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Evidence-based GLP-1 & peptide discussion since 2023
ForumsCOA & Analytical TestingAmino acid analysis for peptide identity — looking for input

Amino acid analysis for peptide identity — looking for input

rick_sfbay Sat, Dec 13, 2025 at 7:06 AM 14 replies 787 viewsPage 1 of 3
rick_sfbay
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Jan 2025
San Francisco, CA
Dec 13, 2025 at 8:31 AM#1
I see a lot of people posting COAs without understanding what they're looking at. This guide will teach you to read a Certificate of Analysis for compounded or research-grade peptides like semaglutide. Bookmarkable reference. What is a COA? A Certificate of Analysis is a document from a testing laboratory reporting the results of quality testing on a specific batch of product. It tells you what's in the vial, how pure it is, and whether it meets specifications. Key sections of a peptide COA: 1. Identity — "Is this actually semaglutide?" - Method: Usually mass spectrometry (ESI-MS or MALDI-TOF) - What to look for: Molecular weight should match semaglutide (MW: 4113.58 Da). The observed mass should be within ±2 Da of theoretical. - Red flag: Mass doesn't match, or identity testing wasn't performed. 2. Purity — "How clean is it?" - Method: HPLC (High Performance Liquid Chromatography) or UPLC - What to look for: Purity ≥ 95% is acceptable. ≥ 97% is good. ≥ 99% is excellent. - How it works: The sample is dissolved and pumped through a column. Different molecules elute at different times. The main peak (semaglutide) is expressed as a percentage of total peak area. - Red flag: Purity below 95%, large unidentified peaks, or no chromatogram provided (just a number without supporting data). 3. Content/Potency — "How much is actually in the vial?" - Method: Quantitative HPLC against a reference standard - What to look for: The measured amount vs. label claim. A 5mg vial should contain 4.5-5.5mg (90-110% of label). Ideally 95-105%. - Red flag: Content below 90% or above 115% of label claim. Under-filling is quality control failure. Over-filling beyond 110% suggests poor manufacturing controls. 4. Sterility (if tested) - Method: USP <71> sterility testing (14-day incubation in growth media) - What to look for: "No growth observed" / "Passes USP <71>" - Red flag: Not tested, or growth detected. NOTE: Most research-grade COAs don't include sterility testing. Only 503B pharmacies are required to test. 5. Endotoxin (if tested) - Method: LAL (Limulus Amebocyte Lysate) test - What to look for: Results should be below 5 EU/kg for injectable products. Often reported as <0.25 EU/mL. - Red flag: Not tested (for injectables) or above threshold. 6. Appearance / Solubility - What to look for: White to off-white powder (lyophilized). Clear and colorless solution (reconstituted/liquid). - Red flag: Yellow discoloration, visible particulates. I'll do a walkthrough of an actual COA in the next post.
47 20mike_mealprep, NicoleRaleigh, james_edin and 44 others
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sarah_TO
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Sep 2024
Toronto, CA
Dec 13, 2025 at 8:48 AM#2
Walkthrough of a real COA (anonymized): Here's what a Janoshik COA for semaglutide typically looks like. I'm using data from a recent community-submitted test: ``` CERTIFICATE OF ANALYSIS Lab: Janoshik Analytical Sample ID: JS-2026-01-4872 Date Received: January 14, 2026 Date Reported: January 21, 2026 Sample Description: White lyophilized powder, labeled "Semaglutide 5mg" TEST METHOD RESULT SPECIFICATION Identity ESI-MS Confirmed MW 4113.58 ± 2 Da (Observed: 4113.4 Da) Purity HPLC-UV 98.3% ≥ 95.0% (220nm) Content HPLC vs. std 4.87 mg 4.50 - 5.50 mg (97.4% of label) Residual Solvents GC-FID < LOQ Meets USP <467> Water Content Karl Fischer 1.2% ≤ 5.0% CONCLUSION: Sample meets all tested specifications. ``` How to interpret this: - Identity confirmed at 4113.4 Da — that's within 0.2 Da of theoretical. This is semaglutide. - 98.3% purity — excellent. The remaining 1.7% is likely minor synthesis impurities (truncated sequences, oxidized variants). Nothing unusual. - 4.87mg content — 97.4% of the labeled 5mg. You're getting slightly less than advertised but well within acceptable range. - Residual solvents below limit of quantitation — good. Means the lyophilization was thorough. - 1.2% water content — normal for lyophilized peptide. Up to 5% is acceptable. This is a solid COA. The product is what it claims to be, at the right purity and potency.
Last edited: Dec 13, 2025 at 12:48 PM
6 5Dr.EndoEP, GraceAZ_72, carl_compliance and 3 others
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mona_PHX
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Dec 2024
Phoenix, AZ
Dec 13, 2025 at 9:05 AM#3
This is incredibly helpful. A few clarifying questions: 1. When the purity says 98.3%, what exactly is the other 1.7%? Is it dangerous? 2. How do I know a COA is real and not fabricated? I've heard vendors can just make fake COAs in Photoshop. 3. What's the difference between a vendor-provided COA (from the manufacturer) and a third-party COA (from Janoshik, etc.)? Which should I trust more?
9 13SallyK_inj, CryptoCarl, MariaRD and 6 others
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Janoshik Analytical — Independent Testing

