High-throughput determination of barbiturates in human plasma using on-line column-switching ultra-fast liquid chromatography–tandem mass spectrometry

A high-throughput method was developed for determinations of eight barbiturates (barbital, allobarbital, phenobarbital, cyclobarbital, amobarbital, secobarbital, thiopental, and thiamylal) in human plasma by on-line column-switching ultra-fast liquid chromatography–tandem mass spectrometry (MS–MS). Plasma samples (100 μl) spiked with the eight barbiturates and 5-(4-methylphenyl)-5-phenylhydantoin (internal standard) were diluted with 300 μl of 13.3 mM 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 onto the analytical column (Phenomenex Onyx monolithic C₁₈ column, 100 mm × 4.6 mm i.d.) by column switching. Quantification was performed by multiple reaction monitoring with negative-ion atmospheric pressure chemical ionization. Good peak separation and peak shapes of the eight drugs were achieved within an analysis time of 3 min, including the extraction time. All drugs spiked into plasma showed recoveries of 80–93 %. The regression equations for the eight drugs showed excellent linearities in the range of 10–5000 ng/ml of plasma, and the limits of detection ranged from 1.0 to 10 ng/ml. The lower and upper limits of quantitation were 10–50 ng/ml and 5000 ng/ml, respectively. Intraday and interday coefficients of variation for all the drugs were not >9.1 %. The accuracies of quantitation were 92.0–108 %. The method was successfully applied to determination of the level of amobarbital in human plasma after its oral administration to a volunteer.