Robust and sensitive quantitation of 18 steroids in human serum using Thermo Scientific TSQ Certis Triple Quadrupole mass spectrometer

Importance of the topic

This work presents a validated, high-throughput LC–MS/MS workflow for simultaneous quantitation of 18 endogenous steroids in human serum. Accurate steroid measurement is critical in clinical research and biomonitoring for assessing endocrine regulation, reproductive health, stress response and diseases. Mass spectrometry-based multiplexed assays overcome limitations of immunoassays by providing superior specificity, sensitivity and dynamic range necessary to quantify low-abundance steroids in complex serum matrices.

Study objectives and overview

The primary objective was to develop and demonstrate a robust, sensitive and high-throughput LC–MS/MS method on the Thermo Scientific TSQ Certis triple quadrupole mass spectrometer for 18 steroid analytes in human serum. Key goals included optimizing ion source and MS conditions for maximal sensitivity, defining limits of quantification (LOQs) and linear ranges, evaluating accuracy and precision using commercial calibrators and QC materials, and demonstrating long-run stability suitable for routine laboratory workflows.

Methodology

Sample preparation:

• Calibration standards prepared by serial dilution of steroid standards in methanol.
• Calibration aliquots combined with isotope internal standard (IS) solution and 0.05% bovine serum albumin (BSA) in PBS to mimic serum matrix.
• Protein precipitation performed with ZnSO4 and ice-cold acetonitrile; extracts reconstituted in 100 µL of 50:50 water:methanol.
• Injection volume: 5 µL.

Chromatography and mass spectrometry:

• UHPLC system: Thermo Scientific Vanquish Horizon.
• Column: Hypersil Gold C18, 2.1 × 100 mm, 1.9 µm.
• Mobile phases: A = 0.2 mM ammonium fluoride in water; B = methanol.
• Flow: 0.300 mL/min; total runtime ~9 minutes per injection using a rapid gradient to separate the steroid panel.
• MS platform: Thermo Scientific TSQ Certis triple quadrupole operated in selected reaction monitoring (SRM) mode.
• Ion source: OptaMax Plus HESI; vaporizer temperature systematically optimized (300–600 °C) for signal enhancement.
• Data acquisition and quantitation: Thermo Scientific TraceFinder software (v5.2) using 1/x weighted linear regression and isotope-dilution calibration.

Internal standards:

• Isotope-labeled analogs were used for most analytes (examples: [2H5]-estradiol, [2H9]-progesterone, [2H3]-DHT, [13C3]-estrone, [2H5]-DHEA, [2H8]-21-DOC, [2H5]-11-DOC, [2H4]-cortisol, etc.) to correct for matrix effects and extraction variability.

Instrumentation used

• Vanquish Horizon UHPLC (Thermo Scientific).
• TSQ Certis triple quadrupole mass spectrometer (Thermo Scientific).
• OptaMax Plus HESI source (Thermo Scientific) with vaporizer temperature control.
• Hypersil Gold C18 analytical column (2.1 × 100 mm, 1.9 µm).
• TraceFinder software v5.2 for data processing and quantitation.

Main results and discussion

Analytical sensitivity and linearity:

• LOQs on column spanned approximately 0.01 to 5.00 pg on column across the 18 steroids, demonstrating sub-picogram to low-picogram sensitivity for many targets.
• Linear concentration ranges extended up to four orders of magnitude for several analytes (e.g., 0.01–20 pg o.c. for testosterone, up to 2000 pg o.c. for select analytes), with coefficients of determination (R²) typically >0.99.

Precision and accuracy:

• Calibration curves used external calibration against a commercial serum calibrator set (6PLUS1 Multilevel) and isotope-dilution quantitation. All analytes showed R² >0.99 over their specified ranges.
• Quality control samples (QC-L, QC-M, QC-H) quantified using these calibrations returned measured concentrations within ±30% of expected values, with %RSD generally <14%.
• At LOQ levels %RSD and %CV of peak area remained acceptably low (typical %RSD at LOQ ranged around 3–12% depending on analyte), supporting reliable quantitation at the low end.

Robustness and long-run stability:

• Method robustness was demonstrated over 311 injections of calibration samples in a 0.05% BSA matrix; internal standard peak area %RSD values remained below 17%, indicating stable MS response and suitability for high-throughput operation.

Source optimization and sensitivity gains:

• Systematic optimization of the HESI vaporizer temperature (300–600 °C) revealed up to two-fold signal increases for most steroids and very large enhancements (6–14×) for water-loss precursors such as DHT, DHEA and pregnenolone.
• A vaporizer temperature of 525 °C was selected as a global optimum balancing signal gains and operational stability.

Representative chromatography and selectivity:

• A 9-minute gradient provided baseline or sufficient separation for the panel; extracted ion chromatograms and SRM transitions (quantifier and qualifier) confirmed selective detection and appropriate ion ratios at LOQ levels.

Key practical benefits and applications

• High sensitivity enables measurement of low-abundance steroids (e.g., estradiol, DHT) that are challenging with immunoassays.
• Short run time (~9 min) and demonstrated long-run stability support high sample throughput needed in clinical and research laboratories.
• Use of isotope-labeled internal standards and external calibrators provides traceable, reproducible quantitation suitable for biomonitoring, pharmacodynamic studies and endocrine disorder research.
• Optimized source conditions (vaporizer temperature) deliver substantial signal improvements for difficult-to-ionize steroids, reducing LOQs and improving confidence at clinical decision limits.

Limitations and considerations

• Calibration and QC assessment used commercial serum calibrators and BSA-based surrogate matrices; real patient matrices may present variable matrix effects requiring in-house verification and possible additional matrix-matched QC.
• Some analytes present at very low physiological levels may still require careful handling to avoid adsorption or loss during prep; robust internal standards mitigate but do not eliminate pre-analytical variability.

Future trends and potential uses

• Broader adoption of optimized ion-source operating windows (e.g., elevated vaporizer temperatures) to improve detection of water-loss or poorly ionizing steroids across platforms.
• Extension of panels to include steroid metabolites and conjugates (sulfates, glucuronides) using complementary sample preparation workflows.
• Integration with automated sample preparation and on-line SPE to further increase throughput and reproducibility in clinical laboratories.
• Application of harmonized isotope-dilution methods and multi-center proficiency testing to support clinical adoption and inter-laboratory comparability.

Conclusions

The presented LC–MS/MS method on the TSQ Certis platform achieves sensitive, precise and robust simultaneous quantitation of 18 serum steroids with sub-picogram to low-picogram on-column LOQs, broad dynamic ranges, and strong long-term stability over hundreds of injections. Optimization of HESI source conditions provided substantial sensitivity gains for several challenging analytes. The workflow is well suited for high-throughput clinical research and biomonitoring applications where selectivity, sensitivity and reproducibility are required.

References

Original work and validation data as reported by Jingshu Guo, Courtney Patterson, Charles Maxey, Neloni Wijeratne, Kerry Hassell (Thermo Fisher Scientific). Thermo Fisher Scientific application note on TSQ Certis triple quadrupole mass spectrometer (2026).