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Steroids Hormone Analysis Service

Gain precise, pathway-level insight into endocrine regulation with LC–MS/MS steroid hormone profiling from Creative Proteomics. Our service quantifies multiple hormones and metabolites in one run, resolving common challenges such as poor isomer separation, matrix interference, and inconsistent recovery.

What We Deliver

Accurate LC–MS/MS quantification with pg/mL sensitivity
Reliable profiling of free and conjugated steroids
Optimized workflows for serum, plasma, urine, and tissue
Internal-standard normalization and complete QC validation

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Methods
Results and Data Analysis
Sample Requirements
FAQ

What are Steroids Hormones?

Steroid hormones are lipophilic messengers derived from cholesterol. They include glucocorticoids, mineralocorticoids, androgens, estrogens, and progestogens. These molecules diffuse across membranes and bind nuclear or membrane receptors. Receptor binding regulates transcription, signaling, and downstream metabolism.

Production occurs mainly in adrenal tissue and gonads. Circulating steroids exist as free molecules or protein-bound pools. Phase I and phase II enzymes convert them to active, inactive, or conjugated forms. Sulfates and glucuronides extend half-life and change polarity, shaping excretion and detection.

Small structural changes create large functional differences. One hydroxyl group can alter receptor affinity and bioactivity. This is why steroid hormone analysis favors LC–MS/MS profiling. The method distinguishes isomers, resolves isobars, and quantifies low-abundance targets.

In research and development, steroid pathways inform mechanism and safety. You can track stress response, fluid balance, and reproductive endpoints. Panels covering precursors, actives, and metabolites reveal pathway bottlenecks and enzyme shifts. Measuring free and conjugated pools clarifies exposure, clearance, and tissue dynamics.

Steroid Hormone Profiling Services in Creative Proteomics

Detailed, Customizable, and Pathway-Driven Sterol Profiling

HPA Axis Hormone Profiling

Supports studies of hormone regulation under stress or metabolic load by monitoring key upstream and downstream changes in adrenal-related pathways.

Androgen Pathway Assessment

Covers major androgens and their related biosynthetic steps to help clarify shifts in hormone levels across physiological or experimental conditions.

Estrogen Metabolism Analysis

Profiles parent estrogens alongside downstream metabolic routes to support comparative studies across sexes, timepoints, or treatment responses.

Free vs Conjugated Hormone Evaluation

Offers selective measurement of free hormones, intact conjugates, or total pools, depending on how exposure or metabolic clearance needs to be interpreted.

Neuroactive Steroid Panel

Designed for brain- or neural-related studies, this panel targets compounds relevant to neurological and behavioral research, with appropriate sample handling options.

Adrenal Steroid Mapping

Targets a broader range of steroid precursors and intermediates involved in adrenal function, offering more complete context for pathway interpretation.

Detectable Steroid Hormones and Related Derivatives (Service Panel)

Below is a representative scope of targets for steroid hormone analysis and LC–MS/MS steroid profiling. Items can be measured as free compounds, intact conjugates, or after controlled hydrolysis, depending on study goals.

Class / Pathway	Representative Analytes (Free)	Conjugates & Related Derivatives
Glucocorticoids & C21 Precursors	Cortisol, Cortisone, Corticosterone, 11-Deoxycortisol, 21-Deoxycortisol, 17-Hydroxyprogesterone, Progesterone, 17-Hydroxypregnenolone, Pregnenolone, Allopregnanolone	Cortisol Sulfate/Glucuronide, Cortisone Sulfate/Glucuronide, THF, Allo-THF, THE
Mineralocorticoids	Aldosterone, 11-Deoxycorticosterone	Aldosterone-18-Glucuronide (where applicable)
C19 Androgens	Testosterone, Dihydrotestosterone (DHT), Androstenedione, Androsterone, Etiocholanolone, 3α-/3β-Androstanediol	Testosterone Sulfate/Glucuronide, DHT Sulfate/Glucuronide
11-Oxygenated Androgens	11β-Hydroxyandrostenedione, 11-Ketoandrostenedione, 11β-Hydroxytestosterone, 11-Ketotestosterone	Corresponding sulfates/glucuronides
Estrogens (C18)	Estrone (E1), Estradiol (E2), Estriol (E3), 2-/4-Hydroxyestrone, 2-/4-Hydroxyestradiol, 2-/4-Methoxyestrogens	E1/E2/E3 Sulfates/Glucuronides, Catechol/Methoxy conjugates
Adrenal Steroids (Δ5 pathway)	DHEA, 7-Hydroxy-DHEA, 7-Oxo-DHEA	DHEA-S and other sulfates
Neurosteroids	Allopregnanolone, Pregnanolone, Tetrahydroprogesterone species	Sulfate/Glucuronide forms where applicable
Phase I Metabolites	5α-/5β-Reduced steroids, 16α-/16β-Hydroxylated species	Conjugated counterparts (sulfates/glucuronides)
Method Controls (Surrogates/IS)	Isotope-labeled analogs covering each class	—

