Determination of the thrombin inhibitor AZD0837 and its metabolites in human bile using mixed mode solid phase extraction and LC–MS/MS

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%.

► Simultaneous analysis of hydrophobic and hydrophilic analytes using mixed mode SPE.
► Bile differs from plasma affecting the choice of extraction procedure.
► The extraction method was fully automated.
► The cycle time was reduced by a high flow rate on a fused core LC column.
► The method was successfully validated for all three analytes.