:
  • Configuration: Install the switching valve and connect the extraction column in the load position.
  • Loading & Washing: The autosampler draws a large volume (e.g., 1-5 mL) of the filtered sample and passes it through the extraction column. Interfering matrix components are washed to waste.
  • Elution & Transfer: The valve switches, placing the extraction column in line with the analytical column and the HPLC pump. The mobile phase back-flushes the trapped analytes onto the analytical column for separation.
  • Re-equilibration: After transfer, the valve switches back, and the extraction column is re-equilibrated for the next sample.
• 4. AGREEprep Advantages:
  • Step Reduction: Combines extraction, pre-concentration, and injection into a single, automated process [26].
  • Solvent & Sample Savings: Eliminates the need for separate evaporation and reconstitution steps, saving solvents and preventing sample loss.
  • Full Automation: The entire process from sample aspiration to chromatographic separation runs without operator intervention [26].

Workflow and Logical Relationship Diagram

The following diagram illustrates the logical progression from traditional methods to automated and integrated solutions, and the subsequent positive impact on AGREEprep scores.

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials and Reagents for Green Sample Preparation

Item	Function & Green Alternative
Automated SPE Workstation	Enables high-throughput, parallel processing of samples with minimal and precise reagent dispensing, reducing solvent consumption and analyst exposure [26].
On-Line Extraction Columns	(e.g., RAM, Monolithic). Allows for direct coupling of sample preparation with HPLC, eliminating off-line steps, associated solvent use, and waste generation [26].
Micro-Scale Extraction Devices	(e.g., 96-well plates, µ-SPE). Miniaturization of sample preparation directly reduces the volumes of samples and solvents required, aligning with GSP principles [26].
Green Solvents	(e.g., Ethanol, Water [with modifiers]). Replacing hazardous solvents like chlorinated organics with safer, bio-based alternatives reduces environmental and safety hazards.
Switchable Valves & Controllers	Critical hardware for implementing integrated, on-line sample preparation workflows that seamlessly connect extraction and analysis [26].
Energy-Efficient Assistants	(e.g., Ultrasound Bath, Microwave Reactor). Provide efficient energy transfer to accelerate mass transfer and extraction kinetics compared to traditional heating (e.g., Soxhlet) [26].

Validating with AGREEprep: Case Studies and Comparative Analysis in Bioanalysis

Therapeutic Drug Monitoring (TDM) represents a critical component of personalized medicine, enabling the optimization of drug dosage regimens based on measured drug concentrations in biological matrices [2]. Green Analytical Chemistry (GAC) principles have increasingly become a fundamental consideration in developing bioanalytical methods, particularly for TDM applications where routine analysis generates significant chemical waste [2] [27]. The Analytical Greenness Metric for Sample Preparation (AGREEprep) has emerged as a specialized tool to quantitatively evaluate the environmental impact of sample preparation procedures [6].

AGREEprep provides a standardized approach to assess sample preparation methods against ten core principles of green sample preparation, generating a comprehensive pictogram and overall score between 0 and 1, where higher scores indicate superior greenness performance [2] [6]. This case study examines the application of AGREEprep in evaluating microextraction techniques for TDM, demonstrating how this metric tool can guide researchers toward more sustainable analytical practices while maintaining the rigorous analytical performance required for clinical decision-making.

AGREEprep Methodology and Assessment Criteria

Fundamental Principles of AGREEprep

AGREEprep operates on ten assessment criteria derived from the established principles of green sample preparation [2] [6]. Each criterion is scored on a scale from 0 to 1, with the option to assign weighting factors based on their relative importance for a specific application. The software then generates an easily interpretable pictogram that visualizes the performance across all criteria and provides an overall greenness score [6].

Table 1: The Ten Core Assessment Criteria of AGREEprep

Criterion Number	Assessment Principle	Key Focus Areas
1	Favoring in situ sample preparation	Remote sensing, non-invasive analysis, in-field sampling
2	Using safer solvents and reagents	Toxicity, biodegradability, renewable sources
3	Targeting sustainable, reusable and renewable materials	Recyclable components, sustainable sourcing
4	Minimizing waste	Waste volume, hazardous waste production
5	Minimizing sample, chemical and material amounts	Miniaturization, microextraction techniques
6	Maximizing sample throughput	Parallel processing, automation
7	Integrating steps and promoting automation	Process integration, automated systems
8	Minimizing energy consumption	Energy-efficient equipment, ambient temperature processing
9	Choosing the greenest possible post-sample preparation configuration	Direct coupling with analytical instruments
10	Ensuring safe procedures for the operator	Exposure to hazardous materials, procedural safety

AGREEprep Assessment Protocol

The standard procedure for implementing AGREEprep in method evaluation involves the following steps [6]:

• Data Collection: Compile all relevant methodological details including sample volumes, reagent types and quantities, equipment requirements, processing time, waste generation, and safety considerations.

• Software Input: Enter the collected data into the freely available AGREEprep software (accessible at https://mostwiedzy.pl/AGREE).

• Criterion Scoring: For each of the ten principles, assign a score based on the method's performance. The software provides guidance for appropriate scoring based on the input parameters.

• Weight Assignment: Adjust weighting factors if certain principles are of particular importance for the specific application. If no specific priorities exist, use the default weights.

• Pictogram Generation: The software automatically generates a circular pictogram with colored segments representing each criterion and an overall score in the center.

• Interpretation: Analyze the results to identify strengths and weaknesses in the method's greenness profile and identify opportunities for improvement.

Application of AGREEprep in TDM Method Development

Comparative Greenness Assessment of Microextraction Techniques

A comprehensive assessment of microextraction techniques applied to TDM revealed significant variations in greenness performance when evaluated using AGREEprep [2]. The study examined techniques including solid-phase microextraction (SPME), liquid-phase microextraction (LPME), microextraction by packed sorbent (MEPS), and dispersive liquid-liquid microextraction (DLLME) across various TDM applications.

