Nonaqueous capillary electrophoresis for analysis of the ethanol consumption biomarker phosphatidylethanol

Abstract

Nonaqueous CE (NACE) methodology was developed for the separation and determination of phosphatidylethanol (Peth), a new biomarker of ethanol intake. Peth is an abnormal phospholipid formed in cell membranes only in the presence of ethanol, via the transphosphatidylation reaction of phospholipase D. The NACE separation medium consisted of 80 mM ammonium acetate in 50% ACN, 33% 2-propanol, 12% hexane and 5% methanol. A stacking effect was obtained by reducing the concentration of ammonium acetate in the separation medium for all injected samples. The LOD was estimated to 1 microM (5.6 fmol) of Peth with conventional UV detection, equalling 0.4 micromol/L blood. Peth was successfully determined in extracts of human blood samples. Separation of Peth from other blood lipids in the lipid extract sample was performed in 5 min. The method facilitates smaller sample volumes and performs about ten times faster compared to earlier chromatographical methods.

Quantification of phosphatidylethanol 16:0/18:1, 18:1/18:1, and 16:0/16:0 in venous blood and venous and capillary dried blood spots from patients in alcohol withdrawal and control volunteers

“…The most used extraction technique is a liquid-liquid extraction (LLE) with hexane [5][6][7][11][12][13][14][15][16][17] (or heptane [8]) after stepwise addition of blood to isopropanol and the internal standard (IS) solution. Some methods added water [6], borate buffer pH 9 [5] or sodium acetate buffer pH 5 [15] to dilute the blood.…”

“…Quantification limits (LLOQ) obtained with these methods ranged between 100-500 ng/mL [4,11], analysing 250 to 300 µL of whole blood. PEths have also been analysed with non-aqueous capillary electrophoresis (CE) coupled to UV [13] detection. Both HPLC-ELSD and CE-UV [13] methods measure the total amount of PEths.…”

“…PEths have also been analysed with non-aqueous capillary electrophoresis (CE) coupled to UV [13] detection. Both HPLC-ELSD and CE-UV [13] methods measure the total amount of PEths. However, LC methods coupled to MS/MS detection allow to obtain much lower LLOQs (between 0.7 and 83 ng/mL, based on the analysis of between 100 and 300 µL of whole blood) and are able to identify and quantify individual molecular species [6-8, 15, 18-20].…”

Molecular Species of the Alcohol Biomarker Phosphatidylethanol in Human Blood Measured by LC-MS

“…An alternative capillary electrophoresis (CE) method with UV detection was recently introduced (21 ), and development of an immunoassay based on a monoclonal PEth antibody has been initiated (22 ).…”

“…Current analytical methods for PEth based on HPLC (19,20 ) and CE (21 ), and any immunoassay targeting the head group (22 ), measure the sum of all PEth species. Measurement of individual species is feasible by LC-MS (18,28 ), depending on the variable length and number of unsaturated bonds of the fatty acid chains.…”