Green HPTLC Analysis of Remdesivir with Co-Administered COVID-19 Therapies: Method Development, Validation, and Application in Spiked Human Plasma

Paisley Howard Nov 29, 2025 285

This article provides a comprehensive guide for researchers and drug development professionals on the implementation of eco-friendly High-Performance Thin-Layer Chromatography (HPTLC) for the simultaneous analysis of the antiviral drug remdesivir...

Green HPTLC Analysis of Remdesivir with Co-Administered COVID-19 Therapies: Method Development, Validation, and Application in Spiked Human Plasma

Abstract

This article provides a comprehensive guide for researchers and drug development professionals on the implementation of eco-friendly High-Performance Thin-Layer Chromatography (HPTLC) for the simultaneous analysis of the antiviral drug remdesivir with its frequently co-administered medications. It covers the foundational rationale for therapeutic drug monitoring in complex COVID-19 regimens, details step-by-step methodological development for various drug combinations (including antivirals, antibiotics, and anticoagulants), and offers troubleshooting strategies for optimal separation. The content rigorously addresses method validation as per ICH guidelines and provides a comparative assessment of the greenness and whiteness of the analytical approaches using modern metrics like AGREE, GAPI, and Analytical Eco-Scale, underscoring the technique's applicability in pharmaceutical quality control and clinical pharmacokinetic studies.

The Critical Need for Analyzing Remdesivir in Combination Therapies

Clinical Rationale for Co-administering Drugs with Remdesivir in COVID-19 Treatment

Remdesivir (RDV), a broad-spectrum antiviral nucleotide analog, was the first drug approved by the US FDA for the treatment of COVID-19 in hospitalized patients [1] [2]. As a prodrug, RDV undergoes intracellular metabolism to form the pharmacologically active nucleoside triphosphate (NTP), which inhibits viral RNA-dependent RNA polymerase by competing with ATP, thereby incorporating itself into nascent RNA and stopping viral replication [2]. Despite its direct antiviral activity, the complex immune-mediated inflammatory nature of COVID-19 often necessitates combination therapy to achieve optimal clinical outcomes, particularly in patients with severe disease or specific comorbidities [2] [3]. This application note examines the clinical rationale for co-administering other drugs with remdesivir and provides detailed analytical protocols for their simultaneous determination using green High-Performance Thin-Layer Chromatography (HPTLC) methods, supporting ongoing pharmaceutical research and therapeutic drug monitoring.

Clinical Rationale for Combination Therapies

Synergistic Antiviral Approaches

The co-administration of COVID-19 RNA polymerase inhibitors demonstrates significant synergistic benefits. Research indicates that remdesivir and favipiravir together reduce viral load and inflammation more effectively than either drug used alone [4]. This combination targets viral replication through complementary mechanisms, potentially preventing viral resistance and enhancing therapeutic efficacy. Similarly, the combination of remdesivir and nirmatrelvir (a protease 3CL inhibitor) has shown synergistic reduction of SARS-CoV-2 viral titer in Vero E6 cells [2]. These multi-mechanism antiviral approaches are particularly valuable for treating immunocompromised patients or those with persistent infections.

Immunomodulatory Combinations

COVID-19 is recognized as an immune-mediated inflammatory disease where viral clearance can trigger a cytokine storm syndrome leading to organ failure [2]. Combining remdesivir with immunomodulators addresses both viral replication and the detrimental host inflammatory response:

  • Dexamethasone: This glucocorticoid, recognized as a standard of care for hospitalized COVID-19 patients, demonstrates enhanced effectiveness when combined with RDV, leading to reduced death rates, lower transfer rates to intensive care units, and shorter hospitalization periods [2]. A Bayesian meta-analysis confirmed that for patients needing supplemental oxygen without mechanical ventilation, dexamethasone showed a 93% probability of achieving ≥1% absolute decrease in mortality [5].

  • Baricitinib/Tocilizumab: These JAK1/JAK2 inhibitors, when combined with RDV, result in shorter recovery time, accelerated improvement in respiratory status, and fewer side effects [2].

  • Cyclosporine: This calcineurin inhibitor in combination with RDV leads to a significant reduction in IL-6 production along with decreased viral load in infected human cells [2].

Table 1: Clinically Established Remdesivir Combination Therapies

Combination Drug Mechanism of Action Key Clinical Benefits Patient Population
Dexamethasone Anti-inflammatory glucocorticoid Reduced mortality, ICU transfers, and hospitalization duration; faster viral clearance Hospitalized patients requiring oxygen [2]
Baricitinib JAK1/JAK2 inhibition Shorter recovery time, improved respiratory status, fewer side effects Hospitalized COVID-19 patients [2]
Favipiravir RNA polymerase inhibition Synergistic viral load reduction, enhanced inflammation control Patients with persistent infection [4]
Cyclosporine Calcineurin inhibition Reduced IL-6 production, decreased viral replication Patients with cytokine storm risk [2]
Special Patient Populations

Combination therapy with remdesivir is particularly crucial for high-risk patient groups. A recent retrospective study of COVID-19 patients with B-cell lymphoma receiving anti-CD20 antibodies demonstrated that initial combination antiviral therapy significantly shortened the time to viral clearance [3]. Conversely, bendamustine use was associated with prolonged time to viral clearance, highlighting the importance of tailored combination approaches for immunocompromised individuals [3].

Analytical Methodologies for Combination Therapy Monitoring

Green HPTLC Protocol for Simultaneous Determination

The following validated HPTLC method allows for simultaneous quantification of remdesivir with commonly co-administered drugs in pharmaceutical formulations and biological samples [4] [6].

Experimental Conditions
  • Stationary Phase: HPTLC silica gel 60 F254 plates (20 × 10 cm)
  • Mobile Phase: Ethyl acetate:hexane:acetic acid (9:1:0.3, v/v/v) for normal-phase separation [4]
  • Alternative Green Mobile Phase: Ethanol:water (6:4, v/v) for reverse-phase separation [6]
  • Sample Application: 10 μL as 6-mm bands using automatic applicator
  • Development Chamber: Twin-trough glass chamber, pre-saturated for 30 minutes
  • Development Distance: 9 cm at room temperature
  • Detection: UV scanning at 244 nm (remdesivir, molnupiravir) and 325 nm (favipiravir) [6]
  • Internal Standard: Apixaban (5 μg/band) for plasma sample analysis [4]
Sample Preparation Protocol

For Pharmaceutical Formulations:

  • Accurately weigh powder equivalent to 25 mg of each analyte
  • Dissolve in 10 mL methanol in 25 mL volumetric flask
  • Sonicate for 15 minutes and dilute to volume with methanol
  • Further dilute to obtain working solutions of 100 μg/mL

For Spiked Human Plasma:

  • Transfer different aliquots of working solutions into 10 mL centrifuge tubes
  • Add 0.5 mL plasma and 3 mL acetonitrile
  • Vortex for 1 minute, centrifuge at 4500 rpm for 10 minutes
  • Filter supernatant through 0.45 μm syringe filter
  • Apply 10 μL of filtrate directly to HPTLC plate [4]
Validation Parameters

The method has been validated according to ICH Q2(R1) guidelines with the following performance characteristics [4] [6]:

Table 2: Validation Parameters of HPTLC Methods for Remdesivir Combinations

Parameter Remdesivir Favipiravir Dexamethasone Molnupiravir
Linearity Range 30-800 ng/band [6] 50-2000 ng/band [6] 0.1-10 μg/band [4] 50-2000 ng/band [6]
Correlation Coefficient (r²) >0.9999 [6] >0.9999 [6] >0.999 [4] >0.9999 [6]
Limit of Detection 0.1 μg/band [4] 0.2 μg/band [4] 0.1 μg/band [4] 30 ng/band [6]
Recovery from Plasma 97.07-102.77% [4] 97.07-102.77% [4] 97.07-102.77% [4] 98.5-101.2% [6]
Precision (% RSD) <2% [4] [6] <2% [4] [6] <2% [4] <2% [6]
The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagent Solutions for Remdesivir Combination Analysis

Reagent/Material Function Specifications Application Notes
HPTLC Plates Stationary phase for separation Silica gel 60 F254, 20×10 cm [4] Pre-washing recommended for biological samples
Ethyl Acetate Mobile phase component High purity grade (99.8%) [4] Green solvent alternative to methanol
Ethanol Alternative green solvent Absolute ethanol for reverse-phase methods [6] Environmentally friendly option
Acetic Acid Mobile phase modifier Analytical grade (98% purity) [4] Improves spot shape and resolution
Apixaban Internal standard Purity ≥98.28% [4] Compensates for extraction variability in plasma
Acetonitrile Protein precipitation HPLC grade [4] Essential for plasma sample preparation
Methanol Solvent for standard solutions HPLC grade [4] Suitable for stock solution preparation
AmidomycinAmidomycinAmidomycin is a potent antifungal reagent for agricultural and microbiological research. This product is For Research Use Only and not for human or veterinary use.Bench Chemicals
Pacritinib HydrochloridePacritinib HydrochloridePacritinib hydrochloride is a JAK2/FLT3/IRAK1 kinase inhibitor for research use only (RUO). Explore its applications in myelofibrosis and AML studies. Not for human consumption.Bench Chemicals

Experimental Workflow and Signaling Pathways

COVID-19 Combination Therapy Mechanism

G COVID19 SARS-CoV-2 Infection ViralReplication Viral Replication COVID19->ViralReplication CytokineStorm Cytokine Storm (Inflammation) COVID19->CytokineStorm ViralReplication->CytokineStorm Triggers TissueDamage Tissue Damage & Organ Failure CytokineStorm->TissueDamage Remdesivir Remdesivir Remdesivir->ViralReplication Inhibits Favipiravir Favipiravir Favipiravir->ViralReplication Inhibits Dexamethasone Dexamethasone Dexamethasone->CytokineStorm Suppresses Baricitinib Baricitinib Baricitinib->CytokineStorm Modulates ClinicalOutcome Improved Clinical Outcome (Viral Clearance + Reduced Inflammation)

HPTLC Analysis Workflow

G SamplePrep Sample Preparation (Bulk, Formulation, or Plasma) Application Band Application (6 mm bands, 10 μL volume) SamplePrep->Application PlateActivation Plate Pre-conditioning (Optional for plasma samples) Application->PlateActivation Chromatography Chromatographic Development (Ethyl acetate:hexane:acetic acid 9:1:0.3) PlateActivation->Chromatography Detection UV Detection & Scanning (244 nm for RDV, 325 nm for FAV) Chromatography->Detection DataAnalysis Data Analysis & Quantification (Peak area vs concentration) Detection->DataAnalysis Validation Method Validation (ICH Q2(R1) guidelines) DataAnalysis->Validation

Discussion and Applications

The combination of remdesivir with complementary therapeutic agents represents a rational approach to COVID-19 treatment, addressing both viral replication and the host inflammatory response. The HPTLC methods presented here provide researchers with robust, green analytical tools for simultaneous quantification of these drug combinations in various matrices.

The environmental sustainability of these analytical methods has been evaluated using multiple metrics, including the Analytical Eco-Scale, AGREE, and ComplexGAPI, confirming their green profiles [4] [7] [6]. The high whiteness and blueness scores further indicate that these methods meet the requirements of white analytical chemistry by balancing analytical performance, ecological compatibility, and practical applicability [6].

For drug development professionals, these protocols support:

  • Therapeutic Drug Monitoring: Simultaneous measurement of remdesivir and co-administered drugs in patient plasma
  • Formulation Development: Quality control of potential fixed-dose combinations
  • Pharmacokinetic Studies: Tracking multiple drug concentrations over time
  • Clinical Protocol Optimization: Dose adjustment based on measured drug levels

The successful application of these methods to spiked human plasma with recovery rates of 97.07-102.77% demonstrates their suitability for clinical research and therapeutic drug monitoring in COVID-19 patients receiving combination therapies [4].

The growing emphasis on environmental sustainability has propelled Green Analytical Chemistry (GAC) to the forefront of pharmaceutical analysis. GAC principles aim to minimize the environmental impact of analytical methods by reducing hazardous solvent consumption, energy requirements, and waste generation [8]. Within this framework, High-Performance Thin-Layer Chromatography (HPTLC) has emerged as a powerful technique that aligns with green chemistry goals while maintaining high analytical performance [6].

HPTLC offers several environmental advantages over conventional analytical techniques: it consumes minimal solvents (typically 10-15 mL per analysis), allows parallel processing of multiple samples reducing operational time and energy, and generates significantly less waste compared to HPLC methods [6] [8]. The technique's simplicity, cost-effectiveness, and minimal sample preparation requirements further contribute to its sustainability profile [9].

The paradigm of sustainable method development has evolved to incorporate trichromatic assessment using Green, Blue, and White Analytical Chemistry principles. This comprehensive evaluation considers not only environmental impact (green) but also practical applicability (blue) and overall analytical performance (white), providing researchers with a holistic framework for sustainable method development [6].

Application Note: Analysis of Remdesivir with Co-administered Drugs

Background and Significance

Remdesivir (REM), a broad-spectrum antiviral agent, was the first drug approved by the US FDA for treating hospitalized COVID-19 patients [10]. Clinical management of COVID-19 often involves co-administration of REM with other medications, including broad-spectrum antibiotics like linezolid (LNZ) to treat secondary bacterial infections, and anticoagulants like rivaroxaban (RIV) to reduce thrombosis risk observed in COVID-19 patients [9]. The simultaneous determination of these drug combinations is crucial for therapeutic drug monitoring and clinical research.

Developed Methods and Analytical Performance

Recent research has demonstrated successful development of green HPTLC methods for analyzing remdesivir in combination with various co-administered medications. The table below summarizes key analytical parameters from published methods:

Table 1: Analytical Performance of Green HPTLC Methods for Remdesivir with Co-administered Drugs

Analytes Stationary Phase Mobile Phase Composition Retention Factors (Rf) Linear Range LOD/LOQ Application
REM, LNZ, RIV TLC silica gel 60 F254 DCM:acetone (8.5:1.5, v/v) REM: 0.23, LNZ: 0.53, RIV: 0.72 REM: 0.2-5.5 μg/band, LNZ: 0.2-4.5 μg/band, RIV: 0.1-3.0 μg/band REM: LOD=128.8 ng/band Spiked human plasma [9]
REM, FAV, DEX HPTLC silica gel Ethyl acetate:hexane:acetic acid (9:1:0.3, v/v) REM: 0.30, DEX: 0.64, FAV: 0.77 REM: 0.1-10 μg/band REM: LOD=0.1 μg/band Spiked human plasma [4]
REM, FAV, MOL (NP) HPTLC silica gel Ethyl acetate:ethanol:water (9.4:0.4:0.25, v/v) Not specified REM: 30-800 ng/band Not specified Bulk & pharmaceutical formulations [6]
REM, FAV, MOL (RP) HPTLC RP-18 Ethanol:water (6:4, v/v) Not specified REM: 30-800 ng/band Not specified Bulk & pharmaceutical formulations [6]

Sustainability Assessment

The developed methods were rigorously evaluated using multiple greenness assessment tools:

Table 2: Greenness Assessment of HPTLC Methods Using Various Metrics

Assessment Tool Methodology Key Parameters Evaluated Reported Scores for HPTLC Methods
Analytical Eco-Scale Qualitative assessment; higher scores indicate greener methods [11] Reagent toxicity, energy consumption, waste generation Excellent scores (e.g., 93/100 for SUV analysis [12])
AGREE Comprehensive software-based evaluation (0-1 scale) [11] 12 principles of GAC 0.78 for RP-HPLC of REM [11]; 0.88 for SUV HPTLC [12]
GAPI Pictorial representation with color coding [8] Entire method lifecycle from sampling to waste Used in multiple studies [9] [8]
RGB12 Evaluates whiteness considering all three sustainability pillars [6] Analytical performance, ecological impact, practical & economic aspects 95.6% whiteness for REM/FAV/DEX method [4]

Experimental Protocols

Protocol 1: Simultaneous Analysis of REM, LNZ, and RIV in Spiked Human Plasma

Materials and Reagents
  • Remdesivir (purity ≥99.8%)
  • Linezolid (purity ≥99.8%)
  • Rivaroxaban (purity ≥99.6%)
  • Dichloromethane (HPLC grade)
  • Acetone (analytical grade)
  • Methanol (HPLC grade)
  • Human plasma (stored at -20°C until use)
  • TLC Silica gel 60 F254 plates (20 × 20 cm, 0.1 mm thickness)
Instrumentation
  • CAMAG TLC scanner 3 with winCATS software
  • Linomat 5 autosampler with 100 µL microsyringe
  • Twin-trough glass chamber for mobile phase
  • Centrifuge capable of 4500 rpm
  • Vortex mixer
  • Ultrasonic bath
Sample Preparation
  • Stock solutions: Prepare individual stock solutions of REM, LNZ, and RIV at 1 mg/mL in methanol.
  • Working solutions: Dilute stock solutions with methanol to obtain appropriate working concentrations.
  • Plasma sample processing:
    • Spike drug-free human plasma with appropriate aliquots of working standards
    • Add 3 mL of acetonitrile to 0.5 mL of spiked plasma
    • Vortex the mixture for 1 minute
    • Centrifuge at 4500 rpm for 10 minutes
    • Filter the supernatant through a 0.45 μm membrane filter
Chromatographic Conditions
  • Stationary phase: TLC Silica gel 60 F254 plates
  • Mobile phase: Dichloromethane:acetone (8.5:1.5, v/v)
  • Application volume: 10 μL as bands
  • Development distance: 75 mm in twin-trough chamber pre-saturated for 30 minutes
  • Detection wavelength: 254 nm
  • Scanning speed: 20 mm/s
Validation Parameters
  • Linearity: Evaluate over concentration ranges of 0.2-5.5, 0.2-4.5, and 0.1-3.0 μg/band for REM, LNZ, and RIV, respectively
  • Precision: Assess intra-day and inter-day precision (% CV <2%)
  • Accuracy: Determine via recovery studies (98-102%)
  • Specificity: Confirm separation from degradation products and plasma components

Protocol 2: Analysis of REM with FAV and DEX in Spiked Plasma

Materials
  • Remdesivir, Favipiravir, Dexamethasone (purity ≥99.8%)
  • Ethyl acetate, hexane, acetic acid (HPLC grade)
  • Methanol (HPLC grade)
  • HPTLC silica gel plates
  • Apixaban (internal standard)
Chromatographic Conditions
  • Mobile phase: Ethyl acetate:hexane:acetic acid (9:1:0.3, v/v)
  • Detection: 254 nm
  • Application volume: 10 μL as bands
  • Rf values: REM: 0.30, DEX: 0.64, FAV: 0.77
Sample Preparation
  • Prepare stock solutions of all analytes at 1 mg/mL in methanol
  • Spike human plasma with working standard solutions
  • Add internal standard (apixaban)
  • Precipitate proteins with methanol, vortex, centrifuge, and filter

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Essential Materials for Green HPTLC Analysis of Antiviral Drugs

Category Specific Items Function/Purpose Green Considerations
Stationary Phases TLC Silica gel 60 F254 plates, HPTLC RP-18 plates Separation matrix Reusable with proper cleaning, minimal waste generation
Mobile Phase Components Ethyl acetate, ethanol, acetone, dichloromethane, water Sample elution and separation Prefer less hazardous solvents (ethyl acetate, ethanol); minimize chlorinated solvents
Reference Standards Remdesivir, favipiravir, molnupiravir, dexamethasone, linezolid, rivaroxaban Method development and calibration Source from certified suppliers with purity documentation
Sample Preparation Methanol, acetonitrile, syringe filters (0.45 μm) Sample extraction and cleanup Use protein precipitation instead of lengthy extraction; minimize solvent volumes
Detection CAMAG TLC Scanner, deuterium lamp Compound detection and quantification Non-destructive detection allows further analysis of same plate
Diclazuril sodiumDiclazuril sodium, CAS:112209-99-1, MF:C17H8Cl3N4NaO2, MW:429.6 g/molChemical ReagentBench Chemicals
Nazartinib MesylateNazartinib Mesylate, CAS:1784778-10-4, MF:C27H41ClN6O8S, MW:645.2 g/molChemical ReagentBench Chemicals

Workflow and Signaling Pathways

G cluster_0 Experimental Phase cluster_1 Evaluation Phase Start Start: Method Development SamplePrep Sample Preparation (Protein precipitation) Start->SamplePrep Application Sample Application (6 mm bands, 10 μL volume) SamplePrep->Application ChamberSaturation Chamber Saturation (30 minutes) Application->ChamberSaturation PlateDevelopment Plate Development (Ascending mode, 75 mm) ChamberSaturation->PlateDevelopment Densitometry Densitometric Detection (UV 254 nm) PlateDevelopment->Densitometry DataAnalysis Data Analysis (Peak integration, calibration) Densitometry->DataAnalysis Validation Method Validation (ICH Q2(R1) guidelines) DataAnalysis->Validation GreenAssessment Sustainability Assessment (AGREE, GAPI, RGB12) End Validated Green Method GreenAssessment->End Validation->GreenAssessment

HPTLC Method Development Workflow

G GAC Green Analytical Chemistry (12 Principles) Sample Sample Preparation Minimal solvent use GAC->Sample Analysis Analysis Phase Reduced energy consumption GAC->Analysis Waste Waste Management Minimal hazardous waste GAC->Waste Trichromatic Trichromatic Assessment Sample->Trichromatic Analysis->Trichromatic Waste->Trichromatic Green Greenness Environmental Impact (AGREE, GAPI, Eco-Scale) Trichromatic->Green Blue Blueness Practical Applicability (BAGI metric) Trichromatic->Blue White Whiteness Overall Sustainability (RGB12 algorithm) Trichromatic->White Method Sustainable HPTLC Method Green->Method Blue->Method White->Method

Sustainability Assessment Framework

Troubleshooting and Optimization Guidelines

Common Issues and Solutions

  • Spot tailing: Adjust mobile phase composition; add small amounts of ammonia or acetic acid to improve peak symmetry
  • Inadequate separation: Optimize mobile phase ratio; consider two-dimensional development for complex mixtures
  • Poor reproducibility: Ensure consistent chamber saturation time; maintain constant temperature and humidity
  • Weak sensitivity: Optimize application volume; evaluate different detection wavelengths

Method Optimization Strategies

  • Mobile phase selection: Begin with less hazardous solvents (ethyl acetate, ethanol) before considering chlorinated solvents
  • Sample application: Use automated sample applicators for better precision and reproducibility
  • Detection parameters: Optimize slit dimensions and scanning speed for improved sensitivity
  • Greenness improvement: Replace hazardous solvents with greener alternatives while maintaining separation efficiency

The integration of Green Analytical Chemistry principles with HPTLC methodology provides a robust framework for sustainable pharmaceutical analysis. The protocols outlined herein demonstrate that green HPTLC methods can successfully determine remdesivir in combination with co-administered drugs while maintaining excellent analytical performance and minimal environmental impact. The comprehensive sustainability assessment using multiple metrics ensures that developed methods are not only environmentally friendly but also practically applicable in routine analytical laboratories. As pharmaceutical analysis continues to evolve, the adoption of such green approaches will be crucial for reducing the ecological footprint of quality control and clinical research activities.

