Validated method for the determination of the ethanol consumption markers ethyl glucuronide, ethyl phosphate, and ethyl sulfate in human urine by reversed-phase/weak anion exchange liquid chromatography-tandem mass spectrometry.
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the ethanol consumption markers ethyl glucuronide (EtG), ethyl phosphate (EtP), and ethyl sulfate (EtS) in human urine was developed. A reversed-phase/weak anion exchange type stationary phase demonstrated particular suitability for the analysis of these highly polar acids. Sample preparation was minimized to centrifugation and dilution of urine prior to injection. The method was validated in the range of 5-750 microg.L(-1) with 1:20 and 1:1000 diluted urine, which corresponds to actual concentration ranges of 0.1-15 and 5-750 mg.L(-1) in undiluted samples. Method validation was carried out using six different lots of human urine. Over the entire calibration range intraday and interday precision (each n = 5, three concentration levels per dilution factor) adopted values between 0.6 and 4.7% and 0.8 and 12.1%, relative standard deviation, respectively. Corresponding accuracy values ranged between 94.2 and 113.5% and 86.6 and 110.9%, respectively. Matrix effects (absolute/relative) were found to be present in minor extent (approximately -30% to +15% MS/MS signal alterations) and were well corrected by the employed isotopically labeled internal standards. The validated assay was applied to urine samples of a drinking study as well as postmortem specimens. It was possible to assess the principal potential of EtP as ethanol consumption marker. Elevated concentration levels were found in real samples; however, EtP seems to be less sensitive compared to the previously known ethanol phase II conjugates EtG and EtS.
Bicker
Wolfgang
W
Christian Doppler Laboratory for Molecular Recognition Materials, Department of Analytical and Food Chemistry, University of Vienna, Währinger Strasse 38, A-1090 Vienna, Austria.
Lämmerhofer
Michael
M
Keller
Thomas
T
Schuhmacher
Rainer
R
Krska
Rudolf
R
Lindner
Wolfgang
W
D000328:Adult; D000428:Alcohol Drinking / Q000652:urine*; D015415:Biomarkers / Q000652:urine; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004355:Drug Stability; D005260:Female; D005554:Forensic Medicine; D005965:Glucuronates / Q000652:urine*; D006801:Humans; D008297:Male; D010710:Phosphates / Q000652:urine; D013463:Sulfuric Acid Esters / Q000652:urine*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D015415:Biomarkers; D005965:Glucuronates; D010710:Phosphates; D013463:Sulfuric Acid Esters; C093924:ethyl glucuronide; C011612:diethyl sulfate