Custom inclusions are available on request. Final panels are aligned to matrix, sensitivity needs, and interpretive endpoints.

Why Choose Our Steroids Hormone Assay?

Wide Quantification Range: Detects hormones from low pg/mL to ng/mL, suitable for both trace and abundant targets.
High Selectivity: Uses 2–3 MRM transitions per compound with retention-time locking to resolve isomers and isobars accurately.
Low Variability: Typical method CVs ≤15%; recovery rates often exceed 85% across matrices.
Flexible Conjugate Profiling: Supports both free and total hormone measurement via intact detection or controlled hydrolysis.
Isotope-Labeled Internal Standards: Included for recovery correction and matrix-effect control, ensuring robust comparability.
Advanced Instrumentation: Performed on QTRAP 6500+, TSQ Altis, or Orbitrap PRM systems; GC–MS/MS available for select workflows.

How Creative Proteomics Provides Steroids Hormone Assay?

Consultation & Panel Design
Define matrices, targets, reporting format, and required LLOQs.
Sample Receipt & Pre-QC
Verify labeling, storage conditions, and matrix compatibility.
Extraction & Cleanup
Protein precipitation and SPE; optional hydrolysis for conjugates.
Chromatography & MS Acquisition
UHPLC separation; targeted MRM or PRM with polarity switching.
Quantification & QC Review
Multi-point calibration, internal standards, system suitability checks.
Reporting & Interpretation
Concentration tables, QC metrics, and pathway-context summaries.

What Methods are Used for Our Steroids Hormone Analysis?

Targeted LC–MS/MS (Primary Method)

Platforms: Agilent 1290 or Thermo Vanquish UHPLC; C18 or phenyl-hexyl columns.
MS: AB Sciex QTRAP 6500+ or Thermo TSQ Altis in MRM mode.
Key choices: ESI or APCI; fast polarity switching; scheduled MRM with 2–3 transitions per analyte; isotope-labeled internal standards; SPE cleanup.
Use when: You need multiplexed quantitation with strong selectivity across serum, urine, tissue, or cell matrices.

LC–HRMS PRM (Selective Confirmation)

Platform: Orbitrap Exploris 480 for accurate-mass PRM.
Key choices: Narrow isolation windows; high resolution; retention-time locking for isomer separation.
Use when: Interferences or isomers challenge MRM-only data and added specificity is required.

GC–MS/MS with Derivatization (For Select Chemotypes)

Platform: Agilent 7890B GC with 7000 series triple quad or 5977B EI detector.
Key choices: MSTFA silylation or oxime formation to improve volatility and response.
Use when: Certain estrogens or reduced steroids perform better with GC separation and EI fragmentation.

SCIEX Triple Quad™ 6500+ (Figure from Sciex)

Thermo Scientific TSQ Altis Triple Quadrupole MS

TSQ Altis Triple Quadrupole MS (Figure from Thermo Scientific)

Orbitrap Exploris 480 (Figure from Thermo Fisher)

Steroids Hormone Analysis Service: Results and Data Analysis

Standard Deliverables

Quantitative Results Table

Absolute concentrations for each steroid (e.g., ng/mL, pg/mL, pmol/mg).
Isotope-labeled internal-standard normalization.
QC fields: %CV, signal-to-noise, transition ratios, retention time.

Chromatograms & Spectra

Extracted-ion chromatograms (EICs) and total-ion chromatograms (TICs).
Representative MS/MS spectra for key targets and qualifiers.

Calibration & Linearity Summary

Full calibration plots with range, regression model, and correlation coefficients.
Curve parameters and back-calculated accuracy for each analyte.