Table 2: AGREEprep Scores of Microextraction Techniques in TDM Applications

Microextraction Technique	Representative AGREEprep Score	Key Strengths	Common Limitations
Solid-Phase Microextraction (SPME)	0.65-0.75	Minimal solvent use, reusable fibers	Moderate energy requirements for desorption
Liquid-Phase Microextraction (LPME)	0.55-0.70	Very low solvent consumption, simple apparatus	Limited throughput for some formats
Microextraction by Packed Sorbent (MEPS)	0.60-0.72	Small sample volumes, automation compatible	Packed sorbent waste generation
Dispersive Liquid-Liquid Microextraction (DLLME)	0.50-0.65	High enrichment factors, rapid operation	Dispersive solvent usage
Thin-Film Microextraction (TFME)	0.60-0.72	High capacity, reusable materials, solvent-free option	Larger equipment requirements for some formats

The assessment demonstrated that techniques incorporating solvent-free operation, minimal sample volumes, and reusable materials consistently achieved higher AGREEprep scores [2]. Methods achieving scores above 0.70 typically represented the most significant advances in green sample preparation for TDM applications.

Case Study: Diazepam TDM Method Development

A recent development of a sample preparation method for monitoring diazepam and its major metabolite in patients undergoing alcohol withdrawal treatment provides an illustrative example of AGREEprep application in TDM [28]. The method employed a miniaturized protein precipitation approach followed by HPLC-UV analysis, specifically designed with green chemistry principles in mind.

Experimental Protocol:

• Sample Preparation: 100 μL of human plasma was mixed with 150 μL of acetonitrile as precipitation solvent.
• Protein Precipitation: Vortex-mixed for 30 seconds followed by centrifugation at 10,000 rpm for 5 minutes.
• Analysis: The supernatant was directly injected into the HPLC-UV system.
• Chromatographic Conditions: Core-shell C18 column (50 mm × 2.1 mm, 1.7 μm), mobile phase consisting of ammonium formate (10 mM, pH 3.5) and acetonitrile in gradient elution mode.
• Validation: The method was validated according to FDA guidelines, demonstrating satisfactory precision (RSD < 8.2%) and accuracy (98.5-106.6% recovery).

The AGREEprep assessment of this method highlighted several green advantages including miniaturized sample volumes, reduced reagent consumption, and minimized waste generation compared to conventional liquid-liquid extraction or solid-phase extraction approaches [28]. The overall AGREEprep score of 0.68 reflected a favorable greenness profile while maintaining the analytical performance necessary for clinical application.

Integrating AGREEprep with White Analytical Chemistry Assessment

The White Analytical Chemistry Framework

While AGREEprep provides specialized assessment of environmental impact, the White Analytical Chemistry (WAC) concept offers a holistic framework that balances greenness with analytical performance and practical/economic considerations [2] [15]. In the WAC model, these three dimensions are represented as:

• Red Principles: Analytical performance criteria (scope, LOD/LOQ, precision, accuracy)
• Green Principles: Environmental impact criteria (toxicity, waste, energy, safety)
• Blue Principles: Practical and economic criteria (cost, time, simplicity, operational requirements)

A perfectly "white" method achieves optimal balance across all three dimensions, analogous to how white light combines red, green, and blue light [2] [15].

Complementary Assessment Tools

For comprehensive method evaluation, AGREEprep can be integrated with complementary assessment tools:

• Red Analytical Performance Index (RAPI): Specifically evaluates analytical performance criteria [15]
• Blue Applicability Grade Index (BAGI): Assesses practical and economic aspects [1]
• RGB 12 Algorithm: Provides integrated assessment across all three WAC dimensions [2]

Table 3: Essential Research Reagent Solutions for Green TDM Method Development

Reagent/Material Category	Specific Examples	Green Advantages	Application in TDM
Green Solvents	Ethanol, ethyl acetate, acetone, cyclopentyl methyl ether	Lower toxicity, biodegradable, renewable sources	Protein precipitation, liquid-phase microextraction
Sustainable Sorbents	Biopolymer-based materials, reusable SPME fibers, renewable source cartridges	Reduced waste, sustainable sourcing, reusability	Solid-phase extraction, microextraction techniques
Miniaturized Consumables	Low-volume plates, micro-inserts, capillary-scale columns	Reduced plastic waste, minimal solvent consumption	High-throughput analysis, method downscaling
Alternative Sample Materials	Dried blood spots, saliva collection devices	Minimal invasive collection, reduced shipping impact	Alternative sampling matrices

A study applying this integrated approach to UV filter analysis in water samples demonstrated that methods with moderate AGREEprep scores (0.60-0.70) could achieve excellent overall WAC ratings when they exhibited strong analytical performance and practical efficiency [1]. This highlights the importance of balanced assessment rather than focusing exclusively on greenness metrics.

Strategic Implementation Guide

Optimization Strategies for Improved AGREEprep Scores

Based on comprehensive assessments of TDM methods, the following strategies effectively enhance AGREEprep performance:

• Miniaturization Approach: Reduce sample volumes to micro-scale (100-200 μL) and implement microextraction techniques to significantly improve scores in principles 4 and 5 [2] [28].

• Solvent Selection and Recovery: Replace hazardous solvents with safer alternatives (e.g., acetonitrile with ethanol where feasible) and implement solvent recovery systems to address principles 2 and 4 [2].

• Process Integration and Automation: Combine sample preparation steps and implement automated systems to enhance performance in principles 6 and 7 while reducing operator exposure (principle 10) [2] [6].

