How to Verify Peptide Purity: Third-Party Testing
A supplier sends you a peptide with a Certificate of Analysis claiming 99.2% purity. The chromatogram looks clean. The mass spec data checks out. But here's the uncomfortable question: do you trust it?
A supplier sends you a peptide with a Certificate of Analysis claiming 99.2% purity. The chromatogram looks clean. The mass spec data checks out. But here's the uncomfortable question: do you trust it?
The supplier manufactured the product, tested it in their own lab, and wrote the report. That's not fraud --- most reputable companies run legitimate quality control. But it is a conflict of interest. The same organization that profits from selling the peptide is also the one certifying its quality.
Third-party testing eliminates that conflict. An independent laboratory with no financial stake in the outcome runs the same analyses --- HPLC, mass spectrometry, and sometimes more --- and delivers an unbiased report. For researchers who need reliable data, clinicians working with compounded peptide therapies, and anyone spending serious money on peptide products, independent verification is the only way to know for certain what's in the vial.
This guide covers the testing methods, the labs that perform them, what the results mean, and how to get your own peptides tested.
Table of Contents
- Why Third-Party Testing Matters
- The Two Tests Every Peptide Needs
- HPLC: Measuring Purity
- Mass Spectrometry: Confirming Identity
- Additional Testing Methods
- What Purity Levels Actually Mean
- Where to Get Peptides Tested
- How to Choose a Testing Lab
- What Third-Party Testing Costs
- How to Submit a Sample for Testing
- Reading Third-Party Results
- Common Problems Third-Party Testing Reveals
- The Bottom Line
- References
Why Third-Party Testing Matters
The peptide market operates in a regulatory gray zone. Many products are sold as "research chemicals" without the oversight that pharmaceutical drugs receive. The FDA has found that up to 40% of online and compounded peptide products contained incorrect dosages or undeclared ingredients during spot-check testing.
That number alone justifies independent verification.
Third-party testing serves three purposes:
- Confirms identity --- The compound in the vial is actually the peptide it claims to be
- Verifies purity --- The peptide meets the stated purity grade with quantified impurities
- Detects contamination --- Heavy metals, endotoxins, residual solvents, or other unintended substances are absent or within safe limits
Without independent testing, you're relying entirely on the supplier's word. For something like BPC-157 or CJC-1295, where the research application demands known concentrations and clean samples, that trust gap can compromise months of work.
The Two Tests Every Peptide Needs
At minimum, peptide verification requires two complementary analyses: HPLC for purity and mass spectrometry for identity. Neither alone is sufficient.
HPLC tells you how clean the sample is. Mass spec tells you whether it's the right molecule. A vial could be 99% pure --- but 99% pure of the wrong peptide. Conversely, mass spec might confirm the identity while the sample is contaminated with 15% synthesis byproducts that HPLC would reveal.
You need both.
HPLC: Measuring Purity
High-Performance Liquid Chromatography remains the gold standard for quantifying peptide purity. The technique separates a dissolved peptide sample through a column packed with hydrophobic material, and a UV detector measures each component as it exits.
How It Works
The peptide sample is dissolved and injected into a reverse-phase C18 column. A gradient of increasing organic solvent (usually acetonitrile) elutes compounds based on their hydrophobicity. The UV detector, set at 214--220 nm where peptide bonds absorb strongly, records each compound's intensity and retention time.
The result is a chromatogram --- a graph where peaks represent individual compounds. The target peptide appears as the dominant peak. Purity is calculated as the area of the main peak divided by the total area of all peptide-related peaks, expressed as a percentage.
What to Look For in HPLC Results
- Purity percentage --- The headline number. Should match or exceed the supplier's claim.
- Chromatogram --- A visual record of the analysis. The main peak should be sharp and symmetrical.
- Retention time --- When the peptide eluted. Consistent retention times across runs confirm reproducibility.
- Impurity peaks --- Any secondary peaks above 0.5% should be noted. Their retention times can hint at what type of impurity they are.
- Baseline quality --- A clean, flat baseline indicates proper instrument function and mobile phase purity.
Limitations of HPLC
HPLC measures relative purity among UV-absorbing compounds. It cannot detect:
- Non-peptide contaminants that don't absorb at 220 nm (salts, water, counterions)
- Co-eluting impurities that overlap with the main peak
- The absolute identity of any peak --- HPLC tells you something is there, not what it is
This is why mass spectrometry is non-negotiable as a companion test.
