How to Read a Peptide Certificate of Analysis (COA)
You just received a vial of peptide for your research. Taped to the box or buried in a download link is a document labeled "Certificate of Analysis." It's packed with numbers, abbreviations, and chromatograms that look like abstract art. Most people glance at the purity percentage and move on.
You just received a vial of peptide for your research. Taped to the box or buried in a download link is a document labeled "Certificate of Analysis." It's packed with numbers, abbreviations, and chromatograms that look like abstract art. Most people glance at the purity percentage and move on.
That's a mistake.
A Certificate of Analysis (COA) is the single most important document attached to any peptide product. It tells you whether the compound in your vial is actually what it claims to be, how pure it is, and whether it's contaminated with anything that could ruin your experiments or, worse, your health. Learning to read one properly takes about 15 minutes. Skipping that step can cost you months of unreliable data.
This guide walks you through every section of a peptide COA, explains what the numbers mean, and shows you how to spot fakes.
Table of Contents
- What Is a Certificate of Analysis?
- Section 1: Product Identification and Batch Information
- Section 2: Identity Confirmation via Mass Spectrometry
- Section 3: Purity by HPLC
- Section 4: Understanding the HPLC Chromatogram
- Section 5: Net Peptide Content vs. Purity
- Section 6: Amino Acid Analysis
- Section 7: Endotoxin Testing
- Section 8: Additional Testing You May Encounter
- Section 9: Methodology and Equipment Details
- How to Spot a Fake or Low-Quality COA
- COA Quick-Reference Checklist
- The Bottom Line
- References
What Is a Certificate of Analysis?
A COA is a formal laboratory document that reports the results of analytical testing performed on a specific batch of peptide. Each COA corresponds to one batch and one batch only. It records what was tested, how it was tested, and whether the results meet defined quality standards.
Think of it as a peptide's report card. A good one includes identity verification, purity data, content measurements, and contamination screening. A bad one lists a purity number with no supporting data --- like a student claiming a 4.0 GPA without any transcripts.
Every COA should be signed or authorized by an analyst, include a unique lot or batch number, and reference the specific testing date. If any of those elements are missing, the document's credibility drops immediately.
Section 1: Product Identification and Batch Information
The header of any COA contains the basics:
- Peptide name and sequence --- The full amino acid sequence of the peptide being tested
- Batch or lot number --- A unique identifier for this specific manufacturing run
- Molecular formula and theoretical molecular weight --- Calculated from the amino acid sequence
- Production or testing date --- When the analysis was performed
- Quantity --- The amount of peptide in the vial
- Storage conditions --- Temperature and handling recommendations
- Salt form --- Usually TFA (trifluoroacetate) or acetate salt
- Physical appearance --- Typically described as "white to off-white lyophilized powder"
Why this matters: The batch number is your traceability link. If you run an experiment in March and need to verify something in September, the lot number lets you trace back to the exact production run. Suppliers who provide identical COAs across different batches are not testing each lot individually --- that is a red flag.
Section 2: Identity Confirmation via Mass Spectrometry
Mass spectrometry (MS) answers the most basic question: is this actually the peptide it claims to be?
The instrument measures the molecular weight of the compound and compares it to the expected weight calculated from the amino acid sequence. The COA will typically report:
- Theoretical mass --- Calculated from the known sequence
- Observed mass --- Measured by the instrument
- Technique used --- Usually ESI-MS (Electrospray Ionization) or MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight)
A passing result means the observed mass matches the theoretical mass within an acceptable tolerance, typically plus or minus 0.5 to 1.0 Daltons for singly charged ions.
Why MS Matters More Than You Think
Here's something most people don't realize: HPLC cannot confirm sequence identity. A peptide missing one amino acid --- a "deletion peptide" --- might still appear 99% pure on an HPLC chromatogram because it separates cleanly from other impurities. But it's the wrong molecule.
Mass spectrometry catches these errors. It can detect:
- Truncated sequences (missing amino acids)
- Deletion peptides
- Oxidation products
- Mislabeled compounds
- Amino acid substitutions
If a COA shows HPLC purity but no MS data, you have no way to confirm you're working with the correct peptide. That's a dealbreaker for any serious application.
ESI-MS vs. MALDI-TOF
ESI-MS produces multiply charged ions, which means it can analyze larger peptides with higher sensitivity. You'll often see peaks labeled as [M+2H]2+ or [M+3H]3+. MALDI-TOF produces mostly singly charged ions ([M+H]+), making the spectra simpler to read. Both are valid for identity confirmation.
Section 3: Purity by HPLC
High-Performance Liquid Chromatography (HPLC) is the gold standard for measuring peptide purity. It separates the peptide sample into its individual components and quantifies each one.
The technique works by pushing the dissolved peptide through a column packed with hydrophobic material (usually C18 silica). Different compounds interact with the column differently and exit at different times. A UV detector at the end measures absorption at 214--220 nm --- the wavelength where peptide bonds absorb strongly --- creating a chromatogram.
What the Purity Number Means
Purity is reported as a percentage: the area of the main peptide peak divided by the total area of all detected peaks. So "98.5% purity" means 98.5% of the UV-detectable peptide material in the sample is the target compound. The other 1.5% consists of peptide-related impurities --- truncated sequences, oxidized forms, or synthesis byproducts.
Purity Benchmarks
| Purity Level | Grade | Suitable For |
|---|---|---|
| 98% or above | High purity | Quantitative assays, structural biology, cell-based studies, binding assays |
| 95--97% | Standard research grade | General in vitro experiments, screening assays |
| 90--95% | Low research grade | Preliminary screening, antibody production |
| Below 90% | Poor quality | Generally unsuitable for reliable research |
For most applications covered on PeptideJournal.org --- including peptides like BPC-157 or semaglutide --- you want to see purity above 95%, and preferably above 98% for anything involving biological assays.
Section 4: Understanding the HPLC Chromatogram
The chromatogram is the actual graph produced by the HPLC analysis. Many COAs include it; the best ones always do.
Reading the Axes
- X-axis (horizontal): Time in minutes. This represents retention time --- when each compound exits the column.
- Y-axis (vertical): Detector response in milli-absorbance units (mAU). This reflects how much UV light the compound absorbed.
What a Good Chromatogram Looks Like
A high-purity peptide produces a single, sharp, symmetrical peak. The baseline before and after the peak should be flat and clean. Minimal secondary peaks indicate few impurities.
Warning Signs in a Chromatogram
| Issue | What It Looks Like | What It Means |
|---|---|---|
| Shoulder peaks | A bump on the side of the main peak | A closely related impurity co-eluting with the target |
| Multiple large secondary peaks | Several distinct peaks besides the main one | Significant impurities or degradation products |
| Peak tailing | The main peak has an asymmetric trailing edge | Column issues or problematic sample interactions |
| Broad main peak | The target peak is wide rather than sharp | Poor chromatographic separation |
| Noisy or drifting baseline | Jagged or sloping flat regions | Detector issues, contaminated mobile phase, or poor equilibration |
Calculating Purity from a Chromatogram
If the main peak area is 2,500,000 and impurity peak areas total 75,000, purity equals (2,500,000 / 2,575,000) x 100 = 97.1%.
Note that early-eluting peaks near the solvent front (the first 1--2 minutes) are often solvent artifacts, not peptide impurities. These should be excluded from the purity calculation. A reputable lab will note this in their methodology.
Section 5: Net Peptide Content vs. Purity
This is the most misunderstood distinction on any COA.
Purity (measured by HPLC) tells you what percentage of the peptide-related material is the target compound. Net Peptide Content (NPC) tells you what percentage of the total vial weight is actual peptide --- as opposed to water, counterions, and residual solvents.
Why a "99% Pure" Peptide Might Only Be 70% Peptide by Weight
After HPLC purification, most peptides are delivered as trifluoroacetate (TFA) salts. TFA molecules bind to every basic amino group --- the N-terminus plus the side chains of lysine, arginine, and histidine residues. These counterions add weight that isn't peptide.
On top of that, lyophilized peptides are hygroscopic. They absorb water from the air. And small amounts of residual solvents from the purification process can persist.
None of these non-peptide components show up on HPLC because they don't absorb UV light at 220 nm. So a peptide can be 99% pure by HPLC while having a net peptide content of only 60--85%.
The Math That Matters for Dosing
To calculate how much actual target peptide you have:
Actual peptide = Gross weight x NPC x HPLC Purity
Example: A vial containing 5 mg gross weight, 84% NPC, and 96% HPLC purity holds 5 x 0.84 x 0.96 = 4.03 mg of the target peptide.
