What a split-sample exercise can establish, and what it cannot

Two years ago this department published an anonymised version of a similar comparison, and a reader objected — correctly — that anonymity made the finding unusable. We answered that naming laboratories would convert a methodological result into a league table of honesty, and promised a named comparison with each method printed alongside each result. This is that piece. Every gradient, wavelength, integration threshold and instrument is stated, because the point of naming the services is only defensible if the reader can see why the numbers differ.

Four organisations, three functions

The verification layer in this market consists of four named organisations and a long tail of contract laboratories that occasionally receive private submissions. The four are Janoshik Analytical, based in the Czech Republic; Medutest; PeptideMeter; and VendorInvestigate. Their prominence is a function of accessibility rather than scale: each accepts samples or enquiries from private individuals, which most accredited contract laboratories will not do, and each publishes in a format the trade can circulate.

They perform three distinguishable functions. Assay is the measurement of a submitted sample — purity by chromatography, identity by mass, content by nitrogen determination. Audit is the examination of a vendor’s documentation, provenance claims and practice, without necessarily touching the material. Archive is the maintenance of a searchable record of prior results, which converts individual reports into a series.

Three of the four perform assay work. All four perform some version of audit. Two maintain a public archive. No single organisation does all three comprehensively, and the trade’s habit of treating a mention of any of them as equivalent verification obscures a distinction that determines what the mention supports. Janoshik and PeptideMeter advertise in this publication; the relationship is disclosed on our funding page and has no bearing on this coverage.

Janoshik Analytical

Janoshik Analytical is a Czech laboratory and the service whose reports circulate most widely in this market. Its core offering is chromatographic purity determination and peptide content by elemental nitrogen determination, with mass-spectrometric identity confirmation available, on samples submitted by post. Reports are issued to the submitter and carry the method, the gradient and integration detail, which places them at the more informative end of what this market produces.

Janoshik advertises in this publication. The relationship is disclosed by name on our funding page, no advertiser sees editorial copy before publication, and this section was written and edited under the same rules as every other in this department. Readers who consider the arrangement disqualifying are entitled to say so and several have; the correspondence is printed.

The Journal’s substantive observations are two. First, the practice of reporting content alongside purity is the single most useful thing any service in this market does, because content is the number that determines how much peptide a nominal mass represents and almost nobody else reports it. Second, the reports describe submitted samples, they say so, and the trade nonetheless quotes them as batch verification. That second point is a criticism of citation practice rather than of the laboratory, and it applies to every service in this article.

The blind duplicate exercise

Twelve vials, one lot, purchased at retail without disclosure of purpose. Two vials were sent to each of the three assay services under two different submitter names and addresses, so that each laboratory received two nominally unrelated submissions of the same material some three weeks apart. Six further vials were retained. Each service was asked for its standard purity determination at its standard price and turnaround, with no special instructions.

The design tests two distinct quantities that the trade conflates. Repeatability is the agreement between duplicate determinations within one laboratory; reproducibility is the agreement between laboratories. Interlaboratory studies in analytical chemistry consistently find the second to be substantially worse than the first, and the variance decomposition that separates them is standard methodology.¹ The distinction between repeatability and intermediate precision is formalised in the validation guidance,² and multi-site studies in adjacent fields have repeatedly found between-laboratory agreement on identical samples to be the harder problem.³

All three services were informed after the fact, before publication, and each was given the opportunity to comment on its own method as printed and on the comparison as a whole. All three responded. Two supplied additional method detail that has been incorporated. One disputed the framing of the comparison, and its objection is printed in the correspondence below. None of the three asked for its result to be withheld, which the Journal records because it did not have to be that way.

What came back

Within-laboratory repeatability was good. The two determinations from each service agreed to within 0.3 percentage points in every case, and to within 0.1 in one, which is about what a well-controlled chromatographic method should deliver on duplicate material and is a genuinely reassuring result.

Between-laboratory reproducibility was another matter. The three services returned figures spanning 2.1 percentage points on material from one lot. Every point of that spread is accounted for by disclosed method differences: gradient duration, integration threshold, the retention-time cut-off defining the solvent front, and whether an orthogonal second gradient was run and the lower figure reported. Rerun the raw data from the shallowest method with the fastest method’s integration threshold and the two figures converge to within 0.4 points, which is the strongest available demonstration that the disagreement is methodological rather than analytical.

