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J.Health Sci., 52(6), 660-665, 2006
Study on the Analysis of Capsaicin Glucuronide in Rat Urine by Liquid Chromatography-Mass Spectrometry after Enzymatic Hydrolysis
Masaaki Noami,*, a, c Kazuo Igarashi,b Fumiyo Kasuya,c Masayuki Ohta,c
Mieko Kanamori-Kataoka,d and Yasuo Setod
aForensic Science Laboratory, Wakayama Pref. Police H. Q., 1 Nishi, Wakayama 640-8313, Japan,
bDepartment of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0212, U.S.A,
cFaculty of Pharmaceutical Sciences, Kobe Gakuin University, 518 Arise, Ikawadanicho, Nishi-ku, Kobe 651-2180, Japan, and
dNational Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan
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 beta-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 beta-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: beta-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, beta-glucuronidase from Ampullaria was found to be suitable for hydrolysis of the conjugate. Under the optimal conditions of Ampullaria beta-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.
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