Peptide Lab Testing Standards: What the Tests Actually Measure
A Certificate of Analysis is only useful if you know what each test detects -- and what the numbers mean. This page explains HPLC, mass spectrometry, and endotoxin testing so you can evaluate any CoA with confidence.
HPLC -- High-Performance Liquid Chromatography
HPLC separates compounds by how they interact with a stationary phase as a mobile solvent carries them through a column. The detector records an absorption peak for each component; the area under the target peak divided by total peak area gives the purity percentage.
1
What it measures: Relative purity -- the percentage of the sample that is the target peptide vs. all other UV-absorbing material (peptide fragments, synthesis byproducts, oxidized variants).
2
What counts as passing: ≥98% purity by HPLC is considered research grade. 95–97.9% is acceptable for most protocols. Below 95% should raise concern about synthesis quality.
3
Limitation: HPLC does not confirm the identity of the compound -- only its relative abundance. A high HPLC purity for the wrong peptide still reads as high purity. Always pair with mass spectrometry.
Mass Spectrometry (MS / LC-MS)
Mass spectrometry ionizes the compound and measures the mass-to-charge ratio (m/z) of the resulting ions. For peptides, the observed molecular weight is compared to the theoretical molecular weight calculated from the amino acid sequence. A match within 0.5–2 Da confirms identity.
1
What it measures: Molecular identity -- whether the compound in the vial is actually the labeled peptide. This is the only test that can catch a completely wrong compound.
2
Reading the result: The CoA will show "Observed MW" and "Theoretical MW" or "Expected MW." A match (within instrument tolerance, typically ±0.5 Da for small peptides, ±2 Da for larger ones) is a pass.
3
What it does not measure: MS does not tell you how much of the compound is present -- only that the correct compound was detected. Always read MS alongside the HPLC purity figure.
Endotoxin Testing (LAL / BET)
The Limulus Amebocyte Lysate (LAL) test detects bacterial endotoxins -- lipopolysaccharide fragments from gram-negative bacteria that cause fever and inflammatory response in mammals. This is especially critical for injectable peptides.
🧫
Pass Threshold
Research injectables: <1 EU/mg is standard. Pharmaceutical grade: <0.5 EU/mg. Above 5 EU/mg is a clear fail for injectable use.
⚠️
Why It Matters
Endotoxin contamination does not affect HPLC purity or MS results. A peptide can show 99% HPLC purity and still have dangerous endotoxin levels.
📋
CoA Reporting
Endotoxin tests show a result in EU/mg with Pass/Fail verdict. A missing endotoxin field on a CoA is a red flag for injectable research.
🔬
No Purity %
Endotoxin CoA lines have no purity percentage -- only EU/mg and Pass/Fail. The absence of a % figure is expected, not an error.
How to Read a Certificate of Analysis
A valid CoA includes: lot number, test date, compound name and sequence, HPLC purity (%), MS observed/theoretical MW, and endotoxin result. What to watch for:
✓
HPLC ≥98%, MS match, Endotoxin <1 EU/mg -- meets research-grade standard for injectable use.
⚠
HPLC 95–97%, no endotoxin test -- borderline quality; acceptable for some in-vitro work, higher risk for injectable protocols.
✗
HPLC <95%, MS mismatch, or no MS result -- do not use. Synthesis quality is inadequate or compound identity is unconfirmed.