Mass Spectrometry Techniques for Steroid Hormone Analysis

Mass spectrometry has become the cornerstone for the accurate measurement of steroid hormones across clinical, preclinical and research settings. The integration of chromatographic separation with tandem mass analysis enables precise quantification of multiple steroids in a single run, overcoming specificity limitations of classical immunoassays. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) remains the workhorse for targeted measurement of androgen, oestrogen, progestogen and corticosteroid panels in serum, plasma, urine and tissue extracts. Advances such as ultra-performance LC, supercritical fluid chromatography and high-resolution mass spectrometry have expanded dynamic range, improved isomer separation and accelerated analysis time. Sample preparation strategies—ranging from simple protein precipitation to enzymatic hydrolysis and solid-phase extraction in 96-well formats—ensure high sensitivity even in low-volume or lipid-rich samples. Derivatisation chemistries enhance ionisation efficiency for poorly ionising molecules such as oestrogens, while multiple reaction monitoring (MRM) affords superior selectivity in complex matrices. Beyond targeted assays, non-targeted “steroidomics” approaches employing high-resolution mass spectrometry are uncovering novel pathway intermediates, facilitating metabolome profiling in endocrine disorders. Collectively, these techniques support diagnostic biochemistry, paediatric and reproductive endocrinology, drug-metabolism studies and environmental steroid monitoring, underscoring global significance and practical utility.

Research from Nature Portfolio

A novel application of supercritical fluid chromatography coupled to tandem mass spectrometry has demonstrated high-throughput profiling of nineteen endogenous steroids in just five minutes from a mere 50 µL of human plasma. This method achieves broad calibration ranges with excellent linearity and sub-15 % precision, providing a rapid, robust platform for simultaneous measurement of estrogens, androgens, progestogens and corticosteroids. The combination of supercritical CO₂ mobile phase and positive-ion electrospray ionisation affords efficient isomer separation and reduced solvent usage, positioning the technique as a promising alternative to conventional LC-MS/MS for routine clinical and research laboratories.

Mass Spectrometry Techniques for Steroid Hormone Analysis publication trend

The graph below shows the total number of articles in mass spectrometry techniques for steroid hormone analysis across all publications each year (not limited to Nature Index journals).

Technical terms

Liquid chromatography–tandem mass spectrometry (LC-MS/MS): An analytical technique combining high-performance liquid separation with sequential mass detection to quantify target compounds with high specificity.

Supercritical fluid chromatography (SFC): A chromatographic method using supercritical CO₂ as the mobile phase, offering rapid separation and reduced solvent consumption, often coupled to MS/MS.

Multiple reaction monitoring (MRM): A mass spectrometric acquisition mode in which predefined precursor-product ion pairs are monitored, enhancing selectivity in complex matrices.

Derivatisation: Chemical modification of analytes to improve ionisation efficiency or chromatographic behaviour, frequently employed for low-abundance steroids such as oestrogens.

Solid-phase extraction (SPE): A sample cleanup technique in which analytes are retained on a sorbent and impurities are washed away, concentrating steroids and reducing matrix effects.

References

1. A Novel Targeted Analysis of Peripheral Steroids by Ultra-Performance Supercritical Fluid Chromatography Hyphenated to Tandem Mass Spectrometry. Scientific Reports (2018).
2. High throughput LC-MS/MS method for steroid hormone analysis in rat liver and plasma – unraveling methodological challenges. Talanta (2023).
3. Non-Steroidal Drug Interferences in a Quantitative Multisteroid LC-MS/MS Assay. Cells (2023).
4. New liquid chromatography tandem mass spectrometry reference data for estradiol show mini-puberty in both sexes and typical pre-pubertal and pubertal patterns. European Journal of Endocrinology (2024).
5. Derivatization of estrogens enhances specificity and sensitivity of analysis of human plasma and serum by liquid chromatography tandem mass spectrometry. Talanta (2015).
6. From a single steroid to the steroidome: Trends and analytical challenges. The Journal of Steroid Biochemistry and Molecular Biology (2020).