Abstract
A new ultra-high performance liquid chromatography-mass spectrometry-mass spectrometry (UHPLC-MS/MS) system has been formulated for the resolution of closely related drugs apigenin (API, a bioflavinoid) and prednisolone (PRD) from their mixture. This developed method comprised of a "BEH (TM) C-18 column (50 mm x 2.1 mm, 1.7 mu m)" using acetonitrile and 0.1% formic acid (35:65 v/v) at a supply rate of 0.25 mL.min(-1) as eluent. It was found that selected eluent provided short run time ( <= 2.5 min) as well as better peak symmetry. Satisfactory values of chromatographic parameters such as resolution (Rs = 2.5), capacity factor (k; 13.6 and 23.4 for API and PRD respectively, selectivity (alpha = 1.72) and number of theoretical plates (N; 3789 and 42,435 for API and PRD respectively) indicate the efficiency of the developed method. The obtained separation was then exploited for the detection and measurement of API in rat plasma sample by means of PRD as an "internal standard" (IS). The eluted compounds in plasma were identified by tandem mass spectrometry by means of tandem quadrupole (TQ) detector ("Waters Corp., Milford, MA") fortified with an "electrospray ionisation (ESI)" source functioning in positive ionization mode. The determination of API in plasma was accomplished by means of "multiple reactions monitoring (MRM)" mode. Assortment of "ionization pairs" (m/z) was displayed in the following manner: API: 270.99 -> 152.9 ("cone voltage" 57 V, "collision energy" 34 V), PRD: 403.172 -> 385.224 ("cone voltage" 42 V, "collision energy" 13 V). The calibration curves followed linearity in concentration range of 05-1000 ng mL(-1) with limit of detection "LOD" and limit of quantification "LOQ" of 7.30 and 22.77 ng mL(-1), respectively. The developed method was validated taking into consideration various test conditions and satisfactory values of various parameters such as linearity (r(2) +/- SD = 0.9995 +/- 0.0005), interday accuracy (88-120%), interday precision % RSD = 3.30-13.65% whereas intraday accuracy (91-118%) intraday precision % RSD = 1.18-5.83) indicated its validity. The validation outcomes fulfilled the standards of united states food and drug administration "USFDA" in addition Scientific Working Group for Forensic Toxicology "SWGTOX" guiding principles and were not beyond the tolerable constraint. The process developed in plasma was efficaciously harnessed in the pharmacokinetic investigation of various formulations of API after oral administration in rats.