High throughput bioanalytical LC/MS/MS determination of benzodiazepines inhuman urine: 1000 samples per 12 hours

The analytical capabilities of liquid chromatography tandem mass spectromet ry for sensitive and highly selective determination of target compounds in complex biological samples makes it well suited for high-throughput analysi s. We report the fast separation of size benzodiazepines isolated from huma n urine via selected reaction monitoring liquid chromatography/mass spectro metry using short dwell times to accommodate fast-eluting chromatographic p eaks. The analytes Fc ere extracted from human urine samples along with the ir deuterium-labeled internal standards by a simple liquid-liquid ex-tracti on in 96-well plates. Using four autosamplers coupled to one chromatographi c column and one tandem mass spectrometer operated in the turbo ion spray m ode with positive ion detection, 1152 samples (12 96-well plates) were anal yzed in less than 12 h. Through an electronic sn;itching box designed and c onstructed in-house, the autosamplers were synchronized with the mass spect rometer so that injections were made as soon as the mass spectrometer was r eady to collect data. Each run required 30 s to complete with another 7-8 s for the data system to load the next data file to be collected. Chromatogr aphic integrity and ion current response remained relatively constant for t he duration of the analyses. The results show acceptable precision and accu racy and demonstrate the feasibility of using fast separations with tandem mass spectrometry for high-throughput analysis of biological samples contai ning multiple analytes.