Evaluation of Analytical Approaches for Compound Identification by LC-MS: a Study of the Alcohol Biomarker Ethyl Glucuronide (EtG) in Urine
Mon 4:00 PM - Track 3: Regulations and Standards
Anders Helander
Karolinska Institutet
*Anders Helander, *Naama Kenan, #Olof Beck.

*Karolinska Institutet, Department of Clinical Neuroscience, Stockholm, Sweden.
#Karolinska Institutet, Department of Medicine, Stockholm, Sweden.
A number of official guidelines regulate requirements for analytical methods used to identify chemical compounds in biological matrices. This study evaluated the accuracy of five electrospray-ionization LC-MS and LC-MS/MS procedures for identification and quantitation of the conjugated minor ethanol metabolite and alcohol biomarker ethyl glucuronide (EtG) in urine. The value of simultaneous measurement of EtG and ethyl sulfate (EtS), another conjugated ethanol metabolite, was also compared. The overall aim was to compare theoretical considerations of analytical selectivity with practical experience for reliable target analysis.

Analysis was carried out on 482 urine specimens following sample cleanup by solid-phase extraction (SPE) or using direct injection of a diluted sample. Based on the results from the routine measurement, samples were selected to cover the entire measuring range for EtG from below the detection limit up to very high concentrations.

SPE followed by LC-MS/MS was demonstrated to be the most selective and sensitive method for urinary EtG and was therefore chosen as the reference method. The urinary EtG results by the different methods showed good overall agreement (r2 = 0.93-0.96). When comparing 5 strict reporting limits in the range 0.10-1.00 mg/L EtG, the agreement of results compared with the reference method (frequency of true positives plus true negatives) was 82-97% for LC-MS/MS, 90-97% for SPE LC-MS, 86-98% for LC-MS, and 86-98% for combined EtG and EtS analysis. Most deviations were attributable to uncertainty in quantitation at concentrations near the cut-points for which the respective results were somewhat above and below, or vice versa, these strict thresholds. However, with direct-injection LC-MS/MS, equally many negative results were due to a product ion ratio outside the ± 20% tolerance limit stated by the guidelines.

Toxicology laboratories aiming to produce scientifically and legally defendable results need to reflect on the specific goal set for application of the test, and select and validate analytical strategies to best fit with the clinical or forensic requirements. The results of this study indicate that analytical methods not fulfilling all requirements of the present official guidelines may also provide reliable compound identification.
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