Abstract Background Phosphatidylethanol (PEth) analysis is crucial for liver organ donor screening due to its high sensitivity and specificity as a biomarker for recent alcohol consumption, with a 2 to 4-weeks’ detection window. The predominant homologues in blood, PEth 16:0/18:1 and PEth 16:0/18:2, are typically quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with a 10 ng/mL cutoff. Extracting PEth is a challenge due to its hydrophobicity and localization within erythrocyte membranes. Different sample preparation techniques involving isopropanol/acetonitrile extraction followed by solid-phase extraction (SPE) or supported liquid extraction (SLE) are commonly deployed to efficiently lyse red blood cells, precipitate proteins, and obtain high analyte recovery with clean extracts. However, each one of them encompass different degrees of procedural complexity and labor intensity. This study compared three different extraction procedures based on multi-well plate or 2 different filter columns for PEth quantification in whole blood by LC-MS/MS. Methods Samples and standards were processed using protein precipitation followed by sample clean-up using Phenomenex Phree filter, MonoSpin Phospholipid spin column S Type, or Waters Ostro phospholipid removal plate as shown in Figure 1, then analyzed by LC-MS/MS on a Waters Xevo TQ-XS. Precision studies included six between-days and three within-day assessments, with two runs occurring 2.5 hours apart. Linearity testing for PEth 18:1 and 18:2 was performed using six concentrations (10, 25, 50, 100, 250, and 1000 ng/mL). The medical decision point was verified through testing of low, mid, and high concentration samples. Specimen stability was analyzed using ten patient samples over 14 days, with =15% CV deviation from baseline as the acceptance criterion. Results Assessment of analytical performance utilizing linearity materials at both the lower and upper boundaries of the measurement range, coupled with recovery studies, demonstrated the superior efficacy of the SPE method. In addition, the plate-based extraction method outperformed the two filter-based techniques in terms of workflow optimization with at least 30-minute shorter processing time and user-friendliness. Specifically, it eliminated the labor-intensive tasks of individual tube handling and labeling, as well as the ergonomically challenging actions of repeated cap removal/replacement and tube centrifugation during wash and elution steps, resulting in an improved operational efficiency and reduced physical strain on technicians. Thus, the plate-based method was chosen as the optimal technique for subsequent experiments. Imprecision for both PEth 16:0/18:1 and 16:0/18:2 by SPE method was 10%. The analytical measurement range of both the homologues was 10-1000 ng/mL. The observed limit of detection and lower limit of quantitation was found to be 2.9 and 10.0 ng/mL for 16:0/18:1 and 2.2 and 10.0 ng/mL for 16:0/18:2, respectively. Negligible carryover (10%) was observed. Stability assessments showed analyte integrity for at least 18 days under refrigerated conditions (4°C), with results 10% of baseline. Conclusion This study demonstrates that analytical efficacy of SPE-based PEth analysis, marked by excellent precision, linearity, and stability. Its wide analytical range, negligible carryover, short processing time, and user-friendliness renders it highly suitable for adoption in routine clinical PEth quantification in diagnostic laboratories.
Yeo et al. (Wed,) studied this question.