Thermo Fisher Scientific has produced an application note describing a robust and sensitive LC-MS/MS workflow for the forensic toxicological analysis of ethyl glucuronide (EtG) and ehtyl sulfate (EtS) in urine using liquid chromatographic separation coupled to a triple quadrupole mass spectrometer.
Introduction
EtG and EtS are well-known biomarkers for monitoring ethanol consumption. Although they are minor metabolites of ethanol, their longer half-lives make them useful for the detection of past alcohol use in forensic research.
Experimental
Equal volumes (50 μL) of urine and internal standard (5000 ng/mL of EtG-d5 and 500 ng/mL of EtS-d5) were mixed and then diluted with 900 μL of water for a 20-fold dilution. A 5 μL sample was injected onto the LC-MS/MS system. Chromatographic separation was performed using a Thermo Scientific™ Vanquish™ Flex HPLC system and MS analysis was carried out on a Thermo Scientific™ TSQ Quantis™ triple quadrupole mass spectrometer equipped with a heated electrospray ionization probe. Data was acquired and processed using Thermo Scientific™ TraceFinder™ software.
Results and Discussion
Limits of quantitation (LOQ) for EtG and EtS were defined as the lowest concentration at which the back-calculated calibrator concentration on the linear calibration curve was within 20% of theoretical, the ion ratio was within 20% of target, and replicate injections have a %RSD of less than 20%. For EtG, the LOQ was 100 ng/mL in authentic urine; for EtS it was 10 ng/mL.
This method demonstrated excellent precision for replicate injections of the QCs of concentrations spanning the calibration range. Precisions for EtG were less than 9.8% and for EtS were less than 4.0%. Additionally, no significant matrix effects were observed in the 17 different lots of authentic urine tested.
The TSQ Quantis triple quadrupole mass spectrometer is shown to provide superb sensitivity and robustness for the analysis of EtG and EtS in urine.
