Quantitation of Endogenous Steroids in Serum Using Dried Blood Spot Serum Separator Card and Triple Quadrupole Mass Spectrometry

Importance of the Topic

Endogenous steroid hormones play a critical role in metabolic regulation, stress response, and reproductive health. Quantitative analysis of these low-level analytes in small-volume samples is challenging. Dried blood spot (DBS) serum separator cards offer a minimally invasive sampling alternative suited for at-home collection, but require sensitive methods to overcome matrix and volume limitations.

Objectives and Overview

This study aimed to develop and validate a robust LC-MS/MS method for quantifying four endogenous steroids in serum using DBS separator cards. Two card formats producing 30 µL and 6 µL of sample were compared. Method performance was evaluated via calibration curves, quality control samples, precision testing, and comparison to established immunoassays.

Methodology

The analytical workflow comprised:

• Creation of six-point calibration curves and three QC levels in stripped human serum
• Sample application to two types of ADx serum separator cards and overnight drying
• Protein precipitation followed by dispersive SPE extraction using DPX XTR tips
• Reconstitution and analysis by HPLC coupled to triple quadrupole mass spectrometry with MRM acquisition

Instrumentation

Key instrumentation used included:

• Shimadzu Nexera XR HPLC system with autosampler and column oven
• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer
• Shim-pack Velox C18 column (100×3.0 mm, 2.7 µm)
• DPX XTR tips packed with Supel Swift HLB sorbent for dSPE cleanup
• ADx DBS serum separator cards providing 30 µL or 6 µL of serum/plasma

Main Results and Discussion

All analytes exhibited linear calibration curves with correlation coefficients (r2) above 0.99. Accuracy for calibrators and QC levels ranged from 80% to 120%. Intra-day and inter-day precision studies yielded RSDs below 8% across all QC levels. Comparison to immunoassay results on real samples showed differences within ±30%, confirming method validity. Both card formats delivered equivalent quantitation performance despite small sample volumes.

Benefits and Practical Applications

The developed method enables:

• Minimally invasive, at-home sample collection via DBS cards
• Accurate quantification of low-concentration steroids in limited-volume samples
• Efficient cleanup and high sensitivity using DPX dispersive SPE and triple quadrupole MS
• Potential integration into clinical, research, and quality control workflows for steroid profiling

Future Trends and Opportunities

Ongoing developments include expanding the analyte panel to additional steroids such as estrone, estradiol, estriol, DHT, and DHEA. Adoption of plasma separator cards with smaller disks may simplify sample handling and reduce logistics. Automation of the DPX cleanup process could further increase sample throughput for large-scale studies.

Conclusion

A sensitive and reliable LC-MS/MS method was successfully established for quantifying endogenous steroids using DBS serum separator cards. The approach demonstrated excellent analytical performance and agreement with immunoassays, supporting its application in clinical diagnostics and research with minimal invasive sampling.