Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC‐ESI‐MS/MS

Abstract

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)--a biomarker for ethanol uptake--has been developed by LC-ESI-MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents-PEth 18:1/18:1, PEth 16:0/16:0-were determined in post-mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g-dose of ethanol. Furthermore, PEth 18:1/16:0 or its isobaric isomer PEth-16:0/18:1 was detected. In comparison to previous high-performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC-MS/MS-method is more sensitive--with a limit of detection below 20 ng/ml--and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC-MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC-MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate.

Quantification of phosphatidylethanol 16:0/18:1, 18:1/18:1, and 16:0/16:0 in venous blood and venous and capillary dried blood spots from patients in alcohol withdrawal and control volunteers

“…Three blood spot volumes (20,35, 50 µL) were tested at low (0.32), intermediate (0.48) and high (0.67) hematocrit levels and at 2 concentrations; low and high. Six spots per concentration and per hematocrit level were prepared and centrally located 3 mm punches (1/DBS) were analysed.…”

“…Because the nonpolar part of PEths tends to interact very strongly with the nonpolar hydrocarbon phase of a reversed phase column, the use of a more polar phase (i.e. C8 [14,18,20], C4 [7,15] or phenyl [16]) instead of a C18 phase allows to decrease the retention of PEths [14]. The use of less polar solvents, such as tetrahydrofuran (index polarity = 4.0), isopropanol (index polarity = 3.9) or methanol (index polarity = 5.1) instead of acetonitrile (index polarity = 5.8) also improved the elution of PEths using a reversed phase column.…”

Application of phosphatidylethanol (PEth) in whole blood in comparison to ethyl glucuronide in hair (hEtG) in driving aptitude assessment (DAA)

“…A direct marker, which has also a potential in the field of abstinence monitoring, is phosphatidylethanol (PEth), as it is formed directly after alcohol intake [13,14].…”

Time Dependence of Elimination of Different PEth Homologues in Alcoholics in Comparison with Social Drinkers

“…Nevertheless, the advantages of mass spectrometry are-when compared to ELSD and UV-a higher specificity and sensitivity, short running times, and the possibility to detect single homologues instead of the sum of all homologues. LC-MS/MS and LC-TOF/MS methods have been developed with much lower limits of detection and running times of only 15 minutes (Gnann et al, 2009;Helander and Zheng, 2009;Tolonen et al, 2005). However, although sensitive and specific analyzing methods are known and homologues of PEth have been identified, the amount of data about the homologues of PEth relative to the background of drinking behaviors is still limited.…”