A rapid, sensitive and speciΓc analytical method was developed and validated to quantify tetrahydrolipostatin (Orlistat, Ro 18-0647) in human plasma in order to provide pharmacokinetic data from clinical trials. This method employs a preliminary plasma protein precipitation step followed by a simple, one-step liquid-liquid extraction procedure to isolate Ro 18-0647 and its pentadeuterated internal standard, Ro from the biological 18-0647-d 5 , matrix. Reconstituted extracts were analyzed by liquid chromatography/ion spray tandem mass spectrometry (LC/MS/MS) in the selected reaction monitoring (SRM) mode. Chromatography was carried out using a 2 mm i.d. Γ 50 mm Deltabond Phenyl column. The eluent was acetonitrile-2 mM ammonium acetate (90 : 10). The retention time of the analyte was 1.2 min and chromatographic run times were less than 1.5 min. No interferences from anticoagulants, collection devices or endogenous constituents of the plasma were observed. The assay has a lower limit of quantitation (LLQ) of 0.20 ng ml-1 plasma and a lower limit of detection (LLD) of 0.10 ng ml-1 plasma, based on 1 ml aliquots. The capability to detect 0.025 ng ml-1 in plasma has also been demonstrated. The calibration graphs were linear from 0.20 to 10 ng ml-1. The assay was initially validated with a linear range of 0.20-1.0 ng ml-1. This range was later extended and validated to an upper level of quantitation of 10 ng ml-1. Intra-and inter-assay precision studies showed a mean variability of less than 10% . The recovery, inter-assay precision and accuracy of the method were within acceptable bioanalytical standards. The assay has been shown to reliably provide automated, unattended sample analysis for ΒΏ150 samples per day. In an additional series of tests, Ro 18-0467 was shown to be stable under conditions that might be encountered during the analysis of samples from clinical trials. This LC/MS/MS assay procedure for Ro 18-0647 in human plasma has proven to be robust, sensitive, speciΓc, accurate and reproducible. This method has been used to analyze over 5000 study samples.