The analytical method developed in this study has proved to be sensitive and accurate for the analysis of EtG and EtS. The lower LOD (0.02 µg/mL) and LOQ (0.05 µg/mL) achieved are particularly useful for quantifying the lower concentrations of EtG and EtS present in blood during pharmacokinetic investigation of EtG and EtS. Also, it is illegal in China to drive with a blood alcohol concentration (BAC) higher than 0.2 mg/mL, and Cmax of BAC (0.22–0.66 mg/mL) at 0.72 g/kg dose of alcohol used in the study is close to that, suggesting that the method might be suitable for monitoring most cases of drunk driving in China.
This study has focused on the concentration-time-curves of alcohol, EtG and EtS in blood of 26 volunteers following a single dose of 0.72 g alcohol/kg, and aimed to establish a method of estimating the time of last drinking. It was found that the time of post-alcohol use could be estimated using the relative concentrations of EtG and EtS. In the preciously studies, Zhao et al.10 once proposed predicting the time of last drinking using the serum concentration ratio of alcohol to EtG. While alcohol is usually metabolized quickly in living body, and the alcohol concentration after death is subject to change due to postmortem redistribution20 and postmortem production21. Therefore, the application of concentration ratios of alcohol to metabolites has great limitations, and as reported in the literature, our study also showed a poor correlation between the concentration ratio of alcohol to its metabolites and time of last drinking. While, despite great individual differences, a good correlation model was obtained from the mean concentration ratio of EtG to EtS in blood and the time of alcohol use, and most of the prediction errors were less than 10%. This would be useful in forensic investigation such as in the case of driving under the influence of alcohol.
Pharmacokinetic parameters of alcohol and EtG, EtS in blood were calculated based on the non-compartment model. For alcohol, the study found that it reached the Cmax of 441.65 ± 113.86 mg/L (0.44 ± 0.11 mg/mL) at 2.02 ± 0.54 h, and compared with the previous studies11, it showed a longer absorption phase. In addition, we found that alcohol in blood of the participants could be detected within 8 h (3–8 h), and the mean elimination half-life of alcohol was 1.24 ± 1.09 h (0.30–4.23 h), with individual differences. For EtG and EtS, in view of their longer detection window, we investigated the concentrations of analytes in blood up to 120 h after drinking, and it was the first time that pharmacokinetics of EtG and EtS in blood were evaluated in a larger number of Chinese population. Compared with EtS, EtG was discovered early and has been most studied10,22,23,24. It was reported that the detection windows of EtG in both serum and urine were longer than those of alcohol25,26,27, and we confirmed that EtG did metabolize slower than alcohol, and the elimination half-life of EtG in blood was 2.56 ± 0.89 h. Additionally, the detection time limit for urinary EtG was reported to depend weakly on the breath ethanol concentrations27, and our study showed no correlation between initial alcohol concentrations and detection limits of EtG, based on the CORREL function, suggesting that the lower alcohol consumption might not affect the detection limit of EtG. EtS was another non-oxidative metabolite of alcohol, and its concentration–time curve was found similar to that of EtG8,18. Georg Schmitt et al.18 once reported that the Cmax of EtS ranged from 0.09 µg/mL to 0.72 µg/mL at 0.52 ± 0.17 g/kg dosage of alcohol, and the Tmax was about 2.94 h, while in our study, the Cmax of EtS was 0.17 µg/mL (ranging from 0.08 µg/mL to 0.28 µg/mL) at 0.72 g/kg dosage, and the Tmax was 3.02 h indicating, interethnic differences. In addition, our study found that both the detection window and Cmax of EtS were lower than those of EtG. However, EtS has been reported to be stable and not susceptible to bacteria28,29suggesting that EtS could provide complementary data in the identification of alcohol ingestion.
