Certificates of analysis: are you reading or merely receiving your COAs?

That record only helps if someone reads it against two other documents: the purchase order, which says what the laboratory asked for, and the label, which says what was actually delivered. Checked at receipt, it can stop a wrong or poorly characterised lot before it reaches an experiment.

Synthetic peptides make a useful example because their certificates often carry several numbers that look alike but measure different things. The same habits apply to most research reagents.

Matching the basics

Start with the identifiers. The product name, and for a peptide the sequence and any modifications, should match the order exactly. The lot or batch number on the COA should match the vial label and the delivery note, character for character. The test date should be plausible: after manufacture, before dispatch, and with any stated retest or expiry date still in the future. The issuing laboratory should be named, with an address, a unique report number and an authorised signatory.

When these do not line up, do not reconcile them by assumption. A transposed digit in a lot number may be a typing error, or it may mean the certificate belongs to a different batch. Quarantine the material: label it as on hold and store it apart from released stock. Ask the supplier, in writing, for a corrected certificate or an explanation, and keep the reply with the receiving record. Until the discrepancy is resolved, the lot is not used.

Reading the purity result

For peptides the headline figure is usually HPLC purity. It is the area of the main peak as a percentage of the total integrated peak area, detected by UV absorbance at a stated wavelength, using a stated column, mobile phase and gradient. Without those conditions, the figure is hard to interpret.

Several things are invisible to that measurement. An impurity that co-elutes with the main peak is counted as product, which is why the specificity of the method matters [1]. Species that absorb weakly or not at all at the detection wavelength are under-represented. Water, inorganic salts and counter-ions such as trifluoroacetate, carried over from synthesis and purification, are not captured by the purity figure [2].

This is why HPLC purity is not net peptide content. Net peptide content is the mass fraction of peptide in the weighed powder, normally determined by amino acid analysis or nitrogen analysis [3]. A lyophilised peptide can show high chromatographic purity while a substantial share of its mass is counter-ion and water. If concentration matters, look for a separate net peptide content value, and request it if missing.

Identity is a separate question

A clean chromatogram shows that one species dominates. It does not show which species that is. A wrong sequence synthesised cleanly would give an equally good purity figure, so identity needs its own evidence [3].

For peptides that evidence is most often mass spectrometry. The certificate should report an observed mass alongside the theoretical mass for the expected sequence, and state whether values are monoisotopic or average, since comparing one with the other produces a false mismatch. Agreement within the stated tolerance is good evidence that a molecule of the expected composition is present.

Mass alone has limits. Leucine and isoleucine have identical masses. A sequence with two residues swapped has the same mass as the intended one, and so does a peptide in which one residue has racemised from the L to the D form. Fragmentation data from tandem mass spectrometry can confirm residue order, although telling leucine from isoleucine needs more specialised approaches, and stereochemistry needs a chiral method or comparison with an authentic reference.

What accreditation covers

Many certificates note that the issuing laboratory is accredited to ISO/IEC 17025 [4]. This means a national accreditation body, UKAS in the United Kingdom, has assessed the laboratory as competent to perform specific tests, operating a management system that covers method validation, equipment calibration, measurement traceability and staff competence [5].

The scope is what matters. Accreditation is granted against a schedule listing the tests and materials covered, and a laboratory may also run tests that sit outside it. Check the laboratory against the accreditation body's public directory, and confirm that the accreditation is current and that the reported test appears in the scope. Accreditation bodies generally require results outside the scope to be clearly identified on an accredited report.

Accreditation speaks to how the laboratory tested the sample it received. It does not show that the sample was representative of the whole lot, that the lot was stored and shipped correctly afterwards, or that the vial on your bench came from the same batch. Those links still rest on the identifiers matched at the start.

A receiving checklist

• Compare product name, sequence or specification, and quantity against the purchase order.
• Match the lot number on the COA, vial label and delivery note.
• Check the test date, retest or expiry date, issuing laboratory, report number and signatory.
• Note the purity method and wavelength, and record purity and net peptide content as separate values.
• Confirm the identity evidence, such as observed against theoretical mass, and note what it cannot resolve.
• If accreditation is claimed, verify its status and scope with the accreditation body.
• Record any discrepancy, quarantine the lot and query the supplier in writing.
• Release the lot only when every item is resolved, and file the COA with the receiving record.

None of this needs extra instrumentation, and it means the certificate has been checked rather than simply received.

References

1. International Council for Harmonisation. ICH Q2(R2): Validation of Analytical Procedures. Adopted 2023.
2. Sikora, K.; Ja?kiewicz, M.; Neubauer, D.; Migo?, D.; Kamysz, W. The role of counter-ions in peptides: an overview. Pharmaceuticals 2020, 13, 442.
3. United States Pharmacopeia. General Chapter <1503> Quality Attributes of Synthetic Peptide Drug Substances.
4. ISO/IEC 17025:2017. General requirements for the competence of testing and calibration laboratories.
5. United Kingdom Accreditation Service (UKAS). Guidance on accreditation and the UKAS directory of accredited organisations, published on the UKAS website.

James Weir (pictured) is Co-Founder, Ultrex Bio