title: Analysis of alcohol markers in hair and the application to authentic hair samples
Creators: name: Phuangphung, Peerayuht
Publisher: name: University of Glasgow
Year: 2019

description: Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) are two direct alcohol markers in hair which are useful for the assessment of abstinence and chronic excessive alcohol consumption according to the 2016 consensus from the Society of Hair Testing (SoHT) for alcohol markers in hair. As these markers are direct alcohol metabolites, there may be variation in the concentrations of these markers between different racial populations. Thus, the aim of this study was to develop and validate liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the quantification of EtG and FAEEs in hair for the purpose of the application to authentic hair samples from the Thai and Scottish populations to evaluate alcohol marker concentrations in these two populations. 15 subjects from Thai post-mortem cases, 30 subjects from Thai living people and 39 subjects from Scottish living patients undergoing alcohol detoxification programmes were recruited in this study. Histories of alcohol consumption in Thai and Scottish subjects were recorded and were categorised from abstinence to alcoholics for each population. Hair samples were collected from the posterior vertex and 0-3 cm hair segments were used for the analysis. Hair samples were washed using dichloromethane (DCM) and methanol (MeOH) twice and by cyclohexane twice for EtG and FAEE analysis, respectively. After washing, hair was left to dry and was finely cut into small pieces. 50 mg of hair were used for the analysis of EtG and FAEEs. For EtG analysis, hair was extracted using dH2O/MeOH and then were further extracted using Oasis® MAX SPE cartridges. For FAEE analysis, hair was extracted using cyclohexane and then further extracted using Bond Elut® NH2 SPE cartridges. 30 µL was used for LC-MS/MS analysis for both EtG and FAEEs. For both methods, reversed phase chromatographic separation was performed on ESI-LC-MS/MS. The two methods were validated following standard guidelines. For EtG, linearity was acceptable over 7-2000 pg/mg (9 calibrators) using 1/x weighted linear regression with r2&gt;0.99 (n=5). The lower limit of quantitation (LLOQ) was 7 pg/mg. Accuracy range for three QC concentrations (30, 200 and 1500 pg/mg) (n=5) was 87.6-114.4%. Intra-day and inter-day precision (n=5) for three QCs was ≤9.8% and ≤10.4%. For FAEEs, linearity was acceptable over 0.025-5.000 ng/mg (8 calibrators) using 1/x2 weighted linear regression with r2&gt;0.99 (n=5). LLOQ of these four FAEEs was 0.025 ng/mg. Accuracy range for three QCs (0.076, 0.400 and 3.800 ng/mg) of four FAEEs was 85.3-114.7%. Intra-day and inter-day precision (n=5) for three QCs of four FAEEs was ≤14.1% and ≤14.2%. The EtG method was applied to the SoHT proficiency testing (PT) authentic hair samples and the analytical results were within acceptable ranges. All Thai people in the abstinence group were negative for EtG (LOD = 5 pg/mg). 15 out of 16 Thai people (93.75%) in the abstinence group had E16:0 ranging from negative (LOD = 0.015 ng/mg) to 0.095 ng/mg which were less than the cut-off concentration of E16:0 at 0.120 ng/mg. In Scottish patients, 10 out of 13 Scottish patients (76.92%) were negative for EtG whereas 12 out of 13 Scottish patients (92.31%) were negative for E16:0. When the chronic excessive alcohol consumption group was considered, 8 out of 10 Thai people (80%) in this group had EtG concentrations &gt;30 pg/mg while 6 out of 10 Thai people (60%) had E16:0 concentrations &gt;0.350 ng/mg. In Scottish patients, 18 out of 26 Scottish people (69%) had EtG concentrations &gt;30 pg/mg whereas 22 out of 26 Scottish people (85%) had E16:0 concentrations &gt;0.350 ng/mg. The concordance rates in the Thai and Scottish populations were 60% and 69.23% in the chronic excessive alcohol consumption groups, respectively. The ratios of the four esters in the Thai and Scottish populations were 8:49:30:12 and 5:50:40:6, respectively. The EtG concentrations in female Scottish patients were significantly less than male Scottish patients (p&lt;0.001). However, the E16:0 concentrations in female Thai and Scottish subjects were not significantly different from male Thai and Scottish subjects (p&gt;0.05). EtG concentrations in the Thai and Scottish populations had a positive correlation with the amount of alcohol consumed (p&lt;0.05) but the correlation in the Thai population was stronger than the Scottish population (r2=0.740 vs r2=0.187). However, E16:0 concentrations in the Thai and Scottish populations did not correlate with the amount of alcohol consumed (p&gt;0.05). EtG and FAEE concentrations in the abstinence and chronic excessive alcohol consumption groups in Thai people were not significantly different from Scottish patients (p&gt;0.05). In conclusion, the majority of EtG and FAEE hair concentrations in the Thai and Scottish populations were in agreement with the cut-off concentrations from the SoHT consensus. In addition, EtG and FAEE concentrations in Thai were not significantly different from Scottish populations both in the abstinence and chronic excessive alcohol consumption groups. Thus, the cut-off concentrations from the SoHT guidelines can be applicable to Thai and Scottish populations.