TY - JOUR
T1 - Development of an LC-MS/MS assay with automated sample preparation for phosphatidylethanol (PEth)– Not your typical clinical marker
AU - Lahr, Richard G.
AU - Sharma, Pragya
AU - Maus, Anthony
AU - Langman, Loralie J.
AU - Jannetto, Paul J.
N1 - Publisher Copyright:
© 2023 Elsevier B.V.
PY - 2023/9/1
Y1 - 2023/9/1
N2 - Phosphatidylethanol (PEth) is a group of phospholipids formed exclusively in the presence of ethanol on the erythrocyte membrane, making it a direct biomarker for long-term ethanol consumption for which a clinical reference interval has been established. Here, we describe an assay for quantitation for two most abundant PEth homologues, PEth 16:0/18:1 and PEth 16:0/18:2, from human whole blood, and present challenges overcome throughout the development process. Since PEth is localized within erythrocyte membranes, a reliable sample preparation technique is an important aspect of PEth analysis. Therefore, various erythrocyte lysing agents for recovery of exogenously spiked standards and controls were evaluated to identify one that performed comparably to the recovery of endogenous analytes found in authentic samples. A supported liquid extraction (SLE) technique was employed for sample cleanup and enrichment which together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis enabled automated sample preparation, appropriate chromatographic resolution, and minimal system carryover. This resulted in a laboratory developed test with an analytical measurement range (AMR) of 10–1000 ng/mL (slope = 0.9902–1.0138, R2 = 0.9958–0.9972), that was precise (intra-day precision: 3.4–4.1%; inter-day precision: 4.4–8.2% over the AMR), accurate when compared with an available external laboratory test (slope = 0.9943–1.0206, R2 = 0.9635–0.9678, no lower decision point interpretation changes), with effective analyte recovery (77.2–83.5%), and established stability characteristics, while chromatographically separating the analytes to ensure no additive effects due to the isotopic distribution of the opposing analyte.
AB - Phosphatidylethanol (PEth) is a group of phospholipids formed exclusively in the presence of ethanol on the erythrocyte membrane, making it a direct biomarker for long-term ethanol consumption for which a clinical reference interval has been established. Here, we describe an assay for quantitation for two most abundant PEth homologues, PEth 16:0/18:1 and PEth 16:0/18:2, from human whole blood, and present challenges overcome throughout the development process. Since PEth is localized within erythrocyte membranes, a reliable sample preparation technique is an important aspect of PEth analysis. Therefore, various erythrocyte lysing agents for recovery of exogenously spiked standards and controls were evaluated to identify one that performed comparably to the recovery of endogenous analytes found in authentic samples. A supported liquid extraction (SLE) technique was employed for sample cleanup and enrichment which together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis enabled automated sample preparation, appropriate chromatographic resolution, and minimal system carryover. This resulted in a laboratory developed test with an analytical measurement range (AMR) of 10–1000 ng/mL (slope = 0.9902–1.0138, R2 = 0.9958–0.9972), that was precise (intra-day precision: 3.4–4.1%; inter-day precision: 4.4–8.2% over the AMR), accurate when compared with an available external laboratory test (slope = 0.9943–1.0206, R2 = 0.9635–0.9678, no lower decision point interpretation changes), with effective analyte recovery (77.2–83.5%), and established stability characteristics, while chromatographically separating the analytes to ensure no additive effects due to the isotopic distribution of the opposing analyte.
KW - Automated extraction
KW - Biomarker
KW - Erythrocytes
KW - Ethanol
KW - Phosphatidylethanol
KW - Recovery
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U2 - 10.1016/j.jchromb.2023.123886
DO - 10.1016/j.jchromb.2023.123886
M3 - Article
C2 - 37714050
AN - SCOPUS:85171345464
SN - 1570-0232
VL - 1229
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
M1 - 123886
ER -