Selective quantification of estrogens in serum with aptamer-affinity and LC-MS/MS analysis

Abstract

In the previous study, we developed an accurate and automated immunologic mass spectrometry (iMS) method for the simultaneous quantification of multiple steroid hormones by target capturing with monoclonal antibodies and LC-MS/MS analysis. However, antibodies are less accessible to a wide variety of low-molecular-weight analytes, which limited the application of iMS method. At the same time, multiple monoclonal antibodies needed also increased the complexity and cost of the iMS assay. In the present study, we developed an aptamer-affinity and LC-MS/MS analysis method with a class-specific aptamer for the selective enrichment and simultaneous determination of estrogens (estrone (E1) and estradiol (E2)) in human serum. The DNA aptamer was coupled to magnetic beads to enrich the target estrogens from serum sample after protein precipitation. The magnetic beads were then washed and eluted with acetonitrile solution, and targets were analyzed with LC-MS/MS. The sample preparation process of enrichment, washing and elution works in an automatic manner. Matrix effect was negligible as the high specificity of target recognition and enrichment by the aptamer. The aptamer-affinity assay for estrogen quantification was validated for sensitivity, linearity, accuracy and precision. The limit of detection (LOD) and quantification (LOQ) were0.01 ng/mLand0.02 ng/mL for E1 and E2, respectively. Satisfactory coefficients of variation (CVs) were obtained, with the intra-batch CVs of5.5 %-7.3 % and 5.2 %-6.0 % for E1 and E2, respectively, and inter-batch CVs of 3.8 %-10.0 % and 5.2 %-6.3 % for E1 and E2, respectively. Recoveries were in the range of99.1 %-107.0 % and 94.9 %-107.7 % for E1 and E2, respectively. Quantification results of 40 serum samples by the aptamer-affinity and solid-phase extraction (SPE) LC-MS/MS method showed good consistency, with the correlation coefficient of 0.93 and 0.98 for E1 and E2, respectively. Clinical application of the aptamer-affinity and LC-MS/MS method was further demonstrated for quantifying serum estrogen levels of 170 pregnant women at different gestational weeks.