title: Determination of the thrombin inhibitor AZD0837 and its metabolites in human bile using mixed mode solid phase extraction and LC-MS/MS.
abstract: A method for the determination of AZD0837 and its two metabolites AR-H069927 and AR-H067637 in human bile was developed and validated. All three analytes and their stable isotope-labeled internal standards were isolated from bile using solid phase extraction on a mixed mode reversed phase/anion exchange column. Elution was done at high ionic strength with 0.125 M ammoniumacetate in 50% methanol. The extraction recoveries were >75%. Due to the high concentration of AR-H067637 a portion of the extract was diluted before injection on to the LC column, while undiluted extract was directly injected for the analysis of AZD0837 and AR-H069927. Chromatographic separation of all three analytes was achieved in a single system utilizing a C18 column based on fused core particle technology at high flow rate. The two metabolites were eluted when a gradient from 30 to 57% methanol was applied while the more hydrophobic pro-drug, AZD0837, eluted during a steeper second gradient from 57 to 80% methanol with the ammonium acetate concentration and acetic acid concentration kept constant at 3.8 mmol/L and 0.1%, respectively. The total cycle time was 3.2 min. Detection was performed using positive electrospray ionization tandem mass spectrometry. The linearity range was 0.02-20 μmol/L for AZD0837 and AR-H069927, and 1-1000 μmol/L for AR-H067637. The repeatability and the overall precision were less than 15% (RSD) and the accuracy was within the interval 93-100%.
authors.affiliation: Regulatory Bioanalysis, Mölndal, Global DMPK-IM, AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden. kristina.duner@astrazeneca.com
authors.forename: Kristina
authors.initials: K
authors.lastname: Dunér
authors.forename: Johan
authors.initials: J
authors.lastname: Hulthe
authors.forename: Mattias
authors.initials: M
authors.lastname: Tranberg
mesh_terms: D000578:Amidines / Q000032:analysis* / Q000302:isolation & purification; D000991:Antithrombins / Q000032:analysis* / Q000302:isolation & purification; D001384:Azetidines / Q000032:analysis* / Q000302:isolation & purification; D001646:Bile / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D016014:Linear Models; D008297:Male; D000432:Methanol / Q000737:chemistry; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: C551586:AZD 0837; D000578:Amidines; D000991:Antithrombins; D001384:Azetidines; D000432:Methanol