Simultaneous quantitation of nine hydroxy-androgens and their conjugates in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.

ABSTRACT: Castration resistant prostate cancer (CRPC), the fatal form of prostate cancer, remains androgen dependent despite castrate levels of circulating testosterone (T) and 5?-dihydrotestosterone (DHT). To investigate mechanisms by which the tumor can synthesize its own androgens and develop resistance to abiraterone acetate and enzalutamide, methods to measure a complete androgen profile are imperative. Here, we report the development and validation of a stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometric (SID-LC-ESI-MS/MS) method to quantify nine human hydroxy-androgens as picolinates, simultaneously with requisite specificity and sensitivity. In the established method, the fragmentation patterns of all nine hydroxy-androgen picolinates were identified, and [13C3]-5?-androstane-3?, 17?-diol and [13C3]-5?-androstane-3?, 17?-diol used as internal standards were synthesized enzymatically. Intra-day and inter-day precision and accuracy corresponds to the U.S. Food and Drug Administration Criteria for Bioanalytical Method Validation. The lower limit of quantitation (LLOQ) of nine hydroxy-androgens is 1.0pg to 2.5pg on column. Diols which have been infrequently measured: 5-androstene-3?, 17?-diol and 5?-androstane-3?, 17?-diol can be determined in serum at values as low as 1.0pg on column. The method also permits the quantitation of conjugated hydroxy-androgens following enzymatic digestion. While direct detection of steroid conjugates by electrospray-ionization tandem mass spectrometry has advantages the detection of unconjugated and conjugated steroids would require separate methods for each set of analytes. Our method was applied to pooled serum from male and female donors to provide reference values for both unconjugated and conjugated hydroxy-androgens. This method will allow us to interrogate the involvement of the conversion of 5-androstene-3?, 17?-diol to T, the backdoor pathway involving the conversion of 5?-androstane-3?, 17?-diol to DHT and the inactivation of DHT to 5?-androstane-3?, 17?-diol in advanced prostate cancer.

SUBMITTER: Zang T 

PROVIDER: S-EPMC5146996 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature