Phosphatidylethanol (PEth) in human blood samples is a marker for alcohol usage. Typically, PEth is detected by reversed-phase liquid chromatography coupled with negative ion tandem mass spectrometry, investigating the fatty acyl anions released from the precursor ion upon collision-induced dissociation (CID). It has been established that in other classes of asymmetric glycerophospholipids, the unimolecular fragmentation upon CID is biased depending on the relative position (known as sn-position) of each fatty acyl chain on the glycerol backbone. As such, the use of product ions in selected-reaction-monitoring (SRM) transitions could be prone to variability if more than one regioisomer is present in either the reference materials or the sample. Here, we have investigated the regioisomeric purity of three reference materials supplied by different vendors, labeled as PEth 16:0/18:1. Using CID coupled with ozone-induced dissociation, the regioisomeric purity (% 16:0 at sn-1) was determined to be 76, 80 and 99%. The parallel investigation of the negative ion CID mass spectra of standards revealed differences in product ion ratios for both fatty acyl chain product ions and ketene neutral loss product ions. Furthermore, investigation of the product ion abundances in CID spectra of PEth within authentic blood samples appears to indicate a limited natural variation in isomer populations between samples, with the cannonical, PEth 16:0/18:1 (16:0 at sn-1) predominant in all cases. Different reference material isomer distributions led to variation in fully automated quantification of PEth in 56 authentic dried blood spot (DBS) samples when a single quantifier ion was used. Our results suggest caution in ensuring that the regioisomeric compositions of reference materials are well-matched with those of the authentic blood samples.

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.