DEVELOPMENT OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY TANDEM MASS-SPECTROMETRIC METHODS FOR THE DETERMINATION OF A NEW OXYTOCIN RECEPTOR ANTAGONIST (L-368,899) EXTRACTED FROM HUMAN PLASMA AND URINE - A CASE OFLACK OF SPECIFICITY DUE TO THE PRESENCE OF METABOLITES

The purpose of this work was to develop HPLC-MS-MS methods for the qua ntification of L-368,899 (1) in human plasma and urine and to evaluate the selectivity of these methods in post-dose samples in the presence of metabolites. Assays were based on double liquid-liquid extraction of the drug and internal standard (I.S., 2) from basified plasma, evap oration of the extracts to dryness, derivatization of the primary amin o groups of 1 and 2 with trifluoroacetic anhydride (TFAA) to form trif luoroacetylated (TFA) analogs, and HPLC analysis using tandem mass spe ctrometer equipped with the heated nebulizer interface as a detector. The derivatization with TFAA was required to eliminate the carryover a nd adsorption problems encountered when underivatized molecules were c hromatographed, and allowed quantitation at low concentration (0.5 ng/ ml) in plasma and urine. Initially, assays in control human plasma and urine were validated in the concentration range of 0.5-75 ng/ml, usin g simplified chromatographic conditions with a 2-min run-time and no s eparation of the drug from I.S.. Quantitation was based on the high se lectivity of detection and multiple reaction monitoring (MRM) using th e precursor-->product ion combinations of m/z 651-->152 and m/z 665--> 425 for the TFA-derivatized 1 and 2, respectively. However, when selec ted post-dose urine samples from a clinical study were analyzed using this assay, the area of the I.S. peak was 4 to 7 times larger than the area of I.S. peak in pre-dose urines, indicating the presence of meta bolites giving rise to the mit 665-->425 LS. peak. A number of metabol ites contributing to the I.S. ion pair were separated from 1 and 2 usi ng a longer analytical column, a weaker mobile phase, and by extending the HPLC run-time to 12 min. Under these new conditions, the modified assays both in plasma and urine were validated in the concentration r ange of 0.5 to 75.0 ng/ml. These assays were selective in the post-dos e urine samples in the presence of metabolites. (C) 1998 Elsevier Scie nce B.V; All rights reserved.