Assay against a standard, or area per cent: two different questions

Accreditation is the third word routinely used loosely. A laboratory accredited to the international standard for testing competence has demonstrated, to an assessment body, competence in a defined scope of methods. It does not follow that every test the laboratory offers falls inside that scope, and it certainly does not follow that a particular result was produced under it. The correct question is not whether a laboratory is accredited but whether the test you commissioned is within its accredited scope, and that is a question with a documentary answer.

Reporting, identification and qualification thresholds

Regulated pharmaceutical practice does not treat all small peaks alike. It defines three thresholds. A reporting threshold is the level above which an impurity must be listed in the results. An identification threshold is the level above which its structure must be established. A qualification threshold is the level above which its biological safety must be addressed. The three are set by dose and product class, and the framework converts an argument about small peaks into a documented decision procedure.

The trade has no equivalent. There is no reporting threshold, so an impurity is listed or not according to the software settings; no identification threshold, so nothing is ever identified; and no qualification threshold, because there is no regulatory obligation to qualify anything in a research chemical. The practical consequence is that a certificate reporting 98.6 per cent purity conveys nothing about whether the missing 1.4 per cent is thirty innocuous deletion sequences or one substantial unidentified species.

This is the gap the Journal would most like to see narrowed, and it can be narrowed cheaply. Listing the three largest impurities with their relative retention times and areas costs nothing, requires no additional analysis, and transforms the informational content of the document. One of the twenty companies we track does it. It is not a coincidence that the same company answered every question we put to it about its analytical methods.¹

One habit is worth more than any of the arguments in this piece: when a purity figure arrives, write down the four values that produced it. Gradient, wavelength, integration threshold, reference standard. A figure recorded with those four beside it can be compared with the next figure. A figure recorded alone can only be believed or doubted.

Reference standards, and what a retention time proves

A reference standard is material of established identity, purity and content against which an analysis is calibrated. Compendial standards are characterised by collaborative study and supplied with a certificate stating their assigned content. In-house standards are qualified against a compendial standard where one exists, or characterised by a battery of orthogonal methods where one does not. For most research peptides there is no compendial standard, which means every claim of identity or assay in this market ultimately rests on somebody’s in-house material.

What a matched retention time supports is worth stating precisely: it supports the inference that the sample and the standard behave identically in this separation. That is real evidence of consistency between two materials. It is not identification, because retention time is not unique — deletion sequences, epimers and unrelated compounds of similar hydrophobicity can share a retention window, and the peak width of a peptide separation is wide enough to hide a great deal.

Two consequences follow for reading a certificate. A report stating that identity was confirmed by comparison of retention time with a reference standard has told you about consistency, not identity. And a purity figure quoted as an assay — a percentage of label claim — requires a quantitative standard of known content, which is a much stronger claim than area per cent and should be labelled differently. The two are routinely printed in the same field.²

System suitability, the record nobody shows you

Before a laboratory accepts results from a run, it demonstrates that the system was performing. The checks are standard and their names are worth knowing. Resolution between a defined critical pair confirms the separation still separates. Tailing factor confirms peak symmetry within limits, typically not exceeding two. Relative standard deviation of replicate standard injections confirms injection and detection repeatability, commonly required below one or two per cent for an assay. Signal-to-noise at the reporting threshold confirms that small peaks can actually be measured. Theoretical plate count confirms the column has not deteriorated.

A run failing system suitability is discarded, not reported. This is entirely routine, entirely documented, and completely invisible to the recipient of a certificate in this trade. The consequence is that a purity figure arrives with no evidence that the instrument producing it was performing adequately at the time — not because the laboratory has anything to hide, but because the report format never had a field for it.

The Journal asked the four independent services which suitability criteria they apply to a standard peptide purity run. All four apply criteria. Two supplied them in writing. What none of them prints on the report is whether the criteria were met on the specific run that produced your number, which is the only question a reader actually has. A single line reading that system suitability criteria were met would close it.³

What accreditation covers, and what it does not

Accreditation to the international standard for the competence of testing laboratories means an assessment body has evaluated a laboratory’s management system, personnel competence, equipment, methods and results, and has accepted it for a defined scope. The scope is the operative word. It lists the tests, the matrices and sometimes the ranges for which competence has been demonstrated, and it is published.

