A study of urinary dolichols as a biological marker for alcohol abuse. Report 1: Quantitation of urinary dolichols by high performance liquid chromatography after BondElutC18 (500 mg) cartridge extraction.

Abstract

This report describes the quantitation of urinary dolichols (Dolichol-90, -95 and -100) by advanced high performance liquid chromatography (HPLC) after BondElutC18 (500 mg) cartridge column extraction. The urine sample (15 ml) mixed with heneicosaprenol as internal standard (IS, 300 ng) was adjusted to pH 3 by 0.1N hydrochloric acid (HCl). BondElutC18 (500 mg) column was preactivated by methanol (4 ml) and distilled water (4 ml), and the urine sample was applied. The column was washed by HCl-sodium acetate buffer (pH 3, 6 ml), distilled water (4 ml) and methanol (4 ml), and then eluted by 2-propanol/dichloromethane/methanol (5/3/2 by vol, 4 ml). After drying the eluted solution under nitrogen gas flow, the residue was dissolved in 60 microliters of HPLC mobile phase, and 20 microliters of the mixture was injected into the HPLC. The HPLC conditions were as follows: column, mu Bondapack C18 (Waters); mobile phase, 2-propanol/methanol (72/28 by vol), 1.0 ml/min; detection, UV at 210 nm. Recovery yields of Dolichol-90, -95 and -100 were in the range of 68-75% and the calibration curve of each dolichol was linear over the range 0.01-1.00 micrograms.