Deceitful dodgy drinkers detected by LC-MS/MS

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  • Published: Jan 10, 2019
  • Author: Ryan De Vooght-Johnson
  • Copyright: Image: milias1987/Shutterstock
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Alcoholism is a deadly addiction

Credit: milias1987/Shutterstock.

Alcoholism is a serious problem in many countries. In the UK, there were over 7,000 deaths specifically due to alcohol consumption in 2016. This is equivalent to about 12 deaths per 100,000 of the population. In addition, alcohol is a contributory factor in many other deaths, such as those caused by various cancers. Treatment of alcoholism may involve the use of various therapies and/or drugs. Proof of abstinence is often required before returning to driving or to potentially hazardous work. However, alcoholics are not always honest about their drinking habits, so objective measures to determine consumption are required. Simply measuring blood alcohol levels may not give an accurate overall picture, since alcohol is metabolised relatively quickly.

One method of determining abstinence from alcohol is by measuring levels of the ethanol metabolite ethyl glucuronide in hair samples. The Antwerp team investigated the detection of an alternative alcohol metabolite, ethyl sulphate, in hair, comparing the results to those found for ethyl glucuronide.

Ethyl sulphate determined in hair samples from alcoholics

Hair samples were washed with water and then with acetone. The samples were dried and then converted to powder using a ball mill. Weighed amounts were placed in a test tube, internal standard (D5-ethyl sulphate) was added, and the hair was ultrasonically extracted with water. The solids were removed by centrifugation, and the ethyl sulphate was absorbed onto a solid-phase extraction cartridge (Oasis WAX cartridge). Oasis WAX cartridges (weak anion exchange) gave far better recovery than Oasis MAX cartridges (strong anion exchange). The cartridge was washed with methanol, and the ethyl sulphate was extracted from the cartridge with a mixture of aqueous ammonia and methanol. The solvent was evaporated, and the residue was taken up into a mixture of 5% methanol and 95% water prior to HPLC.

HPLC employed an Agilent Infinity 1200 instrument fitted with a Waters Atlantis T3 column. The temperature was kept at 40 °C and the flow rate at 0.3 mL/min. Solvent A was water, while solvent B was methanol (both contained 0.1% acetic acid). The solvent was kept at 5% B between 0 and 0.5 min, raised to 20% B between 0.5 and 5.5 min, raised to 98% B between 5.5 and 5.6 min, kept at 98% B between 5.6 and 9.0 min, decreased to 5% B between 9.0 and 9.1 min, and finally kept at 5% B for re-equilibration up to the end of the run at 14.0 min.

Mass spectrometry employed an Agilent 6460 triple quadrupole instrument, using negative electrospray ionisation (ESI). Multiple reaction monitoring (MRM) mode was used for quantitative analysis, with appropriate quantifying and qualifying transitions being found for the ethyl sulphate and the deuterated internal standard.

The method gave a limit of quantification (LOQ) of 5 pg/mg, while also giving acceptable linearity, accuracy and precision. The overall recovery of weighed internal standard was 63%. However, samples of hair from strict teetotallers showed small background levels of ethyl sulphate, possibly from body care products or cosmetics. Further work is needed to determine the source of the ethyl sulphate in these ‘blank’ samples. Samples from alcoholic patients showed much more ethyl sulphate and a significant positive correlation between the levels of ethyl sulphate and ethyl glucuronide, with the latter being determined by a different method. Overall, the levels of ethyl sulphate were higher than those of ethyl glucuronide, meaning that in theory less sensitive instrumentation could be used.

New method suitable for determining if alcoholics have been drinking

The new ethyl sulphate method is suitable for determining whether alcoholics have been drinking, being complementary to the existing ethyl glucuronide method. Allowance has to be made for the weak background signal seen. Ideally, a method should be found that is capable of analysing both ethyl sulphate and ethyl glucuronide in hair, so further work is needed in this field.

Related Links

Drug Testing and Analysis 2018, 10, 1566-1572. Cappelle et al.: "Assessment of ethyl sulphate in hair as a marker for alcohol consumption using liquid chromatography–tandem mass spectrometry."

Alcohol and Alcoholism 2009, 44, 55-61. Helander et al.: "Detection times for urinary ethyl glucuronide and ethyl sulfate in heavy drinkers during alcohol detoxification."

Wikipedia: Triple quadrupole mass spectrometer

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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