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
References
Coleshill, M. J. et al. Rebranding gout: could a name change for gout improve adherence to Urate-Lowering therapy? Ther. Innov. Regul. Sci. 55, 138–141 (2021).
Azevedo, V. F. et al. Critical revision of the medical treatment of gout in Brazil. Rev. Bras. Reumatol. 57, 346–355 (2017).
Global, regional, and national burden of gout, 1990–2020, and projections to 2050: a systematic analysis of the global burden of disease study 2021. Lancet Rheumatol. 6, e507–e517 (2024).
Xia, Y. et al. Global, regional and National burden of gout, 1990–2017: a systematic analysis of the global burden of disease study. Rheumatol. (Oxford). 59, 1529–1538 (2020).
Rai, S. K. et al. The rising prevalence and incidence of gout in British Columbia, canada: Population-based trends from 2000 to 2012. Semin Arthritis Rheum. 46, 451–456 (2017).
Mattiuzzi, C. & Lippi, G. Recent updates on worldwide gout epidemiology. Clin. Rheumatol. 39, 1061–1063 (2020).
Kiadaliri, A. A., Uhlig, T. & Englund, M. Burden of gout in the nordic region, 1990–2015: findings from the global burden of disease study 2015. Scand. J. Rheumatol. 47, 410–417 (2018).
Zhu, B. et al. Trend dynamics of gout prevalence among the Chinese population, 1990–2019: A joinpoint and age-period-cohort analysis. Front. Public. Health. 10, 1008598 (2022).
Roman, Y. M. Moving the needle in gout management: the role of culture, diet, genetics, and personalized patient care practices. Nutrients 14, 2 (2022).
Rashad, A. Y. et al. Towards the development of dual hypouricemic and Anti-inflammatory candidates: Design, Synthesis, stability studies and biological evaluation of some mutual ester prodrugs of Febuxostat-NSAIDs. Bioorg. Chem. 135, 106502 (2023).
Stamp, L. K. & Farquhar, H. Treatment advances in gout. Best Pract. Res. Clin. Rheumatol 35 (2021).
Dehlin, M., Jacobsson, L. & Roddy, E. Global epidemiology of gout: prevalence, incidence, treatment patterns and risk factors. Nat. Rev. Rheumatol. 16, 380–390 (2020).
Zhu, Y., Pandya, B. J. & Choi, H. K. Prevalence of gout and hyperuricemia in the US general population: the National health and nutrition examination survey 2007–2008. Arthritis Rheum. 63, 3136–3141 (2011).
Parisa, N., Kamaluddin, M. T., Saleh, M. I. & Sinaga, E. The inflammation process of gout arthritis and its treatment. J. Adv. Pharm. Technol. Res. 14, 166–170 (2023).
Gullick, D. R., Mott, K. B. & Bartlett, M. G. Chromatographic methods for the bioanalysis of pyrethroid pesticides. Biomed. Chromatogr. 30, 772–789 (2016).
Shen, M. et al. Febuxostat in the treatment of gout patients with low serum uric acid level: 1-year finding of efficacy and safety study. Clin. Rheumatol. 37, 3107–3113 (2018).
Deidda, R., Orlandini, S., Hubert, P. & Hubert, C. Risk-based approach for method development in pharmaceutical quality control context: A critical review. J. Pharm. Biomed. Anal. 161, 110–121 (2018).
Moineau, I. et al. Using analytical quality by design to improve analytical method development in vaccines quality control: application to an optimized quantitative high-performance anion-exchange chromatographic method. J. Chromatogr. B Analyt Technol. Biomed. Life Sci. 1233, 123946 (2024).
Prajapati, P. B., Patel, P. R. & Shah, S. A. Chemometric and DoE-Based analytical quality risk management to HPTLC method for simultaneous Estimation of metronidazole and Norfloxacin. J. Chromatogr. Sci. 61, 428–439 (2023).
Prajapati, P., Tamboli, J. & Mishra, A. Risk and DoE-Based analytical failure mode effect analysis (AFMEA) to simultaneous Estimation of Montelukast sodium and Bilastine by HPTLC method using enhanced AQbD approach. J. Chromatogr. Sci. 60, 595–605 (2022).
Makhija, R., Barik, P., Mehta, A., Ganti, S. S. & Asati, V. Sustainable approaches to analyzing phenolic compounds: a green chemistry perspective. Anal. Sci. 40, 1947–1968 (2024).
Prajapati, P. et al. Application of principal component analysis and DoE-Driven green analytical chemistry concept to liquid chromatographic method for Estimation of Co-formulated Anti-Hypertensive drugs. J. AOAC Int. 106, 1087–1097 (2023).
Lahkar, C. et al. A technique based on infrared spectroscopy for determining sulfanilamide levels sustainably: progress and comparisons of greenness and whiteness using ComplexGAPI, AGREE, and RGB. Spectrochim Acta Mol. Biomol. Spectrosc. 318, 124467 (2024).
