Procurement comparison
Peptide COA Guide: Testing Method Comparison
HPLC (High-Performance Liquid Chromatography) Purity of target peptide vs impurities; separation by retention time through chromatography column Peak area percentage (e.g., 98.2% purity) 0.1% for major impurities; 0.01% for trace contaminants with optimized me
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- HPLC (High-Performance Liquid Chromatography)
- Purity of target peptide vs impurities; separation by retention time through chromatography column
- Peak area percentage (e.g., 98.2% purity)
- 0.1% for major impurities; 0.01% for trace contaminants with optimized methods
- Non-UV-absorbing contaminants (salts, water, buffers); cannot confirm peptide sequence identity—only that something with that retention time is present
- Mass Spectrometry (MS)
- Molecular weight and peptide sequence identity via mass-to-charge ratio measurement
- Found molecular weight vs calculated (e.g., 5136.2 Da found vs 5135.89 Da theoretical)
- Varies by instrument; ±0.5 Da for peptides <3000 Da; ±1–2 Da for 3000–6000 Da
- Relative abundance of impurities—MS detects presence but doesn't quantify how much contamination exists; peptide at 70% purity may still show clean MS if impurities don't ionize
- Amino Acid Analysis (AAA)
- Confirms amino acid composition matches expected sequence; quantifies peptide content by weight
- Peptide content percentage after accounting for water, salts, counterions (e.g., 87% peptide content)
- 1–5% depending on amino acid and hydrolysis method
- Sequence order—AAA confirms the right amino acids are present but not their arrangement; cannot detect transposition errors or D-amino acid substitution
- Endotoxin Testing (LAL Assay)
- Bacterial lipopolysaccharide contamination from synthesis or handling
- Endotoxin units per milligram (e.g., 0.42 EU/mg or <0.1 EU/mg)
- 0.005–0.01 EU/mg with kinetic chromogenic LAL; 0.03 EU/mg with gel-clot method
- Non-endotoxin contaminants like viral particles, fungal toxins, or chemical residues—LAL is bacteria-specific and doesn't detect other biologics
- Peptide Content by Weight
- Actual peptide mass vs total vial weight after subtracting water, salts, and counterions
- Net peptide percentage (e.g., 85% peptide content vs 98% HPLC purity)
- Depends on method—AAA provides 1–2% accuracy; nitrogen analysis less precise at ±5%
- Purity among peptide-related materials—peptide content measures total peptide vs non-peptide material, but a sample can be 85% peptide content with only 90% of that peptide being the correct sequence
- Professional Assessment
- HPLC + MS together provide minimum verification—HPLC for purity, MS for identity. Add AAA for peptide content when dosing accuracy is critical. Endotoxin testing is non-negotiable for in vivo research or cell culture. A COA with only one test type (HPLC alone or MS alone) is insufficient for publication-grade research.