Reproducibility, measured rather than assumed

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.

The sample is not the batch

The most important sentence in every third-party report in this market is the one identifying what was tested, and it always says the same thing: the sample submitted. That phrasing is precise and correct, and the entire trade reads past it. A result obtained on one vial extends to a batch only if the vial is representative, and representativeness is a property of how the vial was selected, not of how carefully it was analysed.

In regulated practice, sampling is a controlled activity in its own right: the accreditation standard treats it as part of the laboratory activity, requiring a documented sampling plan and records of how the portion tested was obtained.¹ Where a laboratory receives a sample it did not draw, the standard expects the report to make clear that the results apply to the sample as received. All four services do this. The market quotes them anyway as though the batch had been tested, which inverts the pharmacopoeial convention that a result on a sample is evidence about a batch only under a stated sampling assumption.²

The practical significance depends on the fill. A batch filled in a single session from a homogeneous bulk solution is likely to be uniform, and a single vial is decent evidence about it. A batch assembled from subdivided bulk, filled across sessions, or blended from more than one synthesis is not, and a single vial is evidence about a vial. Nothing on a report tells a reader which situation applies, because the laboratory does not know either.

Custody begins at the reception desk

What a laboratory can record about a submitted sample begins at the moment of receipt, and the better reports in this market record a surprising amount: the date and time of arrival, the condition of the outer packaging, whether cold-chain materials were present and still cold, whether the vial seal was intact, whether the crimp showed evidence of having been lifted, and the appearance of the cake. Two of the four services record most of this as standard. The others record some of it.

These lines are worth more than they look. A vial arriving with a collapsed cake or a discoloured plug is telling a reader about storage and transit history that no purity figure captures, and a report that notes it has converted an unknown into a datum. Condition on arrival is also the only point in the entire chain at which anybody independent observes the material’s physical state, and it is therefore the only opportunity to catch a transit problem before it is analysed away.

What no laboratory can record is what happened before the package was posted. The submitter’s storage, the route from vendor to submitter, the number of times the vial changed hands, the ambient temperature of a fortnight in a sorting facility: all of it is prior to custody and none of it is knowable. The Journal accordingly cites third-party purity figures with the submission date and, where we know it, the submitter, because those two facts bound what the figure can support.

The submitter pays, and the submitter decides

Reduce the incentive problem to its mechanism and it is very simple. The party who pays for a test receives the report. The report is that party’s property. Nothing obliges them to publish it. Therefore the set of third-party results visible in this market is not the set of results obtained; it is the subset that somebody with a commercial interest chose to make visible.

The size of the resulting distortion is unknown and unknowable from published data, which is precisely the difficulty. If a vendor submits twelve batches over a year and publishes eight, the four unpublished results are invisible, and no amount of scrutiny applied to the eight will recover them. This is the mechanism the research literature calls publication bias, and it is well characterised: the distortion it produces is larger when the number of tests is small, when the cost of a test is high relative to the value of a favourable result, and when nobody records that a test was commissioned.

The accreditation standard for testing laboratories requires a laboratory to identify and manage risks to its impartiality arising from its commercial relationships, and to be able to demonstrate that it has done so.³ That obligation sits on the laboratory and it is the right place for it. What the standard cannot reach is the client’s filing cabinet, and the filing cabinet is where this problem lives.

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.⁴ The distinction between repeatability and intermediate precision is formalised in the validation guidance,⁵ and multi-site studies in adjacent fields have repeatedly found between-laboratory agreement on identical samples to be the harder problem.⁶

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.⁷ 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.⁸

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.⁹

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.¹⁰ What we measured is dispersion around an unknown centre, and the same constraint applies to any quantitation attempted without a matched standard.¹¹

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.

Turnaround, price and the trade-off between them

Across eighteen submissions made by this desk over two years, quoted turnaround ranged from three working days to four weeks and observed turnaround from four days to thirty-one. Prices for a standard purity determination on a single sample ranged over roughly a factor of four, and the addition of peptide content by nitrogen determination roughly tripled the cost of the cheapest purity-only tier.

The relationship between price, turnaround and analytical depth is not a scandal; it is arithmetic. A forty-minute gradient occupies an instrument for three and a half times as long as a twelve-minute one, and if the laboratory runs an orthogonal confirmation that doubles again. A written interpretation occupies an analyst. A reproduced chromatogram occupies nobody but requires that the report be assembled by a person rather than exported by software. Every one of those choices is visible in the price.

The consequence for a reader is that price is a proxy for method depth, and a surprisingly good one. Where a report does not state its gradient — and the cheapest tiers frequently do not — the price paid is the best available indirect evidence about how thorough the determination was. That is an unsatisfactory situation and it is improved by a single line on the report rather than by anybody charging differently.

The report is the product

Across the four services the Journal has catalogued eleven elements that appear on some reports and not others: the compound and lot as declared by the submitter; the date of receipt; the condition of the sample on arrival; the instrument; the column; the gradient; the detection wavelength; the integration threshold; the reproduced chromatogram; a named analyst; and an explicit statement that results apply to the sample as received.

No service omits all of these and none includes all of them on every tier. The best reports in the sector carry nine or ten and are genuinely good documents by any standard — better, in several respects, than the manufacturer certificates they are checking. The thinnest carry three, and a three-element report is a number with a letterhead.

The reporting requirements in the accreditation standard are the natural benchmark here, and they are not demanding: identification of the items tested, the date of receipt, the methods used, the results with units, and a clear statement of what the results apply to.¹² A report meeting that list is checkable. The Journal’s standing request to all four services is a single addition beyond it — print the gradient and the integration threshold — because those two numbers are what make a purity figure comparable with anybody else’s.

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.