• RAJA NAVAMANISUBRAMANIAN Balaji Institute of Pharmaceutical Sciences, Laknepally (V), Narsampet (M), Warangal (R), India
  • SABITHA PANCHAGIRI Department of Pharmaceutical Sciences, UCPSc., Kakatiya University, Warangal, India
  • RAGHUNANDAN NERELLA Balaji Institute of Pharmaceutical Sciences, Laknepally (V), Narsampet (M), Warangal (R), India
  • CHAMUNDEESWARI DURAIPANDIAN Department of Pharmacognosy, Faculty of Pharmacy, Sri Ramachandra University, Chennai, India
  • SHANMUGANATHAN SEETHARAMAN Department of Pharmacognosy, Faculty of Pharmacy, Sri Ramachandra University, Chennai, India


Objective: A simple, selective and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method to estimate repaglinide (REP) in rabbit plasma using rabeprazole (RAB) as an internal standard was developed and validated for various qualifications.

Methods: The chromatographic separation was performed on C18 (2) analytical column (5 μ, 250×4.6 mm) using acetonitrile: 0.05% trifluoroacetic acid in water (55:45, v/v) as mobile phase at the flow rate of 1 ml/min. Validation of the analytical method was performed as per ICH guidelines.

Results: The retention times of REP and RAB were found at ~4.3 and 5.1 min respectively, with adequate system suitability parameters (theoretical plates ≥3619, tailing factor ≤1.38, resolution factor 2.37). The method has linearity over a concentration range of 10 to 1000 ng/ml (r2=0.9987). The results of accuracy (≥98.17%), intra-, inter-day precision (≤2.9%), recovery (101.21±2.09%) and process efficiency (99.77±3.74%) found satisfactory with no matrix effect. The analyte in samples were found stable up to 6 h, 3 freeze-thaw cycles and not more than 2 mo corresponding to bench-top, short and long term stability studies respectively.

Conclusion: The developed RP-HPLC method for estimation of REP in rabbit plasma was developed. The method was found to be rapid, cost-effective and accurate to estimate the REP from the sample matrix. The method can be a most useful tool for in vivo study of REP in the rabbit.

