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 8 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, Robert Wardle, Lisa J Calton
Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, UK
| Short Abstract Care must be taken when developing steroid hormone analytical methods for clinical research, to limit interferences which affect accuracy and precision. MS/MS provides a way to selectively differentiate between these hormones, while chromatography can provide separation of isobaric species. However, sample preparation is the key in providing LC-MS/MS analytical sensitivity by reducing matrix effects. We have developed an LC-MS/MS approach for the analysis of a wide range of steroid hormones. Extraction analytical sensitivity and phospholipid removal capability have been explored, which has shown that for a generic approach to steroid analysis, Oasis® PRiME HLB SPE is the most favourable option in providing optimal LC-MS/MS sensitivity. For Research Use Only, Not for Use in Diagnostic Procedures. |
Long Abstract
Background: The steroid biosynthetic pathway consists of many structurally related steroid hormones. Therefore, care must be taken when developing steroid hormone analytical methods for clinical research, to limit interferences which affect accuracy and precision. MS/MS provides a way to differentiate between these hormones , while the LC dimensions can provide separation of isobaric species. However, sample preparation is the key in providing optimal LC-MS/MS analytical sensitivity by reducing matrix effects. Here we evaluate different sample preparation methodologies to assess the optimum analytical sensitivity for a range of steroid hormones. In addition, as phospholipids are known to contribute to reduced method robustness, we evaluated the phospholipid removal capabilities of each extraction technique.
Methods: Certified reference material for the steroid hormones (adrenal and corticosteroids) were purchased from Cerilliant (Round Rock, TX) and spiked into pooled human serum purchased from Seralab (West Sussex, UK). Samples were extracted in triplicate using protein precipitation, liquid-liquid extraction (LLE), Oasis® MAX SPE, Oasis HLB SPE and Oasis PRiME HLB SPE. Using an ACQUITY UPLC® I-Class system, samples were injected onto a 2.1 x 50 mm Waters® ACQUITY UPLC HSS T3 column using a water/methanol/ammonium acetate gradient and quantified with a Waters Xevo® TQ-S micro mass spectrometer. Phospholipids were assessed by performing a Parent parent scan at m/z 184. Selected lysophosphatidylcholines at m/z 496, 520, 522 and 524 and phosphatidylcholines at m/z 704, 758, 760, 784, 786, 806 and 808 were chosen for phospholipid removal comparisons.
Results: Chromatographic separation of structurally related steroid hormones using the ACQUITY UPLC HSS T3 column was achieved within a 5 minute run time. Resolution of isobaric species was demonstrated, improving the analytical selectivity of the LC-MS/MS method. Sample preparation techniques were compared using the mean peak areas and S/N values obtained for the range of steroids. It was determined that across the range of steroids assessed, Oasis PRiME HLB was the best sample preparation option to use for optimum analytical sensitivity . LLE and Oasis MAX SPE were deemed unsuitable for the analysis of the steroid conjugate, DHEA sulfate. Recovery for this analyte was <2% of that seen for protein precipitation and reverse phase solid phase extract. This analyte is ionised across the workable pH range, and so does not partition well into organic solvent (LLE) or binds irreversibly to anion exchange SPE chemistries (Oasis MAX). The SPE methodologies were shown to provide lower recoveries than LLE, however, it was found that in general SPE reduced the background noise and greatly increased S/N. SPE was also found to provide greater phospholipid removal compared to protein precipitation and LLE techniques. Oasis PRiME HLB SPE, when using acetonitrile as the elution solvent, removed 94% of the phospholipids compared to the protein precipitation trace and 89% compared to the LLE trace. Therefore, it was deemed that Oasis PRiME HLB SPE is a more robust approach to analyze steroid hormones in comparison to other extraction methods.
Conclusions: We have developed an LC-MS/MS approach for the analysis of a wide range of steroid hormones for clinical research. Extraction sensitivity and phospholipid removal capability of the sample preparation methodologies have been explored, which has shown that for a generic approach to steroid analysis, Oasis PRiME HLB SPE is the best option in reducing chromatographic trace background and phospholipid interference, which provides improved analytical sensitivity and selectivity for both conjugated and non-conjugated steroid species. We intend to verify these findings by demonstrating the performance characteristics of a LC-MS/MS Oasis PRiME HLB SPE method for selected steroids hormones.
For Research Use Only, Not for Use in Diagnostic Procedures.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Waters Corporation |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | Waters Corporation |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |