NEW ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ANTI DIABETIC DRUGS BY REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IN BULK AND PHARMACEUTICAL DOSAGE FORMS
DOI:
https://doi.org/10.22159/ajpcr.2026v19i6.58747Keywords:
RP-HPLC, Metformin, Sitagliptin phosphate, Dapagliflozin, International Council for Harmonisation (ICH) guidelines, Quantification and validation.Abstract
Objective: The objective of the present study was to develop and validate reverse phase high-performance liquid chromatography (HPLC) methods for the quantification of Metformin (MET), sitagliptin phosphate (SITA), and dapagliflozin (DAPA) in bulk and pharmaceutical dosage forms, employing three separate methods corresponding to three different binary drug combinations.
Methods: Chromatographic analysis was performed on a C18 column (250×4.6 mm, 5 μm) under isocratic conditions at ambient temperature, with a flow rate of 0.8–1.0 mL/min, injection volume of 10–20 μL, and run time of 10–15 min. Method I: For MET–DAPA, the mobile phase was triethylamine: acetonitrile (55:45, v/v; pH 8.0); Method II: For MET–SITA, 0.1% trifluoroacetic acid (TFA): acetonitrile (60:40, v/v; pH 2.1); and method III: For SITA–DAPA, Tris- hydrochloride: acetonitrile (70:30, v/v; pH 8.1). Detection was carried out at 224 nm. All mobile phases were sonicated for 15–20 min and filtered through a 0.5 μm membrane before use, with HPLC-grade water or methanol and the respective mobile phases used as diluents. Method validation was carried out as per ICH guidelines by assessing specificity, linearity, range, accuracy (% recovery, precision (repeatability and intermediate precision), sensitivity (limit of detection [LOD] and limit of quantification [LOQ]), robustness, and system suitability of the chromatographic method.
Results: MET, SITA, and DAPA at 10 to 60 μg/mL, 20–70 μg/mL, and 20–700 μg/mL, respectively, showed a linear response to the suggested approach. The correlation coefficients (R2 values) for all the analytes were 0.999, and the retention times of MET, SITA, and DAPA were found to be in the acceptable range. The developed chromatographic technique was validated for specificity, linearity, precision, and accuracy (% Recovery) LOD, and LOQ using ICH Q2 (R1) criteria. The analysis results have been validated in accordance with ICH guidelines.
Conclusion: The proposed method was successfully applied for the determination of different analytes and validated as per the ICH guidelines for both bulk and pharmaceutical drugs.
Downloads
References
1. Gaggini M, Sabatino L, Suman AF, Chatzianagnostou K, Vassalle C. Insights into the roles of GLP-1, DPP-4, and SGLT2 at the crossroads of cardiovascular, renal, and metabolic pathophysiology. Cells. 2025;14(5):387. doi: 10.3390/cells14050387, PMID 40072115
2. Kim MJ, Cho YK, Kim S, Moon JY, Jung CH, Lee WJ. Efficacy and safety of combination therapy using SGLT2 and DPP4 inhibitors to treat type 2 diabetes: An updated systematic review and meta-analysis with focus on an Asian subpopulation. Diabetes Obes Metab. 2025;27(9):5019-31. doi: 10.1111/dom.16550, PMID 40555695
3. Liang X, Dai J, Wang F. Sodium glucose co-transporter 2 (SGLT2) inhibitors versus dipeptidyl peptidase-4 (DPP-4) inhibitors and the risk of atrial fibrillation in patients with type 2 diabetes mellitus: A meta-analysis. BMC Cardiovasc Disord. 2025;25(1):59. doi: 10.1186/ s12872-024-04442-5, PMID 39875820
4. Zhang R, Xie Q, Lu X, Fan R, Tong N. Research advances in the anti-inflammatory effects of SGLT inhibitors in type 2 diabetes mellitus. Diabetol Metab Syndr. 2024;16(1):99. doi: 10.1186/s13098-024- 01325-9, PMID 38735956
5. Suzuki Y, Kaneko H, Okada A, Komuro J, Fujiu K, Takeda N. Comparison of incident hypertension between SGLT2 inhibitors vs. DPP4 inhibitors. Hypertens Res. 2024;47(7):1789-96. doi: 10.1038/ s41440-024-01649-z, PMID 38600275
6. Ahmad A, Sabbour H. Effectiveness and safety of the combination of sodium-glucose transport protein 2 inhibitors and glucagon-like peptide-1 receptor agonists in patients with type 2 diabetes mellitus: A systematic review and meta-analysis of observational studies. Cardiovasc Diabetol. 2024;23(1):99. doi: 10.1186/s12933-024- 02192-4, PMID 38500154
7. Gonzalez J, Dave CV. Prescribing trends of SGLT2 inhibitors among HFrEF and HFpEF patients with and without T2DM, 2013-2021. BMC Cardiovasc Disord. 2024;24(1):285. doi: 10.1186/s12872-024- 03961-5, PMID 38816795
8. Nimavat ZR, Patel AN, Gor AP, Ganguly B. Comparison of safety profiles of DPP-4 inhibitors with SGLT-2 inhibitors in type 2 diabetes mellitus: A systematic review. Int J Basic Clin Pharmacol. 2025;14(5):843-9.
