How to read a peptide Certificate of Analysis - Alphapeptide

A COA is the only document a research peptide ships with that contains testable claims. The label is marketing. The product page is marketing. The COA is, in principle, the output of an analytical lab. In practice, COAs vary from rigorous to nearly fictional, and the difference is legible if you know which sections to read.

The lot number is the most important field

A COA is not a statement about a product line. It is a statement about a specific batch — the lot number ties the document to the vial in your hand. If the COA does not name a lot, or if the lot number on the COA does not match the lot number stamped on the vial cap, you have an unverified vial. Every other number on the document is meaningless until lot identity is established.

Reputable suppliers print the lot number in two places: on the COA header and on the vial. Match them before trusting anything else.

HPLC purity — what the percentage means

HPLC purity is the headline number on every COA. It is reported as a percentage — 98%, 99.1%, 99.7%. The percentage refers to the area under the main chromatographic peak, divided by the total area of all peaks detected at the chosen wavelength.

A few things this percentage does not tell you:

• It does not measure water or salt content. HPLC at 220 nm cannot see water. A peptide reported at 99% HPLC purity may still be 30% mass water of hydration and counter-ions. Mass spectrometry and Karl Fischer titration are the reads that catch this.
• It does not catch impurities that co-elute with the target. A peak that looks pure at one method may resolve into two peaks under a different gradient.
• It does not capture chirality. D- and L-isomers may co-elute on standard reverse-phase columns.

A vendor reporting 99%+ purity should also report the wavelength (typically 214 or 220 nm) and the column system. Without those, the percentage is unverifiable.

Mass spectrometry — does the molecule weigh what it should?

Mass spectrometry (usually MALDI-TOF or ESI-MS) reports the observed molecular mass and compares it to the theoretical molecular weight from the sequence. The two numbers should match to within ±1 Da for peptides under ~5 kDa, and ±2–3 Da for larger constructs.

If the observed mass is off by 18 Da, the peptide may be carrying an extra water of hydration; off by 22, sodium adduction; off by 78, a benzyl protecting group that was never removed. Big offsets — 50 Da or more — usually mean a truncation or a wrong sequence, and that is a reject.

Many low-quality COAs report only HPLC and skip MS entirely. A 99% pure peak of the wrong molecule is still the wrong molecule. Insist on MS confirmation for any peptide you intend to cite in a study log.

Appearance and solubility

The "appearance" line is usually filled in as "white to off-white lyophilized powder". This is not noise. A yellow tinge in a peptide that should be white is a sign of oxidation or contamination from the synthesis. A clumped, wet appearance suggests vial-seal failure or moisture ingress.

Solubility is reported as the test result of dissolving the peptide in a specified diluent — often water, BAC water, or a low-percent acetic acid for hydrophobic sequences. "Soluble in water at 1 mg/mL" is a usable claim. "Use BAC water" without a concentration is not.

Peptide content vs net peptide mass

Two numbers that get confused regularly:

• Peptide content (sometimes called "net peptide") is the percentage of the total mass that is actual peptide, with water and counter-ions excluded. A vial labelled 5 mg with 80% peptide content holds 4 mg of peptide and 1 mg of water and salt.
• HPLC purity is the chromatographic purity of the peptide fraction — separate from how much peptide you have.

A peptide can be 99% HPLC pure and only 75% peptide content. That is a real combination, not an error. Reconstitution math should use the labelled vial mass unless the COA reports peptide content, in which case the effective mass is labelled mass × peptide content.

Most research peptides do not report peptide content on the COA. When it is reported, it is the more honest of the two numbers — and the gap between 99% HPLC and 75% peptide content is the difference between a vial that does what its label says and one that quietly under-delivers.

Counter-ion and salt form

Many synthetic peptides are produced as TFA salts — trifluoroacetate counter-ions left over from synthesis. The salt form affects the molecular weight the COA reports. A peptide quoted as "Free base MW: 1419.5 Da, TFA salt MW: 1647.5 Da" is the same molecule with two different mass labels, depending on which form was weighed.

When the salt form is not reported, assume TFA. When research literature and your COA disagree on molecular weight by 100–500 Da, salt form is the most likely cause.

Sterility, endotoxin, microbial limits

Research peptides are not regulated as pharmaceuticals, so most COAs do not report sterility or endotoxin. When they do, the numbers to watch:

• Sterility: pass/fail for USP <71>. A reputable supplier will state the test method.
• Endotoxin: reported in EU/mg. Limits depend on intended research use; most in-vitro work tolerates 1–10 EU/mg comfortably.
• Bioburden: total aerobic count and total yeast/mold count. Numbers under 100 CFU/g are unremarkable; orders of magnitude higher is a reject.

What to do when the COA is missing or thin

A missing COA is a reject. A COA without lot number, MS, or HPLC method details is a near-reject — at minimum, request the analytical raw data before using the material in a study. Suppliers that take a day to respond to "what column did you run this on?" are not running the column.

The COA is a contract about what is in the vial. Reading it carefully is the cheapest QA you have.