Integrated LC–MS/MS bioanalysis for the simultaneous quantification of metformin HCl, pioglitazone HCl, and teneligliptin HBr hydrate in human plasma

Background: Combination therapy is widely prescribed in Type II diabetes mellitus to maintain effective glycemic control. The rising use of multidrug regimens demands selective and reliable bioanalytical methods capable of simultaneously quantifying multiple antidiabetic agents in human plasma for pharmacokinetic and bioequivalence studies. Methodology: A rapid, sensitive, and cost-effective LC–MS/MS method was developed and validated in accordance with ICH M10, USFDA, and EMA guidelines for the simultaneous estimation of metformin hydrochloride, teneligliptin hydrobromide hydrate, and pioglitazone hydrochloride. The assay enabled triple-drug quantification within a single 7-minute chromatographic run, showing an estimated 12–40% reduction in analysis time versus previously reported 8–15-minute single- or dual-analyte methods. Separation was achieved on a Cosmosil CN column (150 × 4.6 mm, 5 μm) using 10 mM ammonium acetate and acetonitrile (40:60 %v/v). Plasma samples were prepared by protein precipitation followed by liquid–liquid extraction, and detection was performed in positive electrospray ionization multiple-reaction-monitoring mode. Results and Discussion: Strong linearity was obtained for all analytes (r² > 0.995). LLOQs were 10.0 ng/mL for metformin, 1.25 ng/mL for teneligliptin, and 5.0 ng/mL for pioglitazone. Metformin-D6 served as the internal standard for metformin, while saxagliptin was used as the internal standard for teneligliptin and pioglitazone to ensure appropriate normalization across chemical classes. Precision remained below 10% CV, recovery was consistent, and stability stayed within ±15% under tested conditions. Reduced runtime and unified multi-analyte detection improved analytical throughput and minimized solvent consumption without compromising regulatory compliance. Conclusion: The validated LC–MS/MS method provides a reliable, resource-efficient platform for concurrent quantification of combined antidiabetic drugs in pharmacokinetic, bioequivalence, and clinical studies.