Peptide Purity Testing: HPLC, Mass Spec and Net Peptide Content

Three numbers get mixed up when people talk about peptide quality: purity, identity and peptide content. They come from different tests and answer different questions. Knowing the difference makes a certificate of analysis much easier to read.

Purity: HPLC

High-performance liquid chromatography (HPLC) separates the components of a sample so each one can be measured. Peptide purity is usually reported from HPLC with UV detection at around 214-220 nm. It is the percentage of the target peptide relative to all the material that absorbs at that wavelength (Bachem; Bio-Synthesis). Impurities are mostly related peptides from synthesis, such as truncated or incomplete sequences. A "98%" purity result means 98% of the UV-absorbing material is the target peptide.

Identity: mass spectrometry

HPLC alone can't confirm what the main peak actually is. Mass spectrometry (MS), often coupled directly to HPLC as HPLC-MS, measures molecular mass to confirm the target peptide's identity (JPT Peptide Technologies). A COA should show both: purity from HPLC and identity from MS.

Net peptide content: a separate number

A lyophilized peptide powder isn't 100% peptide by weight. After synthesis and purification, peptides are typically present as salts with counter-ions such as trifluoroacetate (TFA), and the powder also holds some residual water (JPT Peptide Technologies). Net peptide content is the share of the total weight that is peptide. Bio-Synthesis determines it by nitrogen content analysis. Water and salts don't absorb UV the way peptides do, so HPLC purity doesn't account for them.

The amount of target peptide in a given weight of powder is therefore gross weight × net peptide content × purity (Bio-Synthesis). For example, 10 mg of powder with 80% peptide content and 95% purity contains about 7.6 mg of the target peptide.

What these tests don't cover

Standard HPLC and MS testing doesn't measure sterility, endotoxin levels or residual solvents. Those require separate assays. A COA covers only the tests it lists, for the lot it names.

Zerilyx testing

See the Quality page for the assays reported on Zerilyx certificates, and the Certificates of Analysis page to look up a lot. For help reading a certificate, see How to Read a Peptide COA.