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Abstract Background: Dried Blood Spots (DBS) are an established microsampling technique providing a low-cost approach of collecting, shipping and analyzing samples for clinical research. Ligand-binding assays (LBAs) are often used as the frontline testing methodologies for DBS samples in steroid hormone analysis. Although rapid, the relatively low analytical specificity of the LBAs may necessitate follow-up, using liquid chromatography – tandem mass spectrometry (LC-MS/MS). The challenge is to create a fast, analytically sensitive and selective LC-MS/MS methodology for clinical research.
Methods: Certified androstenedione, 17-OHP, cortisol, 11-deoxycortisol and 21-deoxycortisol reference material purchased from Sigma Aldrich (Poole, UK) were used to create calibrators and QC materials in whole blood; prepared by mixing 50/50 (v/v) red blood cells from BioIVT (West Sussex, UK) and MSG4000 stripped serum from Golden West Biologicals (CA, USA). Blood spots were prepared by adding 50µL whole blood calibrators and QCs to Whatman 903 Protein Saver Blood Spot cards from Sigma Aldrich (Poole, UK) and then left to dry. Two 3mm DBS samples were pre-treated with an internal solution and mixed for 5 minutes. SPE was carried out with a Waters Oasis™ MAX µElution 96-well plate, 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 2.1mm x 50mm CORTECS C18 2.7µm column with pre-column CORTECS C18 2.7µm VanGuard using a water/methanol/ammonium fluoride gradient and quantified with a Xevo™ TQ-S micro mass spectrometer.
Results: The method enabled rapid separation in 1.4 minutes (2.3 minutes injection to injection) for 17-OHP, androstenedione, cortisol, 11-deoxycortisol and 21-deoxycortisol with baseline resolution of steroid isobars. Calibration lines were linear from 0.5 – 500 ng/mL for androstenedione and 11-deoxycortisol; and 1.0 – 500 ng/mL for cortisol, 17-OHP and 21-deoxycortisol with correlation coefficients (r2) >0.99 over five occasions. Coefficients of variation (CV) for total precision and repeatability over five occasions at four concentrations; 2, 5, 50 and 400ng/mL, were ≤ 9.3% (n = 25) with accuracies ranging from 94 – 110%.
Conclusions: The challenge was met by using Ultra Performance Liquid Chromatography (UPLC™) combined with CORTECS™ 2.7µm particle columns to provide UPLC separations at high linear velocities with minimal loss in column performance. This offline automated method demonstrates excellent linearity, analytical sensitivity, precision and accuracy, while providing high sample throughput capabilities for the analysis of androstenedione, 17-OHP, cortisol, 11-deoxycortisol and 21-deoxycortisol in dried blood spots for clinical research purposes.
For Research Use Only, Not for use in diagnostic procedures.
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= Discovery stage.
= Translation stage.
= Clinically available.