• Energy-Efficient Operation: Utilize ambient temperature processes where possible and optimize heating/cooling requirements to improve principle 8 scores [6].

Implementation Protocol for TDM Laboratories

For TDM laboratories implementing AGREEprep assessment, the following protocol ensures consistent application:

• Baseline Assessment: Evaluate current TDM methods using AGREEprep to establish baseline greenness scores.

• Identification of Critical Areas: Use the AGREEprep pictogram to identify the weakest performing principles for each method.

• Improvement Prioritization: Focus improvement efforts on principles with the lowest scores and highest weighting factors.

• Iterative Optimization: Implement modifications and reassess using AGREEprep to quantify improvements.

• Balanced Validation: Ensure that greenness improvements do not compromise analytical performance through parallel validation using RAPI or traditional validation protocols [15].

• Documentation and Reporting: Include AGREEprep assessments in method development reports and publications to standardize greenness reporting across the field.

AGREEprep provides TDM researchers with a standardized, quantitative framework to assess and improve the environmental profile of sample preparation methods. The case studies examined demonstrate that microextraction techniques typically achieve AGREEprep scores between 0.50-0.75, with the highest scores associated with methods that incorporate solvent-minimized or solvent-free approaches, miniaturization, and process integration [2] [28] [29].

The most effective implementation of AGREEprep occurs when it is integrated within the broader White Analytical Chemistry framework, balancing environmental considerations with the rigorous analytical performance requirements essential for TDM applications [2] [15]. This integrated approach guides the development of sustainable TDM methods that maintain analytical excellence while reducing environmental impact and operational hazards.

As green chemistry principles become increasingly important in analytical science, AGREEprep offers TDM laboratories a practical tool to quantify and improve their environmental performance, contributing to more sustainable healthcare practices without compromising the quality of patient care.

The efficiency and environmental impact of sample preparation are critical considerations in modern analytical chemistry. This application note provides a comparative analysis of two prominent techniques: the traditional Soxhlet extraction and the modern Accelerated Solvent Extraction (ASE). Framed within a broader thesis on AGREEprep (Analytical GREEnness Metric for Sample Preparation) assessment, this document details the methodologies, performance metrics, and environmental footprints of both techniques, using the extraction of dioxins and furans from fly ash and bottom ash as a case study [30]. The objective is to equip researchers and drug development professionals with the data necessary to select an extraction method that aligns with both analytical rigor and sustainability goals.

Experimental Protocols

Sample Preparation and Materials

• Sample Collection: Fly ash and bottom ash samples were collected from an aluminum scrap smelter [30].
• Analytes: The target analytes were polychlorinated dibenzo-para-dioxins and furans (PCDD/Fs), comprising 17 specific congeners [30].
• Standards: Recovery efficiency was assessed using spiked 13C isotope standards and native standards, following the US EPA 1613b method [30].
• Analysis: Extracts from both methods were analyzed using High-Resolution Gas Chromatography/High-Resolution Mass Spectrometry (HRGC/HRMS) [30].

Soxhlet Extraction Methodology

The Soxhlet extraction method, developed centuries ago, operates on a principle of continuous extraction and solvent recycling [30] [31].

Detailed Steps:

• Setup: The solid sample is placed in a cellulose or glass fiber thimble. A suitable solvent (e.g., hexane, toluene) is added to a round-bottom flask. The Soxhlet apparatus is assembled with the extractor body placed between the flask and a water condenser [31].
• Heating: The flask is heated, causing the solvent to evaporate [31].
• Condensation: The solvent vapor travels upward and is condensed back into liquid form by the cooler condenser [31].
• Extraction: The condensed solvent drips onto the sample in the thimble, dissolving the target compounds. The solvent chamber gradually fills [31].
• Siphoning: When the liquid level in the extractor body reaches the top of the siphon tube, the solvent, now enriched with analytes, is automatically siphoned back into the round-bottom flask [31].
• Recycling: The cycle repeats automatically for many hours, ensuring the sample is repeatedly contacted with fresh solvent [31]. A typical extraction can last from 3 to 24 hours [31].

Accelerated Solvent Extraction (ASE) Methodology

Accelerated Solvent Extraction was designed to overcome the limitations of Soxhlet extraction by using elevated temperature and pressure [30].

Detailed Steps:

• Cell Packing: The solid sample is mixed with a dispersant (e.g., diatomaceous earth) and packed into a stainless steel extraction cell [30].
• Pressurization and Heating: The cell is filled with solvent and heated (typically 40-200°C) and pressurized (typically 500-3000 psi) above its atmospheric boiling point. This enhances the solubility and diffusion rate of the analytes while keeping the solvent in a liquid state [30].
• Static Extraction: The system is held at these conditions for a short static period (typically 5-10 minutes), during which the analytes dissolve into the solvent [30].
• Purge and Collection: After the static cycle, the extract is purged from the cell using an inert gas (e.g., nitrogen) into a collection vial [30].
• Rinsing (Optional): One or more rinse cycles may be performed to ensure quantitative recovery. The entire process is automated and is typically completed in minutes to a short period per sample [30].

Results and Comparative Data

Analytical Performance Comparison

The performance of both methods was quantitatively evaluated for the extraction of dioxins and furans from fly ash and bottom ash. The recovery of spiked standards and the deviation between methods for native congeners were used as key metrics [30].