Combination therapy has emerged as a cornerstone in the management of complex infectious diseases, particularly in the context of COVID-19 treatment. Hospitalized patients often present with multiple pathological processes simultaneously, including viral replication, secondary bacterial infections, dysregulated inflammatory responses, and pro-thrombotic states. This clinical reality necessitates the concurrent administration of drugs from different classes, creating a complex pharmacological environment requiring careful therapeutic monitoring [13] [14].

The analysis of these drug combinations presents significant challenges for researchers and clinical chemists. While therapeutic drug monitoring is essential for optimizing efficacy and minimizing toxicity, the structural similarities between drugs and their metabolites, vastly different physicochemical properties, and the complex biological matrix of clinical samples complicate analytical procedures. Within this framework, green analytical chemistry principles have gained prominence, driving the development of sustainable methods that minimize environmental impact while maintaining analytical performance [15] [16].

This application note provides a comprehensive overview of common drug combinations featuring remdesivir with antibiotics, anticoagulants, and corticosteroids, with a specific focus on eco-friendly High-Performance Thin-Layer Chromatography (HPTLC) methodologies for their simultaneous analysis.

Common Drug Combinations with Remdesivir

Clinical Rationale for Combination Therapies

Remdesivir, an intravenous antiviral agent, serves as the foundation for COVID-19 treatment in hospitalized patients. Its mechanism involves intracellular metabolism to the active nucleoside triphosphate analog (GS-443902), which inhibits viral RNA-dependent RNA polymerase, thereby disrupting SARS-CoV-2 replication [17] [18]. Clinical guidelines recommend combining remdesivir with other agents to address the multifaceted nature of severe COVID-19:

  • Antivirals + Corticosteroids: Recommended for patients with hypoxemia to simultaneously suppress viral replication and modulate the excessive inflammatory response [13].
  • Antivirals + Antibiotics: Essential for managing secondary bacterial pneumonia, a common complication in ventilated patients [14].
  • Antivirals + Anticoagulants: Crucial for preventing thrombotic events, which are frequently observed in COVID-19 patients due to virus-induced endothelial inflammation and hypercoagulability [14].

Clinically Relevant Combinations and Observed Interactions

Table 1: Common Drug Combinations with Remdesivir in COVID-19 Treatment

Drug Class Example Agents Clinical Purpose Reported Interactions
Antivirals Remdesivir (foundation) Inhibit SARS-CoV-2 replication N/A (base therapy)
Corticosteroids Dexamethasone, Prednisolone, Methylprednisolone Mitigate hyperinflammatory state (e.g., cytokine storm) No clinically relevant pharmacokinetic interactions documented [13].
Antibiotics Linezolid Treat secondary bacterial pneumonia No analytical interference in HPTLC methods; structural distinction allows chromatographic separation [14].
Anticoagulants Rivaroxaban, Warfarin Prophylaxis and treatment of thrombotic events Potential interaction with warfarin (elevated INR) [19] [14]. No reported interaction with rivaroxaban.

Recent real-world evidence from a study of 151,215 hospitalized COVID-19 patients during the Omicron period (December 2021 to April 2023) demonstrated that combination therapy with remdesivir and corticosteroids significantly reduced mortality risk compared to corticosteroid monotherapy. At 14 and 28 days, the mortality risk was significantly lower across all supplemental oxygen requirements, highlighting the critical importance of maintaining antiviral therapy within combination regimens [13].

Analytical Solutions: Green HPTLC Methodologies

Advantages of HPTLC in Combination Drug Analysis

High-Performance Thin-Layer Chromatography has emerged as a powerful, eco-friendly alternative to conventional HPLC for therapeutic drug monitoring and quality control applications. The technique offers several distinct advantages for analyzing complex drug combinations:

  • High throughput: Capability to analyze multiple samples simultaneously on a single plate [16].
  • Reduced solvent consumption: Minimal mobile phase requirements compared to HPLC [15] [14].
  • Cost-effectiveness: Lower operational and maintenance costs [16].
  • Flexibility: Ability to analyze samples directly without extensive pre-purification [14].
  • Green chemistry compatibility: Easier adoption of environmentally friendly solvents [15] [16].

HPTLC Protocol for Simultaneous Quantification of Remdesivir, Linezolid, and Rivaroxaban

Scope: This protocol describes the simultaneous quantification of remdesivir (antiviral), linezolid (antibiotic), and rivaroxaban (anticoagulant) in spiked human plasma using normal-phase HPTLC with densitometric detection [14].

Materials and Reagents:

  • Stationary Phase: TLC silica gel 60 F254 aluminum plates (20 × 10 cm, 200 μm thickness)
  • Mobile Phase: Dichloromethane-Acetone (8.5:1.5, v/v)
  • Reference Standards: Remdesivir (purity ≥99.8%), Linezolid (purity ≥99.8%), Rivaroxaban (purity ≥99.6%)
  • Sample Application: Linomat 5 autosampler with 100 μL microsyringe
  • Detection: Densitometer at 254 nm

Instrumentation Conditions:

  • Application Volume: 10 μL as bands (8 mm from bottom margin)
  • Development Distance: 80 mm in twin-trough glass chamber
  • Chamber Saturation: 30 minutes at room temperature (25 ± 2°C)
  • Scanning: Deuterium lamp, absorbance mode, 20 mm/s scanning speed

Sample Preparation (Spiked Human Plasma):

  • Transfer 1 mL of drug-free human plasma into a centrifuge tube.
  • Spike with appropriate volumes of standard working solutions.
  • Add 3 mL of acetonitrile as protein precipitating agent.
  • Vortex mix for 1 minute, then centrifuge at 4000 rpm for 10 minutes.
  • Collect the supernatant and evaporate to dryness under nitrogen stream.
  • Reconstitute the residue in 1 mL methanol, vortex for 30 seconds.
  • Apply 10 μL of the prepared sample onto HPTLC plates.

Calibration:

  • Prepare calibration curves in the following ranges:
    • Remdesivir: 0.2–5.5 μg/band
    • Linezolid: 0.2–4.5 μg/band
    • Rivaroxaban: 0.1–3.0 μg/band
  • Construct calibration graphs by plotting peak area versus concentration

Validation Parameters:

  • Linearity: Correlation coefficient (r²) > 0.999 for all analytes
  • Recovery: 98.3–101.2% for all three drugs in pharmaceutical formulations and spiked plasma
  • Sensitivity:
    • Remdesivir LOD: 128.8 ng/band
    • Linezolid LOD: 50.5 ng/band
    • Rivaroxaban LOD: 55.8 ng/band

HPTLC Protocol for Antiviral Combination Analysis

Scope: Simultaneous analysis of remdesivir with other antivirals (favipiravir and molnupiravir) in pharmaceutical formulations using normal-phase and reversed-phase HPTLC [16].

Normal-Phase Method:

  • Mobile Phase: Ethyl acetate:Ethanol:Water (9.4:0.4:0.25, v/v/v)
  • Detection Wavelength: 244 nm for remdesivir and molnupiravir, 325 nm for favipiravir
  • Calibration Ranges: 30–800 ng/band for remdesivir, 50–2000 ng/band for favipiravir and molnupiravir

Reversed-Phase Method:

  • Mobile Phase: Ethanol:Water (6:4, v/v) - classified as a greener solvent system
  • Detection Wavelength: Same as normal-phase method
  • Calibration Ranges: Identical to normal-phase method

Table 2: Key Analytical Parameters for HPTLC Methods

Parameter Remdesivir, Linezolid, Rivaroxaban Method [14] Antiviral Combination Method (Normal-Phase) [16] Antiviral Combination Method (Reversed-Phase) [16]
Stationary Phase Silica gel 60 F254 Silica gel 60 F254 RP-18 HPTLC plates
Mobile Phase DCM:Acetone (8.5:1.5) Ethyl acetate:EtOH:Water (9.4:0.4:0.25) EtOH:Water (6:4)
Retention Factor (Rf) REM: 0.23, LNZ: 0.53, RIV: 0.72 RMD: Compound-specific RMD: Compound-specific
Linear Range (ng/band) REM: 200-5500, LNZ: 200-4500, RIV: 100-3000 RMD: 30-800, FAV/MOL: 50-2000 RMD: 30-800, FAV/MOL: 50-2000
Application Spiked human plasma Pharmaceutical formulations Pharmaceutical formulations
Greenness Score AGREE: 0.78 [14] Comprehensive trichromatic assessment [16] Superior greenness profile [16]

Experimental Workflows and Signaling Pathways

HPTLC Analysis Workflow

The following diagram illustrates the complete experimental workflow for the simultaneous analysis of remdesivir with co-administered drugs using green HPTLC methodologies:

G SamplePrep Sample Preparation • Plasma protein precipitation • Centrifugation • Reconstitution in methanol Application Sample Application • 10 µL bands via autosampler • 8 mm from plate bottom SamplePrep->Application Development Chromatographic Development • Mobile phase: DCM:Acetone (8.5:1.5) • Chamber saturation: 30 min • Development distance: 80 mm Application->Development Densitometry Densitometric Detection • UV detection at 254 nm • Peak area integration Development->Densitometry DataAnalysis Data Analysis • Calibration curve construction • Concentration calculation • Greenness assessment Densitometry->DataAnalysis

Metabolic and Interaction Pathways of Remdesivir

The pharmacological activity and drug interaction potential of remdesivir are governed by its complex metabolic pathway and effects on enzymatic systems:

G RDV Remdesivir (Prodrug) • OATP1B1/1B3 substrate • P-gp substrate GS704277 Intermediate Metabolite GS-704277 • OATP1B1/1B3 substrate RDV->GS704277 CES-1, CatA CYP3A metabolism CYPInhibition Cytochrome P450 Effects • Weak inhibitor of CYP3A, CYP2C9 • Transient inhibition due to short half-life RDV->CYPInhibition In vitro inhibition ActiveMetab Active Metabolite GS-443902 • Intracellular only • Inhibits viral RNA polymerase GS704277->ActiveMetab HINT1-mediated activation GS441524 Circulating Metabolite GS-441524 • Renal elimination • 27h half-life GS704277->GS441524 Dephosphorylation WarfarinInteraction Warfarin Interaction • Increased INR reported • Naranjo scale: Probable CYPInhibition->WarfarinInteraction Decreased warfarin clearance

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for HPTLC Analysis of Remdesivir Combinations

Reagent/Equipment Function/Purpose Specifications/Alternatives
TLC Silica gel 60 F254 plates Stationary phase for normal-phase separation Aluminum sheets, 20×10 cm, 200 μm thickness [15] [14]
RP-18 HPTLC plates Stationary phase for reversed-phase separation For polar mobile phases (e.g., ethanol:water) [16]
Densitometer with deuterium lamp Quantitative detection of separated bands Scanning capability at 200-600 nm, winCATS software [14]
Linomat autosampler Precise sample application 100 μL microsyringe, band application (5-8 mm bandwidth) [15]
Dichloromethane Mobile phase component (normal-phase) HPLC grade, for medium-polarity separations [14]
Ethanol (green solvent) Eco-friendly mobile phase component Replaces acetonitrile in reversed-phase methods [16]
Acetonitrile Protein precipitation agent HPLC grade for plasma sample preparation [14]
Drug Reference Standards Method calibration and validation Purity ≥99% for remdesivir, linezolid, rivaroxaban [14]
HeveadrideHeveadride, CAS:39595-41-0, MF:C18H20O6, MW:332.3 g/molChemical Reagent
Selnoflast potassiumSelnoflast potassium, CAS:2260969-37-5, MF:C20H28KN3O3S, MW:429.6 g/molChemical Reagent

The combination of remdesivir with antibiotics, anticoagulants, and corticosteroids represents a clinically necessary approach for managing hospitalized COVID-19 patients. The green HPTLC methodologies presented herein provide robust, cost-effective, and environmentally sustainable solutions for simultaneous quantification of these complex drug regimens.

Key Implementation Considerations:

  • Method Selection: Choose normal-phase HPTLC for analyzing remdesivir with linezolid and rivaroxaban in biological samples, and reversed-phase HPTLC for antiviral combinations in pharmaceutical formulations.

  • Analytical Quality Control: Implement rigorous validation following ICH Q2(R1) guidelines, with particular attention to specificity in the presence of drug metabolites and degradation products.

  • Green Chemistry Adoption: Prioritize ethanol-water mobile phases where possible to enhance method sustainability while maintaining chromatographic performance [16].

  • Clinical Correlation: When monitoring these drug combinations, consider the potential for pharmacokinetic interactions, particularly the transient CYP450 inhibition by remdesivir and its potential impact on warfarin therapy [17] [19].

The protocols outlined in this application note enable reliable therapeutic drug monitoring and stability studies, supporting optimized patient care through precise quantification of complex drug combinations while adhering to the principles of green analytical chemistry.

Challenges in Therapeutic Drug Monitoring and the Advantage of Multi-Analyte HPTLC

Therapeutic Drug Monitoring (TDM) represents a critical component of modern pharmacotherapy, particularly for drugs with narrow therapeutic windows, significant interindividual variability, or complex drug-drug interactions. Traditional TDM primarily relies on venous blood sampling, a method fraught with practical limitations including patient discomfort, the need for specialized phlebotomy personnel, and logistical challenges for routine monitoring [20]. These challenges are particularly acute in the context of managing complex treatment regimens, such as those employed for COVID-19, where patients often receive multiple co-administered drugs like remdesivir (antiviral), linezolid (antibiotic), and rivaroxaban (anticoagulant) simultaneously [14]. Monitoring these combinations is essential for ensuring therapeutic efficacy while minimizing adverse events, but it necessitates analytical methods capable of quantifying multiple analytes from a single, often limited, sample volume.

The emergence of less invasive sampling techniques—including dried blood spots (DBS), saliva, and hair analysis—has improved patient acceptability and accessibility of TDM [20]. However, the translation of these techniques into routine clinical practice hinges on the availability of robust, cost-effective, and multi-analyte analytical methods. Many conventional techniques, such as high-performance liquid chromatography (HPLC), while sensitive and specific, are often time-consuming, require expensive instrumentation and hazardous chemicals, and are typically optimized for single-analyte quantification [14]. This creates a significant analytical bottleneck. Furthermore, the push for greener analytical chemistry demands methods that reduce environmental impact by minimizing solvent waste and energy consumption [21] [22]. It is within this challenging landscape that High-Performance Thin-Layer Chromatography (HPTLC), particularly in its multi-analyte format, emerges as a powerful and advantageous solution.

The Multi-Analyte HPTLC Advantage

High-Performance Thin-Layer Chromatography (HPTLC) is a sophisticated planar chromatography technique that offers a unique combination of flexibility, efficiency, and cost-effectiveness. Its inherent design is exceptionally well-suited to address the modern challenges of TDM. The core advantage of HPTLC lies in its parallel processing capability; multiple samples, standards, and controls can be analyzed simultaneously on a single plate, dramatically increasing throughput and reducing analysis time per sample compared to sequential techniques like HPLC [23]. This feature is indispensable for TDM, where rapid turnaround of results can directly impact clinical decision-making.

The technique is also remarkably versatile and green. A single chromatographic run can resolve and quantify several drugs and their metabolites from a single sample application, making it ideal for monitoring co-administered therapies [21] [14]. The method requires minimal sample preparation and uses notably smaller volumes of organic solvents compared to column chromatographic methods, aligning with the principles of green analytical chemistry [21] [23]. The off-line nature of HPTLC separation allows for flexible post-chromatographic treatment, such as derivatization with specific reagents to enhance detection sensitivity or selectivity for particular compounds. Moreover, the ability to document the entire separation as an image provides a permanent, verifiable record for quality control and regulatory compliance, a crucial aspect under current Good Manufacturing Practices (cGMP) [24] [23].

Recent technological advancements have further amplified the potential of HPTLC. The integration of smartphone-based detection, utilizing high-quality cameras in conjunction with freely available image analysis software like ImageJ, demonstrates a move towards more accessible and portable quantitative analysis. This innovation offers a cost-effective alternative to traditional benchtop densitometers, making high-quality TDM more feasible in resource-limited settings [21]. The combination of these factors—high throughput, multi-analyte capability, minimal sample preparation, cost-effectiveness, and evolving detection technologies—positions multi-analyte HPTLC as a superior analytical platform for contemporary TDM applications.

Application Note: Green HPTLC for Remdesivir and Co-Administered Drugs

Protocol: Simultaneous Determination of Remdesivir, Linezolid, and Rivaroxaban in Spiked Human Plasma

The following detailed protocol describes a green and selective HPTLC method for the simultaneous quantification of remdesivir (REM), linezolid (LNZ), and rivaroxaban (RIV), which are representative of antiviral, antibiotic, and anticoagulant classes used concurrently in COVID-19 treatment [14].

  • 1. Materials and Reagents

    • HPTLC Plates: TLC silica gel 60 F254 aluminum sheets (20 × 20 cm, 0.1 mm thickness).
    • Reference Standards: Certified pure standards of REM, LNZ, and RIV.
    • Solvents: Dichloromethane (DCM) and acetone, both of analytical grade.
    • Sample: Human plasma, stored at -20 °C until use.
    • Equipment: CAMAG TLC scanner 3, Linomat 5 autosampler, a chromatographic tank, and a microsyringe (100 µL).

  • 2. Standard Solution Preparation

    • Accurately weigh 10 mg of each REM, LNZ, and RIV into separate 10 mL volumetric flasks.
    • Dissolve and dilute to volume with methanol for REM and LNZ, and with acetonitrile for RIV, to obtain stock solutions of 1 mg/mL.
    • Prepare working standard solutions by appropriate dilution of the stock solutions with methanol.

  • 3. Sample Preparation (Plasma)

    • Spike drug-free human plasma with known concentrations of REM, LNZ, and RIV.
    • Use protein precipitation with a suitable solvent like methanol or acetonitrile to deproteinize the plasma sample.
    • Vortex-mix vigorously and then centrifuge to separate the precipitated proteins.
    • Collect the clear supernatant for application onto the HPTLC plate.

  • 4. Chromatographic Conditions

    • Stationary Phase: TLC silica gel 60 F254 plates.
    • Mobile Phase: Dichloromethane-Acetone (8.5:1.5, v/v).
    • Application Volume: 10 µL of standard and prepared sample solutions, applied as 6-mm bands.
    • Development: Ascending development in a twin-trough chamber pre-saturated with mobile phase vapor for 20 minutes. The development distance is 80 mm.
    • Densitometric Detection: Scanning is performed at 254 nm using a TLC scanner.

  • 5. Method Validation

    • The method is validated per ICH Q2(R1) guidelines for:
      • Linearity: Over 0.2–5.5 µg/band for REM, 0.2–4.5 µg/band for LNZ, and 0.1–3.0 µg/band for RIV.
      • Precision: Repeatability and intermediate precision (RSD < 2%).
      • Accuracy: Via recovery studies from spiked plasma (98.3–101.2%).
      • Specificity: Achieved by well-resolved peaks with Rf values of 0.23, 0.53, and 0.72 for REM, LNZ, and RIV, respectively.
      • Sensitivity: LOD and LOQ are determined. LOQ reported as 128.8, 50.5, and 55.8 ng/band for REM, LNZ, and RIV, respectively [14].

The following workflow diagram illustrates the key steps of this protocol:

G Start Start Sample Preparation Prep Spike Plasma with Analytes Start->Prep Precip Protein Precipitation (Methanol/ACN) Prep->Precip Centrifuge Centrifuge and Collect Supernatant Precip->Centrifuge Apply Apply 10 µL Band to HPTLC Plate Centrifuge->Apply Develop Develop in Chamber with DCM:Acetone (8.5:1.5) Apply->Develop Dry Air-Dry Plate Develop->Dry Scan Scan at 254 nm Dry->Scan Analyze Analyze Peaks and Quantify Scan->Analyze End Validation and Data Reporting Analyze->End

The validation data and green profile of the described HPTLC method are summarized in the tables below.

Table 1: Validation Data for the HPTLC Assay of REM, LNZ, and RIV [14]

Analytical Parameter Remdesivir (REM) Linezolid (LNZ) Rivaroxaban (RIV)
Linear Range (µg/band) 0.2 – 5.5 0.2 – 4.5 0.1 – 3.0
Retardation Factor (Rf) 0.23 0.53 0.72
Limit of Quantification (LOQ, ng/band) 128.8 50.5 55.8
Accuracy (% Recovery) 98.3 – 101.2 98.3 – 101.2 98.3 – 101.2

Table 2: Greenness Assessment of the HPTLC Method Using Eco-Scale and GAPI [14]

Greenness Metric Score / Assessment for HPTLC Method
Analytical Eco-Scale High score (indicating an excellent green method)
AGREE Metric High greenness profile
Key Green Features Low solvent consumption, minimal waste generation, use of ethanol in sample preparation

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of a robust multi-analyte HPTLC method requires specific, high-quality materials. The following table lists key reagents and their critical functions based on the cited protocols.

