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HPLC/MS is a linear technique characterized by serial injection and analysis of individual samples. Parallel-format high-throughput screens for druglike properties present a significant analytical challenge. Analysis speed and system ruggedness are key requirements for bioanalysis of thousands of samples per day. The tasks involved in LC/MS analysis are readily divided into three areas, sample preparation/liquid handling, LC/MS method building/sample analysis, and data processing. Several automation and multitasking strategies were developed and implemented to minimize plating and liquid handling errors, reduce dead times within the analysis cycle, and allow for comprehensive review of data. Delivering multiple samples to multiple injectors allows the autosampler time to complete its wash cycles and aspirate the next set of samples while the previous set is being analyzed. A dual-column chromatography system provides column cycling and peak stacking and allows rapid throughput using conventional LC equipment. Collecting all data for a compound into a single file greatly reduces the number of data files collected, increases the speed of data collection, allows rugged and complete review of all data, and provides facile data management. The described systems have analyzed over 40 000 samples per month for two years and have the capacity for over 2000 samples per instrument per day.

Transformation of prostaglandin D2 to 9cv,11,8-(15S)-trihydroxyprosta-(5Z,13E)-dien-l-oic acid (9a,11j3-prostaglandin F2): A unique biologically active prostaglandin produced enzymatically in vivo in humans (11-ketoreductase/11-epiprostaglandin Communicated by Charles R. Park, April 17, 1985 ABSTRACTIn vitro studies examining the metabolic transformation of prostaglandin D2 (PGD2) by human liver were conducted. PGD2 was found to be converted by a NADPH-dependent enzyme in the 100,000 x g supernatant of human liver exclusively to a single more polar compound that had a mass spectrum essentially the same as that of prostaglandin F2a (PGF2,). However, this compound could be chromatographically separated from PGF2. and failed to form a butylboronate derivative. The structure of this compound was established as 9a,llfi-(15S)-trihydroxyprosta-(5Z,13E)-dien-1-oic acid (9a,11P-PGF2) by comparison of its chromatographic and mass spectral characteristics with authentic 9a,11P-PGF2. This compound was found to be biologically active by demonstrating increases in blood pressure in rats in a dose-related fashion following intravenous administration. By using a mass spectrometric assay, levels of this compound in plasma and urine from a normal volunteer were 6 pg/ml and 982 ng/24 hr, respectively. In a patient with systemic mastocytosis associated with overproduction of PGD2, urinary excretion of 9a,11f-PGF2 was 6634 ng/24 hr and a circulating plasma level as high as 490 ng/ml was found during a severe episode of systemic mast cell activation. 9a,11-PGF2 is structurally a unique prostaglandin, is enzymatically formed, is produced in vivo in humans, and is biologically active.Recently we investigated the metabolism of prostaglandin D2 (PGD2) in a normal male volunteer and found that PGD2 is converted predominantly to PGF-ring compounds (1). These data provided presumptive evidence for 11-ketoreductase activity in vivo in humans analogous to evidence for this pathway of metabolism of PGD2 described in the monkey (2). Furthermore, almost all of the PGF-ring urinary metabolites of PGD2 isolated failed to form a butylboronate derivative when treated with n-butylboronic acid, suggesting that the C-9 and C-li hydroxyl groups of these metabolites were not coplanar (3). Intact PGF2 was also excreted into the urine as a metabolite of PGD2 and did not react with n-butylboronic acid. We have now undertaken in vitro studies of the conversion of PGD2 to PGF2 and report the finding that PGD2 is converted stereospecifically to 9a,11,8-(15S)-trihydroxyprosta-(5Z,13E)-dien-l-oic acid (9a,11, by an enzyme in the 100,000 X g supernatant of human liver, that this compound is present in plasma and urine from humans, and that this compound is biologically active. EXPERIMENTAL PROCEDURESMaterials and Methods. Unlabeled PGD2, PGF2,, and 9a,11i-PGF2 were the generous gifts of John Pike and Gordon Bundy of Upjohn. Synthesis of 9a,11f3-PGF2 was accomplished with modifications of procedures described previously (4). The synthetic 9a,li,-PGF2 contains...

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.