How to Read a Peptide Certificate of Analysis (COA)

A certificate of analysis (COA) is the only objective evidence of what is in a vial. A well-built COA lets you verify identity, purity and traceability in under five minutes. A weak one gives you a number and nothing to check it against. This guide walks through each section and what to look for.

The header: identity and traceability

The top of a COA should identify the material unambiguously:

• Product name and amino acid sequence
• Molecular formula and theoretical molecular weight
• CAS number, where one exists
• Batch (lot) number, matching the vial label
• Manufacture date, test date and retest or expiry date
• Name of the testing laboratory and an authorising signature

If the batch number on the COA does not match the vial, the document does not describe your material.

HPLC purity

Reversed-phase high-performance liquid chromatography (RP-HPLC) separates the target peptide from synthesis by-products: truncated sequences, deletion sequences, incompletely deprotected species and oxidised forms. Detection is usually UV absorbance at 214–220 nm, where the peptide bond absorbs.

Purity is calculated as the area of the main peak divided by the total area of all integrated peaks. Research-grade peptides are commonly specified at ≥98%.

What to check:

• The chromatogram is attached, not just a percentage.
• Method details are listed: column, mobile phases, gradient and detection wavelength.
• All peaks are integrated, including small ones near the main peak.
• A plausible result. A reported 100.0% is a warning sign; real chromatograms show minor impurities.

HPLC purity does not confirm identity. A wrong peptide can be 99% pure.

Mass spectrometry: identity

Mass spectrometry (MS) confirms that the main component has the expected mass. Electrospray ionisation (ESI) and MALDI-TOF are the common methods.

ESI typically produces multiply charged ions. A peptide with a theoretical mass of 1,419.5 Da, such as BPC-157, appears as [M+2H]²⁺ at roughly m/z 710.8. A good COA reports the observed ions, the deconvoluted mass, and the theoretical value side by side. Agreement within about ±1 Da (average mass) is typical for routine identity testing.

For blends, expect one confirmed mass for each component.

Purity vs peptide content

These two numbers answer different questions and are often confused.

Measure	Question it answers	Typical method	Typical range
HPLC purity	Of the peptide material, how much is the target sequence?	RP-HPLC	≥98%
Net peptide content	Of the total powder mass, how much is peptide?	Amino acid analysis, nitrogen (CHN) analysis	~70–90%

The remainder of the powder is counter-ions, bound water and residual salts. Example: a vial labelled 10 mg with 99% purity and 80% net peptide content contains about 10 × 0.80 × 0.99 ≈ 7.9 mg of the target peptide. For experiments where molar concentration matters, correct for content.

Counter-ions: TFA vs acetate

Trifluoroacetic acid (TFA) is used to cleave peptides from the synthesis resin and in HPLC mobile phases. It remains as a counter-ion on basic groups: lysine, arginine, histidine and the free N-terminus. Peptides with many basic residues can carry substantial TFA by mass.

TFA has been reported to affect cell proliferation in vitro (Cornish et al., 1999). For cell-based work, an acetate or hydrochloride salt is often preferred, produced by ion exchange after purification. A complete COA states the counter-ion, and ideally the residual TFA content measured by ion chromatography or ¹⁹F NMR.

Endotoxin

Endotoxins are lipopolysaccharides from Gram-negative bacteria. They are heat-stable and survive lyophilisation. They can confound cell culture and in vivo animal studies by triggering inflammatory signalling unrelated to the peptide.

Endotoxin is measured by the Limulus amebocyte lysate (LAL) test described in USP ⟨85⟩, and reported in EU/mg. Not every research COA includes it; if your model is sensitive to inflammatory signalling, ask for it.

Other tests you may see

• Water content (Karl Fischer titration)
• Residual solvents (gas chromatography)
• Appearance and solubility (white to off-white lyophilised powder; clear solution in a stated solvent)
• Sterility (USP ⟨71⟩), relevant only for material supplied as sterile

Batch numbers and independent verification

Every vial should carry a batch number that maps to exactly one COA. You can look up any Purity Peptides batch at /coa. Independent third-party testing is stronger evidence than in-house testing, because the laboratory has no stake in the result. Where possible, the COA should name the laboratory so it can be contacted.

Red flags

• A purity percentage with no chromatogram or mass spectrum
• Purity reported as exactly 100%
• No batch number, or the same COA used for several batches
• An observed mass with no theoretical value, or one that does not match
• No test date, no laboratory name, no signature
• Inconsistent fonts, misaligned fields or visible editing
• A blend COA that reports a single purity and a single mass

Quick checklist

1. Batch number matches the vial.
2. HPLC chromatogram attached; purity ≥98% and plausible.
3. Observed mass matches theoretical mass.
4. Counter-ion stated.
5. Test date recent relative to the batch.
6. Laboratory identified.

References

• United States Pharmacopeia. General Chapter ⟨85⟩ Bacterial Endotoxins Test. USP–NF.
• United States Pharmacopeia. General Chapter ⟨71⟩ Sterility Tests. USP–NF.
• International Council for Harmonisation. ICH Q2(R2): Validation of Analytical Procedures. 2023.
• Cornish J, Callon KE, Lin CQ, et al. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. Am J Physiol. 1999;277(5):E779–E783.
• US Food and Drug Administration. ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin. Guidance for Industry. 2021.