Development and Validation of Sensitive, Specific and Stability-Indicating LC-MS/MS Method for Simultaneous Quantification of Metformin and Becalutamide in Human Plasma

Abstract

Metformin, an oral anti-hyperglycaemic drug belongs to the biguanide class and is traditionally known as an insulin sensitizer which lowers the blood glucose levels by activating AMPK (adenosine monophosphate protein kinase). Metformin has shown anti-neoplastic effects in various tumour models, including prostate, ovarian, breast, colorectal and endometrial cancers, by activating AMPK, which in turn blocks the mTOR/S6 kinase pathway and inhibits insulin/insulin-like growth factor (IGF)-mediated cellular proliferation. Bicalutamide, is a non-steroidal anti-androgen class agent, binds to androgen receptors, thus blocking the effects of androgenic hormones such as testosterone and dihydrotestosterone. Previous reports on in vivo and in vitro studies suggests the combination of metformin and biclutamide dramatically decreases prostate cancer cell proliferation more than either drug alone. This work was undertaken to examine the effect of metformin on bicalutamide pharmacokinetic parameters, which may further aid in dose adjustment when administered concurrently to treat prostate cancer. So, for the simultaneous quantitation of metformin and becalutamide in spiked human plasma by LC-MS/MS method was used. The preconcentration of the analyte was done using Protein precipitation using acetonitrile. Separations were done on Agilent 1290 infinity II LC system paired with an Agilent 6470 triple quadrupole mass spectrometer with positive and negative ionization modes. The mobile phase comprised 0.1% acetic acid in MilliQ water as solvent A and 100% methanol as solvent B with gradient flow at 0. 6 mL/min. column, column oven and degasser with a Zorbax Eclipse plus C8 column (4.6 × 100 mm, 3.5 µm) was utilized. The retention times for metformin, bicalutamide and the IS (propranolol and tolbutamide) were 3.6 min, 4.7 min, 5.9 min and 6.1 min, respectively. For the samples, subsequent Q1/Q3 transitions: bicalutamide, ESI^– m/z 427.2 > 184.5 (10 eV); metformin, ESI⁺ m/z 130.10 > 60.2 (12 eV); tolbutamide, ESI^– m/z 269.09 > 170 (16 eV); and propranolol, ESI⁺ m/z 260.2 > 183.0 (32 eV). Data acquisition and instrument control were operated with Mass Hunter workstation (version 10.1). The established method was validated by following USFDA bioanalytical guidelines, Linear regression equations for metformin and biclutamide were 0.0822x + 0.00848 and 0.09588 x + 0.02635, respectively, with (1/x) weighting factor, the regression coefficients for metformin and biclutamide are 0.9963 and 0.9971, respectively. Mean extraction recoveries for metformin was 85.04% and for biclutamide was 86.23% all QCs. Solifenacin’s stability in plasma was confirmed through rigorous testing, including bench-top exposure (17 h), injector residence, six freeze-defrost cycles and long-term storage at –20 ± 5 ºC for over 7 days. The compound exhibited consistent integrity under all conditions. The analytical method underwent full validation and met qualification standards, supporting its reliability for routine bioanalysis and pharmacokinetic profiling in biological matrices.