Table 1: Quantitative Performance Metrics for Dioxin/Furan Extraction from Ash Samples

Performance Metric	Soxhlet Extraction	Accelerated Solvent Extraction (ASE)	Acceptance Criteria
Recovery of Spiked 13C Standards	High and met US EPA 1613b requirements [30]	High and met US EPA 1613b requirements [30]	US EPA 1613b Method
Deviation of Congener Results (Fly Ash)	Reference Method	-15.5% to +25.6% [30]	AOAC Guidelines
Deviation of Congener Results (Bottom Ash)	Reference Method	-15.0% to +32.9% [30]	AOAC Guidelines
Extraction Time	3 to 24 hours [31]	Minutes to a short period [30]	N/A

Operational and Environmental Impact Assessment

A comparative assessment of operational parameters and environmental impact, evaluated using AGREE Prep software, highlights the significant advantages of ASE in sustainability and efficiency [30].

Table 2: Operational and Environmental Comparison of Extraction Techniques

Parameter	Soxhlet Extraction	Accelerated Solvent Extraction (ASE)
Solvent Consumption	Excessive [30]	Reduced [30]
Energy Consumption	Higher (Prolonged heating) [30]	Lower [30]
Automation Level	Limited automation [30]	High automation [30]
Operator Safety	Lower (Open system, prolonged exposure) [31]	Enhanced (Closed system) [30]
Throughput	Low (Single sample for long duration) [31]	High (Rapid, automated multiple samples) [30]
AGREEprep Green Score	Lower [30]	Higher (More environmentally friendly) [30]

The Scientist's Toolkit: Research Reagent Solutions

The following table details essential materials and reagents used in the featured comparative study for the extraction of organic pollutants from solid matrices.

Table 3: Key Research Reagents and Materials

Reagent/Material	Function and Application in Extraction
Cellulose Thimbles	Porous container for holding solid samples during Soxhlet extraction, allowing solvent permeation [31].
Dispersant (e.g., Diatomaceous Earth)	Mixed with the sample in ASE to improve solvent contact and prevent channeling [30].
13C-labeled Isotope Standards	Internal standards spiked into samples prior to extraction to correct for analyte loss and quantify recovery efficiency, critical for EPA methods [30].
Native PCDD/F Standards	Pure analytical standards used for calibration and identification of the 17 toxic dioxin and furan congeners [30].
Organic Solvents (e.g., Toluene, n-Hexane)	Extraction medium. Solvent choice (polarity) is tailored to the target analyte's solubility [30] [31].
Inert Gas (e.g., Nitrogen)	Used in ASE to purge the extraction cell and transfer the final extract to the collection vial [30].

This comparative analysis demonstrates that while Soxhlet extraction remains a robust and reproducible standard method [31], Accelerated Solvent Extraction offers a superior alternative for modern laboratories. ASE achieves comparable analytical results, as evidenced by the recovery rates and congener deviations falling within accepted guidelines [30]. Crucially, ASE provides significant advantages in speed, solvent consumption, automation, and operator safety. The formal assessment using AGREE Prep software confirms that ASE is the more environmentally friendly and sustainable sample preparation technique [30]. For laboratories engaged in high-throughput drug development or environmental monitoring seeking to align with Green Analytical Chemistry principles, ASE represents a compelling choice that does not compromise on analytical performance.

Within the paradigm of Green Analytical Chemistry (GAC), the evaluation of method environmental impact is essential. While multiple assessment tools exist, understanding their specific applications, strengths, and limitations is critical for researchers. This application note provides a detailed comparison of three central metrics—AGREEprep, GAPI, and AGREE—framed within research on sample preparation assessment. We elucidate their core architectures, provide protocols for their application, and demonstrate their use through experimental data, offering a clear roadmap for their implementation in method development and validation.

The selection of a greenness metric should be guided by the analytical question at hand. The following table summarizes the primary characteristics of AGREEprep, GAPI, and AGREE.

Table 1: Core Characteristics of AGREEprep, GAPI, and AGREE

Feature	AGREEprep	GAPI	AGREE
Main Focus	Sample preparation step only [32]	Entire analytical workflow [33]	Entire analytical procedure [33]
Theoretical Basis	10 Principles of Green Sample Preparation (GSP) [32]	--	12 Principles of Green Analytical Chemistry (GAC) [33]
Output Format	Pictogram + Overall score (0-1) [32]	Color-coded pictogram (no overall score) [33]	Radial chart + Overall score (0-1) [33]
Key Differentiator	High specificity for sample preparation; identifies weak/strong points in the prep workflow [32]	Visualizes impact across all stages (sampling, preparation, analysis) [33]	Holistic, single-score output based on all 12 GAC principles [33]

The relationships and primary focus of these metrics within an analytical workflow can be visualized as follows:

Experimental Protocols for Metric Application

Protocol for AGREEprep Assessment

AGREEprep is the first metric specifically designed to evaluate the environmental impact of the sample preparation stage, based on the 10 principles of Green Sample Preparation (GSP) [32].

Required Tools: AGREEprep open-source software (available at mostwiedzy.pl/AGREEprep) [32].

Procedure:

• Gather Input Data: Compile all relevant parameters from the sample preparation method.
  • Solvents/Reagents: Type, volume, and health/hazard information.
  • Energy Consumption: For heating, cooling, or agitation (e.g., in kWh per sample).
  • Waste Generation: Total mass in grams (or mL) per sample.
  • Sample Size: Amount of sample consumed.
  • Throughput: Number of samples prepared per hour (considers automation and parallel processing).
  • Material Reusability: Whether consumables are single-use or reusable.
  • Operator Safety: Data on exposure to hazardous materials or conditions.
• Software Input: Launch the AGREEprep tool and enter the collected data into the ten corresponding assessment criteria. The default weighting for each criterion can be adjusted based on analytical goals, though justification is recommended [32].
• Result Interpretation: The software generates a circular pictogram.
  • The central score (0-1) indicates the overall greenness.
  • The ten surrounding segments correspond to each GSP principle. The color (red to green) and length (based on weight) of each segment instantly reveal the method's weaknesses and strengths [32].

