Using supported liquid extraction together with cellobiohydrolase chiral stationary phases-based liquid chromatography with tandem mass spectrometry for enantioselective determination of acebutolol and its active metabolite diacetolol in spiked human plasma

Abstract

A high through-put, sensitive, and enantioselective LC-MS/MS-based bioanalytical method was developed and validated for the simultaneous determination of individual acebutolol (AC) and its active metabolite-diacetolol (DC) enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP). Systematic optimization of chromatographic conditions including organic content, buffer concentration, and pH of mobile phases was conducted to improve the through-put for the direct separation of both AC and DC on CBH column during method development. Complete baseline separation of enantiomeric AC and DC was achieved within 1.5 min with a LC flow rate of 0.9 ml/min under method validation conditions. To further improve the assay through-put, supported liquid extraction (SLE) in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.0500-50.0 ng/ml for each AC and DC enantiomer using 0.100 ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed <or=4.5% relative standard deviation (R.S.D.) and -8.7 to 5.6% relative error (R.E.) for individual AC and DC enantiomers.