A COA, or Certificate of Analysis, must refer to a specific batch. HPLC (high-performance liquid chromatography, a purity-testing method) purity, mass spectrometry and mass content do not duplicate one another. Each answers a different question about the sample.

Three lines that are often confused

Result What it shows What it cannot prove on its own
HPLC purity The share of the chromatogram signal occupied by the main peak under a stated method Exact molecular mass, sequence or amount of substance in the vial
MS (mass spectrometry) or LC (liquid chromatography)-MS Whether the observed mass matches the expected mass of the molecule Purity by peak area or mass fraction of the substance
Mass content The quantitatively assigned amount of target substance in the sample The full impurity profile without separate chromatographic analysis

HPLC separates components in a mixture. On a COA, the main peak is usually assigned to the target substance and the other peak areas represent impurities visible to that method. The result depends on the column, mobile phase, wavelength and integration settings. A 99% figure without the chromatogram and method description is therefore less useful than a complete report.

MS measures mass-to-charge ratio and allows the observed mass to be compared with a calculated value. It is a strong mass check, but equal mass does not always mean equal structure. Isomers and some modifications can require additional identity methods.

Mass content is not the same as HPLC purity. HPLC purity commonly expresses relative peak area. Mass content expresses the quantitatively assigned fraction of target substance in the material. Water, residual salts and other non-target components can affect that value even when HPLC purity is high.

Endotoxin: a separate line on the COA

Endotoxin sits on a COA separately from HPLC purity and mass content, because it answers a different question. It is a lipopolysaccharide from the outer membrane of gram-negative bacteria, and chromatographic purity does not reveal it. A sample can show a high HPLC main peak and still contain endotoxin introduced during synthesis or purification [1].

Endotoxin is measured by the LAL test (gel-clot, chromogenic or turbidimetric variant, harmonised across pharmacopoeias [3]). The result is recorded in EU/mg or EU/mL. A blank field in this line does not mean zero, it means the parameter was not measured.

Why the net peptide content can be lower than the weighed amount

The weight of powder in a vial is not the mass of the target peptide. The counterion from synthesis (for example trifluoroacetic acid or acetate) and residual water are part of the total material mass but are not peptide. So the quantitatively assigned net peptide content is often below the total material mass [4].

Molecular identity is confirmed separately, usually by LC-ESI-MS with impurity profiling [2]. That check shows what the substance is, while net content shows how much target compound the material contains.

A one-minute COA check

  1. Match the lot number on the COA, label and verification page. If it is absent or different, the report cannot be assigned to the sample.
  2. For HPLC, read the numeric result together with the chromatogram and method conditions.
  3. For MS, compare expected and observed mass. It helps when the report explains charge state or adduct.
  4. Do not turn one number into three conclusions. HPLC purity, MS identity and mass content are separate checks.

What a readable COA contains

A readable report is tied to a lot, names the methods and shows primary results. If a laboratory supplies only a percentage, ask for the chromatogram, mass spectrum and an explanation of how mass content was assigned.

For Longeva batches, the lot number can be checked on the verification page. A COA confirms characteristics of the tested sample. It does not replace a method description, explain analytical limits or automatically apply to another batch.

The general context of COAs and research-use status is covered separately: research-use peptides and what a COA confirms and why origin and the COA matter more than price. This page is deliberately limited to a few key analytical readouts of a COA and does not claim to be a full list of every method.

Certificates of analysis for our batches: COA archive.

Sources

  1. D'Hondt M, et al. Related impurities in peptide medicines. Journal of Pharmaceutical and Biomedical Analysis, 2014. 10.1016/j.jpba.2014.06.012
  2. De Spiegeleer B, et al. Impurity profiling quality control testing of synthetic peptides using liquid chromatography-photodiode array-fluorescence and liquid chromatography-electrospray ionization-mass spectrometry: The obestatin case. Analytical Biochemistry, 2008. 10.1016/j.ab.2008.02.014
  3. Tamura H, et al. Outstanding Contributions of LAL Technology to Pharmaceutical and Medical Science: Review of Methods, Progress, Challenges, and Future Perspectives in Early Detection and Management of Bacterial Infections and Invasive Fungal Diseases. Biomedicines, 2021. 10.3390/biomedicines9050536
  4. Colalto C. Aspects of complexity in quality and safety assessment of peptide therapeutics and peptide-related impurities. A regulatory perspective. Regulatory Toxicology and Pharmacology, 2024. 10.1016/j.yrtph.2024.105699