Validation of HPLC-ESI-MS/MS Protocol to Analyze EtG
in Hair for Assessment of Chronic Excessive Alcohol
Use in Thailand in Conjunction with AUDIT
Thiwaphorn Thananchai MS*, Anongphan Junkuy MS*,
Phunnapa Kittirattanapaiboon MD**, Pongruk Sribanditmongkol MD, PhD*
Affiliation :
* Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
** Bureau of Mental Health Service Administration, Department of Mental Health,
Ministry of Public Health, Nonthaburi, Thailand
Background : Hair analysis for chronic excessive alcohol (ethanol) use has focused on ethyl glucuronide (EtG), a minor
metabolite of ethanol. Preferred methods have involved high-performance liquid chromatography (HPLC) combined with
tandem mass spectrometry (MS/MS) in line with an electrospray ionization (ESI) source. EtG analysis in hair has not yet
been introduced to Thailand.
Objective : To validate an in-house HPLC-ESI-MS/MS hair analysis protocol for EtG and to apply it to a field sample of
alcohol drinkers to assess different risk levels of alcohol consumption as measured by the Alcohol Use Disorders Identification
Test (AUDIT).
Material and Method: Validation procedures followed guidelines of the US Food and Drug Administration, the European
Medicines Agency, and the Scientific Working Group for Forensic Toxicology. One hundred twenty subjects reported
consuming alcohol during a 3-month period prior to enrollment. After taking the Thai-language version of AUDIT,
subjects were divided on the basis of test scores into low, medium, and high-risk groups for chronic excessive alcohol
use.
Results : The protocol satisfied the international standards for selectivity, specificity, accuracy, precision, and calibration
curve. There was no significant matrix effect. Limits of detection and quantification (LOD/LOQ) were set at 15 pg of EtG
per mg of hair. The protocol was not able to detect EtG in low-risk subjects (n = 38). Detection rates for medium-risk
(n = 42) and high-risk subjects (n = 40) were 14.3% and 85%, respectively. The median of EtG concentration between these
two groups were significantly different. Sensitivity and specificity were both more than 90% when EtG concentrations of
high-risk subjects were compared with the 30 pg/mg cutoff recommended by the Society of Hair Testing (SoHT) for diagnosing
chronic excessive alcohol consumption, based on an average ethanol daily intake greater than 60 g.
Conclusion : The in-house protocol for EtG analysis in hair was validated according to international standards. The protocol
is a useful tool for evaluating risk for chronic excessive drinking as defined by AUDIT scores. It strongly predicted the
highest level of risk, although it was inadequate for assessing lower levels of risk.
Keywords : HPLC-ESI-MS/MS, hair analysis, chronic excessive alcohol use, EtG, AUDIT, Thailand
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