title: Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for determination of GHB, precursors and metabolites in different specimens: Application to clinical and forensic cases.
abstract: Gamma-hydroxybutyric acid (GHB) acts as a precursor and metabolite of the inhibitory central nervous system (CNS) neurotransmitter gamma-aminobutyric acid (GABA). Sodium salt of GHB has been used as a medication for narcolepsy and alcohol withdrawal. Moreover, GHB and its precursor gamma-butyrolactone (GBL), are illegal recreational drugs of abuse. A procedure based on ultra-high-performance liquid chromatography tandem mass spectrometry has been developed and validated in plasma, urine, cerebrospinal fluid and hair for acute and chronic exposure to GHB and in seized preparations coming from black market. In biological matrices, GHB was investigated together with its glucuronide (GHB-Gluc) as a potential marker of exposure, GABA as endogenous precursor and metabolite and GBL as eventual exogenous precursor. GBL was sought together with GHB in illegal preparations. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and an isocratic elution with two solvents: 0.1% formic acid in water and pure methanol. Multiple reaction monitoring (MRM) was used. The method was linear for all analytes under investigation from limit of quantification (LOQ) to 500μgmL-1plasma, urine and cerebrospinal fluid, from LOQ to 100ngmg-1hair and from LOQ to 10mgmL-1illicit preparations with good correlation coefficients (r2=0.99) for all substances. Recovery of analytes under investigation was always higher than 75% and intra-assay and inter-assay precision and accuracy were always better than 15%. The validated method was then successfully applied to real specimens from either forensic (one post-mortem urine sample taken from a GHB fatal intoxication case) or clinical cases (cerebrospinal fluid, plasma and hair samples collected from narcoleptic patients under sodium oxybate treatment). Finally, illicit preparations, seized by police forces were also checked for GHB amount and eventual presence of prodrug GBL.
authors.affiliation: Unit of Forensic Toxicology (UoFT), Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Sapienza University of Rome, V. Le Regina Elena 336, 00161 Rome, Italy. Electronic address: fra.busardo@libero.it.
authors.forename: Francesco Paolo
authors.initials: FP
authors.lastname: Busardò
authors.affiliation: Unit of Forensic Toxicology (UoFT), Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Sapienza University of Rome, V. Le Regina Elena 336, 00161 Rome, Italy.
authors.forename: Chrystalla
authors.initials: C
authors.lastname: Kyriakou
authors.affiliation: National Centre on Addiction and Doping, Istituto Superiore di Sanità, V. le Regina Elena 299, 00161 Rome, Italy.
authors.forename: Emilia
authors.initials: E
authors.lastname: Marchei
authors.affiliation: National Centre on Addiction and Doping, Istituto Superiore di Sanità, V. le Regina Elena 299, 00161 Rome, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Pacifici
authors.affiliation: Faculty of Health and Medical Sciences, Department of Drug Design and Pharmacology, Jagtvej 162, University of Copenhagen, 2100 Copenhagen, Denmark.
authors.forename: Daniel Sejer
authors.initials: DS
authors.lastname: Pedersen
authors.affiliation: National Centre on Addiction and Doping, Istituto Superiore di Sanità, V. le Regina Elena 299, 00161 Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Pichini
mesh_terms: D015107:4-Butyrolactone / Q000737:chemistry; D002555:Cerebrospinal Fluid / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D044707:Forensic Sciences / Q000379:methods; D006197:Hair / Q000737:chemistry; D006801:Humans; D006885:Hydroxybutyrates / Q000737:chemistry*; D013287:Illicit Drugs / Q000032:analysis; D008297:Male; D000432:Methanol / Q000737:chemistry; D008875:Middle Aged; D010949:Plasma / Q000737:chemistry; D012978:Sodium Oxybate / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D005680:gamma-Aminobutyric Acid / Q000737:chemistry
chemical_list: D006885:Hydroxybutyrates; D013287:Illicit Drugs; C111420:4-hydroxybutyric acid; D005680:gamma-Aminobutyric Acid; D012978:Sodium Oxybate; D015107:4-Butyrolactone; D000432:Methanol
keywords: Gamma-aminobutyric acid; Gamma-butyrolactone; Gamma-hydroxybutyric acid; Gamma-hydroxybutyric acid glucuronide; Ultra-high performance liquid chromatography tandem mass spectrometry; pharmacotoxicology