Methylene Blue (Methylthioninium Chloride) Reference Standard — CAS 61-73-4, C₁₆H₁₈ClN₃S

1. What this material is

This page describes methylene blue supplied as an analytical reference material and laboratory reagent: a weighed quantity of a single identified substance, intended as the point of comparison against which another sample is measured, or as the defined-quality reagent in a method whose output is a number.

That second role is what makes this compound unlike most of the catalogue. Methylene blue is not only an analyte; it is an instrument. It is the colour-forming reagent in the classical spectrophotometric determination of sulfide in water [33] and in current work on sulfane sulfur [32]; it is a redox indicator, blue in the oxidised state and colourless as the reduced leuco form; it is a photoredox catalyst in synthetic photochemistry [25]; and it is a constituent of the Romanowsky stains on which a century of haematological diagnosis rests [19]. In each of those roles the number produced at the end depends on the quality of the dye at the beginning, and a reagent that is part water and part azure dyes does not fail loudly. It produces a slightly wrong slope, indefinitely.

The compound is also, separately, an authorised medicinal substance — in Poland and across the European Union as Proveblue and in the United States as ProvayBlue, indicated for acquired methaemoglobinaemia [6]. Nothing offered here is that medicine. The material on this page is a laboratory chemical, supplied on the terms in section 16.

Three things decide whether a given bottle of methylene blue can serve either purpose, and none of the three is settled by a percentage printed on a label. Which hydrate is in the bottle determines what a weighed mass means (section 3). How the purity was assayed determines whether the closely related azure dyes were counted as impurity or silently folded into the result (section 4). At what concentration and wavelength the absorbance was read determines whether a photometric check means anything at all (section 6). Those are three specific fields on a certificate, and they are exactly the three that a technical-grade dye supplier has no reason to fill in.

The terms on which this and every other reference material here is supplied are collected in the reference standards category.

2. Identity and registry codes

Two rows carry more weight than they look. Covalently bonded units: 2 is the registry stating in its own vocabulary that this is a salt — an organic cation and a chloride ion, not one covalent molecule. Everything in section 3 follows from that single integer. And the identity of SMILES and isomeric SMILES is the machine-readable way of saying that there is no stereochemistry to record, which section 7 then confirms from the four stereodescriptor counters.

One naming point deserves its own paragraph, because it is the shortest route to the wrong substance. C.I. 52015 is a Colour Index number. It identifies a dye in the index of colouring matters used by the textile, ink and staining trades. It is not a food-additive authorisation, it is not an E number, and it says nothing about pharmacopoeial quality. Methylene blue has no E number; figures resembling one circulate in commercial listings and appear to be garbled renderings of the Colour Index number. The blue food colours in the European system are E131 Patent Blue V, E132 indigotine and E133 Brilliant Blue FCF, and this substance is none of the three. The nomenclature of the methylene blue, azure and violet family has been confused for more than a century, and has been reviewed as such [18] — which is the practical reason to anchor identity on CAS number and InChIKey rather than on a trade name or a colour name.

3. Three records, three masses

Ask a registry for methylene blue and you are answered by three different objects, each with its own molecular formula, its own mass and its own hash key. They are not three descriptions of one thing. They are three things, and a quantity weighed out on a balance belongs to exactly one of them.

The arithmetic that follows is the whole of the problem. The trihydrate is heavier than the anhydrous salt by a factor of 373.90 / 319.85 = 1.1690, which is 16.9 %. One thousand milligrams of the trihydrate contains 855.4 mg of anhydrous-equivalent salt, and 760.6 mg of the cation that actually carries the colour and the redox chemistry. A calibration curve prepared by weighing trihydrate and calculating with the anhydrous mass is wrong by nearly a fifth, in a direction that looks entirely plausible on the plot.

Why the name on the bottle does not settle it

Salt and solvate ambiguity is not unique to this compound, but methylene blue is unusually bad at signalling which form it is. Where a product is called procaine hydrochloride, the counter-ion is written into the name and no one calculates with the free base by accident. Where a compound is normally traded as a specific salt, as with sildenafil and its citrate, the mass difference is large enough that the convention is fixed and visible. Here the same word, methylene blue, is applied indifferently to the anhydrous chloride, to the trihydrate, to material of unstated water content, and in older commercial practice to the zinc chloride double salt — a form named in the manufacturing description on the registry record itself [1] and central to an entire series of measurements on commercial samples [14]. The registry even notes plainly that the substance forms double salts.

