How to Read a Peptide COA: Every Field on a Certificate of Analysis

A peptide certificate of analysis (COA) is a lab report for one specific production lot. Knowing how to read a peptide COA means checking four things in order: the lot number matches your vial, purity was measured by high-performance liquid chromatography (HPLC), identity was confirmed by mass spectrometry (MS), and a named, accredited lab ran the tests on a stated date.

This guide walks through each field of a typical report using two catalog peptides as worked examples. BPC-157 is a 15-residue peptide (GEPPPGKPADDAGLV, C62H98N16O22, 1,419.5 g/mol). TB-500 is the 43-residue, N-terminally acetylated thymosin β4 sequence (C212H350N56O78S, 4,963.4 g/mol); the U.S. Food and Drug Administration (FDA) uses the name TB-500 for a seven-residue fragment (Ac-LKKTETQ) (FDA, 2026).

What is a peptide certificate of analysis?

A peptide certificate of analysis is a document issued for a single lot, also called a batch, of synthetic peptide. It records which tests were run on a sample from that lot, by which laboratory, on what date, with what method and with what measured value. A COA describes the lot it names and nothing else, so its value depends on the link between the report and the vial in hand.

Synthetic peptides are commonly built by solid-phase peptide synthesis (SPPS), one amino acid at a time, and each step can leave closely related by-products. A review by D'Hondt et al. (2014) groups peptide-related impurities into synthesis-related forms (deletions, insertions, racemization, incomplete removal of protecting groups, oxidation, dimers and residual trifluoroacetate) and degradation products such as diketopiperazine, pyroglutamate and succinimide forms. The same review notes that such impurities can distort early research findings, which is why a lot-specific COA matters to a laboratory.

How to read a peptide COA, field by field

Every field on a peptide COA answers a specific question. The table below lists the fields found on a complete report, what each one tells you, and what to check before relying on it.

Read the fields in three groups. The identity fields at the top tie the report to a molecule and a lot. The analytical fields in the middle carry the evidence. The composition fields at the bottom explain what else is in the powder besides the target peptide.

What does HPLC purity measure?

HPLC purity is the share of the UV-detected material in a peptide sample that elutes as the main peak. Peptide purity is usually measured by reversed-phase HPLC, in which a hydrophobic column (often C18, meaning octadecyl-bonded silica) and a gradient of water and acetonitrile separate species by hydrophobicity. The mobile phases typically carry an acid modifier such as trifluoroacetic acid at 0.1% (Sikora et al., 2020).

The detector reads UV absorbance at about 214 to 220 nm, where the peptide bond absorbs. That choice lets the method detect peptides through their backbone, including sequences such as BPC-157 and TB-500 that contain no tryptophan or tyrosine. Kuipers and Gruppen (2007) used absorbance at 214 nm to quantify peptides separated by reversed-phase HPLC and showed that the response depends on amino acid composition.

Purity is reported as area percent: the main peak's integrated area divided by the total integrated area of all peaks, multiplied by 100. A chromatogram that supports a high purity figure shows retention time on the x-axis, absorbance on the y-axis, one dominant main peak, and small impurity peaks before or after it, each integrated. Area percent has limits that every reader of a peptide COA should keep in mind:

• It counts only what the detector sees. Water and inorganic salts do not appear as peptide peaks, and counter-ion signals are not counted as peptide species, so HPLC purity says nothing about how much of the powder is peptide.
• Different species absorb differently. Because UV response depends on composition, area percent approximates, rather than equals, the mass fraction of each peptide species.
• Co-eluting impurities hide under the main peak. An impurity that leaves the column at the same time as the target is counted as product, one reason mass spectrometry is run alongside HPLC.
• Method details matter. A purity value without the column, gradient and wavelength cannot be compared with another lab's value.

How does mass spectrometry confirm peptide identity?

Mass spectrometry confirms peptide identity by measuring the mass-to-charge ratio (m/z) of ions formed from the sample and comparing the observed mass with the theoretical mass calculated from the sequence. A common setup is electrospray ionization (ESI), often coupled to liquid chromatography (LC-MS).

Small peptides often appear as the protonated molecule, written [M+H]+. For BPC-157, the monoisotopic mass calculated from C62H98N16O22 is 1,418.70 Da, so [M+H]+ is expected near m/z 1,419.71, and a doubly charged [M+2H]2+ ion may appear near m/z 710.36.

Larger peptides pick up several protons at once. Electrospray spectra of large molecules show a series of peaks from multiply charged ions, each differing by one charge from its neighbors (Fenn et al., 1989). Instrument software deconvolutes that series into one neutral mass, which is the number to compare with the catalog value.

TB-500, which carries nine lysine residues, would be expected to give a ladder such as [M+4H]4+ near m/z 1,241.9, [M+5H]5+ near m/z 993.7 and [M+6H]6+ near m/z 828.2. These values use the average-mass convention, (average molecular weight + n × 1.007 Da) ÷ n, with the catalog value of 4,963.4 g/mol: for [M+5H]5+, (4,963.4 + 5 × 1.007) ÷ 5 = 993.7.