LOD/LOQ (Matrix-Matched)

Compound-specific LOD and LOQ determined in the study matrix.
Notes on recovery, carryover checks, and ion-suppression assessment.

Calibration curves with confidence bands and residuals, including LLOQ indicators for steroids.

Calibration performance for representative steroids. Curves show 95% CIs; residuals verify model fit. LLOQs and back-calculated accuracy confirm low-level quantitation.

Overlay of quantifier and qualifier chromatograms in matrix showing baseline-resolved isomers and matched ion ratios

Selectivity in study matrix. Quantifier/qualifier traces meet ion-ratio and RT criteria; isomeric targets are baseline-resolved with matching MS/MS fragments

Optional Visual & Bioinformatic Outputs

Profile heatmapsshow side-by-side steroid patterns across groups or timepoints
PCA (Principal Component Analysis)reveals multivariate trends and highlights outliers
Pathway mappingtraces precursors, actives, and metabolites along steroidogenesis routes (on request)
Ratio metricssuch as precursor/product ratios support mechanistic interpretation
Volcano plots & bar chartsvisualize group differences and fold changes
Customizable thresholdsavailable for p-values, FDR, and fold-change cutoffs

Data Formats

Excel (.xlsx): annotated raw and processed tables, metadata, and QC.
PDF report: figures, interpretation notes, and summary conclusions.
Raw files (on request): vendor formats (e.g., Agilent .d, Thermo .raw).
Graphics: PNG, SVG, or high-resolution TIFF ready for publication.

Explore our Lipidomics Solutions brochure to learn more about our comprehensive lipidomics analysis platform.

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What Our Steroids Hormone Analysis Used For

Endocrine Research

Tracks hormone biosynthesis and conversion rates to understand regulatory pathways and enzyme kinetics.

Reproductive Biology

Quantifies androgen and estrogen profiles to study hormone regulation in gametogenesis and developmental processes.

Pharmacology & Drug Metabolism

Evaluates drug–hormone interactions, metabolic stability, and pathway modulation in preclinical models.

Environmental and Ecotoxicology Research

Detects steroid-like compounds and endocrine-active substances in environmental or biological samples.

Stress and Metabolic Studies

Monitors adrenal responses and steroid balance under nutritional, environmental, or physiological stress.

Translational & Systems Biology

Integrates steroid data with transcriptomics or metabolomics to reveal network-level regulation and pathway crosstalk.

Sample Requirements for Steroids Hormone Analysis Solutions

Sample Type	Recommended Volume / Amount	Container	Preservatives / Additives	Storage Conditions	Shipping Instructions	Special Notes
Serum / Plasma	≥ 200 µL (preferably 300 µL)	1.5 mL low-bind microtube or cryovial	None; avoid heparin and EDTA when possible	Store at −80 °C; avoid repeated freeze–thaw	Ship on dry ice in secondary sealed bag	Record anticoagulant if used; avoid hemolysis and lipemia
Urine	≥ 1 mL	Sterile polypropylene tube	Optional: pH ≈ 6–7 buffer if extended storage	Store at −20 °C or below	Ship frozen with ice packs or dry ice	Mix thoroughly; note collection time and dilution factor
Tissue (fresh or frozen)	30–50 mg wet weight	Cryovial, RNase/DNase-free	None	Snap-freeze in liquid N₂; store at −80 °C	Ship on dry ice; label species + tissue type	Record tissue source and region; avoid fixatives
Cell Pellet	≥ 1 × 10⁶ cells	Cryovial	None	Store at −80 °C immediately after centrifugation	Ship on dry ice	Wash twice with PBS; remove medium residues
Cell Lysate / Extract	≥ 100 µL	Low-bind microtube	Keep buffer MS-compatible; avoid detergents, glycerol > 5%, or > 200 mM salt	Store at −80 °C	Ship on dry ice	Include buffer composition; note protein concentration if available
Culture Medium / Supernatant	≥ 500 µL	Sterile polypropylene tube	None	Store at −20 °C or below	Ship frozen	Filter or centrifuge to remove cells/debris
Saliva (research only)	≥ 500 µL	Salivette or polypropylene tube	None	Store at −20 °C	Ship frozen	Collect unstimulated samples; avoid food, caffeine, and nicotine 1 h before collection

General Guidelines:

Avoid hemolysis and protein precipitation prior to extraction.
Label each tube with sample ID, matrix type, and date.
Minimize freeze–thaw cycles (≤ 2 times recommended).
Use polypropylene containers—glass may adsorb hydrophobic steroids.