Water content is therefore a specification field, not a footnote. The comparison worth making inside this catalogue is alpha-GPC, where hygroscopicity is the first thing anyone handling the material learns and where a mass without a water figure is understood to be provisional. Methylene blue is the same problem wearing a more confident label: the dye looks stable, dry and definite, and the difference between its hydrates is nonetheless larger than most impurity specifications.

4. What a purity figure on a dye does not say

Dyes carry a measurement tradition of their own, and it does not line up with the one used for reference standards. The traditional figure is dye content: the fraction of the powder that is colouring matter, determined by titration or by absorbance against a reference. Dye content says nothing about which colouring matter. A sample can be 98 % dye and still contain several dyes.

For this particular dye that gap is occupied by a specific, named family. Successive demethylation of methylene blue gives azure B (trimethyl), azure A (dimethyl), azure C (monomethyl) and finally thionine; the related methylene violet belongs to the same chemical and nomenclatural cluster [18]. These compounds are produced during synthesis, they are produced on storage, and they are produced in vivo as metabolites. They are also, unhelpfully for anyone hoping to separate concerns, the intended constituents of the Romanowsky stains, where the ratio of methylene blue to azure B is the parameter that makes the stain work [19].

The azures are not inert ballast. Azure B inhibits acetylcholinesterase and butyrylcholinesterase in its own right, and it is at the same time a metabolite of methylene blue [20]. That combination is the hardest possible case for an impurity specification: the same species that must be limited in the vial is also the species expected to appear downstream in any biological experiment, so its presence cannot be dismissed as irrelevant and cannot be attributed unambiguously to one origin.

The certification that does exist

There is an established scheme for exactly this problem, and it is worth knowing about before comparing suppliers. The Biological Stain Commission operates a certification programme in which submitted dye lots are assayed and tested for performance, and certified lots carry the Commission’s mark with a lot number; the analytical procedures used have been published in the open literature [17]. A Commission-certified lot answers a question about staining performance and dye composition. It is not the same question as chromatographic purity for a reference standard, and neither is it the same question as compliance with a pharmacopoeial monograph, which fixes identity tests, an assay and specified limits. Three different certificates, three different meanings, all of them legitimately describing “methylene blue”.

This is why a purity number is only half a specification anywhere in this catalogue. On modafinil the qualifier by HPLC is doing real work, because it names an area-per-cent measurement with a separation behind it. On a dye the same bare number can mean dye content, and dye content is compatible with several dyes at once. And where a substance has a pharmacopoeial monograph in wide use — paracetamol is the obvious example — the monograph, not the supplier, defines what the assay means. For methylene blue, monographs exist [10] and the registry itself records the pharmacopoeial identity and purity procedures: infrared absorption against a reference for identity, and visible absorption spectrophotometry at 663 nm against standards for chemical purity, alongside an older titrimetric assay for the substance in drug matrices [1].

5. The chromophore is not an identity test

Most compounds in this catalogue are difficult to detect. This one is the opposite: a deeply coloured cation with an absorption maximum in the red, visible to the eye at micromolar concentrations and quantifiable on any visible-range photometer. The temptation that follows is to treat colour as identity. It is not.

Every member of the phenothiazinium family absorbs in the same region and looks blue in solution: azure A, azure B, azure C, thionine, toluidine blue O, methylene violet. They differ by degrees of N-methylation on the same tricyclic core, and each successive demethylation shifts the maximum by tens of nanometres, not hundreds. A visible spectrum therefore constrains the family and does not select the member — and this is precisely the family that co-occurs with methylene blue in real samples for the reasons set out in section 4.

The catalogue supplies two useful contrasts here. For bromantane a single halogen isotope pattern settles identity from a mass spectrum with no chromatography at all — the molecule announces itself. For mebicar the opposite extreme holds: no useful chromophore, no ionisable group, nothing for a conventional detector to see. Methylene blue is the third case, and the least intuitive one. It is trivially detectable and stubbornly hard to confirm, because everything it is likely to be confused with is equally visible and equally blue.

6. Why photometry at the absorption maximum is not linear

The second trap follows directly from the first. Having decided to quantify a strongly absorbing dye by absorbance, an analyst reaches for a molar absorptivity and Beer’s law. For methylene blue that relationship holds only in a narrow window, and it fails in a way that produces smooth, believable, wrong numbers.

