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 2 mg/L and 20-150 mg/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 20-50 mg/L. The limits of detection were approximately 0.5 mg/L for GHB and GBL, and 0.02 mg/L for 1,4-BD in ante-mortem blood. The corresponding lower limits of quantification were less than 1 mg/L for GHB and GBL, and less than 0.1 mg/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.
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