Three checks make the MS line on a peptide COA meaningful:

1. The theoretical value is printed beside the observed value, with the ion type stated.
2. The mass convention is clear. Catalog molecular weights are average masses, while high-resolution instruments often report monoisotopic masses. BPC-157 can correctly read about 0.8 Da below its 1,419.5 g/mol catalog weight for that reason alone.
3. Unexpected masses are explained. Common shifts point to specific impurities: a missing proline in BPC-157 lowers the mass by 97.05 Da, oxidation of the single methionine in TB-500 adds 15.99 Da, and loss of the N-terminal acetyl group of TB-500 removes 42.01 Da.

Mass spectrometry cannot separate molecules of identical mass. A racemized impurity, in which one residue has flipped from the L to the D form, has the same mass as the target peptide, so it has to be separated chromatographically to be seen. That is why a peptide COA reports HPLC purity and MS identity together.

What do water content, counter-ion and net peptide content show?

A lyophilized peptide powder contains more than peptide. The powder also holds bound water, the counter-ion that pairs with the peptide's charged groups, and sometimes residual salts or solvents. The composition fields on a peptide COA explain the gap between the mass of powder in a vial and the mass of peptide.

Water content is often measured by Karl Fischer titration. Counter-ion content can be measured by ion chromatography, which Sikora et al. (2020) describe as the first-choice technique, or by fluorine-19 nuclear magnetic resonance (19F NMR) for trifluoroacetate. Peptides with basic groups that are purified with trifluoroacetic acid are obtained as trifluoroacetate (TFA) salts unless the counter-ion is exchanged for acetate or chloride. Some laboratories specify acetate or chloride salts for cell-based work (Sikora et al., 2020).

Net peptide content is the percentage of the powder's mass that is peptide, determined by methods such as amino acid analysis or elemental nitrogen analysis. In CCQM-K115, an international peptide purity comparison coordinated by the Bureau International des Poids et Mesures (BIPM), a synthetic human C-peptide material was assigned 801.8 mg/g of the target peptide, with peptide-related impurities, trifluoroacetate and water making up most of the remainder (Josephs et al., 2017). Net peptide content and HPLC purity are separate numbers.

Which optional tests appear on some peptide COAs?

Some peptide COAs add tests beyond purity, identity and composition. Each optional test should name its method and its acceptance limit.

• Endotoxin. Bacterial endotoxin is commonly measured with the Limulus amebocyte lysate (LAL) test, an FDA-accepted method described in United States Pharmacopeia (USP) chapter 85, Bacterial Endotoxins Test (FDA, 2012).
• Residual solvents. Gas chromatography can quantify solvents left from synthesis and purification, as in the CCQM-K115 study. The Q3C(R9) guideline from the International Council for Harmonisation (ICH) classifies solvents by toxicity and places acetonitrile, a common HPLC solvent, in Class 2 (ICH, 2024).

What are the red flags on a peptide COA?

Weak peptide COAs tend to fail in recognizable ways. Any of the following is a reason to ask questions before using the material:

• No lot number, or a lot number that does not match the vial.
• One generic COA reused across lots. If every lot of a product shows the same test date, the same purity to the decimal and an identical chromatogram, the report likely describes one sample, not your lot.
• Purity without a chromatogram. A bare percentage cannot be checked.
• A mass that does not match the sequence. Compare the observed mass with a value calculated from the catalog formula, allowing for the difference between monoisotopic and average mass.
• No laboratory named, or a lab with no address, phone number or way to confirm the report.
• A test date before the lot existed, such as a report dated earlier than the lot's manufacture.
• Edited images. Mismatched fonts, misaligned text, blurred numbers beside sharp ones, or a chromatogram that looks pasted in are signs that a report was altered.

How do lot matching and COA verification work?

Lot matching turns a generic document into evidence about the vial in hand. Each production lot gets a unique identifier, the identifier is printed on every vial from that lot, and the COA carries the same identifier. A match connects the vial to the tested sample; a mismatch means the report says nothing about that vial.

To match a PeppedCo vial, read the lot number on the label and enter it in the lookup at /lab-testing/#verify. The lot number is printed on every vial, and the COA for each released lot is published on the Lab Testing page.

To verify a peptide COA with the issuing lab:

1. Note the lab name, report or sample number and test date from the COA.
2. Find the lab's contact details independently, from its own website rather than from the COA.
3. Ask the lab to confirm that it issued that report number, for that sample and lot, on that date. Use the lab's online report lookup if it offers one.
4. Check accreditation separately. According to the International Laboratory Accreditation Cooperation (ILAC), accreditation bodies keep directories of accredited laboratories that can be searched by name and scope, and accreditation certificates list the scope. ISO/IEC 17025:2017 sets requirements for the competence, impartiality and consistent operation of laboratories (ISO, 2017), but accreditation applies only to the methods on the lab's scope.

A COA is one part of evaluating a seller; How to Vet a Research Peptide Supplier covers the rest. Handling after delivery also affects whether a tested lot stays as described, as explained in How to Store Lyophilized Peptides. For identity and regulatory details on one of the example peptides, see What Is BPC-157? Sequence, Salt Forms and Regulatory Status.

Sourcing BPC-157 and TB-500 for laboratory research

Every PeppedCo lot is third-party tested before release: HPLC for purity and mass spectrometry for identity. The purity release threshold is 98.0% or higher by HPLC, and the measured result is on the lot's COA. Full spec sheets for BPC-157 and TB-500 are on each product page.