title: Simultaneous determination of a selective adenosine 2A agonist, BMS-068645, and its acid metabolite in human plasma by liquid chromatography-tandem mass spectrometry--evaluation of the esterase inhibitor, diisopropyl fluorophosphate, in the stabilization of a labile ester-containing drug.
abstract: BMS-068645 is a selective adenosine 2A agonist that contains a methyl ester group which undergoes esterase hydrolysis to its acid metabolite. To permit accurate determinations of circulating BMS-068645 and its acid metabolite, blood samples must be rapidly stabilized at the time of collection. A sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of BMS-068645 and its acid metabolite in human plasma has been developed and validated using diisopropyl fluorophosphate (DFP) as the esterase inhibitor to prevent BMS-068645 from converting to its acid metabolite. The D(5)-stable isotope labeled analogs of BMS-068645 and its metabolite were used as the internal standards (IS). Analytes and IS in plasma containing 20 mM DFP were acidified and extracted into methyl tert-butyl ether. The liquid-liquid extraction effectively eliminated the strong matrix effect caused by the esterase inhibitor. The chromatographic separation was achieved on a Waters Atlantis C18 column with a run time of 4 min. Detection was performed on a Sciex API 4000 with positive ion electrospray mode (ESI/MS/MS), monitoring the ion transitions m/z 487>314 and 473>300 for BMS-068645 and its acid metabolite, respectively. The method was validated over the range from 0.020 to 10.0 ng/mL for BMS-068645 and 0.050 to 10.0 ng/mL for its acid metabolite. Inter- and intra-run precision for the quality control samples during validation were less than 8.7% and 4.0%, respectively, for the two analytes. The assay accuracy was within +/-5.4% of the nominal values. The esterase inhibitor effectively stabilized BMS-068645 during blood collection and storage. Blood collection tubes containing DFP were easily prepared and used at the clinical sites and could be stored at -30 degrees C for 3 months. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability and ruggedness to support the analysis of human plasma samples in pharmacokinetic studies.
authors.affiliation: Bioanalytical Sciences Department, Pharmaceutical Research Institute, Bristol-Myers Squibb, Route 206 and Province Line Road, Lawrenceville, NJ 08640, USA. jianing.zeng@bms.com
authors.forename: Jianing
authors.initials: J
authors.lastname: Zeng
authors.forename: David
authors.initials: D
authors.lastname: Onthank
authors.forename: Paul
authors.initials: P
authors.lastname: Crane
authors.forename: Steve
authors.initials: S
authors.lastname: Unger
authors.forename: Naiyu
authors.initials: N
authors.lastname: Zheng
authors.forename: Stephanie
authors.initials: S
authors.lastname: Pasas-Farmer
authors.forename: Mark
authors.initials: M
authors.lastname: Arnold
mesh_terms: D000480:Alkynes / Q000097:blood* / Q000493:pharmacokinetics; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004950:Esterases / Q000037:antagonists & inhibitors*; D004952:Esters; D006801:Humans; D007531:Isoflurophate / Q000737:chemistry*; D011684:Purine Nucleosides / Q000097:blood* / Q000493:pharmacokinetics; D058906:Purinergic P1 Receptor Agonists; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000480:Alkynes; C521541:BMS-068645; D004791:Enzyme Inhibitors; D004952:Esters; D011684:Purine Nucleosides; D058906:Purinergic P1 Receptor Agonists; D007531:Isoflurophate; D004950:Esterases