Retatrutide, the 39-amino-acid peptide conjugated to a C20 fatty diacid (LY3437943, CAS 2381089-83-2), is a unimolecular triple agonist at GLP-1R, GIPR, and GCGR, currently under investigation as a premium obesity and metabolic-disease pharmacotherapy. Its sequence incorporates substantial non-native modifications to optimise receptor cross-talk, which increases both its therapeutic potential and the risk of synthesis-related defects. In this article, we outline how a research-grade lab can decode a Retatrutide CoA using HPLC peptide analysis and mass spectrometry verification, with emphasis on interpreting the Retatrutide molecular weight and spotting red flags in Janoshik-style reports.
Why Retatrutide Is Chemically More Complex
Retatrutide is a 39-residue sequence with a C-terminal C20 acyl tail, giving a theoretical molecular weight of approximately 4731.33 Da and a molecular formula of C221H342N46O68. This places it at the upper end of complexity for synthetic peptides, with multiple charged residues, a long hydrophobic stretch, and a fatty-acid moiety that complicates solubility and folding during solid-phase synthesis.
Compared with Tirzepatide (39 residues, C18-diacid) or Semaglutide (31 residues, C18-diacid), Retatrutide's sequence contains more non-canonical residues and a longer acyl chain, which magnifies the risk of truncated sequences, mis-acylations, and racemisation at individual coupling steps. Each step in Retatrutide synthesis compounds residual error rates, meaning even small per-step impurities (e.g., 0.5–1%) can yield a measurable burden of deletion, truncation, or D-amino-acid isomer impurities detectable by HPLC and mass spectrometry.
HPLC Analysis: Purity and the "Main Peak"
High-performance liquid chromatography (HPLC), typically reversed-phase (RP-HPLC), is the standard method for peptide purity assessment. In a Retatrutide CoA, the "main peak" represents the elution profile of the full-length, correctly folded peptide under the chosen gradient and column conditions; its area-under-curve relative to the total chromatogram is reported as the purity (often ≥98–99% for research-grade material).
Common impurities in Retatrutide synthesis include:
A well-characterised Retatrutide batch should show a single dominant peak with minimal shoulders, and the report should explicitly state whether impurities are "identified" (e.g., via MS) or "unidentified" (requiring orthogonal techniques such as amino-acid analysis or LC-MS/MS for full attribution).
Mass Spectrometry: Identity and Charge-State Signatures
Mass spectrometry, especially electrospray-ionisation mass spectrometry (ESI-MS), is the primary tool for identity confirmation of Retatrutide. The reported Retatrutide molecular weight is 4731.33 Da; the monoisotopic mass computed by PubChem is 4728.47 Da, and common experimental values cluster around 4731.4 Da, reflecting adducts and ionisation conditions.
In an ESI-MS spectrum, Retatrutide is typically observed as a series of multiply-charged ions:
| Ion Species | Observed m/z | Charge State |
|---|---|---|
| [M+3H]3+ | ≈ 1577.4 | z = 3 |
| [M+4H]4+ | ≈ 1183.0 | z = 4 |
| [M+5H]5+ | ≈ 946.4 | z = 5 |
These charge-state peaks must align with the theoretical mass within a narrow tolerance (e.g., ±0.05% or ±2–3 Da depending on instrument resolution and calibration). A clean spectrum will show a dominant cluster of these peaks, with minimal satellite ions from adducts (Na⁺, K⁺) or in-source fragments.
A compliant CoA must:
- → Report the observed vs. theoretical mass and note the ionisation mode (ESI-MS or MALDI-TOF).
- → Confirm that the pattern of charge-state peaks is consistent with the expected z = 3, 4, 5 envelope and that the derived neutral mass reconstructs closely to 4731.33 Da.
Discrepancies in mass or a missing or distorted charge-state envelope (e.g., dominant [M+2H]2+ instead of higher charge states) are strong indicators of either incorrect identity, severe adduction, or contamination.
Red Flags in Supplier CoAs and "Janoshik-Style" Reports
Third-party testing, including services comparable to Janoshik, has become a de facto standard for verifying peptide quality. However, malicious or negligent suppliers sometimes reuse, forge, or "recycle" test reports. For a Retatrutide CoA, watch for:
•Batch codes, sample IDs, or submission dates do not align with the vial's lot number or shipping records.
•The same "unique task number" or verification key appears tied to multiple, chemically distinct products on the lab's public verification portal.
•Chromatograms or mass spectra are pixelated, cropped, or visibly compressed, especially around the main peak and charge-state region.
•Fonts differ between the header (e.g., "Retatrutide CoA") and the body text, suggesting copy-pasting from different source documents.
•A lab-branded report claims a "unique key" or "task number" but this cannot be validated on the lab's official verification page, or the verified result shows a different compound or mass.
•The report lacks a verifiable URL, QR code, or hash that links to raw data or a public-results portal, which is typical for accredited peptide-testing services.
These issues are strong red flags in any Retatrutide CoA, whether labelled as a "Janoshik report" or from another vendor-affiliated lab.
Why Independent Verification Matters: Astrea Lab Batch AL-2026-001
At Astrea Lab, we do not rely on our synthesis lab's internal CoA. We send blind samples from Batch AL-2026-001 directly to independent, third-party facilities like Janoshik Analytical. This ensures that the HPLC and Mass Spectrometry data we publish is completely decoupled from the manufacturing process, eliminating the risk of forged or recycled reports.
Our Verification Protocol
Blind Sample Dispatch
Samples from Batch AL-2026-001 are sent to Janoshik Analytical without disclosure of expected results, synthesis origin, or internal purity estimates.
Independent HPLC/MS Analysis
Janoshik performs RP-HPLC purity assessment and ESI-MS identity confirmation independently, generating a report with a publicly verifiable task number.
Cross-Validation Against Theoretical Values
The observed molecular weight and charge-state envelope are compared against the theoretical Retatrutide molecular weight (4731.33 Da) and the z = 3, 4, 5 charge-state pattern.
Public CoA Publication
The full, unredacted Janoshik report — including the chromatogram, mass spectrum, and verification key — is published on astrealab.nl 24 hours prior to batch release.
This approach ensures that the liquid in the vial genuinely corresponds to the Retatrutide CoA labelled on the packaging — and that every researcher who orders from Astrea Lab can independently verify the report using Janoshik's public verification portal.
References
- [1]Si, P., et al. "Structural insights into the triple agonism at GLP-1R, GIPR and GCGR by retatrutide." Cell Discovery. 2024; 10:80.
- [2]Wang, L., et al. "Retatrutide — A Game Changer in Obesity Pharmacotherapy." Pharmacology Research & Perspectives. 2025; 13(3):e1219.
- [3]"Retatrutide (LY3437943)." PubChem Compound Summary. CID 171390338. National Library of Medicine.
- [4]Janoshik Testing Laboratory. Verification Portal. https://janoshik.com/verification/
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Pre-Release Access
Batch AL-2026-001 — Awaiting Janoshik Certification
Join the pre-release waitlist to receive the full CoA, HPLC chromatogram, and mass spectrum 24 hours before public release.
Join the Pre-Release WaitlistNo institutional licence required. Open to independent researchers.