Steroid Hormone Analysis Service

What Are Steroid Hormones?

Steroid hormones are a group of lipophilic signaling molecules synthesized primarily from cholesterol. They exert their effects by binding to specific intracellular or membrane-bound receptors, regulating gene transcription, protein synthesis, and cellular activity. These hormones are categorized into distinct classes—glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, and their biosynthetic precursors—and play vital roles in maintaining metabolic balance, reproductive function, immune modulation, and stress adaptation.

Because steroid hormones can diffuse across membranes and have relatively short half-lives, their concentrations fluctuate rapidly in response to environmental, developmental, or physiological stimuli. This dynamic nature makes them valuable indicators in biochemical, physiological, and molecular studies.

Why Conduct Steroid Hormone Analysis?

Accurate steroid hormone analysis is fundamental for understanding biochemical pathways, evaluating endocrine function, and investigating physiological adaptations.

Our clients typically seek steroid hormone profiling to:

• Map Biosynthetic Pathways – Quantify hormone intermediates and end products for pathway characterization.
• Assess Hormonal Balance – Detect shifts in multiple hormones to study regulatory feedback mechanisms.
• Support Mechanistic Studies – Correlate hormone levels with gene expression, proteomics, or metabolomics data.
• Compare Experimental Conditions – Identify hormone-level changes across treatments, genotypes, or environmental conditions.

Creative Proteomics enables researchers to overcome common challenges such as low hormone abundance, matrix interference, and the need for simultaneous multi-hormone detection.

Comprehensive Steroid Hormone Analysis Service by Creative Proteomics

• Targeted LC-MS/MS and GC-MS/MS Quantification – Ultra-sensitive measurement of specific steroid hormones, ideal for validating biomarkers or confirming pathway intermediates.
• Comprehensive Multi-Hormone Panels – Simultaneous profiling of glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, and their key precursors in a single run.
• Custom Hormone Analysis Design – Tailored target lists and detection parameters to address unique project requirements, from developmental studies to stress physiology.
• Stable Isotope-Labeled Internal Standards – Ensuring accuracy by compensating for matrix effects across all sample types.
• Pathway Mapping and Comparative Analysis – Linking hormone quantification results to KEGG/HMDB pathways for functional interpretation and experimental comparisons.

List of Detected Steroid Hormones and Related Analytes

Why Choose Our Steroid Hormone Analysis Service: Key Advantages

• Ultra-Sensitive Quantification – Limits of detection down to 2–5 pg/mL for most steroid hormones, enabling accurate profiling of low-abundance analytes.
• High Analytical Precision – Multi-point calibration with R² ≥ 0.995; intra- and inter-batch CV ≤ 10% to ensure data reproducibility.
• Comprehensive Hormone Panel – Simultaneous detection of >20 steroid hormones across glucocorticoid, mineralocorticoid, androgen, estrogen, and progestogen pathways.
• Stable Isotope Dilution – Isotope-labeled internal standards for each target to correct matrix effects and guarantee absolute quantification.
• Integrated Bioinformatics Output – Includes hormone ratio calculations, pathway-level mapping (KEGG/HMDB), and comparative statistical analysis.

Steroid Hormone Assay Technical Details & Coverage

Our steroid hormone assays are performed on high-sensitivity LC-MS/MS and GC-MS/MS platforms to deliver the selectivity and sensitivity required for accurate hormone quantification. Compared with single-stage LC-MS or GC-MS, tandem MS/MS allows selective monitoring of precursor-to-product ion transitions, reducing matrix interference and enabling reliable quantification of structurally similar compounds at pg/mL levels.

Instrumentation:

• LC-MS/MS – Sciex QTRAP® 6500+, Thermo TSQ Altis, Waters Xevo TQ-S, coupled with UHPLC systems using reverse-phase C18 columns (2.1 × 100 mm, 1.7 μm).
• GC-MS/MS – Agilent 7890B GC with 7000D Triple Quadrupole for volatile or derivatized steroids.

Detection Mode: Multiple Reaction Monitoring (MRM) with polarity switching to maximize analyte coverage.

Example Transitions: Testosterone (m/z 289 → 97), Estradiol (m/z 271 → 145), Cortisol (m/z 363 → 121), Progesterone (m/z 315 → 97).

LOD: Typically 2–5 pg/mL for most analytes; Dynamic Range: 5–6 orders of magnitude.

By combining LC-MS/MS for thermolabile or polar hormones and GC-MS/MS for volatile derivatives, our method ensures full coverage of glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, and their key precursors and metabolites.

SCIEX Triple Quad™ 6500+ (Figure from Sciex)

Waters Xevo TQ-s (Figure from Waters)

7890B GC system (Figure from Agilent)

Vanquish UHPLC systems(Figure from Thermo)

Step-by-Step Workflow for Steroid Hormone Analysis

1

Consultation & Project Confirmation

• Discuss research goals, sample types, and analytical requirements with our technical experts.
• Determine assay scope (target hormones, detection platform, quantitative/qualitative approach).
• Provide a customized quotation and confirm project timeline.