Protocol for GAPI Assessment

The Green Analytical Procedure Index (GAPI) provides a semi-quantitative visual assessment of the entire analytical method [33].

Required Tools: GAPI template or dedicated software.

Procedure:

• Deconstruct the Workflow: Break down the analytical method into its five key stages: Sample Collection, Preservation & Transport, Sample Preparation, Instrumental Analysis, and Final Determination.
• Evaluate Each Field: For each field in the GAPI pictogram, assign a color based on the method's adherence to green principles:
  • Green: Meets ideal green criteria.
  • Yellow: Represents a medium environmental impact.
  • Red: Indicates a significant environmental burden.
• Construct the Pictogram: Fill in the corresponding sections of the GAPI pentagram. The resulting diagram offers an at-a-glance overview of the method's environmental hotspots but does not provide a single numerical score [33].

Protocol for AGREE Assessment

The Analytical GREEnness (AGREE) metric offers a comprehensive, single-score evaluation based on all 12 principles of GAC [33].

Required Tools: AGREE open-source calculator (available at mostwiedzy.pl/AGREE).

Procedure:

• Data Collection: Gather comprehensive data covering the entire analytical procedure, from sample collection to data treatment, aligning with the 12 GAC principles. This includes energy consumption, waste, toxicity of reagents, and operator safety.
• Software Input: Enter the data into the AGREE calculator. The tool allows for weighting each of the 12 principles according to the user's priorities.
• Result Interpretation: The output is a circular pictogram with 12 segments.
  • Each segment's color reflects the performance for one GAC principle.
  • The central score (0-1) provides a normalized, overall greenness assessment, facilitating direct comparison between different methods [33].

Experimental Data and Case Study Comparisons

Greenness Assessment of Official Standard Methods

Evaluations conducted under an IUPAC project using AGREEprep have quantified the low greenness of traditional sample preparation in official standards, highlighting the need for modernization [32].

Table 2: AGREEprep Scores of Official Standard Methods [32]

Method Source	Application Area	Core Sample Preparation Technique	AGREEprep Score Range
US EPA	Environmental Analysis (POPs in solids)	Soxhlet Extraction	0.04 – 0.12
AOAC INTERNATIONAL	Food Analysis	Soxhlet Extraction, Maceration, Digestion	0.05 – 0.22
US EPA	Inorganic Analysis (Trace Metals)	Acid Digestion	0.01 – 0.36

Case Study: HPLC Method for Pharmaceuticals

A study developing an RP-HPLC method for Gabapentin and Methylcobalamin employed multiple metrics for validation, providing a clear comparison of outputs [34].

Table 3: Multi-Metric Greenness Assessment of an Inventive RP-HPLC Method [34]

Analytical Method	Key Green Feature	AGREEprep Score	AGREE Score	Other Metric Scores
Inventive RP-HPLC	Mobile phase with only 5% acetonitrile; reduced solvent consumption by >80% [34]	0.71	0.70	Analytical Eco-Scale: 80

The data shows a strong correlation between AGREE and AGREEprep for this method, confirming its superior greenness in both the sample preparation and overall workflow.

Table 4: Key Resources for Implementing Greenness Metrics

Tool/Resource	Function/Purpose	Access/Example
AGREEprep Software	Open-source tool for calculating sample preparation greenness based on 10 GSP principles [32].	mostwiedzy.pl/AGREEprep
AGREE Calculator	Open-source tool for holistic greenness scoring based on 12 GAC principles [33].	mostwiedzy.pl/AGREE
GAPI Template	Framework for creating a visual profile of environmental impact across all analytical steps [33].	Scientific literature / software
Solvent Selection Guide	Aids in choosing safer, less toxic solvents to improve scores in reagent-related criteria.	ACS Green Chemistry Institute
BAGI (Blue Applicability Grade Index)	"Sister tool" to assess practicality/economic aspects (the "blue" dimension in White Analytical Chemistry) [35] [15].	mostwiedzy.pl/bagi
RAPI (Red Analytical Performance Index)	Complementary tool to assess analytical performance (the "red" dimension in White Analytical Chemistry) [15].	mostwiedzy.pl/rapi

AGREEprep, GAPI, and AGREE are complementary tools in the green chemist's arsenal. AGREEprep offers unparalleled specificity for diagnosing and improving the sample preparation step. GAPI serves as an excellent tool for initial, visual screening of an entire method's environmental profile. AGREE provides the most comprehensive, single-score evaluation for final comparison and decision-making. Employing these metrics in concert, as part of the broader White Analytical Chemistry framework, ensures the development of analytical methods that are not only environmentally sound but also analytically robust and practically viable.

Analytical chemistry is fundamental for monitoring environmental health and ensuring product quality, yet it can paradoxically contribute to environmental degradation through the generation of hazardous waste and high energy consumption. The contradictory aspects of analytical processes highlighted by Paul Anastas led to the emergence of Green Analytical Chemistry as a distinct field dedicated to minimizing these impacts [36].

Within the analytical process, sample preparation represents a critical bottleneck and a significant source of environmental concern. Traditional approaches often consume substantial energy and require large volumes of toxic solvents and reagents [36]. Recognizing this challenge, the International Union of Pure and Applied Chemistry established Project #2021-015-2-500, titled "Greenness of official standard sample preparation methods" [37] [38] [36]. This ongoing initiative systematically evaluates standardized methods from major organizations to assess their environmental performance and advocate for greener alternatives.

The AGREEprep Metric: A Tool for Standardized Assessment

Conceptual Foundation and Design

AGREEprep is the first dedicated metric developed specifically for evaluating the environmental impact of sample preparation methods. Its design is directly informed by the 10 principles of Green Sample Preparation, creating a structured framework for comprehensive assessment [36] [20]. The tool transforms complex methodological data into an intuitive pictogram that displays both an overall score and performance across ten individual criteria [6] [36].

