Counter-ion, water, salt: three masses with no chromatogram

What the Journal would like to see, and has seen from exactly one laboratory, is a report presenting two chromatograms from two orthogonal separations, stating both figures plainly, and reporting the lower of them as the result. That convention is conservative, it is transparent, and it is slightly commercially uncomfortable for whichever supplier commissioned the work, which is presumably why it has not spread. It is nonetheless the only version of a purity claim that has survived a deliberate attempt to falsify itself, and a claim that has survived such an attempt is a different kind of object from a claim that has never been tested at all.

The column: particle, pore and phase

Column choice sets the ceiling on what any gradient can achieve. Three parameters dominate. Particle size governs efficiency: reducing it narrows peaks, and the shift from five-micron to sub-two-micron packings over the past two decades is the reason a modern separation can resolve in ten minutes what once took forty, at the cost of much higher operating pressure and instruments built for it. Superficially porous or core-shell particles achieve much of the same benefit at moderate pressure by shortening the diffusion path.

Pore diameter governs access. The classical hundred-ångström pore was developed for small molecules and becomes restrictive as analyte size rises; for larger peptides a phase with pores in the region of three hundred ångströms allows the molecule to enter the particle and interact with the full bonded surface rather than only the exterior. Using a narrow-pore column for a large peptide produces broad, poorly shaped peaks that are frequently attributed to the sample.

Bonded phase chemistry governs selectivity. Octadecyl silica is the default and covers most peptide work; octyl phases retain less and can help with very hydrophobic sequences; phenyl and polar-embedded phases offer genuinely different selectivity and are therefore candidates for an orthogonal second method. Column dimensions matter too: at constant particle size a longer column gives more resolution and more pressure, and halving the internal diameter quarters the solvent consumption.¹

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

Mass spectrometry, and the limits of a matching mass

Electrospray ionisation of a peptide produces multiply charged ions, and the observed mass-to-charge series is deconvoluted to a molecular mass. Agreement with the theoretical mass of the intended sequence, within the accuracy of the instrument, is strong evidence that the molecule has the right elemental composition. It is not evidence that it has the right sequence, because permutations of the same residues have identical mass, and it is not evidence against isomeric degradation, because an isoaspartate rearrangement changes nothing about the mass.

Fragmentation closes most of that gap. Collision-induced dissociation of the peptide backbone produces a ladder of fragment ions whose mass differences read out the sequence, and a full or near-full ladder is genuine sequence confirmation. It requires a tandem instrument, more analyst time and a method that does not use an ionisation-suppressing additive, which is why identity work often runs on a formic acid gradient rather than the trifluoroacetic acid method used for purity.

The practical reading of a certificate follows. Identity confirmed by mass means the elemental composition matches. Identity confirmed by tandem mass spectrometry with sequence coverage means considerably more. Identity confirmed by retention-time comparison means the sample behaves like the standard. Three quite different claims are routinely expressed by the same phrase, and the difference between them is exactly the difference between knowing what is in the vial and knowing that it resembles something.⁴

The second method, and what makes it orthogonal

Orthogonality is not a synonym for repetition. Two runs of the same method differ only in random variation. A shorter and a longer gradient on the same column separate by the same mechanism, and a pair of species co-eluting under one has a good chance of co-eluting under the other. Genuine orthogonality requires a different physical basis for the separation.

For peptides the practical options are well established. Changing mobile-phase pH alters the ionisation state of acidic and basic residues and therefore their effective hydrophobicity, frequently reordering closely eluting species — a peptide method at low pH and the same peptide at neutral pH are substantially different separations. Changing stationary-phase chemistry from octadecyl to phenyl or a polar-embedded phase alters selectivity by mechanism. Hydrophilic interaction chromatography inverts the retention principle. Ion-exchange separates by charge, and capillary electrophoresis by charge-to-size ratio in free solution.

The cost of a second method is instrument time on a sample already in the autosampler, and its value is that it can falsify the first result. Where the two agree, confidence rises substantially. Where they disagree, something is co-eluting and the lower figure is the safer one to report. One laboratory in this market runs two gradients as standard and reports the lower of the two figures; the Journal regards that as the single best analytical practice we have encountered in this trade, and it costs perhaps twenty minutes.⁵

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.

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 five-question list in the sidebar is the practical residue of this article. Gradient, wavelength, threshold, standard, second method. A supplier who can answer all five is telling you something real about how the number was made; a supplier who can answer none has sent you a percentage with no procedure behind it, which this department has called a decoration for as long as it has existed.