Study on the Analysis of Capsaicin Glucuronide in Rat Urine by Liquid Chromatography-Mass Spectrometry after Enzymatic Hydrolysis

Abstract

We established a method for determining capsaicin glucuronide in rat urine samples using liquid chromatography-mass spectrometry combined with enzymatic hydrolysis. Capsaicin was not detected in urine samples of rats administered capsaicin intraperitoneally (i.p., 2 and 4 mg/kg), but after hydrolysis with β-glucuronidase, extraction with methanol and solid-phase extraction, capsaicin was clearly detected by liquid chromatography-mass spectrometry with electrospray ionization. The limit of detection was obtained to be 0.1 ng/ml in urine sample matrix. The conditions for the enzymatic hydrolysis of the conjugate were optimized for β-glucuronidases from Ampullaria, Escherichia coli, bovine liver, Helix pomatia and Patella vulgata. The optimal conditions among those examined [pH (3.3-9.0), temperature (37-70°C) and enzyme amount (50-2500 U)] for 1 ml of the urine sample were as follows: β-glucuronidase from Ampullaria (pH 4.2, 45°C, 500 U), from Escherichia coli (pH 6.0-7.2, 37°C, 500 U), from bovine liver (pH 5.0, 45°C, 500 U), from Helix pomatia (pH 5.0, 60°C, 1250 U) and from Patella vulgata (pH 3.8, 45-60°C, 2500 U). Among the enzymes examined, β-glucuronidase from Ampullaria was found to be suitable for hydrolysis of the conjugate. Under the optimal conditions of Ampullaria β-glucuronidase, the incubation of 1 ml of urine sample for 90 min was sufficient for hydrolysis of capsaicin glucuronide in the urine sample. The urinary recovery values of capsaicin collected for 0-48 hr after administration of 2 and 4 mg/kg capsaicin to rats were 1.4 and 1.1%, respectively.