Mass Spectrometry: Confirming Identity
Mass spectrometry (MS) measures the molecular weight of a compound with high precision. For peptide verification, it answers the question: does the measured molecular weight match the expected weight calculated from the amino acid sequence?
ESI-MS (Electrospray Ionization)
ESI-MS is the most common technique for peptide identity confirmation. It ionizes the peptide by spraying it through a charged needle, producing multiply charged ions. This allows analysis of peptides across a wide mass range with high sensitivity.
You'll see results reported as charge states: [M+H]+, [M+2H]2+, [M+3H]3+. The instrument software deconvolutes these to calculate the actual molecular weight.
MALDI-TOF
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry is faster and produces simpler spectra, predominantly generating singly charged [M+H]+ ions. It's often preferred for routine identity checks because the data is easier to interpret.
What Mass Spec Catches
| Problem | How MS Detects It |
|---|---|
| Wrong peptide entirely | Molecular weight doesn't match expected value |
| Deletion peptide (missing amino acid) | Mass is lower by the weight of the missing residue |
| Truncated sequence | Mass corresponds to a shortened version of the target |
| Oxidation | Mass is 16 Da higher (addition of one oxygen atom) |
| Deamidation | Mass is 1 Da higher (asparagine to aspartic acid conversion) |
| TFA adducts | Mass is 114 Da higher per TFA molecule |
| Scrambled sequence | Same mass but different fragmentation pattern (requires MS/MS) |
Mass accuracy should be within 0.1% of the theoretical value, or within 0.5--1.0 Daltons for standard ESI-MS. Anything outside that range demands explanation.
Additional Testing Methods
Beyond HPLC and MS, some situations call for deeper analysis.
LC-MS (Liquid Chromatography-Mass Spectrometry)
LC-MS couples HPLC separation with mass spectrometric detection in a single run. Each peak in the chromatogram gets a mass spectrum, which means you can identify every impurity --- not just quantify it. This is the most informative single test available for peptide characterization.
Amino Acid Analysis (AAA)
AAA hydrolyzes the peptide into individual amino acids and quantifies each one. It confirms the amino acid composition matches the expected sequence and determines net peptide content. Particularly useful when precise dosing matters.
Capillary Electrophoresis (CE)
CE separates peptides based on charge-to-size ratio in an electric field. It's an orthogonal method to HPLC --- meaning it separates compounds by a different physical principle --- which can catch impurities that co-elute in reverse-phase chromatography.
Endotoxin Testing (LAL Assay)
Detects bacterial lipopolysaccharide contamination using the Limulus Amebocyte Lysate test. Critical for peptides destined for cell culture or animal studies. Standard research-grade limits are below 1 EU per microgram for general use and below 0.1 EU per microgram for sensitive applications.
What Purity Levels Actually Mean
Not all purity numbers are created equal. Here's what the grades mean in practice.
| Purity | Grade | Real-World Meaning |
|---|---|---|
| 99%+ | Ultra-high purity | Pharmaceutical-grade quality. Suitable for the most demanding applications. Rare and expensive for custom peptides. |
| 98--99% | High purity | Excellent for quantitative research, binding assays, structural studies, and cell-based work. |
| 95--98% | Standard research grade | Adequate for most in vitro experiments, screening, and general research. |
| 90--95% | Economy grade | Acceptable for preliminary work, antibody production, or applications where impurities are tolerable. |
| Below 90% | Low quality | Significant impurity burden. Results may be compromised by contaminating peptides. |
Context matters. A semaglutide sample for pharmacokinetic research demands 98%+ purity. A peptide being used to generate polyclonal antibodies might work fine at 90%.
Also understand: purity is relative to the method. A peptide that tests at 97% on one HPLC method might test at 95% on a different column, gradient, or wavelength. Comparing purity numbers across labs requires knowing the method details.
Where to Get Peptides Tested
Several independent laboratories specialize in peptide analysis. Here are the most established options as of 2025.
Established Third-Party Labs
Janoshik Analytical --- Widely regarded as the gold standard in the peptide research community. Provides HPLC purity analysis and mass spectrometry identity confirmation. COAs include QR codes for online verification, allowing you to scan and confirm results against their database.