Methylene blue self-associates in solution. The monomer band near 664 nm is accompanied by a dimer band near 610–615 nm, and the ratio between them changes with concentration. The kinetics of the monomer–dimer equilibrium in water were characterised by flash methods in 1979 [23]; the same equilibrium has since been shown in alcohols, with dimers and higher aggregates and a critical aggregation concentration determined separately for methanol, ethanol, propanol and butanol [24]; and the aggregation state has been shown to change the compound’s behaviour as a photoredox catalyst, which is a functional consequence rather than a spectroscopic curiosity [25]. Ionic strength, temperature and the presence of surfactants or polyanions all shift the equilibrium further.

Nor is there a single authoritative wavelength to anchor on. The registry record carries “Absorption max: 668, 609 nm” in one entry and “UV absorption maxima 688 and 609 nm” in another, both attributed to the same trihydrate [1]. The pharmacopoeial purity procedure reads at 663 nm [1][10]. That is three different numbers for the position of one band, inside sources a laboratory would reasonably treat as authoritative. We report the disagreement rather than choosing a value, because choosing one would hide exactly the thing a buyer needs to know.

We do not print a molar absorptivity on this page for the same reason. A single figure without the conditions attached would be a number that fails silently, and we could not verify a primary value under stated conditions to the standard the rest of this page is held to. The comparison inside the catalogue is instructive: for apigenin, a flavone with a well-behaved ultraviolet spectrum, an absorptivity travels reasonably well between laboratories because the molecule does not stack appreciably at working concentrations. For a cationic dye that dimerises, it does not.

7. Structure and stereochemistry

The molecule is a planar tricyclic phenothiazinium cation: a central thiazine ring flanked by two benzene rings, with a dimethylamino group at position 3 and another at position 7, and the positive charge delocalised across the conjugated system. The chloride ion balances it. That planarity is the structural reason for the stacking described in section 6, and modern crystallography of a zinc chloride complex of the dye makes the π–π interaction explicit [13].

The stereochemistry is stated by the registry without ambiguity. Quoted verbatim, for all three records [1][2][3]:

The compound is achiral. There are no enantiomers, there is no optical rotation to specify, chiral chromatography has nothing to resolve, and any claim about a dextrorotatory or laevorotatory methylene blue is false by construction. Two independent confirmations sit alongside the counters: the isomeric SMILES is identical to the canonical SMILES, and the middle block of the InChIKey is UHFFFAOYSA in all three records, which is the standard hash for a structure with no stereochemical layer.

This is worth stating plainly because it removes a whole class of specification that other products in this catalogue genuinely need. Tadalafil carries two defined stereocentres and therefore four stereoisomers of identical exact mass, and for that compound the certificate has to say which one is in the vial. Here the certificate has nothing to say on the subject, and a supplier who volunteers an enantiomeric purity figure for methylene blue is describing something that does not exist. The specification burden for this substance sits entirely on form, water and dye composition instead.

8. Methods that work and methods that fail

The descriptors point at the method before any experiment does. This is a permanent cation with a formal positive charge delocalised over the ring system, no hydrogen-bond donors, four acceptors, one rotatable bond and a computed partition coefficient of 2.2 for the free cation [3]. It is not a neutral molecule whose retention can be tuned with mobile-phase pH, and it is not a weak base that can be switched between forms. It is charged at every pH, and it sticks to things.

Two practical warnings that do not fit in a table. First, this dye adsorbs onto surfaces — glass, plastic, filter membranes, tubing and column frits — and at trace concentrations the loss is not negligible; standards prepared in a glass volumetric flask and transferred through a syringe filter can arrive at the detector visibly weaker than intended. Second, it is a redox indicator, so it participates in the chemistry of its own sample: mild reducing agents convert it to the colourless leuco form, and the colour returns on exposure to air. Anything that reduces it — ascorbate, thiols, dithionite, some biological matrices — will quietly change an absorbance reading. That is the reverse of the situation for L-DOPA, where the analyte oxidises visibly and the analyst learns to protect it; here the colour change runs in the other direction and can be mistaken for dilution error.