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hyun_seoul
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1,456
Jul 2024
Seoul, KR
Dec 13, 2025 at 9:22 AM#4
Let me tackle these: 1. The "other 1.7%": In peptide synthesis, common impurities include: - Deletion sequences — the peptide chain missing one or more amino acids - Oxidized variants — methionine residues oxidized during synthesis or handling - Truncated sequences — synthesis stopped before completion - Deamidation products — asparagine converting to aspartate - TFA (trifluoroacetic acid) — residual counterion from purification - Acetic acid salt forms — if the peptide was acetate-exchanged At 98.3% purity, the 1.7% impurities are at very low levels. For injectable peptides, the concern is whether any single impurity exceeds 0.5% and whether it's been identified. Reputable labs will flag any individual impurity above 0.5% on the chromatogram. At sub-1% levels each, these synthesis byproducts are generally considered safe for research purposes (and pharmaceutical-grade GLP-1 products have similar impurity profiles — they're just more thoroughly characterized). 2. Fake COAs: This is a real problem. Ways to verify: - Contact the lab directly. Janoshik has a verification page where you can enter the sample ID to confirm it's genuine. Use it. - Check for consistency. A fake COA often has mismatched fonts, wrong test methods, or results that are suspiciously "perfect" (e.g., exactly 100.0% purity — real analytical data has decimal variation). - Look for the chromatogram. A real COA usually includes the actual HPLC chromatogram image. Faking a realistic chromatogram is much harder than faking a table of numbers. - Batch-specific data. A real COA has a unique sample ID and batch number. If a vendor uses the same COA for every batch, it's likely fake. 3. Vendor vs. third-party COA: Always prioritize third-party testing. A vendor-provided COA is the manufacturer grading their own homework. It may be legitimate, but there's an inherent conflict of interest. Third-party labs like Janoshik, Finnrick, or Vanta have no financial stake in the product passing. That independence is what makes their results credible. Best case scenario: the vendor provides their own COA AND a community member independently tests the same batch at a third-party lab, and the results agree. That's the gold standard of verification.
9 9BariatricNurseD, MASHdoc_SA, GenomicsKate and 6 others
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pam_stl
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Oct 2024
St. Louis, MO
Dec 13, 2025 at 9:39 AM#5
Great post Annie. I want to add some detail on reading HPLC chromatograms since the OP mentioned them: What you're looking at: An HPLC chromatogram is a graph with time (minutes) on the X-axis and detector response (absorbance units or millivolts) on the Y-axis. Each "peak" represents a compound eluting from the column. For semaglutide, expect: - Main peak at a retention time typically between 12-18 minutes (depends on the method). This is the semaglutide. - Small peaks before the main peak — these are more polar impurities (hydrophilic, elute faster) - Small peaks after the main peak — these are less polar impurities or oxidized forms - Solvent front at ~2-3 minutes — ignore this, it's just the injection solvent What "98.3% purity by HPLC" means: The area under the main semaglutide peak represents 98.3% of the total area of ALL peaks. This is "area percent" or "area normalization" purity. Important caveat: HPLC purity depends heavily on the method. A method that uses a short column, fast gradient, or UV detection at 280nm might miss impurities that would show up with a longer column, slower gradient, or detection at 220nm. This is why comparing purity numbers across different labs requires knowing the method details. A "99.5% pure" result from one lab is not necessarily better than "97.8% pure" from another if they're using different methods. The lab using the more sensitive method might simply be detecting more impurities that the other lab missed.
42 3RegAffairsDC, BiostatsBrad, PeptideSynthNJ and 39 others
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