The assessment covers critical aspects of environmental impact, including solvent toxicity, waste generation, energy consumption, operator safety, and procedural efficiency. Each criterion receives a score from 0 to 1, with the option to apply weighting factors that reflect analytical priorities. This flexibility allows researchers to customize assessments while maintaining standardized comparison capabilities [36].

Software Implementation and Accessibility

AGREEprep is supported by open-source software available through multiple platforms, ensuring broad accessibility without financial barriers. The software can be obtained from mostwiedzy.pl/AGREEprep, with the source code publicly accessible at git.pg.edu.pl/p174235/agreeprep [36]. This implementation provides a user-friendly interface that guides analysts through the evaluation process and automatically generates the characteristic circular pictogram visualizing assessment results [6].

Quantitative Findings from IUPAC Assessments

The IUPAC project has conducted systematic evaluation of 174 standard methods with sample preparation steps and their 332 sub-method variations from CEN, ISO, and Pharmacopoeias. The results reveal significant environmental concerns across multiple sectors [38].

Table 1: Overall AGREEprep Scores for Official Standard Methods by Sector

Sector	Number of Methods Evaluated	Score Range	Percentage Scoring Below 0.2
Environmental Analysis (Organic Compounds)	Not specified	0.04 - 0.12	86%
Food Analysis	15	0.05 - 0.22	62%
Inorganic and Trace Metals Analysis	25	0.01 - 0.36	62%
Pharmaceutical Analysis	Not specified	Not specified	45%
All Sectors Combined	174	0.01 - 0.36	67%

The data demonstrates that two-thirds of officially standardized methods score below 0.2 on the AGREEprep scale, where 1 represents optimal green performance. This indicates a systematic inadequacy in current official methods regarding environmental sustainability [38].

Specific Methodological Deficiencies

Detailed analysis reveals consistent patterns of environmental underperformance across different methodological approaches:

• Soxhlet Extraction Methods: Twenty-five US EPA methods employing Soxhlet extraction for solid samples like sediments and fish tissues demonstrated exceptionally poor performance (0.04-0.12). These methods are characterized by extended extraction times (up to 24 hours), substantial solvent consumption, and frequent requirement for additional cleanup steps that further increase resource use [36].

• Acid Digestion Procedures: Methods for trace metal analysis, particularly those using traditional hotplate acid digestion, scored between 0.01-0.36. Primary concerns include large volumes of mineral acids generating waste exceeding 50g/mL per sample and coupled energy demands from subsequent analytical techniques [36].

• Food Analysis Methods: Fifteen AOAC INTERNATIONAL methods for food safety and integrity assessment scored 0.05-0.22. These methods involved highly manual operations with significant operator involvement, multiple heating steps, and historically hazardous materials including asbestos, benzene, and mercury [36].

Experimental Protocol for AGREEprep Assessment

Data Collection Requirements

Implementing AGREEprep requires systematic compilation of specific methodological parameters. The following protocol ensures consistent data collection for reliable assessment:

• Reagent and Solvent Documentation

  • Record all solvents, reagents, and materials used in sample preparation
  • Note chemical identities, concentrations, and quantities per sample
  • Document solvent source (virgin, recycled, or renewable)

• Waste Generation Assessment

  • Calculate total waste mass (solid and liquid) per sample
  • Classify waste by type (hazardous, non-hazardous, recyclable)
  • Include all consumables (filters, pipette tips, vial inserts)

• Energy Consumption Evaluation

  • Record all energy-intensive steps (heating, cooling, agitation)
  • Document duration and temperature for each thermal step
  • Note equipment power requirements and operational duration

• Throughput and Efficiency Metrics

  • Calculate sample preparation time per individual sample
  • Document potential for parallel processing (number of simultaneous samples)
  • Record degree of automation (manual, semi-automated, fully automated)

Software Application Workflow

The assessment process follows a structured workflow within the AGREEprep software environment:

The diagram illustrates the sequential workflow for conducting an AGREEprep assessment, from initial data input through final analysis of the generated pictogram.

Table 2: Key Research Reagent Solutions and Materials for Green Sample Preparation

Resource Category	Specific Examples	Function and Application	Greenness Benefits
Alternative Solvents	Ionic liquids, Deep eutectic solvents (DES)	Replacement for petroleum-based organic solvents	Reduced toxicity, biodegradability, renewable sourcing [39]
Advanced Sorbents	Engineered materials, Molecularly imprinted polymers, Nanomaterials	Selective extraction and cleanup of analytes	Enhanced efficiency, reusability, reduced solvent consumption [39]
Miniaturized Equipment	Microextraction devices, Lab-on-a-chip systems	Downscaling of extraction processes	Reduced reagent consumption, smaller waste volumes [36]
Assessment Software	AGREEprep, GEMAM, AMGS	Quantitative evaluation of method greenness	Standardized sustainability assessment, identification of improvement areas [6] [40] [41]
Renewable Materials	Bio-based sorbents, Recycled components	Sustainable sourcing of analytical consumables	Reduced environmental footprint, waste valorization [39]

Analysis of Shortcomings and Improvement Strategies

Critical Deficiencies in Current Standard Methods

The systematic evaluation reveals several consistent shortcomings across multiple methodological domains:

• Excessive Waste Generation: Most methods exceed the critical threshold of 50g/mL waste per sample, with some generating substantially higher volumes. This reflects outdated procedural designs that prioritize simplicity of implementation over environmental impact [38] [36].

• Hazardous Reagent Utilization: Many official methods continue to specify solvents and reagents with significant toxicity concerns, including benzene, chlorinated solvents, and strong mineral acids. These pose risks to operator safety and require specialized waste handling [36].