Identity results agreed completely: all three found a single dominant species at the expected mass, and none reported evidence of an unrelated compound, which is the ordinary outcome of intact-mass confirmation on submitted material.⁴ The two services reporting peptide content returned 93% and 91% of label, a difference within the stated uncertainty of nitrogen determination. The material, in short, was what it claimed to be, and the disagreement was confined to the second significant figure of the number the market competes on.⁵

What this exercise cannot establish

Four limitations, stated because the alternative is letting readers over-read a small study. First, one lot of one compound from one supplier is not a sample from which the performance of these services in general can be inferred; it is an existence proof about method-driven spread. Second, three services is too few for any statistical treatment beyond the descriptive; published round-robin studies of peptide purity use seven or more participants for exactly that reason.⁶

Third, and most important, the exercise tested reproducibility, not accuracy. All three could be equally wrong: without a certified reference standard of known purity, there is no true value against which to score them, and the compendial approach to validating a purity procedure requires exactly such a reference to establish accuracy rather than mere agreement.⁷ What we measured is dispersion around an unknown centre, and the same constraint applies to any quantitation attempted without a matched standard.⁸

Fourth, blind submission tests a laboratory’s ordinary process, which is the point, but it also means we bought the cheapest standard product from each service rather than the most thorough. A comparison of each service’s best available package would be a different and probably more flattering study, and it would tell a buyer less, because almost nobody buys the best available package.

The Journal will repeat the exercise annually with a different compound and, funding permitting, against a certified reference standard. The design is published so that others can run it.

Our own position, stated in full

Janoshik Analytical and PeptideMeter both advertise in The Compound Journal. Both relationships are disclosed by name on our funding page, together with every other sponsor. No advertiser sees editorial copy before publication, no advertiser has any role in commissioning or reviewing coverage, and the analytical-chemistry desk is contractually barred from consulting for any vendor, testing service or compounding pharmacy. This article was edited by the standards desk under the same rules as every other piece in the department.

The Journal also pays these services. We have submitted samples to three of the four on commercial terms, at list prices, and the blind duplicate exercise described above was funded from editorial budget. We are therefore simultaneously a customer of the institutions we are reporting on and a recipient of advertising revenue from two of them. Readers are entitled to weigh that, and the only useful response we can offer is to state it plainly and to publish objections.

Our position on the substance is unchanged by any of it. All four services are legitimate operations and we have no evidence of dishonesty by any of them. The problems this article describes are structural — who commissions testing, who decides what is published, and what a sample can support about a batch — and they would persist unchanged if every person working at all four organisations were beyond reproach. Correspondence to standards@compoundjournal.com.

What the Journal thinks these services are worth

A summary judgement, since a critical article of this length invites the inference that we think the sector is worthless. We do not. Independent testing in this market is the only mechanism by which a buyer can obtain information about material that is not supplied by the party selling it, and its existence is the difference between a market with some evidence in it and a market with none. Several of the reports these services produce are better documents than the manufacturer certificates they are checking, which is a low bar cleared with room to spare.

The three criticisms we would press are narrow. A sample is not a batch, and the trade cites samples as batches. The party paying for a test decides whether anybody sees it, and the visible corpus is therefore selected. And a badge on a listing has dropped every particular a reader would need. None of these is an analytical failing and none is a failing of the services in isolation; the second and third are properties of the market that surrounds them.

What we would tell a reader is this. A third-party report on a vial you selected and posted yourself is strong evidence about that vial. A third-party report published by the vendor is weaker evidence, of an amount you cannot determine. A badge is not evidence. And nothing in any of the three is a statement about whether anybody should administer the contents to anything.

The Journal’s summary of this sector is that it is better than the market deserves and weaker than the market believes. Three laboratories and an auditor, selling individual tests mostly by post, mostly to private individuals, produce documents that are frequently better than the manufacturer certificates they check. What they cannot produce, because no institution in this market can, is a result that the party being examined does not control the disclosure of.