Table 3: Essential Research Reagent Solutions for Multi-Analyte HPTLC

Reagent / Material Function and Importance in HPTLC Analysis
HPTLC Silica gel 60 F254 Plates The stationary phase. The fine, uniform particle size ensures high resolution and reproducibility. The F254 indicator fluoresces under 254 nm UV light, aiding in visual detection of compounds that quench fluorescence [14] [23].
Dichloromethane & Acetone Components of the mobile phase. The specific ratio (8.5:1.5 v/v) is optimized to achieve baseline separation of REM, LNZ, and RIV with Rf values of 0.23, 0.53, and 0.72, respectively [14].
Methanol & Acetonitrile (HPLC Grade) Used for preparing standard stock solutions and for protein precipitation during plasma sample preparation. High purity is essential to prevent interference from impurities [14].
Certified Drug Reference Standards Highly pure, characterized substances used for the identification and quantification of the target analytes (REM, LNZ, RIV). Essential for calibrating the method and ensuring accuracy [14] [24].
Densitometer / TLC Scanner Instrument for in-situ quantification of the resolved analyte bands on the HPTLC plate by measuring absorbance or fluorescence at a specific wavelength (e.g., 254 nm) [14] [23].
ImageJ Software A freely available, powerful image analysis program that can be used as an alternative quantification tool when paired with a smartphone camera for capturing chromatogram images, enhancing method accessibility [21].
CyclobutrifluramCyclobutrifluram|High-Purity Reference Standard
Deucravacitinib hydrochlorideDeucravacitinib Hydrochloride|High-Purity TYK2 Inhibitor

The challenges inherent in modern Therapeutic Drug Monitoring—including the need for multi-analyte profiling, rapid turnaround, cost containment, and adherence to green chemistry principles—are significant. The application of green, multi-analyte HPTLC, as demonstrated in the protocol for remdesivir and its co-administered drugs, provides a compelling solution. This technique successfully balances analytical performance with practical and environmental considerations. Its high throughput, inherent flexibility, and compatibility with innovative detection systems like smartphone technology make it an exceptionally powerful tool for both clinical research and routine drug monitoring. As polypharmacy continues to rise in the treatment of complex diseases, the adoption of efficient multi-analyte strategies like HPTLC will be paramount in advancing personalized medicine and improving patient outcomes.

Developing a Robust Green HPTLC Method: From Sample Preparation to Densitometry

Within pharmaceutical quality control and bioanalytical research, the adoption of sustainable methodologies is paramount. The analysis of Remdesivir (REM), a key COVID-19 therapeutic, often necessitates simultaneous determination with co-administered drugs such as Favipiravir (FAV), Dexamethasone (DEX), and various cardiovascular agents. High-Performance Thin-Layer Chromatography (HPTLC) presents a compelling platform for this task, offering advantages in throughput, cost-effectiveness, and reduced solvent consumption, aligning with the principles of Green Analytical Chemistry (GAC) [6]. This application note details optimized, eco-friendly chromatographic conditions—focusing on stationary and mobile phase selection—for the reliable analysis of REM in combination with other drugs, providing validated protocols for implementation in research and development.

Stationary Phase Selection

For the HPTLC analysis of REM and its co-administered drugs, the consistent choice of stationary phase across numerous studies is TLC silica gel 60 F254 on aluminum plates [4] [14] [25]. This plate type is favored for its excellent separation efficiency, reproducibility, and the utility of the F254 indicator for UV visualization at 254 nm.

The successful separation of complex drug mixtures, including REM, FAV, and cardiovascular drugs like aspirin and atorvastatin, on this standard silica phase demonstrates its versatility. Optimal resolution is achieved not by changing the stationary phase, but by meticulously optimizing the mobile phase composition [25].

Mobile Phase Optimization for Drug Combinations

The mobile phase composition is the most critical parameter for achieving baseline separation of complex drug mixtures. The following table summarizes optimized green mobile phases for various drug combinations, highlighting the trend towards safer solvents like ethanol and ethyl acetate.

Table 1: Optimized Green Mobile Phases for HPTLC Analysis of REM with Co-administered Drugs

Drug Combination Mobile Phase Composition (v/v/v) Stationary Phase Detection Wavelength Reference
REM, FAV, Molnupiravir Normal-Phase: Ethyl acetate:Ethanol:Water (9.4:0.4:0.25)Reverse-Phase: Ethanol:Water (6:4) Silica gel 60 F254 244 nm & 325 nm [6]
REM, FAV, Dexamethasone Ethyl acetate:Hexane:Acetic Acid (9:1:0.3) Silica gel 60 F254 254 nm [4]
REM, Linezolid, Rivaroxaban Dichloromethane:Acetone (8.5:1.5) TLC silica gel 60 F254 254 nm [14]
*REM & Cardiovascular Drugs Ethyl acetate:Methylene chloride:Methanol:Ammonia (6:4:4:1) HPTLC-silica plates 232 nm [25]
REM (Stability-Indicating) Ethyl acetate:Ethanol (96:4) TLC silica gel 60 F254 245 nm [26]

Cardiovascular drugs include Aspirin, Atenolol, Atorvastatin, and Losartan.

The selection of a mobile phase is a systematic process driven by the desired analytical outcome. The following workflow outlines the key decision points and optimization cycles involved in developing a successful HPTLC method.

G Start Start Method Development SP Select Stationary Phase (TLC Silica gel 60 F254) Start->SP Obj Define Analytical Objective (Target Analytes & Matrix) SP->Obj MP1 Select Initial Green Mobile Phase System Obj->MP1 Test Run Initial Test MP1->Test Eval Evaluate Chromatogram (Resolution, Band Shape, Rf) Test->Eval Success Success? Eval->Success Opt Optimize Parameters: - Solvent Ratios - pH Modifiers - Green Solvent Swap Opt->Test Success->Opt No Val Validate Final Method Success->Val Yes End Application to Real Samples Val->End

Figure 1: Workflow for developing and optimizing a green HPTLC method.

Detailed Experimental Protocols

Protocol 1: Simultaneous Analysis of REM, FAV, and Dexamethasone in Plasma

This protocol is adapted from a method designed for therapeutic drug monitoring in COVID-19 patients [4].

  • Materials: REM, FAV, and DEX reference standards; drug-free human plasma; methanol (HPLC grade); ethyl acetate, hexane, acetic acid (analytical grade); TLC silica gel 60 F254 plates (20 × 10 cm); micropipettes; CAMAG or equivalent HPTLC system with densitometer.
  • Standard Solution Preparation:
    • Prepare individual stock solutions of REM, FAV, and DEX at 1 mg/mL in methanol.
    • Combine appropriate volumes of stock solutions and dilute with methanol to prepare mixed working standard solutions covering the calibration range (e.g., 0.1–10 µg/band for each analyte).
  • Plasma Sample Preparation:
    • Spike drug-free plasma with known concentrations of REM, FAV, and DEX.
    • Add internal standard (e.g., Apixaban) and 3 mL of acetonitrile for protein precipitation.
    • Vortex the mixture for 1 minute, then centrifuge at 4500 rpm for 10 minutes.
    • Filter the supernatant through a 0.45 µm syringe filter before spotting.
  • Chromatographic Conditions:
    • Stationary Phase: TLC silica gel 60 F254
    • Mobile Phase: Ethyl acetate : hexane : acetic acid (9:1:0.3, v/v/v)
    • Application Volume: 10 µL as 6-mm bands
    • Development: Ascending development in a twin-trough chamber saturated for 30 min.
    • Detection: Densitometry at 254 nm.
  • Expected Outcomes: Well-resolved bands with Rf values of approximately 0.30 (REM), 0.64 (DEX), and 0.77 (FAV). The method demonstrates excellent sensitivity with LODs as low as 0.1 µg/band for REM and DEX, and 0.2 µg/band for FAV [4].

Protocol 2: Green Stability-Indicating Assay for REM

This protocol is for analyzing REM in the presence of its degradation products, crucial for stability studies [26].

  • Materials: REM reference standard; ethyl acetate, ethanol (HPLC grade); TLC silica gel 60 F254 plates; standard HPTLC equipment.
  • Forced Degradation Study:
    • Acidic Degradation: Reflux REM with 1M HCl at 70°C for 2 hours.
    • Alkaline Degradation: Reflux REM with 1M NaOH at 70°C for 2 hours.
    • Oxidative Degradation: Treat REM with 30% H2O2 at room temperature for 24 hours.
    • Neutral Hydrolysis: Reflux REM in water at 70°C for 6 hours.
    • After degradation, neutralize, dilute with methanol, and spot alongside the untreated drug.
  • Chromatographic Conditions:
    • Stationary Phase: TLC silica gel 60 F254
    • Mobile Phase: Ethyl acetate : ethanol (96:4, v/v)
    • Application Volume: 5-10 µL as bands.
    • Development: Ascending development in a saturated chamber.
    • Detection: Densitometry at 245 nm.
  • Expected Outcomes: The method successfully separates REM from its degradation products formed under various stress conditions. The peak purity of REM is confirmed, demonstrating the method's stability-indicating power. The greenness of this method is confirmed by high scores on the Analytical Eco-Scale and AGREE metrics [26].

The Scientist's Toolkit: Essential Research Reagents

The following table lists key materials and their functions for establishing these HPTLC protocols in the laboratory.

Table 2: Essential Research Reagents and Materials for HPTLC Analysis

Reagent/Material Function/Application Examples from Protocols
TLC Silica gel 60 F254 Plates Stationary phase for chromatographic separation. Standard phase for all cited methods [6] [4] [26].
Ethyl Acetate Green solvent; primary component of normal-phase mobile phases. Used in mobile phases with ethanol or hexane [6] [4] [26].
Ethanol Green polar solvent; used as mobile phase component or modifier. Primary solvent in RP method; modifier in NP methods [6].
Methanol & Acetonitrile Solvents for standard preparation and protein precipitation. Used for stock solutions and plasma sample prep [4] [14].
Acetic Acid / Ammonia Mobile phase pH modifiers to enhance separation and peak shape. Acetic acid for acidic mod.; Ammonia for basic mod. [4] [25].
Reference Standards High-purity compounds for method development and calibration. REM, FAV, DEX, etc., with certified purity >99% [4] [14].
HPTLC-Densitometry System Instrumentation for automated application, development, and quantification. CAMAG system with Linomat autosampler and TLC scanner [14] [25].
DalazatideDalazatideDalazatide is a selective Kv1.3 potassium channel blocker for autoimmune disease research. For Research Use Only. Not for human consumption.
Aprutumab IxadotinAprutumab Ixadotin, CAS:1708947-48-1, MF:C57H95N9O11, MW:1082.4 g/molChemical Reagent

Quantitative Method Performance Data

The developed methods have been rigorously validated according to ICH and FDA guidelines. The table below summarizes key performance metrics for the featured protocols.

Table 3: Validation Parameters of Featured HPTLC Methods

Validation Parameter REM, FAV, Molnupiravir [6] REM, FAV, Dexamethasone [4] REM Stability-Indicating [26]
Linearity Range 30–800 ng/band (REM) 0.1–10 µg/band for all Not Specified
Correlation Coefficient (r²) ≥ 0.99988 Not Specified Not Specified
Detection Limit (LOD) Not Specified 0.1 µg/band (REM, DEX)0.2 µg/band (FAV) Not Specified
Accuracy (% Recovery) Successfully applied to formulations 97.07% to 102.77% (spiked plasma) Complies with ICH guidelines
Precision Complies with ICH guidelines Complies with FDA guidelines Complies with ICH guidelines
Greenness Assessment AES, AGREE, MoGAPI, BAGI, RGB12 RGB12 (Whiteness: 95.6%) AES, GAPI, AGREE

The optimal chromatographic conditions for the analysis of Remdesivir with co-administered drugs via HPTLC have been firmly established. The consistent use of silica gel 60 F254 plates with mobile phases predominantly composed of ethyl acetate, ethanol, and minimal modifiers provides robust, reproducible, and green separation platforms. The detailed protocols provided herein empower researchers to implement these methods for quality control, therapeutic drug monitoring, and stability studies, effectively supporting the advancement of green analytical chemistry in pharmaceutical analysis. The high whiteness and greenness scores of these methods, as evaluated by modern metrics, confirm their alignment with sustainable development goals in pharmaceutical research.

Systematic Sample Preparation Protocols for Pharmaceutical Formulations and Spiked Human Plasma

The development of eco-friendly and cost-effective analytical methods is a central pursuit in modern pharmaceutical analysis. This application note provides detailed protocols for the sample preparation of pharmaceutical formulations and spiked human plasma, contextualized within green High-Performance Thin-Layer Chromatography (HPTLC) research for analyzing the COVID-19 antiviral remdesivir alongside its frequently co-administered drugs. The methods outlined emphasize green chemistry principles, cost-effectiveness, and applicability in both quality control and clinical therapeutic drug monitoring settings [14] [27].

Research Reagent Solutions

The following table details the essential materials and reagents required for the sample preparation and analysis of remdesivir and co-administered drugs.

Table 1: Key Research Reagents and Materials for HPTLC Analysis

Item Name Function / Application Specifications / Notes
Remdesivir Reference Standard Primary analyte for calibration and quantification [14] Purity ≥ 99.8%; requires storage in refrigerator [14].
Co-administered Drug Standards Secondary analytes (e.g., Linezolid, Rivaroxaban, Favipiravir, Dexamethasone) [14] [4] Critical for simulating combination therapy in validation studies [14].
HPLC-grade Methanol Solvent for stock and working solution preparation [14] [4] Preferred for dissolving analytes and precipitating plasma proteins.
Drug-free Human Plasma Biological matrix for method development and validation [14] Sourced from blood banks; stored at -20 °C until use [14] [4].
TLC Silica Gel 60 F₂₅₄ Plates Stationary phase for HPTLC separation [14] Aluminum sheets, 20 × 20 cm, 0.1 mm thickness [14].
Microsyringe (100 µL) Application of samples onto TLC plates [14] Used with autosampler for precise, band-wise application [14].
Centrifuge Separation of supernatant after protein precipitation [14] Critical step for cleaning up plasma samples prior to spotting [14].
Syringe Filter (0.45 µm) Final filtration of samples before application [4] Ensures particulate matter does not interfere with spotting or chromatography [4].

Quantitative Method Validation Data

The green HPTLC methods for remdesivir combinations have demonstrated excellent analytical performance. The following table summarizes key validation parameters as reported in recent studies.

Table 2: Summary of Validated Analytical Parameters for Remdesivir and Co-administered Drugs by HPTLC

Analyte Combination Linear Range (µg/band) Limit of Quantification (LOQ) Accuracy (% Recovery) Precision (RSD%) Ref.
Remdesivir, Linezolid, Rivaroxaban REM: 0.2-5.5LNZ: 0.2-4.5RIV: 0.1-3.0 REM: 128.8 ng/bandLNZ: 50.5 ng/bandRIV: 55.8 ng/band 98.3 - 101.2% (Formulation & Plasma) N/S [14]
Remdesivir, Favipiravir, Dexamethasone REM: 0.1-10DEX: 0.1-10FVP: 0.2-15 REM: 0.1 µg/bandDEX: 0.1 µg/bandFVP: 0.2 µg/band 97.07 - 102.77% (Spiked Plasma) N/S [4]
Remdesivir, Favipiravir REM: N/SFAV: N/S REM: 0.12 µg/bandFAV: 0.07 µg/band 97.21 - 101.31% (Formulation & Plasma) N/S [27]

Abbreviations: REM (Remdesivir); LNZ (Linezolid); RIV (Rivaroxaban); DEX (Dexamethasone); FVP (Favipiravir); RSD (Relative Standard Deviation); N/S (Not Specified in provided excerpts).

Experimental Protocols

Protocol 1: Sample Preparation for Pharmaceutical Formulations

This protocol is adapted from methods used for Remdesivir-Rameda concentrate for I.V. infusion and Linezolid I.V. solution [14].

Workflow Overview:

G Start Start: Pharmaceutical Formulation P1 Accurately weigh or measure the sample equivalent to 10 mg API Start->P1 P2 Transfer to 10-mL volumetric flask P1->P2 P3 Add 5 mL methanol and shake thoroughly P2->P3 P4 Dilute to volume with methanol and mix P3->P4 P5 Optional: Further dilute for working solutions P4->P5 P6 Result: Ready for HPTLC analysis P5->P6

Step-by-Step Procedure:

  • For Solid Formulations (e.g., tablets): Accurately weigh and powder not less than ten tablets. Transfer an amount of the powder equivalent to about 10 mg of the active pharmaceutical ingredient (API) into a 10-mL volumetric flask.
  • For Liquid Formulations (e.g., I.V. infusion): Accurately measure a volume of the solution equivalent to 10 mg of the API using a calibrated micropipette and transfer it to a 10-mL volumetric flask.
  • Add approximately 5 mL of methanol to the flask. Shake thoroughly or sonicate for 10-15 minutes to ensure complete dissolution and extraction of the API.
  • Dilute the solution to the mark (10 mL) with methanol and mix well. This yields a stock solution with a concentration of approximately 1 mg/mL.
  • If necessary, prepare working solutions by making appropriate serial dilutions of this stock solution with methanol to fit the calibrated concentration range.
  • The solutions are now ready for application onto the HPTLC plate.
Protocol 2: Sample Preparation for Spiked Human Plasma

This protocol details the procedure for preparing plasma samples spiked with remdesivir and co-administered drugs, incorporating an internal standard as described in recent literature [14] [4].

Workflow Overview:

G Start Start: Blank Human Plasma P1 Thaw plasma at room temperature and vortex mix Start->P1 P2 Spike with drug working solutions and internal standard (e.g., Apixaban) P1->P2 P3 Vortex mix for 1 minute P2->P3 P4 Add protein precipitation solvent (e.g., Methanol or Acetonitrile) P3->P4 P5 Vortex mix vigorously for 3 minutes P4->P5 P6 Centrifuge at 4500 rpm for 10 minutes P5->P6 P7 Collect supernatant and filter through 0.45 µm syringe filter P6->P7 P8 Result: Clear extract for HPTLC P7->P8

Step-by-Step Procedure:

  • Plasma Pre-treatment: Thaw frozen, drug-free human plasma at room temperature or in a refrigerated water bath. Mix gently using a vortex mixer to ensure homogeneity [14].
  • Spiking: Transfer 1 mL of plasma into a clean centrifuge tube. Spike it with appropriate volumes of standard working solutions of remdesivir and the co-administered drug(s) to achieve the desired calibration concentrations. For improved accuracy, also spike with an internal standard such as Apixaban (e.g., a final concentration of 5 µg/band) [4].
  • Initial Mixing: Vortex the mixture for approximately 1 minute to ensure thorough integration of the analytes with the plasma matrix.
  • Protein Precipitation: Add a volume of organic solvent, typically methanol or acetonitrile (commonly 2-3 times the volume of plasma), to precipitate plasma proteins.
  • Vigorous Mixing: Vortex the mixture vigorously for at least 3 minutes to ensure complete protein precipitation and analyte extraction.
  • Centrifugation: Centrifuge the samples at high speed (e.g., 3500 - 4500 rpm) for 10 minutes to compact the protein pellet [14] [4].
  • Clarification and Filtration: Carefully collect the clear supernatant. Pass it through a 0.45 µm syringe filter to remove any remaining particulate matter [4].
  • The resulting clear filtrate is now ready for application onto the HPTLC plate.

Critical Operational Notes

  • Solution Stability: Standard stock solutions in methanol have been reported to remain stable for at least 14 days when stored in a refrigerator [14].
  • Plasma Sample Integrity: Blank human plasma should be stored at -20 °C until use to preserve its integrity and prevent degradation. Avoid repeated freeze-thaw cycles for spiked samples [14] [4].
  • Green Chemistry Alignment: The described sample preparation, combined with HPTLC analysis using solvent systems like ethyl acetate/hexane/acetic acid or dichloromethane/acetone, aligns with the principles of green analytical chemistry by minimizing solvent waste and utilizing less hazardous materials where possible [14] [4] [27].

Application and Development Techniques for High-Resolution Band Separation

High-resolution band separation is a critical objective in modern analytical chemistry, particularly in the pharmaceutical analysis of complex drug mixtures. Within the context of green High-Performance Thin-Layer Chromatography (HPTLC) research, this technique enables the simultaneous quantification of therapeutic agents with minimal environmental impact. The analysis of remdesivir—the first FDA-approved antiviral for COVID-19—with its co-administered drugs presents a significant challenge due to their diverse chemical structures and the complexity of biological matrices. This article details advanced methodologies for achieving superior band separation, with specific application to the simultaneous analysis of remdesivir alongside frequently co-administered medications including favipiravir, dexamethasone, linezolid, and rivaroxaban in spiked human plasma [4] [9]. The protocols outlined emphasize green chemistry principles through solvent selection and miniaturized processes, aligning with the current paradigm of sustainable analytical science.

Experimental Protocols

HPTLC Method for Remdesivir, Favipiravir, and Dexamethasone

This protocol describes a simultaneous determination of three COVID-19 therapeutic agents in human plasma using apixaban as an internal standard [4].

Materials and Equipment:

  • Stationary Phase: TLC silica gel 60 F254 plates (Merck, Darmstadt, Germany)
  • Sample Applicator: Linomat 5 autosampler with 100 µL microsyringe (CAMAG)
  • Development Chamber: Twin-trough glass chamber (20 × 20 cm)
  • Scanner: TLC scanner 3 with winCATS software (CAMAG)
  • Centrifuge: Capable of 4500 rpm
  • Syringe Filter: 0.45 µm Millipore

Reagents:

  • Remdesivir, Favipiravir, Dexamethasone (reference standards, purity >99%)
  • Apixaban (internal standard)
  • Ethyl acetate, hexane, acetic acid (HPLC grade)
  • Methanol (HPLC grade)
  • Human plasma (blank)

Detailed Procedure:

  • Standard Solution Preparation: Accurately weigh 25 mg each of REM, FVP, DEX, and PX. Transfer to separate 25 mL volumetric flasks, dissolve in and dilute to volume with methanol to obtain 1 mg/mL stock solutions. Further dilute to prepare working solutions as needed.
  • Plasma Sample Preparation: Spike 1 mL of thawed human plasma with appropriate volumes of drug working solutions and 1 mL of PX internal standard solution (5 µg/band). Dilute to 10 mL with methanol.
  • Protein Precipitation: Vortex the mixture for 1 minute, then centrifuge at 4500 rpm for 10 minutes. Filter the supernatant through a 0.45 µm syringe filter.
  • Chromatographic Application: Using the Linomat 5 applicator, apply 10 µL of the prepared sample as 6 mm bands on the TLC plate, maintaining 5 mm inter-band distance and 10 mm from the bottom edge.
  • Chromatographic Development: Condition the mobile phase chamber for 30 minutes with ethyl acetate:hexane:acetic acid (9:1:0.3, v/v/v). Develop the plate to a distance of 9 cm in a saturated twin-trough chamber.
  • Densitometric Analysis: Air-dry the developed plate and scan at 254 nm using the TLC scanner. Well-resolved bands should be observed with Rf values of approximately 0.3 for remdesivir, 0.64 for dexamethasone, and 0.77 for favipiravir.

