^(15)N标记S-苄基-L-半胱氨酸含量及其同位素丰度检测

Detection of Content and Isotope Abundance of ^(15)N-S-benzyl-L-cysteine

建立了一种测定发酵液中15N标记S-苄基-L-半胱氨酸(SBC)含量的高效液相色谱法(HPLC),该方法采用Cosmosil C18(5μm,150 mm×4.6 mm)色谱柱为分离柱,2,4-二硝基氟苯为衍生剂,0.05 mol/L乙酸钠缓冲溶液(pH=6.5,含10 mL/L N,N-二甲基甲酰胺)与V(乙腈)∶V(水)=3∶1为流动相,梯度洗脱,流速1.0 mL/min,检测波长为350 nm,柱温为30℃;并采用微量高温燃烧-质谱法对15N标记SBC样品的同位素丰度进行了检测。结果显示,SBC浓度为0.2~1.0 g/L时,与其峰面积呈良好的线性关系,y=3×106x-4.16×104,R2=0.998 6,平均加标回收率97.30%,检测限为3.95×10-4g/L,测定结果的相对标准偏差为0.55%;15N标记SBC丰度检测时,最小称样量为2 mg,最佳反应温度为530℃,反应时间为3 h;15N丰度测定值的相对标准偏差为0.17%。

A HPLC method for determination of S-benzyl-L-cysteine in fermentation broth was established.Cosmosil C18 column（5 μm,150 mm×4.6 mm） was used with 2,4-dinitrofluorobenzene as derivatization reagent and 0.05 mol/L sodium acetate buffer（pH=6.5,including 10 mL/L N,N-dimethylformamide） and V（acetonitrile）∶V（water）=3∶1 as mobile phase.The content was detected at a flow rate of 1.0 mL/min（gradient）,the detection wavelength was 350 nm,column temperature was 30 ℃.The calibration curves were linear in the range of 0.2-1.0 g/L（R^2=0.998 6）,the average recovery rates were 97.30%,the minimum limit was 3.95×10^-4 g/L,the relative standard deviation of results was 0.55%.Microscale high temperature combustion-MS method was also described to detect the isotope abundance of ^15N-SBC.The minimum consumption of ^15N-SBC product was 2 mg,the optimal combustion temperature was 530 ℃ and reaction time was 3 h.The RSD of results was 0.17%.