Identity test: Is it really the peptide you ordered?

HPLC Purity in Peptides: Why a Single Peak Isn't Enough

An HPLC purity certificate (high-performance liquid chromatography) tells you how large the main peak is compared to all other peaks. This method separates substances in a sample and answers the question of how much of a single substance is present, but it does not provide proof of that substance's exact identity.

A peptide can have a purity of 98% and still be the wrong substance. In analytical chemistry, purity is considered necessary but not sufficient. Even a sharp peak at the expected retention time - the moment the substance exits the separation column - does not guarantee that it is the right molecule.

Confirming Peptide Identity: Mass Spectrometry and LC-MS/MS

The identity of a peptide is checked using mass spectrometry (MS), which measures the molecular mass. Common methods include ESI-MS (electrospray ionization), MALDI-TOF (laser desorption with time-of-flight analyzer), and LC-MS/MS (liquid chromatography with tandem mass spectrometry).

While ESI-MS generates multiply charged ions of the peptide that produce a characteristic pattern, and MALDI-TOF simply confirms the molecular mass, LC-MS/MS is considered the gold standard. This method directly verifies the amino acid sequence and rules out isobaric peptides - those that have the same mass but a different structure.

Dangers in the Gray Market: Documented Mislabeling of Peptides

Reports on laboratory analyses of peptide vials show that nearly 30% of samples had issues such as contamination. These data come from a television report about a commercial laboratory and summarize various analytical problems, but they do not constitute a peer-reviewed scientific study.

Scientific literature and regulatory authorities warn of a growing market for counterfeit peptide biopharmaceuticals, including substances in preclinical or clinical development. The risk of substitution is real: a vial can contain a completely different peptide than the label promises, even if the content is highly pure.

Limits of Identity Testing: What a Result Tells You

An identity result confirms that the molecular mass (via MS) or the amino acid sequence (via LC-MS/MS) matches the target peptide. It rules out major synthesis errors, but it does not provide information about purity, the actual content of the active substance, or safety-relevant residues.

For a complete picture, further analyses are needed: RP-HPLC for purity, amino acid analysis for content, and the LAL test for endotoxins. Additionally, GC-MS checks for solvent residues, and ICP-MS checks for heavy metals or elemental impurities. An identity certificate only clarifies the 'what', not the safety or quantity.

COA Without Identity Proof: An Incomplete Analysis Certificate

An analysis certificate (COA) that only states HPLC purity leaves the identity of the substance unconfirmed. The ICH guidelines (International Council for Harmonisation) require two separate identification methods for peptide active ingredients, because a single measurement is often not enough for a definitive proof.

Without an identity method, a COA does not even meet the minimum standard. Given known cases of mislabeling in the gray market, a purity-only certificate offers no protection against mix-ups. A safe analysis package must include identity, purity, content, and safety as complementary tests.

HPLC Purity in Peptides: Why a Single Peak Isn't Enough

Confirming Peptide Identity: Mass Spectrometry and LC-MS/MS

Dangers in the Gray Market: Documented Mislabeling of Peptides

Limits of Identity Testing: What a Result Tells You

COA Without Identity Proof: An Incomplete Analysis Certificate

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