HPLC法测定血浆中尿嘧啶和二氢尿嘧啶的含量

目的：建立人血浆中内源性尿嘧啶（U）和二氢尿嘧啶（UH2）的HPLC测定方法，为通过UH2／U比值评价体内二氢嘧啶脱氢酶活性提供方法学基础。方法：采用Shim-packCLC—ODS柱（6mm×150mm，5μm），以0．05mol·L^-1磷酸盐缓冲液（pH值为4．0）为流动相，测定U的检测波长为260nm，测定UH2的检测波长205nm。以含3％牛血清白蛋白（BSA）的水溶液配制标准样品和质控样品。以溴尿嘧啶为内标，采用叔丁基甲醚／正丙醇（75：25）为提取溶剂进行两次提取，氮气吹干、复溶后进样分析。结果：U在5—200μg·L^-1的范围内线性良好（r=0．9995，n=6），测定的批内变异RSD在1．73％-3．45％之间，批间变异RSD在1．76％～2．53％之间，回收率为96．15％-105．09％；UH2在10-400μg·L^-1的范围内线性良好（r=0．9996，n=6），测定的批内变异RSD在3．67％-3．80％之间，批间变异RSD在3．40％～5．74％之间，回收率为101．51％～104．06％。结论：本方法灵敏度高，操作简便易行，为测定体内尿嘧啶和二氢尿嘧啶，进而评价二氢嘧啶脱氢酶活性提供方法学基础。

Establishment of HPLC assay for determination of uracil and dihydrouracil

Objective： To develop high-performance liquid chromatographic （HPLC） method for the determination of endogenous uracil and its metabolite dihydrouracil in human plasma and for indirect estimation of dihydropyrimidine dehydrogenase activity. Methods： A Shim-pack CLC - ODS column was selected and the mobile phase was 0.05 mol · L^-1 phosphate buffer at pH 4.0 with a flow rate of 1.0 mL · min^-1. The detection wavelength for uracil was 260 nm, and for dihydrouracil was 205 nm. With bromouracil as the internal standard, 300 μL plamsa was extracted by terthutyl methyl ether/n-propyl alcohol. The organic layer was evaporated to dryness under nitrogen stream at room temperature, and the residual was reconstituted with water. Results： A linearity was obtained from 5 to 200 μg · L^-1 for uracil with a good correlation coefficient （r = 0.999 5, n = 6）. The intra-day validations was 1.73% - 3.45%, and the inter-day validations was 1.76% - 2.53%. The mean recoveries were 96.15% - 105.09%. A linearity was obtained from 10 to 400 μg · L^-1 for dihydrouraeil with a good correlation coefficient （r = 0.999 6, n = 6）. The intra-day validations was 3.67% - 3.80%, and the inter-day validations was 3.40% - 5.74%. The mean recoveries were 101.51% - 104.06%. Conclusion： The method was sensitive, specific and simple. It is suitable for the determination of endogenous uracil and dihydrouraeil in human plasma and for the investigation of individual variation in dihydropyrimidine dehydrogenase activity.