The older methods of testing alcohol involving state markers like carbohydrate deficient transferring, gamma-glutamyl-transpeptidase and mean corpuscular volume are only helpful in detecting long term or chronic alcohol consumption in a person and thus become ineffective with small doses or short term alcohol tests.
Other more recent methods which incorporate urine test to detect alcohol largely depend and tend getting affected with various non-relevant factors like age, gender etc making the procedure rather crippled. Moreover, since alcohol gets metabolized within hours of time, detection becomes obscure, and on the contrary sometimes due to in vitro fermentation, alcohol may remain in the urine for much longer periods of time providing false and disguised results of a recent consumption.
However in contrast, EtG (ethyl glucuronide) which is an immediate metabolite of ethanol is associated only with genuine alcohol consumptions, remains in the urine even after ethanol is completely metabolized and can be traced well within five days, thus it becomes an accurate and a reliable marker for detecting recent alcohol consumption by an accused.
Although EtG is known since as early as 1950, convincing findings were actually introduced in the year 2001 through the studies made by Dr. Gregory Skipper from USA and Dr. Fredrich Wurst from Switzerland. The study confirmed EtG to be a more reliable and a sensitive marker than urine alcohol. Dr Skipper acknowledges urine EtG to be very effective in monitoring professionals having little or no abstinence.
The EtG Detection Procedure
EtG is produced through the fusion of activated glucuronic acid and ethanol and with the help of membrane-bound mitochondrial UDP glucuronyl transferase, representing 0.02% and 0.04% in humans.
The molecular formula and weight of EtG are C8H14O7 and 222 g/mol respectively.
It is water soluble, non-volatile and a stable marker having a melting point of 150o Celsius.
Urine test to detect alcohol by detecting EtG in a urine sample is normally performed with the following steps:
Standard HHS rules are taken into consideration during the specimen collection under the strict supervision of medical personnel avoiding any possible adulteration or manipulation.
The LOD (Limit of Determination) for urinary alcohol being 0.02%, an aliquot of 3 ml of urine specimen is frozen and delivered for EtG detection.
0.2 ml of urine sample is mixed with 0.5 ml of methanol and 10 μl aqueous solution of internal standard (D5-EtG, 10 μg/ml, Medichem, Germany.)
Through centrifugation (14000 RPM, @ 4o Celsius for 10 min.) the supernatant is evaporated until dry with the help of vacuum concentrator.
It’s redissolved and diluted relative to creatinine concentration using 0.1 % HCOOH (v/v);
Precisely, the above step is done by a factor of 2 for creatinine concentration less than 50 mg/dl, with a factor of 4 for creatinine concentration exceeding 50 mg up to 100 mg and by a factor of 6 and 8 for creatinine concentrations 100-200 mg /dl and above 200 mg respectively.
An aliquot of 10 μl of the above processed specimen is injected into an LC-MS (Liquid chromatography-mass spectrometry) system.
The LC-MS system basically comprises of a triple quadruple mass spectrometer accompanied with a turbo-ion-spray interface which is further coupled to a LC system. The introduced specimen is analyzed through multi-reaction inspections, using 221/75 amu for EtG and 226/75 amu for D5/EtG with intermediate imitated transitions to avoid possible cross-talks.