Development and application of electrospray-active derivatization reagents for hydroxysteroids

New derivatization reagents, 1-(2,4-dinitro-5-fluorophenyl)-4-methylpiperazine (PPZ) and 4-(4-methyl-1-piperazyl)-3-nitrobenzoyl azide (APZ), were developed for the liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) determination of steroids having a hydroxy group. PPZ reacted with a phenolic hydroxy group in estrogens. After quaternarization of the PPZ-estrogens with methyl iodide, the resulting derivatives provided more than a 2000-fold higher sensitivity compared to the intact estrogens. After derivatization of steroids having an alcoholic hydroxy group (5-ene-steroids or 5α-reduced steroids) with APZ followed by methylation, their detection responses increased more than 500 times. These derivatization procedures coupled with LC–ESI-MS/MS were successfully used for the determination of estrogens in the serum and prostatic 5α-dihydrotestosterone.