If you're following a reconstitution protocol and need precise dosing, this calculation matters. Assuming the gross weight equals the peptide weight can lead to underdosing by 15--40%.
How NPC Is Measured
Net peptide content is determined through amino acid analysis (AAA), elemental analysis (CHN), or UV spectrophotometry. AAA requires less material but is less precise. Elemental analysis needs milligram quantities but delivers more accurate results.
Section 6: Amino Acid Analysis
Amino acid analysis (AAA) breaks the peptide into its individual amino acid components and quantifies each one. This confirms the peptide contains the correct amino acids in the correct ratios.
Results typically show each amino acid alongside expected and observed values. Acceptable tolerance is usually plus or minus 10% of the theoretical composition.
Not every COA includes AAA --- it's more common for custom synthesis orders, GMP-grade products, or premium research peptides. But when present, it provides an additional layer of identity confirmation beyond mass spectrometry.
AAA also doubles as one method for determining net peptide content, making it a dual-purpose test.
Section 7: Endotoxin Testing
Endotoxins are lipopolysaccharides (LPS) found on the outer membrane of gram-negative bacteria. They're potent inflammatory triggers that can cause fever, tissue injury, and toxic shock in animals. In cell culture, even low levels of endotoxin contamination produce non-specific immune responses that confound results.
The LAL Assay
Endotoxin testing uses the Limulus Amebocyte Lysate (LAL) assay, derived from horseshoe crab blood. The test detects endotoxin activity measured in Endotoxin Units (EU) rather than weight, because endotoxin potency varies depending on the bacterial source and molecular characteristics.
One EU is roughly equivalent to 0.1--0.2 nanograms of endotoxin per milliliter.
Acceptable Levels
| Application | Typical Endotoxin Limit |
|---|---|
| General in vitro research | Less than 1 EU per microgram |
| Cell-based assays | Less than 0.1 EU per microgram |
| In vivo animal research | Less than 0.1 EU per microgram or less than 0.25 EU/mL |
| Pharmaceutical injectable (non-intrathecal) | 5 EU per kilogram body weight |
When Endotoxin Testing Is Present (and When It's Not)
Most standard research-use-only (RUO) COAs do not include endotoxin testing. It's an additional test that some vendors offer for a premium. If you're planning any cell-based or in vivo work, the absence of endotoxin data means you're flying blind on contamination status.
For peptides intended for subcutaneous injection protocols, endotoxin data becomes especially relevant. A contaminated batch could cause injection site reactions that have nothing to do with the peptide itself.
Section 8: Additional Testing You May Encounter
Water Content (Karl Fischer Titration)
Measures the percentage of water in the lyophilized sample. Important for calculating true peptide content and assessing storage stability. Typical values range from 2--8%.
Residual Solvent Analysis
Detects leftover acetonitrile, TFA, or other solvents from the purification process. GMP-grade COAs report these values against ICH Q3C solvent limit guidelines.
Counterion Content (Ion Chromatography)
Quantifies the TFA or acetate counterions bound to the peptide. This directly affects net peptide content calculations.
Optical Rotation
Measures the chirality of the peptide to confirm correct stereochemistry. Important for peptides containing D-amino acids or specific chiral centers.
Bioburden
A count of viable microorganisms in the sample. Relevant for sterile applications.
The Kaiser Test
Detects free amino groups. In solid-phase synthesis, free amines can cause unintended amino acid additions. A negative Kaiser test on the final product confirms proper synthesis completion.
Section 9: Methodology and Equipment Details
A credible COA includes the testing methodology: what equipment was used, what parameters were applied, and what standards were referenced.
For HPLC, look for:
- Column specifications --- Typically C18 reverse-phase, 4.6 mm x 250 mm, 5 micrometer particle size
- Detection wavelength --- 214 or 220 nm for peptide bond detection
- Mobile phase composition --- Usually water and acetonitrile with a TFA modifier
- Gradient program --- How the solvent ratio changes over time (a 1% per minute acetonitrile increase is standard)
- Flow rate --- Commonly 1.0 mL/min
Without methodology details, you cannot compare results across suppliers or independently evaluate the analysis. A purity number without a method is scientifically incomplete.
How to Spot a Fake or Low-Quality COA
The peptide market includes bad actors. Some suppliers generate fabricated COAs or recycle old data across multiple batches. Here's what to watch for:
Red Flags
- Suspiciously round numbers --- Real analytical results have decimal places. "99.00% purity" on every batch is not how chemistry works.
