Determination Of Gamma-hydroxybutyric Acid Research Articles

Determination of Gamma-Hydroxybutyric Acid in Urine by Solid Phase Extraction and Gas Chromatography—Mass Spectrometry

ABSTRACTGamma-hydroxybutyric acid has a short half-life in the human body and may be determined at less than 8 h in blood and 12 h in urine. Consequently, the sensitivity of the instrumentation and the extraction procedure for gamma-hydroxybutyric acid from biological samples are important factors in its determination. In this study, solid-phase extraction was compared to the commonly used liquid–liquid extraction with analysis by gas chromatography—mass spectrometry. Solid-phase extraction enhanced the recovery of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L gamma-hydroxybutyric acid. The recovery for gamma-hydroxybutyric acid by solid-phase extraction was 64.5–70.9% compared to 32.6–47.5% by liquid–liquid extraction. Solid-phase extraction also offered shorter analysis time, smaller sample quantities and solvent volumes, and better separation. Solid phase extraction for gamma-hydroxybutyric acid analysis in urine was fully validated and the results were satisfactory. Both extraction methods were used to analyze urine from an individual that was sexually assaulted. Significant differences were observed in the concentrations of the gamma-hydroxybutyric acid with values of 148.07 and 233.15 µg/mL by liquid–liquid extraction and solid-phase extraction, respectively.

Determination of gamma-hydroxybutyric acid in biofluids using a one-step procedure with “in-vial” derivatization and headspace-trap gas chromatography–mass spectrometry

A headspace-trap gas chromatography–mass spectrometry (HS-trap GC–MS) method was developed to determine GHB, a low molecular weight compound and drug of abuse, in various biological fluids. Combining this relatively novel and fully automated headspace technique with “in-vial” methylation of GHB allowed for a straightforward approach. One single method could be used for all biofluids (urine, plasma, serum, whole blood or lyzed blood), requiring only 100μl of sample. Moreover, our approach involves mere addition of all reagents and sample into one vial. Following optimization of headspace conditions and trap settings, validation was performed. Although sample preparation only consists of the addition of salt and derivatization reagents directly to a 100μl-sample in a HS-vial, adequate method sensitivity and selectivity was obtained. Calibration curves ranged from 5 to 150μg/ml GHB for urine, from 2 to 150μg/ml for plasma, and from 3.5 to 200μg/ml for whole blood. Acceptable precision and accuracy (<13% bias and imprecision) were seen for all quality controls (QC's) (LLOQ-level, low, medium, high), including for the supplementary serum- and lyzed blood-based QC's, using calibration curves prepared in plasma or whole blood, respectively. Incurred sample reanalysis demonstrated assay reproducibility, while cross-validation with another GC–MS method demonstrated that our method is a valuable alternative for GHB determination in toxicological samples, with the advantage of requiring only 100μl and minimal hands-on time, as sample preparation is easy and injection automated.

Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid

Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD+. With an optimal NAD+ concentration of 3.7 mmol/L in the reaction, the enzyme yields a K(M) of 1.0 mmol/L for GHB and a Vmax of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively.

Determination of gamma-hydroxybutyric acid in serum and urine by headspace solid-phase dynamic extraction combined with gas chromatography–positive chemical ionization mass spectrometry

Gamma-hydroxybutyric acid is an emerging drug of abuse. Beside relaxation and euphoria it causes hypnosis and unconsciousness. Therefore the substance is misused as recreational drug and at drug-facilitated sexual assaults. An automated and effortless method for quantitation of gamma-hydroxybutyric acid in serum and urine was optimized and validated. Five hundred microliters sample volume are used for both matrices. The acid catalyzed conversion of gamma-hydroxybutyric acid to the corresponding gamma-butyrolactone is applied. Furthermore the method is based on headspace solid-phase dynamic extraction coupled with gas chromatography–mass spectrometry. The extraction process is performed by repeated aspiration and ejection of the headspace through a steel cannula which is coated on the inside with a polydimethylsiloxane sorbent. Thus absorption of analyte molecules by the sorbent is achieved. The influence of parameters as sorbent type, incubation temperature, number of extraction strokes, injection port temperature and injection flow speed on extraction recovery was investigated. The validation revealed good accuracy with a bias less than ±5%. Intra- and interday precision determined at 10, 50 and 150 μg/ml for each matrix were in following ranges: 1.96–3.49% (intraday, serum), 2.38–4.31% (intraday, urine), 2.33–5.13% (interday, serum) and 2.53–5.64% (interday, urine). The method provided good linearity between 2 and 200 μg/ml yielding coefficients of determination R 2 ≥ 0.9985. Limit of detection were determined at 0.16 μg/ml for serum and 0.17 μg/ml for urine, respectively. This method exhibits a fast, solvent-free and widely automated extraction process. It has been applied to toxicological routine analysis and therapeutic drug monitoring successfully.

Determination of GHB in urine and serum by LC/MS using a simple one-step derivative

A sensitive and specific method for the determination of gamma-hydroxybutyric acid (GHB) in urine and serum is described. Prior to quantification by LC/MS in a Pauli-type ion trap, the molecule is converted by a fast and simple one-step procedure into its n-butyl ester derivative. Hexa-deutero GHB has been used as internal standard.

1H NMR Analysis of GHB and GBL: Further Findings on the Interconversion and a Preliminary Report on the Analysis of GHB in Serum and Urine

A 1H nuclear magnetic resonance (1H NMR) method for the determination of gamma-hydroxybutyric acid (GHB) and gamma-hydroxybutyrolactone (GBL) in human serum and urine using spiked samples has been developed. The method gives linear responses (correlation coefficients of 0.99 or greater) over the concentration range 0.01 mg/mL to 4.0 mg/mL in urine and 0.3 mg/mL to 2.0 mg/mL in serum. No sample pretreatment is required. Studies of the chemical interconversion of GBL and GHB showed hydrolysis of GBL to be rapid at pH 11.54, slower and less complete (30% hydrolysis) at pH 2.54 and slowest at pH 7.0, reaching 30% hydrolysis in about 40 days. No esterification of GHB was observed at any pH.

Determination of gamma-hydroxybutyric acid (GHB) in plasma and urine by headspace solid-phase microextraction and gas chromatography/positive ion chemical ionization mass spectrometry.

A new method for the qualitative and quantitative analysis of gamma-hydroxybutyric acid (GHB) in plasma and urine samples is described. It involves the conversion of GHB to gamma-butyrolactone (GBL), its subsequent headspace solid-phase microextraction (SPME), and detection by gas chromatography/positive ion chemical ionization mass spectrometry (GC/PICI-MS), using D(6)-GBL as internal standard. The assay is linear over a plasma GHB range of 1-100 microg/mL (n = 5, r = 0.999) and a urine GHB range of 5-150 microg/mL (n = 5, r = 0. 998). Relative intra- and inter-assay standard deviations, determined for plasma and urine samples at 5 and 50 microg/mL, are all below 5%. The method is simple, specific and reasonably fast. It may be applied for clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring.