Selected Reaction Monitoring LC-MS Determination of Idoxifene and Its Pyrrolidinone Metabolite in Human Plasma Using Robotic High-Throughput, Sequential Sample Injection

The generation of large numbers of samples during early drug discovery has increased the demand for rapid and selective methods of analysis. Liquid chromatographytandem mass spectrometry (LC-MS-MS), because ofits sensitivity, selectivity, and robustness, has emerged as a powerful tool in the pharmaceutical industry for many analytical needs. This work presents a high-throughput selected reaction monitoring LC-MS bioanalytical method for the determination of idoxifene, a selective estrogen receptor modulator, and its pyrrolidinone metabolite in clinical human plasma samples. The described method uses short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite within 10 s/sample. Sequential injections were accomplished with a 215/889 multiple probe liquid handier (Gilson, Inc.), which aspirates eight samples simultaneously and performs its rinse cycle parallel to sample injection, resulting in minimum lag time between injections. This highthroughput method was applied to the determination of idoxifene and its metabolite in clinical human plasma samples. Sample preparation employed liquid/liquid extraction in the 96-well format Method validation included determination of intra and interassay accuracy and precision values, recovery studies, autosampler stability, and freeze-thaw stability. The LOQ obtained was 10 ng/mL for idoxifene and 30 ng/mL for the metabolite. Using idoxifene-d5 as an internal standard, idoxifene showed acceptable accuracy and precision values at QC level 1 (QC1, 15 ng/mL), level 2 (QC2, 100 ng/mL), and level 3 (QC3, 180 ng/mL) (85.0% accuracy ± 12.0% precision, 95.1 ± 4.9%, and 90.3 ± 4.7%, respectively). The pyrrolidinone metabolite also showed acceptable accuracy and precision values (using no internal standard for quantitation) at QCI (60 ng/mL), QC2 (100 ng/mL), and QC3 (180 ng/mL) (104.9 ± 14.4%, 91.1 ± 13.0%, and 90.8 ± 12.2%, respectively). The validated method was applied to the analysis of 613 human clinical plasma samples. An average run time of 23 s/sample (~37 min/ 96-well plate or over 3700 sample/day) was achieved. The successful validation presented indicates that rapid methods of analysis can efficiently and reliably contribute to the fast sample turnaround required for high sample number generating processes.