RAPID QUANTITATIVE-DETERMINATION OF A COLLAGENASE INHIBITOR AND ITS MAJOR METABOLITE BY ONLINE LIQUID-CHROMATOGRAPHY WITH IONSPRAY TANDEM MASS-SPECTROMETRIC DETECTION

A rapid, sensitive and specific analytical method has been developed a nd validated to quantify the collagenase inhibitor thyl)-4-methylvaler yl]-N1,3-dimethyl-L-valin-amide (I) and its major metabolite (II) from plasma and urine. This method employs an automated solid-phase extrac tion procedure to isolate the analytes and the internal standard from the biological matrix. Reconstituted extracts were analyzed by HPLC-io nspray MS-MS. Chromatography was performed on a 4.6 mm I.D. reversed-p hase guard column. The retention times of the analytes and the interna l standard were approximately 1.3 min. The assay has a limit of quanti fication of 5 ng/ml plasma and a limit of detection of 1 ng/ml, based on 100-mu l plasma aliquots. No sample-drying step was required. The s tandard curves were linear from 5 to 5000 ng/ml using weighted linear regression analysis (1/y(2)). The intra- and inter-assay precision wer e better than +/- 10% with intra- and inter-assay accuracies between 9 5 and 105%. This new HPLC-MS-MS assay procedure for I and II in plasma and urine has proven to be specific, sensitive, accurate and robust, and is being used routinely for the analysis of I in pre-clinical and clinical trial samples. Up to 200 unknowns may be analyzed each 24 h p er analyst.