• Application Note

Rapid UPLC-MS/MS Dried Blood Spot Analysis of Steroid Hormones for Clinical Research

Rapid UPLC-MS/MS Dried Blood Spot Analysis of Steroid Hormones for Clinical Research

  • Dominic Foley
  • Heather A. Brown
  • Lisa J. Calton
  • Waters Corporation
Blood spot self test on filter

For research use only. Not for use in diagnostic procedures.

This is an Application Brief and does not contain a detailed Experimental section.


A rapid UPLC-MS/MS method for the analysis of steroid hormones in dried blood spots is demonstrated using the Xevo TQ-S micro Mass Spectrometer.


Rapid separation and analysis of steroid hormones in dried blood spots was achieved using the ACQUITY UPLC I-Class System/Xevo TQ-S micro Mass Spectrometer and CORTECS Column.


Dried blood spots (DBS) are an established micro sampling technique providing a low-cost approach of collecting, shipping, and analyzing samples for clinical research. Ligand-binding assays (LBAs) are the established 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 multiplexed LC-MS/MS analysis of steroid hormones panels provides greater detail about the underlying enzyme activity, compared with single-analyte LBAs, which is important for the assessment of biomarkers in clinical research. The challenge is to create a LC-MS/MS methodology which separates key analytes from matrix and isobaric interferences, while maximizing throughput. 

UltraPerformance Liquid Chromatography (UPLC) on the ACQUITY UPLC I-Class System enables high pressure chromatographic separations in short analysis times. Combining the ACQUITY UPLC with a CORTECS 2.7 µm particle Column provides UPLC separations at high linear velocities without a loss in column performance, particularly in regard to critical  pair separations.

Results and Discussion

Through utilization of the ACQUITY UPLC I-Class System and the CORTECS Column technology, separation of cortisol, 21-deoxycortisol, 11-deoxycortisol, androstenedione, and 17-hydroxyprogesterone (17-OHP) from other endogenous steroid hormones was achieved within a 2.3 minute injection cycle time (Figure 1). Analytically sensitive detection between 0.5–1.0 ng/mL in DBS samples was achieved for the steroid hormones using the Xevo TQ-S micro, following extraction employing Oasis MAX µElution SPE on the Tecan Freedom Evo 100 Liquid Handling System (LHS).

Chromatographic selectivity of the CORTECS Column Figure 1. Chromatographic selectivity of the CORTECS C18 2.7 µm Column for the separation of cortisol, 11-deoxycortisol, 21-deoxycortisol, androstenedione, and 17-OHP from other endogenous steroid hormones.

Sample preparation and UPLC-MS/MS analysis

Using the LHS, 150 µL internal standard was added to two 3 mm DBS samples, shaken for 5 minutes and diluted with water. The Oasis MAX SPE µElution Plate was conditioned with methanol prior to 700 µL supernatant being applied. This was followed by a wash with 1% (v/v) ammonia in 10% (v/v) acetonitrile (aq). Samples were eluted with 70% acetonitrile and diluted with water. 25 µL of extracted sample was injected on the ACQUITY UPLC I-Class System utilizing a 0.05 mM Ammonium fluoride/methanol gradient and a 2.7 µm, 2.1 mm x 50 mm CORTECS C18 Column with VanGuard Pre-Column. Detection was performed using Multiple Reaction Monitoring (MRM) on the Xevo TQ-S micro. The complete analytical workflow is shown in Figure 2.

Analytical workflow Figure 2. Analytical workflow for the analysis of DBS samples using Oasis MAX µElution SPE and UPLC-MS/MS analysis.

DBS 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.

Signal/Noise (S/N) at the LLOQ calibrators are shown in Figure 3, demonstrating the analytical sensitivity of the method from two 3 mm dried blood spots.

Total precision and repeatability of the method for analyzing the steroid hormones over five occasions was ≤9.3% CV (Table 1).

LLOQ chromatograms Figure 3. LLOQ chromatograms for cortisol, 21-deoxycortisol, 11-deoxycortisol, androstenedione, and 17-OHP in DBS samples.
Total precision and repeatability Table 1. Total precision and repeatability over five occasions for 17-OHP, androstenedione, cortisol, 11-deoxycortisol, and 21-deoxycortisol.


A UPLC-MS/MS method for the separation and detection of steroid hormones in dried blood spots has been developed for clinical research purposes. The benefits of this method include:

  • Rapid separation of steroid hormones within 1.4 minutes (2.3 minutes injection to injection) with baseline-resolution of steroid isobars, using the CORTECS 2.7 μm Column.
  • Improved method analytical sensitivity, laboratory efficiency, reproducibility, and reduced sample-handling time by using an automated, offline, Oasis MAX μElution SPE protocol for the extraction of steroid hormones from the DBS samples.
  • Extraction and analysis of 384 samples in less than 18 hours.
  • Using two 3 mm blood spots, the method was analytically sensitive (0.5–1 ng/mL) and reproducible (<9.3% CV).


We would like to thank Susan Johnston and colleagues from NHS Glasgow and Clyde for the provision of 17-OHP DBS calibrators for use during initial investigations.

720006551, April 2019

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