Quantitative analysis of simvastatin and its β-hydroxy acid in human plasma using automated liquid–liquid extraction based on 96-well plate format and liquid chromatography-tandem mass spectrometry

• Published in: Journal of pharmaceutical and biomedical analysis 2004-01, Vol.34 (1), p.175-187
• Main Authors: Zhang, Nanyan, Yang, Amy, Rogers, John Douglas, Zhao, Jamie J
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Automated liquid–liquid extraction; Biological and medical sciences; Chromatography, Liquid - methods; Drug Evaluation, Preclinical - instrumentation; Drug Evaluation, Preclinical - methods; Fundamental and applied biological sciences. Psychology; General pharmacology; Human plasma; Humans; Liquid chromatography-tandem mass spectrometry; Mass Spectrometry - methods; Medical sciences; Pharmacology. Drug treatments; Simvastatin; Simvastatin - analogs & derivatives; Simvastatin - blood; Simvastatin - chemistry
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.japna.2003.08.016

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 β-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×2 mm i.d., 5 μm) and detected by tandem mass spectrometry with a TurboIonspray interface. Stable isotope-labeled SV and SVA, 13 CD 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.