Semysim, F. A., Hussain, B. K., Hussien, M. A., Azooz, E. A. & Snigur, D. Assessing the greenness and environmental friendliness of analytical methods: Modern approaches and recent computational programs. Crit. Rev. Anal. Chem. https://doi.org/10.1080/10408347.2024.2304552 (2024).
Katamesh, N. S., Abbas, A. E. F. & Mahmoud, S. A. Four chemometric models enhanced by Latin hypercube sampling design for quantification of anti-COVID drugs: sustainability profiling through multiple greenness, carbon footprint, blueness, and whiteness metrics. BMC Chem. 18, 54 (2024).
Patel, R. & Kotadiya, R. Stability-indicating green HPLC method for fixed-dose tablets containing remogliflozin etabonate and teneligliptin: an AQbD approach. Drug Dev. Ind. Pharm. https://doi.org/10.1080/03639045.2024.2400199 (2024).
Gurumukhi, V. C. et al. Quality-by-design based fabrication of febuxostat-loaded nanoemulsion: statistical optimization, characterizations, permeability, and bioavailability studies. Heliyon 9, e15404 (2023).
Premsagar, K. M. et al. Development of a gradient method for sulfamethoxazole, trimethoprim, isoniazid, and pyridoxine hydrochloride in rabbit plasma through QbD-driven investigation. Sci Rep 14, 25806 (2024).
Patel, R. B., Patel, N. M., Patel, M. R. & Solanki, A. B. Optimization of robust HPLC method for quantitation of ambroxol hydrochloride and roxithromycin using a DoE approach. J. Chromatogr. Sci. 55, 275–283 (2017).
Wu, B., Li, Q., Wang, L., Chen, F. & Jiang, J. Development and validation of bioanalytical methods to support clinical study of disitamab Vedotin. Bioanalysis 16, 385–400 (2024).
Zeng, W. & P Bateman, K. Quantitative LC-MS/MS. 1. Impact of points across a peak on the accuracy and precision of peak area measurements. J. Am. Soc. Mass. Spectrom. 34, 1136–1144 (2023).
Iwamoto, N., Shimada, T., Terakado, H., Hamada, A. & Validated LC-MS/MS analysis of immune checkpoint inhibitor nivolumab in human plasma using a fab peptide-selective quantitation method: nano-surface and molecular-orientation limited (nSMOL) proteolysis. J. Chromatogr. B Analyt Technol. Biomed. Life Sci. 1023–1024, 9–16 (2016).
Iwamoto, N. et al. Fully validated LCMS bioanalysis of bevacizumab in human plasma using nano-surface and molecular-orientation limited (nSMOL) proteolysis. Drug Metab. Pharmacokinet. 31, 46–50 (2016).
Barone, R. et al. Development and validation of a fast UPLC-MS/MS screening method for the detection of 68 psychoactive drugs and metabolites in whole blood and application to post-mortem cases. J. Pharm. Biomed. Anal. 228, 115315 (2023).
Hailat, M., Al-Ani, I., Hamad, M., Zakareia, Z. & Abu Dayyih, W. Development and validation of a method for quantification of favipiravir as COVID-19 management in spiked human plasma. Molecules 26, 13 (2021).
Prajapati, P. et al. Whiteness, redness, blueness and greenness profile assessment and design of experiments approach to microwave-aided sensitive and green spectrofluorophotometric determination of Baclofen. Luminescence 39, e4911 (2024).
Prajapati, P., Pulusu, V. S. & Shah, S. White analytical chemistry-driven stability-indicating concomitant chromatographic Estimation of thiocolchicoside and aceclofenac using response surface analysis and red, green, and blue model. J. Sep. Sci. 46, 2300139 (2023).
Han, D. G. et al. Pharmacokinetic evaluation of metabolic drug interactions between repaglinide and celecoxib by a bioanalytical HPLC method for their simultaneous determination with fluorescence detection. Pharmaceutics 11, 13 (2019).
Prajapati, P. et al. Green LC-MS/MS method for in-vivo pharmacokinetics of mirabegron-encapsulated nanostructured lipid carriers in rat plasma: integrating white analytical chemistry and analytical quality by design approach. Sustain. Chem. Pharm. 39, 101523 (2024).
Prajapati, P., Prajapati, B., Pulusu, V. S. & Shah, S. Multivariate analysis and response surface modeling to green analytical Chemistry–Based RP-HPLC-PDA method for chromatographic analysis of vildagliptin and remogliflozin etabonate. J. AOAC Int. 106, 601–612 (2023).
Shao, J., Cao, W., Qu, H., Pan, J. & Gong, X. A novel quality by design approach for developing an HPLC method to analyze herbal extracts: A case study of sugar content analysis. PLoS One. 13, e0198515 (2018).
Liu, Q. D., Qin, K. M., Shen, B. J., Cai, H. & Cai, B. C. Optimization of the processing technology of fructus arctii by response surface methodology. Chin. J. Nat. Med. 13, 222–231 (2015).
