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

Francesco Paolo Busardò, Chrystalla Kyriakou, Emilia Marchei, Roberta Pacifici, Daniel Sejer Pedersen, Simona Pichini

Research output: Contribution to journalArticle

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 500 μg mL−1 plasma, urine and cerebrospinal fluid, from LOQ to 100 ng mg−1 hair and from LOQ to 10 mg mL−1 illicit 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.

Original languageEnglish
Pages (from-to)123-131
Number of pages9
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume137
DOIs
Publication statusPublished - Apr 15 2017

Fingerprint

High performance liquid chromatography
Metabolites
Tandem Mass Spectrometry
Mass spectrometry
High Pressure Liquid Chromatography
4-Butyrolactone
Cerebrospinal fluid
Hair
Cerebrospinal Fluid
formic acid
Urine
Street Drugs
Plasmas
gamma-Aminobutyric Acid
Assays
Sodium Oxybate
4-hydroxybutyric acid
Narcolepsy
Glucuronides
Prodrugs

Keywords

  • Gamma-aminobutyric acid
  • Gamma-butyrolactone
  • Gamma-hydroxybutyric acid
  • Gamma-hydroxybutyric acid glucuronide
  • pharmacotoxicology
  • Ultra-high performance liquid chromatography tandem mass spectrometry

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

Cite this

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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 500 μg mL−1 plasma, urine and cerebrospinal fluid, from LOQ to 100 ng mg−1 hair and from LOQ to 10 mg mL−1 illicit 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.",
keywords = "Gamma-aminobutyric acid, Gamma-butyrolactone, Gamma-hydroxybutyric acid, Gamma-hydroxybutyric acid glucuronide, pharmacotoxicology, Ultra-high performance liquid chromatography tandem mass spectrometry",
author = "Busard{\`o}, {Francesco Paolo} and Chrystalla Kyriakou and Emilia Marchei and Roberta Pacifici and Pedersen, {Daniel Sejer} and Simona Pichini",
year = "2017",
month = "4",
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T1 - Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) for determination of GHB, precursors and metabolites in different specimens

T2 - Application to clinical and forensic cases

AU - Busardò, Francesco Paolo

AU - Kyriakou, Chrystalla

AU - Marchei, Emilia

AU - Pacifici, Roberta

AU - Pedersen, Daniel Sejer

AU - Pichini, Simona

PY - 2017/4/15

Y1 - 2017/4/15

N2 - 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 500 μg mL−1 plasma, urine and cerebrospinal fluid, from LOQ to 100 ng mg−1 hair and from LOQ to 10 mg mL−1 illicit 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.

AB - 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 500 μg mL−1 plasma, urine and cerebrospinal fluid, from LOQ to 100 ng mg−1 hair and from LOQ to 10 mg mL−1 illicit 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.

KW - Gamma-aminobutyric acid

KW - Gamma-butyrolactone

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KW - Gamma-hydroxybutyric acid glucuronide

KW - pharmacotoxicology

KW - Ultra-high performance liquid chromatography tandem mass spectrometry

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