DEVELOPMENT AND VALIDATION OF AN ANALYTICAL QUALITY BY DESIGN-DRIVEN REVERSED PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF METFORMIN AND ITS IMPURITY
DOI:
https://doi.org/10.22159/ajpcr.2026v19i6.58823Keywords:
RP-HPLC, Impurity profiling, QBD, ICH GuidelinesAbstract
Objectives: The occurrence of impurities such as 1-Methylbiguanide Hydrochloride (Impurity E) is a major safety and regulatory issue of the widely used antidiabetic drug metformin (MTF) hydrochloride. The objective of the current study was to design and develop a rapid, sensitive, and robust reversed-phase high-performance liquid chromatography (RP-HPLC) methodology to simultaneously estimate MTF and its impurity E in an analytical quality by design (AQbD) methodology, as required by International Conference on Harmonization (ICH) Q2(R1), Q3A, Q3B, and Q14 requirements.
Methods: The method was developed with Design-Expert software (version 13) by implementing a central composite design to investigate how key method parameters (including mobile phase composition and pH) affect chromatographic responses. The separation was performed by using a HiQ Sil C18 column (250 × 4.6 mm, 5 μm) and the mobile phase with orthophosphoric acid (0.1 M) to adjust the pH (3.6) and a 1.0 mL/min flow rate. The method was validated as specified in ICH Q2(R2) under linearity, accuracy, precision, robustness, specificity, Limit of detection (LOD), and limit of quantification (LOQ).
Results: The developed technique in the concentration range of 25–150 μg/mL of MTF and 0.2–1.2 μg/mL of Impurity E was found to be very linear with a correlation coefficient (r2=0.999). Accuracy studies revealed mean percentage recoveries ranging from 99.81 to 100.02% for MTF and 98.20–100.00% for Impurity E, with % relative standard deviation values below 2%. Both analytes demonstrated intra-day and inter-day precision with a <2.0% of RSD, indicating good repeatability and intermediate precision. MTF was determined to have LOD and LOQ of 4.08 μg/mL and 12.35 μg/mL, respectively, and Impurity E LOD and LOQ of 0.0305 μg/mL and 0.0925 μg/mL, respectively. Robustness studies showed that there was no great impact of small intentional changes in the chromatographic conditions.
Conclusion: A straightforward, sensitive, precise, and robust AQbD-based RP-HPLC procedure was effectively developed and confirmed to determine MTF and its impurity E simultaneously. The procedure meets the existing regulatory requirements and can be used in daily quality control and impurity profiling of MTF in pharmaceutical dosage forms.
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References
1. Musmade BD, Baraskar ML, Ghodke VN. Bhope SG, Padmanabhan S, Lohar KS. Impurity profiling method development and validation of metformin hydrochloride and teneligliptin hydrobromide hydrate in their combination tablet dosage form by using RP-HPLC with UV/PDA detector. Future J Pharm Sci. 2021;7(1):218.
2. Tiwari AT, Mishra B, Mishra S. Compilation of scientific data for newly CDSCO-approved antidiabetic drugs in last five years: A review. Int J Pharm Qual Assur. 2024 Jun;15(2):1072-80. doi: 10.25258/ ijpqa.15.2.79
3. Shah S, Kotadiya R. A novel RP-HPLC approach for simultaneous determination of dapagliflozin, linagliptin, and metformin in pharmaceutical formulations. BMC Chem. 2025;19(1):257. doi: 10.1186/s13065-025-01620-0, PMID 40887637
4. Xiao T, Hu X, Chen Y, Lin H. Quantitative determination of potential genotoxic impurities in metformin hydrochloride and empagliflozin tablets through ultra-performance liquid chromatographymass spectrometry. J Pharm Biomed Anal. 2024;241:116000. doi: 10.1016/j. jpba.2024.116000, PMID 38280234
5. Pandian SM, Thanikachalam PV, Kaliyappan D, Kalusalingam A. A comprehensive analysis of market forms and analytical methods for metformin formulations. Biomed Chromatogr. 2025;39(4):e70028. doi: 10.1002/bmc.70028, PMID 40071511
6. Sha’at M, Spac AF, Stoleriu I, Bujor A, Cretan MS, Hartan M. Implementation of QbD approach to the analytical method development and validation for the estimation of metformin hydrochloride in tablet dosage forms by HPLC. Pharmaceutics. 2022;14(6):1187. doi: 10.3390/ pharmaceutics14061187, PMID 35745760
7. Manoel JW, Primieri GB, Bueno LM, Wingert NR, Volpato NM, Garcia CV. The application of quality by design in the development of the liquid chromatography method to determine empagliflozin in the presence of its organic impurities. RSC Adv. 2020;10(12):7313-20. doi: 10.1039/c9ra08442h, PMID 35493885.
8. Polyakova EB, Sabirzyanov DR, Prozorova NA, Foteeva AB. Physicochemical properties and methods for the determination of metformin hydrochloride (A review). Pharm Chem J. 2022;55(10):1119-25. doi: 10.1007/s11094-021-02546-2
9. Magdy MA, Abdelrahman MM, Mohamed DG, Ahmed AB. Sequential separation of anti-diabetic drugs in the presence of melamine as impurity using chromatographic methods. BMC Chem. 2025;19(1):26. doi: 10.1186/s13065-025-01385-6, PMID 39871319, PMCID 11773764.
10. Magdy G, Al-Enna AA, Belal F, El-Domany RA, Abdel-Megied AM. Analytical quality-by-design approach for development and validation of HPLC method for the simultaneous estimation of omarigliptin, metformin, and ezetimibe: Application to human plasma and dosage forms. BMC Chem. 2023;17(1):45. doi: 10.1186/s13065-023-00955-w, PMID 37147652
11. Kłaczkow G, Anuszewska EL. Determination of impurities in medical products containing metformin hydrochloride. Acta Pol Pharm. 2010;67(6):593-8. PMID 21229873.