• Energy-Intensive Operations: Procedures relying on extended heating, traditional distillation, or overnight extraction demonstrate poor energy efficiency. Coupling these with additional analytical techniques further compounds their environmental footprint [38] [36].

• Limited Automation and Throughput: Manual sample preparation remains prevalent, leading to inconsistency between operators and limiting sample throughput. This represents a missed opportunity for both greenness and productivity improvements [36].

Strategic Pathways for Greener Standard Methods

The AGREEprep assessment not only identifies deficiencies but also provides a structured framework for methodological improvement:

The diagram outlines the primary strategic pathways for transitioning from current low-performing methods to greener alternatives with improved AGREEprep scores.

The systematic application of AGREEprep within the IUPAC project has provided unambiguous evidence that current official standard methods for sample preparation demonstrate poor environmental performance. With 67% of evaluated methods scoring below 0.2 on the AGREEprep scale, there is an urgent need for modernization of standardized protocols [38].

The findings serve as both a critical assessment of current practices and a constructive roadmap for future method development. By identifying specific deficiencies and providing quantitative metrics for improvement, AGREEprep enables the systematic transition toward more sustainable analytical practices without compromising analytical performance [36].

The ongoing IUPAC initiative represents a crucial step toward aligning analytical standardization with broader sustainability goals, highlighting the essential role of metrology in environmental protection. Widespread adoption of greenness assessment tools like AGREEprep will be instrumental in driving this necessary transition across industrial, regulatory, and research applications [38] [36].

In modern analytical chemistry, particularly within drug development, the environmental impact of an analytical method is only one dimension of a complete evaluation. The White Analytical Chemistry (WAC) concept posits that a truly optimal method balances three primary attributes: greenness (environmental impact), redness (analytical performance), and blueness (practicality and economy) [15] [42]. A method that excels in only one dimension may be unsuitable for routine application; for instance, an extremely green method might be analytically inadequate for the intended purpose, while a high-performing method might be too costly or hazardous for regular use [3]. Green Sample Preparation (GSP), as assessed by the AGREEprep metric, focuses specifically on the environmental footprint of the sample preparation stage [43] [6]. However, to be adopted in regulated industries like pharmaceutical development, this greenness must be balanced with robust analytical performance and high practicality.

This Application Note provides a structured framework and detailed protocols for the simultaneous application of AGREEprep with the Red Analytical Performance Index (RAPI) and the Blue Applicability Grade Index (BAGI). This integrated approach allows researchers and scientists to visualize and quantify the trade-offs between sustainability, functionality, and operational viability, facilitating holistic decision-making in analytical method development and selection [15] [3].

The Scientist's Toolkit: Essential Metric Tools and Software

The following tools are fundamental for implementing the integrated assessment described in this protocol.

Table 1: Essential Software Tools for Integrated Method Assessment

Tool Name	Primary Focus	Assessment Output	Access Information
AGREEprep	Greenness of Sample Preparation	Pictogram & Score (0-1)	https://mostwiedzy.pl/rapi [6]
RAPI	Analytical Performance ("Redness")	Pictogram & Score (0-100)	https://mostwiedzy.pl/rapi [15]
BAGI	Practicality & Economy ("Blueness")	Pictogram & Score (25-100)	https://mostwiedzy.pl/bagi [15]

Integrated Analytical Protocol: A Step-by-Step Workflow

This protocol outlines the procedure for extracting anthocyanins from purple corn, a model bioactive compound, using two alternative techniques. The process is designed to generate the necessary data for a subsequent comprehensive assessment using AGREEprep, RAPI, and BAGI [44].

Materials and Reagents

• Analytical Standards: Cyanidin chloride, Cyanidin-3-glucoside chloride [44].
• Plant Material: Purple corn (Zea mays L.) powder [44].
• Solvents: Ethanol (EtOH), Methanol (MeOH), o-Phosphoric acid (o-PA), Acetonitrile (ACN), all analytical grade [44].
• Water: Milli-Q water or equivalent purified water [44].
• Equipment: High-Performance Liquid Chromatography (HPLC) system coupled with UV or tandem Mass Spectrometry (MS) detection [44].
• Extraction Equipment: Pressurized Liquid Extraction (PLE) system (e.g., Dionex ASE) and Ultrasonic Bath or Probe Sonicator for Ultrasound-Assisted Extraction (UAE) [44].

Detailed Experimental Procedures

Pressurized Liquid Extraction (PLE)

• Sample Preparation: Homogenize 0.5 g of purple corn powder with 1.5 g of diatomaceous earth using a mortar and pestle [44].
• Cell Loading: Load the mixture into a 5 mL stainless-steel PLE cell pre-equipped with cellulose filters at the bottom [44].
• Extraction: Perform extraction using a food-grade solvent mixture of 2% o-PA in EtOH/water (1:1, v/v). The optimized PLE conditions are [44]:
  • Temperature: 95 °C
  • Pressure: 1500 psi
  • Static Time: 3 minutes
  • Cycles: 1
• Extract Collection: Collect the extract (~5 mL), dilute it 1:9 with a compatible mobile phase, and filter (0.22 µm) prior to chromatographic analysis [44].

Ultrasound-Assisted Extraction (UAE)

• Sample Preparation: Weigh 1.0 g of purple corn powder into a 50 mL centrifuge tube [44].
• Extraction: Add 10 mL of the extraction solvent (2% o-PA in EtOH/water, 1:1, v/v) [44].
• Sonication: Place the tube in an ultrasonic bath and sonicate for 10 minutes at a controlled temperature of 30 °C [44].
• Separation: Centrifuge the mixture at 5000 rpm for 5 minutes to separate the supernatant [44].
• Extract Collection: Carefully collect the supernatant, and filter (0.22 µm) prior to chromatographic analysis [44].

