Quantitative Analysis of Steroid Hormones Using Triple Quadrupole LC/MS/MS

Significance of the Topic

The precise quantitation of steroid hormones in human serum is central to clinical research, diagnosis and therapy monitoring. These bioactive molecules regulate metabolism, signaling and neurological functions, and their dysregulation is linked to endocrine disorders, cancer and neurological diseases. A robust analytical method enables accurate measurement at low concentrations and supports advances in personalized medicine.

Study Objectives and Overview

This application note describes a workflow for the simultaneous quantitation of 13 steroid hormones in human serum using triple quadrupole liquid chromatography–tandem mass spectrometry (LC/MS/MS). The aim was to develop a streamlined pretreatment procedure based on supported liquid extraction (ISOLUTE SLE+) and to validate analytical performance in terms of linearity, recovery and matrix effects.

Methodology and Sample Preparation

Serum samples (100 μL) were diluted with ultrapure water and loaded onto ISOLUTE SLE+ plates. After absorption, steroids were eluted with dichloromethane, evaporated to dryness, and reconstituted in mobile phase before LC/MS/MS analysis. Key steps:

1. Dilution of serum with water and mixing.
2. Application to SLE+ plate and absorption (5 min).
3. Elution with dichloromethane (2× 900 μL) under gravity and pressure.
4. Evaporation at 40 °C and reconstitution in water/methanol buffer.

Used Instrumentation

• LC System: Shimadzu Nexera X2 with Shim-Pack FC-ODS column (75×2 mm, 3 µm) at 40 °C.
• Mobile Phase: 5 mM ammonium formate in water (A) and methanol (B); gradient from 35 % to 95 % B over 18 min; flow rate 0.3 mL/min.
• MS Detector: Shimadzu LCMS-8050 triple quadrupole with ESI source; interface temperature 370 °C; heat block 450 °C; DL 110 °C; nebulizer, drying and heating gas flows optimized.
• MRM Transitions: Specific precursor/product ion pairs for each steroid and corresponding internal standards.

Main Results and Discussion

Calibration curves for all 13 hormones exhibited excellent linearity (r2 ≥ 0.997) across concentration ranges spanning sub-nanogram to 100 ng/mL. Recovery rates for 12 analytes ranged from 80 % to 107 %, and matrix factors indicated moderate ion suppression but were consistent and correctable. Implementation of an ion-pair reagent (25 mM dibutyl ammonium acetate) during extraction enhanced recovery of the sulfated steroid DHEAS, demonstrating the method’s flexibility to address challenging analytes.

Benefits and Practical Applications

This approach offers:

• High sensitivity and wide dynamic range for simultaneous steroid profiling.
• Efficient removal of proteins, phospholipids and salts via SLE+, reducing matrix interference.
• Reproducible recovery and matrix factor data to support quantitative reliability.
• Applicability in clinical and research laboratories for endocrine studies, therapeutic monitoring and biomarker discovery.

Future Trends and Applications

Advances may include integration of automated sample prep platforms for higher throughput, expansion to additional steroid metabolites and conjugates, and coupling with high-resolution mass spectrometry for untargeted profiling. Emerging sorbent materials and microextraction techniques could further reduce sample volume and improve recovery, supporting personalized diagnostics and large-scale epidemiological studies.

Conclusion

The described triple quadrupole LC/MS/MS method, combined with ISOLUTE SLE+ pretreatment, provides a robust, sensitive and reproducible platform for quantifying a broad panel of steroid hormones in human serum. Its validated performance underscores its suitability for clinical research and routine laboratory applications.