Analysis of Multiclass Steroids in Serum Using Agilent Ultivo Triple Quadrupole LC/MS

Importance of the Topic

The quantitative determination of steroid hormones at trace levels in serum is critical for clinical research, diagnostics, and therapeutic monitoring. Steroids regulate metabolism, immune response, and reproductive functions, and their accurate measurement supports disease diagnosis and precision medicine.

Aims and Overview of the Study

This work presents a streamlined workflow for simultaneous quantitation of 15 steroid analytes in human serum. It combines automation-ready supported liquid extraction (SLE) on 96-well plates with a fast 14-minute LC separation and Agilent Ultivo triple quadrupole LC/MS in multiple reaction monitoring (MRM) mode. Performance was benchmarked using certified reference samples from ChromSystems.

Methodology and Instrumentation

The sample preparation uses Agilent Chem Elut S 2 mL 96-well plates on a positive pressure manifold for rapid, reproducible extraction of steroids from 350 µL serum aliquots spiked with deuterated internal standards. Elution is achieved by sequential gravity additions of ethyl acetate/MTBE. Extracts are evaporated and reconstituted in 70/30 water/methanol mobile phase.

Used Instrumentation

• Agilent 1260 Infinity II LC system with binary pump, multisampler at 6 °C, multicolumn compartment, and inline 0.3 µm filter
• Poroshell 120 EC-C18 column (3.0×50 mm, 2.7 µm) with guard column, 45 °C
• Mobile phases: water and methanol, each with 0.5 mM ammonium fluoride; 14 min gradient at 0.5 mL/min
• Agilent Ultivo LC/TQ (G6465B) with Jet Stream ESI source; MRM acquisition optimized by MassHunter Optimizer

Main Results and Discussion

Limits of detection (MDL) ranged from 8 ng/L for aldosterone to 43,500 ng/L for DHEAS, all suitably below lowest calibrator levels. Calibration curves over six levels exhibited excellent linearity (R²>0.995). Precision for retention time (%RSD<0.4%) and response ratio (%RSD<10%) met acceptance criteria. Accuracy across calibrators fell between 85–115%. Recovery from three QC levels (LQC, MQC, HQC) ranged 82–112% with intraday repeatability ≤12% and interday reproducibility ≤10%. Carryover after the highest calibrator was below 0.05% relative to L6 and <3% relative to L1.

Benefits and Practical Applications

• High specificity and sensitivity for multiplexed steroid profiling in clinical and research laboratories
• Automation-friendly SLE reduces manual steps and improves throughput compared to liquid–liquid extraction
• Short chromatographic runtime increases sample throughput while maintaining separation of isobaric compounds
• Robust performance for routine QA/QC with certified reference materials ensures reliable data

Future Trends and Applications

Advances may include further miniaturization of sample preparation, integration with high-throughput robotic platforms, and extension to additional steroid metabolites. Coupling with high-resolution MS could enhance structural elucidation and broaden target panels for metabolic or endocrine studies.

Conclusion

The described SLE-LC/TQ workflow provides a rapid, sensitive, and reproducible approach for simultaneous quantitation of 15 steroids in serum. Its automation readiness and validated performance make it a valuable tool for clinical research and routine laboratory analysis.

Reference

• Greaves RF, et al. A Guide to Understanding the Steroid Pathway: New Insights and Diagnostic Implications. Clin Biochem. 2014;47:5–15.
• Ketha H, et al. Clinical Applications of LC/MS Sex Steroid Assays: Evolution of Methodologies in the 21st Century. Curr Opin Endocrinol Diabetes Obes. 2014;21:217–226.
• Couchman L, et al. Challenges and Benefits of Endogenous Steroid Analysis by LC/MS/MS. Bioanalysis. 2011;3(22):2549–2572.