Simultaneous quantification of 2′,3′,5′-tri-O-acetyl-N6-(3-hydroxylaniline)adenosine and its principal metabolites in hamster blood by LC-MS/MS and its application in pharmacokinetics study

2′,3′,5′-Tri-O-acetyl-N6-(3-hydroxylaniline)adenosine (IMM-H007, once called WS070117) is being developed as a novel anti-hyperlipidemia agent for its high efficacy and low toxicity. In this study, a sensitive and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was established for the simultaneous quantification of IMM-H007 and its two major metabolites (3S,4R,5R)-2-(hydroxymethyl)-5-(6-((3-hydroxyphenyl)amino)-9H-purin-9-yl)tetrahdrofuran-3,4-diol (M1) and ((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((3-hydroxyphenyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate (MP) in hamster blood. An analogue of IMM-H007, WS070119 was used as the internal standard. Blood samples were prepared by a simple protein precipitation with acetonitrile. The chromatographic separation was performed on a ReproSil-Pur 120C18 column (3 μm, 2 mm × 100 mm) with a gradient mobile phase of methanol/water containing 0.1% formic acid (v/v) in a flow rate of 0.2 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) in positive ion selective reaction monitoring (SRM) mode. The monitored transitions were 486.2 → 228.1 for IMM-H007, 360.0 → 228.0 for M1, 440.0 → 228.0 for MP and 374.1 → 242.0 for the internal standard, respectively. Satisfactory linearity was obtained for the analytes over the range of 1-500 ng/mL for IMM-H007, 2-1000 ng/mL for M1 and 10-5000 ng/mL for MP. The lower limits of the quantification (LLOQs) were 1 ng/mL for IMM-H007, 2 ng/mL for M1 and 10 ng/mL for MP. The intra-day and inter-day precisions (RSD, %) of the analytes were within 14.2%, and the accuracy (RE, %) ranged from −9.4% to 10.7%. The average recoveries of the analytes were more than 80.0%. The analytes were proved to be stable during given storage, preparation, and analytic procedures. The method was successfully applied to the pharmacokinetic study in hamsters after oral administration of IMM-H007.