Third-Party Peptide Testing Explained: HPLC, LC-MS, Endotoxin, Sterility

Why testing matters more than the number

"99% pure" on a product page tells you nothing without knowing what test produced the number, what the impurities look like, and whether the material is actually the peptide on the label. Here's what each line on a legitimate peptide COA measures.

HPLC purity

High-Performance Liquid Chromatography separates the peptide from truncated sequences, oxidized side chains, and manufacturing byproducts. A reversed-phase column (typically C18) with an acetonitrile/water/TFA gradient is standard.

What to look for on the report:

• Method: column, gradient, flow rate, detection wavelength (usually 214 nm).
• Chromatogram: the main peak should be sharp and dominant. Multiple peaks near the retention time are a bad sign.
• Integration: area-percent of the main peak versus everything else. ≥99% is research-grade; ≥95% is acceptable for early screening.

Mass spectrometry (LC-MS / MALDI-TOF)

HPLC tells you how pure. Mass spec tells you what. The observed mass should match the theoretical monoisotopic mass of the target peptide within instrument tolerance (typically ±1 Da for LC-MS, better for high-resolution MS).

Mass spec catches wrong peptides entirely, deletion sequences (missing amino acids), and post-synthetic modifications (oxidation, deamidation).

Endotoxin (LAL or rFC)

Bacterial endotoxin (lipopolysaccharide, LPS) is a byproduct of gram-negative bacterial contamination. It's not removed by sterile filtration and it invalidates most cell-based research at low concentrations.

Standard tests:

• LAL (Limulus Amebocyte Lysate) — the classic horseshoe-crab-derived assay.
• rFC (recombinant Factor C) — a synthetic alternative with equivalent sensitivity.

Research-grade limit: <10 EU/mg. Cell-culture-grade should be lower.

Sterility

Direct inoculation or membrane filtration into growth media (fluid thioglycollate + soybean-casein digest), incubated 14 days. Reports pass/fail.

Moisture (Karl Fischer titration)

Residual water in lyophilized powder. High moisture (>5%) shortens shelf life and can cause peptide hydrolysis. Well-lyophilized product is typically <3%.

Residual solvents

GC or HS-GC quantifies leftover solvents from manufacturing and purification — TFA, acetonitrile, DMF. Class 2 and Class 3 solvent limits are set by ICH Q3C.

What a complete COA looks like

A trustworthy Certificate of Analysis includes: product name, sequence, lot number, manufacture date, retest date, appearance, HPLC purity + chromatogram, LC-MS mass + spectrum, endotoxin result, sterility result, moisture content, residual solvents, and the signature of the QC lead.

See PX1's per-lot reports at /purity-reports and /endotoxin-reports.