9. Physicochemical data, with the disagreements left visible

Methylene blue has been in continuous industrial and clinical use since the 1870s, so the natural expectation is a complete and settled set of constants. The registry does not deliver one. Some quantities are recorded several times with different values; others have no entry at all. Below is what exists, attributed per value, with the contradictions left in place.

Dissociation constants: the values are real, but they are not where they look

The dissociation-constant section of the record appears at first to be empty: it holds two pointers to an external compilation rather than inline numbers. Followed through, that compilation returns three experimental values, each with a temperature, a method and a primary citation:

Two observations follow. First, these constants describe protonation of the already cationic dye, not deprotonation of a neutral acid: the species carried in ordinary aqueous solution is the cation, and none of these equilibria offers a handle for controlling chromatographic retention at working pH. Second, the third value belongs to an excited state, which is a different physical object from the ground state and cannot be used interchangeably with it. A separate number, approximately 3.14, appears in the environmental-fate narrative on the same record; that one is explicitly labelled an estimate and should never be quoted as a measurement.

Compared with the emptier corners of this catalogue — chlodantane is the extreme case, where the registry holds almost nothing measured at all — methylene blue is data-rich. The difficulty here is the opposite one: there is enough data for two sources to disagree, and enough history for estimates to have been repeated until they look like measurements.

10. Spectra: what is deposited, what is missing

Deposited spectral data for this compound is unusually well attributed — instrument, technique, and in one case the catalogue and lot number of the sample measured. It is also unevenly distributed between the anhydrous and trihydrate records, which is worth knowing before searching for a reference spectrum and concluding that none exists.

What remains genuinely missing after both records are searched is worth stating precisely, because it defines what a well-characterised lot can add. There is no ¹³C NMR spectrum on either record, no two-dimensional NMR, no tandem mass spectrometry data and no Raman spectrum — established by enumerating every branch of the spectral sections rather than by keyword search, so this is an absence in the record, not a failure of the query. For a compound with this depth of literature, the gap in the free public record is real, and a lot accompanied by an assigned proton and carbon spectrum, a product-ion spectrum and a solid-phase infrared of the declared hydrate carries information that cannot simply be looked up.

One further caution about infrared identity. The pharmacopoeial identity test is infrared absorption compared with a reference [1], and the deposited infrared data was measured on a KBr pellet and on an ATR film cast from chloroform. Those are two different sample preparations, and neither is guaranteed to match a spectrum recorded on a different hydrate of the same salt. Comparing an ATR spectrum of your trihydrate against a KBr reference of anhydrous material and calling the differences instrument variation is a mistake this substance invites.

11. Hazard classification and how broad its basis is

Read those rows together, because they say something more interesting than a single pictogram. This is a broad classification base by the standards of a research chemical — hundreds of company reports rather than the single filing that stands behind many catalogue substances. Breadth, however, is not unanimity. The percentages are fractions of the 273 company reports, not of the 18 notifications — 91.6 % of 18 would not be a whole number, while 91.6 % of 273 is the 251 reports the record itself names. Read that way they disagree substantially: barely a quarter of the reports carry the serious-eye-damage hazard that is the one classified at Danger level, one in ten carries the aquatic hazard, and roughly one in twelve asserts that the substance is not hazardous under GHS at all. A safety data sheet built from any one of those positions is defensible, and two suppliers can therefore hand you materially different documents for the same substance without either being wrong.

The absence of a harmonised classification was measured, not assumed. The ECHA substance record for methylthioninium chloride carries an empty index-number field; the same query against formaldehyde, a substance with a well-known Annex VI entry, returns index number 605-001-00-5 [4]. The instrument fires, so the empty field is a real absence. In practice this means no legally binding classification applies across the European Union, the notified classification is what suppliers have chosen to declare, and the national entry with the reproductive-toxicity statement should be read into your own risk assessment rather than ignored because it sits outside the ECHA aggregate.

12. Regulatory status

13. What the literature is about

The literature on methylene blue is enormous, and its size is a trap for anyone using publication counts as a proxy for how well a specific question has been studied. A bibliographic query on the name returns figures in the hundreds of thousands, dominated by work in which the dye is a model pollutant for adsorption and photocatalysis studies — papers about activated carbon and titanium dioxide, not about methylene blue itself. Filtered for the questions this page turns on, the picture inverts sharply: the core literature on the composition of commercial preparations is thin and old, centred on a series of papers published between 1963 and 1979 [14][15][16]. That combination — a vast literature that does not address the practical question, and a small old literature that does — is exactly why a modern, well-documented lot is worth something.

