Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography–electrospray ionization tandem mass spectrometry

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M + H]+ as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC–ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0 pg/ml, 96.0% and 9.9% for estrone, and 0.5 pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.