Liquid chromatography : mass spectrometry study of two biochemical alcohol markers

Abstract

The interest in biochemical alcohol markers for detecting acute and chronic alcohol consumption has expanded greatly during recent years. The development and application of laboratory tests identifying early problematic drinking and monitoring abstinence have the potential of reducing the healthcare costs and suffering associated with alcohol misuse. Laboratory tests which are sensitive enough to detect single alcohol intake include ethanol, ethyl glucuronide (EtG) and 5-hydroxytryptophol glucuronide (GTOL). The overall aim of the present work was development of sensitive and specific liquid chromatography-mass spectrometry (LC-MS) methods for direct quantification of EtG and GTOL in urine, and to apply these for clinical studies.

EtG is a minor direct metabolite of ethanol, and is present for some time after ethanol is eliminated. A simple analytical procedure was developed based on direct injection of diluted urine into the LC-MS system. EtG was found to be stable in urine with no breakdown or artificial formation on storage in room temperature. Presence of ethanol in urine did not result in any artificial formation. EtG was not detectable in urine samples collected after abstinence from alcohol. EtG remains in the urine for many hours after ethanol itself has been eliminated. Thus, testing urine for the presence of EtG provides a mean for determination of recent alcohol consumption. Expressing urinary EtG as a ratio to creatinine should be recommended in routine clinical use to compensate for urine dilution. The method fulfils the need for a simple and reliable assay to be used as a routine test of recent alcohol intake.

GTOL is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin (5-HT) metabolite. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. An LC-MS procedure, including solid-phase extraction for direct quantification of GTOL was developed. The method was highly correlated with an established gas chromatography-MS method for urinary 5-HTOL (r2 = 0.99, n = 70; mean 5-HTOL/GTOL = 1.10). This was the first direct assay for quantification of GTOL in urine, suitable for routine application.

In clinical use, GTOL is expressed as a ratio to the main 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA), in order to compensate for variations in urine dilution and 5-HT turnover. A fully validated and robust LC-MS/MS method for measurement of urinary GTOL and 5-HIAA, based on direct injection was developed. The method was capable of measuring endogenous GTOL and 5-HIAA levels in urine that agreed with literature data. The method was applied and compared with a new developed enzyme-linked immunosorbent assay (ELISA) for GTOL in clinical material. Determination of GTOL by ELISA showed 82% sensitivity in detecting positive samples, compared to the LC-MS/MS method. When 10 alcoholic patients were followed during detoxification, the GTOL/5-HIAA ratio gave a median detection time of 39 hours, while EtG was detectable for a median of 65 hours. The lower sensitivity of the urinary GTOL/5-HIAA ratio compared with EtG for recent drinking may be clinically useful, in cases where the EtG test provides an unwanted high sensitivity for intake of only small amounts of alcohol or unintentional ethanol exposure. The present work demonstrated the potential of developing robust and selective methods for quantification of analytes in urine using electrospray ionization LC-MS and LC-MS/MS with minimal sample preparation.