RP-HPLC测定正常人血清中的瓜氨酸

Determination of citrulline in human serum by reversed phase HPLC

目的采用反向HPLC法测定人血清中的瓜氨酸含量。方法以2,4-二硝基氟苯(DNFB)为衍生化试剂,柱前衍生;采用Amethyst C18-H色谱柱(250 mm×4.6 mm,5μm),流动相A为乙腈-水溶液(1:1),流动相B为50 mmol·L^-1磷酸二氢钾加1‰磷酸的缓冲盐溶液,梯度洗脱,检测波长360 nm,柱温30℃,流速1 mL·min^-1。结果在该实验条件下,瓜氨酸在30 min内的分离度良好,0.25~100μg·mL^-1瓜氨酸与峰面积呈良好的线性关系,精密度的RSD=1.98%,供试样品48 h内稳定,重复性的RSD=1.55%,平均加样回收率为87.45%,RSD=1.47%;在此方法基础上测定正常人血清中瓜氨酸的含量为33.08±6.46μmol·L^-1。结论所用方法稳定可靠、高效简洁、灵敏度高、成本低。

OBJECTIVE To determine citrulline in human serum by RP-HPLC.METHODS 2,4-dinitrofluorobenzene(DNFB)served as derivative reagent.Amethyst C18-H chromatographic columns(250 mm×4.6 mm,5μm)was used.Mobile phase A was as follows:acetonitrile-water solution(1:1),mobile phase B:50 mmol·L^-1 of potassium dihydrogen phosphate and 1‰ phosphate buffer salt solution with gradient elution.The detection wavelength was 360 nm,with column temperature of 30℃ and flow rate of 1 mL·min^-1.RESULTS Under this experimental condition,citrulline was separated completely within 30 minutes.A good linear relationship was obtained within 0.25-100μg·mL^-1.Precision was 1.98%.Serum samples stability was 1.21%.Repeatability was 1.55%.Average recovery rate was 87.45%,and RSD was 1.47%.Determined concentration range of serum citrulline in normal Chinese human beings was carried out,and the result was 33.08±6.46μmol·L^-1.CONCLUSION The method is stable and reliable,as well as of high efficiency,high detection sensitivity and low cost.