气相色谱-质谱联用辅助鉴定细菌内毒素标准品菌种来源的纯度

Analysis of the Impurity of Bacterium Source of Standard Endotoxin by Gas Chromatography/Mass Spectrometry

为分析内毒素标准品菌种来源的纯度,建立了采用气相色谱-质谱联用法、以N,O-双三甲基硅三氟乙酰胺作为硅烷化试剂对待测物进行衍生、对内毒素标准品所含的3-羟基脂肪酸种类进行检测的方法。色谱柱为DB-5(60m×0.25mm i.d.);载气为氦气,恒压,柱前压206kPa;进样器温度250℃;柱箱初始温度为90℃,保持5m in,以5℃/m in的升温速率升至280℃,保持5m in。进样量为1μL,不分流进样。质谱离子源为电子轰击离子源(E I),离子源温度为250℃,接口温度为280℃。通过对内毒素标准品和大肠杆菌、绿脓杆菌、去离子水中3-羟基脂肪酸种类的比较,探讨了通过3-羟基脂肪酸种类来辅助判断内毒素标准品菌种来源的问题。结果显示,来源为大肠杆菌的9 000EU/支内毒素国家标准品中只含有3-羟基十四烷酸;20EU/支的内毒素工作标准品中除含有3-羟基十四烷酸外,还含有3-羟基十二烷酸,说明其含有非大肠杆菌的细菌。

To analyse the impurity of bacterium source of standard endotoxin, 3-hydroxy fatty acid species in different endotoxin standards was determined by gas chromatography/mass spectrometry （GC/MS） using N, O-his （trimethylsilyl）trifluoroacetamide as the silanizing reagent. GC/MS analysis was performed using a gas chromatograph equipped with a 60 m × 0.25 mm i.d. DB-5 fused silica capillary column and an injector at 250 ℃. Helium was used as the carrier gas under a constant pressure of 206 kPa. The oven was programmed at a rate of 5 ℃/min from 90 ℃ （ held for 5 min） to 280 ℃ （ held for 5 min）. The sample size was 1 μL. The transfer line was kept at 280 ℃. The quadrupole mass spectrometer was operated in electron impact （EI） ionization mode, and the temperature of the source was kept at 250 ℃. The kind of 3-hydroxy fatty acids in 9 000 EU/tube national standard endotoxin, 20 EU/tube working standard endotoxin, escherichia coli, pseudomonas aeruginosa and deionized water were determined to study the purity of bacterium source of the standard endotoxin. It was shown that 9 000 EU/tube endotoxin standard and escherichia coli only contained 3-hydroxytetradecanoic acid. There was 3-hydroxydodecanoic acid in 20 EU/tube working standard endotoxin, which indicated the presence of impurity of bacterium source.