How to read a peptide certificate of analysis (HPLC, mass spec and purity)

A certificate of analysis (COA) is the document that links a specific batch of peptide to the tests run on it. A good one lets a researcher confirm that the material in the vial is the compound on the label, and tells them how pure it is. A poor one is just decoration. This guide explains what each part of a typical peptide COA means and how to tell the difference.

Start with the batch details

Before looking at any result, check that the document identifies exactly what was tested. It should name the peptide, give its sequence or molecular formula, and show a lot (batch) number that matches the number on your vial. Without a lot number, a COA cannot be tied to the material you actually received.

• Product name, sequence or molecular formula, and theoretical molecular weight
• Lot or batch number, and the manufacture or test date
• The name of the laboratory that ran the tests, and whether it holds an accreditation such as ISO/IEC 17025
• The methods used, and the specification each result is compared with

HPLC: what the purity percentage means

High-performance liquid chromatography (HPLC) separates a sample into its components as it passes through a column. The detector, usually measuring ultraviolet light absorbed by the peptide bonds around 214 to 220 nanometers, draws a chromatogram: a line with a peak for each component. The headline purity figure is normally the area of the main peak as a percentage of the area of all peaks.

That makes the number a useful measure of how much of the detected material is the target peptide, compared with related impurities such as truncated or modified sequences. It is not a measure of the weight of peptide in the vial, because the detector cannot see water, salts or counter-ions, and different molecules absorb light differently.

• Look at the chromatogram itself, not just the percentage. A single dominant peak with a clean baseline is what you want to see.
• Check that the method is stated: column type, gradient, detection wavelength and run time.
• A figure such as 99.2% is an area percentage under that method. A different method can give a different number for the same batch.

Mass spectrometry: confirming identity

HPLC tells you how clean a sample is, but not what it is. Mass spectrometry (MS) measures the mass of the molecules, which confirms identity. Peptides usually carry several charges in the instrument, so the spectrum shows a series of peaks for the same molecule, and the software converts them to a single molecular weight.

A good COA shows the observed mass next to the theoretical mass. For standard instruments they should agree to within a fraction of a dalton. A match supports the identity of the peptide, though full sequence confirmation needs further tests such as tandem MS or amino acid analysis.

Other results you may see

Purity and identity are the core of a COA, but several other results help describe the material, and a thorough report may include some of them.

• Appearance: peptides are usually supplied as a white to off-white lyophilized (freeze-dried) powder.
• Net peptide content: the proportion of the powder that is actually peptide, as opposed to water and counter-ions. It is commonly well below 100%.
• Water content, often measured by Karl Fischer titration.
• Counter-ion: most synthetic peptides are supplied as a trifluoroacetate (TFA) or acetate salt.
• Endotoxin, measured in endotoxin units per milligram, when the material is intended for cell-based work.

Red flags

Most problems with a COA are visible once you know what to look for. Treat any of the following as a reason to ask questions before relying on the material.

• No lot number, or a lot number that does not match your vial.
• No laboratory named, or only an in-house test with no independent verification.
• The same chromatogram or spectrum appearing on certificates for different products.
• A purity figure with no chromatogram, method or date to support it.
• Results that are perfectly round or identical from batch to batch.

Questions to ask any supplier

Reading a COA is only part of due diligence. It is reasonable to ask a supplier which laboratory tests each batch, whether certificates are issued per lot rather than per product, and how long they keep retained samples. Clear, specific answers are a good sign. Vague ones are worth noting.

All products sold by Peptides Biotech are intended for laboratory research use only and are not for human or veterinary consumption. You can browse available certificates on our certificates of analysis page.