高效液相色谱法测定人血浆中内源性尿嘧啶和二氢尿嘧啶含量

Determination of endogenous uracil and dihydrouracil in plasma by HPLC

目的：建立准确测定内源性尿嘧啶和二氢尿嘧啶血药浓度的高效液相色谱法。方法：以氟尿嘧啶（5-Fu）为内标，醋酸乙酯-异丙醇混合液（85：15）为提取溶剂；色谱柱Diamonsil C18柱（250mm×4．6mm，5μm）；流动相A-0．01mol·L^-1磷酸二氢钾缓冲液（pH5．5），B-乙腈，梯度洗脱；流速为0．8mL·min^-1；柱温为4℃；检测波长为204nm（0-14．5min），254nm（14．5～35min）。结果：尿嘧啶和二氢尿嘧啶线性范围为8-500μg·L^-1，线性回归方程分别为C（UH2）=61．7608Y＋0．5065，r=0．9998；C（U）=95．2011Y-3．0640，r=0．9993，（n=7）。最低检测质量浓度均为5μg·L^-1。尿嘧啶方法回收率为99．3％～107．0％，二氢尿嘧啶方法回收率为95．0％～98．3％。尿嘧啶日内RSD小于6．5％，日间RSD小于11．7％，二氢尿嘧啶日内RSD小于9．2％，日间RSD小于12．4％。结论：本方法可用于内源性尿嘧啶和二氢尿嘧啶血药浓度的常规监测。

OBJECTIVE To establish an HPLC method for determining uracil（U）and dihydrouracil（UH2 ）in human plasma. METHODS A mixture composed of ethyl acetate and isopropyl alcohol （85 ： 15） was used for extraction, and 5-Fluorouracil （5-FU） was chosen as the internal standard. Diamonsil C18 column （250 mm× 4. 6mm,5μm） was used. The mobile phase of gradient elution was composed of 0. 01 mol·L^-1 KH2 PO4 buffer （pH 5.5） and acetonitrile. UV detection wavelength was set at 204 nm （0-14. 5 min） and 254 nm（14. 5-35 min）. RESULTS A good linear calibration curves were obtained for U and UH2 between the range 8μg·L^-1 to 500μg·L^-1. The calibration equation of UH2 and U were C（UH2） = 61. 760 8Y＋ 0. 506 5, r= 0. 999 8; C（U） = 95. 201 1Y- 3. 064 0, r = 0. 999 3 （n = 7）. The detect limitation were 5μg·L^-1. The recovery of U and UH2 were 99.3%-107. 0% and 95.0%-98.3%, respectively. The RSD within day and between days were less than 6. 5% and 11.7% for U, and less than 9. 2% and 12. 4% for UH2. CONCLUSION This method can be used for routine clinical determination of endogenous U and UH2 plasma concentration.