Abstract
The purpose of this work was to develop HPLC–MS–MS methods for the quantification 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, evaporation of the extracts to dryness, derivatization of the primary amino groups of 1 and 2 with trifluoroacetic anhydride (TFAA) to form trifluoroacetylated (TFA) analogs, and HPLC analysis using tandem mass spectrometer equipped with the heated nebulizer interface as a detector. The derivatization with TFAA was required to eliminate the carryover and adsorption problems encountered when underivatized molecules were chromatographed, 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, using simplified chromatographic conditions with a 2-min run-time and no separation of the drug from I.S.. Quantitation was based on the high selectivity of detection and multiple reaction monitoring (MRM) using the precursor→product ion combinations of m/z 651→152 and m/z 665→425 for the TFA-derivatized 1 and 2, respectively. However, when selected 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 metabolites giving rise to the m/z 665→425 I.S. peak. A number of metabolites contributing to the I.S. ion pair were separated from 1 and 2 using 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 range of 0.5 to 75.0 ng/ml. These assays were selective in the post-dose urine samples in the presence of metabolites.
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