Rapid and highly sensitive analysis of benzodiazepines and tandospirone in human plasma by automated on-line column-switching UFLC-MS/MS.

Abstract

A high-throughput method was developed for the detection of 31 benzodiazepine drugs and tandospirone in human plasma by on-line column-switching ultra-fast liquid chromatography-tandem mass spectrometry. Plasma samples (100μl) spiked with the 32 drugs and oxazepam-d5 (internal standard) were diluted with 300μl of 13.3mM ammonium acetate/acetonitrile (33:67, v/v). After centrifugation and filtration, the clear supernatant was injected directly onto the extraction column (Oasis HLB cartridge column). The following procedure was fully automated. The analytes retained on the extraction column were eluted by backflushing of the extraction column and introduced into an analytical column (SUMIPAX ODS D-Swifter column, 30mm×3.0mm i.d.; particle size 2μm) by column switching. Quantification was performed by multiple reaction monitoring with positive-ion electrospray ionization. Distinct peaks appeared for each drug and the internal standard on each channel within 7min, including the extraction time. All drugs spiked into plasma showed recoveries of 83-95%. The regression equations for the 32 drugs showed excellent linearities in the range of 50-2000pg/ml of plasma and the limits of detection ranged from 20 to 50pg/ml. The lower and upper limits of quantitation were 50-100ng/ml and 2000pg/ml, respectively. Intra- and interday coefficients of variation for none of the drugs were greater than 13.6%. The accuracies of quantitation were 87-112%. The multiple reaction monitoring information-dependent acquisition of enhanced product ions method enabled the quantification and confirmation of diazepam, triazolam, and lorazepam obtained from actual plasma.