Peptides and drug tests: how anti-doping labs detect them

Peptide hormones — GHRPs, GHRH analogs, gonadotropins, EPO, recombinant growth hormone, GLP-1 analogs, and related compounds — are on the World Anti-Doping Agency (WADA) Prohibited List and are also of interest to clinical toxicology laboratories. They are not, however, on routine workplace drug-testing panels. Detecting them requires dedicated instrumentation, dedicated sample preparation, and dedicated reference materials.

This article describes how those detection workflows actually work. It is intended for chemists, lab personnel, journalists, athletes' support staff, and anyone trying to understand the analytical literature. It is not a guide to evading detection, a guide to administration, or a guide to clinical use. CertikLabs does not publish dosing or prescribing information.

Small-molecule drugs of abuse are present in urine at concentrations from hundreds of ng/mL to low µg/mL, are chemically distinct from anything the body produces in significant quantity, and persist for hours to days. They yield reliably to immunoassay screening and GC-MS or LC-MS confirmation.

Peptide hormones are a different analytical problem:

• Concentration. Circulating levels are typically picomolar to low nanomolar — three to six orders of magnitude lower than small-molecule drugs.
• Half-life. Most administered peptides are cleared from plasma within minutes to hours; the urinary window is often shorter than the screening interval.
• Background. Many target peptides are structurally similar to, or identical to, endogenous proteins. Discriminating exogenous from endogenous is itself an analytical question.
• Matrix. Urine and plasma contain abundant salts, proteins, and small molecules that suppress ionization and contaminate columns.

The standard response is aggressive enrichment up-front, a high-resolution mass spectrometer downstream, and a two-stage screening-and-confirmation workflow that allows the screen to be sensitive at the cost of specificity.

A raw urine or plasma sample is not directly compatible with LC-MS analysis of low-abundance peptides. Three enrichment strategies are common, often used in combination:

• Solid-phase extraction (SPE) on weak-cation-exchange or mixed-mode polymeric sorbents (e.g., Oasis MCX, HLB) removes salts and matrix and concentrates the target peptide by 10× to 100×. This is the default first step for low-molecular-weight peptides such as GHRPs, GHRH analogs, and synthetic peptide hormones below ~5 kDa.
• Immunoaffinity capture uses antibody-coated magnetic beads or monolithic columns to selectively bind the target before elution. It is the preferred enrichment for endogenous proteins such as EPO, hCG, and growth hormone because it pulls a single analyte out of an otherwise impossible matrix.
• Tryptic or chymotryptic digestion is used for larger proteins (recombinant erythropoietin, growth hormone, IGF-1 in some workflows). The intact protein is digested to signature peptides whose accurate masses and product ions are then used as surrogates for the parent.

Internal standards — typically stable-isotope-labeled (¹³C, ¹⁵N) analogs of the target peptide — are spiked in before extraction. They co-elute with the target and correct for recovery, ion suppression, and instrument drift, and are the basis for quantitative reporting.

Once enriched, the extract is separated on a short reverse-phase column (typically C18, 2.1 mm × 50–150 mm, sub-2-µm particles) under a fast acetonitrile gradient with 0.1% formic acid as the ion-pairing modifier. The eluent enters a high-resolution mass spectrometer — Orbitrap or Q-TOF — via electrospray ionization.

For target peptides on a published prohibited list, screening is performed in one of two modes:

• Parallel reaction monitoring (PRM) / targeted MS/MS. The instrument cycles through a scheduled list of precursor m/z values, isolates each, fragments it, and records the full product-ion spectrum at high resolution. PRM is fast, sensitive (low pg/mL for many GHRPs), and quantitative against the isotope-labeled internal standard.
• Data-independent acquisition (DIA / SWATH). The instrument cycles through fixed precursor windows that together cover the whole m/z range, recording fragment spectra for every species. DIA captures unanticipated peptides retrospectively, at some cost to per-target sensitivity.

A screen positive is any signal that matches the target on retention time and at least one diagnostic product ion above a defined signal-to-noise threshold. False-positive rates are explicitly tolerated; confirmation handles specificity.

A screen-positive sample is re-analyzed on the same or a second instrument under stricter conditions. The reportable identification criteria, as published in WADA Technical Documents (TD2023IDCR and successors), require all of the following:

• Retention time within ±0.1 minute (or 1%, whichever is smaller) of the reference standard run in the same sequence.
• Accurate mass of the precursor and of at least two diagnostic product ions within 5 ppm (high-resolution) or within defined Da tolerances on lower-resolution instruments.
• Relative ion abundances of the diagnostic product ions within defined tolerance windows of the reference standard.
• Signal-to-noise ≥ 3 for each diagnostic ion in the extracted ion chromatogram.

For sequence-confirmable peptides, MS/MS fragmentation generates a b- and y-ion series that reads the residue sequence directly; matching the de novo sequence to the declared structure is the strongest possible identification. Confirmation reports are reviewed by a second qualified analyst before the result is released.

