Quantitation of endogenous steroids in serum using plasma separation cards and triple quadruple mass spectrometry

Significance of the topic

Endogenous steroids are key regulators of metabolic pathways, stress response, and reproductive functions. Accurate measurement of these hormones at low concentrations in small-volume samples is critical for clinical diagnostics, research applications, and quality control in pharmaceutical and biomedical laboratories.

Objectives and Study Overview

This study aimed to develop and evaluate a sensitive and accurate LC-MS/MS method for quantifying selected endogenous steroids in serum using plasma separation cards. The work compared results obtained via triple quadrupole mass spectrometry with conventional ELISA immunoassays to assess correlation, accuracy, and precision across cortisol, DHEAs, testosterone, and progesterone.

Methodology

Sample collection utilized Telimmune DUO plasma separation cards producing 6 µL of whole blood and yielding about 3 µL of separated plasma per spot. Calibration standards, quality controls, and serum samples were applied to the cards, dried, and extracted using DPX XTR dispersive SPE tips packed with 20 mg Swift HLB sorbent following a Bind-Wash-Elute protocol. Extracts were analyzed by LC-MS/MS under MRM mode.

Used Instrumentation

• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with IonFocus design
• Shimadzu Nexera XR HPLC system (pump, autosampler, column oven)
• Shim-pack Velox C18 column (100 mm × 3.0 mm I.D., 2.7 µm)
• DPX XTR tips with 20 mg Supel HLB sorbent (DPX170357)
• Telimmune DUO plasma separation cards
• Absorbance microplate reader set at 450 nm for ELISA assays

Main Results and Discussion

Six-point calibration curves were established for each analyte with three QC levels. Calibration ranges were 5–1250 ng/mL for cortisol, 40–10,000 ng/mL for DHEAs, and 0.1–25 ng/mL for both testosterone and progesterone. All analytes demonstrated linear responses (R² > 0.99) using 1/C weighting without forcing the zero point. Accuracy across calibrators and QCs ranged between 80% and 120%.

Comparison with ELISA for testosterone showed percent differences below 15% and a strong correlation coefficient (r² = 0.9932) across five independent samples, confirming the method’s reliability.

Benefits and Practical Applications

The approach offers a minimally invasive sampling workflow with significantly reduced sample volumes and eliminates manual punch steps required for traditional dried blood spots. High sensitivity, robust accuracy, and strong agreement with immunoassays make this method suitable for clinical research, therapeutic monitoring, and high-throughput screening applications.

Future Trends and Potential Applications

Future work will expand the method to cover a broader panel of steroids and other low-abundance biomarkers. Advances may include automation of the dispersive SPE workflow, integration with point-of-care devices, and adaptation to other biological matrices to support decentralized testing and personalized medicine.

Conclusion

A highly sensitive and precise LC-MS/MS method for steroid quantitation from plasma separation cards was established, demonstrating excellent linearity, accuracy, and strong correlation with ELISA immunoassays. The protocol streamlines sample preparation, reduces volume requirements, and is well-suited for research and QC laboratories.

References

No specific literature references were provided in the original text.