Development and validation of a bioanalytical hplc-dad method for ketoprofen quantification in rat plasma and brain tissue: application to pharmacokinetic studies and tissue distribution analysis
DOI:
https://doi.org/10.14393/BJ-v42n0a2026-79041Keywords:
Chromatography, NSAIDs, Solvent extraction.Abstract
Ketoprofen (KTP) is used as an anti-inflammatory agent and has been studied as a potential treatment for neurodegenerative diseases. This study developed and validated a sensitive high-performance liquid chromatography with diode-array detection method for quantifying KTP in rat plasma and brain tissue. Sample preparation was performed via solvent extraction assisted by protein precipitation, using acetonitrile acidified with 10% HCl in plasma or brain homogenate (1:10, v/v). Chromatographic separation was carried out on a Waters C18 reversed-phase column (150 × 4.6 mm, 5 μm) with an isocratic mobile phase composed of water, 0.3% triethylamine, acetonitrile, and methanol (50:40:10, v/v/v). The aqueous phase was adjusted to pH 3.5 with orthophosphoric acid, and the flow rate was set at 1 mL/min. Detection wavelengths were set at 255 nm for KTP and 355 nm for the internal standard, with a total run time of 5 min. The method demonstrated lower limits of quantification of 0.5 μg/mL for plasma and 0.25 μg/mL for brain tissue, with linearity ranges of 0.5–20 μg/mL and 0.25–8 μg/mL, respectively, and correlation coefficients above 0.999. Intra- and interday accuracy and precision ranges were within ±15% for both matrices, in accordance with the official guidelines of Brazil’s National Health Surveillance Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. The method also demonstrated adequate selectivity, an absence of endogenous interferences, and consistent recovery in plasma and brain tissue. Stability studies confirmed the analyte content under different storage and processing conditions. Hence, the validated method proved sensitive, precise, accurate, and selective, supporting its application in preclinical pharmacokinetic and tissue-distribution research on KTP.
References
ALLEGRINI, A., et al. Fast HPLC Method for the Determination of Ketoprofen in Human Plasma Using a Monolithic Column and its Application to a Comparative Bioavailability Study in Man. Arzneimittelforschung. 2011, 59, 135-140. https://doi.org/10.1055/s-0031-1296376
AMIN, A.S. Spectrophotometric determination of piroxicam and tenoxicam in pharmaceutical formulations using alizarin. Journal of Pharmaceutical and Biomedical Analysis. 2002, 29, 729-736. https://doi.org/10.1016/S0731-7085(02)00035-3
ATTWA, M. W., et al. An ultra-fast validated green UPLC-MS/MS method for the quantification of osimertinib in human liver microsomes: Screening for ADME parameters and in vitro metabolic stability. Acta Chromatographica. 2025, 37, 515-530. https://doi.org/10.1556/1326.2024.01300
CERIONI, A., et al. Validation of a Headspace Gas Chromatography with Flame Ionization Detection Method to Quantify Blood Alcohol Concentration (BAC) for Forensic Practice. Chemosensors. 2024, 12, 133. https://doi.org/10.3390/chemosensors12070133
CHOI, J.-S., JIN, M.J. e HAN, H.-K. Intestinal absorption characteristics of ketoprofen in rats. Biopharmaceutics & Drug Disposition. 2006, 27, 17-21. https://doi.org/10.1002/bdd.479
CZUB, M.P., STEWART, A.J., SHABALIN, I.G. e MINOR, W. Organism-specific differences in the binding of ketoprofen to serum albumin. IUCrJ. 2022, 9, 551-561. https://doi.org/10.1107/S2052252522006820
DAVID, A., et al. A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatography-nanoelectrospray ionisation mass spectrometry. Journal of Chromatography A. 2014, 1365, 72-85. https://doi.org/10.1016/j.chroma.2014.09.001