12. Kutty SV, Shahla TS, Shahshad PP, Shifa VS. Innovative analytical method development and validation for metformin using simulated salivary fluid: A preformulation approach. J Appl Spectrosc. 2025;92(4):889-96. doi: 10.1007/s10812-025-01984-0
13. Sakhare RS, Pekamwar SS, Mohkare DP. Development and validation of stability indicating HPTLC method for the determination of metformin hydrochloride and benfotiamine in bulk and combined dosage form. Indian J Pharm Educ Res. 2017;51(2):S8-16.
14. Patel KY, Dedania ZR, Dedania RR, Patel U. QbD approach to HPLC method development and validation of ceftriaxone sodium. Future J Pharm Sci. 2021;7(1):141. doi: 10.1186/s43094-021-00286-4
15. Giri PT, Chabukswar AR, Jagdale SC, Chindhe SA. QBD (quality by design) approach: Development and validation of an RP-HPLC method for estimating imeglimin HCL and its ketone impurity. 2025;17(4):241-53.
16. Orlandini S, Pinzauti S, Furlanetto S. Application of quality by design to the development of analytical separation methods. Anal Bioanal Chem. 2013;405(2-3):443-50. doi: 10.1007/s00216-012-6302-2, PMID 22941176
17. Yu LX, Amidon G, Khan MA, Hoag SW, Polli J, Raju GK. Understanding pharmaceutical quality by design. AAPS J. 2014;16(4):771-83. doi: 10.1208/s12248-014-9598-3, PMID 24854893, PMCID 4070262
18. El-Sayed HM, Abdellatef HE, Hendawy HA, El-Abassy OM, Ibrahim H. DoE-enhanced development and validation of eco-friendly RP-HPLC method for analysis of safinamide and its precursor impurity: QbD approach. Microchem J. 2023;190:108730. doi: 10.1016/j. microc.2023.108730
19. Rmandić M, Rašević M, Stanković J, Zečević M, Otašević B, Protić A. Development and robust optimization of the RP-HPLC method for the determination of Riociguat and its four impurities in tablets using the AQbD approach. J Pharm Biomed Anal. 2025;258:116742. doi: 10.1016/j.jpba.2025.116742, PMID 39951829
20. Kasagić-Vujanović I, Jančić-Stojanović B. Quality by Design oriented development of hydrophilic interaction liquid chromatography method for the analysis of amitriptyline and its impurities. J Pharm Biomed Anal. 2019;173:86-95. doi: 10.1016/j.jpba.2019.05.026, PMID 31125948
21. Radić I, Runje M, Babić S. Development of an analytical method for the determination of pimavanserin and its impurities applying analytical quality by design principles as a risk-based strategy. J Pharm Biomed Anal. 2021;201:114091. doi: 10.1016/j.jpba.2021.114091, PMID 33964725
22. Dave V, Patel PU. Patel, and technology, Analytical QbD assisted RP-HPLC method development and validation for estimation of anti-diabetic drug remogliflozin in bulk and its pharmaceutical dosage form. Res J Pharm Technol. 2025;18(7):3244-9. 23. Upadhyay J, Padhiyar S, Jivani K, Patel TB, Bhavsar VP, Prajapati V. Quality by design (QbD) based development and validation of RP-HPLC method for simultaneous estimation of metformin and teneligliptin in bulk and their pharmaceutical formulation. Indian J Pharm Edu Res. 2025;59(2s):s689-704. doi: 10.5530/ ijper.20253717.
24. 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. 2024;50(8):750-62. doi: 10.1080/03639045.2024.2400199, PMID 39226128
25. Bhatt DA, Rane SI. QbD approach to analytical RP-HPLC method development and its validation. Int J Pharm Pharm Sci. 2011;3(1):179-87.
26. Thakur D, Kaur A, Sharma S. Application of QbD based approach in method development of RP-HPLC for simultaneous estimation of antidiabetic drugs in pharmaceutical dosage form. J Pharm Investig. 2017;47(3):229-39. doi: 10.1007/s40005-016-0256-x
27. Attimarad M, Venugopala KN, Islam MM, Shafi S, Altaysan AI. Rapid simultaneous quantitative analysis of hypoglycemic agents by RP HPLC: Development, validation and application to medicine. IJPER. 2022;56(2):564-72. doi: 10.5530/ijper.56.2.79
28. Gedawy A, Al-Salami H, Dass CR. Development and validation of a new analytical HPLC method for simultaneous determination of the antidiabetic drugs, metformin and gliclazide. J Food Drug Anal. 2019;27(1):315-22. doi: 10.1016/j.jfda.2018.06.007, PMID 30648585
29. Gujjar NR, Hadagali MD, Yallur BC, Itigimatha N. Novel and stability indicating RP-HPLC method development and validation for the determination of metformin HCl: A potential drug against diabetic disorder, in pure and pharmaceutical dosage forms. Pharm Chem J. 2024;58(8):1332-8. doi: 10.1007/s11094-024-03278-9.
30. Shaikh A, Devakumar D, Radhika S. Analytical method development of a stability-indicating RP-HPLC technique for simultaneous estimation of metformin and sitagliptin in combined dosage forms. Vascular and Endovascular Review. 2025;8(18s):232-44.
31. Siddique W, Zulcaif, Umar H, Usmani SJ, Waqas M, Gul MJ. Method development and validation of metformin HCl and Dapagliflozin by using RP-HPLC. AChrom. 2025;37(2):176-82. doi: 10.1556/1326.2024.01226
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