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

Abstract

The generation of large numbers of samples during early drug discovery has increased the demand for rapid and selective methods of analysis. Liquid chromatography-tandem mass spectrometry (LC-MS-MS), because of its 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 handler (Gilson, Inc.), which aspirates eight samples simultaneously and performs its rinse cycle parallel to sample injection, resulting in minimum lag time between injections. This high-throughput 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 QC1 (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 (approximately 37 min/ 96-well plate or over 3,700 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.

Rapid detection and identification of N-acetyl-L-cysteine thioethers using constant neutral loss and theoretical multiple reaction monitoring combined with enhanced product-ion scans on a linear ion trap mass spectrometer

“…Therefore, analytical methods have to be developed to screen blood or urine samples for unknown chemicals or known drugs and their metabolites for compound identification to calculate ultimate exposure levels [2]. Over recent years, tandem LC-MS/MS has been extensively applied to quantify compounds with known structures using transitions for the sensitive multiple reaction monitoring (MRM) methods [3].…”

Using short columns to speed up LC–MS quantification in MS binding assays

“…Although the Shimadzu SIL HT(A) autosampler works comparably fast the "overhead" time to the chromatographic cycle time of 18 s produced by the autosampler (including a rinsing step) could not be reduced below 39 s. It should be emphasized, however, that this "overhead" time can be further reduced employing an even faster autosampler or by the use of two (or more) autosamplers [18][19][20]. Alternatively, omitting the rinsing step of the autosampler, at the expense of an enhanced carryover, may be considered to this end as well.…”

Quantitative strategies to fuel the merger of discovery and hypothesis-driven shotgun proteomics

“…In fact, the development of selective reaction monitoring (SRM) was largely pioneered by those interested in detecting these types of compounds in highly complex and typically unfractionated samples [46][47][48][49][50][51]. The use of SRM for the robust detection of a multitude of compounds has been reported in human plasma [49,[52][53][54] and other complex human samples [55,56]. Indeed, the use of isotope-labeled internal standards using SRM is well established for the robust and high-throughput analysis of metabolites in complex human samples.…”