Alcohol metabolism is influenced by many factors including the amount of alcohol consumption, age, gender, race, and genetic variability30, among which genetic difference between individuals has been considered a major contributor to the pharmacokinetic variation of alcohol. Gudrun31 and Lostia32 reported that the concentrations of alcohol, EtG, and EtS would increase as the dosage of alcohol intake increases, and differ in individuals with the same dosage of alcohol. A homogenous sample was obtained in our study, since the 26 Chinese Han people recruited were young and healthy, and all of them were free from somatic or psychiatric illness, alcohol drinking, and regular medication. Although effects of these parameters can not be judged out, the results obtained in this study do suggest the association of the diversified pharmacokinetics of alcohol in a Chinese population with genetic difference between individuals. To further elucidate the mechanism underlying the observed differences, we need to investigate the metabolic enzymes phenotyping of the 26 Chinese participants in future studies.
Race is another important element affecting the result of alcohol pharmacokinetics. Alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2) and CYP2E1 are three major ethanol-metabolizing enzymes. It was reported that the allele frequencies in Chinese populations (76.7%, 15.6%, and 28.9% respectively) were obsevably higher than those in European groups (0%, 0%, and 5.1% respectively)33and a strongly protective variant in ALDH2 is essentially only found in Asians34. Uridine diphosphate-glucuronosyltransferases (UGT) and sulfotransferases (SULT) are two enzymes responsible for EtG and EtS formation. It was reported that genes coding for these enzymes have considerable polymorphism35, while few studies reported the interethnic difference of the allele frequencies of UGT and SULT. Apart from the dosage, here results showed a possible influence of race on the formation of EtG and EtS. Further studies on the influence of race on the glucuronidation and sulfonation of alcohol should be considered in the next studies.
Factors affecting the metabolism of alcohol might affect the ratio of EtG/EtS, and then affect the outcome of time estimation. In this study, we estimated the drinking time of volunteers based on the individual value of EtG/EtS at each time point (data was not shown here), and it was found that there was still a great individual difference, especially for the application of extreme values like min and max EtG/EtS. Generally, the calculation of ratios can counteract the influence of dosage, but this is not the case when the dosage is too large, which is due to the report that sulphation is more saturable than glucuronidation at higher concentrations of substrate. And our study also showed an influence of dosage on the time estimation, especially when the metabolic ability was also relatively poor or strong. As shown in Table 7, the min values of observed time were obtained from a volunteer with the lowest dosage of alcohol due to his lighter weight, at the same time, the volunteer had not only a poor ability of alcohol metabolism (tmax= 3 h), but a lower level of EtG (Cmax= 0.18 μg/mL) and higher level of EtS (Cmax= 0.20 μg/mL). Conversely, this was also true for the max values of observed time. Here the estimated model to calculate the period of time after drinking was only based on a unique dosage of 0.72 g/kg, and the facts affecting the metabolism of alcohol, such as genotypes of metabolic enzyme, were also not considered, therefore, further studies on the influence of these factors affecting alcohol metabolism on the time estimation could also be needed.
Additionally, cumulative effects caused by drinking cumulatively can also affect the calculation of last drinking. Here we simulated the drinking pattern at a normal meal of Chinese, namely, all volunteers were required to finish drinking within half an hour. In fact, drinking back and forth for more than half an hour is a rather normative drinking situation for most recreational alcohol drinkers. In this condition, there should be a cumulative addition of EtG and EtS concentrations in blood originating from these different drinking periods, and it is not possible to use the proposed model. Therefore, more studies on the impact of this for the calculation of last drinking should also be done in the next step.
In summary, the current study established a method of estimating the time of last drinking using the relationship between concentration ratio of EtG to EtS in blood and the time of alcohol use, and once further validated, this novel discovery will provide a useful analytical tool for monitoring alcohol use by Chinese motorists on the road. Additionally, we investigate the pharmacokinetics of alcohol and EtG, EtS in blood of Chinese population, and obtained the pharmacokinetic parameters of the targets. The sensitive LC–MS/MS approach developed and validated in the study can be applied in drink driving and other forensic cases when alcohol is involved, and the long detection windows of EtG and EtS support their use as useful markers for the detection of alcohol consumption .