How Janoshik verifies peptide purity

A technical walkthrough of the Janoshik Analytical certificate-of-analysis process — the orthogonal methods (HPLC-UV, mass spectrometry, peptide content by weight) used to qualify every research peptide batch Aurex ships, and how to read the resulting report.

Who Janoshik is and why labs use them

Janoshik Analytical is a private EU-based analytical laboratory that has, over the last several years, become the de-facto third-party QC provider for the research-peptide market. Their operating model is narrow by design: they do not sell peptides, they do not manufacture peptides, and they do not offer white-label branding to vendors. They only run assays. That independence is what makes their certificate-of-analysis documents useful as a check on vendor claims — the lab has no commercial incentive to inflate a purity number.

For a researcher running in-vitro work, the relevant questions for any incoming peptide vial are three: is the molecule in the vial the sequence that is printed on the label (identity), is it a clean sample or is it contaminated with related impurities (purity), and how much of the vial mass is actually peptide versus water and counter-ions (content). Janoshik's standard peptide report is designed to answer each of those questions with a separate, orthogonal assay.

HPLC-UV: the purity number

High-performance liquid chromatography with UV detection (HPLC-UV) is the first assay most researchers look at on a COA, and it is the origin of the purity percentage. In reversed-phase HPLC, the peptide sample is injected onto a C18 column and eluted with a gradient of water and acetonitrile, typically with 0.1% trifluoroacetic acid or formic acid as a mobile-phase modifier. Species elute off the column at different retention times based on hydrophobicity, and a UV detector records their absorbance at 214 nm — the wavelength where the amide bond in the peptide backbone absorbs most strongly, giving roughly sequence-independent sensitivity.

The reported purity number is the area under the main peak divided by the total integrated area of every peak in the chromatogram, expressed as a percentage. A clean synthesis produces a single dominant peak with a symmetric gaussian profile; a problematic synthesis shows shoulder peaks, front-edge truncations, or late-eluting oxidation products. Limits of detection at 214 nm commonly reach 0.05–0.1% for related-substance impurities, which is why research-peptide COAs typically round purity to a tenth of a percent. The general QC framework for synthetic peptides — HPLC purity combined with mass-spec identity and amino-acid analysis — is discussed in detail in D'Hondt et al. (J Pharm Biomed Anal 2014, PMID: 24858305) and in the scientific guidelines for peptide impurity control.

Mass spectrometry: the identity check

HPLC tells you the sample is clean; it cannot tell you whether the clean peak is the correct molecule. That is the job of mass spectrometry. Janoshik's standard peptide report uses electrospray-ionization mass spectrometry (ESI-MS) to measure the molecular mass of the main HPLC peak. The researcher compares the observed mass against the theoretical monoisotopic or average mass of the declared sequence and confirms the match within the instrument's mass-accuracy tolerance.

For a peptide such as BPC-157 (theoretical monoisotopic mass 1418.68 Da for the free acid, C₆₂H₉₈N₁₆O₂₂), an ESI-MS reading within a few decimal places of that value confirms identity. A mismatch of 14, 16, or 28 Da is the signature of common synthesis byproducts — an extra methyl group, an oxidation, or a formylation respectively — and would be disqualifying. Peptide MS identity is the load-bearing check against the worst-case fraud scenario of a structurally different molecule being sold under the correct label. An accessible reference on ESI-MS for peptides is Banerjee & Mazumdar (Int J Anal Chem 2012, DOI: 10.1155/2012/282574).

Peptide content by weight

The third number on a complete peptide COA is peptide content by weight, and it is the number most often misread by first-time buyers. A freeze-dried peptide vial is not 100% peptide. It contains the peptide itself, trifluoroacetate or acetate counter-ions left over from the final purification step, residual bound water, and trace solvents. A vial labeled 5 mg typically contains 5 mg of total lyophilized mass; the peptide content number tells the researcher how much of that 5 mg is actually the peptide of interest.

Net peptide content is quantified separately from HPLC purity — commonly by nitrogen-based methods, amino-acid analysis, or quantitative NMR — and it is independent of the purity number. A sample can be 99% pure by HPLC (almost no related impurities) and simultaneously be only 80% peptide by weight (because 20% of the mass is counter-ion and water). Both numbers are legitimate and both should appear on the COA. For research protocols where mass dosing matters, the peptide-content number is the one that drives the math.

What a good COA looks like

A credible Janoshik report is a single-page PDF with a unique document reference number, a date of analysis, the sample name as submitted, and clearly labeled sections for each assay. The HPLC section shows a chromatogram image with retention time, column identity, mobile-phase gradient, detection wavelength, and the integrated peak table. The mass-spec section shows the m/z spectrum and the deconvoluted mass with the theoretical mass for comparison. The peptide-content section states the content percentage and the method used. A report that is missing the chromatogram image, the mass spectrum, or the document reference number should be treated as suspect.

Limits of what a COA proves

A COA is an analytical document. It is not a sterility certificate, an endotoxin certificate, or a GMP compliance statement. It does not authorize human use, and it is not a substitute for the additional quality controls that apply to clinical-grade pharmaceutical material. For in-vitro research, the HPLC + MS + content triad is the right level of rigor and is consistent with how synthetic peptides are characterized in the peer-reviewed literature. For anything beyond in-vitro research, additional controls not covered by a standard COA are required, and research-grade material is not appropriate.

How Aurex integrates Janoshik into the batch workflow

Every Aurex batch is tested twice. The manufacturing lab runs the initial QC, and then a retained sample is shipped to Janoshik for independent re-verification. Only batches that pass both checks are released to the ship queue. The Janoshik reference number is printed on the vial label and encoded in the batch QR code, so a researcher can pull the exact document that corresponds to the vial in hand from the batch lookup tool. If any batch fails the independent re-verification, Aurex reships free under the purity-replacement guarantee and the affected lot is recalled.

Frequently asked questions