High-Throughput LC-MS/MS Analysis of Phosphatidylethanol in Whole Blood

• Simple protein precipitation sample prep streamlines workflow.
• Fast 3.5-minute gradient cycle time supports faster sample throughput.
• Easy-to-prepare mobile phases are quick to make consistently.

Phosphatidylethanol (PEth) is a group of phospholipids formed through an enzymatic reaction between ethanol and phosphatidylcholine on cell membranes. Among the multiple homologues of PEth, PEth-16:0/18:1 is the predominant molecule extracted from human erythrocytes. Analysis of phosphatidylethanol 16:0/18.1 is an important biomarker of alcohol consumption, and it can be measured in whole blood with a detection window of up to 3-4 weeks. Previously, PEth was considered a biomarker for high and sustained alcohol consumption, but with the application of highly sensitive LC-MS/MS techniques, it is now possible to use PEth concentration in blood to differentiate chronic drinking from social drinking or as a marker of absolute abstinence.

The LC-MS/MS method shown here was developed using a Raptor FluoroPhenyl column because of the column’s unique selectivity and retention mechanisms. Specific and sensitive analysis of phosphatidylethanol 16:0/18:1 in whole blood was achieved using a simple protein precipitation sample preparation procedure and a fast 3.5-minute LC cycle time. The high-quality chromatographic results that were achieved in a short analysis time make this approach ideal for labs needing a low-cost, high-throughput PEth method for monitoring alcohol consumption.

	Peaks	tR (min)	Conc.	Precursor Ion	Product Ion
1.	Phosphatidylethanol-d5 (PEth-d5) 16:0/18:1	1.58	0.4 μM	706.6	281.3
2.	Phosphatidylethanol (PEth) 16:0/18:1	1.58	0.5 μM	701.5	255.3

Column	Raptor FluoroPhenyl (cat.# 9319A52)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	40 °C
Standard/Sample
Conc.:	0.5 μM in blood
Inj. Vol.:	2 µL
Mobile Phase
A:	Water, 5 mM ammonium acetate
B:	9:1 Methanol:2-propanol
	Time (min) Flow (mL/min) %A %B 0.00 0.5 30 70 1.00 0.5 20 80 2.00 0.5 20 80 2.50 0.5 0 100 2.51 0.5 30 70 3.50 0.5 30 70

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC
Sample Preparation	Procedure provided by Redhot Diagnostics: The fortified blood sample (50 µL) was mixed with 50 µL of internal standard (0.4 µM PEth-d5 in 2-propanol) and 150 µL of 4:1 2-propanol:tetrahydrofuran. The mixture was vortexed for 20 seconds at 3000 rpm and centrifuged for 10 minutes at 4300 rpm. The supernatant (2 µL) was injected for analysis.

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