Derivatization-assisted LC-MS/MS method for simultaneous quantification of endogenous gamma-hydroxybutyric acid and its metabolic precursors and products in human urine

The accurate, precise, and robust quantification of endogenous biomarkers is a challenging task because of the presence of significantly low levels of endogenous compounds in biological samples, the absence of analyte-free matrix-matched calibrators, and sample instability due to in-vitro production or degradation of the analytes. Gamma-hydroxybutyric acid (GHB), a compound often used in drug-facilitated crimes, is a human neurotransmitter produced during both the biosynthesis and metabolism of gamma-aminobutyric acid (GABA). Evidently, proving GHB intoxication through the quantification of GHB and its metabolites in biological samples is not straightforward. This study aimed to develop a sensitive and accurate quantitative method for the simultaneous determination of endogenous GHB and its metabolic precursors and products (glutamic acid, GABA, succinic acid, 2,4-dihydroxybutyric acid, 3,4-dihydroxybutyric acid, glycolic acid, and succinylcarnitine) in human urine using LC-MS/MS. For this purpose, chemical derivatization with benzoyl chloride was employed to improve the sensitivity to glutamic acid and GABA. Synthetic urine was used to prepare calibrators, and the validity of this approach was fully demonstrated, particularly focusing on the instability issues. The validation results proved the method to be selective, sensitive, accurate, and precise, with acceptable linearity within calibration ranges. Moreover, our results regarding the in-vitro production or degradation of metabolites highlight the effects of handling and storage conditions of urine samples. Finally, this effective analytical method is expected to be useful in studying the relationship between GHB intoxication and metabolic alterations and, thus, discovering practical biomarkers for GHB ingestion.