An evaluation of washing and extraction techniques in the analysis of ethyl glucuronide and fatty acid ethyl esters from hair samples

• An extensive comparison was performed on the decontamination and extraction of FAEEs and EtG.
• Decontamination with DCM and methanol removed relatively much FAEEs (10% of total).
• Decontamination with DCM and methanol removed an average amount of EtG (50% of total).
• FAEEs extraction was most efficient with heptane/DMSO and water was best for EtG.
• Extraction of EtG and FAEEs with methanol for 30 min was a good compromise for both.

Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) are alcohol metabolites measured in hair and are after a decade of research thought to be the best markers in hair to indicate alcoholism and abstinence Forensic Sci. Int. 218 (2012) 2. A great body of work concerning EtG and FAEEs detection in hair has been performed. However, no recent extensive comparison has been made concerning washing and extraction procedures. This work shows that the washing procedure of dichloromethane followed by a methanol rinse of the hair sample removes more than 16% of the FAEEs and 50% of the total EtG that is present in and on the hair. A review of ten washing protocols (where the removal is categorised: high, medium or low) showed that a relatively high percentage of FAEEs was removed and “medium” amount of EtG compared to the other washing protocols. This work shows promising results for the extraction of the FAEEs and the combined extraction of FAEEs and EtG by using 30 min of sonication with methanol. More FAEEs were recovered from hair with methanol than with any other extraction solvent including the commonly used dimethyl sulfoxide/heptane mixture. When the sonication time was increased a higher percentage of transesterification of the FAEEs was observed, the extraction was “dirtier” as solids and a colour change was observed whereas the extraction efficiency did not increase. Therefore, washing the hair sample with dichloromethane and methanol followed by an addition of 1 ml of methanol and sonication for 30 min to extract the FAEEs and EtG from hair is recommended for FAEEs as well as for the combined analysis of EtG and FAEEs. A linear calibration curve (r2 > 0.99) was obtained for all analytes.