Journal of Forensic Sciences | 2019
Letter to the Editor—Hair Ethanol Glucuronide (EtG) and Washout Effects: Can and Should Pre‐Analytical Washout Effects be Assessed During the Method Validation Phase?
Abstract
Sir, Ethanol glucuronide (EtG) is a non-oxidative metabolite of ethanol that can accumulate in hair. It has been used for a variety of both clinical and non-clinical applications as a biomarker of long-term alcohol consumption (~3 months; 1–4). In this letter to the editor, we would like to raise a conundrum concerning the analysis of this alcohol biomarker in hair and the possibility of assessing washout effects during the pre-analytical stage. This came to our attention after recent publications highlighted this effect (5). To briefly summarize, Luginb€uhl and et al. (5) found that washout effects induced by water can have a significant effect on the quantification of EtG in hair. This was found for both chlorinated (swimming pool water with chlorine content: 0.30–0.40 mg/L, pH 7.15–7.35) and deionized water, with losses of approximately ~50%. To investigate washout effects, the authors conducted two experiments. In their first experiment, hair samples initially positive for EtG were incubated/mixed with either chlorinated or deionized water for a period of 0 to up to 10 h. After 2 h, reductions of 20% were reported and this decreased further to nearly 50% by 10 h. In their second experiment, 20 hair samples were compared with and without incubation in chlorinated water for 10 h. Similar findings as their first experiment were observed (around losses of close to half). The authors concluded that the washout effects seen were directly induced by water and subsequently can affect the quantitation of EtG in hair. The dilemma we would like to raise to readers is whether washout effects can also occur during the sample preparation stage and whether or not this should be assessed as part of method validation. The method employed for the experiments described above was stated to be in accordance to the German Society of Toxicological and Forensic Chemistry (GTFCh) for method validation (6). The first step of this protocol was to remove contaminants from the hair samples and this involved hair washes in water followed by acetone and dichloromethane. After hair pulverization following the washing procedure, EtG was then extracted by water using a combination of mixing, sonication, and solid phase extraction. Specifically concerning the water wash, the same volume of water was used as in the incubation experiments (4 mL) but for a significantly lower amount of time (4 min vs. 10 h max). In this instance, it is likely that negligible losses would occur but as this procedure was used to remove “contaminants”, we cannot discern if there were in fact reductions in EtG content arising from this step. Other validated methods also report the use of a water washing step (7–10) whilst others have used only organic solutions (namely dichloromethane and methanol; 9,11–14). In some of these methods, different volumes were used and the length of time for the washing process was either longer (up to hours), shorter, or not specified. In the study conducted by Bossers et al. (9) specifically investigating the effect of washout effects during the sample preparation stage, they observed that even organic solutions can induce washout with effects of up to 50% in losses. The main aim of the washing process is to decontaminate the hair sample (15,16). This is based on the underlying notion of how drugs and their metabolites in general become incorporated into hair (15,17,18) and the need to remove exogenous sources of potential interference. Due to this, two common approaches include the preparation of standards and controls by spiking blank powdered hair, and the addition of the internal standard (IS) after the washing and pulverization step. Hence, recovery experiments and potential compensatory IS losses alongside EtG during the washing process cannot be gauged (as the spiking solutions and IS cannot be incorporated in the same manner as endogenous hair EtG). In the article by Luginb€uhl et al. (5), their incubation experiments can essentially be viewed as a significantly extensive pre-wash of hair samples in water. As reductions in concentrations of EtG were observed with longer incubation times, an interesting question arises over the washing steps used and their ability to extract EtG during this time (whilst the hair samples are still intact). Another common theme in the methods listed previously for hair EtG analysis is the use of a water extraction process following washing and hair pulverization, most likely attributing to the polar nature of EtG and its ability to get into solution with water (7). Although an organic, methanol has the ability to dissolve polar compounds and is one of the common solvents used for hair washing described in these methods. Consequently, its ability to extract EtG during this process can occur as well (as supported by Bossers et al., [9]) and this may be the medium as to how washout effects have occurred. Whilst not re-simulating actual real-life events, experiments during method validation investigating hair EtG washing steps and the solvents used (following in line with Bossers et al., (9) and Luginb€uhl et al.’s (5) research into washout effects) may also be considered to determine the extent it may have on analytical quantitation. In addition, comparing multiple short-term exposures as well as extended exposures may also offer different insights into how washout effects arise as we can only speculate on the former. The next question is whether or not this is actually possible. As the washing steps are employed to remove exogenous interferences, an experiment comparing an authentic hair sample washed and not washed can have differing interpretations (if differing results are found) with the possibility of contamination alongside washout a potential cause. To overcome this, to some extent the accuracy of the analytical method can be compared to other laboratories with enrollment in proficiency testing offered by the GTFCh (ARVECON GmbH, Germany). To ensure that the washout procedure does not significantly affect the analytical method, comparisons should be made against authentic hair samples, rather than spiked samples. It should be noted however that as all participating laboratories will most likely have their own hair decontamination procedure, inadvertently varying degrees of washout effects will also be experienced. This means that results from proficiency testing schemes on authentic hair samples will have no “assigned value” unlike for instance a spiked sample and therefore it can be argued that the mean and/or median result of all participants may not necessarily be the most accurate one. To our knowledge, there is no harmonized reference method for the analysis of EtG in hair. Future attempts to do so,