The strands below are described as facts about published work. They are not claims about this article and not a description of any use of it.

Structure and physical chemistry

The pentahydrate crystal structure was solved in 1973 [12]; a complex of the dye with zinc(II) chloride was characterised crystallographically in 2026, with the π–π stacking interactions described explicitly [13]. The self-association behaviour that section 6 rests on runs from kinetic work on the monomer–dimer equilibrium in water [23] through aggregation in alcohols [24] to the effect of aggregation state on photoredox catalysis [25]. The excited-state acid–base behaviour was studied by flash spectroscopy in 1967 [26], and ground-state protonation as early as 1951 [27].

Dye chemistry, staining and nomenclature

Preparative separation of thionine, azure A, azure B and methylene blue was published in 1963 [16]. The azure B content of commercial samples was measured in 1979 [14], and material of low azure B content was prepared from commercial medicinal-grade dye in 1978 [15]. The certification procedures used for biological stains have been published in full [17]. The nomenclature of the family has been reviewed as a subject in its own right [18], and the chemistry of Romanowsky staining has been reviewed recently from a chemical rather than a histological perspective [19].

Analytical chemistry

The most directly useful papers are two residue methods: a tandem mass spectrometry method that determines methylene blue together with azure A, B and C [21], and an earlier method reporting quantification limits and recoveries in the same matrix class [22]. Beyond residues, the compound appears throughout analytical chemistry as a reagent: the classical spectrophotometric determination of hydrogen sulfide in natural waters is the methylene blue method [33], and the technique remains in the current toolkit for sulfane sulfur [32].

Pharmacology and clinical literature

This strand is reported here strictly as bibliography, and it is the reason the framing of this page is laboratory-only rather than anything else. Published work establishes that the compound is a potent monoamine oxidase inhibitor [28]; a clinical case report describes serotonin syndrome following its administration during cardiac surgery [29]; a haematology note warns about haemolysis in the context of glucose-6-phosphate dehydrogenase deficiency [30]. Azure B, the principal related dye and metabolite, inhibits acetylcholinesterase and butyrylcholinesterase [20]. A 2022 review examines the compound as a diagnostic agent and medication and concludes that its basic toxicological characterisation remains incompletely established despite well over a century of use [31], and a review of its history and pharmacology sets that longevity in context [34]. Research-stage work under a development name investigated the compound’s effect on tau protein aggregation, which is why that name appears among the registry synonyms [1].

Two things follow for a laboratory. First, this is not an innocuous dye to be handled casually because it is old and familiar. Second, the interaction and deficiency issues in that literature belong to clinical practice with an authorised medicine, and are cited here to explain why the article on this page is supplied strictly as a reagent and not for any other purpose.

14. Handling and storage

15. What we certify and what we do not

16. Terms of supply

This material is supplied as a laboratory reagent and analytical reference material, for in-vitro laboratory use by qualified personnel in an appropriately equipped facility. It is not a medicinal product, not a dietary supplement, not a food, feed or cosmetic ingredient, not a food colour, and it is not supplied for administration to humans or animals in any form or by any route.

By ordering, the purchaser confirms that they are a professional user acquiring the material for laboratory purposes; that they will handle it in accordance with section 14 and their own institutional risk assessment; that they will not administer it to humans or animals, nor supply it to any person who intends to; and that they are responsible for compliance with all laws applicable at the destination. That last point deserves emphasis for this substance in particular, because the same molecule is an authorised medicine subject to pharmaceutical law in the same jurisdictions where it is also an ordinary industrial dye. The article supplied here is neither the medicine nor the industrial dye: it is a reagent, and the documentation that accompanies it describes the lot as such.

Nothing on this page is medical advice, nor an offer of a medicinal product. Statements in sections 12 and 13 describe medicines authorised elsewhere and the published research literature; they describe neither this article nor any use of it. The full terms applying to every reference material in this catalogue are set out in the reference standards category.

17. Questions and answers

Which form am I buying, anhydrous or trihydrate?

The lot documentation states the form and, where determined, the water content. This matters more here than for almost anything else in the catalogue: the anhydrous chloride is 319.85 g·mol⁻¹ and the trihydrate is 373.90, so 1000 mg of trihydrate corresponds to 855.4 mg of anhydrous-equivalent salt. Calculating a molarity from a weighed mass without knowing the form introduces a 16.9 % error.