EL ORCHE, A., et al. Advancing Bioanalytical Method Validation: A Comprehensive ICH M10 Approach for Validating LC-MS/MS to Quantify Fluoxetine in Human Plasma and Its Application in Pharmacokinetic Studies. Molecules. 2024, 29, 4588. https://doi.org/10.3390/molecules29194588
GNIAZDOWSKA, E., et al. Replicates Number for Drug Stability Testing during Bioanalytical Method Validation-An Experimental and Retrospective Approach. Molecules. 2022, 27, 457. https://doi.org/10.3390/molecules27020457
GNIAZDOWSKA, E., et al. How does the order of sample analysis influence the matrix effect during LC-MS bioanalysis? Journal of Chromatography B. 2023, 1227, 123800. 10.1016/j.jchromb.2023.123800
GOMEZ-RIOJA, R., et al. Recommendation for the design of stability studies on clinical specimens. Clinical Chemistry and Laboratory Medicine (CCLM). 2023, 61, 1708-1718. https://doi.org/10.1515/cclm-2023-0221
GRANERO, G.E. e AMIDON, G.L. Possibility of enterohepatic recycling of ketoprofen in dogs. International Journal of Pharmaceutics. 2008, 349, 166-171. https://doi.org/10.1016/j.ijpharm.2007.08.005
HOLLANDER, E. M., et al. Development and validation of an ultra-performance liquid chromatography–tandem mass spectrometry method to quantify the small molecule inhibitors adagrasib, alectinib, brigatinib, capmatinib, crizotinib, lorlatinib, selpercatinib, and sotorasib in human plasma. Biomedical Chromatography. 2024, 38(10), e5986. https://doi.org/10.1002/bmc.5986
HARISH, V., et al. Bioanalytical Method Development, Validation and Stability Assessment of Xanthohumol in Rat Plasma. Molecules. 2022, 27, 7117. https://doi.org/10.3390/molecules27207117
JAYAWICKREME, D.K., et al. Luteolin for neurodegenerative diseases: a review. Pharmacological Reports. 2024, 76, 644-664. https://doi.org/10.1007/s43440-024-00610-8
KNYCH, H.K., et al. Ketoprofen in horses: Metabolism, pharmacokinetics, and effects on inflammatory biomarkers. Drug Testing and Analysis. 2024, 16, 289-302.https://doi.org/10.1002/dta.3543
KUCZYNSKA, J. e NIERADKO-IWANICKA, B. New uses of ketoprofen - a review of studies from 2015 to 2021. Current Issues in Pharmacy and Medical Sciences. 2022, 35, 16-20. https://doi.org/10.2478/cipms-2022-0004
LAMBARKI, L.Z., et al. Comparison of approaches for assessing detection and quantitation limits in bioanalytical methods using HPLC for sotalol in plasma. Scientific Reports. 2025, 15, 5472. https://doi.org/10.1038/s41598-024-83474-5
LIU W, Li X, Li N, Mi Z, Li N, Che J. UPLC-MS/MS method for Icariin and metabolites in whole blood of C57 mice: development, validation, and pharmacokinetics study. Frontiers in Pharmacology. 2023, 14, 1195525. https://doi.org/10.3389/fphar.2023.1195525
LORIER, M., et al. Stereoselective Pharmacokinetics of Ketoprofen After Oral Administration of Modified-Release Formulations in Caucasian Healthy Subjects. European Journal of Drug Metabolism and Pharmacokinetics. 2016, 41, 787-793. https://doi.org/10.1007/s13318-015-0313-2
MAHDAVIJALAL, M., PETIO, C., STAFFILANO, G., MANDRIOLI, R. e PROTTI, M. Innovative Solid-Phase Extraction Strategies for Improving the Advanced Chromatographic Determination of Drugs in Challenging Biological Samples. Molecules. 2024, 29, 2278. https://doi.org/10.3390/molecules29102278
PÉHOURCQ, F., LAGRANGE, F., LABAT, L. e BANNWARTH, B. Simultaneous Measurement of Flurbiprofen, Ibuprofen, and Ketoprofen Enantiomer Concentrations in Plasma Using L-Leucinamide as the Chiral Coupling Component. Journal of Liquid Chromatography. 1995, 18, 3969-3979. https://doi.org/10.1080/10826079508013739
