How we verify peptides

Reversed-phase HPLC coupled to ESI-TOF-MS is the workhorse of our analysis. The instrument runs a single chromatographic separation — peptides partition by hydrophobicity across a C18 column — with two detectors in series: a UV detector at 214 nm & an ESI-TOF mass spectrometer.

The UV detector at 214 nm records the peptide bond absorption of every peak in the chromatogram. Peak areas yield the purity percentage directly: the main peak divided by the sum of all peaks. This is the industry-standard purity figure reported on every analytical report.

The ESI-TOF-MS detectorreceives the same eluent & measures the mass of ions as they exit the column. For the main chromatographic peak, this gives the observed molecular weight to within <5 ppm of the theoretical monoisotopic mass — unambiguous identity confirmation. For impurity peaks, it identifies what those impurities actually are: deletion sequences (slightly lower MW), oxidized variants (+16 Da on Met or Trp), deamidated residues (+1 Da), or truncated sequences. This is information a UV-only chromatogram cannot provide.

Our purity quantification is consistent with ICH Q2(R1) analytical validation guidelines, covering specificity, linearity, range, & precision.

Nuclear magnetic resonance spectroscopy provides structural information that mass spectrometry cannot. While MS confirms the molecular weight, NMR resolves the chemical environment of individual atoms, detecting structural isomers, epimers, incorrect disulfide connectivity, & non-UV-active impurities that would pass undetected in a chromatogram.

We record NMR spectra as an orthogonal check on every sample. The outcome (consistent, inconclusive, or inconsistent with the expected structure) is reported alongside the MS & LC data in the report. A result where MS confirms identity but NMR flags an inconsistency is a meaningful finding that no single-method service would catch.

Peptides synthesized in non-GMP environments can carry lipopolysaccharide (LPS) endotoxins from gram-negative bacteria. Even at sub-microgram concentrations, endotoxins trigger potent immune responses: fever, inflammation, & in high doses, septic shock. Standard MS & NMR analysis cannot detect endotoxins because they are not the peptide compound itself.

We use the Limulus Amebocyte Lysate (LAL) assay, the gold standard for endotoxin detection in pharmaceutical & research contexts. Results are reported in EU/mg (endotoxin units per milligram of peptide). A result above 1 EU/mg is a meaningful contamination signal for research use; above 5 EU/mg warrants serious caution.

Synthesis reagents, solvents, & equipment can introduce trace metal contaminants into the final peptide product. Lead, arsenic, cadmium, & mercury are particularly concerning: they accumulate in tissue, interfere with enzymatic processes, & are toxic at very low chronic exposures. A peptide that passes identity & purity checks by MS may still carry hazardous metal residues.

We analyze for the four most clinically significant heavy metals using inductively coupled plasma mass spectrometry (ICP-MS), which achieves detection limits in the parts-per-billion range. Results are reported per element in µg/g (ppm) against the ICH Q3D guideline thresholds for research use, giving you a clear, quantitative picture of elemental safety.

Most vendor peptide COAs are based on reversed-phase HPLC with UV detection only. HPLC-UV is fast & inexpensive, which is why it dominates. It is also insufficient as a standalone method.

What HPLC-UV cannot tell you. UV detection measures how much light each chromatographic peak absorbs at a fixed wavelength. It produces a purity figure — the fraction of total UV signal from the main peak — but it cannot confirm what that main peak actually is. Two peptides with the same amino acid composition but different sequences may co-elute. A D-amino acid substitution at a single residue is completely invisible. Non-UV-active impurities do not register at all. You get a number, not a confirmation.

What the MS detector adds.By coupling ESI-TOF-MS to the same HPLC run, every peak that the UV detector sees is also mass-analysed in real time. The main peak's molecular weight is confirmed to <5 ppm — ruling out mis-labelled or substituted compounds. Impurity peaks are identified by their mass rather than inferred from retention time alone. This is the critical difference between "purity by HPLC" & "purity by RP-HPLC-MS": in the latter, you know what each peak is.

What NMR adds. ESI-TOF-MS confirms the molecular weight but cannot resolve structural isomers, epimers (D vs. L amino acid configurations), incorrect disulfide connectivity, or modifications that leave mass unchanged. NMR probes the chemical environment of individual atoms & is the only routine method that catches these classes of defect. A sample that passes RP-HPLC-MS but fails NMR is a meaningful finding — precisely what a UV-only COA would miss entirely.

• Quantity: Minimum 2 mg lyophilized powder for full analysis across MS & NMR experiments.
• Format: Sealed vial or resealable bag inside the collection pouch we provide.
• Storage: Keep frozen until you insert the sample. Room temperature for shipping is acceptable for up to 72 h but our shipping is usually much faster.
• Anonymity: The vial is pre-labelled with your anonymous token before it reaches you. Do not add your name or any other identifier. The labs receive samples labelled with tokens only.

1. 1
   Register online

   Fill in the submission form & pay securely via Stripe. You receive an anonymous token & we send you a collection envelope within 1-2 business days.

2. 2
   Receive your envelope

   A tamper-evident collection envelope arrives by post. Your anonymous token is printed on the label. No name, no address on the vial.

3. 3
   Insert & post back

   Place your sample vial in the envelope, seal it, & post it back to us.

4. 4
   Handed to the lab

   We collect your sample & hand it directly to the lab. Analysis takes 1-3 business days from receipt.

5. 5
   Result published

   We format the analytical result & publish it to your account. You receive an email notification & can view the full report in your dashboard, with the option to share publicly.