新型荧光衍生试剂用于尿样中痕量游离雌二醇和雌三醇的反相高效液相色谱-荧光检测和质谱定性

合成了新型高灵敏度的荧光标记试剂10-乙基吖啶酮-2-磺酰氯(EASC)。采用EASC柱前衍生化实现了雌二醇(E2)和雌三醇(E3)的反相高效液相色谱(RP-HPLC)-荧光分析及柱后质谱鉴定。试剂EASC比丹磺酰氯(DNS-Cl)具有更高的紫外、荧光和质谱检测灵敏度,其荧光发光强度是丹磺酰氯的1000倍以上。EASC与E2和E3在NaHCO3缓冲液(pH 10.5)中,于60 ℃下反应3 min即可获得稳定的荧光产物,最大激发波长(λex)和最大发射波长(λem)分别为270 nm和430 nm。所建立的方法具有良好的重现性,线性回归系数大于0.9990;检出限(S/N=3)为31 fmol 和40 fmol。对实际根田鼠尿样中的雌二醇和雌三醇含量进行了测定，结果令人满意。

A novel fluorescence reagent for the analysis of trace free oestradiol and oestriol in urine by reversed-phase high performance liquid chromatography with fluorescence detection and mass spectrometry identification

A new fluorescent derivatization reagent, 10-ethyl-9-oxo-9,10-dihydroacridine-2-sulfonyl chloride (EASC), was synthesized. A pre-column derivatization with EASC and reversed-phase high performance liquid chromatographic (RP-HPLC) method with fluorescence （FL） detection and mass spectrometry （MS） identification was performed for the trace analysis of oestradiol (E2) and oestriol (E3) in urine. This reagent shows higher sensitivities in ultraviolet （UV）, FL and MS detection than those of dansyl chloride (DNS-Cl), and the fluorescence intensity of EASC is 1000 times higher than that of DNS-Cl. The results of derivatization indicated that the derivatives can be obtained by the labeling reaction of EASC with estradiol (E2) and estriol (E3) in the presence of NaHCO3 buffer (pH 10.5) at 60 ℃ for 3 min. The excitation wavelength (λex) and emission wavelength (λem) were 270 nm and 430 nm, respectively. The established method exhibited excellent reproducibility and recovery. The calibration curves were linear with regression coefficients over0.9990, and the detection limits (S/N=3) were 40, 31 fmol for the studied compounds. The practical applicability of the method was demonstrated by analyzing trace of free oestradiol and oestriol in the urine of root voles.