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Gamma-hydroxybutyric acid (GHB), used as a therapeutic and an illegal anesthetic, is a human neurotransmitter produced during gamma-aminobutyric acid (GABA) biosynthesis and metabolism. Potential biomarker metabolites of GHB intoxication have been identified previously; however, reference concentrations have not been set due to the lack of clinical study data. Urinary profiling of endogenous GHB and its biomarker metabolites in urine samples (n = 472) of 206 healthy females was performed based on differences in age and time of sample collection using liquid chromatography-tandem mass spectrometry following validation studies. The unadjusted and creatinine-adjusted urinary concentrations ranges were obtained after urinary profiling. The creatinine-adjusted concentrations of glutamic and succinic acids and succinylcarnitine significantly increased, whereas that of glycolic acid significantly decreased with advancing age. Significant inter-day variation of GABA concentration and intra-day variation of 3,4-dihydroxybutyric acid and succinylcarnitine concentrations were observed. The urinary concentrations of 2,4-dihydroxybutyric acid, succinic acid, and 3,4-dihydroxybutyric acid showed the highest correlation with that of GHB. Data from this study suggest population reference limits to facilitate clinical and forensic decisions related to GHB intoxication and could be useful for identification of biomarkers following comparison with urinary profiles of GHB-administered populations.

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