title: Determination of a novel substance P inhibitor in human plasma by high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometric detection using single and triple quadrupole detectors.
abstract: Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2R)-[1(R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3(S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant (I) and internal standard (II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid-liquid extraction with methyl-t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific's Javelin BDS C-8 2 mm x 4.6 mm 3 microm guard column coupled to BDS C-8 50 mm x 4.6 mm 3 microm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor --> ion combinations of m/z 535 --> 277 and 503 --> 259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.
authors.affiliation: Merck Research Laboratories, Sumneytown Pike, WP75A-303, West Point, PA 19486, USA. marvin_constanzer@merck.com
authors.forename: M L
authors.initials: ML
authors.lastname: Constanzer
authors.forename: C M
authors.initials: CM
authors.lastname: Chavez-Eng
authors.forename: J
authors.initials: J
authors.lastname: Dru
authors.forename: W F
authors.initials: WF
authors.lastname: Kline
authors.forename: B K
authors.initials: BK
authors.lastname: Matuszewski
mesh_terms: D000077608:Aprepitant; D001274:Atmospheric Pressure; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods; D009025:Morpholines / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013373:Substance P / Q000037:antagonists & inhibitors*
chemical_list: D009025:Morpholines; D000077608:Aprepitant; D013373:Substance P