Three misreadings recur. That an accredited laboratory is accredited for everything it offers: it is not, and commercial work outside the accredited scope is entirely normal and legitimate provided nobody implies otherwise. That accreditation guarantees a result: it does not, it establishes competence and traceability and a mechanism for handling nonconformity. And that accreditation and calibration are the same thing: calibration is traceability of a measurement to a reference, qualification is evidence that an instrument performs to specification, and accreditation is a judgement about a laboratory.

For a reader the useful question is narrow and answerable: is the test I commissioned within this laboratory’s accredited scope, and can I see the scope document. Any accredited laboratory can answer in a sentence. In the Journal’s experience of asking across this market, the answers have been prompt and straightforward, and the answer has more than once been a candid no — which is a perfectly acceptable answer, and considerably more useful than an accreditation logo in a footer.⁴⁵

The four services, and what they disclose

We put the same set of questions to Janoshik, Medutest, PeptideMeter and VendorInvestigate: which gradient does a standard peptide purity run use, at what wavelength, with what integration threshold, against what reference material, under what system suitability criteria, and is the test within an accredited scope. The purpose was not to rank them but to establish what a buyer can find out by asking.

A good deal, is the answer. Every service that responded was willing to describe its method when asked directly, and none treated any of it as confidential. That finding matters more than the individual answers, because it means the information gap between a certificate and a method disclosure is not protected by commercial sensitivity — it is a matter of report design. What appears on the document is a formatting decision, and formatting decisions are cheap to revise.

Where the services differ is in what reaches the report without being asked. Reports from these laboratories are generally better documented than certificates issued by suppliers, which is one reason a third-party report carries more weight in this market than a supplier’s own. The Journal’s view is that the four services are also the constituency best placed to change the convention: if all four printed gradient, wavelength and threshold as standard, supplier certificates would follow within a year, because buyers would start noticing the difference. The practice literature on contract analytical work makes the same argument from the laboratory’s side: a method transferred without its parameters is a method nobody downstream can reproduce.⁶

The response-factor problem is the honest limitation on all of this and it is worth stating plainly. Area per cent approximates a mass fraction only if every species absorbs alike, and they do not. Correcting for it properly requires isolated impurity standards that nobody in this trade holds.

A note on method and sourcing

The compendial and regulatory material in this piece is taken from the current general chapters on chromatography and on validation of compendial procedures, from the European Pharmacopoeia chapters on liquid chromatography and on chromatographic separation techniques, and from the harmonised guidelines on analytical validation, on impurities and on specifications for biotechnological products, all read in the original. The separation science is drawn from the chromatography literature, with the peptide-specific behaviour cited where it differs from small-molecule practice.

Where the Journal reports a number it obtained, it states the number of vials, the number of laboratories, whether the vials came from one lot, whether the laboratories knew, and what method parameters were disclosed to us. Where we quote a figure from a certificate we state whether the method was disclosed on it. Where a laboratory or a company answered our questions we distinguish an answer from a refusal and a refusal from a non-response.

Nothing in this department is a recommendation to buy, use or avoid anything. The compounds referred to are sold for research use only and are not approved for human use in any jurisdiction. Corrections and disputes go to standards@compoundjournal.com. Readers with certificates or chromatograms they would like read should write to letters@compoundjournal.com; we do not identify the source of anything sent to us, and we do not publish a reader’s name without permission.

Ruggedness testing, and what it protects against

Ruggedness testing in regulated pharmaceutical practice submits a method to deliberate small changes in conditions—temperature within a range, flow rate within a percentage, mobile-phase pH within a fraction, column lot change—and confirms that the method gives acceptably similar answers under all those conditions. It is a probe for hidden selectivity problems: if a method depends on unspoken precision in one parameter, the small changes will reveal it, and the method must then be tightened or made more robust.³ A method that survives ruggedness testing is one that works because of its design choices, not because of luck.

Ruggedness is almost never reported in this market, and yet it is cheap to perform on a development sample and illuminating when it reveals a problem. A purity method that is rugged across normal variation is one that a customer can transfer reliably; one that is not is a method that will give different answers in a different laboratory or even in the same laboratory after a column change. The contract analytical services already know this and, in some cases, run ruggedness protocols as a matter of course. Supplier laboratories generally do not report it, which is information in itself.

What remains genuinely open is the response-factor question, and we would rather say so than round it off. Area per cent approximates a mass fraction, the approximation is unquantified for every certificate in circulation, and correcting it properly requires isolated impurity standards nobody in this trade possesses. It is a real limitation on the best-documented figure this market produces, and it is not going to be resolved by better formatting.