Gottardo Rossella, Bortolotti Federica, Trettene Maristella, De Paoli Giorgia, Tagliaro Franco
Department of Medicine and Public Health, Unit of Forensic Medicine, University of Verona, Policlinico, 37134 Verona, Italy.
J Chromatogr A. 2004 Oct 8;1051(1-2):207-11.
The present work was aimed at the development of a capillary electrophoretic analysis of gamma-hydroxybutyric acid (GHB) using electrospray ion trap mass spectrometry to achieve the direct and unequivocal detection of this analyte in human urine. Optimized capillary electrophoretic conditions were: injection, 20 s at 0.5 psi (1 psi = 6894.76 Pa); buffer electrolyte, 12.5 mM ammonium formate adjusted to pH 8.35 with diethylamine; fused silicacapillary: 100 cm x 50 microm i.d.; separation voltage, 25 kV (forward polarity) + 0.5 psi; room temperature. Electrospray and mass spectrometric conditions were: drying gas and nebulizing gas (nitrogen) at flow rate 3 l/min, temperature 250 degrees C, nebulizer pressure: 10 psi; sheath liquid solution: methanol-water (90:10) containing 0.1% ammonia delivered at 3 microl/min; spray voltage 3.5 kV. Mass spetrometric detection was carried out in the selected ion monitoring mode of negative molecular ions at 103 m/z for GHB and 115 m/z for maleic acid (I.S.). Under these conditions the baseline separation of GHB and the I.S. was obtained. The selectivity of the analysis allowed for direct injection of unextracted urine, previously diluted 1:4 with water. Linearity was assessed in the GHB concentration range from 80 to 1280 microg/ml in urine. Analytical sensitivity (as limit of detection) resulted about 5 microg/ml in water and 20 microg/ml in original urine. Analytical precision was fairly acceptable with R.S.D. values lower than 5% for migration times and 18% for quantitation in real samples, in both intra day and day-to-day experiments. On these grounds, the developed method can be adopted for rapid identification of acute intoxications from GHB in humans.
本研究旨在开发一种利用电喷雾离子阱质谱对γ-羟基丁酸（GHB）进行毛细管电泳分析的方法，以实现对人尿中该分析物的直接、明确检测。优化后的毛细管电泳条件为：进样，0.5 psi（1 psi = 6894.76 Pa）下进样20 s；缓冲电解质，12.5 mM甲酸铵，用二乙胺调至pH 8.35；熔融石英毛细管：100 cm×50 μm内径；分离电压，25 kV（正向极性）+ 0.5 psi；室温。电喷雾和质谱条件为：干燥气和雾化气（氮气）流速3 l/min，温度250℃，雾化器压力：10 psi；鞘液溶液：甲醇-水（90:10），含0.1%氨，流速3 μl/min；喷雾电压3.5 kV。质谱检测在负离子的选择离子监测模式下进行，GHB的质荷比为103 m/z，马来酸（内标）的质荷比为115 m/z。在这些条件下，实现了GHB与内标的基线分离。该分析方法的选择性允许直接进样未提取的尿液，尿液事先用水按1:4稀释。在尿液中GHB浓度范围为80至1280 μg/ml时评估了线性关系。分析灵敏度（作为检测限）在水中约为5 μg/ml，在原始尿液中为20 μg/ml。无论是日内实验还是日间实验，实际样品中迁移时间的相对标准偏差（R.S.D.）值低于5%，定量的R.S.D.值低于18%，分析精密度相当令人满意。基于这些结果，所开发的方法可用于快速鉴定人体中GHB急性中毒情况。
