B-161 Liquid chromatography/mass spectrometry analysis of ethyl glucuronide and ethyl sulfate using a perfluorophenyl stationary phase

Abstract

Abstract Background Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol that are excreted in urine. These two metabolites are often tested for to determine if an individual has recently consumed alcohol. EtG and EtS are very polar analytes, which make them difficult to chromatograph using reversed-phase chromatography. These analytes often elute early, which can lead to irreproducible results. When EtG and EtS are analyzed by LC/MS with a mobile phase that contains formic acid, the signal of these two analytes can be suppressed. This leads to increased LOQ and cutoffs. We examined the perfluorophenyl (PFP) stationary phase for its utility in analyzing EtG and EtS. This stationary phase has been shown to have greater retention of polar compounds than C18 reversed-phase columns. In addition, we compared the effect of formic acid to acetic acid on the separation. Methods Urine samples containing EtG and EtS were diluted 10:1, using 50mM acetic acid or 0.1% formic acid, into a 96-well microtiter filter plate containing a 0.2micron filter. Internal standards were added to the microtiter plate containing the urine samples. The samples were filtered through the filter plate and analyzed by LC/MS using the PFP column (50 x 2.7micron) with a 50mM acetic acid or 0.1% formic acid/acetonitrile mobile phase. The analytes were detected using negative mode ionization. The total analysis time was 4.5minutes. Results EtG and EtS showed improved retention using the PFP column. Retention times for EtG and EtS were 0.75 and 1.20 minutes respectively using the PFP column, compared to 0.4 and 0.3 minutes using the C18 column. The greater retention time improved the LOQ for both analytes.The LOQ for EtS and EtG were determined to be 125 and 250 ng/mL, respectively. Greater retention of EtG was observed by switching the modifier from formic acid to acetic acid. The retention time increased from 0.45 to 0.75 minutes. There was less signal suppression using acetic acid in place of formic acid. Improvement in peak shape was observed when the injection volume was reduced to 5microliters. The method was validated over a range of 250 to 10000 ng/mL for EtG and 125 to 5000 ng/mL for EtS. Acceptable linearity was obtained for both analytes. Accuracy was performed at three concentration levels. All three levels demonstrated acceptable accuracy with the difference between the measured and expected concentration being with in 20% of each other. Conclusions The PFP stationary can be used to test EtG and EtS. This stationary phase gave greater retention of EtS and EtG. The use of acetic acid improved the limit of detection by decreasing the ion suppression, which decreased the limit of detection. The assay was accurate and precise for both analytes, demonstrated in the validation.