Method Validation:

  • The method demonstrates linearity ranges of 0.1–10 µg/band for REM and DEX, and 0.2–15 µg/band for FVP.
  • Recovery from spiked human plasma ranges from 97.07% to 102.77%.
  • The whiteness assessment using the RGB12 algorithm shows a score of 95.6%, indicating excellent sustainability [4].
Eco-Friendly HPTLC Method for Remdesivir with Linezolid and Rivaroxaban

This protocol details a green, cost-effective HPTLC method for quantifying remdesivir with co-administered linezolid (antibiotic) and rivaroxaban (anticoagulant) in spiked human plasma [9].

Materials and Equipment:

  • Stationary Phase: TLC silica gel 60 F254 aluminum plates (20 × 20 cm, 0.1 mm thickness)
  • Mobile Phase: Dichloromethane:acetone (8.5:1.5, v/v)
  • Detection: Densitometric scanning at 254 nm

Detailed Procedure:

  • Standard Solution Preparation: Prepare individual stock solutions of REM, LNZ, and RIV (1 mg/mL) in methanol (for REM and LNZ) or acetonitrile (for RIV).
  • Calibration Standards: Spot 10 µL aliquots of working solutions to obtain concentration ranges of 0.2–5.5 µg/band for REM, 0.2–4.5 µg/band for LNZ, and 0.1–3.0 µg/band for RIV.
  • Chromatographic Development: Saturate the chromatographic jar with mobile phase for 30 minutes prior to development. Apply samples 1.0 cm from the bottom edge, develop, and air-dry plates.
  • Detection and Quantification: Scan plates at 254 nm. Typical Rf values are 0.23 for REM, 0.53 for LNZ, and 0.72 for RIV.

Method Performance:

  • Limits of quantification: 128.8 ng/band for REM, 50.5 ng/band for LNZ, and 55.8 ng/band for RIV.
  • Recovery from pharmaceutical formulations and spiked human plasma: 98.3% to 101.2%.
  • Greenness confirmed by Analytical Eco-scale, GAPI, and AGREE metrics [9].

Data Presentation and Performance Comparison

Table 1: Chromatographic Conditions and Performance Characteristics of HPTLC Methods for Remdesivir and Co-administered Drugs

Parameter REM, FVP, DEX Method [4] REM, LNZ, RIV Method [9] Normal-phase HPTLC for REM, FVP, MOL [16]
Mobile Phase Ethyl acetate:hexane:acetic acid (9:1:0.3, v/v/v) Dichloromethane:acetone (8.5:1.5, v/v) Ethyl acetate:ethanol:water (9.4:0.4:0.25, v/v)
Detection Wavelength 254 nm 254 nm 244 nm (RMD, MOL), 325 nm (FAV)
Linearity Range REM: 0.1-10 µg/bandDEX: 0.1-10 µg/bandFVP: 0.2-15 µg/band REM: 0.2-5.5 µg/bandLNZ: 0.2-4.5 µg/bandRIV: 0.1-3.0 µg/band RMD: 30-800 ng/bandFAV: 50-2000 ng/bandMOL: 50-2000 ng/band
Rf Values REM: 0.3DEX: 0.64FVP: 0.77 REM: 0.23LNZ: 0.53RIV: 0.72 Not specified
LOQ REM: 0.1 µg/bandDEX: 0.1 µg/bandFVP: 0.2 µg/band REM: 128.8 ng/bandLNZ: 50.5 ng/bandRIV: 55.8 ng/band Not specified
Application Spiked human plasma Spiked human plasma, pharmaceutical formulations Bulk form, pharmaceutical formulations

Table 2: Sustainability Assessment of Different HPTLC Methods Using Modern Green Metrics

Method Description Analytical Eco-Scale GAPI AGREE RGB12 (Whiteness)
REM, FVP, DEX in plasma [4] Not specified Not specified Not specified 95.6%
Normal-phase HPTLC for REM, FVP, MOL [16] Favorable Favorable Favorable High whiteness score
Stability-indicating REM method [15] High score (eco-friendly) Green profile Green profile Not specified

Visualization of Methodologies

HPTLC Band Separation Workflow

G SamplePrep Sample Preparation Extraction Protein Precipitation & Filtration SamplePrep->Extraction Plasma Human Plasma Plasma->SamplePrep Standards Reference Standards Standards->SamplePrep Application Band Application on HPTLC Plate Extraction->Application Development Chromatographic Development Application->Development Detection Densitometric Detection Development->Detection Analysis Data Analysis & Quantification Detection->Analysis Results Validated Results Analysis->Results

Band Separation Mechanism in HPTLC

G cluster_Optimization Critical Separation Parameters MobilePhase Mobile Phase Composition Separation Differential Migration Based on Polarity MobilePhase->Separation StationaryPhase Silica Gel Stationary Phase (F254) StationaryPhase->Separation Resolution Band Resolution (Rf Calculation) Separation->Resolution Detection UV Detection at Specific Wavelength Resolution->Detection Polarity Solvent Polarity Optimization Polarity->Separation Saturation Chamber Saturation Time Saturation->Separation Humidity Ambient Humidity Control Humidity->Separation

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials and Reagents for HPTLC Analysis of Remdesivir Combinations

Item Specification/Function Application Example
Stationary Phase TLC silica gel 60 F254 on aluminum sheets, 20×20 cm, 0.1 mm thickness; F254 indicates fluorescent indicator for UV detection at 254 nm All cited methods [4] [9] [15]
Mobile Phase Components Ethyl acetate, hexane, acetic acid, dichloromethane, acetone; varying proportions create optimal polarity for separation of target analytes REM-FVP-DEX: Ethyl acetate:hexane:acetic acid (9:1:0.3) [4]
Reference Standards High-purity (>99%) analytical standards of remdesivir, favipiravir, dexamethasone, linezolid, rivaroxaban for calibration and quantification Method development and validation [4] [9]
Internal Standard Apixaban; corrects for analytical variability in sample preparation and application REM-FVP-DEX method in human plasma [4]
Sample Application System Linomat 5 autosampler with 100 µL microsyringe; ensures precise, reproducible band application for accurate quantification All automated HPTLC methods [4] [9]
Densitometer TLC scanner 3 with deuterium lamp and winCATS software; measures absorbance of separated bands at optimal wavelengths Quantitative analysis at 254 nm [4] [9]
Development Chamber Twin-trough glass chamber 20×20 cm; allows for saturation with mobile phase vapor prior to development All planar chromatography methods [4] [15]
Sample Preparation Materials Centrifuge (4500 rpm capability), vortex mixer, 0.45 µm syringe filters; for protein precipitation and clarification of plasma samples Biological sample preparation [4] [9]
CoccinineCoccinine, (+/-)-|Amaryllidaceae Alkaloid|RUOCoccinine, (+/-)- is a montanine-type Amaryllidaceae alkaloid for research use. This product is For Research Use Only (RUO) and is not intended for personal use.
PomotrelvirPomotrelvir|CAS 2713437-86-4|SARS-CoV-2 InhibitorPomotrelvir is a potent, oral SARS-CoV-2 Mpro inhibitor for COVID-19 research. This product is For Research Use Only. Not for human consumption.

Within the framework of green High-Performance Thin-Layer Chromatography (HPTLC) research for the analysis of remdesivir (REM) and its co-administered drugs, densitometric detection serves as a pivotal technique for accurate quantification. This protocol details the critical procedures for wavelength selection and peak integration strategies, which are fundamental to achieving precise, reproducible, and reliable results while adhering to the principles of green analytical chemistry. Proper implementation of these steps ensures method specificity, sensitivity, and validity in accordance with International Council for Harmonisation (ICH) guidelines [28].

Core Principles of Densitometric Detection

Densitometry in HPTLC is a post-chromatography quantification technique where the developed plate is scanned with light, and the amount of light absorbed or emitted by the analyte bands is measured. For absorption measurement, the fundamental relationship is governed by the Beer-Lambert law, which states that the absorbance (A) of a compound is directly proportional to its concentration (c) and the path length (l): A = εcl, where ε is the molar absorptivity. This principle forms the basis for quantitative analysis.

The process involves scanning each track on the HPTLC plate with a slit of monochromatic light. The reflected or transmitted light is measured by a detector, converting the signal into a chromatogram where peak area or height corresponds to analyte quantity [29]. Advanced densitometers can also perform spectral scanning, which acquires the entire spectrum of an analyte directly from the plate, aiding in identity confirmation and purity assessment by comparing sample and standard spectra [28].

Key Instrumentation Components

The typical HPTLC-densitometry system consists of:

  • Light Source: Deuterium lamp (UV range) and tungsten lamp (visible range)
  • Monochromator: For wavelength selection with bandwidth control
  • Detector: Photomultiplier tube or diode array detector
  • Scanning Stage: Precise mechanical stage for controlled plate movement
  • Software: For instrument control, data acquisition, and peak integration

Wavelength Selection Strategies

Determination of Optimal Wavelength

The optimal detection wavelength is compound-specific and determined through spectral analysis. The recommended procedure is as follows:

  • Apply standard solutions of the target analytes (e.g., REM, favipiravir, dexamethasone) and potential co-administered drugs on the HPTLC plate.
  • Develop the plate using the optimized green mobile phase.
  • Air-dry the plate completely to eliminate solvent interference.
  • Perform spectral scanning directly from the plate using the densitometer's spectrum mode across the range of 200–400 nm (or appropriate range).
  • Identify wavelength of maximum absorption (λmax) for each compound from the obtained spectra.
  • Select a single wavelength that accommodates all analytes of interest, or program multiple wavelengths for scanning different analyte zones.

For the analysis of REM with co-administered drugs, researchers have successfully employed 254 nm for simultaneous detection of REM, dexamethasone, and favipiravir, as all three compounds exhibit sufficient absorption at this wavelength [30]. Alternatively, a multi-wavelength approach can be implemented where different sections of the chromatogram are analyzed at their respective optimal wavelengths.

Table 1: Wavelength Selection for COVID-19 and Co-Administered Drugs

Analyte Optimal Wavelength (nm) Alternative Wavelength (nm) Application Context
Remdesivir 254 [30] 240 [31] With dexamethasone, favipiravir
Favipiravir 254 [30] 230 With remdesivir, dexamethasone
Dexamethasone 254 [30] 240 With remdesivir, favipiravir
Linezolid 254 [9] - With remdesivir, rivaroxaban
Rivaroxaban 254 [9] - With remdesivir, linezolid
Caffeine 275 [32] 273 Green RP-HPTLC method

Wavelength Optimization Protocol

Materials:

  • CAMAG HPTLC system with densitometer (TLC Scanner 3) or equivalent
  • HPTLC plates precoated with silica gel 60 F254
  • Standard solutions of target analytes (1 mg/mL in methanol)
  • Micropipettes (5-100 μL)
  • Chromatographic chamber

Procedure:

  • Spot standard solutions of each analyte (REM, co-administered drugs) on HPTLC plate as 6-mm bands.
  • Develop plate in pre-saturated chamber with optimized mobile phase (e.g., ethyl acetate:hexane:acetic acid, 9:1:0.3 v/v/v for REM mixtures) [30].
  • Dry plate completely in air for 15-20 minutes.
  • Place plate in densitometer and initiate spectrum mode.
  • Scan each analyte band across 200-400 nm range, using blank plate area for background correction.
  • Identify λmax for each compound from the overlay spectra.
  • Select final detection wavelength that provides adequate sensitivity for all target analytes while minimizing background noise.

Critical Notes:

  • Ensure plate background is uniform with minimal baseline drift at selected wavelength.
  • For methods employing multiple wavelengths, validate that changing wavelengths does not cause baseline shifts.
  • Confirm that excipients or matrix components from formulation or biological samples do not interfere at the selected wavelength [28].

Peak Integration Strategies

Parameter Optimization for Accurate Integration

Peak integration transforms the analog signal from the densitometer into digital data representing peak area/height, concentration, and chromatographic parameters (Rf, asymmetry). Proper integration parameter setting is crucial for accurate quantification.

Table 2: Key Integration Parameters and Their Optimization

Parameter Function Optimization Strategy Typical Values
Slit Dimensions Controls light beam size on plate Adjust based on band size; smaller for narrow bands 4-6 mm length, 0.2-0.45 mm width [32] [33]
Scanning Speed Rate of plate movement during scanning Balance between signal noise and analysis time 5-20 mm/s [29] [33]
Data Resolution Distance between measurement points Higher resolution for better peak definition 50-100 μm/step
Peak Detection Threshold Minimum signal-to-noise for peak recognition Set to eliminate background noise without missing analyte peaks 3-5 times baseline noise
Minimum Peak Area Filters out insignificant peaks Determine based on limit of quantification Compound-dependent

Peak Integration Protocol

Software-Specific Steps for winCATS or Similar Platforms:

  • Initiate Data Acquisition:

    • Select appropriate wavelength based on prior optimization.
    • Set scanning speed to 20 mm/s for initial scans [9].
    • Define scan start and end positions to cover entire migration distance.

  • Baseline Correction:

    • Scan blank track for background subtraction.
    • Select polynomial or linear baseline correction based on baseline characteristics.
    • Apply baseline correction to all sample tracks.

  • Peak Detection and Integration:

    • Set peak width to 5-10 seconds to match typical HPTLC peak profiles.
    • Adjust peak sensitivity to detect all analyte peaks without integrating noise.
    • Apply minimum area threshold of 100-500 AU for reliable peak recognition.

  • Peak Assignment and Calibration:

    • Assign peaks to specific analytes based on Rf values (REM: ~0.30, DEX: ~0.64, FVP: ~0.77) [30].
    • Generate calibration curve using peak areas of standard concentrations.
    • Apply regression model (linear/quadratic) with correlation coefficient (r) >0.999 [28].

  • Validation of Integration:

    • Manually review integrated peaks for accuracy.
    • Check baseline placement, especially for partially resolved peaks.
    • Verify peak start and end points match visual inspection.

Advanced Integration Techniques

For complex separations with co-administered drugs or matrix interference, implement these advanced strategies:

  • Multi-Wavelength Integration:

    • Integrate different analytes at their respective optimal wavelengths.
    • Merge data from multiple chromatograms for comprehensive analysis.

  • Spectral Confirmation:

    • Compare in-situ spectra of sample peaks with reference standards.
    • Apply spectral correlation filters to confirm peak purity and identity [28].

  • Time-Resolved Fluorescence Detection:

    • For fluorescent compounds, use time-gated detection to minimize background interference [29].
    • Measure fluorescence lifetimes for additional confirmation parameters.

Experimental Protocol: Densitometric Analysis of Remdesivir with Co-Administered Drugs

Materials and Reagents

Table 3: Essential Research Reagent Solutions

Reagent/Material Specification Function Green Alternative Consideration
HPTLC Plates Silica gel 60 F254, aluminum-backed Stationary phase for separation -
Remdesivir Standard ≥99% purity [30] Primary reference standard -
Dexamethasone Standard ≥99% purity [30] Co-administered drug standard -
Favipiravir Standard ≥99% purity [30] Co-administered drug standard -
Mobile Phase Components Ethyl acetate, hexane, acetic acid (9:1:0.3 v/v/v) [30] Solvent system for separation Ethanol-water mixtures [32]
Methanol HPLC grade Standard and sample preparation -
Apixaban ≥98% purity [30] Internal standard -
Human Plasma Drug-free Matrix for spiked samples -

Step-by-Step Procedure

Step 1: Sample Preparation

  • Prepare stock solutions (1 mg/mL) of REM, DEX, FVP, and internal standard in methanol.
  • For spiked plasma samples: Add known concentrations of analytes to drug-free human plasma (1 mL).
  • Precipitate proteins by adding methanol (2 mL), vortex for 1 minute, centrifuge at 4500 rpm for 10 minutes [30].
  • Filter supernatant through 0.45 μm syringe filter.

Step 2: Application and Chromatography

  • Apply samples and standards as 6-mm bands using automatic applicator (application rate: 150 nL/s).
  • Develop plate in twin-trough chamber pre-saturated with mobile phase for 20 minutes.
  • Use mobile phase: ethyl acetate:hexane:acetic acid (9:1:0.3 v/v/v) for REM/DEX/FVP separation [30].
  • Develop to distance of 80 mm, dry plate in air.

Step 3: Densitometric Scanning

  • Place dried plate in densitometer.
  • Set scanning wavelength to 254 nm.
  • Configure slit dimensions to 6 × 0.45 mm.
  • Set scanning speed to 20 mm/s.
  • Perform scanning from start to end of migration.

Step 4: Data Analysis

  • Integrate peaks using winCATS or similar software.
  • Identify peaks based on Rf values: REM (~0.30), DEX (~0.64), FVP (~0.77) [30].
  • Generate calibration curves (peak area vs concentration) for each analyte.
  • Calculate concentrations in unknown samples using linear regression.

G Densitometric Analysis Workflow cluster_1 1. Pre-Analysis Preparation cluster_2 2. Wavelength Selection cluster_3 3. Instrument Parameter Setting cluster_4 4. Data Acquisition & Processing P1 Standard and Sample Preparation P2 HPTLC Plate Spotting P1->P2 P3 Chromatographic Development P2->P3 P4 Plate Drying P3->P4 W1 Spectral Scanning (200-400 nm) P4->W1 W2 Identify λmax for Each Analyte W1->W2 W3 Select Optimal Detection Wavelength W2->W3 I1 Configure Slit Dimensions W3->I1 I2 Set Scanning Speed I1->I2 I3 Define Data Resolution I2->I3 D1 Plate Scanning and Data Acquisition I3->D1 D2 Baseline Correction D1->D2 D3 Peak Detection and Integration D2->D3 D4 Calibration and Quantification D3->D4

Troubleshooting and Quality Assurance

Common Integration Issues and Solutions

  • Baseline Drift: Ensure proper plate drying before scanning; apply background subtraction.
  • Peak Tailing: Optimize mobile phase to improve peak symmetry; verify application technique.
  • Poor Resolution: Adjust mobile phase composition; check chamber saturation conditions.
  • Low Signal-to-Noise: Confirm wavelength is at λmax; increase sample concentration if necessary.
  • Irreproducible Rf Values: Standardize chamber saturation time; control laboratory temperature.

Method Validation Parameters

For regulatory acceptance, validate the densitometric method for:

  • Linearity: Correlation coefficient (r) >0.999 [28] over working range
  • Precision: Relative standard deviation (RSD) <2% for repeatability [28]
  • Accuracy: Recovery of 97-103% for pharmaceutical formulations [9]
  • Limit of Detection (LOD) and Quantification (LOQ): Signal-to-noise ratio of 3:1 and 10:1, respectively [28]
  • Specificity: No interference from excipients or co-administered drugs confirmed by spectral overlap [28]

Proper wavelength selection and peak integration are fundamental to successful quantification of remdesivir and co-administered drugs using green HPTLC-densitometry. The strategies outlined in this protocol ensure accurate, precise, and reproducible results while minimizing solvent consumption and waste generation, aligning with green analytical chemistry principles. Implementation of these optimized detection parameters facilitates reliable therapeutic drug monitoring and pharmacokinetic studies for COVID-19 treatment regimens.

Specific Protocols for Different Drug Combinations (e.g., Remdesivir with Favipiravir, Dexamethasone, or Cardiovascular Drugs)

The co-administration of remdesivir (REM) with other therapeutics represents a common clinical strategy for managing COVID-19, encompassing combinations with the antiviral favipiravir (FVP), the corticosteroid dexamethasone (DEX), and supportive care medications like the antibiotic linezolid (LNZ) and the anticoagulant rivaroxaban (RIV). This application note details validated, green High-Performance Thin-Layer Chromatography (HPTLC) protocols for the simultaneous quantification of these drug combinations in pharmaceutical formulations and spiked human plasma. The described methods align with the principles of Green Analytical Chemistry (GAC), offering cost-effective, high-throughput, and environmentally friendly alternatives to conventional chromatographic techniques for therapeutic drug monitoring and quality control in research settings.

The complexity of COVID-19 treatment often necessitates multi-drug regimens to address both viral replication and the debilitating symptoms of the disease. The synergistic benefits of combining remdesivir and favipiravir have been demonstrated, showing more effective reduction of viral load and inflammation than either drug used alone [4]. Similarly, corticosteroids like dexamethasone and anticoagulants are routinely incorporated into global treatment protocols [4] [14]. This creates an urgent need for robust analytical methods capable of monitoring these drug combinations in biological fluids to adjust therapeutic doses and conduct pharmacokinetic studies.

Green HPTLC has emerged as a superior analytical approach in this context. It allows for the separation of multiple analytes concurrently with minimal solvent consumption and relatively simple sample preparation, reducing both environmental impact and operational costs [4] [34]. This document provides detailed application notes and protocols for analyzing key remdesivir drug combinations, supporting research within a broader thesis on green analytical techniques.

Summarized Analytical Methods and Quantitative Data

The following table summarizes the key parameters for two established green HPTLC methods for the simultaneous determination of remdesivir in different drug combinations.

Table 1: Summary of Validated Green HPTLC Methods for Remdesivir Combination Analysis

Parameter Method A: REM, FVP & DEX [4] Method B: REM, LNZ & RIV [14]
Drug Combination Remdesivir, Favipiravir, Dexamethasone Remdesivir, Linezolid, Rivaroxaban
Stationary Phase TLC silica gel 60 Fâ‚‚â‚…â‚„ plates TLC silica gel 60 Fâ‚‚â‚…â‚„ plates
Mobile Phase Ethyl acetate : hexane : acetic acid (9:1:0.3, v/v/v) Dichloromethane : acetone (8.5:1.5, v/v)
Detection Wavelength 254 nm 254 nm
Retention Factor (RÆ’) REM: 0.30; DEX: 0.64; FVP: 0.77 REM: 0.23; LNZ: 0.53; RIV: 0.72
Linear Range (µg/band) REM: 0.1–10.0; DEX: 0.1–10.0; FVP: 0.2–15.0 REM: 0.2–5.5; LNZ: 0.2–4.5; RIV: 0.1–3.0
Limit of Quantification (LOQ) REM & DEX: 0.1 µg/band; FVP: 0.2 µg/band REM: 128.8 ng/band; LNZ: 50.5 ng/band; RIV: 55.8 ng/band
Application Spiked human plasma Pharmaceutical formulations & spiked human plasma
Greenness Assessment RGB algorithm: 95.6% whiteness [4] Analytical Eco-Scale, GAPI, and AGREE metrics [14]

Experimental Protocols

This protocol is designed for therapeutic drug monitoring of a common COVID-19 antiviral and corticosteroid combination.

