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 of lack of specificity due to the presence of metabolites

被引:48
作者
Matuszewski, BK [1 ]
Chavez-Eng, CM [1 ]
Constanzer, ML [1 ]
机构
[1] Merck Res Labs, W Point, PA 19486 USA
来源
JOURNAL OF CHROMATOGRAPHY B | 1998年 / 716卷 / 1-2期
关键词
oxytocin; L-368,899;
D O I
10.1016/S0378-4347(98)00317-X
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 mit 665-->425 LS. 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. (C) 1998 Elsevier Science B.V; All rights reserved.
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页码:195 / 208
页数:14
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