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Peptide Testing Methods Explained

A step-by-step laboratory method used by an independent peptide testing lab: sample prep, HPLC purity, mass spec identity, and supporting assays.

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The 6-Step Method to Test Peptides

  1. 1

    Prepare the sample

    Weigh 100-500 ug of lyophilized peptide. Reconstitute in a compatible aqueous or acetonitrile buffer. See our peptide reconstitution and storage guide for solvent selection.

  2. 2

    Run reverse-phase HPLC

    Inject onto a C18 column with a water/acetonitrile + 0.1% TFA gradient. Detect at 214 nm. See HPLC vs mass spec for method selection.

  3. 3

    Calculate purity

    Purity % = target peak area / total peak area × 100. Learn how to read the resulting report in how to read a peptide COA.

  4. 4

    Confirm identity by mass spectrometry

    Run LC-MS or ESI-MS and match observed mass to the theoretical monoisotopic mass. Details: molecular analysis.

  5. 5
  6. 6

    Issue the Certificate of Analysis

    A complete COA includes chromatograms, mass spectra, calculated purity, impurity table, and pass/fail against specification. See third-party verification.

HPLC-UV

Purity quantification at 214 nm, 0.1% detection.

LC-MS Identity

Matches observed mass to theoretical monoisotopic mass.

Validated Methods

Validated methods, defensible data, audit-ready COA.

Prefer to outsource? Here's how

How to Test Peptides FAQ

1) Dissolve 100-500 ug of lyophilized peptide in a compatible solvent. 2) Inject onto a reverse-phase HPLC C18 column with a water/acetonitrile + 0.1% TFA gradient. 3) Detect at 214 nm and integrate the chromatogram to calculate purity. 4) Confirm identity with LC-MS or ESI-MS against the theoretical monoisotopic mass. 5) Run supporting assays (Karl Fischer, LAL, ICP-MS) as needed. 6) Compile the chromatogram, mass spectrum, and results into a signed Certificate of Analysis.

At minimum an HPLC system with UV detector (for purity) and a mass spectrometer (LC-MS or ESI-MS) for identity confirmation. Full analytical panels also use Karl Fischer titrators, LAL kinetic readers, ICP-MS instruments, and GC-MS. This is why most researchers outsource to a specialty peptide testing lab rather than trying to test peptides in-house.

No. Peptide purity and identity are chromatography and mass spectrometry measurements — they cannot be performed at home. Test strips and color kits sold online do not measure purity. Send your sample to an independent third-party lab.

100-500 ug of lyophilized peptide is enough for HPLC purity and mass spec identity. Full COA panels including moisture and endotoxin typically need 1-5 mg. Ship lyophilized samples overnight with an ice pack.

Standard turnaround at ACS Peptide Testing Labs is 5-7 business days from sample receipt. Large CROs typically take 4-6 weeks.

HPLC purity testing runs $150-$300 per sample. A full COA panel with HPLC, mass spec identity, moisture, and endotoxin is typically $400-$700. Volume discounts apply at 5+ samples.

Purity testing (HPLC-UV) tells you what percentage of the sample is the target peptide vs. impurities. Identity testing (LC-MS) tells you whether the peak is actually your peptide. You need both — a 99% pure something is not the same as 99% pure your peptide.

For research use, an independent lab is strongly recommended because it guarantees method validation, traceability, and audit-ready data. For 503A/503B compounding pharmacies and regulatory submissions, documented method validation is effectively required.

Get Your Peptides Tested

Ship your sample to our independent peptide testing lab. Full COA in 5-7 business days.

(561) 513-8435cpierre@acslabtest.com