Confounders of Serum Phosphatidylethanol: Role of Red Blood Cell Turnover and Cirrhosis.

Purpose: Ethyl glucuronide (EtG), ethyl sulfate (EtS) and phosphatidylethanol (PEth) are considered specific direct biomarkers for detecting alcohol consumption. However, PEth, which is produced in red blood cells (RBC), varies considerably between patients for unknown reasons. We here studied various confounders of PEth elimination including fibrosis after alcohol withdrawal. Patients and Methods: EtG, EtS and PEth together with routine laboratory and clinical parameters were studied in 100 Caucasian heavy drinkers prior and after alcohol detoxification. In addition, fibrosis stage and degree of steatosis were assessed by transient elastography (Fibroscan, Echosens, Paris). Results: All three biomarkers were highly correlated (0.61-0.72) with initial serum alcohol levels, but only PEth correlated with daily alcohol consumption. After alcohol withdrawal, PEth significantly decreased within 6.1 days from 1708 to 810 ng/mL (half-life varied from 1.6 to 15.2 days). Both levels of serum alcohol but also EtG and EtS were higher in patients with liver cirrhosis as compared to patients without fibrosis despite comparable alcohol consumption suggesting a decreased alcohol elimination in patients with cirrhosis. PEth was also elevated in cirrhosis but not significantly. In contrast, PEth elimination rate was significantly higher in patients with enhanced RBC turnover and signs of alcohol-mediated hemolytic anemia with elevated ferritin, LDH and increased mean corpuscular volume (MCV). Conclusion: We here demonstrate that alcohol elimination is decreased in patients with liver cirrhosis. In patients with cirrhosis, PEth levels are both affected in opposite directions by enhanced red blood cell turnover and elevated alcohol levels. Our data have important implications for the use and interpretation of PEth in the clinical setting.

Phospholipid metabolites of GHB as potential biomarkers in whole blood: Synthesis, analytics, and in vitro formation of homolog 16:0/18:1.

Gamma-hydroxybutyric acid (GHB) is a common drug of abuse, and the detection of a consumption or administration is a longstanding research objective in clinical and forensic toxicology. However, until now, the short detection window of GHB could not be enlarged by the use of GHB metabolites. Therefore, new biomarkers for the detection of a GHB intake are needed. In analogy to phosphatidylethanols as long-time biomarkers of ethanol, phospholipids with GHB might represent a promising compound class. While the availability of reference compounds often represents a bottleneck in clinical and forensic toxicological research, two phospholipids-phosphatidyl-GHB (16:0/18:1) and its isomer phosphatidyl beta-hydroxybutyric acid (16:0/18:1)-were successfully synthesized by a new highly versatile synthetic route. Structural characterization data, together with 1 H-, 13 C-, and 31 P-NMR and high-resolution mass spectrometry (HRMS) spectra, are reported. Subsequently, a HPLC-MS/MS method was established for the determination of both compounds (limits of detection [LOD] ≤ 2 ng/ml), and the formation of these metabolites was investigated in two in vitro experiments. The formation of phosphatidyl-GHB (16:0/18:1) was observed in an incubation experiment by converting phosphatidylcholine (16:0/18:1) and GHB with phospholipase D and in whole blood samples spiked with 50 mM GHB, respectively. Therefore, phosphatidyl-GHB (16:0/18:1) might represent a valuable new metabolite of GHB with the potential for an extension of the detection window as GHB biomarker.

Simultaneous Determination of 5- and 6-APB in Blood, Other Body Fluids, Hair and Various Tissues by HPLC--MS-MS.

5-(2-Aminopropyl)benzofuran (5-APB) and 6-(2-aminopropyl)benzofuran (6-APB) are benzofuran analogues of amphetamine and belong to the category of new psychoactive substances. Despite already published fatal 5- and 6-APB intoxication after consumption of both substances in most cases, no sensitive method for the simultaneous detection and quantification of these new psychoactive compounds in human blood samples has yet been developed. Therefore, an easy and fast sample preparation and specific high-performance liquid chromatography and tandem mass spectrometry methods for the determination of both substances in blood were established and validated. In a fatal intoxication in 2017 at the Institute of Forensic and Traffic Medicine in Heidelberg, Germany, concentrations of 850 (5-APB) and 300 ng/mL (6-APB) were determined in peripheral blood. Besides, other body fluids (central blood, urine and bile), hair and various tissues were examined to verify the presence of both compounds and to gain first insights into their distribution. In this publication, we show a method for the simultaneous determination of 5- and 6-APB in human samples by a chromatographic method and to investigate their distribution in the human body.

Development and validation of an analytical method for the simultaneous determination of the alcohol biomarkers ethyl glucuronide, ethyl sulfate, N-acetyltaurine, and 16:0/18:1-phosphatidylethanol in human blood.

As alcohol is the most common addictive substance worldwide, it is inevitable to advance the established research. New and more substantial analytical methods can be applied to reply to complex questions in legal or forensic contexts. Therefore, an analytical method for the simultaneous determination of four different alcohol biomarkers-ethyl glucuronide, ethyl sulfate, N-acetyltaurine, and 16:0/18:1-phosphatidylethanol-in human blood was developed, validated, and verified. Despite the different chemical properties of the analytes, a specific determination via HPLC-MS/MS was achieved using a novel type of a Phenomenex Luna® Omega Sugar column. Furthermore, all criteria for a successful validation were fulfilled according to forensic guidelines. The method proved to be linear and demonstrates selectivity and sufficient sensitivity for every biomarker. LODs obtained with this method of 2.6 ng/ml (EtG), 4.7 ng/ml (EtS), 12.5 ng/ml (NAcT), and 6.9 ng/ml (PEth) were in an acceptable range for routine applications, and the stability of all analytes over a range of 12 h is given. The verification of the new developed method was performed with authentic samples. Thus, whole blood and postmortem samples were analyzed to obtain information about the drinking behavior, which can answer complex questions regarding alcohol consumption.