Comprehensive characterization of in vitro and in vivo metabolites of 2',3',5'‑tri‑O‑acetyl‑N‑(3‑hydroxyphenyl) adenosine and study of the metabolites distribution in rats by combined methods of HPLC-DAD, off-line cryoNMR, and HPLC-QTOFMS.

The compound 2',3',5'‑tri‑O‑acetyl‑N‑(3‑hydroxyphenyl) adenosine (also known as IMM-H007) is a new adenosine analogue that displays anti-hyperlipidaemic activity in many preliminary studies. To clarify its biotransformation process, in vitro and in vivo metabolic patterns of IMM-H007 in rat liver microsomes (RLMs), urine, feces, serum, and various tissues were investigated using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD), off-line cryogenically cooled probe nuclear magnetic resonance (cryoNMR), and high-performance liquid chromatography quadrupole TOF MS (HPLC-QTOFMS) measurements. A total of 21 metabolites were detected and identified based on accurate mass measurements, diagnostic product ions, and 1D and 2D NMR data. All of the 21 metabolites were detected in vivo besides the 7 ones (LM1-3, LM4a-b, LM5, LM6 (M8)) in vitro. Among them, eight metabolites were phase I metabolites composed of the hydrolysis products LM1-3, LM4a, LM4b, LM5 and M7-8, and hydrolysis and hydroxylation products M6. Others were phase II metabolites including glucuronidation products M2, M4, M9, M11a-c, and M12a-c; and sulfation products M3, M5, and M10. Notably, 14 metabolites (LM1-3, LM4a-b, LM5, M9-10, M11a-c, M12a-c) were unreported before and the distribution of IMM-H007 and its all metabolites was reported for the first time. The results revealed IMM-H007 was metabolized mainly in the small intestine and serum, kidney, stomach, small and large intestines were important samples for metabolites presence. This work improves understanding of the metabolism, distribution, and excretion of IMM-H007, and demonstrates the HPLC/HPLC-MS/off-line cryoNMR approach can be applied for detection and identification of metabolites in complex biological matrices.