MZ Biolabs --- Offers standard HPLC and mass spectrometry analysis. COAs can be verified through their online platform, similar to Janoshik's verification system.
Chromate --- Provides analytical testing with verifiable online results. QR codes link to verification pages where researchers confirm document authenticity.
Finnrick --- An independent testing platform specifically built for the peptide market. They've tested over 4,500 samples from more than 169 vendors, creating a large comparative database of quality information.
Professional Analytical Labs
Peptide Test --- U.S.-based testing performed in Michigan by scientists with experience in commercial quality control of FDA-regulated pharmaceuticals. Method validations follow United States Pharmacopeia (USP) and National Formulary (NF) standards.
Ethos Analytics --- ISO/IEC 17025-accredited laboratory offering specialized peptide purity and quantitation services. Accreditation means their methods have been independently audited for accuracy, precision, and reliability.
Creative Proteomics --- Provides peptide purity analysis using analytical reverse-phase HPLC combined with mass spectrometry. Also offers amino acid analysis and other specialized testing.
ResolveMass Laboratories --- Offers a full suite of peptide characterization including purity, molecular weight, sequence verification, modification analysis, and stability testing using LC-MS, HPLC, NMR, and amino acid analysis.
How to Choose a Testing Lab
Not all labs are equal. Here are the factors that matter most.
Independence
The lab should have zero financial relationship with peptide vendors. If a "third-party lab" is owned by, funded by, or exclusively contracted with the supplier whose product you're testing, the results carry the same conflict of interest as in-house testing.
Accreditation
ISO/IEC 17025 accreditation is the international standard for testing and calibration laboratories. It means the lab's methods have been validated for accuracy, precision, specificity, and detection limits according to internationally recognized protocols. Labs without accreditation may still produce quality work, but accreditation provides documented assurance.
Verification Capability
Can you independently verify the results? Labs that offer QR codes linking to online verification databases, or that allow you to call and confirm specific batch results, add a layer of accountability that static PDF reports do not.
Methods Offered
At minimum, the lab must offer both HPLC and mass spectrometry. If they only run HPLC without MS, you cannot confirm identity. If they only run MS without HPLC, you cannot quantify purity. Beware any lab that offers one without the other.
Reporting Quality
A good lab report includes:
- Raw data (chromatograms, mass spectra)
- Methodology details (column, gradient, wavelength, instrument)
- Clear pass/fail criteria
- Analyst signatures and dates
- Unique sample and report identifiers
Reports that show only a percentage without supporting data are insufficient.
What Third-Party Testing Costs
Testing costs vary based on the analyses requested and the lab's pricing structure.
| Test | Typical Cost Range (2025) |
|---|---|
| HPLC-UV purity analysis | $300--$800 per sample |
| LC-MS (combined HPLC + mass spec) | $600--$1,500 per sample |
| Amino acid analysis | $400--$900 per sample |
| Peptide mapping | $1,200--$2,500 per sample |
| NMR spectroscopy | $1,000--$3,000 per sample |
| Endotoxin testing (LAL) | $150--$400 per sample |
| Full characterization panel | $800--$5,000 per sample |
For most users, an LC-MS analysis ($600--$1,500) provides the best value: both purity and identity confirmation in a single run.
Some community-focused labs offer lower prices for individual researchers, while pharmaceutical-grade testing from ISO-accredited facilities sits at the higher end of these ranges.
Is It Worth the Cost?
Consider what you're protecting. If you're running a study using a peptide that costs $200 per vial and spending weeks on experiments, a $600 verification test that catches a purity problem before you waste time and materials pays for itself immediately. For peptide therapy patients using compounded products, the peace of mind alone may justify the expense.
How to Submit a Sample for Testing
The process is straightforward.
Step 1: Choose Your Lab and Tests
Contact the lab or visit their website to determine which tests you need and their sample requirements. Most labs need 1--5 mg of peptide for a standard HPLC + MS panel.
Step 2: Prepare Your Sample
- Keep the peptide in its original vial if possible
- Store frozen or refrigerated until shipping
- Do not reconstitute unless the lab specifically requests a solution
- Label the sample clearly with the peptide name, expected molecular weight, and your contact information
Step 3: Ship the Sample
Use cold shipping (ice packs or dry ice) for temperature-sensitive peptides. Most labs provide shipping instructions. Standard ground shipping works for lyophilized peptides that are stable at room temperature for short periods, but overnight shipping with cold packs is safer.
