title: 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.
authors.affiliation: Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Tamae
authors.forename: Michael
authors.initials: M
authors.lastname: Byrns
authors.forename: Brett
authors.initials: B
authors.lastname: Marck
authors.forename: Elahe A
authors.initials: EA
authors.lastname: Mostaghel
authors.forename: Peter S
authors.initials: PS
authors.lastname: Nelson
authors.forename: Paul
authors.initials: P
authors.lastname: Lange
authors.forename: Daniel
authors.initials: D
authors.lastname: Lin
authors.forename: Mary-Ellen
authors.initials: ME
authors.lastname: Taplin
authors.forename: Steven
authors.initials: S
authors.lastname: Balk
authors.forename: William
authors.initials: W
authors.lastname: Ellis
authors.forename: Larry
authors.initials: L
authors.lastname: True
authors.forename: Robert
authors.initials: R
authors.lastname: Vessella
authors.forename: Bruce
authors.initials: B
authors.lastname: Montgomery
authors.forename: Ian A
authors.initials: IA
authors.lastname: Blair
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D000728:Androgens / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000728:Androgens
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