Detection of ethyl glucuronide/ethyl suphate in urine of sexual assault cases

Jeffery Hackett and Albert A Elian outline this process which uses an anion exchange solid phase extraction technique and LC-MS/MS analysis.

Of all the analyses carried out by forensic toxicology laboratories, the determination of ethanol is probably the most common. Most forensic toxicology laboratories measure parent and metabolite concentrations of a variety of drugs in biological samples. However, the measurement of the metabolites of ethanol is rarely performed.

In this study, samples of urine taken from completed and signed out cases (ethanol related sexual assault) were analysed for the concentrations of ethyl glucuronide (EtG) and ethyl suphphate (EtS) using solid phase extraction (SPE) and LC-MS/MS. This study was initiated to develop a simple, robust method that would assist forensic analysts in the quantification of EtG/EtS in urine samples. The method is based on the employment of anion solid phase extraction to produce clean extracts for analysis by liquid chromatography-tandem mass spectrometry. The method was then applied to genuine cases to determine the concentrations of both EtG/EtS where the ethanol in the urine sample was negative, but had been suspected.

Chemicals and equipment

EtG, EtS (1mg/mL in methanol), EtG-d5, EtS-d5 (0.1mg/mL in methanol), were obtained from Lipomed (Cambridge MA). Strong anion exchange/hydrophobic solid phase extraction (SPE) columns - CUQAX156 were obtained from UCT Inc (Bristol PA). Methanol, acetic acid (glacial), formic acid, and acetonitrile (LC/MS grade) were obtained from Fisher Scientific (Pittsburgh, PA).  De-ionised (DI) water was generated in house.

Liquid chromatography was performed in isocratic mode on Diamond Hydride LC column (100mm x 2.1mm (4μm)) obtained from Microsolv (Eatontown, NJ) using a mobile phase consisting of DI water w/0.1 per cent formic acid/acetonitrile w/0.1 per cent formic acid; (50: 50) at a flow rate of  0.35mL/minute.The instrumentation used was a Shimadzu Prominence HPLC system. The HPLC column was maintained at 40°C throughout the analyses. Tandem mass spectrometry was performed on an AB Sciex 3200 Qtrap (Foster City, CA) in negative multiple reaction mode (MRM). The mass spectrometric conditions are shown in Table 1.

Method

SPE of urine samples (calibrators, controls, and test) was performed on  CUQAX156 anion exchange columns pre-conditioned with methanol (3mL) followed by DI water (3mL) prior to sample loading. 0.5mL urine samples (containing deuterated analogues of EtG/EtS) were diluted with 4mL of DI water and mixed. After loading the samples onto the SPE columns, the cartridges were washed with DI water and methanol (3mL of each, respectively). Each SPE column was dried and eluted with 2 x 3mL of methanol containing 6 per cent acetic acid. The samples were then evaporated to dryness at 40°C under nitrogen. The residues were dissolved in 100μL of the mobile phase. 10μL injections were used for each analysis.

Results

The limits of detection and quantification for this method were determined to be 10ng/mL and 50ng/mL, respectively for EtG/EtS. The method was found to be linear from 50ng/mg to 1000 ng/mL (r2>0.999). Recoveries were found to be greater than 95 per cent for all the compounds. Interday and Intraday analysis of the EtG/EtS were found to <8 per cent and <10 per cent, respectively. Matrix effects were determined to be < 6 per cent. Concentrations of EtG and EtS from the urine samples are shown in Table 2. (Note: Where EtG/EtS concentrations exceeded the upper limit of the curve, the samples were diluted and re-extracted according to the method) (Ethanol concentrations: negative).

Conclusion

As the forensic community becomes more aware of EtG/EtS as measureable metabolites, the use of this procedure for the analysis of EtG/EtS will be of great use to analysts in the field of forensic urine analysis as it demonstrates the use of SPE extraction followed by LC-MS/MS analysis to provide data on EtG/EtS in sexual assault cases. This new method provides data on these analytes where the ethanol concentrations in the urine samples are low (ie, below the laboratory cut off threshold) or negative.

For more information, www.scientistlive.com/elab

Jeffery Hackett is Senior Forensic Toxicologist, UCT Inc, Bristol, PA, USA. www.unitedchem.com. Albert A Elian is Technical Leader, Massachusetts State Police Crime Laboratory, Sudbury, MA, USA.

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