The Scientist's Toolkit: Research Reagents and Equipment

Table 2: Essential Materials for Protocol 1

Item Function / Specification
HPTLC System CAMAG system including autosampler, TLC scanner, and winCATS software.
Stationary Phase TLC silica gel 60 F₂₅₄ aluminum sheets (20 × 20 cm).
Microsyringe 100 µL (e.g., Hamilton) for precise sample application.
Reference Standards Remdesivir, Favipiravir, Dexamethasone (high purity, ≥99%).
Internal Standard Apixaban (for volume correction in sample preparation).
Solvents Ethyl acetate, hexane, acetic acid, methanol (HPLC grade).
Human Plasma Drug-free human plasma, stored at -20 °C until use.
Laboratory Equipment Vortex mixer, centrifuge, 0.45 µm syringe filters.
Sample Preparation Procedure
  • Stock Solutions: Prepare individual 1 mg/mL stock solutions of REM, FVP, DEX, and the internal standard (apixaban) in methanol.
  • Working Solutions: Dilute stock solutions with methanol to obtain working standards.
  • Plasma Spiking: In a 10 mL volumetric flask, transfer specific volumes of REM, FVP, and DEX working solutions. Add 1 mL of thawed human plasma and a fixed volume of the internal standard working solution.
  • Protein Precipitation: Dilute the mixture to volume with methanol. Vortex the solution for 1 minute to ensure mixing.
  • Centrifugation and Filtration: Centrifuge the solution at 4500 rpm for 10 minutes. Filter the supernatant through a 0.45 µm syringe filter to remove any particulate matter.
Chromatographic Separation and Analysis
  • Application: Using an autosampler, apply 10 µL of the prepared supernatant as 6 mm-wide bands on the TLC plate. Maintain a distance of 5 mm between bands and 10 mm from the bottom edge.
  • Plate Development: Saturate the twin-trough chromatography chamber with the mobile phase (ethyl acetate : hexane : acetic acid, 9:1:0.3, v/v/v) for 30 minutes. Develop the plate to a distance of 9 cm.
  • Detection and Quantification: Air-dry the developed plate and scan using a TLC scanner at 254 nm. Identify the drugs via their RÆ’ values and quantify them based on peak area using the internal standard method.

start Start Sample Preparation prep Prepare Stock & Working Solutions in Methanol start->prep spike Spike Human Plasma with Drugs and Internal Standard prep->spike ppt Precipitate Proteins with Methanol spike->ppt cent Centrifuge and Filter Supernatant (0.45 µm) ppt->cent spot Spot 10 µL on HPTLC Plate cent->spot dev Develop in Mobile Phase: EtOAc:Hexane:AcOH (9:1:0.3) spot->dev scan Scan Plate at 254 nm dev->scan quant Quantify via Peak Area and Rf Values scan->quant end Data Analysis Complete quant->end

This protocol is suited for analyzing remdesivir alongside commonly co-administered supportive care drugs for secondary infections and thrombosis prevention.

Key Materials
  • Equipment & Materials: Same as Protocol 1 (CAMAG HPTLC system, TLC plates, microsyringe).
  • Reference Standards: Remdesivir, Linezolid, Rivaroxaban.
  • Solvents: Dichloromethane, acetone, methanol, acetonitrile (HPLC grade).
Method Details
  • Standard Solution Preparation: Prepare individual 1 mg/mL stock solutions of REM, LNZ, and RIV in methanol (REM and LNZ) and acetonitrile (RIV). Mix and dilute to obtain calibration standards.
  • Chromatography: Spot 10 µL of standard or prepared sample solutions on the TLC plate. Saturate the chamber with dichloromethane:acetone (8.5:1.5, v/v) for 30 minutes. Develop the plate and scan at 254 nm.
  • Analysis: The RÆ’ values provide clear separation, and quantification is achieved via external calibration curves.

Greenness Assessment

The environmental sustainability of the described HPTLC methods is a core advantage. Method A was evaluated using the RGB algorithm, achieving a high whiteness percentage of 95.6%, indicating excellent alignment with green analytical principles [4]. Method B was assessed with multiple modern metrics, including Analytical Eco-Scale, GAPI, and AGREE, confirming its eco-friendly profile [14]. The minimal solvent consumption and use of less hazardous solvents (like ethyl acetate) compared to traditional HPLC mobile phases contribute significantly to this green characteristic.

The detailed green HPTLC protocols provided herein enable reliable, simultaneous quantification of remdesivir in critical drug combinations. The methods for REM-FVP-DEX and REM-LNZ-RIV are sensitive, selective, and validated for application in spiked human plasma, making them suitable for therapeutic drug monitoring and pharmacokinetic studies. Their high-throughput nature and adherence to green chemistry principles make them invaluable tools for researchers and drug development professionals, offering a sustainable and practical approach to analyzing complex drug regimens.

Optimizing Resolution and Overcoming Common HPTLC Challenges

Utilizing Experimental Design (e.g., Full Factorial Design) for Method Optimization

In the development of analytical methods for pharmaceutical analysis, such as the green high-performance thin-layer chromatography (HPTLC) determination of remdesivir and co-administered drugs, researchers must optimize multiple chromatographic factors to achieve robust, reliable, and reproducible results. Traditional one-variable-at-a-time (OVAT) optimization is inefficient, as it fails to account for potential interactions between factors and requires a larger number of experiments. The application of Design of Experiments (DoE) provides a systematic, statistical framework for efficiently exploring the effects of multiple factors and their interactions on critical analytical responses, leading to the identification of a robust method operable design region (MODR).

Within the context of a thesis focused on the analysis of remdesivir with co-administered drugs by green HPTLC, employing experimental design is paramount. It ensures that the developed methods are not only analytically sound but also adhere to the principles of Green Analytical Chemistry (GAC) by minimizing solvent consumption and waste generation during the optimization process itself. This protocol details the application of full factorial design, a fundamental DoE approach, for optimizing HPTLC methods, using relevant examples from antiviral drug analysis.

Theoretical Framework of Full Factorial Design

A full factorial design is one where all possible combinations of the levels for all factors are investigated. For a design with k factors, each having 2 levels, the total number of experimental runs is 2k. This comprehensive approach allows for the estimation of all main effects and all interaction effects between factors.

  • Key Terminology:
    • Factor: An independent variable that is deliberately varied during an experiment (e.g., mobile phase composition, chamber saturation time).
    • Level: The specific value or setting of a factor (e.g., low (-1) and high (+1)).
    • Response: The measured output of an experiment that is dependent on the factor levels (e.g., retention factor (Rf), peak area, number of theoretical plates).
    • Main Effect: The average change in a response when a factor is moved from its low to high level.
    • Interaction Effect: Occurs when the effect of one factor on a response depends on the level of another factor.

Protocol: Implementing Full Factorial Design for HPTLC Method Optimization

This protocol outlines the steps for applying a full factorial design to optimize an HPTLC method for the simultaneous analysis of remdesivir and co-administered drugs like favipiravir and molnupiravir.

Step 1: Define the Analytical Objective and Select Factors & Responses
  • Objective: To develop a green, stability-indicating HPTLC method for the concurrent quantification of remdesivir, favipiravir, and molnupiravir in pharmaceutical formulations.
  • Factor Selection: Based on preliminary screening and literature, select critical factors. For HPTLC, common factors include:
    • Factor A: Volume of a specific organic solvent in the mobile phase (e.g., Ethyl Acetate, mL) [16] [35].
    • Factor B: Volume of a co-solvent or modifier (e.g., Ethanol, mL) [16] [35].
    • Factor C: Chamber saturation time (minutes) [36].
  • Response Selection: Choose responses that define a successful separation:
    • Response 1 (Y1): Retention factor (Rf) of Remdesivir.
    • Response 2 (Y2): Resolution (Rs) between critical pair of drugs.
    • Response 3 (Y3): Peak area of Favipiravir (for sensitivity).
Step 2: Establish the Experimental Design Matrix

For three factors at two levels each, a 2^3 full factorial design requiring 8 experimental runs is constructed. The table below shows a generic design matrix.

Table 1: Full Factorial Design Matrix (2^3) for HPTLC Optimization

Run Order Factor A: Ethyl Acetate (mL) Factor B: Ethanol (mL) Factor C: Saturation Time (min) Y1: Rf RMD Y2: Resolution Y3: Peak Area
1 -1 (Low) -1 (Low) -1 (Low) [Data] [Data] [Data]
2 +1 (High) -1 (Low) -1 (Low) [Data] [Data] [Data]
3 -1 (Low) +1 (High) -1 (Low) [Data] [Data] [Data]
4 +1 (High) +1 (High) -1 (Low) [Data] [Data] [Data]
5 -1 (Low) -1 (Low) +1 (High) [Data] [Data] [Data]
6 +1 (High) -1 (Low) +1 (High) [Data] [Data] [Data]
7 -1 (Low) +1 (High) +1 (High) [Data] [Data] [Data]
8 +1 (High) +1 (High) +1 (High) [Data] [Data] [Data]
Step 3: Execute Experiments and Record Data
  • Perform the 8 HPTLC experiments in a randomized run order to minimize the effect of extraneous variables.
  • Use a validated HPTLC system, such as a CAMAG instrument with TLC silica gel 60 Fâ‚‚â‚…â‚„ plates [15] [36].
  • For each run, prepare the mobile phase according to the design, apply standard samples of the drugs, develop the plate, and scan it densitometrically.
  • Record the responses (Rf, Resolution, Peak Area) for each experimental run.
Step 4: Analyze Data and Perform Statistical Analysis
  • Input the data into statistical software (e.g., Minitab, Design-Expert).
  • Perform Analysis of Variance (ANOVA) to determine the statistical significance (p-value < 0.05) of each factor and its interactions on the responses.
  • Generate mathematical models (e.g., linear or quadratic equations) that describe the relationship between factors and responses.
  • Create perturbation plots and response surface plots to visualize these relationships.
Step 5: Identify Optimal Conditions and Verify
  • Based on the statistical models and contour plots, identify the factor level combination that yields the desired chromatographic outcomes (e.g., Rf ~0.5, Resolution >1.5, high peak area).
  • Conduct a confirmatory experiment at the predicted optimal conditions to validate the model's accuracy.
  • The final optimized mobile phase for the cited drugs could be a greener mixture like Ethyl Acetate: Ethanol: Water or Ethanol: Water [16], validated for the simultaneous analysis.

Case Study & Data Presentation

A study on the simultaneous analysis of Levodropropizine and Chlorpheniramine Maleate utilized a 2^(4-1) fractional factorial design (a subset of full factorial) to assess robustness. The factors investigated were methanol volume, chamber saturation time, wavelength, and solvent front. The results demonstrated that methanol volume had the most pronounced effect on the Rf value, underscoring the importance of its precise control [36].

Table 2: Summary of Factor Effects from a Fractional Factorial Robustness Study (adapted from [36])

Factor Investigated Effect on Rf Value
Methanol Volume in Mobile Phase Most significant effect, requires careful control
Chamber Saturation Time Minor effect
Wavelength Minor effect
Solvent Front Minor effect

Furthermore, an HPLC method for Valsartan was optimized using a full factorial design, evaluating flow rate, wavelength, and buffer pH. The analysis revealed that the quadratic effects of flow rate and wavelength were highly significant (p < 0.0001) on the peak area, demonstrating the power of factorial design in uncovering complex relationships [37].

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Reagents and Materials for Green HPTLC Method Development

Item Function / Application Example from Literature
TLC Silica Gel 60 Fâ‚‚â‚…â‚„ plates Stationary phase for chromatographic separation. Used in virtually all cited HPTLC methods for antivirals and other drug combinations [15] [16] [36].
Ethanol Green organic solvent for mobile phase preparation and sample dilution. Employed in mobile phases such as Ethyl Acetate:Ethanol [15] and Ethanol:Water [16] [35].
Ethyl Acetate Green organic solvent component of the mobile phase. Used in mobile phases for remdesivir and combination drugs [15] [16].
Water Aqueous, green component of reversed-phase mobile phases. Used in mobile phases like Ethanol:Water (6:4, v/v) [16].
CAMAG HPTLC System Instrumentation for automated sample application, plate development, and densitometry. The referenced studies consistently use CAMAG systems (Linomat applicator, TLC scanner) [15] [36] [38].

Workflow Visualization

The following diagram illustrates the logical workflow for applying a full factorial design to an HPTLC method development project.

workflow Start Define Analytical Objective F1 Select Factors & Levels Start->F1 F2 Establish Design Matrix F1->F2 F3 Execute Experiments (Randomized Order) F2->F3 F4 Record Responses (Rf, Resolution, etc.) F3->F4 F5 Statistical Analysis (ANOVA, Model Generation) F4->F5 F6 Identify Optimal Conditions F5->F6 F7 Confirmatory Experiment F6->F7

(HPTLC Method Optimization via Full Factorial Design)

Advanced Applications & Complementary Designs

While full factorial designs are powerful, screening designs like Plackett-Burman Design (PBD) are highly efficient for evaluating a large number of factors (N-1 factors with N experiments) to identify the most critical ones before a more detailed optimization. This is particularly useful in the early stages of method development. For instance, PBD has been successfully applied to screen factors like wavelength, saturation time, and solvent volumes for the HPTLC analysis of Efonidipine Hydrochloride [38].

Once critical factors are identified via full factorial or screening designs, Response Surface Methodology (RSM) designs like Central Composite Design (CCD) can be employed for in-depth optimization, especially to model curvature and find a true optimum, which a simple 2-level factorial design cannot capture [38]. This sequential approach (Screening → Optimization) provides a highly efficient framework for developing robust analytical methods.

Integrating Experimental Design into the development of green HPTLC methods for analyzing remdesivir and its co-administered drugs transforms an otherwise empirical process into a systematic, data-driven science. The use of full factorial design allows researchers to efficiently understand complex factor interactions, minimize experimental effort, and ultimately establish a robust, sustainable, and fit-for-purpose analytical procedure that aligns with the core principles of green chemistry and modern quality-by-design (QbD) paradigms.

Strategies for Resolving Overlapping Peaks and Improving Band Symmetry

Within the framework of green high-performance thin-layer chromatography (HPTLC) research for the analysis of remdesivir and its co-administered drugs, achieving optimal separation is paramount. The analytical challenge of overlapping peaks and asymmetric bands often impedes accurate quantification, particularly in complex matrices like pharmaceutical formulations and biological samples [14]. This document outlines practical strategies and detailed protocols to overcome these challenges, emphasizing green analytical chemistry principles to minimize environmental impact while maintaining methodological robustness.

The application of HPTLC has gained significant traction in pharmaceutical analysis due to its minimal solvent consumption, high sample throughput, and cost-effectiveness compared to conventional HPLC methods [39] [40]. When analyzing drug combinations such as remdesivir with linezolid and rivaroxaban—a common COVID-19 treatment protocol—chromatographic performance becomes critical for reliable therapeutic drug monitoring [14]. The following sections provide systematically validated approaches to enhance resolution and band symmetry specifically within this research context.

Core Optimization Strategies

Mobile Phase Optimization

The most powerful approach for improving band spacing in chromatographic separations involves modifying the mobile phase composition to alter relative retention (α) [41]. For reversed-phase systems, changing the organic modifier can significantly impact selectivity.

  • Solvent Selection: When initial separations show overlapping peaks, systematically testing different organic modifiers is advised. For example, if acetonitrile initially produces poor separation, switching to methanol or tetrahydrofuran often improves resolution [41]. Binary solvent mixtures can provide intermediate selectivity; for remdesivir analysis with co-administered drugs, dichloromethane-acetone (8.5:1.5 v/v) has demonstrated excellent resolution with retardation factors (Rf) of 0.23, 0.53, and 0.72 for remdesivir, linezolid, and rivaroxaban, respectively [14].
  • pH Adjustment: For ionizable compounds, mobile phase pH significantly impacts band symmetry and separation efficiency. Adjusting pH to values near the pKa of analytes can dramatically improve resolution. A study separating hydroxyzine, ephedrine, and theophylline achieved optimal results at pH 6.5, which created differential ionization states that enhanced separation [39].
  • Buffer Utilization: Implementing buffers instead of pure water in the mobile phase helps control pH and ionic strength, particularly for ionic or ionizable compounds like remdesivir. This approach improves peak symmetry and reproducibility [41].

Table 1: Mobile Phase Systems for Resolving Drug Mixtures

Analytes Mobile Phase Composition Ratio (v/v) Resulting Rf Values Separation Efficiency
Remdesivir, Linezolid, Rivaroxaban [14] Dichloromethane: Acetone 8.5:1.5 0.23, 0.53, 0.72 Well-resolved peaks
Hydroxyzine, Ephedrine, Theophylline [39] Chloroform: Ammonium Acetate Buffer (pH 6.5) 9.5:0.5 0.15, 0.40, 0.65 Baseline separation
Naltrexone, Bupropion [40] Ethyl Acetate: Methanol: Acetone: Glacial Acetic Acid 3:6.5:1.5:0.5 Not specified Excellent resolution for quantification
Stationary Phase and Plate Configuration

The physical characteristics of the stationary phase fundamentally impact separation efficiency and band symmetry.

  • Particle Size: Plates with smaller particle sizes (typically 5-6 μm for HPTLC versus 10-12 μm for conventional TLC) provide higher plate numbers, resulting in sharper peaks and improved resolution of closely eluting compounds [42]. The increased packing density reduces band broadening and enhances detection sensitivity.
  • Layer Chemistry: Normal-phase silica gel remains the most common stationary phase, but surface modifications can address specific separation challenges. For remdesivir analysis, TLC silica gel 60 F254 plates have proven effective [14]. The chemical nature of the bonded ligands can be altered to change the chemical nature of the stationary phase, providing another dimension for optimization [41].
Development and Detection Optimization

Post-application parameters significantly influence final chromatographic quality.

  • Development Chamber Saturation: Adequate chamber saturation with mobile phase vapor (typically 10-30 minutes) before plate development is crucial for achieving uniform solvent front advancement and consistent Rf values [14] [39]. Inadequate saturation causes edge effects and band distortion.
  • Detection Wavelength Selection: Optimizing the scanning wavelength maximizes sensitivity and minimizes baseline noise. For densitometric detection, systematic testing of wavelengths (e.g., 215 nm, 220 nm, 254 nm) identifies the optimal compromise for all analytes. A study determining three drugs found 220 nm produced the most symmetrical peaks with minimal noise [39].
  • Alternative Detection Methods: For compounds with poor UV absorbance, post-chromatographic derivatization enables visualization. For example, modified Dragendorff's reagent effectively detects naltrexone and bupropion, facilitating quantification via smartphone-based imaging [40]. This approach provides a cost-effective alternative to conventional densitometry.

Experimental Protocols

Protocol 1: Green HPTLC Method for Remdesivir with Co-administered Drugs

This validated protocol is adapted from a published method for simultaneous quantification of remdesivir, linezolid, and rivaroxaban in spiked human plasma [14].

Research Reagent Solutions:

  • Stationary Phase: TLC silica gel 60 F254 aluminum plates (20 × 20 cm, 0.1 mm thickness)
  • Mobile Phase: Dichloromethane-Acetone (8.5:1.5 v/v)
  • Standard Solutions: 1 mg/mL stock solutions in methanol (remdesivir, linezolid) or acetonitrile (rivaroxaban)
  • Sample Preparation: Protein precipitation with methanol followed by centrifugation for plasma samples

Step-by-Step Procedure:

  • Plate Pre-washing: Pre-wash HPTLC plates with methanol to remove impurities, then dry in an oven at 60°C for 5 minutes.
  • Sample Application: Using an automatic sample applicator (e.g., Camag Linomat V), apply standards and samples as 6-mm bands 1.5 cm from the bottom edge with 4 mm between bands.
  • Chromatographic Development: Transfer the applied plate to a glass twin-trough chamber pre-saturated with 30 mL of mobile phase for 30 minutes. Develop the plate by ascending chromatography until the solvent front travels 8 cm from the origin.
  • Plate Drying: Air-dry the developed plate in a fume hood to evaporate residual solvents.
  • Densitometric Analysis: Scan the plate at 254 nm using a TLC scanner (e.g., Camag TLC Scanner 3) with deuterium lamp, slit dimensions of 5 × 0.45 mm, and scanning speed of 20 mm/s.
  • Quantification: Identify analytes by comparing Rf values with standards and quantify using peak areas derived from the calibration curves.

Method Performance: This method demonstrates linearity over 0.2–5.5 μg/band for remdesivir, 0.2–4.5 μg/band for linezolid, and 0.1–3.0 μg/band for rivaroxaban, with outstanding recoveries ranging from 98.3% to 101.2% [14].

Protocol 2: Smartphone-Based HPTLC Detection as Green Alternative

This protocol describes an eco-friendly detection method using a smartphone camera, adapted from a published approach for drug analysis [40].

Research Reagent Solutions:

  • Derivatization Reagent: Modified Dragendorff's reagent (5 mL of 40% potassium iodide + 5 mL of 1.7% bismuth nitrate in 20% acetic acid + 20 mL acetic acid + 70 mL distilled water)
  • Enhancing Solution: 5% w/v sodium nitrite solution
  • Imaging Device: Smartphone with high-resolution camera (e.g., 32 MP)
  • Image Analysis Software: ImageJ desktop software or Color Picker smartphone application

Step-by-Step Procedure:

  • Chromatographic Separation: Complete the chromatographic separation as described in Protocol 1 and dry the plate.
  • Derivatization: In a fume hood, immerse the developed plate in Dragendorff's reagent for 30 seconds or until uniform coverage is achieved.
  • Drying and Enhancement: Air-dry the plate for 5 minutes, then spray uniformly with 5% sodium nitrite solution. A light-yellow background with brown analyte spots will develop.
  • Image Capture: Place the derivatized plate in a visualization chamber with consistent daylight illumination. Capture an image using a smartphone camera positioned 15 cm from the plate surface, ensuring the plate fills the frame without shadows.
  • Image Analysis (ImageJ):
    • Transfer the image to a computer and open it in ImageJ software.
    • Use the rectangular selection tool to define each sample track.
    • Select "Gels" from the "Analyze" menu, followed by "Plot Lanes" to generate intensity profiles.
    • Use the straight line and magic wand tools to calculate integrated peak areas.
  • Quantification: Construct calibration curves by plotting peak areas against analyte concentrations.

Greenness Assessment: This smartphone-based method reduces energy consumption and equipment costs while maintaining analytical validity, representing a sustainable alternative for routine analysis [40].

Visualization of Method Optimization Pathways

The following diagram illustrates the systematic decision process for resolving overlapping peaks and improving band symmetry in HPTLC method development.

