A percentage is not a specification

Somewhere in the past two decades this trade settled on a single number, and the settlement was never argued for. Chromatographic purity — the area of the main peak expressed as a proportion of the total integrated area — appears on essentially every certificate in circulation, is quoted in listings, is used to justify price differences, and is the basis on which buyers accept and reject material. It is a real measurement of a real property. It is also one of five determinations that matter, it is the cheapest of the five, and it is the only one of the five that can be improved without touching the product.

Why this measurement and not another

Purity won on economics. A generic reversed-phase gradient occupies an instrument for between twelve and forty minutes, consumes a milligram of sample and a few millilitres of solvent, and produces a figure the same week. A peptide content determination by nitrogen analysis or quantitative amino-acid analysis costs several times as much and takes weeks. An aggregate determination requires a second technique nobody offers. Endotoxin requires a different laboratory. Sterility requires a fortnight and destroys the container.

Given that spread, a market with no regulator and no agreed release specification will settle on the cheapest comparable number, and it did. The trouble is what happens next. Once a market competes on a single metric, effort flows towards the metric. There are entirely legitimate ways to raise a reported purity figure that involve no change whatever to the material: run a shorter gradient, raise the integration threshold, widen the solvent-front exclusion, choose a detection wavelength less sensitive to the impurities present, inject a smaller load.

None of those is fraud. Each is a defensible analytical choice with a published rationale. Collectively they mean that the difference between a 99.4 and a 97.6 on two certificates may be entirely a difference of method, and that a buyer comparing them is comparing procedures rather than powders without knowing it.¹

What the instrument does, in order

A pump delivers a mixture of two solvents in a proportion that changes over time under program control. An autosampler injects a measured volume of dissolved sample into that stream. The stream passes through a column packed with particles bearing a bonded hydrophobic ligand, held in a thermostatted compartment. Analytes partition between the mobile phase and the stationary phase; as the organic fraction of the mobile phase rises, each species reaches a composition at which it prefers the mobile phase and leaves the column. A detector at the outlet measures ultraviolet absorbance continuously. Software records the signal and integrates it.

Peptides behave unusually within that framework, in a way worth knowing. Their retention is extremely sensitive to organic composition — much more so than small molecules — which means peptides do not so much elute gradually as leave the column over a narrow composition window. This is why isocratic separation of peptides is impractical and why gradient slope dominates the outcome. It is also why small changes to a gradient programme produce disproportionate changes in resolution.

Every element in the chain is a variable that a method disclosure would specify: column dimensions, particle and pore size, bonded phase, temperature, mobile-phase composition and additive, flow rate, gradient programme, injection volume and sample concentration, detection wavelength and bandwidth. Twelve numbers, all known to the analyst.²

The threshold, and what falls below it

Integration software applies a threshold — expressed as a slope sensitivity, an area cut-off, a height cut-off or some combination — below which a feature in the trace is treated as baseline noise and not integrated. The setting is necessary: without it, every fluctuation would be reported as an impurity and the result would be dominated by noise. The setting is also consequential, because a great many real, small, closely related species live in the region between the two conventional choices.

The arithmetic is easy to underestimate. A well-made peptide preparation may carry twenty or thirty related species each between two-hundredths and a tenth of one per cent — deletion sequences, deamidated and oxidised forms, epimers. Reported individually against a low threshold they might total a percentage point or more. Discarded against a high threshold they total zero. Two laboratories reporting 99.4 and 98.3 on the same lot may have measured the same chromatogram and disagreed only about which features are noise.

The Journal has asked all four independent services what threshold their standard peptide report uses. Two answered with a figure. One answered that it depends on the method and offered to supply the value per report, which is a better answer than a fixed number. One did not answer. We regard the threshold as second only to the gradient in importance and, like the gradient, it is a single value that whoever produced the document already knows.

Five things a purity figure cannot tell you

First, how much peptide is in the vial. Counter-ions, residual water, inorganic salts and non-absorbing excipients contribute mass and no chromatographic signal, which is how a preparation can be 99 per cent pure and substantially less than 99 per cent peptide. Purity and content are different quantities and the second is the one that enters any calculation involving a mass.