- Identical results across batches --- If the COA for lot #2024-001 shows the exact same numbers as lot #2024-037, the testing was not performed individually.
- No chromatogram --- A purity percentage without a chromatogram is a claim without evidence.
- No MS data --- Without mass spectrometry, identity is unverified. You might have a 99% pure sample of the wrong peptide.
- Missing batch or lot number --- No traceability means no accountability.
- No methodology section --- If they don't tell you how the testing was done, you can't evaluate whether it was done correctly.
- PDF metadata inconsistencies --- A "fresh" PDF creation date for an old batch suggests the document was recently generated rather than archived from actual testing.
- Generic or template appearance --- COAs that look like marketing materials rather than lab reports often are exactly that.
How to Verify
Some third-party labs now include QR codes on their COAs that link to online verification pages. Labs like Janoshik, MZ Biolabs, and Chromate offer this feature. If you can scan a code and confirm the results against the lab's database, the COA is likely legitimate.
You can also submit your peptide for independent third-party testing to verify the supplier's claims.
COA Quick-Reference Checklist
Use this checklist to evaluate any peptide COA in under two minutes:
| Element | Present? | What to Check |
|---|---|---|
| Batch/Lot Number | Required | Unique to this specific production run |
| Peptide Sequence | Required | Matches your order |
| Testing Date | Required | Recent and batch-specific |
| MS Identity | Required | Observed mass within 0.5--1.0 Da of theoretical |
| HPLC Purity | Required | Above 95% for research; above 98% for sensitive work |
| Chromatogram | Strongly recommended | Single sharp peak, clean baseline |
| Methodology | Strongly recommended | Column, wavelength, gradient, flow rate specified |
| Net Peptide Content | Recommended | Reported via AAA, elemental analysis, or UV |
| Salt Form | Recommended | TFA or acetate noted |
| Endotoxin Level | Context-dependent | Required for cell-based or in vivo applications |
| Storage Conditions | Recommended | Temperature and light protection specified |
The Bottom Line
A COA is only as useful as your ability to read it. The purity percentage gets all the attention, but identity confirmation through mass spectrometry, net peptide content for accurate dosing, and endotoxin levels for biological applications matter just as much.
The best COAs include HPLC chromatograms, MS data, methodology details, and batch-specific information. The worst ones show a single number on a branded template with no supporting data.
Before using any peptide --- whether for research applications or under medical supervision at a peptide therapy clinic --- take five minutes to review the COA. Check the mass spec data first (is this the right molecule?), then the HPLC purity (how clean is it?), then the net peptide content (how much is actually in the vial?). Those three data points tell you 90% of what you need to know.
The document exists for your protection. Use it.
References
- AmbioPharm. "What data is provided on the Certificate of Analysis (CoA)?" https://www.ambiopharm.com/faq/data-provided-on-certificate-of-analysis/
- Bachem. "Quality Control of Amino Acids and Peptides: A Guide." https://www.bachem.com/knowledge-center/peptide-guide/quality-control-of-amino-acids-and-peptides/
- Creative Proteomics. "RP-HPLC Peptide Purity Analysis." https://www.creative-proteomics.com/peptidomics/rp-hplc-peptide-purity-analysis.html
- Iris Biotech. "Net content and purity, two key parameters in peptide synthesis." https://iris-biotech.de/en/blog/net-content-and-purity-two-key-parameters-in-peptide-synthesis.html
- Iris Biotech. "How to Decipher a Certificate of Analysis (CoA)." https://iris-biotech.de/global/blog/how-to-decipher-a-certificate-of-analysis-coa.html
- Sarin VK, et al. "HPLC Analysis and Purification of Peptides." Methods in Molecular Biology. PMC7119934. https://pmc.ncbi.nlm.nih.gov/articles/PMC7119934/
- GenScript. "Peptide Accupep+QC Testing." https://www.genscript.com/accupep_quality.html
- Ethos Analytics. "Peptide Purity and Quantitation Testing." https://www.ethosanalytics.io/peptide-purity-and-quantitation
- Thermo Fisher Scientific. "Overview of Endotoxin Testing Methods." https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-purification/endotoxin-quantitation-removal/overview-of-endotoxin-testing-methods.html
- AmbioPharm. "What is Net Peptide Content?" https://www.ambiopharm.com/faq/what-is-net-peptide-content/