2-Hydrazino-1-methylpyridine: a highly sensitive derivatization reagent for oxosteroids in liquid chromatography–electrospray ionization-mass spectrometry

A derivatization reagent, 2-hydrazino-1-methylpyridine, was developed for the liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) of oxosteroids. The reagent quantitatively reacted with oxosteroids at 60 °C within 1 h and the resulting derivatives of the mono-oxosteroids provided a 70–1600-fold higher sensitivity compared to intact steroids. However, HMP was unsuitable for di-oxosteroids, such as androstenedione and progesterone. The developed derivatization procedure was applied to the LC–ESI-MS analysis of 5α-dihydrotestosterone in human prostate, and allowed the reproducible quantification of nanogram/gram level of the androgen with a 10-mg sample.