Determination of serotonin metabolites in urine sample by liquid chromatography-mass spectrometry as biomarkers of recent alcohol intake: Implication for aircraft accident investigation.

Background: Determination of ethanol levels in aircraft accident victims constitutes an important part of investigation. However, postmortem production of alcohol by microbial fermentation is known to interfere with the results. Distinguishing postmortem produced alcohols from antemortem ingested is very important in interpretation of results. Ratio of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA) metabolites of serotonin, has known to provide a convenient, rapid, and reliable solution as antemortem ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio after ingestion of alcohol (5-HTOL/5-HIAA = >15 pm/nm).

Methods: Triple quadruple (QQQ) liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization positive mode has been used for development of single tube multiple reaction monitoring (MRM) method for simultaneous quantification of 5-HTOL and 5-HIAA in urine. Deglucuronidation of 5-HTOL glucuronide in urine by beta-glucuronidase followed by simple sample preparation has been adopted. Examination of the ratio on urine samples from 15 individuals after consumption of 60 and 90 ml of whiskey has been carried out at different time interval.

Results: A single method for analysis of both the analytes was developed with sensitivity of 50 ppb and recovery of around 80-90%. Examination of the ratio on urine samples revealed that the ratio was >15 in all groups consuming 60 ml and 90-ml whiskey up to 12 h after alcohol ingestion.

Conclusion: This is a unique highly sensitive single LC-MS method, which has been developed for simultaneous estimation of both 5-HTOL and 5-HIAA on same instrument for proving antemortem alcohol ingestion with high degree of sensitivity and specificity.