Step 4: Receive and Review Results
Turnaround times range from 3--10 business days depending on the lab and tests requested. Results typically arrive as a PDF report with chromatograms, spectra, and summary data.
Reading Third-Party Results
When your results arrive, check these elements in order:
1. Identity Confirmation (Mass Spec)
Does the observed molecular weight match the theoretical weight? A match within 0.5--1.0 Da confirms identity. If the mass is off by the weight of an amino acid residue (57--187 Da depending on the amino acid), you may have a deletion or truncation product.
2. Purity Assessment (HPLC)
Compare the third-party purity to the supplier's claim. Some variance is normal --- different methods can produce slightly different numbers. A discrepancy of 1--2% is common and usually acceptable. A discrepancy of 5% or more suggests either the supplier inflated their numbers or the sample degraded between testing and delivery.
3. Impurity Profile
Look at what the impurities are, not just how much there is. A 2% impurity that's a closely related peptide analog is very different from a 2% impurity that's an unknown compound. LC-MS data can often identify the major impurities by their molecular weights.
4. Chromatogram Quality
The chromatogram should show a clean separation with a sharp main peak. Compare it visually to the supplier's chromatogram if one was provided. Major differences in peak shape or retention time warrant further investigation.
Common Problems Third-Party Testing Reveals
Based on published data from independent testing platforms, here are the most frequently encountered issues.
Purity Lower Than Claimed
The most common finding. A supplier claims 98% purity; independent testing shows 91%. This happens when suppliers test under optimized conditions that minimize apparent impurities, or when they simply fabricate numbers.
Wrong Peptide
Less common but more serious. The vial is labeled as one peptide but mass spectrometry reveals a different molecular weight entirely. This can result from labeling errors, cross-contamination during manufacturing, or deliberate substitution.
Degradation Products
The peptide may have been correctly synthesized but degraded during storage or shipping. Oxidation (common with methionine-containing peptides) and deamidation (common with asparagine residues) create new peaks that reduce apparent purity.
Underdosing
The vial claims to contain 5 mg but amino acid analysis reveals only 3.2 mg of actual peptide content. This can reflect either deliberate underfilling or a failure to account for net peptide content in labeling.
Contamination
Endotoxin contamination, heavy metals, or residual solvents above acceptable limits. Particularly concerning for anyone following injection protocols.
The Bottom Line
Supplier COAs are a starting point, not the final word. Third-party testing adds an independent verification layer that protects your research, your health, and your investment.
At minimum, any peptide intended for biological research or therapeutic use should be verified by an independent lab running both HPLC and mass spectrometry. The cost is modest compared to the consequences of using a misidentified, impure, or contaminated product.
The best suppliers welcome independent verification. If a vendor discourages third-party testing or refuses to provide batch-specific COAs for verification, that tells you everything you need to know about their confidence in their own product.
Trust, but verify. Then verify again.
References
- Verified Peptides. "Why Laboratory Validation Matters in Peptide Quality." https://verifiedpeptides.com/knowledge-hub/why-laboratory-validation-matters-in-peptide-quality/
- SeekPeptides. "Peptide Testing Labs: Your Complete Guide to Third-Party Verification." https://www.seekpeptides.com/blog/articles/peptide-testing-labs-guide
- Ethos Analytics. "Peptide Purity and Quantitation Testing." https://www.ethosanalytics.io/peptide-purity-and-quantitation
- Creative Proteomics. "Peptide Purity Analysis Service." https://www.creative-proteomics.com/services/peptide-purity-analysis-2.htm
- Peptide Test. https://peptidetest.com/
- GenScript. "Peptide Accupep+QC Testing." https://www.genscript.com/accupep_quality.html
- ResolveMass Laboratories. "Cost of Peptide Analysis Service." https://resolvemass.ca/cost-of-peptide-analysis-service/
- Sarin VK, et al. "HPLC Analysis and Purification of Peptides." PMC7119934. https://pmc.ncbi.nlm.nih.gov/articles/PMC7119934/
- Peptard. "How to Verify Peptide Purity: Understanding HPLC, Mass Spectrometry." https://www.peptard.com/blog/how-to-verify-peptide-purity/
- Vici Health Sciences. "Analytical Testing for Peptide Formulations." https://vicihealthsciences.com/analytical-testing-for-peptide-formulations/