Androgenic biomarker profiling in human matrices and cell culture samples using high throughput, electrospray tandem mass spectrometry

Authors

  • John H. Wilton,

    Corresponding author
    1. PK/PD Core Resource, Roswell Park Cancer Institute, Buffalo, New York
    • Correspondence to: John H. Wilton, PhD, PK/PD Core Resource, CGP L1-140, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY.

      E-mail: john.wilton@roswellpark.org

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    • Senior Mass Spectroscopist.
  • Mark A. Titus,

    1. Department of Urology, Roswell Park Cancer Institute, Buffalo, New York
    Current affiliation:
    1. David H. Koch Center, M.D. Anderson Cancer Center, University of Texas, Houston, TX
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    • Associate Professor.
  • Eleni Efstathiou,

    1. David H. Koch Center, M.D. Anderson Cancer Center, University of Texas, Houston, Texas
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    • Associate Professor.
  • Gerald J. Fetterly,

    1. PK/PD Core Resource, Roswell Park Cancer Institute, Buffalo, New York
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    • PK/PD Core Director.
  • James L. Mohler

    1. Department of Urology, Roswell Park Cancer Institute, Buffalo, New York
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    • Senior Vice President for Translational Research.

  • Conflicts of interest: No potential conflicts of interest were disclosed.

Abstract

BACKGROUND

A high throughput, high pressure liquid chromatographic (HPLC) method with triple quadrupole mass spectral detection (LC/MS/MS) was validated for the measurement of 5 endogenous androgens in human plasma and serum and applied to various in vivo and in vitro study samples to pursue a better understanding of the interrelationship of the androgen axis, intracrine metabolism, and castration-recurrent prostate cancer (CaP).

METHODS

A Shimadzu HPLC system interfaced with a Sciex QTRAP 5500 mass spectrometer with electrospray ionization was used with inline column-switching. Samples were liquid/liquid extracted and chromatographed on a Luna C18(2) column at 60°C with a biphasic gradient using a 15-min run time.

RESULTS

The method was validated for five androgens in human plasma and serum, and applied to four sets of samples. Plasma (n = 188) and bone marrow aspirate (n = 129) samples from patients with CaP, who received abiraterone acetate plus prednisone for up to 945 days (135 weeks), had undetectable androgens after 8 weeks of treatment. Plasma dehydroepiandrosterone (DHEA) concentrations were higher in African Americans than Caucasian Americans with newly diagnosed CaP. Analysis of prostate tumor tissue homogenates demonstrated reproducible testosterone (T) and dihydrotestosterone (DHT) concentrations with a minimal sample size of ∼1.0–2.0 mg of tissue. Finally, cell pellet and media samples from the LNCaP C4-2 cell line showed conversion of T to DHT.

CONCLUSION

The proposed LC/MS/MS method was validated for quantitation of five endogenous androgens in human plasma and serum, and effectively profiles androgens in clinical specimens and cell culture samples. Prostate 74:722–731, 2014. © 2014 Wiley Periodicals, Inc.

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