Development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization/selected reaction monitoring/mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of keto-androgens in human serum

Abstract

Prostate cancer is the most frequently diagnosed form of cancer in males in the United States. The disease is androgen driven and the use of orchiectomy or chemical castration, known as androgen deprivation therapy (ADT) has been employed for the treatment of advanced prostate cancer for over 70 years. Agents such as GnRH agonists and non-steroidal androgen receptor antagonists are routinely used in the clinic, but eventually relapse occurs due to the emergence of castration-resistant prostate cancer. With the appreciation that androgen signaling still persists in these patients and the development of new therapies such as abiraterone and enzalutamide that further suppresses androgen synthesis or signaling, there is a renewed need for sensitive and specific methods to quantify androgen precursor and metabolite levels to assess drug efficacy. We describe the development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of serum keto-androgens and their sulfate and glucuronide conjugates using Girard-T oxime derivatives. The method is robust down to 0.2-4pg on column, depending on the androgen metabolite quantified, and can also quantify dehydroepiandrosterone sulfate (DHEA-S) in only 1μL of serum. The clinical utility of this method was demonstrated by analyzing serum androgens from patients enrolled in a clinical trial assessing combinations of pharmacological agents to maximally suppress gonadal and adrenal androgens (Targeted Androgen Pathway Suppression, TAPS clinical trial). The method was validated by correlating the results obtained with a hydroxylamine derivatization procedure coupled with tandem mass spectrometry using selected reaction monitoring that was conducted in an independent laboratory.

Trial registration: ClinicalTrials.gov NCT00298155.

Keywords: 5α-androstane-3,17-dione; 5α-dihydrotestosterone; A; ADT; Adione; Androgen metabolome; CD-FBS; CRPC; CV; DHEA; DHEA-G; DHEA-S; DHT; ELISA; Epi-T; GnRH; HSD; LC; LLOQ; Mass spectrometry; ND; NS; PSA; Prostate cancer; QC; RIA; SID-LC/ESI/SRM/MS; T; TAPS; TFA; Targeted Androgen Pathway Suppression clinical trial; UDP glucuronosyltransferase; UGT; androgen deprivation therapy; androsterone; castration resistant prostate cancer; charcoal dextran stripped fetal bovine serum; coefficient of variation; dehydroepiandrosterone; dehydroepiandrosterone glucuronide; dehydroepiandrosterone sulfate; enzyme-linked immunosorbent assay; epitestosterone; gonadotropin releasing hormone; hydroxysteroid dehydrogenase; liquid chromatography; lower limit of quantification; m/z; mass to charge ratio; not detected; not significant; prostate specific antigen; quality control; radioimmunoassay; stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry; testosterone; trifluoroacetic acid; Δ(4)-AD; Δ(4)-androstene-3,17-dione.

Copyright © 2013 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

Representative ion-chromatograms of androgen standards 100 pg on column. Channels (from top to bottom) correspond to mass transition 400.4–341.2 for Δ4-AD Girard T derivative (the chemical structure of which is depicted in the figure); mass transition 402.4–343.2 for DHEA, T, Adione and Epi-T Girard T derivatives; mass transition 404.4–345.2 for DHT and A Girard T derivatives; mass transition 405.4-346.2 for 13C3-T and mass transition 407.4-348.2 for 13C3-DHT.

Fig. 2

Calibration curves for quantifying each androgen metabolite using calibrators in the range from 2 to 1000 pg using CDS-FBS as a matrix. Formulas for each slope are displayed along with r2 values for each analyte.

Scheme 1

The androgen metabolome is critical for male development and is therapeutically targeted in advanced prostate cancer. The metabolites in black represent the classical pathway for T and DHT biosynthesis, which act as potent ligands for the androgen receptor. The metabolites in green represent the “backdoor pathway” for T and DHT biosynthesis. The red stars indicate the keto-androgens quantified using the Girard T derivatization, SID-LC-ESI-MS/MS method described in this paper. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Scheme 2

The work flow describing the quantification of conjugated and unconjugated keto-androgens using the Girard T derivatization and SID-LC-ESI-MS/MS method.