Understanding Purity Testing

HPLC-MS (High-Performance Liquid Chromatography-Mass Spectrometry) is the gold standard for compound purity verification, referenced extensively in USP monographs and ICH Q7 guidelines for active pharmaceutical ingredient characterization.

How it works: Reversed-phase HPLC separates sample components by flowing them through a C18 stationary phase column under gradient elution conditions (typically water/acetonitrile with 0.1% TFA). Each component elutes at a characteristic retention time determined by its hydrophobicity. The eluate then enters an electrospray ionization (ESI) mass spectrometer, which measures each component's mass-to-charge ratio (m/z), confirming molecular identity via exact monoisotopic mass.

UV detection: Analytical HPLC typically monitors at 214nm (peptide bond absorption, universal detection) and 280nm (aromatic residue absorption — tryptophan, tyrosine, phenylalanine). The 214nm trace provides the purity percentage, while 280nm helps identify specific impurity profiles in sequences containing aromatic amino acids.

Reading a COA: A rigorous Certificate of Analysis includes: purity percentage calculated from integrated peak areas (target: 98% or higher), retention time chromatogram showing the main peak and any impurity peaks, mass spectrum with the observed [M+H]+ or [M+2H]2+ ion confirming molecular identity within 0.02 Da of theoretical mass, endotoxin assay results (LAL method, acceptable threshold typically less than 0.5 EU/mg), and lot-specific synthesis and testing dates.

What purity percentage means: A purity of 99.2% indicates that 99.2% of the sample by integrated UV peak area at 214nm is the target compound. The remaining 0.8% typically consists of truncated sequences (from incomplete coupling during SPPS), deletion products, oxidized variants (particularly methionine sulfoxide), or residual TFA salts from purification. Each impurity peak should be individually characterized below 0.5% for research-grade material.

Red flags to watch for: Purity below 95% for research-grade compounds, missing mass spectrometry data (HPLC alone cannot confirm identity), generic methodology descriptions that omit column specifications or gradient conditions, COAs without batch numbers or dates, in-house testing without independent verification, and purity claims that lack supporting chromatogram images.

At AUREX, every batch is tested by independent third-party laboratories — never in-house — to eliminate conflicts of interest in purity reporting. All analytical methods follow USP chapter 621 guidelines for chromatographic analysis. You can verify any batch number on our website to view the complete analytical package.