New Improved UPLC-MS/MS Method for Reliable Determination of Clarithromycin in Human Plasma to Support a Bioequivalence Study

An improved, highly sensitive UPLC-MS/MS method has been developed for the determination of clarithromycin in human plasma. For sample preparation, liquid-liquid extraction with n-hexane: methyl tert-butyl ether (20:80, v/v) mixture was carried out using clarithromycin 13C-d3 as the internal standard (IS). Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column was used for chromatography with methanol-5.0 mM ammonium formate, pH 3.0 (78:22, v/v) as the mobile phase under isocratic conditions. The analysis time was 1.5 min. Quantitation of analyte was done by tandem mass spectrometer using electrospray ionization in the positive mode. The precursor → product ion transitions monitored for clarithromycin and IS were m/z 748.9 → 158.1 and m/z 752.8 → 162.0 respectively. The method was validated over a dynamic concentration range of 0.80-1600 ng/mL with correlation coefficient (r) ≥ 0.9998. The mean extraction recovery of clarithromycin was 96.2 % across six quality control levels. Intra-batch and inter-batch accuracy and precision (% CV) ranged from 96.8 to 103.5 % and 1.28 to 4.85 % respectively. Stability of clarithromycin in plasma was evaluated under different conditions like bench top, auto sampler, dry and wet extract, freeze-thaw and long term. The present method was successfully applied to a bioequivalence study in 20 healthy subjects who received single oral dose 250 mg clarithromycin tablet formulation. The reproducibility of the method was investigated by reanalysis of 100 incurred samples. Keyword: Clarithromycin, clarithromycin 13C-d3, UPLC-MS/MS, human plasma, sensitive, bioequivalence