Method Evaluation: Analysis of Phosphatidylethanol (PEth) in Human Whole Blood by LC-MS/MS
Abstract
The method established in this study provides accurate analysis of phosphatidylethanol (PEth) using a simple protein precipitation procedure and a fast 3.5 minute LC-MS/MS gradient run. Specific, sensitive analysis of PEth 16:0/18:1 in whole blood was obtained due to the unique selectivity and retention of the Raptor FluoroPhenyl column.
Introduction
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 (palmitic acid/oleic acid) (Figure 1) is the predominant molecule extracted from human erythrocytes and can be measured in whole blood as a specific biomarker of alcohol consumption with a detection window of up to 3-4 weeks. Previously, PEth was considered a biomarker for high, 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 use it as a marker of absolute abstinence. In this study, a fast chromatographic analysis was developed using a Raptor FluoroPhenyl column. Specific and sensitive measurement of PEth-16:0/18:1 in whole blood was achieved with a combination of a simple protein precipitation sample preparation and fast 3.5-minute LC cycle time.
Figure 1: Structure of PEth-16:0/18:1
Experimental
Calibration Standards and Quality Control Samples
PEth-free, pooled human whole blood (BioIVT) was fortified with PEth-16:0/18:1 (RedHot Diagnostics AB) to prepare calibration standards and QC samples. The linearity ranges were 0.025–4.0 µM (18–2810 ng/mL). Three QC levels were prepared at 0.075, 0.75, and 2.5 µM. The fortified standard and QC samples were subjected to the following sample preparation procedure.
Sample Preparation
Using a sample preparation procedure described by RedHot Diagnostics, the 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 was injected for analysis.
Instrument Conditions
LC-MS/MS analysis of PEth in human whole blood was performed on a Waters ACQUITY UPLC coupled to a Xevo TQ-S MS/MS system. Instrument conditions were as follows and analyte transitions are provided in Table I.
| Analytical column: | Raptor FluoroPhenyl 2.7 µm, 50 mm x 2.1 mm (cat.# 9319A52) |
| Mobile phase A: | Water, 5 mM ammonium acetate |
| Mobile phase B: | 9:1 Methanol:2-propanol |
| Gradient: | Time (min) %B |
| 0.00 70 | |
| 1.00 80 | |
| 2.00 80 | |
| 2.50 100 | |
| 2.51 70 | |
| 3.50 70 | |
| Flow rate: | 0.5 mL/min |
| Injection volume: | 2 µL |
| Column temp.: | 40 °C |
| Ion mode: | Negative ESI |
| Compound | Precursor Ion | Product Ion Quantifier | Product Ion Qualifier |
| PEth-16:0/18:1-d5 | 706.6 | 281.3 | - |
| PEth-16:0/18:1 | 701.5 | 255.3 | 281.3 |
Results and Discussion
Chromatographic Performance
A fast 3.5-minute chromatographic analysis (Figure 2) was achieved with injection of supernatant after a simple protein precipitation procedure. Specific and sensitive analysis of phosphatidylethanol 16:0/18:1 in whole blood was obtained due to the unique selectivity and retention mechanisms of the Raptor FluoroPhenyl column. No carryover was observed from the analysis of blank blood injected immediately after the highest concentration standard (Figure 3). Consistent chromatographic performance (retention, peak shape, and sensitivity) was observed across 500 continuous injections, demonstrating very good method robustness (Figure 4).
Figure 2: Fortified Human Whole Blood at 0.5 µM
Figure 3: Blank Human Whole Blood
Figure 4: Robust Analysis of Phosphatidylethanol on a Raptor FluoroPhenyl Column (Whole Blood at 0.5 µM)
Linearity
Using 1/x weighted linear regression, PEth-16:0/18:1 showed acceptable linearity with r2 values of 0.999 or greater, and %deviations from the nominal concentrations of <10% (Figure 5). The established limit of quantitation was 0.025 µM (~18 ng/mL) in blood.
Figure 5: Standard Curve
Accuracy and Precision
Precision and accuracy analyses were performed on three different days. The accuracy of the method was demonstrated by recovery values being within 5% of the nominal concentration for all QC levels. The %RSD was 0.117–1.33% and 2.30–5.08% for intraday and interday, respectively, indicating acceptable method precision (Table II).
Table II: Accuracy and Precision of QC Samples for PEth Analysis
| Analyte | QC-1 (0.0750 µM) | QC-2 (0.750 µM) | QC-3 (2.50 µM) | ||||||
| Avg. Conc. (µM) | Avg. Accuracy (%) | Precision (%RSD) | Avg. Conc. (µM) | Avg. Accuracy (%) | Precision (%RSD) | Avg. Conc. (µM) | Avg. Accuracy (%) | Precision (%RSD) | |
| PEth-16:0/18:1 | 0.0760 | 101 | 5.08 | 0.755 | 101 | 2.30 | 2.56 | 102 | 4.50 |
Conclusion
The method developed here for the analysis of phosphatidylethanol using a Raptor FluoroPhenyl column was specific and sensitive for PEth-16:0/18:1 in human whole blood. Accurate, reproducible results were achieved with a simple protein precipitation procedure and a fast 3.5 minute run time, making this method suitable for low-cost, high-throughput analysis to monitor alcohol consumption.
Acknowledgements
The authors would like to thank RedHot Diagnostics for providing reference materials for standard and QC preparations, as well as for their collaboration efforts with the sample preparation procedures.