Dominic Foley (Presenter)
Waters Corporation
Bio: Dominic Foley holds a BSc in Biochemistry from the University of Manchester and is a Senior Application Scientist working at Waters MS Headquarters in Wilmslow, UK. Having joined the company in September 2013, he has specialized in the development of LC-MS/MS clinical research applications, with a particular focus on steroid hormone analysis. Dominic has worked the mass spectrometry industry for 9 years, but started his career with a well-known CRO in the north of the UK where he worked as a LC-MS/MS method development and validation scientist.
Authorship: Dominic Foley, Lisa Calton
Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, UK
| Short Abstract Here we evaluate an offline automated method for the measurement of serum androgens; testosterone, androstenedione and dehydroepiandrosterone sulfate (DHEAS), and serum corticosteroids: 17-hydroxyprogesterone (17-OHP), cortisol, 11-deoxycortisol and 21-deoxycortisol, enabling steroid profiling for the investigation of metabolic dysfunction biomarkers for clinical research. An LC-MS/MS method was developed using a novel Solid Phase Extraction (SPE) sorbent, reducing sample preparation time and removing more matrix interference in comparison to other sample preparation techniques. Chromatographic resolution between structurally related steroid species was achieved. This offline automated method demonstrates excellent linearity, analytical sensitivity, selectivity, precision and accuracy, while providing high sample throughput capabilities. |
Long Abstract
Background: Here we evaluate an offline automated method for the measurement of serum androgens; testosterone, androstenedione and dehydroepiandrosterone sulfate (DHEAS), and serum corticosteroids: 17-hydroxyprogesterone (17-OHP), cortisol, 11-deoxycortisol and 21-deoxycortisol, enabling steroid profiling for the investigation of metabolic dysfunction biomarkers for clinical research. An LC-MS/MS 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 other sample preparation techniques. Chromatographic resolution between structurally related steroid species was achieved. This is critical in steroid analysis, as MS/MS Multiple Reaction Monitoring (MRM) alone cannot provide the analytical selectivity required to obtain highly precise and accurate data, particularly at low physiological concentrations.
Methods: Certified testosterone, androstenedione, DHEAS, cortisol, 11-deoxycortisol and 21-deoxycortisol 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). Serum samples purchased from UK NEQAS (Birmingham, UK) for testosterone, androstenedione, DHEAS, 17-OHP and cortisol were analyzed and concentrations were compared to the EQA MS mean for each steroid hormone.
100 µL serum 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, while providing phospholipid removal and allowing direct injection of the SPE eluate. Offline automated extraction was performed using a Tecan® Freedom Evo 100. Using an ACQUITY UPLC® I-Class system, samples were injected onto a 2.1 x 50 mm Waters ACQUITY UPLC HSS T3 column with a pre-column T3 VanGuard™ using a water/methanol/ammonium acetate/formic acid gradient and quantified with a Waters Xevo® TQ-S micro mass spectrometer.
Results: Using a parent scan at m/z 184 to detect phospholipids containing the phosphocholine polar head group structure, the Oasis PRiME HLB SPE sorbent demonstrated >97% phospholipid depletion compared to protein precipitation. The developed method was shown to be linear for the serum androgens and corticosteroids. No significant carryover was observed from high concentrations serum samples into serum blanks. A 1:5 dilution was successfully performed on over-range samples for the serum steroids with accuracies ranging from 97-107% and CVs < 7%. Coefficients of variation (CV) for total precision and repeatability on five separate days for low, mid and high QC samples were all ≤ 9.1% (n = 30) for all analytes. Analytical sensitivity investigations performed over three occasions demonstrate a CV < 20% at 0.10 nmol/L for testosterone, 0.09 nmol/L for androstenedione, 0.19 nmol/L for 17-OHP, 0.011 µmol/L for DHEAS, 0.35 nmol/L for cortisol, and 0.72 nmol/L for 11-deoxycortisol and 21-deoxycortisol. S/N (PtP) calculations at each of these concentrations were > 10:1. Matrix Factor experiments demonstrate the internal standard compensates for ion suppression observed in the method, with accuracies of 93 – 100% and CVs < 3% for the serum steroids. The method has shown to be analytically selective through separation of isobaric steroid species and matrix specific interferences such as albumin, triglycerides and bilirubin that could affect accuracy and imprecision. Excellent agreement between this analytical method and the EQA LC-MS mean values have been demonstrated with mean method bias of -0.1%, -5.1%, 5.2%, -7.1% and -1.4% for testosterone, androstenedione, 17-OHP, DHEAS and cortisol, respectively.
Conclusions: We have successfully quantified serum androgens and corticosteroids using SPE with LC-MS/MS for clinical research purposes. This offline automated method demonstrates excellent linearity, analytical sensitivity, selectivity, precision and accuracy, while providing high sample throughput capabilities.
For Research Use Only, Not for use in diagnostic procedures.
References & Acknowledgements:
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IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |