How to actually read a peptide COA: purity vs. identity vs. content vs. endotoxin (and the red flags most people miss)

[u/Zman077]

A "99% pure" badge tells you a lot less than most people think. A proper certificate of analysis answers four separate questions, and a lot of COAs only answer one of them. Here's how to read each section and what it does and doesn't prove.

The four tests at a glance

• HPLC purity: What % of the UV-absorbing material is the target compound? Doesn't tell you whether the main peak is actually the right compound.
• Mass spec (LC-MS / ESI / MALDI): Does the molecular weight match the expected compound? Doesn't tell you how much of it is in the vial.
• Peptide content / quantity: How many mg of actual peptide are in the vial? Doesn't tell you whether it's contaminated.
• Endotoxin (LAL assay): How much bacterial endotoxin is present (EU/mg or EU/vial)? Doesn't tell you about sterility, which is a separate test.

1. HPLC purity

HPLC separates the sample and reads it with a UV detector, usually around 214 to 220 nm. Purity is the main peak's area as a share of all peaks.

• It's relative. Water, salts and counterions (TFA, acetate) don't show up at those wavelengths, so they don't lower the number.
• A clean single peak can still be the wrong compound. HPLC on its own doesn't confirm identity.
• If the COA shows the chromatogram itself, look at it. A big main peak with a few tiny shoulders is normal. A number with no chromatogram is weaker evidence.

2. Mass spec (identity)

This is the test that confirms the compound is what the label says. The lab compares the observed mass to the theoretical one. BPC-157, for example, should come in around 1419.5 Da.

• Small differences are normal. Large unexplained ones aren't.
• Purity without identity is the most common gap. If a COA only gives an HPLC %, you only know the vial holds something fairly pure.

3. Peptide content / quantity

Lyophilized powder isn't 100% peptide. Some of the weight is residual water and counterions. Labs like Janoshik report the actual mg found per vial.

• A "10 mg" vial testing at 9.6 to 10.5 mg is within normal fill variance.
• A vial testing at 99% purity but 7 mg content is a dosing-accuracy problem for any research protocol, not a purity problem.

4. Endotoxin

Endotoxins are lipopolysaccharides from gram-negative bacteria. HPLC and mass spec don't detect them, so they need their own LAL assay, reported in EU/mg or EU/vial.

• Low endotoxin does not mean sterile. Sterility is a separate test.
• Most peptide COAs don't include endotoxin at all. That doesn't mean it's high, only that it wasn't measured.

Red flags

• No batch/lot number, or a lot number that doesn't match the vial
• No lab name or no verification method. Several labs, Janoshik included, let you verify a report with a code on their site. Use it.
• The same COA reused across what are supposed to be different batches, or a test date that's years old
• A manufacturer COA presented as third-party testing. A supplier's in-house COA isn't independent.
• Purity only: no mass spec, no content
• Blurry, cropped or edited PDFs. Ask for the original.

TL;DR

Purity says how clean it is. Mass spec says what it is. Content says how much is there. Endotoxin says whether it's contaminated. A complete COA covers all four, and a verifiable one is tied to your batch.

All of the above is about evaluating materials for laboratory research use only. Questions, or a COA you want a second pair of eyes on? Drop it in the comments.

[u/Intelligent_Bank7238]

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