Analysis of Serum Testosterone, Androstenedione and DHEAS for Clinical Research

Program: Abstracts - Orals, Poster Previews, and Posters
Session: SUN 203-235-Steroid Hormone Actions, Biosynthesis and Metabolism (posters)
Bench to Bedside
Sunday, April 3, 2016: 1:15 PM-3:15 PM
Exhibit/Poster Hall (BCEC)

Poster Board SUN 231
Laura Owen*1, Dominic Foley2, Brian G. Keevil3, Michelle Wills4 and Lisa Calton2
1MAHSC, 2Waters, Wilmslow, United Kingdom, 3University Hospital of South Manchester, Manchester, United Kingdom, 4Waters
Background: Here we evaluate a LC-MS/MS method for the measurement of serum testosterone, androstenedione and dehydroepiandrosterone sulfate (DHEAS) enabling investigation of metabolic dysfunction for clinical research.  An analytical method was developed using a novel Solid Phase Extraction (SPE) sorbent in 96-well plate format, reducing sample preparation time and removing more matrix interference in comparison to standard reverse phase SPE formats.  Chromatographic resolution between structurally related steroid species was achieved. 

Methods: Certified testosterone, androstenedione and DHEAS reference material purchased from Cerilliant (Round Rock, TX) were used to create calibrators and QC materials in stripped pooled serum purchased from Golden West Biologicals (Temecula, CA).  EQA samples obtained from UK NEQAS (Birmingham, UK) were analyzed for all analytes and compared to the mass spectrometry mean concentrations.  Serum samples were analyzed using the newly developed method and results were compared to an independent LC-MS/MS method for testosterone, androstenedione and DHEAS.  All samples were pre-treated with internal standard, methanol and water.  SPE was carried out with a Waters® Oasis® PRiME HLB µElution 96-well plate, which negated the need for conditioning and equilibration of the sorbent and allowed direct injection of the SPE eluate.  Offline automated extraction was performed using a Tecan Freedom Evo 100.  Samples were quantified with a Waters Xevo TQD mass spectrometer.

Results: The method was shown to be linear from 0.17 – 69 nmol/L for testosterone and androstenedione, and 0.14 – 54 µmol/L for DHEAS.  Coefficients of variation (CV) for total precision and repeatability on 5 separate days for low, mid and high QC samples were all ≤ 8.2% (n = 30) for all analytes.  Analytical sensitivity investigations demonstrate a CV < 20% at 0.17 nmol/L for testosterone and androstenedione, and 0.14 µmol/L for DHEAS.  The method has shown to be analytically selective through separation of isobaric steroid species and other matrix-bourne interferences, that could affect accuracy and imprecision.  Excellent agreement between this analytical method and the EQA MS mean values has been demonstrated for testosterone, androstenedione and DHEAS, with mean method bias within ±5.8%.  In addition, excellent agreement between this method and an independent LC-MS/MS method has been demonstrated for the analysis of testosterone, androstenedione and DHEAS, with mean method bias within ±6.3%.

Conclusions: We have successfully quantified serum testosterone, androstenedione and DHEAS using SPE with LC-MS/MS for clinical research purposes. This offline automated method demonstrates excellent linearity, precision and accuracy, while providing high sample throughput capabilities.

For Research Use Only, Not for Use in Diagnostic Procedures.

Disclosure: DF: Employee, Waters. MW: Employee, Waters. LC: Employee, Waters. Nothing to Disclose: LO, BGK

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