2

Sample Submission

• Prepare samples according to our Sample Requirements guidelines.
• Label samples clearly with unique identifiers and relevant metadata.
• Ship samples under recommended conditions (e.g., dry ice at -80 °C) to ensure integrity.

3

Sample Reception & Quality Check

• Upon arrival, samples are logged into our secure tracking system.
• Initial quality assessment is performed (volume, condition, contamination check).
• Samples are stored under optimal conditions until processing.

4

Sample Preparation & Extraction

• Utilize validated protocols for the extraction and purification of steroid hormones from various matrices (serum, plasma, urine, tissue, cells, etc.).
• Incorporate internal standards to ensure accuracy and consistency.

5

Instrumental Analysis (GC-MS/MS or LC-MS/MS)

• Perform highly sensitive and specific detection using advanced GC-MS/MS or LC-MS/MS platforms.
• Apply optimized chromatographic separation and mass spectrometric conditions tailored for steroid hormones.

6

Data Processing & Quantification

• Process raw spectra to identify and quantify target hormones.
• Apply internal standard calibration and correction for matrix effects.
• Calculate detection limits (LOD/LOQ) and ensure data quality via QC samples.

7

Quality Control & Validation

• Evaluate signal stability, peak shape, retention time, and repeatability.
• Confirm results with standard curves and replicate analyses if necessary.

8

Data Analysis & Visualization (Optional)

• Conduct statistical analyses (t-test, ANOVA, PCA, PLS-DA).
• Generate professional visual outputs such as heatmaps, volcano plots, and correlation networks.

How to Prepare Your Samples for Steroid Hormone Quantification

From Lab to Discovery: Applications of Steroid Hormone Testing

Pharmaceutical & Biotechnology Research

Support drug discovery, efficacy evaluation, and safety assessment for hormone-related compounds.

Sports Science & Anti-Doping

Detect exogenous hormone use and study performance-related physiological changes.

Metabolic and Stress Response Studies

Investigate hormone fluctuations in metabolism, energy regulation, and stress adaptation.

Reproductive Biology Research

Monitor hormone dynamics in developmental, fertility, and endocrinology studies.

Veterinary & Animal Science

Assess reproductive status and endocrine functions in livestock, companion animals, and wildlife.

Environmental & Ecotoxicology Studies

Evaluate the impact of endocrine-disrupting chemicals on organisms and ecosystems.

Deliverables: What You Get from Our Steroid Hormone Analysis Service

GC-MS/MS chromatogram with labeled steroid hormone peaks (A–D).

LC-MS/MS chromatogram highlighting five target hormones (E–I) with high-resolution separation.

PCA scatter plot (PC1 vs PC2) showing clear separation of Control, Treatment A, and Treatment B with 95% confidence ellipses.

Volcano plot highlighting significantly altered steroid hormones (|log2FC| ≥ 1, p < 0.05).

Estrogen–Microbiota Link Revealed by Steroid Hormone Profiling

Background

Sex differences in gut microbiota arise from both sex hormones and sex chromosomes. Disentangling their roles requires models that decouple gonadal sex from XX/XY complement.

Objective

Determine whether sex hormones or sex chromosomes primarily shape gut microbiota, and test whether Akkermansia muciniphila responds to estrogen.

Model & Methods

• Four-core genotype (FCG) mice (XX or XY with ovaries; XX or XY with testes), 15–16 weeks; n=6–8/group.
• Gonadectomy to deplete circulating sex steroids.
• Plasma steroid quantification: LC-MS/MS (ESI-MRM) multi-hormone panel (Creative Proteomics); key readouts included estradiol, progesterone, testosterone and precursors/metabolites.
• Microbiota profiling: 16S rRNA sequencing; α/β diversity analyses.
• In vitro assay: growth of A. muciniphila ± β-estradiol (2.2–10 μM).

Key Findings

• Hormone depletion confirmed: ovariectomy lowered estradiol and progesterone in females (P<0.001/0.03); castration markedly reduced testosterone in males (P<0.0001).
• Diversity shifts: female α-diversity increased after gonadectomy regardless of XX/XY; male β-diversity differed between XX and XY.
• A. muciniphila is estrogen-responsive: abundance decreased in gonadectomized females vs intact (P<0.0001). β-estradiol dose-dependently increased A. muciniphila growth in vitro (P<0.001).
• Conclusion: Sex hormones override sex chromosomes in shaping gut microbiota in this model; estrogen positively associates with Akkermansia.

Plasma steroids after gonadectomy. Ovariectomy lowers female estradiol; aldosterone shows XX>XY effect. Castration reduces male testosterone; estradiol/aldosterone unchanged. Two-way ANOVA; n=4–6/group; *P<0.05.

Impact for Researchers.

• Integrating steroid profiling (LC-MS/MS) with microbiome readouts can reveal hormone–microbe interactions that underlie metabolic and immune phenotypes.
• Supports study designs probing estrogen signaling, microbiota modulation, and pathway-level mechanisms in preclinical models.

Reference

1. Sakamuri, Anil, et al. "Sex hormones, sex chromosomes, and microbiota: identification of Akkermansia muciniphila as an estrogen-responsive bacterium." Microbiota and host 1.1 (2023).