Recombinant human growth hormone (rhGH), erythropoietin (EPO), and human chorionic gonadotropin (hCG) are essentially identical in sequence to the endogenous proteins. Mass-spectrometric sequence matching cannot distinguish them. Three orthogonal approaches are used:

• Isoform ratio (the GH isoforms test). Endogenous pituitary GH is secreted as a mixture of 22 kDa and 20 kDa isoforms plus oligomers; recombinant preparations are pure 22 kDa monomer. Two immunoassays with differential isoform specificity yield a ratio that is altered by rhGH administration.
• Biomarker signature (the GH biomarkers test). Serum IGF-1 and the N-terminal pro-peptide of type III pro-collagen (P-III-NP) are downstream of GH action. A validated multivariate score, age- and sex-corrected, identifies recent exogenous GH even after the GH itself has cleared.
• Charge-profile electrophoresis (the EPO test). Endogenous EPO and recombinant epoetins differ in glycosylation pattern and therefore in isoelectric focusing or SAR-PAGE migration. The banding pattern is read against reference standards. Sequence-level MS workflows are an emerging complement.

For hCG, both immunoassay and LC-MS quantification of the β-subunit are used, with reference cut-offs that depend on sex and clinical context.

Published method-validation data from WADA-accredited laboratories indicate the following representative figures — specific values vary by laboratory, instrument, and matrix:

• Small synthetic GHRPs and GHRH analogs in urine by LC-HRMS: lower limits of detection in the 10–100 pg/mL range; detection windows of hours to a few days depending on the compound and dose.
• Insulin and insulin analogs in plasma by immunoaffinity-LC-HRMS: low pg/mL detection of synthetic analogs; sequence-confirmable B-chain product ions discriminate analogs from human insulin.
• rhGH by isoforms immunoassay: detection windows of ~24–36 hours post-administration; the biomarkers panel extends the practical detection window to ~2 weeks.
• Recombinant EPO and analogs by charge-profile or SAR-PAGE: detection windows of days for short-acting agents, longer for continuous erythropoiesis receptor activators.

These figures are stated to convey the analytical state of the art. They are not, and should not be read as, guidance about when a given compound is or is not detectable in any particular individual.

Every assay described above depends on a characterized reference standard of the target peptide. The retention-time window, the diagnostic product ions, the isotope-labeled internal standard, and the validation curve are all anchored to a reference material whose identity, purity, and content have been independently established. This is the same kind of analytical package CertikLabs issues for research peptide batches — HPLC purity, LC-MS identity against the theoretical monoisotopic mass, amino-acid analysis for true content, and counter-ion characterization. See peptide characterization for the underlying methods.

The detection workflow and the verification workflow are not the same thing, but they share a common analytical backbone: high-resolution mass spectrometry against a calibrated reference.

Readers who want the underlying technical documents and peer-reviewed methods should consult:

• WADA Technical Documents on minimum required performance levels (TD MRPL), identification criteria (TD IDCR), and the prohibited list, published at wada-ama.org.
• Thomas A. et al., on LC-HRMS detection of GHRPs and small peptide hormones, Analytical and Bioanalytical Chemistry and Drug Testing and Analysis.
• Such-Sanmartín G. et al., on insulin and insulin-analog detection by immunoaffinity-MS.
• Reichel C. et al., on SAR-PAGE detection of recombinant erythropoietins.

CertikLabs does not perform anti-doping testing. This article is a reference summary of how those workflows are constructed in the published literature.

References last updated Jun 10, 2026. External links verified on Jun 10, 2026.

1. World Anti-Doping Agency. TD2023IDCR: Minimum Criteria for Chromatographic-Mass Spectrometric Confirmation of the Identity of Analytes for Doping Control Purposes (Version 1.1). 14 February 2023. wada-ama.org.
   Accessed on Jun 10, 2026.
2. World Anti-Doping Agency. TD2023MRPL: Minimum Required Performance Levels for Detection and Identification of Non-Threshold Substances. 2023. wada-ama.org.
   Accessed on Jun 10, 2026.
3. World Anti-Doping Agency. International Standard: Prohibited List (2025). wada-ama.org.
   Accessed on Jun 10, 2026.
4. Thomas A, et al. Determination of growth hormone releasing peptides (GHRP) and their major metabolites in human urine for doping controls by means of liquid chromatography mass spectrometry. Anal Bioanal Chem. 2011;401(2):507-516. doi.org/10.1007/s00216-011-4702-3.
   Accessed on Jun 10, 2026.
5. Thevis M, Thomas A, Schänzer W. Doping control analysis of selected peptide hormones using LC-MS(/MS). Forensic Sci Int. 2011;213(1-3):35-41. doi.org/10.1016/j.forsciint.2011.08.015.
   Accessed on Jun 10, 2026.
6. Thomas A, Benzenberg L, Bally L, Thevis M. Facilitated Qualitative Determination of Insulin, Its Synthetic Analogs, and C-Peptide in Human Urine by Means of LC-HRMS. Metabolites. 2021;11(5):309. doi.org/10.3390/metabo11050309.
   Accessed on Jun 10, 2026.
7. Memdouh S, et al. Advances in the detection of growth hormone releasing hormone synthetic analogs. Drug Test Anal. 2021;13(11-12):1871-1887. doi.org/10.1002/dta.3183.
   Accessed on Jun 10, 2026.
8. Reichel C, et al. SARCOSYL-PAGE: a new method for the detection of MIRCERA- and EPO-doping in blood. Drug Test Anal. 2009;1(11-12):494-504. doi.org/10.1002/dta.97.
   Accessed on Jun 10, 2026.
9. Reihlen P, Blobel M, Kempkes R, Reichel C, Völker Schänzer E, Majer B, Schänzer W. Optimizing SAR-PAGE. Drug Test Anal. 2015;8(5-6):418-425. doi.org/10.1002/dta.1915.
   Accessed on Jun 10, 2026.