Why do two CAS numbers appear for methylene blue?

Because there are two substances. 61-73-4 is the anhydrous chloride; 7220-79-3 is the trihydrate, and it has its own registry record, its own environmental-database identifier and its own toxicology dataset [2][35][36]. Eleven further CAS numbers are carried on the anhydrous record as deprecated, so a supplier document quoting an unfamiliar number is not automatically wrong — but it needs checking against the record rather than being copied forward.

Is a pentahydrate real, or is that a typographical error?

Real. The pentahydrate has a single-crystal structure solved and published in 1973 [12], and the European substance record for this dye even carries a French pentahydrate name among its IUPAC name variants [4]. At least three hydration states of this salt are documented.

What does a bare purity percentage on a dye actually mean?

Without a stated method, very little. The historical dye figure is dye content, the fraction of the powder that is colouring matter, which is compatible with several different dyes being present at once. Chromatographic purity expressed as area per cent with a separation from azure A, B and C is a different and much more informative number. Ask which one the figure is, and against what method.

What are the azures, and why do they matter?

Azure B, azure A and azure C are successively demethylated relatives of methylene blue, ending at thionine. They arise in synthesis, on storage and as metabolites. The azure B content of commercial samples was measured directly in 1979 [14], and a procedure for lowering it in commercial medicinal-grade material was published the year before [15]. Azure B is not inert: it inhibits acetylcholinesterase and butyrylcholinesterase [20].

Can I confirm identity from the visible spectrum?

No, and this is the central analytical point about the compound. Every member of the phenothiazinium family — the azures, thionine, toluidine blue O, methylene violet — absorbs in the same region and looks blue. A visible spectrum constrains the family and does not select the member. Separation with mass detection does [21]; infrared against an authentic reference of the same solid form is the pharmacopoeial identity test [1].

Which wavelength should I use?

Whichever your method specifies, and state it. There is no single canonical figure: the registry carries 668 and 609 nm in one entry and 688 and 609 nm in another for the same trihydrate, while the pharmacopoeial purity procedure reads at 663 nm [1][10]. Report the wavelength, the concentration, the solvent, the temperature and the path length with any absorbance you quote.

Why is my calibration curve not linear?

Because the dye dimerises, and the fraction present as monomer changes as you dilute. The monomer band near 664 nm and the dimer band near 610 nm shift in relative intensity with concentration; the equilibrium has been characterised kinetically in water [23] and shown to extend to dimers and higher aggregates in alcohols [24], and the aggregation state even changes the compound’s catalytic behaviour [25]. Work within a narrow, stated concentration range, or model the equilibrium.

Do you supply a molar absorptivity?

No. A single figure without conditions attached would fail silently for exactly the reason in the previous answer, and we do not print numbers we cannot attribute to a primary measurement under stated conditions. Determine it on your own material, at your own working concentration, and record the conditions alongside it.

Is the compound chiral?

No. All four stereodescriptor counters are zero on all three registry records, the isomeric SMILES is identical to the canonical SMILES, and the InChIKey middle block is the standard no-stereochemistry hash. Chiral chromatography has nothing to resolve here, and any optical rotation quoted for methylene blue is fabricated by construction.

Is this the same thing as the medicine?

No. Methylthioninium chloride is an authorised medicinal substance — in Poland and the European Union as Proveblue, authorised through the centralised procedure [6][11], and in the United States as ProvayBlue for acquired methaemoglobinaemia [1]. The article supplied here is a laboratory reagent, is covered by no marketing authorisation, and carries none of the guarantees attached to a licensed medicinal product.

Does methylene blue have an E number?

No. It has a Colour Index number, C.I. 52015, which identifies a dye in the index of industrial colouring matters and is not a food authorisation. In the United States system it is listed as a colour additive whose permitted use is recorded as “None”, with former use limited to external drugs and external cosmetics [1]. Its food-related entries concern food-contact sanitising solutions, which is the opposite of a food additive.

Is it a controlled substance, or on the anti-doping list?

It is not scheduled in the Polish narcotic, psychotropic or new-psychoactive schedules [7], and it does not appear by name on the 2026 anti-doping list [9]. Both negatives were established with positive controls firing in the same documents, and in both cases raw text matching produced false positives — every apparent anti-doping hit was a methylenedioxy- compound, and the apparent Polish hit was the phrase methylene group inside an unrelated structural definition.

