How to Read a Peptide COA (Certificate of Analysis): Complete Guide

A Certificate of Analysis (COA) is your primary tool for verifying peptide quality before use in research. Yet many researchers struggle to interpret these technical documents, missing critical quality indicators or falling for falsified reports.

This comprehensive guide breaks down every section of a peptide COA, explains what each test measures, and teaches you to spot both quality peptides and potential red flags that suggest contamination, degradation, or outright fraud.

What Is a Certificate of Analysis?

A Certificate of Analysis is an official document issued by a quality control laboratory that verifies a product meets specified standards. For research peptides, COAs document:

• Identity confirmation — proof the peptide is what it claims to be
• Purity levels — percentage of target peptide vs. impurities
• Physical characteristics — appearance, solubility, pH
• Batch information — lot number, synthesis date, expiration
• Testing methods — analytical techniques used for verification

Reputable peptide suppliers provide batch-specific COAs for every product. These documents serve as quality assurance, allowing researchers to verify they're working with properly synthesized, sufficiently pure compounds.

Key Sections of a Peptide COA

Understanding each section of a COA helps you extract maximum information about peptide quality. Here's what to look for:

1. Header Information

The top of a COA should include:

• Product name — common name and/or catalog number
• Lot/batch number — unique identifier for this synthesis run
• Date of manufacture — when the peptide was synthesized
• Date of analysis — when testing was performed
• Expiration date — stability-based use-by date
• Quantity — amount in the batch

2. Product Specifications

This section lists expected parameters and actual test results:

3. Analytical Test Results

This is the most important section—actual quality data from laboratory testing:

Understanding HPLC Results

High-Performance Liquid Chromatography (HPLC) is the gold standard for peptide purity analysis. Here's how to interpret these results:

How HPLC Works

HPLC separates a sample into components based on their chemical properties. The peptide solution is pushed through a column, and different molecules exit (elute) at different times. A detector measures what comes out, producing a chromatogram—a graph showing peaks at different retention times.

Reading an HPLC Chromatogram

A quality COA often includes the actual HPLC chromatogram image. Here's what to look for:

• Main peak — the target peptide (should dominate the graph)
• Retention time — when the main peak elutes (typically noted in minutes)
• Peak area percentage — relative area under each peak indicates purity
• Minor peaks — represent impurities (synthesis byproducts, degradation products)

HPLC Purity Standards

Mass Spectrometry: Confirming Identity

While HPLC tells you how pure your peptide is, mass spectrometry (MS) confirms you have the correct peptide. This is crucial—a peptide could be 99% pure but be the wrong molecule entirely.

Types of Mass Spectrometry

• ESI-MS (Electrospray Ionization) — most common for peptides, good for larger molecules
• MALDI-TOF — Matrix-Assisted Laser Desorption/Ionization, excellent for peptide identification
• LC-MS — combines HPLC separation with MS detection for comprehensive analysis

Interpreting MS Results

The COA should report:

• Theoretical (calculated) mass — expected molecular weight based on sequence
• Observed (experimental) mass — what the instrument actually measured
• Mass accuracy — deviation between theoretical and observed

For peptide identity confirmation, the observed mass should match the theoretical mass within ±1 Dalton (Da). Modern instruments often achieve accuracy within 0.1 Da.

Common Mass Spectrometry Annotations

You may see notation like [M+H]⁺ or [M+2H]²⁺. These indicate:

• [M+H]⁺ — molecular mass plus one proton (singly charged)
• [M+2H]²⁺ — molecular mass plus two protons (doubly charged, mass appears halved)
• [M+Na]⁺ — sodium adduct (adds 22 Da to observed mass)

Understanding these notations helps you verify the reported mass correctly reflects your peptide's molecular weight.

Additional Quality Tests

Beyond HPLC and MS, comprehensive COAs may include:

Amino Acid Analysis (AAA)

This test hydrolyzes the peptide into individual amino acids and quantifies each. Results appear as ratios (e.g., Ala 3.0, Gly 2.0 for a peptide with 3 alanines and 2 glycines). AAA verifies the peptide contains the correct amino acids in proper proportions.

Endotoxin Testing (LAL Test)

Limulus Amebocyte Lysate testing detects bacterial endotoxins. This is especially important for peptides intended for cell culture or in vivo research, where endotoxins could trigger inflammatory responses and confound results.

Water Content (Karl Fischer)

Measures residual moisture in lyophilized peptides. High water content (>10%) can accelerate degradation and affects accurate weighing for dose preparation.

Acetate/TFA Content

Peptides synthesized using standard methods often contain counterions (acetate or trifluoroacetate salts). This affects net peptide content—a vial labeled 5mg may contain less actual peptide if counterion content is high.

Red Flags: Spotting Fake or Low-Quality COAs

Unfortunately, not all COAs are legitimate. Here's how to identify potential problems:

Warning Signs of Fraudulent COAs

Questions to Ask Your Supplier

• Can I get a batch-specific COA for my exact lot number?
• Who performed the testing (in-house or third-party)?
• Can you provide the raw HPLC chromatogram file?
• What is your QC laboratory's accreditation status?
• How often do you re-test stored inventory?

Practical COA Checklist

Use this checklist when reviewing peptide COAs:

COA Terminology Glossary

Frequently Asked Questions

Related Resources

For more information on peptide quality and handling:

• Understanding Peptide Purity: HPLC & Testing Guide
• How to Reconstitute Peptides
• How to Store Peptides Properly
• How to Tell If Peptides Have Gone Bad
• What Are Peptides? Beginner's Guide
• Best Peptides for Beginners