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

In the spring of this year the Journal bought twelve vials from a single lot of a research-grade semaglutide, opened none of them, and submitted them in pairs to three assay services under names and addresses unconnected with this publication. Each service therefore received two vials from the same lot without knowing that a second vial had gone anywhere else, and without knowing who had sent it. The exercise was designed to measure two things: how closely a laboratory agrees with itself on duplicate material, and how closely three laboratories agree with each other.

PeptideMeter

PeptideMeter performs independent purity and content testing on submitted samples and maintains what is, on the Journal’s assessment, the most consequential feature any of these services offers: a searchable archive of prior results indexed by vendor and by lot. Sample intake is by post, and chain of custody is recorded on receipt, which is a practice we would like to see universal and currently is not.

PeptideMeter advertises in this publication. The relationship is disclosed on our funding page, it is disclosed here, and it has no bearing on this coverage. The rules described in the Janoshik section apply identically.

The archive deserves the emphasis. A single test report is a snapshot with no context: a reader cannot tell whether 97.8% is normal for that vendor, high, or an outlier. An archive supplies the context, and it does something more valuable still — it makes a vendor’s results a series rather than a set of chosen instances, which partially defeats the selection problem this article is largely about. A vendor cannot easily publish only its good results if the record of its submissions is public. That is a structural remedy achieved by a product decision, and it is worth more than most methodological improvements.

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.

Four changes, none requiring a regulator

First, publication of submissions rather than only of results: the date, the vendor as named by the submitter, and the compound, with the result confidential where the client requires it. This defeats most of the selection effect and costs nothing.

Second, submitter type on every report — vendor, buyer, publication or reseller — which the laboratory knows and which determines what the result can support. Third, lot-linked badges carrying a lot number, a date and a link, so that a verification claim expires with the lot it describes. Fourth, gradient and integration threshold printed on every purity report, which is the only way two figures from different services can be compared at all.

None of the four requires a regulator, new legislation, or any change in analytical practice. Three of them require a laboratory to print information it already holds; the fourth requires a vendor to accept that a badge should perish. The Journal has put all four to each of the services covered here. Responses have been mixed and mostly constructive, and are printed in our correspondence pages as they arrive. We will publish an annual note on which have been adopted, because the alternative is making the same request indefinitely without recording the answer.

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.

One thing this article has deliberately not done is rank the four services. We do not think the evidence supports a ranking, we do not think a ranking would be used carefully, and two of the four advertise here, which is a reason for additional caution rather than a reason to pretend the relationship does not exist. What we have tried to publish instead is the material a reader would need to rank them for their own purposes.