Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry

Alissa Renne, Lindi Luo, Jonathan Jarow, William W Wright, Terry R. Brown, Haolin Chen, Barry R Zirkin, Marlin D. Friesen

Research output: Contribution to journalArticle

Abstract

An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17bestradiol (E2), and 5a-androstan-3a,17b-diol (3a-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-mL samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E2. Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E2 and 3a-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.

Original languageEnglish (US)
Pages (from-to)691-698
Number of pages8
JournalJournal of Andrology
Volume33
Issue number4
DOIs
StatePublished - Jul 2012

Fingerprint

Tandem Mass Spectrometry
Isotopes
Steroids
High Pressure Liquid Chromatography
Dihydrotestosterone
Electrospray Ionization Mass Spectrometry
Solid Phase Extraction
Environmental Exposure
Reverse-Phase Chromatography
Radioimmunoassay
Testosterone
Chlorides
Mass Spectrometry
Hormones
Pharmaceutical Preparations

Keywords

  • Androgen
  • Steroidogenesis
  • Testis

ASJC Scopus subject areas

  • Urology
  • Reproductive Medicine
  • Endocrinology, Diabetes and Metabolism
  • Endocrinology

Cite this

Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry. / Renne, Alissa; Luo, Lindi; Jarow, Jonathan; Wright, William W; Brown, Terry R.; Chen, Haolin; Zirkin, Barry R; Friesen, Marlin D.

In: Journal of Andrology, Vol. 33, No. 4, 07.2012, p. 691-698.

Research output: Contribution to journalArticle

Renne, Alissa ; Luo, Lindi ; Jarow, Jonathan ; Wright, William W ; Brown, Terry R. ; Chen, Haolin ; Zirkin, Barry R ; Friesen, Marlin D. / Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry. In: Journal of Andrology. 2012 ; Vol. 33, No. 4. pp. 691-698.
@article{e5065618a5834f2cb70386b035008e8e,
title = "Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry",
abstract = "An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17bestradiol (E2), and 5a-androstan-3a,17b-diol (3a-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-mL samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E2. Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E2 and 3a-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.",
keywords = "Androgen, Steroidogenesis, Testis",
author = "Alissa Renne and Lindi Luo and Jonathan Jarow and Wright, {William W} and Brown, {Terry R.} and Haolin Chen and Zirkin, {Barry R} and Friesen, {Marlin D.}",
year = "2012",
month = "7",
doi = "10.2164/jandrol.111.014977",
language = "English (US)",
volume = "33",
pages = "691--698",
journal = "Journal of Andrology",
issn = "0196-3635",
publisher = "American Society of Andrology Inc.",
number = "4",

}

TY - JOUR

T1 - Simultaneous quantification of steroids in rat intratesticular fluid by HPLC-isotope dilution tandem mass spectrometry

AU - Renne, Alissa

AU - Luo, Lindi

AU - Jarow, Jonathan

AU - Wright, William W

AU - Brown, Terry R.

AU - Chen, Haolin

AU - Zirkin, Barry R

AU - Friesen, Marlin D.

PY - 2012/7

Y1 - 2012/7

N2 - An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17bestradiol (E2), and 5a-androstan-3a,17b-diol (3a-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-mL samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E2. Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E2 and 3a-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.

AB - An isotope dilution mass spectrometry method has been developed for the simultaneous measurement of picolinoyl derivatives of testosterone (T), dihydrotestosterone (DHT), 17bestradiol (E2), and 5a-androstan-3a,17b-diol (3a-diol) in rat intratesticular fluid. The method uses reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Following derivatization of 10-mL samples of testicular fluid with picolinoyl chloride hydrochloride, the samples were purified by solid phase extraction before analysis. The accuracy of the method was satisfactory for the 4 analytes at 3 concentrations, and both inter- and intraday reproducibility were satisfactory for T, DHT, and E2. Measurements of intratesticular T concentrations in a group of 8 untreated adult rats by this method correlated well with measurements of the same samples by radioimmunoassay. As in men, there was considerable rat-to-rat variability in T concentration, despite the fact that the rats were inbred. Although its levels were more than an order of magnitude lower than those of T, DHT was measured reliably in all 8 intratesticular fluid samples. DHT concentration also varied from rat to rat and was highly correlated with T levels. The levels of E2 and 3a-diol also were measurable. The availability of a sensitive method by which to measure steroids accurately and rapidly in the small volumes of intratesticular fluid obtainable from individual rats will make it possible to examine the effects, over time, of such perturbations as hormone and drug administration and environmental toxicant exposures on the intratesticular hormonal environment of exposed individual males and thereby to begin to understand differences in response between individuals.

KW - Androgen

KW - Steroidogenesis

KW - Testis

UR - http://www.scopus.com/inward/record.url?scp=84866358647&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84866358647&partnerID=8YFLogxK

U2 - 10.2164/jandrol.111.014977

DO - 10.2164/jandrol.111.014977

M3 - Article

C2 - 22016356

AN - SCOPUS:84866358647

VL - 33

SP - 691

EP - 698

JO - Journal of Andrology

JF - Journal of Andrology

SN - 0196-3635

IS - 4

ER -