Pena-Pereira, F., Wojnowski, W. & Tobiszewski, M. AGREE - Analytical greenness metric approach and software. Anal. Chem. 92, 10076–10082 (2020).
Płotka-Wasylka, J. & Wojnowski, W. Complementary green analytical procedure index (ComplexGAPI) and software. Green Chem. 23, 8657–8665 (2021).
Nowak, P. M. & Kościelniak, P. What color is your method? Adaptation of the Rgb additive color model to analytical method evaluation. Anal. Chem. 91, 10343–10352 (2019).
Hicks, M. B. et al. Making the move towards modernized greener separations: introduction of the analytical method greenness score (AMGS) calculator. Green Chem. 21, 1816–1826 (2019).
Kang, Y. J., Jeong, H. C., Kim, T. E. & Shin, K. H. Bioanalytical method using Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPL-CHRMS) for the detection of metformin in human plasma. Molecules 25, 20 (2020).
Dong, H. et al. Liquid chromatography-tandem mass spectrometry simultaneous determination and pharmacokinetic study of fourteen alkaloid components in dog plasma after oral administration of Corydalis bungeana Turcz extract. Molecules 23, 20 (2018).
Heo, H. M., Jo, H. W., Chang, H. R. & Moon, J. K. Development of simultaneous analytical method for imidazolinone herbicides from livestock products by UHPLC-MSMS. Foods 11, 21 (2022).
Al-Wasidi, A. S., Ahmed, H. A., Alshammari, M. F. A., Nafee, S. S. & Mohamed, M. A. Cutting-edge HPLC and MCR techniques for synchronically quantifying anticholinergic drugs in the presence of C12 and C14 homologs: robust application to green and white chemistry. Arch. Pharm. (Weinheim). 357, e2400256 (2024).
Płotka-Wasylka, J. A new tool for the evaluation of the analytical procedure: green analytical procedure index. Talanta 181, 204–209 (2018).
Mostafa, A. Insights into the sustainability of liquid chromatographic methods for favipiravir bioanalysis: a comparative study. RSC Adv. 14, 19658–19679 (2024).
Sinzervinch, A., Torres, M. S. I. & Kogawa, C. A. Tools to evaluate the eco-efficiency of analytical methods in the context of green and white analytical chemistry: A review. Curr. Pharm. Des. 29, 2442–2449. https://doi.org/10.2174/0113816128266396231017072043 (2023).
Boutkhoum, O., Hanine, M., Boukhriss, H., Agouti, T. & Tikniouine, A. Multi-criteria decision support framework for sustainable implementation of effective green supply chain management practices. Springerplus 5, 664 (2016).
Veerendra, Y. V. S., Brahman, P. K., Mankumare, S. D., Ch, J. & C, V. K. Evaluation of analytical greenness metric for an eco-friendly method developed through the integration of green chemistry and quality-by-design for the simultaneous determination of nebivolol hydrochloride, Telmisartan, Valsartan, and amlodipine besylate. Heliyon 10, e35376 (2024).
Ražić, S., Arsenijević, J., Đogo Mračević, S., Mušović, J. & Trtić-Petrović, T. Greener chemistry in analytical sciences: from green solvents to applications in complex matrices. Current challenges and future perspectives: a critical review. Analyst 148, 3130–3152 (2023).
Guo, L., Shi, Y., Li, K. W., Yan, J. & Xu, R. K. Using an inexpensive RGB color sensor for field quantitative assessment of soil accessible Cu(Ⅱ). Environ. Pollut. 344, 123348 (2024).
Deka, M. K. et al. Development of three UV-spectroscopic methods for simultaneous Estimation of raloxifene and aspirin in pharmaceutical dosage form: whiteness and greenness assessment with application of ComplexGAPI, AGREE, and RGB. Green. Anal. Chem. 8, 100088 (2024).
Pujol-Cano, N. et al. Near-infrared fluorescence cholangiography at a very low dose of indocyanine green: quantification of fluorescence intensity using a colour analysis software based on the RGB color model. Langenbecks Arch. Surg. 407, 3513–3524 (2022).
Jankech, T. et al. Current green capillary electrophoresis and liquid chromatography methods for analysis of pharmaceutical and biomedical samples (2019–2023) - A review. Anal. Chim. Acta. 1323, 342889 (2024).
Mohamed, H. M. & Lamie, N. T. Analytical Eco-Scale for assessing the greenness of a developed RP-HPLC method used for simultaneous analysis of combined antihypertensive medications. J. AOAC Int. 99, 1260–1265 (2016).
Starlin, Z., Harahap, Y. & Sitepu, S. Method validation of acrylamide in dried blood spot by liquid Chromatography-tandem mass spectrometry. Pak J. Biol. Sci. 23, 1321–1331 (2020).
Abdelhamid, N. S., Magdy, M. A., Anwar, B. H. & Farid, N. F. US FDA-validated TLC method with four greenness assessment evaluations for simultaneous determination of prednisolone and Esomeprazole in spiked human plasma. Biomed. Chromatogr. 36, e5343 (2022).
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
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.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
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
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-36517-y