Method Validation for Performance (RAPI) Assessment

To generate data for the RAPI metric, a full method validation must be conducted for each extraction technique (PLE and UAE) according to regulatory guidelines (e.g., ICH, FDA) [15]. The following validation parameters should be established:

• Specificity/Selectivity: Confirm no interference from the matrix at the retention time of the analytes [44].
• Linearity: Prepare a calibration curve with at least 5 concentration levels. The coefficient of determination (R²) should be ≥ 0.999 [44].
• Limit of Detection (LOD) and Quantification (LOQ): Determine via signal-to-noise ratio or standard deviation of the response [44].
• Precision: Evaluate through repeatability (intra-day, n=6) and intermediate precision (inter-day, different analyst) expressed as % Relative Standard Deviation (%RSD). The obtained RSD should be ≤ 5.4% [44].
• Accuracy: Perform a recovery study by spiking the sample with known analyte concentrations (e.g., 50, 100, 150 mg/kg). Recovery should be between 97-102% [44].
• Robustness: Assess the method's capacity to remain unaffected by small, deliberate variations in procedural parameters (e.g., temperature, sonication time).

Data Analysis and Multi-Metric Evaluation Workflow

After completing the experiments and validation, the collected data is used to populate the three assessment tools. The following workflow diagrams the integrated evaluation process.

Executing the AGREEprep Assessment

AGREEprep evaluates the sample preparation step against 10 principles of Green Sample Preparation [6].

• Input Data Collection: Gather quantitative and qualitative data for the PLE and UAE methods, including:
  • Amount of waste generated (mL or g per sample)
  • Types and hazards of chemicals used
  • Energy consumption per sample (kW·h)
  • Degree of automation and integration
  • Scale of operation (e.g., sample amount, solvent volume)
• Software Input: Enter the collected data into the open-source AGREEprep software.
• Output Interpretation: The software generates a score between 0 and 1 and a circular pictogram. A higher score (closer to 1) indicates a greener sample preparation process [6] [44].

Executing the RAPI Assessment

RAPI assesses the method's analytical performance based on 10 key validation criteria [15].

• Input Data Collection: Use the results from the method validation (Section 3.3) to score the method on criteria such as:
  • Repeatability and Intermediate Precision
  • Accuracy (Trueness)
  • Limit of Detection (LOD) & Limit of Quantification (LOQ)
  • Linearity range & Robustness
• Software Input: Enter the scores into the dedicated RAPI software.
• Output Interpretation: The tool outputs a score from 0 to 100 and a star-shaped pictogram. A higher score indicates superior analytical performance [15].

Executing the BAGI Assessment

BAGI evaluates the practical and economic aspects of the entire analytical method [15].

• Input Data Collection: Gather data on 10 practicality criteria, including:
  • Cost of equipment and analysis
  • Sample throughput (samples per hour)
  • Operational simplicity and skill requirements
  • Safety for the operator
  • Availability and portability of equipment
• Software Input: Enter the data into the BAGI software.
• Output Interpretation: The result is a score between 25 and 100 and a blue-tone pictogram. A higher score denotes better practicality and applicability for routine use [15] [44].

Case Study: Integrated Assessment of PLE vs. UAE for Anthocyanin Extraction

The following table synthesizes the typical results from applying the integrated metric framework to the PLE and UAE protocols described above, based on a published study [44].

Table 2: Comparative Integrated Assessment of PLE and UAE Methods

Assessment Metric	Pressurized Liquid Extraction (PLE)	Ultrasound-Assisted Extraction (UAE)	Interpretation & Trade-off Analysis
AGREEprep Score	0.73 [44]	0.76 [44]	Both methods are quite green. UAE has a slight edge, likely due to lower energy consumption and minimal waste generation [44].
RAPI Score	~85-90 (Estimated)	~80-85 (Estimated)	PLE likely scores higher due to superior performance in LOD, precision, and robustness afforded by its controlled, high-pressure environment [44].
BAGI Score	77.5 [44]	72.5 [44]	PLE is more practical in a high-throughput lab due to higher automation and throughput. UAE is simpler and requires lower equipment cost [44].
Overall Balance	Performance & Practicality Leader	Greenness & Cost Leader	PLE offers better performance for routine use, while UAE is an excellent, greener choice for labs with budget constraints.

The data from the three metrics can be visualized to provide an at-a-glance comparison of the two methods, illustrating the core concept of White Analytical Chemistry.

The integration of AGREEprep, RAPI, and BAGI provides a powerful, holistic framework for evaluating analytical methods. This protocol demonstrates that no single metric is sufficient for method selection. For drug development professionals, this tripartite assessment is invaluable. It enables objective selection of methods that are not only environmentally sustainable (AGREEprep) but also capable of delivering reliable, reproducible data that meets strict regulatory standards (RAPI) while remaining cost-effective and practical for deployment in quality control laboratories (BAGI). By adopting this integrated approach, researchers can systematically navigate the complex trade-offs between greenness, performance, and practicality, ultimately accelerating the development of safer, more efficient, and sustainable analytical workflows.

Conclusion

AGREEprep represents a significant advancement in the objective quantification of sustainability in sample preparation, a critical and often resource-intensive step in analytical workflows. Its systematic, ten-principle approach provides researchers and drug development professionals with a clear roadmap to not only assess but also significantly improve the environmental profile of their methods. The future of sustainable science lies in holistic evaluation, where tools like AGREEprep for greenness are used in concert with metrics for analytical performance and practicality. As the field moves forward, the widespread adoption of such metrics will be crucial for driving the innovation and regulatory shifts needed to make biomedical and clinical research more environmentally responsible. Embracing this tool empowers scientists to make informed decisions that align analytical excellence with planetary health.

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