PENG, L., et al. Design of experiment techniques for the optimization of chromatographic analysis conditions: A review. ELECTROPHORESIS. 2022, 43, 1882-1898. https://doi.org/10.1002/elps.202200072
QIU, H.-X., LIU, J., KONG, H., LIU, Y. e MEI, X. Isobolographic analysis of the antinociceptive interactions between ketoprofen and paracetamol. European Journal of Pharmacology. 2007, 557, 141-146. https://doi.org/10.1016/j.ejphar.2006.11.017
RAJAMOHAN, R., et al. Enhancing ketoprofen’s solubility and anti-inflammatory efficacy with safe methyl-β-cyclodextrin complexation. Scientific Reports. 2024, 14, 21516. https://doi.org/10.1038/s41598-024-71615-9
SATYAVERT, et al. Development and validation of bioanalytical method for the determination of hydrazinocurcumin in rat plasma and organs by HPLC-UV. Journal of Chromatography B. 2020, 1156, 122310. https://doi.org/10.1016/j.jchromb.2020.122310
SHENDY, M. K., et al. Comparative insights to microwave and ultrasound as extraction tools for on-spot protein denaturation and HPLC-UV determination of favipiravir in human plasma: Application to real sample and storage stability. Journal of Chromatography A. 2025, 1759, 466229. https://doi.org/10.1016/j.chroma.2025.466229
SINHA, S., et al. Development and Validation of a Simple HPLC–UV-Based Bioanalytical Method for Estimation of Acalabrutinib in Rat Plasma and Its Application in Evaluation of Drug-Loaded Nanocrystal Formulation. Separation Science Plus. 2024, 7, e202400110. https://doi.org/10.1002/sscp.202400110
TANG, Y., et al. Investigation of Transdermal Drug Delivery and In Vivo Pharmacokinetics of Choline Ketoprofen Ionic Liquid. ACS Materials Au. 2026, 6, 128-139. https://doi.org/10.1021/acsmaterialsau.5c00109
TELEANU, D.M., et al. An Overview of Oxidative Stress, Neuroinflammation, and Neurodegenerative Diseases. International Journal of Molecular Sciences. 2022, 23, 5938. https://doi.org/10.3390/ijms23115938
TYUMINA, E., SUBBOTINA, M., POLYGALOV, M., TYAN, S. e IVSHINA, I. Ketoprofen as an emerging contaminant: occurrence, ecotoxicity and (bio)removal. Frontiers in Microbiology. 2023, 14, 1200108. https://doi.org/10.3389/fmicb.2023.1200108
VAZVAEI-SMITH, F., Wickremsinhe, E., Woolf, E. et al. ICH M10 Bioanalytical Method Validation Guideline-1 year Later. AAPS J 26, 103 (2024). https://doi.org/10.1208/s12248-024-00974-y
VOZNIUK, O., et al. A Fast HPLC/UV Method for Determination of Ketoprofen in Cellular Media. ChemistryOpen. 2024, 13, e202300147. https://doi.org/10.1002/open.202300147
WOZIŃSKI, M., et al. Modification of gradient HPLC method for determination of small molecules’ affinity to human serum albumin under column safety conditions: Robustness and chemometrics study. Journal of Pharmaceutical and Biomedical Analysis. 2024, 239, 115916. https://doi.org/10.1016/j.jpba.2023.115916
XI, M.M., ZHANG, S.Q., WANG, X.Y., FANG, K.Q. e GU, Y. Study on the characteristics of pectin-ketoprofen for colon targeting in rats. International Journal of Pharmaceutics. 2005, 298, 91-97. https://doi.org/10.1016/j.ijpharm.2005.04.012
EUROPEAN MEDICINES AGENCY (EMA). ICH M10 on bioanalytical method validation - Scientific guideline. 2023. Disponível em: https://www.ema.europa.eu/en/ich-m10-bioanalytical-method-validation-scientific-guideline. Access in: 28 Jan. 2025.
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Camila Oliveira Pacheco, Jamily Rosa Langendorf , Joane Guterres Ferreira , Pedro Santos da Silva , Tamara Ramos Maciel, Sandra Elisa Haas
This work is licensed under a Creative Commons Attribution 4.0 International License.