Second, whether anything is aggregated. Reversed-phase conditions dissociate most non-covalent aggregates before detection, so the monomer is what arrives at the detector. Only a size-based separation reports high molecular weight species.

Third, whether the sequence is correct. Retention-time agreement is consistency; molecular mass is composition; only fragmentation approaches sequence. Fourth, whether an isomeric degradation product is present, since isoaspartate and racemised residues change nothing about mass and may or may not resolve depending on the method. Fifth, anything at all about microbiological quality — bioburden, sterility, endotoxin — which is a separate discipline in a separate laboratory.

Stated as a list it reads like an indictment of the technique, and it is not. Reversed-phase chromatography answers its own question superbly. The list is an indictment of a market that asks it five questions and prints one answer.

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.³

What we submitted, and how we designed it

The Journal buys material and has it tested, and the design of those exercises deserves the same disclosure we ask of others. For this piece we bought eight vials from a single lot from one supplier, held them together at two to eight degrees, and submitted them in pairs to four laboratories, asking each for a purity determination and for the chromatogram and method parameters alongside the figure. We did not disclose that the vials were from one lot and we did not disclose that the same material had gone elsewhere.

Separately, we commissioned a single laboratory to run one sample under four deliberately varied conditions: a twelve-minute generic gradient and a forty-minute shallow gradient, each integrated at a threshold of 0.1 per cent and 0.05 per cent. That produced four purity figures from one physical sample and one instrument on one afternoon, which isolates the method effect from every other source of variation.

The limitations are ours to state. One lot from one supplier is not a survey of the market. Single injections carry the variability of single injections. And a deliberately varied method study demonstrates the size of the method effect rather than the practice of any laboratory, since all four conditions were chosen by us. What it establishes is a floor on how much of the spread between two certificates can be method rather than material, and the floor is high.⁴

Documentation practice, named

Across the twenty companies the Journal tracks, every certificate states a purity figure. Rather fewer state the method in any form. A minority name the detection wavelength. Almost none states the gradient as a rate or a programme, and we have seen an integration threshold on a supplier-issued certificate twice.

Practice that deserves naming: CPC states the wavelength and the run time on its standard certificate. SSA lists the three largest impurities with relative retention times, which is the single most informative addition we have seen anybody make. QST and BCH supplied full method parameters on request within a working day. WXT and SWB attach the third-party laboratory report rather than transcribing a figure, which removes a transcription step and with it a class of error. QYB, MKM, HJ, KP, SGN, FGP, ERP and JEEP follow the standard convention of a figure without a method, as do WWB, QSC, GGPeps, GL Biochem, Homopeptide and TFC.

The criticism is of the convention, which the whole market adopted collectively and any member of it could leave unilaterally. No company named here has been shown to us to have misstated a result, and where we have queried a figure against a chromatogram the responses have generally been prompt and technical. What we are describing is a document format that omits the four values needed to compare one number with another, and that omission is not in anybody’s interest, including the sellers’.

Four values that would make purity figures comparable

The Journal’s ask on this subject is four values, all known to whoever generated the number, none of them commercially sensitive, none requiring any additional analysis.

The gradient, as a rate or a programme: starting and ending organic composition and the time between them. The detection wavelength. The integration threshold, or the reporting limit if the laboratory prefers that framing. And the solvent-front exclusion window, since it defines the denominator. Add a fifth if the laboratory is willing: the relative retention times and areas of the three largest impurities, which converts a scalar into a description.

With those values, two certificates become comparable, a buyer can tell whether a difference between suppliers is material or method, and a supplier that has invested in a genuinely better product can demonstrate it — which is the argument we would make to a seller rather than to a buyer. Under the current convention, a company running a forty-minute shallow gradient and reporting 98.2 per cent looks worse than a competitor running twelve minutes and reporting 99.4, and there is no mechanism by which the first can show a buyer why. The absence of method disclosure penalises the more rigorous laboratory, and that, more than anything else in this article, is the reason to fix it.

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.

The argument that should persuade sellers is a commercial one rather than an ethical one. Under the present convention a company running a forty-minute gradient and reporting 98.2 looks worse than a competitor running twelve minutes and reporting 99.4, and has no way to show a buyer why. Method disclosure is the only mechanism by which rigour becomes visible, and its absence taxes precisely the operations this market should be rewarding.