Keywords: HPLC-UV, Repaglinide, Bioanalytical, Validation, Rabbit plasma


1. Marbury TM, Ruckle JL, Hatorp V, Andersen MP, Nielsen KK, Huang WC, et al. Pharmacokinetic of repaglinide in subjects with renal impairment. Clin Pharmacol Ther 2000;67:7-15.
2. Scott LJ. Repaglinide. Drugs 2012;72:249-72.
3. Kassem AA, El-Alim SHA, Basha M, Salama A. Phospholipid complex enriched micelles: a novel drug delivery approach for promoting the antidiabetic effect of repaglinide. J Pharm Sci 2017;99:75-84.
4. Stephan D, Winkler M, Kuhner P, Russ U, Quast U. Selectivity of repaglinide and glibenclamide for the pancreatic over the cardiovascular KATP channels. Diabetologia 2006;49:2039-48.
5. Jirovsky D, Bartosova Z, Skopalova J, Maier V. Electrochemical characterization of repaglinide and its determination in human plasma using liquid chromatography with dual-channel coulometric detection. J Chrom B 2010;878:3243-8.
6. Hatorp V, Huang WC, Strange P. Repaglinide pharmacokinetics in healthy young adult and elderly subjects. Clin Ther 1999;21:702–10.
7. Greischel A, Beschke K, Rapp H, Roth W. Quantitation of the new hypoglycaemic agent AG-EE388ZW in human plasma by automated high-performance liquid chromatography with electrochemical detection. J Chrom B 1991;568:246-52.
8. Yao J, Shi YQ, Li ZR, Jin SH. Development of a RP-HPLC method for screening potentially counterfeit anti-diabetic drugs. J Chrom B 2007;853:254-9.
9. Venkatesh P, Harisudhan T, Choudhury H, Mullangi R, Srinivas NR. Simultaneous estimation of six anti-diabetic drugs glibenclamide, gliclazide, glipizide, pioglitazone, repaglinide and rosiglitazone: development of a novel HPLC method for use in the analysis of pharmaceutical formulations and its application to human plasma assay. Biomed Chrom 2006;20:1043-8.
10. Ruzilawati AB, Abd Wahab MS, Imran A, Ismail Z, Gan SH. Method development and validation of repaglinide in human plasma by HPLC and its application in pharmacokinetic studies. J Pharm Biomed Anal 2007;43:1831-5.
11. Zhang J, Gao F, Guan X, Sun Y, Gu J, Fawcett JP. Determination of repaglinide in human plasma by high-performance liquid chromatography-tandem mass spectrometry. Acta Pharm Sin B 2011;1:40-5.
12. Wang M, Miksa IR. Multi-component plasma quantitation of anti-hyperglycemic pharmaceutical compounds using liquid chromatography-tandem mass spectrometry. J Chrom B 2007;856:318–27.
13. Sharma K, Pawar G, Yadam S, Giri S, Rajagopal S, Mullangi R. LC-MS/MS-ESI method for simultaneous quantitation of metformin and repaglinidie in rat plasma and its application to pharmacokinetic study in rats. Biomed Chrom 2013;27:356-64.
14. Ho EN, Yiu KC, Wan TS, Stewart BD, Watkins KL. Detection of anti-diabetics in equine plasma and urine by liquid chromatography-tandem mass spectrometry. J Chrom B 2004;811:65-73.
15. Gandhimathi M, Ravi TK, Renu SK. Determination of repaglinide in pharmaceutical formulations by HPLC with UV detection. Anal Sci 2003;19:1675-7.
16. Berecka A, Gumieniczek A, Hopkala H. Development and validation of a new high-performance liquid chromatography method for the determination of gliclazide and repaglinide in pharmaceutical formulations. J AOAC Int 2006;89:319-25.
17. Joshi SS, Nahire RR, Shastri NR, Surendranath KV, Satish J. Validated stability-indicating RP-HPLC UV method for simultaneous determination of metformin and repaglinide. Acta Chrom 2012;24:419-43.
18. Soni LK, Narsinghani T, Jain M. Development and validation of RP-HPLC method for simultaneous estimation of metformin hydrochloride and repaglinide in tablet dosage form. J Liq Chromatogr Relat Technol 2012;35:385-92.
19. Yin K, Meng X, Dong P, Ding T, Shen L, Zhang L, et al. A simple, rapid, economical, and practical method for the determination of efavirenz in plasma of Chinese AIDS patients by reverse-phase high-performance liquid chromatography with ultraviolet detector. BioSci Trends 2014;8:227-34.
20. Ma Z, Shayeganpour A, Brocks DR, Lavasanifar A, Samuel J. High-performance liquid chromatography analysis of curcumin in rat plasma: application to the pharmacokinetics of polymeric micellar formulation of curcumin. Biomed Chrom 2007;21:546–52.
21. ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2 (R1). International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Geneva; 2005. p. 1-13.
22. Guidance for industry: bioanalytical method validation, U. S. Department of health and human services, Food and Drug administration, Centre for Drug Evaluation and Research (CDER) Centre for Veterinary Medicine (CVM); 2018. p. 1-41.
23. Jimmerson LC, Zheng JH, Bushman LR, MacBrayne CE, Anderson PL, Kiser JJ. Development and validation of a dried blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry. J Chrom B 2014;944:18–24.
24. Bansal S, DeStefano A. Key elements of bioanalytical method validation for small molecules. AAPS J 2007;9:109-14.
25. Shanmugasundaram P, Bijithra C, Ragan G, Sankar A, Sumithra M. Analyitcal method development and validation of reversed-phase high-performance liquid chromatography for the determination of modafinil in bulk and pharmaceutical dosage forms. Asian J Pharm Clin Res 2016;9:177-81.
26. Ashok P, Narenderan ST, Meyyanathan SN, Babu B, Vadivelan R. Development and validation of a RP-HPLC method for estimation of telmisartan in human plasma. Int J Appl Pharm 2019;11:237-40.
27. Mukti AA, Jannah F, Nurrochmad A, Lukitaningsih E. Development and validation method for quantitative determination of ciprofloxacin in human plasma and its application in bioequivalence test. Asian J Pharm Clin Res 2016;9:89-95.
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