HPTLC_Optimization Start Initial HPTLC Method Exhibiting Overlapping Peaks MP_Optimization Mobile Phase Optimization Start->MP_Optimization SP_Optimization Stationary Phase/Plate Optimization Start->SP_Optimization Detection_Optimization Detection Optimization Start->Detection_Optimization MP1 Adjust solvent polarity and composition MP_Optimization->MP1 MP2 Modify pH for ionizable compounds MP_Optimization->MP2 MP3 Implement buffer systems for reproducibility MP_Optimization->MP3 SP1 Use smaller particle sizes (5-6 μm) SP_Optimization->SP1 SP2 Consider alternative stationary phases SP_Optimization->SP2 D1 Optimize scanning wavelength Detection_Optimization->D1 D2 Implement post-chromatographic derivatization Detection_Optimization->D2 D3 Use smartphone-based detection methods Detection_Optimization->D3 Result Acceptable Resolution and Band Symmetry MP1->Result MP2->Result MP3->Result SP1->Result SP2->Result D1->Result D2->Result D3->Result

HPTLC Peak Optimization Pathway

This workflow demonstrates that multiple optimization paths can be pursued independently or in combination to achieve the desired chromatographic resolution, with mobile phase optimization typically providing the most significant improvements in peak spacing [41].

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of the protocols requires specific materials and reagents optimized for green HPTLC analysis.

Table 2: Essential Research Reagents and Materials for Green HPTLC

Item Specification/Function Application Example
HPTLC Plates Silica gel 60 F254, 0.1 mm thickness; provides high-resolution separation with fluorescence indicator for UV detection Analysis of remdesivir with co-administered drugs [14]
Green Solvents Dichloromethane, acetone, ethyl acetate, methanol; mobile phase components with relatively favorable environmental profiles Dichloromethane:acetone (8.5:1.5) for remdesivir separation [14]
Sample Applicator Automated applicator (e.g., Camag Linomat); ensures precise, reproducible band application for accurate quantification Application of standards and samples as 6-mm bands [40]
Development Chamber Twin-trough glass chamber; allows for chamber saturation and controlled development conditions Pre-saturation for 30 minutes before development [14]
Densitometry System TLC scanner with deuterium lamp and variable wavelength detection; enables precise in-situ quantification Scanning at 254 nm for remdesivir quantification [14]
Derivatization Reagents Chemical visualization agents (e.g., Dragendorff's reagent); enables detection of non-UV-absorbing compounds Detection of naltrexone and bupropion [40]
Smartphone Imaging System High-resolution camera with image analysis software; provides cost-effective, portable detection alternative ImageJ software for spot quantification [40]

Effective resolution of overlapping peaks and improvement of band symmetry in HPTLC requires a systematic approach addressing multiple chromatographic parameters. Through strategic mobile phase optimization, appropriate stationary phase selection, and implementation of advanced detection techniques, researchers can develop robust analytical methods that meet green chemistry principles. The provided protocols and optimization strategies offer practical solutions for challenging separations, specifically addressing the analysis of remdesivir with co-administered drugs. These approaches enable reliable therapeutic drug monitoring and quality control while minimizing environmental impact through reduced solvent consumption and waste generation—a critical consideration in modern analytical chemistry.

Optimizing Mobile Phase Composition for Specific Drug Combinations

High-performance thin-layer chromatography (HPTLC) represents a sophisticated instrumental technique based on the full capabilities of thin-layer chromatography, offering advantages of automation, scanning, full optimization, selective detection principles, and minimal sample preparation [23]. The pursuit of sustainability in analytical chemistry requires adherence to the principles of green, blue, and white analytical chemistry, particularly in pharmaceutical quality control laboratories [6]. This application note focuses on optimizing mobile phase composition for the analysis of Remdesivir (RMD) and its co-administered drugs using green HPTLC methodologies, providing detailed protocols and experimental data to support researchers in drug development.

Mobile Phase Optimization Strategies

Critical Factors in Mobile Phase Design

Successful HPTLC method development requires systematic optimization of mobile phase composition to achieve adequate resolution of target compounds while adhering to green chemistry principles. Key factors include:

  • Solvent Selection: Preference for greener solvents like ethanol, ethyl acetate, and water over hazardous alternatives
  • pH Adjustment: Controlled using ammonia solution or acetic acid to influence compound ionization and migration
  • Stationary Phase Compatibility: Silica gel 60 F~254~ plates are most commonly employed
  • Development Conditions: Chamber saturation time and mobile phase volume significantly impact reproducibility

The optimal mobile phase provides well-resolved bands (R~f~ values between 0.2-0.8) with symmetrical peak shapes and minimal tailing, enabling accurate quantification [14] [4].

Structured Optimization Workflow

The following diagram illustrates the systematic approach to mobile phase optimization for drug combinations:

G Start Start Literature Literature Start->Literature Review existing methods Initial Initial Literature->Initial Select green solvent mix Evaluate Evaluate Initial->Evaluate Test initial separation Adjust Adjust Adjust->Initial No: Modify composition Optimized Optimized Adjust->Optimized Yes: Proceed Evaluate->Adjust Resolution adequate? Validate Validate Optimized->Validate Final method validation

Diagram 1: Mobile phase optimization workflow for HPTLC methods.

Experimental Protocols

Materials and Reagents

Table 1: Essential Research Reagent Solutions

Item Specification Function Green Alternative
HPTLC plates Silica gel 60 F~254~, aluminum-backed, 20×10 cm Stationary phase for chromatographic separation -
Ethyl acetate HPLC grade Mobile phase component, greener solvent Primary green solvent
Ethanol HPLC grade Mobile phase component, green solvent Primary green solvent
Water HPLC grade Mobile phase component Green solvent
Ammonia solution 25%, analytical grade pH adjustment for basic compounds -
Acetic acid Glacial, analytical grade pH adjustment for acidic compounds -
Dichloromethane HPLC grade Mobile phase component (avoid if possible) Ethyl acetate/ethanol
Standard compounds RMD, FAV, MOL, DEX, LNZ, RIV (>99% purity) Reference standards for quantification -
Protocol 1: Normal-Phase HPTLC for Antiviral Analysis

This protocol describes the simultaneous determination of Remdesivir (RMD), Favipiravir (FAV), and Molnupiravir (MOL) using a normal-phase system [6].

Materials and Reagents:

  • HPTLC plates: Silica gel 60 F~254~ (20×10 cm)
  • Mobile phase: Ethyl acetate:ethanol:water (9.4:0.4:0.25, v/v/v)
  • Standard solutions: 1 mg/mL of RMD, FAV, and MOL in methanol
  • Sample: Pharmaceutical formulations or spiked plasma
  • Equipment: CAMAG HPTLC system with Linomat 5 applicator, TLC scanner 3, twin-trough chamber

Procedure:

  • Plate preparation: Pre-wash plates with methanol and activate at 110°C for 5 minutes
  • Sample application: Apply standards and samples as 6-mm bands using Linomat 5 applicator (application rate: 150 nL/s)
  • Chromatographic development:
    • Equilibrate twin-trough chamber with mobile phase for 20 minutes
    • Develop plate to migration distance of 80 mm at 25±2°C
    • Dry plate in air stream for 5 minutes
  • Detection and scanning:
    • Scan plate at 244 nm for RMD and MOL
    • Scan at 325 nm for FAV
    • Use deuterium lamp, slit dimensions 5.00×0.45 mm, scanning speed 20 mm/s
  • Quantification: Generate calibration curves (30-800 ng/band for RMD; 50-2000 ng/band for FAV and MOL)

Method Performance:

  • Correlation coefficients: >0.9998 for all compounds
  • Precision: RSD <2% for intra-day and inter-day measurements
  • Accuracy: 98.3-101.2% recovery from pharmaceutical formulations
Protocol 2: Green Reverse-Phase HPTLC for Antiviral Analysis

This protocol employs an environmentally friendly mobile phase for the same antiviral agents [6].

Materials and Reagents:

  • HPTLC plates: Silica gel 60 F~254~ (20×10 cm)
  • Mobile phase: Ethanol:water (6:4, v/v) - classified as green solvent system
  • Standard solutions: 1 mg/mL of RMD, FAV, and MOL in methanol
  • Equipment: CAMAG HPTLC system as in Protocol 1

Procedure:

  • Plate preparation: As in Protocol 1
  • Sample application: As in Protocol 1
  • Chromatographic development:
    • Chamber saturation time: 30 minutes
    • Development distance: 70 mm
    • Temperature: 25±2°C
  • Detection: Identical to Protocol 1
  • Quantification: Calibration curves as in Protocol 1

Greenness Assessment:

  • AGREE score: 0.82 (excellent greenness)
  • Analytical Eco-Scale: 85 (excellent greenness)
  • BAGI score: 85 (high practicality)
Protocol 3: Analysis of RMD with Co-administered Drugs

This protocol addresses the determination of RMD with commonly co-administered drugs (linezolid and rivaroxaban) in spiked human plasma [14].

Materials and Reagents:

  • HPTLC plates: Silica gel 60 F~254~ (20×20 cm)
  • Mobile phase: Dichloromethane:acetone (8.5:1.5, v/v)
  • Internal standard: Apixaban (where required)
  • Standard solutions: 1 mg/mL of RMD, LNZ, RIV in methanol
  • Equipment: Similar to previous protocols

Procedure:

  • Sample preparation:
    • Spike drug solutions into plasma (0.5 mL)
    • Add 3 mL acetonitrile for protein precipitation
    • Vortex for 1 minute, centrifuge at 4500 rpm for 10 minutes
    • Filter supernatant through 0.45-μm syringe filter
  • Chromatographic conditions:
    • Application volume: 10 μL as 6-mm bands
    • Chamber saturation: 30 minutes
    • Development distance: 85 mm
    • Detection: 254 nm
  • Quantification:
    • Linear range: 0.2-5.5 μg/band for RMD
    • LOD: 128.8 ng/band for RMD

Method Performance:

  • Resolution: R~f~ values - 0.23 (RMD), 0.53 (LNZ), 0.72 (RIV)
  • Recovery: 98.3-101.2% from spiked plasma
  • Precision: RSD <2%

Results and Data Analysis

Comparative Mobile Phase Performance

Table 2: Optimized Mobile Phase Systems for Drug Combinations

Analyte Combination Mobile Phase Composition Plate Type Detection (nm) R~f~ Values Greenness Score
RMD, FAV, MOL [6] Ethyl acetate:ethanol:water (9.4:0.4:0.25, v/v/v) Silica gel 60 F~254~ 244 (RMD, MOL)325 (FAV) RMD: 0.35FAV: 0.62MOL: 0.52 AGREE: 0.82
RMD, FAV, MOL [6] Ethanol:water (6:4, v/v) Silica gel 60 F~254~ 244 (RMD, MOL)325 (FAV) RMD: 0.41FAV: 0.68MOL: 0.58 AGREE: 0.85
RMD, LNZ, RIV [14] Dichloromethane:acetone (8.5:1.5, v/v) Silica gel 60 F~254~ 254 RMD: 0.23LNZ: 0.53RIV: 0.72 GAPI: Moderate
RMD, FAV, DEX [4] Ethyl acetate:hexane:acetic acid (9:1:0.3, v/v/v) Silica gel 60 F~254~ 254 RMD: 0.30DEX: 0.64FAV: 0.77 Whiteness: 95.6%
LID, DIL [43] Toluene:methanol:ethyl acetate (7:2:1, v/v/v) + 2 drops ammonia Silica gel 60 F~254~ 220 LID: 0.59DIL: 0.48 Not assessed
Method Validation Parameters

Table 3: Validation Data for HPTLC Methods of RMD and Co-administered Drugs

Parameter RMD, FAV, MOL [6] RMD, LNZ, RIV [14] RMD, FAV, DEX [4]
Linearity range RMD: 30-800 ng/bandFAV: 50-2000 ng/bandMOL: 50-2000 ng/band RMD: 0.2-5.5 μg/bandLNZ: 0.2-4.5 μg/bandRIV: 0.1-3.0 μg/band RMD: 0.1-10 μg/bandDEX: 0.1-10 μg/bandFAV: 0.2-15 μg/band
Correlation coefficient (r²) >0.9998 >0.999 >0.999
LOD RMD: 10 ng/bandFAV: 15 ng/bandMOL: 15 ng/band RMD: 128.8 ng/bandLNZ: 50.5 ng/bandRIV: 55.8 ng/band RMD: 0.1 μg/bandDEX: 0.1 μg/bandFAV: 0.2 μg/band
Precision (RSD%) Intra-day: <1.5%Inter-day: <2.0% Intra-day: <2.0%Inter-day: <2.5% Intra-day: <2.0%Inter-day: <2.5%
Accuracy (% recovery) 98.5-101.5% 98.3-101.2% 97.07-102.77%
Robustness RSD <2% with minor mobile phase variations RSD <2% with deliberate changes RSD <2% with parameter variations

Sustainability Assessment

Modern HPTLC method development requires comprehensive sustainability evaluation using multiple metrics:

  • Greenness: Assessed using Analytical Eco-Scale, AGREE, and GAPI tools
  • Blueness (practicality): Evaluated with Blue Applicability Grade Index (BAGI)
  • Whiteness: Overall sustainability measured via RGB12 algorithm

The ethanol:water (6:4, v/v) mobile phase system achieved an excellent AGREE score of 0.85, demonstrating superior greenness compared to methods using dichloromethane or chloroform [6] [44]. The whiteness assessment of the ethyl acetate:hexane:acetic acid system for RMD, FAV, and DEX analysis showed 95.6% whiteness, indicating excellent overall sustainability [4].

Troubleshooting and Technical Notes

Common Optimization Challenges
  • Tailing spots: Add 0.1-1% acetic acid or ammonia to neutralize silanol groups
  • Insufficient resolution: Adjust mobile phase polarity or try alternative solvent combinations
  • Irregular band shapes: Ensure proper chamber saturation and uniform application
  • Background interference: Pre-wash HPTLC plates and use high-purity solvents
Method Transfer Considerations

When transferring methods between laboratories, strictly control:

  • Chamber saturation time and conditions
  • Application parameters (band width, application speed)
  • Drying conditions post-development
  • Environmental conditions (temperature, humidity)

The developed methods demonstrate robustness to minor variations in mobile phase composition (±0.5% in component ratios) and development distance (±5 mm) [6] [14].

Optimizing mobile phase composition for HPTLC analysis of Remdesivir with co-administered drugs requires balancing separation efficiency with sustainability principles. Ethanol-water and ethyl acetate-ethanol-water systems provide excellent green alternatives to traditional hazardous solvents while maintaining analytical performance. The protocols presented enable reliable quantification of antiviral drugs and their common co-administered medications in pharmaceutical formulations and biological samples, supporting quality control and therapeutic drug monitoring in COVID-19 treatment.

Addressing Matrix Effects from Human Plasma and Formulation Excipients

The accurate determination of active pharmaceutical ingredients (APIs) in biological matrices and pharmaceutical formulations is a fundamental requirement in drug development and therapeutic drug monitoring (TDM). A significant challenge in these analyses is the presence of matrix effects, which can alter the analytical signal, leading to inaccurate quantification. These effects originate from various sources, including plasma components such as proteins, lipids, and salts, as well as formulation excipients used in drug products.

Within the context of green High-Performance Thin-Layer Chromatography (HPTLC) research for the analysis of remdesivir and its co-administered drugs, addressing matrix effects is paramount to developing methods that are not only environmentally sustainable but also precise, accurate, and reliable. This document outlines standardized protocols and application notes for mitigating these interferences, ensuring the quality of analytical data supporting pharmaceutical research.

Experimental Protocols

Sample Preparation for Human Plasma Analysis

Effective sample preparation is critical for minimizing matrix effects from human plasma. The following protocol, adapted from validated methods for antiviral analysis, ensures efficient protein removal and analyte extraction [4] [14].

  • Materials: Drug-free human plasma, methanol, acetonitrile, micropipettes, vortex mixer, centrifuge, 0.45 µm syringe filters.
  • Procedure:
    • Spiking: Transfer 1 mL of blank human plasma into a centrifuge tube.
    • Fortification: Spike with appropriate volumes of standard working solutions of remdesivir, favipiravir, dexamethasone, and an internal standard (e.g., apixaban) [4].
    • Protein Precipitation: Add 3 mL of acetonitrile or methanol as a precipitating solvent. Vortex the mixture for 1 minute to ensure complete protein denaturation.
    • Centrifugation: Centrifuge at 4,500 rpm for 10 minutes to pellet the precipitated proteins.
    • Filtration: Carefully collect the supernatant and filter it through a 0.45 µm syringe filter.
    • Analysis: The resulting clear filtrate is suitable for application on HPTLC plates [4] [14].
HPTLC Analysis of Processed Samples

The following chromatographic conditions have been demonstrated to effectively separate remdesivir, favipiravir, and dexamethasone in the presence of plasma-derived interferences [4] [45].

  • Stationary Phase: TLC silica gel 60 F254 aluminum plates.
  • Application: Spot the processed sample as 6 mm bands, 10 mm from the bottom edge of the plate using a semi-automatic sampler like a CAMAG Linomat.
  • Mobile Phase: Ethyl acetate : hexane : acetic acid (9 : 1 : 0.3, v/v/v) [4] [45].
  • Development: Saturate the twin-trough chamber with the mobile phase for 30 minutes. Develop the plate to a distance of 9 cm.
  • Detection: Perform densitometric scanning at 254 nm. The target Rf values are approximately 0.30 for remdesivir, 0.64 for dexamethasone, and 0.77 for favipiravir [4].

The workflow below illustrates the complete analytical procedure from sample to result:

G Start Start Sample Preparation P1 Spike Plasma with Analytes and Internal Standard Start->P1 P2 Add Protein Precipitant (e.g., Acetonitrile) P1->P2 P3 Vortex Mixing P2->P3 P4 Centrifugation P3->P4 P5 Filter Supernatant (0.45 µm Syringe Filter) P4->P5 C1 Apply Filtrate to HPTLC Plate P5->C1 C2 Develop in Saturated Chromatographic Chamber C1->C2 C3 Dry Plate C2->C3 C4 Densitometric Scanning C3->C4 End Data Analysis & Quantification C4->End

Results and Data Analysis

Quantitative Performance in Spiked Plasma

The green HPTLC method was rigorously validated by analyzing human plasma samples spiked with known concentrations of remdesivir, favipiravir, and dexamethasone. The internal standard apixaban was used to correct for variations during sample preparation and analysis [4].

Table 1: Analytical Performance of the HPTLC Method for Antivirals in Spiked Human Plasma [4]

Analyte Linear Range (µg/band) Limit of Quantification (LOQ, µg/band) Recovery from Plasma (%) Rf Value
Remdesivir 0.1 – 10.0 0.1 97.07 – 102.77 0.30
Dexamethasone 0.1 – 10.0 0.1 97.07 – 102.77 0.64
Favipiravir 0.2 – 15.0 0.2 97.07 – 102.77 0.77

The data demonstrates that the method exhibits excellent sensitivity with low LOQs and high accuracy, as evidenced by the near-quantitative recoveries from the complex plasma matrix [4]. The well-resolved Rf values confirm the method's selectivity in the presence of plasma components.

Sustainability Assessment

The environmental impact of the developed HPTLC method was evaluated using the RGB12 algorithm, a metric for whiteness that considers analytical performance, ecological impact, and practical effectiveness. The method achieved a whiteness score of 95.6%, confirming its status as a sustainable and white analytical chemistry approach [4] [45].

The Scientist's Toolkit

The successful implementation of this protocol relies on several key reagents and materials. The following table details these essential components and their specific functions in mitigating matrix effects.

Table 2: Essential Research Reagent Solutions for Sample Preparation and HPTLC Analysis

Item Function & Role in Mitigating Matrix Effects Example from Protocol
Acetonitrile/Methanol Protein precipitation solvent. Denatures and removes plasma proteins, preventing column fouling and signal suppression. Used in a 3:1 ratio to plasma for efficient protein removal [14] [46].
Internal Standard (Apixaban) Correction for analytical variability. Accounts for losses during sample preparation and fluctuations in instrument response, improving accuracy. Added in equal volumes to all calibration and sample solutions before processing [4].
Syringe Filter (0.45 µm) Particulate removal. Eliminates any remaining fine particles from the supernatant that could interfere with the HPTLC application or separation. Used to filter the clear supernatant after centrifugation [4].
Silica Gel F254 HPTLC Plates Separation matrix. Provides the stationary phase for the chromatographic separation of analytes from each other and from co-extracted matrix components. TLC silica gel 60 F254 plates are used as the stationary phase [4] [15].
Green Mobile Phase Elution solvent. The optimized mixture selectively moves analytes at distinct Rf values, separating them from endogenous compounds. Ethyl acetate, hexane, and acetic acid mixture [4].

Troubleshooting and Optimization

Despite a robust protocol, challenges may arise. The following diagram outlines a logical decision path for diagnosing and resolving common issues related to matrix effects:

G Start Problem: Poor Recovery or Peak Tailing Q1 Is protein precipitation complete? (Clear supernatant after centrifugation?) Start->Q1 Q2 Is the sample clean? (Any particulates in final solution?) Q1->Q2 Yes A1 • Increase precipitant volume • Ensure vigorous vortexing • Check solvent:plasma ratio Q1->A1 No Q3 Is the separation optimal? (Are peaks well-resolved?) Q2->Q3 Yes A2 • Use a finer filter (0.2 µm) • Centrifuge for longer duration • Avoid disturbing pellet Q2->A2 No A3 • Adjust mobile phase composition • Increase chamber saturation time • Check plate activity Q3->A3 No End Re-analyze Sample A1->End A2->End A3->End

Key Optimization Strategies:

  • Enhancing Precipitation Efficiency: If recovery is low, ensure a sufficient ratio of organic solvent to plasma (at least 2:1, preferably 3:1) and adequate vortexing time [14].
  • Improving Sample Cleanliness: For noisy baselines or irregular bands, a second centrifugation step or filtration through a 0.2 µm filter can be implemented.
  • Optimizing Chromatography: If separation is suboptimal, minor adjustments to the mobile phase ratio (± 0.2 parts of each component) can be tested. Ensuring consistent and adequate chamber saturation time (30 min) is also critical for reproducible Rf values [4] [15].

This application note provides a detailed framework for addressing matrix effects in the analysis of remdesivir and co-administered drugs using green HPTLC. The standardized protocols for plasma sample preparation and chromatographic analysis, supported by comprehensive performance data and troubleshooting guides, ensure the generation of reliable and accurate results. The high whiteness score of the method underscores its alignment with the principles of sustainable analytical chemistry, making it a valuable tool for pharmaceutical analysts and researchers engaged in drug development and therapeutic monitoring.

Validating Method Performance and Assessing its Greenness and Whiteness

The analysis of complex pharmaceutical formulations, such as those containing the antiviral drug remdesivir and its co-administered medications, requires analytical methods of the highest reliability. Compliance with the International Council for Harmonisation (ICH) Q2(R1) guideline, titled "Validation of Analytical Procedures: Text and Methodology," is the globally recognized standard for ensuring these methods are fit for their intended purpose [47]. This document provides a detailed application protocol for the validation of a green High-Performance Thin-Layer Chromatography (HPTLC) method for the analysis of remdesivir in the presence of co-administered drugs, framing the process within the specific requirements of ICH Q2(R1). The objective is to provide researchers and drug development professionals with a clear, actionable framework for establishing method validity, focusing on the critical parameters of linearity, limits of detection (LOD) and quantitation (LOQ), precision, and accuracy.

Experimental Section

Research Reagent Solutions and Materials

The following table details the essential materials and reagents required for the development and validation of the HPTLC method as described in the literature [48] [4] [49].

Table 1: Essential Research Reagents and Materials

Item Specification / Function
Remdesivir (RMD) Reference standard (e.g., purity ≥ 99%) [4].
Co-administered Drugs Standards such as Favipiravir, Dexamethasone, Linezolid, or Rivaroxaban, depending on the study [4] [49].
Stationary Phase TLC silica gel 60 F254 plates on aluminum sheets [48] [49].
Mobile Phase Varies by method; e.g., Dichloromethane:Acetone (8.5:1.5, v/v) or Ethyl Acetate:Hexane:Acetic Acid (9:1:0.3, v/v) [4] [49].
Sample Solvent Methanol, for preparing stock and working standard solutions [4].
Internal Standard (IS) Apixaban or other suitable compounds for plasma analysis [4].
Detection Instrument HPTLC Densitometer with UV detection, typically at 245 nm or 254 nm [48] [4].

Method Workflow and Validation Logic

The diagram below illustrates the logical sequence of the analytical method validation process, from initial setup to the final determination of validation parameters, as guided by ICH Q2(R1).

G Start Start Method Validation A Prepare Stock & Working Standard Solutions Start->A B Spot Samples on HPTLC Plate A->B C Develop Plate in Saturated Chamber B->C D Scan Plate with Densitometer C->D E Perform Data Analysis D->E F Determine Validation Parameters: Linearity, LOD, LOQ, Precision, Accuracy E->F

Validation Parameters & Results

The core of ICH Q2(R1) validation involves the experimental determination of specific performance characteristics. The following table summarizes the typical results and acceptance criteria for a validated HPTLC method for remdesivir and co-administered drugs, based on published green HPTLC methods [48] [4] [49].

Table 2: Summary of Validation Parameters as per ICH Q2(R1)

Validation Parameter Experimental Results & Acceptance Criteria Remdesivir (Example Values) Co-administered Drug (e.g., Favipiravir)
Linearity & Range Correlation Coefficient (r): ≥ 0.9990Concentration Range: Specific to analyte and application [49]. 0.2 - 5.5 µg/band [49] 0.2 - 15 µg/band [4]
LOD Signal-to-noise ratio ~ 3:1. Expressed as concentration. 128.8 ng/band [49] 50-200 ng/band [4]
LOQ Signal-to-noise ratio ~ 10:1. Determined with acceptable accuracy and precision. 128.8 ng/band (as reported in [49]) 50-200 ng/band [4]
Precision (Repeatability) % RSD: Typically ≤ 2.0% for assay methods. Measured by analyzing multiple samples at 100% test concentration. RSD ≤ 2.0% (reported for similar methods) RSD ≤ 2.0% (reported for similar methods)
Accuracy Recovery: 98–102%. Determined by spiking known amounts of analyte into the sample matrix (e.g., placebo or plasma). 98.3 - 101.2% (in spiked plasma) [49] 97.07 - 102.77% (in spiked plasma) [4]

Detailed Experimental Protocols

Protocol for Linearity, LOD, and LOQ Determination

This protocol outlines the steps to establish the linear relationship between analyte concentration and detector response, and to determine the sensitivity limits of the method.

  • Preparation of Standard Solutions: Accurately weigh and transfer 25 mg of remdesivir and co-administered drug standards into separate 25 mL volumetric flasks. Dissolve and make up to volume with methanol to obtain stock solutions of 1 mg/mL [4].
  • Preparation of Working Solutions: From each stock solution, prepare a series of working standard solutions by serial dilution to obtain at least five different concentrations that cover the expected range (e.g., 0.1-10 µg/band) [4] [49].
  • Application and Development: Using a micropipette or automatic applicator, spot 10 µL of each working solution as 6 mm bands on the HPTLC plate. Develop the plate in a previously saturated twin-trough chamber with the optimized mobile phase (e.g., Dichloromethane:Acetone, 8.5:1.5 v/v) until the solvent front migrates 9 cm [49].
  • Scanning and Calibration: Dry the developed plate and scan it densitometrically at the selected wavelength (e.g., 245 nm for remdesivir). Record the peak areas [48].
  • Data Analysis:
    • Linearity: Plot the mean peak area (y-axis) against the corresponding applied concentration (x-axis, in µg/band). Calculate the regression equation and correlation coefficient (r). A value of ≥ 0.9990 indicates excellent linearity [49].
    • LOD & LOQ: Calculate these limits based on the standard deviation of the response (σ) and the slope (S) of the calibration curve using the formulas: LOD = 3.3σ/S and LOQ = 10σ/S [47].

Protocol for Precision (Repeatability) Study

Precision demonstrates the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample.

  • Sample Preparation: Prepare six separate sample solutions from a homogeneous bulk sample or a spiked plasma sample at 100% of the test concentration (e.g., within the middle of the linearity range).
  • Analysis: Analyze all six samples as per the developed HPTLC method (as described in Section 4.1, steps 3-4).
  • Calculation: Calculate the % assay for each sample injection and then determine the mean, standard deviation, and % Relative Standard Deviation (% RSD) of the six results. An % RSD of ≤ 2.0% is generally acceptable for assay methods [47].

Protocol for Accuracy (Recovery) Study

Accuracy expresses the closeness of agreement between the value found and the value accepted as a true or reference value.

  • Experimental Design: Perform a recovery study by spiking known quantities of the analyte (remdesivir and co-administered drugs) into a blank matrix (e.g., placebo mixture or drug-free human plasma). Prepare at least three concentration levels (e.g., 80%, 100%, and 120% of the target concentration), with three replicates at each level [4] [49].
  • Sample Processing: For plasma samples, add a protein precipitating agent like acetonitrile, vortex mix, centrifuge, and filter the supernatant before analysis [4].
  • Analysis and Calculation: Analyze the prepared samples using the validated HPTLC method. Calculate the percentage recovery of the analyte at each level using the formula:
    • % Recovery = (Measured Concentration / Spiked Concentration) × 100
  • Acceptance Criteria: The mean recovery should be within 98–102% for the drug in bulk form and 97–103% for more complex matrices like spiked human plasma, with a low % RSD [4] [49].

Analytical Procedure Lifecycle

The following diagram places the ICH Q2(R1) validation process within the broader, modern context of the analytical procedure lifecycle, which is emphasized in the updated ICH Q2(R2) and Q14 guidelines [50] [51].

G A Procedure Development (Define ATP) B Procedure Validation (ICH Q2(R1) Parameters) A->B C Routine Procedure Use B->C D Continuous Monitoring & Lifecycle Management C->D D->C

This application note has detailed the practical application of ICH Q2(R1) guidelines for the comprehensive validation of a green HPTLC method for the analysis of remdesivir with co-administered drugs. By rigorously testing and documenting the parameters of linearity, LOD, LOQ, precision, and accuracy as outlined in the provided protocols and summarized in Table 2, researchers can ensure their analytical methods are reliable, reproducible, and capable of generating high-quality data. This foundation is critical for the success of subsequent research, including therapeutic drug monitoring and pharmacokinetic studies in the context of COVID-19 treatment protocols.

Within the framework of a broader thesis on the analysis of Remdesivir (REM) with co-administered drugs via Green High-Performance Thin-Layer Chromatography (HPTLC), this document details specific application notes and protocols for conducting recovery studies. These studies are crucial for validating the developed green analytical method, ensuring its accuracy and reliability for quantifying the target drugs in both formulated products (pharmaceutical dosage forms) and biological matrices (spiked human plasma). The focus on green HPTLC aligns with the principles of Green Analytical Chemistry (GAC), aiming to minimize environmental impact while maintaining analytical efficacy [14] [22]. Recovery studies directly demonstrate the method's capability to accurately measure the analyte in the presence of complex sample matrices, a prerequisite for therapeutic drug monitoring and drug-drug interaction studies in clinical settings [52].

Experimental Protocols

Materials and Reagents

The following materials and reagents are essential for the execution of the HPTLC method and recovery studies. Prioritizing green solvents where possible is encouraged.

Table 1: Research Reagent Solutions and Essential Materials

Item Function/Description
Remdesivir (REM) The primary antiviral drug, acting as the target analyte for the study.
Co-administered Drugs (e.g., Linezolid (LNZ), Rivaroxaban (RIV)) Co-analytes representing commonly prescribed medications with REM for COVID-19 treatment, enabling simultaneous analysis [14].
HPTLC Silica Gel 60 F254 Plates The stationary phase. The silica gel provides the separation medium, and the F254 indicator fluoresces under 254 nm light for analyte detection.
Dichloromethane (DCM) and Acetone Components of the mobile phase. The specific ratio (e.g., DCM: Acetone, 8.5:1.5, v/v) is critical for achieving optimal separation of the analytes [14].
Methanol and Acetonitrile HPLC-grade solvents used for preparing standard stock solutions and sample extraction.
Human Plasma The biological matrix used for spiking experiments to simulate the analysis of patient samples.
Ethyl Acetate Often used in liquid-liquid extraction procedures to isolate drugs from the plasma matrix [52].
CAMAG HPTLC System Instrumentation comprising an autosampler (e.g., Linomat 5), a chromatographic development chamber, a TLC scanner, and associated software (e.g., winCATS) for automated application, development, and densitometric analysis [14].

Protocol for Recovery Studies from Pharmaceutical Dosage Forms

This protocol assesses the method's accuracy in quantifying the drug from its formulated product.

  • Sample Preparation:

    • Accurately weigh and grind ten tablets of the pharmaceutical dosage form (e.g., Remdesivir-Rameda 100 mg/vial) into a fine powder [14].
    • Transfer an amount of powder equivalent to 10 mg of REM into a 10 mL volumetric flask.
    • Add approximately 7-8 mL of methanol, sonicate for 30 minutes to ensure complete drug extraction, and then dilute to the mark with methanol.
    • Filter the solution using Whatman filter paper (grade No. 1), discarding the initial portion of the filtrate.

  • Spiking and Analysis:

    • Prepare a series of solutions from the filtered sample solution at different concentration levels (e.g., 80%, 100%, 120%) within the linear range of the method.
    • Spot 10 µL of each solution in triplicate on the HPTLC plate alongside standard solutions of known concentration.
    • Develop the plate using the pre-optimized mobile phase (e.g., DCM: Acetone, 8.5:1.5, v/v) in a chamber saturated for 30 minutes.
    • Air-dry the plate and scan at the selected wavelength (e.g., 254 nm).

  • Calculation:

    • The recovery percentage is calculated using the formula: Recovery (%) = (Found Concentration / Theoretical Concentration) × 100
    • The results should fall within the acceptance criteria of 98-102% [14], demonstrating the method's accuracy for quality control of dosage forms.

Protocol for Recovery Studies from Spiked Human Plasma

This protocol validates the method's performance for bioanalytical applications, assessing its ability to accurately measure the drug in a biological matrix.

  • Plasma Sample Preparation and Extraction:

    • Spiking: Add known volumes of REM and co-administered drug working standards to 100 µL of drug-free human plasma to achieve concentrations within the linear range (e.g., for REM, a range covering its reported Cmax of 4420 ng/mL) [22].
    • Protein Precipitation: Add 200 µL of acetonitrile to the spiked plasma sample to precipitate proteins. Vortex the mixture vigorously for 30 seconds [53].
    • Centrifugation: Centrifuge the sample at 4000 rpm for 30 minutes to separate the precipitated proteins [53].
    • Collection: Collect the clear supernatant, which contains the extracted analytes.

  • Analysis:

    • Spot the extracted supernatant (e.g., 10 µL) onto the HPTLC plate. A blank plasma extract should also be spotted to check for endogenous interference.
    • Develop and scan the plate as described in Section 2.2.

  • Calculation and Assessment:

    • Calculate the recovery percentage using the formula mentioned in Section 2.3.2.
    • The recovery should be consistent, precise, and fall within the acceptable range (e.g., 98-101.2% as reported in similar studies) [14], confirming that the sample preparation effectively isolates the analyte with minimal matrix interference.

Data Presentation and Analysis

The quantitative data from recovery studies must be presented clearly to facilitate evaluation. The following table structure is recommended for summarizing results.

Table 2: Summary of Recovery Data for REM and Co-administered Drugs in Spiked Human Plasma This table presents a model dataset based on validation guidelines [14] [22].

Analyte Spiked Concentration (ng/band) Found Concentration (Mean ± SD, ng/band) Recovery (%) RSD (%)
Remdesivir (REM) 0.5 0.502 ± 0.02 100.4 1.2
2.5 2.48 ± 0.06 99.2 1.5
5.0 5.05 ± 0.08 101.0 0.9
Linezolid (LNZ) 0.5 0.495 ± 0.015 99.0 1.8
2.0 2.02 ± 0.05 101.0 1.4
4.0 3.97 ± 0.07 99.3 1.1
Rivaroxaban (RIV) 0.2 0.198 ± 0.008 99.0 2.0
1.0 1.01 ± 0.03 101.0 1.9
2.5 2.48 ± 0.05 99.2 1.3

SD: Standard Deviation; RSD: Relative Standard Deviation (a measure of precision).

Workflow and Greenness Assessment

The entire analytical process, from sample preparation to data analysis, can be visualized as a coherent workflow. Furthermore, the environmental impact of the method should be formally evaluated using recognized greenness assessment tools.

G HPTLC Analysis Workflow start Sample & Standard Preparation spot Spot Application on HPTLC Plate start->spot develop Chromatographic Development spot->develop scan Densitometric Scanning develop->scan analyze Data Analysis & Quantification scan->analyze report Report Results & Greenness Assessment analyze->report

Diagram 1: HPTLC Analysis Workflow

The greenness of the analytical method should be investigated using metrics such as the Analytical GREEnness (AGREE) calculator or the Green Analytical Procedure Index (GAPI) [52] [14]. These tools provide visual outputs that highlight the environmental performance of the method across multiple criteria, including waste production, energy consumption, and toxicity of reagents, aligning with the core thesis on green HPTLC research.

Greenness Assessment Using AGREE, Analytical Eco-Scale, and GAPI Metrics

The integration of Green Analytical Chemistry (GAC) principles into pharmaceutical analysis represents a critical advancement in sustainable science. Within this framework, the greenness assessment of analytical methods has become a mandatory practice, supported by specialized metrics that evaluate environmental impact, safety, and practicality. For researchers focusing on the analysis of remdesivir with co-administered drugs, particularly using green High-Performance Thin-Layer Chromatography (HPTLC) approaches, understanding and applying these metrics is essential for validating both analytical and environmental performance [54] [55].

This protocol details the application of three cornerstone greenness assessment tools—AGREE, Analytical Eco-Scale, and GAPI—within the context of a broader thesis investigating green HPTLC methods for remdesivir combinations. These metrics provide complementary perspectives: AGREE offers a comprehensive quantitative score based on all 12 GAC principles, Analytical Eco-Scale employs a penalty-based system to calculate an overall environmental impact score, and GAPI delivers a detailed visual profile of environmental impacts across the entire analytical procedure [54] [56] [55].

Fundamental Principles

Greenness assessment metrics translate the theoretical principles of GAC into practical evaluation tools. The foundation of these metrics rests on common objectives: minimizing hazardous chemical usage, reducing energy consumption, enhancing operator safety, and implementing proper waste management protocols [54] [56] [55]. The ideal green analytical method incorporates miniaturization, uses alternative solvents with low toxicity, aims for high sample throughput, and minimizes overall waste generation [56].

Comparative Characteristics of Assessment Tools

The table below summarizes the core characteristics, advantages, and limitations of the three primary metrics discussed in this protocol.

Table 1: Comparison of Key Greenness Assessment Metrics

Metric Type of Output Basis of Assessment Key Advantages Reported Limitations
AGREE(Analytical Greenness) Numerical score (0-1) & pictogram 12 Principles of GAC Comprehensive; user-friendly software; easy method comparison [55] Does not fully account for pre-analytical processes [55]
Analytical Eco-Scale Numerical score (0-100) Penalty points for non-green aspects Simple calculation; direct method comparison; encourages transparency [55] Lacks visual component; relies on expert judgment for penalties [56] [55]
GAPI(Green Analytical Procedure Index) Qualitative pictogram (5-color scale) Entire analytical process steps Comprehensive visual identification of high-impact stages [55] No overall score; some subjectivity in color assignment [56] [55]

Experimental Protocols for Greenness Assessment

Protocol 1: Assessment Using the AGREE Metric

The AGREE metric evaluates methods against all 12 principles of GAC, providing a unified score via freely available software [56] [55].

3.1.1 Step-by-Step Procedure:

  • Download Software: Access the AGREE calculator from https://mostwiedzy.pl/AGREE.
  • Input Method Parameters: Enter detailed information about the analytical method across all 12 principles, including sample preparation, derivatization, reagent types and amounts, instrumentation energy consumption, and waste generation quantities [55].
  • Generate Output: The software produces a circular pictogram with 12 segments and an overall score from 0 (not green) to 1 (excellent greenness) [55].

3.1.2 Application Example: An HPTLC method for remdesivir, linezolid, and rivaroxaban using a mobile phase of dichloromethane:acetone achieved an AGREE score of 0.78, indicating a high level of greenness [14] [49]. The method's strengths included minimal sample preparation and the absence of derivatization, though the use of dichloromethane incurred a penalty [14].

Protocol 2: Assessment Using the Analytical Eco-Scale

The Analytical Eco-Scale is a quantitative tool that assigns penalty points to non-green method aspects, subtracted from a base score of 100 [55].

3.2.1 Step-by-Step Procedure:

  • Establish Baseline: Begin with a perfect score of 100.
  • Assign Penalty Points: Deduct points for hazardous reagents, energy consumption, and waste generation.
    • Reagents: Penalties based on amount and hazard (e.g., 1-5 points).
    • Waste: Penalties based on volume and hazard (e.g., 1-5 points).
    • Energy: Penalize high energy consumption (>1.5 kWh/sample).
    • Occupational hazards: Penalize non-closed systems and corrosiveness [55].
  • Calculate Final Score: Subtract total penalties from 100. Scores >75 are considered excellent green methods, 50-75 are acceptable, and <50 are inadequate [55].

3.2.2 Application Example: An RP-HPLC method for remdesivir analysis using methanol:acetonitrile:water mobile phase achieved an Analytical Eco-Scale score of 79, classifying it as an excellent green method [11].

Protocol 3: Assessment Using the GAPI Metric

GAPI provides a qualitative visual assessment using a five-color pictogram to represent environmental impact across the analytical lifecycle [55].

3.3.1 Step-by-Step Procedure:

  • Identify Process Stages: Map the entire analytical procedure from sample collection to final detection.
  • Evaluate Each Step: For each of the five sections in the GAPI pictogram (sample collection/preservation, sample preparation, reagents/solvents, instrumentation, waste generation), assign a color based on environmental impact: green (low), yellow (medium), or red (high) [55].
  • Construct Pictogram: Fill in the GAPI template to create a visual profile of the method's greenness.

3.3.2 Application Example: The previously mentioned HPTLC method for remdesivir with co-administered drugs was evaluated using GAPI, which visually highlighted the environmental impact of each step, particularly noting the solvent choices in the mobile phase [14] [49].

Greenness Assessment Workflow

The following diagram illustrates the logical workflow for conducting a comprehensive greenness assessment using the three complementary metrics.

G Start Start Greenness Assessment Method Define Analytical Method Parameters Start->Method AGREE AGREE Assessment (12 GAC Principles) Method->AGREE EcoScale Analytical Eco-Scale (Penalty Point System) Method->EcoScale GAPI GAPI Assessment (Pictogram Profile) Method->GAPI Compare Compare & Synthesize Results AGREE->Compare EcoScale->Compare GAPI->Compare Report Final Greenness Profile Compare->Report

Case Study: Green HPTLC Analysis of Remdesivir with Co-administered Drugs

A reported green HPTLC method simultaneously quantifies remdesivir with co-administered medications linezolid and rivaroxaban in spiked human plasma [14] [49]. The method employs TLC silica gel plates with a mobile phase of dichloromethane:acetone (8.5:1.5, v/v) and detection at 254 nm.

Greenness Assessment Results

The method was evaluated using all three metrics, with results summarized below.

Table 2: Greenness Assessment Results for HPTLC Method of Remdesivir with Co-administered Drugs

Assessment Metric Score/Result Interpretation & Key Findings
AGREE 0.78 High level of greenness. Strengths: Miniaturization, no derivatization. Weakness: Use of dichloromethane [14] [49].
Analytical Eco-Scale Not explicitly reported for this method, but an RP-HPLC method for remdesivir scored 79 [11]. Score >75 indicates excellent green method.
GAPI Favorable profile with some amber/red elements for solvents [14] [49]. Visual tool highlighting solvent choice as the main environmental concern.

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of green HPTLC methods for remdesivir analysis requires specific materials and reagents selected with GAC principles in mind.

Table 3: Essential Research Reagents and Materials for Green HPTLC Analysis

Item Function/Application Green Considerations
TLC Silica Gel 60 F254Aluminum sheets Stationary phase for separation Standard, commercially available plates with minimal packaging waste [14].
Dichloromethane (DCM) Mobile phase component Penalized in green metrics; consider alternative solvents for improved greenness [14].
Acetone Mobile phase component Less hazardous than many chlorinated solvents; preferred green choice [14].
Methanol Standard solution preparation Common solvent with relatively lower toxicity compared to alternatives like acetonitrile [14].
Human Plasma Biological matrix for method validation Ethical sourcing; proper disposal according to biohazard protocols [14].
Microsyringe (100 µL) Sample application onto HPTLC plates Enables precise, minimal volume application, reducing reagent consumption [14].

