Challenges of LC-MS/MS ethyl glucuronide analysis in abstinence monitoring of liver transplant candidates

BackgroundUrinary ethyl glucuronide (EtG) has emerged as the biomarker of choice for alcohol abstinence monitoring in forensic toxicology and is now used in the listing decision process for liver transplantations (LTs) in the German transplant program. However, EtG analysis in this patient group is challenging due to severely impaired liver function, renal failure, co-morbidities and multidrug regimens. The aim of our study was to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based EtG analysis for a precise abstinence monitoring in transplant candidates.MethodsEtG and ethyl sulfate (EtS) were analyzed by a commercial LC-MS/MS assay in 1787 spot urine samples of 807 patients (>85% from the Department of Hepatology) using a combination of quantifier and two qualifier mass transitions for each analyte. Influences of bacterial contamination, kidney and liver function were investigated.ResultsTwo hundred and sixty-four urine samples had elevated (≥0.5 mg/L) EtG concentrations when only analyzing one quantifier mass transition. Eleven results (4.2%) were found to be false positive after combining three mass transitions for EtG quantification and verification with parallel analysis of EtS. Decreased kidney function was associated with a significantly higher rate of positive EtG samples. One of the false positive results was caused by bacterial metabolism.ConclusionsMultimorbid pre-transplant patients have a high risk of individual analytical disturbances of EtG results obtained by LC-MS/MS. Therefore, EtG and EtS should always be measured by a combination of one quantifier and two qualifiers each and evaluated together.

Fully Automated Determination of Phosphatidylethanol 16:0/18:1 and 16:0/18:2 in Dried Blood Spots

Purpose Direct alcohol markers are widely applied during abstinence monitoring, driving aptitude assessments and workplace drug testing. The most promising direct alcohol marker was found to be phosphatidylethanol (PEth). Compared to other markers it shows a long window of detection due to accumulation in blood. To facilitate and accelerate the determination of PEth in DBS, we developed a fully automated analysis approach. Methods The validated and novel online-SPE-LC-MS/MS method with automated sample preparation using a CAMAG DBS-MS 500 system reduces manual sample preparation to an absolute minimum, only requiring calibration and quality control DBS. Results During the validation process, the method showed a high extraction efficiency (>88%), linearity (correlation coefficient >0.9953), accuracy and precision (within ±15%) for the determination of PEth 16:0/18:1 and PEth 16:0/18:2. Within a run time of about 7 min, the two monitored analogs could be baseline separated. A method comparison in liquid whole blood of 28 authentic samples from alcohol use disorder patients showed a mean deviation of less than 2% and a correlation coefficient of >0.9759. The comparison with manual DBS extraction showed a mean deviation of less than 8% and a correlation coefficient of >0.9666. Conclusions The automated analysis of PEth in DBS can provide a fast and accurate solution for abstinence monitoring. In contrast to the manual extraction of PEth in DBS, no laborious sample preparation is required with this automated approach. Furthermore, the application of the internal standard by a spray module can compensate for extraction bias and matrix effects.