High-Throughput Quantitative LC-MS/MS Analysis of Barbiturates in Human Urine

We hereby present a fast, high-throughput, and clinical LC-MS/MS assay for the simultaneous analysis of barbiturates in human urine. It is deployed as a quantitative assay for phenobarbital, butalbital, pentobarbital/amobarbital, and secobarbital, as well as for confirmations following positive immunoassay drug screens in patient urine. Briefly, urine specimens are processed via dilute and shoot, i.e., by mixing the sample with 20 times volume of internal standard reagent and injecting 50 μL of that mixture into the analytical instrument. Chromatographic separation is performed using a reversed-phase C18 column in a mobile-phase system doped with <1 mM ammonium fluoride. Mass spectrometric detection occurs via negative-mode electrospray ionization multiple reaction monitoring in the TSQ Quantiva triple-quadrupole instrument. All the analytes in the mixture are detected and quantified simultaneously with respect to internal calibration in the range 20-2500 ng/mL. However, the assay cannot distinguish pentobarbital from amobarbital, which are isobaric analytes. Nonetheless, the assay is sensitive, robust, and amenable to harmonization with other assays that employ barbiturate cutoffs in the range of 20-150 ng/mL.