Metabolite pattern analysis (HPLC coupled to on-line radioactivity detection). The HPLC systems used for metabolite pattern analysis consisted of autosamplers (LC PAL, CTC Analytics, Zwingen, Switzerland), HPLC pumps (G1312A, Agilent Technologies, Waldbron, Germany), degassers (G1379A, Agilent Technologies), and radioactivity detectors (LB 507B or LB 509, Berthold, Bad Wildbad, Germany) equipped with solid-phase scintillation cells (YG 150 S4D). The separation was additionally monitored by diode array detectors (G1315A, Agilent Technologies). The software used was Chromelion, version 6.2 (Dionex, Idstein, Germany). Samples were analyzed on 70 × 4-mm Nucleosil C100–5 C18 HD columns protected by 8 × 4-mm Nucleosil 100 C18 HD guard columns (both 5-μm particle size; Marcherey-Nagel, Düren, Germany). Metabolites were separated with a gradient of aqueous ammonium formate (0.02 M, pH 8, adjusted with ammonium hydroxide: mobile phase A) versus ammonium formate in methanol (0.02 M, pH 8, adjusted with ammonium hydroxide: mobile phase B) at a flow rate of 0.8 ml/min (gradient: 3% B at 0 min, linear to 20% B at 20 min, linear to 85% B at 31.5 min). With a signal to noise ratio = 2, the HPLC systems were linear (r2 ≥ 0.99) over the range of 425 to 88,935 dpm (absolute amount injected on column), respectively, as assessed by triplicate injections of dabigatran at various concentrations. The inaccuracy and imprecision of the method were ≤7.9 and ≤15%, respectively.
