Synthesis, characterisation and detection of γ-hydroxybutyrate salts

Sodium, potassium, magnesium and calcium salts of gamma-hydroxybutyrate have been synthesised from gamma-butyrolactone and the corresponding group 1 or 2 hydroxide. Although the group 2 salts are non-hygroscopic, FT-IR spectroscopy and elemental analysis revealed them to be hydrated. X-ray powder diffraction was found to be a quick, non-destructive method of discriminating between the four salts. The Smith and the chlorophenol red/modified Schweppes reagent presumptive colour tests gave positive results regardless of the salt tested. Microcrystalline tests for NaGHB were in accordance with previous literature reports, but results for the other three salts were not reliable.     

Enzymatic detection of γ-hydroxybutyrate using aldo-keto reductase 7A2

Gamma-hydroxybutyrate (GHB) is a prescribed medication as well as a drug of abuse. Its detection in various matrices for in-field forensic scientists remains a challenge. We have developed an assay that uses aldo-keto reductase 7A2 (AKR7A2) for the specific determination of GHB in various drinks. AKR7A2 was purified using Ni-affinity chromatography. The Michaelis-Menten constant for the GHB oxidation reaction was 10 mM, and the minimum detection limit was 4 mM. Ethanol was not a substrate for AKR7A2. In a coupled reaction with NADP(+), phenazine methosulfate (PMS), and 2,6-dichlorophenolindophenol, various beverages (orange juice, milk, soda, and numerous alcoholic drinks) containing GHB turned from blue to light yellow. In a second coupled reaction where diaphorase replaced PMS, the presence of GHB also caused the expected change of color in various beers.     

A sandwich enzyme-linked immunosorbent assay for human seminal γ-glutamyl transpeptidase

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting human seminal γ-glutamyl transpeptidase (γ-GTP) using a combination of anti-seminal γ-GTP monoclonal antibodies. These monoclonal antibodies did not react with human ovary or uterus in immunohistochemical study. Optimal assay condition, resulting in a sensitive assay with a low background, is presented. The detection limit of this assay was estimated to be 1 ng/ml of seminal γ-GTP corresponding to about 100 000 times dilution of seminal sample. This ELISA was specific for seminal γ-GTP, without cross-reactivity to renal or hepatic gg-GTP, normal blood serum, non-coital vaginal fluid or saliva. The recovery of seminal γ-GTP added to various biological fluids were also examined.     

Detection of human seminal γ-glutamyl transpeptidase in stains using sandwich ELISA

A sensitive and specific sandwich ELISA for human seminal γ-glutamyl transpeptidase (γ-GTP) was developed using a combination of monoclonal antibodies, SG1 and SG3, which we produced. For semen identification in forensic samples, we modified the assay so as to be more sensitive and to establish efficient extracting conditions. After testing the extracting abilities of several detergents, CHAPS and deoxy-BIGCHAP were chosen as the solubilizer. Polystyrene beads coated with SG1 were incubated with samples extracted by the detergents, and further with biotinylated SG3, followed by peroxidase-labeled streptavidin. γ-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal γ-GTP equivalent to 10⁷ times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal γ-GTP was detected in other biological stains such as blood, saliva and vaginal smear. The extract of a 500 fold diluted seminal stain, 8 months old, showed the detection limit. Seminal γ-GTP was detectable even in 14-year-old stains.     

Rapid screening for the detection and differentiation of γ-hydroxybutyrate using ion chromatography

The analysis of gamma-hydroxybutyric acid (GHB) is problematic because it is hygroscopic, it lacks a good UV chromophore, and it undergoes heat-induced cyclization. This paper presents a new method utilizing ion-exchange chromatography (IC) with conductivity detection. The simple sample preparation, rapid analysis time, and inorganic anion detection capabilities are all advantages over the current methods. The detection of inorganic salts (formed during GHB synthesis) gives insight into the synthetic route utilized and can aid in drug seizure comparison. The developed method has a detection limit for GHB anions of 0.57 mg/L and chloride of 0.22 mg/L. A comparison of this technique with a current gas chromatography-mass spectrometry technique is presented, and a t-test found that the two methods' results are not statistically different at the 99.9% confidence level demonstrating the merits of this fast, simple, and informative IC method as a routine screening tool.     

A hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry method for the simultaneous determination of γ-hydroxybutyrate and its precursors in forensic whole blood

A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of γ-hydroxybutyric acid (GHB), γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in human ante-mortem and post-mortem whole blood. The blood proteins were precipitated using a mixture of methanol and acetonitrile, and the extract was cleaned-up by passage through a polymeric strong cation exchange sorbent. Separation of the analytes and their structural isomers was obtained using a column with a zwitterionic stationary phase. Matrix-matched calibrants, combined with isotope dilution, were used for quantitative analysis. GHB was determined in both positive and negative ion modes. The relative intra-laboratory reproducibility standard deviations were better than 10% and 6% for blood samples at concentrations of 2mg/L and 20-150mg/L, respectively. The mean true extraction recoveries were 80% for GHB and greater than 90% for GBL and 1,4-BD at concentration levels of 20-50mg/L. The limits of detection were approximately 0.5mg/L for GHB and GBL, and 0.02mg/L for 1,4-BD in ante-mortem blood. The corresponding lower limits of quantification were less than 1mg/L for GHB and GBL, and less than 0.1mg/L for 1,4-BD. GBL was unstable in whole blood freshly preserved with a sodium fluoride oxalate mixture, but the stability could be improved significantly by preservation with a sodium fluoride citrate EDTA mixture.