The integration of AGREE, Analytical Eco-Scale, and GAPI metrics provides a robust, multi-faceted framework for evaluating the environmental sustainability of analytical methods. For thesis research focusing on green HPTLC analysis of remdesivir with co-administered drugs, these tools offer validated protocols to demonstrate methodological greenness alongside traditional validation parameters. The complementary nature of these assessments—combining quantitative scoring (AGREE, Eco-Scale) with visual profiling (GAPI)—delivers a comprehensive sustainability evaluation that aligns with the growing imperative for environmentally responsible analytical science.

Whiteness Evaluation Using RGB12 and BAGI Algorithms

In modern pharmaceutical analysis, the principles of Green Analytical Chemistry (GAC) have evolved into a more comprehensive framework known as White Analytical Chemistry (WAC). While GAC primarily focuses on the ecological impact of analytical methods, WAC strives to harmonize three equally important pillars: analytical performance (Red), environmental impact (Green), and practical/economic feasibility (Blue). The concept draws inspiration from the RGB color model, where the balanced combination of red, green, and blue light produces the perception of white light [57]. Similarly, an ideal "white" analytical method demonstrates a perfect balance between these three attributes.

The transition from singular green assessment to comprehensive whiteness evaluation addresses a critical gap in sustainable method development. Traditional green metrics often overlooked whether methods remained functionally viable for routine application in quality control laboratories, particularly those with limited resources [58]. The WAC framework ensures that methods are not only environmentally benign but also analytically sound and practically applicable, supporting the broader goals of sustainable development in pharmaceutical sciences.

To operationalize this concept, two complementary algorithms have been developed: the RGB 12 algorithm for whiteness assessment and the Blue Applicability Grade Index (BAGI) for blueness evaluation. Together, these tools provide researchers with a standardized approach to quantify and compare the overall sustainability profile of analytical methods, enabling informed decisions during method development and selection processes [58].

Theoretical Foundations of RGB12 and BAGI Algorithms

The RGB12 Algorithm

The RGB12 algorithm provides a quantitative assessment framework based on the 12 principles of White Analytical Chemistry, which serve as an expansion and refinement of the original 12 principles of Green Analytical Chemistry. Each of the 12 principles is evaluated across the three RGB domains, resulting in a balanced score that reflects the method's overall "whiteness" [57].

The algorithm produces a whiteness percentage score ranging from 0% to 100%, with higher values indicating more ideal methods that successfully balance all three dimensions. This percentage offers a convenient single-value parameter that simplifies method comparison and selection [57]. The calculation involves systematic scoring of each principle across the red (analytical), green (ecological), and blue (practical) dimensions, with the final whiteness percentage representing the degree of harmony among these aspects.

Recent applications in pharmaceutical analysis demonstrate the utility of this approach. For instance, an HPTLC method for analyzing COVID-19 therapeutic protocols achieved a whiteness percentage of 95.6% using the RGB12 algorithm, indicating excellent balance across all domains [4]. Similarly, a method for analyzing anti-Helicobacter pylori therapy scored 88.9% on the RGB12 scale [58].

The BAGI Algorithm

The Blue Applicability Grade Index (BAGI) complements the RGB12 algorithm by specifically evaluating the practical and economic aspects of analytical methods. This tool focuses on parameters critical for routine implementation, particularly in resource-limited settings [58].

BAGI assesses multiple practical dimensions including:

  • Instrumentation requirements and availability
  • Analysis time and throughput
  • Cost per analysis
  • Sample preparation complexity
  • Operator skill requirements
  • Safety considerations
  • Energy consumption

The algorithm generates a score out of 100, with higher values indicating superior practical applicability. For example, an HPTLC-densitometric method for quantifying anti-Helicobacter pylori drugs achieved a high BAGI score of 90, confirming its excellent applicability for routine quality control [58].

Table 1: Key Characteristics of Whiteness Assessment Algorithms

Algorithm Evaluation Focus Scoring System Output Range Ideal Profile
RGB12 Holistic whiteness (Red, Green, Blue aspects) 12 principles of WAC 0-100% Higher percentage indicates better balance
BAGI Practicality and economic feasibility (Blue aspect) Multiple practical parameters 0-100 points Higher score indicates better applicability

Experimental Protocols for Whiteness Assessment

RGB12 Assessment Procedure

The implementation of the RGB12 algorithm follows a systematic protocol designed to ensure comprehensive and reproducible evaluation:

Step 1: Method Characterization Thoroughly document all aspects of the analytical method, including instrumentation, reagents, sample preparation steps, energy consumption, waste generation, analytical performance characteristics, and operational requirements.

Step 2: Principle-by-Principle Evaluation Score each of the 12 WAC principles across the three RGB dimensions using a standardized scoring system. For each principle, assign separate scores for analytical (red), ecological (green), and practical (blue) performance.

Step 3: Data Integration Input the individual scores into the RGB12 calculation algorithm. The specific calculation methodology involves weighted summation of the individual scores, though the exact mathematical formula may vary based on implementation.

Step 4: Results Interpretation Interpret the final whiteness percentage according to established benchmarks:

  • >90%: Excellent whiteness
  • 80-90%: Good whiteness
  • 70-80%: Acceptable whiteness
  • <70%: Requires improvement

Step 5: Comparative Analysis Compare the obtained whiteness percentage with alternative methods to determine the most sustainable approach for the specific analytical application.

BAGI Assessment Procedure

The BAGI evaluation follows a complementary protocol focused on practical aspects:

Step 1: Parameter Identification Identify all relevant practical parameters for the specific analytical context, including equipment availability, analysis time, cost factors, and operational complexity.

Step 2: Quantitative Scoring Assign scores for each parameter based on standardized criteria. For example, analysis time might be scored based on the number of samples processed per hour, while cost would be evaluated relative to alternative methods.

Step 3: Weighted Calculation Apply appropriate weighting factors to each parameter based on its relative importance in the specific application context, then calculate the overall BAGI score.

Step 4: Applicability Assessment Interpret the final BAGI score according to established ranges, with scores above 85 indicating excellent applicability for routine use.

G start Start Whiteness Assessment method_char Method Characterization Document all method parameters start->method_char principle_eval Principle-by-Principle Evaluation Score 12 WAC principles across RGB domains method_char->principle_eval data_integration Data Integration Input scores into RGB12 algorithm principle_eval->data_integration results_interp Results Interpretation Calculate whiteness percentage data_integration->results_interp comparative Comparative Analysis Compare with alternative methods results_interp->comparative bagi_start BAGI Assessment Evaluate practical parameters comparative->bagi_start param_id Parameter Identification Equipment, time, cost, complexity bagi_start->param_id quantitative Quantitative Scoring Score each practical parameter param_id->quantitative weighted Weighted Calculation Apply weighting factors quantitative->weighted applicability Applicability Assessment Interpret final BAGI score weighted->applicability decision Method Selection Choose optimal balanced method applicability->decision

Figure 1: Workflow for Comprehensive Whiteness and Applicability Assessment

Application in Pharmaceutical Analysis: Remdesivir and Co-administered Drugs

Case Study: HPTLC Analysis of COVID-19 Therapeutic Protocol

A recent application of whiteness assessment in pharmaceutical analysis involved the development of an HPTLC method for simultaneous determination of remdesivir (REM), favipiravir (FVP), and dexamethasone (DEX) in human plasma. This combination represents a common COVID-19 therapeutic protocol where therapeutic drug monitoring is crucial [4].

The analytical method employed ethyl acetate, hexane, and acetic acid (9:1:0.3, by volume) as the mobile phase system with detection at 254 nm. The method successfully resolved the three compounds with Rf values of 0.30 for REM, 0.64 for DEX, and 0.77 for FVP. Validation studies demonstrated excellent sensitivity with quantitation limits as low as 0.1 µg/band for REM and DEX, and 0.2 µg/band for FVP [4].

Whiteness assessment using the RGB12 algorithm yielded a remarkable whiteness percentage of 95.6%, indicating nearly ideal balance across analytical, ecological, and practical dimensions. The method's excellent performance across all three domains positioned it as a superior sustainable alternative to previously reported methods for therapeutic drug monitoring in COVID-19 patients [4].

Case Study: Eco-friendly Spectrofluorimetric Determination of Remdesivir

Another exemplary application involved the development of an eco-friendly spectrofluorimetric method for determination of remdesivir in the presence of its metabolite in human plasma. The method utilized the intrinsic fluorescence properties of REM, achieving a linear response within the range of 3.0–120.0 ng/mL at 428.3 nm using first-order derivative spectrofluorimetry [10].

The method demonstrated high sensitivity with detection and quantification limits of 1.12 and 3.67 ng/mL, respectively, making it suitable for therapeutic drug monitoring of COVID-19 patients. The environmental sustainability was comprehensively evaluated using GAPI, AGREE, and RGB12 metrics, confirming its green and eco-friendly characteristics while maintaining excellent analytical performance [10].

Table 2: Whiteness Assessment Results for Reported Analytical Methods

Analytical Method Analytes Matrix RGB12 Score (%) BAGI Score Key Strengths
HPTLC-densitometry [4] REM, FVP, DEX Human plasma 95.6% N/R Excellent sensitivity, green solvents
HPTLC-densitometry [58] OMZ, TNZ, CLR Dosage forms 88.9% 90 Cost-effective, minimal sample preparation
Spectrofluorimetry [10] REM, metabolite Human plasma N/R N/R High sensitivity, green solvents
HPTLC-densitometry [59] VON, ASP Laboratory-prepared tablets N/R N/R First chromatographic method for this combination

N/R: Not explicitly reported in the cited literature

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of whiteness assessment requires specific reagents, materials, and instrumentation. The following table summarizes key components used in the referenced studies:

Table 3: Essential Research Reagents and Materials for Green HPTLC Analysis

Item Specification Function Green Considerations
HPTLC Plates Silica gel 60 F254, 20×20 cm Stationary phase for separation Reusable in some applications, minimal material usage
Mobile Phase Components Ethyl acetate, ethanol, ethyl acetate:hexane:acetic acid (9:1:0.3) Solvent system for compound separation Preferred over hazardous solvents like chloroform or acetonitrile
Reference Standards REM, FVP, DEX (purity >99%) Method development and validation Minimal quantities required due to high sensitivity
Sample Application 100 µL microsyringe, Linomat applicator Precise sample positioning Automated application reduces human error and variability
Detection System Densitometric scanner with deuterium lamp Quantitative analysis after separation Minimal energy consumption compared to HPLC systems
Sample Preparation Methanol, acetonitrile Solvent for standard solutions Green solvent selection based on GSST assessment

The implementation of RGB12 and BAGI algorithms for whiteness assessment represents a significant advancement in sustainable pharmaceutical analysis. These tools provide researchers with a standardized framework to develop methods that balance analytical excellence, ecological responsibility, and practical feasibility. The case studies involving remdesivir and co-administered drugs demonstrate that highly white methods achieving scores above 90% are attainable without compromising analytical performance.

As the pharmaceutical industry continues to embrace sustainability principles, the integration of whiteness assessment during method development will become increasingly important. The RGB12 and BAGI algorithms offer a practical pathway toward this goal, enabling researchers to quantify and optimize the sustainability profile of their analytical methods while maintaining the rigorous standards required for pharmaceutical quality control and therapeutic drug monitoring.

Comparative Analysis with Other Analytical Techniques (HPLC, Spectrofluorimetry)

The analysis of complex pharmaceutical formulations and biological samples, particularly for therapeutics like remdesivir (REM) and its co-administered drugs, demands analytical techniques that are selective, sensitive, and environmentally sustainable. The principles of Green Analytical Chemistry (GAC) are increasingly becoming a central consideration in method development within quality control and bioanalytical laboratories. High-performance thin-layer chromatography (HPTLC) is re-emerging as a powerful technique that aligns with these principles. This application note provides a comparative analysis of HPTLC against two other widely used techniques—High-Performance Liquid Chromatography (HPLC) and spectrofluorimetry—for the analysis of REM, with a specific focus on greenness, cost-effectiveness, and applicability in therapeutic drug monitoring.

Quantitative Comparison of Analytical Techniques

The table below summarizes the key analytical performance and greenness metrics for HPTLC, HPLC, and Spectrofluorimetry methods as reported in recent literature for the analysis of REM and co-administered drugs.

Table 1: Comparative Analysis of Techniques for Remdesivir and Co-administered Drug Assay

Feature Green HPTLC RP-HPLC Spectrofluorimetry
Representative Analytic(s) REM, FAV, DEX, LNZ, RIV [14] [4] REM (Raw Materials) [11] REM & Metabolite (GS-441524) [10]
Linear Range 0.1 - 5.5 µg/band for REM [14] [4] Not specified in detail 3.0 - 120.0 ng/mL [10]
Limit of Quantification (LOQ) 128.8 ng/band for REM [14] 0.06 µg/mL [11] 3.67 ng/mL [10]
Analysis Time High throughput; multiple samples in parallel (~15-20 min) [60] Sequential analysis; ~2.2 min retention time for REM [11] Rapid, but sample preparation may be needed
Solvent Consumption Very Low (µL per sample) [16] High (mL per minute of runtime) [11] Low (mL per sample)
Greenness Score (AGREE) 0.78 (Similar HPTLC method) [14] 0.78 (for a specific RP-HPLC method) [11] Evaluated as "green" via GAPI, AGREE, RGB12 [10]
Key Advantage Cost-effective, high throughput, minimal solvent waste [49] [14] High reproducibility and widespread availability [11] Excellent sensitivity for native fluorescent compounds [10]
Primary Limitation Lower sensitivity vs. some techniques Higher solvent consumption and cost Limited to fluorescent analytes or derivatization

Detailed Experimental Protocols

Protocol 1: Green HPTLC for Remdesivir with Co-administered Drugs

This protocol is adapted from methods for the simultaneous quantification of REM, linezolid (LNZ), and rivaroxaban (RIV) in spiked human plasma [49] [14].

Research Reagent Solutions

Table 2: Essential Materials and Reagents for HPTLC

Reagent/Solution Function Specification
TLC Silica gel 60 F254 plates Stationary phase for chromatographic separation Aluminum sheets, 20 × 20 cm, 0.1/0.25 mm thickness
Dichloromethane (DCM) and Acetone Mobile phase components HPLC or Analytical Grade
Remdesivir, Linezolid, Rivaroxaban Reference standards Purity ≥ 99.6%
Methanol, Acetonitrile Solvent for stock standard solutions HPLC Grade
CAMAG TLC scanner with winCATS software Densitometric detection and data analysis Deuterium lamp, scanning speed 20 mm/s
CAMAG Linomat autosampler Precise application of samples as bands 100 µL microsyringe
Step-by-Step Procedure
  • Standard Solution Preparation: Accurately weigh 10 mg each of REM, LNZ, and RIV reference standards. Transfer to separate 10 mL volumetric flasks, dissolve, and make up to volume with methanol (for REM and LNZ) or acetonitrile (for RIV) to obtain 1 mg/mL stock solutions. These can be stored refrigerated for up to 14 days. Prepare working solutions by appropriate dilution with methanol.
  • Sample Preparation (Spiked Plasma): Transfer aliquots of working solutions into centrifuge tubes. Add 0.5 mL of drug-free human plasma. Precipitate proteins by adding 3 mL of acetonitrile, vortex for 1 minute, and centrifuge. Filter the supernatant through a 0.45 µm membrane filter before application on the HPTLC plate [10] [14].
  • Chromatographic Separation:
    • Spotting: Using the Linomat autosampler, apply 10 µL of the processed sample or standard as 6 mm bands on the HPTLC plate, 10 mm from the bottom and edge.
    • Development: Saturate a twin-trough glass chamber with the mobile phase (DCM:Acetone, 8.5:1.5, v/v) for 30 minutes. Develop the plate to a distance of 8 cm.
    • Drying: Air-dry the developed plate at room temperature.
  • Detection & Quantification: Scan the plate densitometrically at 254 nm using a deuterium lamp. The typical retardation factors (Rf) are approximately 0.23 for REM, 0.53 for LNZ, and 0.72 for RIV [14].
  • Calibration: Construct calibration curves by plotting the peak area against the corresponding concentration (µg/band) for each drug.
Protocol 2: Spectrofluorimetric Determination of Remdesivir and its Metabolite

This protocol describes a stability-indicating method for REM in the presence of its alkaline-induced degradation product (AKDP, metabolite GS-441524) in human plasma [10].

Research Reagent Solutions

Table 3: Essential Materials and Reagents for Spectrofluorimetry

Reagent/Solution Function Specification
Remdesivir reference standard Primary analyte Purity ~100%
Sodium Hydroxide (NaOH) & Hydrochloric Acid (HCl) For forced degradation of REM to produce AKDP 1 N solutions
Acetonitrile Protein precipitating agent in plasma samples HPLC Grade
SHIMADZU RF-6000 Spectrofluorometer Fluorescence measurement Equipped with a 150 W Xenon lamp
Step-by-Step Procedure
  • Preparation of Alkaline-Induced Degradation Product (AKDP): Reflux 25 mg of pure REM with 25 mL of 1 N NaOH in a water bath at 100°C for 2 hours. After cooling, neutralize the solution with 1 N HCl and dilute to 100 mL with distilled water to obtain a stock solution of 250 µg/mL [10].
  • Calibration Curve Construction: From a REM working standard solution (100 ng/mL), transfer aliquots (30-1200 ng) into a series of 10 mL volumetric flasks. Dilute to volume with distilled water to achieve a concentration range of 3-120 ng/mL. Record the fluorescence spectra between 246 and 600 nm after excitation at 245 nm.
  • Derivative Spectra Generation: Use the instrument's software to convert the zero-order spectra into first-order derivative (D1) spectra with a Δλ of 8 and a scaling factor of 100.
  • Measurement: Measure the peak amplitude of the D1 spectrum at 428.3 nm. Construct a calibration curve by plotting this amplitude against the corresponding REM concentration.
  • Sample Analysis (Spiked Plasma): Spike human plasma with REM, precipitate proteins with acetonitrile (as in section 3.1.2), and dilute the supernatant with distilled water to fall within the linear range. Follow steps 2-4 to determine the REM concentration using the regression equation.
Protocol 3: Green RP-HPLC for Remdesivir in Raw Materials

This protocol is suited for the quantification of REM in raw material and drug product quality control [11].

Research Reagent Solutions

Table 4: Essential Materials and Reagents for RP-HPLC

Reagent/Solution Function Specification
Methanol, Acetonitrile, Water Mobile phase components HPLC Grade
Remdesivir reference standard Primary analyte High Purity
RP-HPLC System Chromatographic separation and analysis Isocratic or gradient pump, UV/ PDA detector
Step-by-Step Procedure
  • Mobile Phase Preparation: Prepare a mixture of methanol, acetonitrile, and water in the ratio 10:10:80 (v/v/v). Filter and degas the solution.
  • Standard Solution: Prepare a stock solution of REM and dilute with the mobile phase or a compatible solvent to the required concentration range.
  • Chromatographic Conditions:
    • Column: Reverse-phase C18 column (e.g., 150 mm x 4.6 mm, 5 µm).
    • Mobile Phase: Methanol:Acetonitrile:Water (10:10:80, v/v/v).
    • Flow Rate: 1.0 mL/min.
    • Detection: UV detection at a suitable wavelength (e.g., 244 nm).
    • Injection Volume: 10-20 µL. Under these conditions, REM elutes at approximately 2.2 minutes [11].
  • System Suitability & Quantification: Inject the standard solutions to ensure the system is suitable (theoretical plates, tailing factor). The concentration of REM in unknown samples is calculated based on the peak area response from the calibration curve.
HPTLC Workflow for Antiviral Analysis

The following diagram illustrates the streamlined workflow of the green HPTLC method, highlighting its high-throughput capability and minimal solvent consumption.

Start Start Sample Analysis Spot Spot Samples on HPTLC Plate Start->Spot Develop Develop Plate in Saturated Chamber Spot->Develop Dry Air-Dry Plate Develop->Dry Scan Scan with Densitometer Dry->Scan Analyze Analyze Peaks & Quantify Scan->Analyze Result Final Quantitative Result Analyze->Result

Greenness Assessment of Analytical Techniques

This diagram compares the three techniques against the 12 principles of White Analytical Chemistry (WAC), demonstrating the superior overall sustainability profile of the HPTLC method.

HPTLC HPTLC P1 Sample Throughput HPTLC->P1 P2 Analysis Time HPTLC->P2 P3 Cost per Analysis HPTLC->P3 P6 Waste Generation HPTLC->P6 P7 Miniaturization HPTLC->P7 P11 Methodology Simplicity HPTLC->P11 HPLC HPLC P8 Automation Potential HPLC->P8 P9 Analytical Performance HPLC->P9 Fluor Fluor Fluor->P2 Fluor->P9 P12 Direct vs. Derivatization Fluor->P12 P4 Operator Safety P5 Energy Consumption P10 Chemical Toxicity

The comparative analysis confirms that Green HPTLC presents a compelling alternative to HPLC and spectrofluorimetry for the simultaneous analysis of remdesivir and co-administered drugs, particularly in a bioanalytical context. While RP-HPLC offers robust performance and is well-established in quality control, its environmental footprint is higher. Spectrofluorimetry provides exceptional sensitivity for specific applications but lacks the multi-analyte capability without additional steps. The high-throughput, minimal solvent use, cost-effectiveness, and excellent greenness credentials of HPTLC make it a highly suitable and sustainable choice for therapeutic drug monitoring and pharmaceutical analysis, aligning perfectly with the modern principles of Green and White Analytical Chemistry.

Conclusion

Green HPTLC has firmly established itself as a cost-effective, sensitive, and environmentally sustainable analytical platform for the simultaneous quantification of remdesivir and its co-administered drugs. The successful application of these methods in complex matrices like spiked human plasma underscores their significant potential for therapeutic drug monitoring and pharmacokinetic studies in COVID-19 patients on multi-drug regimens. The excellent greenness and whiteness scores validate their alignment with the principles of sustainable and white analytical chemistry. Future directions should focus on applying these methods in large-scale clinical settings, expanding their use to monitor drug-drug interactions, and adapting the platforms for the analysis of new antiviral therapies and their metabolites, thereby solidifying the role of green HPTLC in modern bioanalytical and clinical research.

References