Phosphatidylethanol (Peth) in Dried Blood Spots: Development, validation and comparison between LC-MS/MS and QTOF methods

Abstract

Validation of Phosphatidylethanol (Peth) in Dried Blood Spot (DBS) technique as a biomarker for routine diagnosis of alcohol abuse. According to recent studies, alcohol abuse in the population is increasing, forcing clinical and forensic toxicology laboratories to work towards effective and quick-turnout results techniques. In this context, detecting Peth, a relatively new biomarker, with the Dried Blood Spots, a micro sampling technique based on a capillary blood drop collection on a filter paper, may be the procedure of choice. Peth is a group of phospholipids serving as direct, non-oxidative alcohol biomarkers, only formed within the human body when ethanol is present. Peth concentrations provide information about a person's drinking behavior and offer a longer alcohol detection window up to 1 month. It is applicable as a complement to Ethyl glucuronide (EtG) for the better distinguishing of alcohol abusers from social drinkers. Blank whole blood drops were spiked with Peth 16:0–18:1 at six concentration levels (20, 50, 100, 200, 300, 500 ng/mL) in three working sessions along five days. 30 μl of blood was deposited on the DBS card and left for 3 h drying at room temperature. After liquid extraction with 1 mL of hexane, the supernatant was transferred into a fresh tube prior to evaporating the solvent under a nitrogen flow at room temperature. The dry residue was reconstituted in 30 μl of acetonitrile and then injected. The detection of Peth was achieved with both UHPLC-QTOF-HRMS and UHPLC-MS/MS. Statistical evaluation of several validation parameters was executed, and the values obtained from the two different acquisition methods were found to be similar. Intra- and inter-day precision and accuracy were found to be lower than ±20% in both methods. The LOD and LOQ calculated with the Hubaux-Vos approach were 19.8 and 39.7 ng/ml for, respectively, the QTOF and the MS/MS acquisition. Recovery, matrix effect and stability parameters from the analysis of Peth and its respective internal standards Peth-D5 were evaluated. A moderate ion suppression was observed, with final values between −2.7 and −14% (tested at the 20 ng/ml level of concentration) and the method showed an extraction efficiency higher than 30% (tested at the 500 ng/mL level of concentration). The two methods were also tested with real samples within an ongoing project and their performance was compared. The extraction method and the DBS technique proved effective for the detection of Peth, with excellent results ensuring rapid assessment of the biomarker levels in whole blood. The precision calculated in the lower concentration range showed a slight upward deviation at 20 and 50 ng/ml, which is negligible when an individual is assessed for chronic alcohol abuse (in this case, tentative cut-offs are set in the range of 200–270 ng/ml). Due to the higher sensitivity obtained, the QTOF mass spectrometer proved adequate to be used for abstinence assessment in delicate clinical or forensic cases for example with newborns, organ transplant and driving relicensing. Peth in DBS combined with high resolution instrumental analysis results show great potential for routine and research analysis. Thanks to this study, a larger detection window is opened thus providing the forensic toxicology laboratories an accurate, easy, sustainable, and fast technique for the challenge of alcohol abuse diagnosis.