title: Quantitative analysis of simvastatin and its beta-hydroxy acid in human plasma using automated liquid-liquid extraction based on 96-well plate format and liquid chromatography-tandem mass spectrometry.
abstract: An assay based on automated liquid-liquid extraction (LLE) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed and validated for the quantitative analysis of simvastatin (SV) and its beta-hydroxy acid (SVA) in human plasma. A Packard MultiProbe II workstation was used to convert human plasma samples collected following administration of simvastatin and quality control (QC) samples from individual tubes into 96-well plate format. The workstation was also used to prepare calibration standards and spike internal standards. A Tomtec Quadra 96-channel liquid handling workstation was used to perform LLE based on 96-well plates including adding solvents, separating organic from aqueous layer and reconstitution. SV and SVA were separated through a Kromasil C18 column (50 mm x 2 mm i.d., 5 microm) and detected by tandem mass spectrometry with a TurboIonspray interface. Stable isotope-labeled SV and SVA, 13CD(3)-SV and 13 CD(3)-SVA, were used as the internal standards for SV and SVA, respectively. The automated procedures reduced the overall analytical time (96 samples) to 1/3 of that of manual LLE. Most importantly, an analyst spent only a fraction of time on the 96-well LLE. A limit of quantitation of 50 pg/ml was achieved for both SV and SVA. The interconversion between SV and SVA during the 96-well LLE was found to be negligible. The assay showed very good reproducibility, with intra- and inter-assay precision (%R.S.D.) of less than 7.5%, and accuracy of 98.7-102.3% of nominal values for both analytes. By using this method, sample throughput should be enhanced at least three-fold compared to that of the manual procedure.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, Merck & Co, Inc, WP14-1101, West Point, PA 19486, USA. rena_zhang@merck.com
authors.forename: Nanyan
authors.initials: N
authors.lastname: Zhang
authors.forename: Amy
authors.initials: A
authors.lastname: Yang
authors.forename: John Douglas
authors.initials: JD
authors.lastname: Rogers
authors.forename: Jamie J
authors.initials: JJ
authors.lastname: Zhao
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D019821:Simvastatin / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry
chemical_list: C063287:simvastatin acid; D019821:Simvastatin