Abstract

Amplifex Diene reagent was employed to derivatize estradiol (E2) to enhance the analyte signal at low picogram concentrations. This derivatization enabled measurement of E2 (and other estrogens) in ESI+ mode, earlier retention times for analytes than other methods, avoidance of MS harmful ammonium fluoride in mobile phases, and an LLOQ below 1 pg/mL. The sample preparation workflow involved liquid–liquid extraction followed by Amplifex Diene derivatization for 10 min at ambient temperature. Samples were chromatographed using a standard C18 column and analyzed using a SCIEX 6500⁺ mass spectrometer. The assay calibrators were prepared in-house, traceable to certified reference materials, and ranged from 1.29 to 624 pg/mL. A method comparison to samples from the CDC HoSt program yielded a correlation coefficient of 0.9858 and bias of −1.37%. The LLOQ using certified reference material was 0.66 pg/mL. The intra-run precision was <9.00% for low- and high-level samples, whereas the inter-run precision was 15.2 and 5.43% for low- and high-level samples, respectively. No interference from other clinically relevant steroids was found. Amplifex Diene derivatized E2 and estrone (E1) was found to be stable for over 6 months, both refrigerated and frozen.

中文翻译：

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灵敏且特异的ESI阳性LC-MS / MS方法可在3分钟内定量测定人血清中的雌激素

摘要

使用Amplifex Diene试剂衍生化雌二醇（E2），以在低皮克浓度下增强分析物信号。这种衍生化可以在ESI +模式下测量E2（和其他雌激素），比其他方法更早地保留分析物，避免在流动相中使用MS有害的氟化铵，并且LLOQ低于1 pg / mL。样品制备工作流程涉及液-液萃取，然后在环境温度下将Amplifex Diene衍生化10分钟。使用标准C18色谱柱色谱分离样品，并使用SCIEX 6500 ⁺质谱仪。该检测校准物是在实验室内部制备的，可追溯至认证的参考物质，范围为1.29至624 pg / mL。与来自CDC HoSt程序的样本进行的方法比较得出相关系数为0.9858，偏差为-1.37％。使用认证参考物质的LLOQ为0.66 pg / mL。低水平和高水平样品的内部运行精密度分别小于9.00％，而低水平和高水平样品的内部运行精密度分别为15.2和5.43％。没有发现其他临床相关类固醇的干扰。发现Amplifex Diene衍生的E2和雌酮（E1）在冷藏和冷冻后均稳定6个月以上。