Simultaneously Determination of Fifteen Estrogens and Metabolites in Human Serum by an Efficient Liquid Chromatography-Tandem Mass Spectrometry Method

Abstract

Determination of female hormones and their metabolites in human serum is one of the most critical steps in human physiological and pathological diagnosis, such as in risk assessment for certain cancers. There are a number of concerns on sample derivatization, method specificity, sensitivity, separation efficiency and long elution time, although a large number of methods using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for determination of estrogens and metabolites have been published before. We successfully developed and validated an efficient Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method for determination of fifteen estrogens and metabolites in human serum. The estrogens and metabolites were derivatized with dansyl chloride and analyzed by LC-MS/MS using positive Electrospray Ionization (ESI+) and a Multiple Reaction Monitoring (MRM) mode. A Phenomenex Synergi Hydro-RP 2.5 μm 80 Å column (100 × 2.0 mm) at 60°C was eluted with mobile phases consisting of acetonitrile, methanol, water and formic acid at a flow rate of 0.4 mL/min. All of the fifteen dansyl derivatives of estrogen and metabolites were separated by LC with retention time differences between peaks ≥ 0.2 min. The recoveries of the fifteen estrogens and metabolites from hydrolysis and derivatization were in a range of 74.4-95.6% at 30-801 pg/mL level. The interday accuracies of the dansyl derivatives were in a range of 91.7-109.6% with a precision of 2.7-12.6%RSD. The LC-MS/MS system had linear responses to these derivatives within the calibration range of 12-10980 pg/mL with regression coefficient r2 ≥ 0.9934. The Limits of Quantification (LOQ) of these derivatives were in a range of 5.3-71.1 pg/mL in human serum. In comparison to a typical published method, our method provided a more efficient LC separation using only one-third of the elution time, and a similar or slightly better sensitivity.