Is the GHS classification reliable?

It is broad and it is not unanimous. The aggregate rests on 273 company reports from 18 notifications, but only 91.6 % report the acute oral hazard, 25.6 % the serious eye damage that drives the Danger signal word, and 8.1 % of reports say the substance meets no GHS criteria at all [1]. There is no harmonised European classification — the ECHA record carries no Annex VI index number [4] — so suppliers may lawfully classify differently, and a separate national classification adds reproductive and organ-toxicity statements that the ECHA aggregate does not.

Is there a reference NMR spectrum?

A proton spectrum exists, but it is filed against the trihydrate record rather than the anhydrous one [2], which is why a search of the anhydrous record alone concludes that none exists. No carbon spectrum is deposited against either record, and there is no two-dimensional NMR, no tandem mass spectrometry and no Raman data on either. Those are genuine gaps in the free public record.

What should a good certificate for this substance contain?

Six things: the form supplied and its water content with the method used; the assay figure with its method named; a statement of how azure A, B and C were assessed; the identity test performed and against what reference; the conditions of any photometric measurement quoted; and the lot number tying all of it together. Section 15 sets out which of those we assert and which we do not.

References

Registry and regulatory sources

1. National Center for Biotechnology Information. 2026. “PubChem Compound Summary for CID 6099, Methylene Blue.” PubChem. https://pubchem.ncbi.nlm.nih.gov/compound/6099.
2. National Center for Biotechnology Information. 2026. “PubChem Compound Summary for CID 104827, Methylene Blue Trihydrate.” PubChem. https://pubchem.ncbi.nlm.nih.gov/compound/104827.
3. National Center for Biotechnology Information. 2026. “PubChem Compound Summary for CID 4139, Methylthioninium Cation.” PubChem. https://pubchem.ncbi.nlm.nih.gov/compound/4139.
4. European Chemicals Agency. 2026. “Substance Information — Methylthioninium Chloride, EC 200-515-2, Registry Entry 100.000.469.” ECHA. https://chem.echa.europa.eu/100.000.469.
5. World Health Organization Collaborating Centre for Drug Statistics Methodology. 2026. “ATC/DDD Index — V03AB17 Methylthioninium Chloride.” https://atcddd.fhi.no/atc_ddd_index/.
6. Urząd Rejestracji Produktów Leczniczych, Wyrobów Medycznych i Produktów Biobójczych. 2026. “Rejestr Produktów Leczniczych — Methylthioninium Chloride Proveblue.” https://rejestry.ezdrowie.gov.pl/rpl/search/public.
7. Minister Zdrowia. 2024. “Obwieszczenie w sprawie wykazu substancji psychotropowych, środków odurzających oraz nowych substancji psychoaktywnych.” Dziennik Ustaw. https://isap.sejm.gov.pl/.
8. European Commission. 2002. “Commission Decision 2002/657/EC Implementing Council Directive 96/23/EC Concerning the Performance of Analytical Methods and the Interpretation of Results.” EUR-Lex. https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32002D0657.
9. World Anti-Doping Agency. 2026. “World Anti-Doping Code International Standard: Prohibited List 2026.” https://www.wada-ama.org/en/prohibited-list.
10. United States Pharmacopeial Convention. n.d. “Methylene Blue Injection, Veterinary.” USP-NF. https://doi.org/10.31003/uspnf_m3290_07_01.
11. European Medicines Agency. 2026. “Methylthioninium Chloride Proveblue — European Public Assessment Report.” https://www.ema.europa.eu/en/medicines/human/EPAR/methylthioninium-chloride-proveblue.

Structure and solid-state chemistry

12. Marr, H. E., J. M. Stewart, and M. F. Chiu. 1973. “The Crystal Structure of Methylene Blue Pentahydrate.” Acta Crystallographica Section B 29 (4): 847–853. https://doi.org/10.1107/s0567740873003432.
13. Sabirov, V. K., and M. X. Kadirova. 2026. “Synthesis, Crystal Structure, and π–π-Stacking Interactions in a Complex of Zinc(II) Chloride with Methylene Blue.” Journal of Structural Chemistry 67 (5): 1174–1185. https://doi.org/10.1134/s0022476626050197.

Dye composition, certification and nomenclature

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