title: High-throughput salting-out assisted liquid/liquid extraction with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid chromatography.
abstract: Simvastatin (SS) is an effective cholesterol-lowering medicine, and is hydrolyzed to simvastatin acid (SSA) after oral administration. Due to SS and SSA inter-conversion and its pH and temperature dependence, SS and SSA quantitation is analytically challenging. Here we report a high-throughput salting-out assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for simultaneous LC-MS/MS analysis of SS and SSA. The sample preparation of a 96-well plate using SALLE was completed within 20 min, and the SALLE extract was diluted and injected into an LC-MS/MS system with a cycle time of 2.0 min/sample. The seamless interface of SALLE and LC-MS eliminated drying down step and thus potential sample exposure to room or higher temperature. The stability of SS and SSA in various concentration ratios in plasma was evaluated at room and low (4 degrees C) temperature and the low temperature (4 degrees C) was found necessary to maintain sample integrity. The short sample preparation time along with controlled temperature (2-4 degrees C) and acidity (pH 4.5) throughout sample preparation minimized the conversion of SS-->SSA to < or = 0.10% and the conversion of SSA-->SS to 0.00% The method was validated with a lower limit of quantitation (LLOQ) of 0.094 ng mL(-1) for both SS and SSA and a sample volume of 100 microL. The method was used for a bioequivalence study with 4048 samples. Incurred sample reproducibility (ISR) analysis of 362 samples from the study exceeded ISR requirement with 99% re-analysis results within 100+/-20% of the original analysis results.
authors.affiliation: Department of Drug Analysis, Abbott Laboratories, Abbott Park, IL 60064, USA. jun.zhang@abbott.com
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Ramona
authors.initials: R
authors.lastname: Rodila
authors.forename: Eric
authors.initials: E
authors.lastname: Gage
authors.forename: Mathew
authors.initials: M
authors.lastname: Hautman
authors.forename: Leimin
authors.initials: L
authors.lastname: Fan
authors.forename: Linda L
authors.initials: LL
authors.lastname: King
authors.forename: Huaiqin
authors.initials: H
authors.lastname: Wu
authors.forename: Tawakol A
authors.initials: TA
authors.lastname: El-Shourbagy
mesh_terms: D000097:Acetonitriles / Q000737:chemistry*; D053000:Analytic Sample Preparation Methods; D001774:Blood Chemical Analysis / Q000379:methods*; D002138:Calibration; D005591:Chemical Fractionation / Q000379:methods*; D055598:Chemical Phenomena; D002853:Chromatography, Liquid; D004355:Drug Stability; D057166:High-Throughput Screening Assays; D006801:Humans; D015203:Reproducibility of Results; D012492:Salts / Q000737:chemistry*; D019821:Simvastatin / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics; D013696:Temperature; D013810:Therapeutic Equivalency; D013997:Time Factors
chemical_list: D000097:Acetonitriles; D012492:Salts; C063287:simvastatin acid; D019821:Simvastatin; C032159:acetonitrile