9. Pamu S, Patyar S, Thakkalapally L. Sitagliptin with piperine pharmacodynamic and pharmacokinetic studies in normal and diabetic rabbits. Curr Drug Metab. 2025;20(3):393-401. doi: 10.2174/15748855 18666230815163226
10. McGuire DK, Shih WJ, Cosentino F, Charbonnel B, Cherney DZ, Dagogo-Jack S. Association of SGLT2 inhibitors with cardiovascular and kidney outcomes in patients with type 2 diabetes: A meta-analysis. JAMA Cardiol. 2021;6(2):148-58. doi: 10.1001/ jamacardio.2020.4511, PMID 33031522
11. Brunton LL, Hilal-Dandan R, Knollmann BC. Goodman and Gilman’s the Pharmacological Basis of Therapeutics. 14th, 15th ed. New York: McGraw-Hill Education; 2023.
12. Ferrannini E, Ramos SJ, Salsali A, Tang W, List JF. Dapagliflozin monotherapy in type 2 diabetic patients with inadequate glycemic control by diet and exercise: A randomized, double-blind, placebo-controlled, phase 3 trial. Diabetes Care. 2010;33(10):2217-24. doi: 10.2337/dc10-0612, PMID 20566676
13. Neal B, Perkovic V, Mahaffey KW, De Zeeuw D, Fulcher G, Erondu N. Canagliflozin and cardiovascular and renal events in type 2 diabetes. N Engl J Med. 2017;377(7):644-57. doi: 10.1056/ nejmoa1611925, PMID 28605608
14. McMurray JJ, Solomon SD, Inzucchi SE, Køber L, Kosiborod MN, Martinez FA. Dapagliflozin in patients with heart failure and reduced ejection fraction. N Engl J Med. 2019;381(21):1995-2008. doi: 10.1056/nejmoa1911303, PMID 31535829
15. Khatik MA, Ghule SD, Vengurlekar S, Jain SK. Development and validation of RP-HPLC method for the simultaneous estimation of sitagliptin phosphate and dapagliflozin in bulk and marketed formulations with forced degradation studies. J Neonatal Surg. 2024;13:1082-10.
16. Sunkara N, Maneesha KN, Lavanya B, Arunkumar S. UV spectrophotometric method development and validation of sitagliptin in bulk and pharmaceutical dosage form. Int J Pharm Chem Res. 2017;3(3):577-80.
17. Patel NS, Patel BH. Development and validation of stability indicating RP-HPLC method for the simultaneous estimation of dapagliflozin propanediol and metformin hydrochloride in tablet dosage form. Int J Pharm Sci Drug Res. 2020;12(6):660-7. doi: 10.25004/ ijpsdr.2020.120609
18. Kang YJ, Jeong HC, Kim TE, Shin KH. 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. 2020 Oct 11;25(20):4625. doi: 10.3390/molecules25204625.
19. Liévin S. ICH Q2(R2) Validation of Analytical Procedures: An Overview of the Revised Guideline; 2024 Mar 20.
Published
How to Cite
Issue
Section
Copyright (c) 2026 Suri CR
This work is licensed under a Creative Commons Attribution 4.0 International License.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.