Eliminating Carryover in the Analysis of Alcohol Biomarkers in Whole Blood on Raptor C8 by LC-MS/MS

Abstract

The method herein provides a robust and accurate analysis of the two most predominant phosphatidylethanol (PEth) homologues in whole blood using a simple protein precipitation. PEth compounds are prone to carryover, and steps were taken when developing the chromatographic gradient to eliminate it using Restek's C8 column. The analytical method achieves baseline separation between the two compounds, detection limits down to 10 ng/mL, and no carryover. 

Introduction

Phosphatidylethanol (PEth) is a group of phospholipids exclusively formed in cell membranes when in the presence of ethanol. As such, these compounds are adequate biomarkers for alcohol use, having a much longer half-life than ethanol in blood, and can be used to distinguish drinking patterns with a window of detection from 2-4 weeks. Among the multiple homologues of these compounds, PEth 16:0/18:1 (palmitic acid/oleic acid-POPEth), is the predominant compound (approximately 37% of total PEth) while the second most abundant PEth compound is 16:0/18:2 (palmitic acid/linoleic acid - PLPEth) which accounts for 25% of total PEth in blood [1]. In this application, a workflow was developed for the quantitative analysis of POPEth and PLPEth that uses a simple sample preparation protocol followed by LC-MS/MS analysis.

Experimental

Bovine blood was used as a surrogate matrix to ensure POPEth- and PLPEth-free blood. Bovine blood was fortified with POPEth and PLPEth to prepare calibration standards and QC samples. The linearity ranges were 10-1000 ng/mL, and three QC levels were prepared at 15, 100, and 500 ng/mL in whole blood. The fortified samples and QC samples were subjected to the following sample preparation procedure.

Sample Preparation

This sample preparation procedure follows the protocol outlined by RedHot Diagnostics. 50 µL of whole blood was mixed with 50 µL of internal standard (50 ng/mL) and 150 µL of 4:1 isopropyl alcohol:tetrahydrofuran. The mixture was vortexed for approximately 20 seconds and centrifuged for 10 minutes at 4300 rpm. The supernatant was transferred to an LC vial (cat.# 21143) with a vial insert (cat.# 21776), capped (cat.# 24498), and injected onto the LC-MS/MS instrument.

Instrument Conditions

LC-MS/MS analysis of PEth in whole blood was performed on a Shimadzu 8060 LC-MS/MS system. The method conditions are outlined in Table I and the analyte transitions are provided in Table II.

 

 

Results and Discussion

Chromatographic Performance

The specific and sensitive analysis of PEth 16:0/18:1 and 16:0/18:2 was achieved using a Raptor C8 column. No carryover was observed from the analysis of blank blood injected immediately after the highest concentration standard (Figure 1). Consistent chromatographic performance was observed across three different lots of columns (Figure II and Table 3) and across 500 consecutive injections (Figure 2 and Table III) demonstrating exceptional robustness of the method. Ion suppression was assessed by a 500 ppb infusion of POPEth and PLPEth post-column while simultaneously injecting whole bovine blood blank extract on the analytical column while running the analytical method. The chromatogram from these experiments is overlaid with an injection of a blood sample spiked at 1000 ng/mL to show both analytes do not elute within a matrix suppression or enhancement zone (Figure 4).

 

 

 

 

 

 

Linearity

A weight of 1/x linear regression was used for calibration of the compounds and showed acceptable linearity with R2 values of 0.996 or greater. The established limit of quantitation was 10 ng/mL in whole blood.

 

Accuracy and Precision

Accuracy and precision were assessed over three days. The accuracy of the method was determined by the evaluation at low, medium, and high spiked levels, and the values were all within 10% of the nominal concentration of all QC levels. The %RSD was 1.93–14.2% and 0.310–4.64% for intra- and inter-day, respectively, indicating acceptable method precision. A summary of accuracy and precision can be seen in Table V. 

 

Conclusion

The method developed within provides a straightforward protocol for the analysis of POPEth and PLPEth in whole blood using a Raptor C8 column. This workflow provides accurate and reproducible results with a simple protein precipitation procedure making it suitable for a low-cost, high-throughput analysis to monitor alcohol consumption.

Acknowledgements

The authors would like to thank RedHot Diagnostics for providing their collaboration efforts with the sample preparation procedure.

References

1. A. Helander, Y. Zheng, Molecular species of the alcohol biomarker phosphatidylethanol in human blood measured by LC-MS, Clin Chem. 55 ( 7) (2009) 1395-405. https://doi.org/10.1373/clinchem.2008.120923

This method has been developed for research use only; it is not suitable for use in diagnostic procedures without further evaluation.