FAQs for Steroids Hormone Analysis Service

What is the most accurate way to measure steroid hormones?

Targeted LC–MS/MS is widely regarded as the reference approach because it separates isomers, minimizes cross-reactivity, supports multiplex panels, and delivers high specificity at low concentrations. These advantages are emphasized in endocrine best-practice reviews and method papers.

Can multiple steroid hormones be quantified in one run?

Yes. LC–MS/MS enables multiplexed panels that quantify many steroids simultaneously, improving throughput and comparability across samples while maintaining selectivity.

Do you measure free and conjugated forms separately?

We can measure free hormones, intact conjugates (sulfates/glucuronides), or total pools after controlled hydrolysis; this is a common LC–MS/MS strategy used to interpret biosynthesis and clearance.

How is data accuracy maintained across batches and labs?

Accuracy and comparability rely on matrix-matched calibration, isotope-labeled internal standards, QC samples, and participation in recognized standardization frameworks (e.g., CDC hormone standardization and reference procedures).

What are the main challenges in steroid testing, and how are they handled?

Typical issues include matrix effects, isobaric overlap, and isomer separation. Optimized extraction/SPE, chromatographic resolution, qualifier-ion ratios, and—when needed—PRM or orthogonal methods address these challenges.

Is LC–MS/MS better than immunoassay for low-level steroids?

For low concentrations and isomeric interferences, LC–MS/MS generally shows higher specificity and accuracy than immunoassays; consensus statements recommend standardization to ensure reliable results.

Are saliva measurements reliable for research use?

Validated LC–MS/MS methods can establish saliva reference ranges and quantify multiple steroids with appropriate sensitivity, offering a convenient, non-invasive matrix when matched to study goals.

Why do some labs emphasize participation in standardization programs?

External standardization and reference measurement procedures reduce between-laboratory bias and improve long-term comparability of steroid data—important for multi-site or longitudinal research.

How do you confirm selectivity when isomers co-elute?

We combine chromatographic separation with multiple qualifier transitions and retention-time criteria; if necessary, high-resolution PRM or GC–MS/MS confirmation is applied to verify identity.

Can results integrate with other omics analyses?

Yes. Quantitative steroid panels measured by LC–MS/MS are frequently integrated with transcriptomics or metabolomics to contextualize enzyme activity and pathway crosstalk. (General LC–MS multiplexing and pathway applications support this integration.)

What influences the choice of matrix (serum vs urine vs saliva)?

Decision factors include target concentration range, conjugation state, and study design. Serum/plasma support broad panels; urine is useful for excreted metabolites; saliva offers non-invasive sampling of bioavailable fractions when methods are validated.

What about standardization for testosterone and estradiol in particular?

CDC programs provide reference procedures and standardization materials to verify calibration and improve trueness for these high-impact analytes.

Publications

White matter lipid alterations during aging in the rhesus monkey brain. GeroScience, 2024. https://doi.org/10.1007/s11357-024-01353-3
Characterization of Dnajc12 knockout mice, a model of hypodopaminergia. bioRxiv, 2024. https://doi.org/10.1101/2024.07.06.602343
Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation. The FASEB Journal, 2023. https://doi.org/10.1096/fj.202201088R
Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction. EMBO Journal, 2019. https://doi.org/10.15252/embj.201899558
Evidence for phosphate-dependent control of symbiont cell division in the model anemone Exaiptasia diaphana. mBio, 2024. https://doi.org/10.1128/mbio.01059-24
The olfactory receptor Olfr78 promotes differentiation of enterochromaffin cells in the mouse colon. EMBO reports, 2024. https://doi.org/10.1038/s44319-023-00013-5
Metabolomic Studies in Girls With Central and Peripheral Precocious Puberty. Fabad Journal of Pharmaceutical Sciences, 2023. https://doi.org/10.55262/fabadeczacilik.1344851
Prospective randomized, double-blind, placebo-controlled study of a standardized oral pomegranate extract on the gut microbiome and short-chain fatty acids. Foods, 2023. https://doi.org/10.3390/foods13010015

* Our services can only be used for research purposes and Not for clinical use.

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