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Development of a green chemistry based bioanalytical method using response surface methodology to analyze febuxostat and indomethacin in rabbit plasma
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  • Published: 17 January 2026

Development of a green chemistry based bioanalytical method using response surface methodology to analyze febuxostat and indomethacin in rabbit plasma

  • Akramul Ansary1,
  • Piyongsola2,
  • Biprojit Paul1,
  • Koushik Nandan Dutta3,
  • Bhargab Jyoti Sahariah4,
  • Manoj Kumar Deka4,
  • Amit kumar Das4 &
  • …
  • Manish Majumder1,5 

Scientific Reports , Article number:  (2026) Cite this article

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We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Subjects

  • Bioanalytical chemistry
  • Cheminformatics

Abstract

This research established an environmentally friendly and sustainable approach to measure Febuxostat and Indomethacin levels in rabbit plasma samples, utilizing isocratic liquid chromatography guided by green analytical chemistry principles and Analytical Quality by Design (AQbD) methodology. Chromatographic separation was performed on an Eclipse Plus C18 column (25 cm x 5 cm,4.6 μm), using a binary mobile phase of ethanol and 50 mM potassium dihydrogen orthophosphate (pH 4.5) in a 66:34 ratio, delivered at 0.8 mL/min for 15 min. Resolution and asymmetry factors were designated Critical Analytical Attributes (CAAs). Control Noise Experimentation (CNX) screening identified flow rate, mobile phase pH, and ethanol concentration as significant contributors to CAAs variability. Subsequent optimization utilizing Central Composite Design (CCD) refined the Critical Method Parameters (CMPs) to ensure optimal performance. Chromatographic analysis revealed Febuxostat and Indomethacin retention times of 4.41 and 7.35 min, respectively. The method’s greenness and analytical quality were assessed using AGREE, ComplexGAPI, RGB, and AMGS tools. Validation studies confirmed linearity (R2: 0.9959 for Febuxostat, 0.9981 for Indomethacin) within 200–4600 ng/mL, alongside successful precision, accuracy, recovery, and stability evaluations at concentrations of 250, 750, 1500, and 3000 ng/mL.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Abbreviations

FEB:

Febuxostat

IND:

indomethacin

HPLC:

High Performance Liquid Chromatography

AQbD:

Analytical Quality by Design

MODR:

Method Operable Design Region

DoE:

Design of Experiments

GAC:

Green Analytical Chemistry

AGREE:

Analytical Greenness Calculator

ComplexGAPI:

Green Analytical Procedure Index

RGB:

Red, Green, Blue

AMGS:

Analytical Method Greenness Score

CCD:

Central Composite Design

QC:

Quality control

LLOQ:

Lower limit of quantification

LQC:

Low-quality control

MQC:

Medium-quality control

HQC:

High-quality control

IAEC:

Institutional Animal Ethical Committee

LOD:

Limit of detection

LOQ:

Limit of quantification

CS:

Colour Score

C&S:

Cause and Effect

MB:

Method Brilliance

LAV:

Lowest Acceptable Value

LSV:

Lowest Satisfactory Value

CNX:

Control-Noise-Experimentation

Factor A:

pH

Factor B:

Ethanol concentration

Factor C:

Flow rate

R1:

Resolution (Rs)

R2:

Asymmetric factor (As)

R1(FEB):

Resolution of Febuxostat

R1(IND):

Resolution of Indomethacin

R2(FEB):

Asymmetric factor of Febuxostat

R2(IND):

Asymmetric factor of Indomethacin

R:

Redness (Analytical performance)

R1:

Scope of application

R2:

LOD (R2.1) & LOQ (R2.2)

R3:

Precision

R4:

Accuracy

G:

Greenness (Safety and eco- friendliness)

G1:

Toxicity of the reagents

G2:

Amount of reagents and waste

G3:

Consumption of energy and waste

G4:

Direct impact (Occupational hazards and no of Genetically Modified Organism)

B:

Blueness (Productivity/ Practical effectiveness)

B1:

Cost-effectiveness

B2:

Time efficiency

B3:

Requirements: sample consumption (B3.1) & Advanced instruments (B3.2)

B4:

Operational simplicity: Mini-automatization (B4.1) & Portability (B4.2)

SD:

Standard Deviation

%RE:

Percentage of Relative Error

CV:

Coefficient of variation

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Acknowledgements

The authors are thankful to NETES Institute of Pharmaceutical Science, Mirza, for providing all the research facilities to carry out the research work.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, NETES Institute of Pharmaceutical Science, Nemcare Group of Institution, Mirza, Kamrup, Assam, 781125, India

    Akramul Ansary, Biprojit Paul & Manish Majumder

  2. Department of Pharmacology, NETES Institute of Pharmaceutical Science, Nemcare Group of Institution, Mirza, Kamrup, Assam, 781125, India

    Piyongsola

  3. Department of Pharmacognosy, NETES Institute of Pharmaceutical Science, Nemcare Group of Institution, Mirza, Kamrup, Assam, 781125, India

    Koushik Nandan Dutta

  4. Department of Pharmaceutics, NETES Institute of Pharmaceutical Science, Nemcare Group of Institution, Mirza, Kamrup, Assam, 781125, India

    Bhargab Jyoti Sahariah, Manoj Kumar Deka & Amit kumar Das

  5. School of Pharmacy, Sainath University, Ranchi, Jharkhand, India

    Manish Majumder

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Contributions

Akramul Ansary: Data curation, Methodology Visualization, Investigation; Piyongsola, Biprojit Paul: Data curation, Visualization, Methodology, Investigation; Amit kumar Das: Writing- Original draft preparation, Investigation, Software; Koushik Nandan Dutta: Original draft preparation, Investigation, Software; Bhargab Jyoti Sahariah, Manoj Kumar Deka: Supervision, Writing- Reviewing and Editing; Manish Majumder: Conceptualization, Supervision, writing, Reviewing and Editing.

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Correspondence to Manish Majumder.

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Ansary, A., Piyongsola, Paul, B. et al. Development of a green chemistry based bioanalytical method using response surface methodology to analyze febuxostat and indomethacin in rabbit plasma. Sci Rep (2026). https://doi.org/10.1038/s41598-026-36517-y

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  • Received: 16 December 2024

  • Accepted: 13 January 2026

  • Published: 17 January 2026

  • DOI: https://doi.org/10.1038/s41598-026-36517-y

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Keywords

  • Febuxostat
  • Indomethacin
  • Analytical quality by design
  • Green analytical chemistry
  • Validation
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