title: 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.
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.
authors.affiliation: Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: B K
authors.initials: BK
authors.lastname: Matuszewski
authors.forename: C M
authors.initials: CM
authors.lastname: Chavez-Eng
authors.forename: M L
authors.initials: ML
authors.lastname: Constanzer
mesh_terms: D000089:Acetic Anhydrides; D000818:Animals; D001892:Camphanes / Q000097:blood* / Q000378:metabolism / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D005463:Fluoroacetates; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010879:Piperazines / Q000097:blood* / Q000378:metabolism / Q000652:urine*; D011786:Quality Control; D018045:Receptors, Oxytocin / Q000037:antagonists & inhibitors*; D012680:Sensitivity and Specificity
chemical_list: D000089:Acetic Anhydrides; D001892:Camphanes; D005463:Fluoroacetates; D010879:Piperazines; D018045:Receptors, Oxytocin; C017958:trifluoroacetic anhydride; C086206:L 368899