Stability of γ-Hydroxybutyrate in Blood Samples from Impaired Drivers after Storage at 4°C and Comparison of GC-FID-GBL and LC-MS-MS Methods of Analysis.

The stability of γ-hydroxybutyrate (GHB) was determined in 50 blood samples from impaired drivers after storage at 4 °: C for up to 12 months. GHB was determined in whole blood by gas chromatography-flame ionization detector (GC-FID) after conversion into γ-butyrolactone (GBL) and results were compared with LC-MS-MS. Both analytical methods showed a linear response (R(2) > 0.99) to GHB concentrations from 2 to 250 mg/kg. The mean decrease in concentration after storage was 4.8 mg/kg, with extreme changes of +13 mg/kg or -29 mg/kg. Results by the GC-FID-GBL method (y-variate) and the LC-MS-MS method (x-variate) were highly correlated (R(2) = 0.974). The regression equation was y = 0.85x + 2.2 and residual standard deviation (SD) was 7.8 mg/kg. The y-intercept (2.2 mg/kg) was not significantly different from zero (P > 0.05), although the slope of the regression line (0.85) differed from unity (P < 0.001), indicating a proportional bias of 15%. The LC-MS-MS method tended to give higher results than the GC-FID-GBL method. The mean difference (bias) was 12 mg/kg (P < 0.001). The SD of individual differences was 11.3 mg/kg and 95% limits of agreement were -11 to +33 mg/kg. The results of this study show that concentrations of GHB in whole blood are stable during storage at 4 °: C for up to 6 months.

Simultaneous determination of γ-Hydroxybutyrate (GHB) and its analogues (GBL, 1.4-BD, GVL) in whole blood and urine by liquid chromatography coupled to tandem mass spectrometry.

A simple liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous identification and quantification of γ-hydroxybutyrate (GHB), γ-butyrolactone (GBL), 1.4-butanediol (1.4-BD), and γ-valerolactone (GVL) in whole blood from forensic cases. The sample preparation of whole blood involved protein precipitation by acidic methanol. Urine samples were diluted and evaluated in relation to a control at the cutoff concentration. Hexadeutero GHB (GHB-d(6)) was used as the internal standard. Separation was achieved by reversed-phase chromatography, and detection was by MS-MS in MRM mode. The linear range for all compounds was from 1.0 to 100 mg/kg in whole blood with a limit of quantification of about 1 mg/kg. The method was validated with regards to selectivity, recovery, accuracy and precision, and stability. The method is currently applied to investigations on suspected drug-facilitated sexual assaults, driving under the influence of drugs, and general intoxication with these substances.