柱前荧光衍生-高效液相色谱法测定尿中雌二醇

Determination of 17α-Estradiol and 17β-Estradiol in Urine by High Performance Liquid Chromatography with Pre-column Derivatization

建立了对硝基苯甲酰氯柱前荧光衍生 高效液相色谱法测定尿中 1 7α 和 1 7β 雌二醇的方法。尿样经盐酸水解、固相萃取柱浓缩、净化分离后 ,在无水条件下与对硝基苯甲酰氯反应生成荧光产物 ,再用高效液相色谱 荧光检测器测定。流动相为乙腈 水 (5 5∶45 )。线性范围为 7.0× 1 0 - 3～ 1 0mg L ,检出限均为 7.0× 1 0 - 3mg L,相对标准差分别为 0 .93 %～ 1 .5 %和 0 .88%～ 1 .3 %。平均回收率分别为 83 .5 %和 88.2 %。

A high performance liquid chromatographic method for the determination of 17α-estradiol and 17β-estradiol with fluorimetric detection after precolumn derivatization with p -nitrobenzoyl chloride was developed. Estradiols in urine were hydrolyzed with 0.5 mol/L HCl, and then enriched and cleaned-up by ENV-18 C_ 18 solid phase extraction column. Phenomenex C_ 18 (250 mm×4.6 mm) column was used. Mobile phase consisted of acetonitrile-water (55∶45, V/V ) with a flow rate of 1.0 mL/min. Fluorescence detection was done with the excitation at 282 nm and emission at 315 nm .The linear ranges of 17α-estradiol and 17β-estradiol were found to be 7.0×10 -3 mg/L～10 mg/L, and the mean recoveries of 17α-estradiol and 17β-estradiol were 83.5% and 88.2% respectively. Their limits of detection both were 7.0×10 -3 mg/L. This method has been applied to the determination of 17α-estradiol and 17β-estradiol in urine with satisfactory results.