Methylene Blue

1 Identity

Small-molecule phenothiazinium dye salt - not a peptide and not peptide-adjacent by structure; carried in this catalog because it circulates in the same channel. It is simultaneously an established biological stain, a redox indicator, an aquaculture and aquarium chemical, an analytical reagent, and the active ingredient of an FDA-approved drug product. Basic Blue 9, Color Index 52015, Swiss blue, CAS 61-73-4 for the anhydrous chloride and CAS 7220-79-3 for the trihydrate (both commonly published, not independently verified here). Not synonyms and each a separate article with its own formula and mass: the demethylated homologs Azure B, Azure A, Azure C and thionine.

Sequence

Not applicable - not a peptide, no residues, no sequence, no stereocenter. The structural statement that replaces a sequence: a tricyclic phenothiazine core, oxidized at the ring sulfur to the thiazinium cation, bearing dimethylamino groups at the 3 and 7 positions; the molecule is a permanent cation and is supplied as a chloride salt. There is no chiral center, so no chiral method applies. The molecule carries four N-methyl groups, and they are what the purity specification is built around: successive loss of methyl groups gives Azure B (three methyls), Azure A (two), Azure C (one) and thionine (none), which is why demethylated homolog content, and Azure B content in particular, is the defining purity attribute of this article rather than a generic related-substances line.

Molecular formula

C16H18ClN3S (anhydrous chloride). Trihydrate C16H24ClN3O3S, which is the form most bulk material actually is. Cation alone C16H18N3S+. Reduced (leuco) form C16H19N3S. Homologs: Azure B chloride C15H16ClN3S; Azure A chloride C14H14ClN3S; Azure C chloride C13H12ClN3S; thionine chloride C12H10ClN3S.

Average mass

319.85 Da anhydrous chloride (C16H18ClN3S); the formula on IUPAC 2021 abridged conventional atomic weights gives 319.851. Chloride is 11.1 percent of that by mass. Trihydrate C16H24ClN3O3S = 373.90, of which 14.5 percent is water of crystallization - SO A gross weight of trihydrate quoted against the anhydrous molecular weight overstates the dye content by about 17 percent, and that is before any adsorbed moisture. Homologs, each from its stated formula: Azure B chloride 305.82 (-14.03, one methylene); Azure A chloride 291.80; Azure C chloride 277.77; thionine chloride 263.74. Reduced leuco form 285.41.

Monoisotopic mass

319.09100 Da neutral anhydrous chloride ion pair. The species actually observed in positive-mode electrospray is the intact cation at m/z 284.1216 with no adduct chemistry required, because the molecule is already charged - the chloride is not in that ion, and 'mass confirms 284' is therefore equally true of the anhydrous salt, the trihydrate and the free cation from any source. Homolog cations, for a method that must separate them: Azure B 270.1060; Azure A 256.0903; Azure C 242.0747; thionine 228.0590 - each successive demethylation is exactly -14.0157 Da monoisotopic. Trihydrate 373.12269 as the neutral formula unit. All values follow from the stated formulas.

Salt / variant note

The four methyl groups are the article, and each one lost is A different saleable dye. (1) Methylthioninium chloride anhydrous, C16H18ClN3S, 319.85 / cation 284.1216. (2) trihydrate C16H24ClN3O3S, 373.90 - the form most bulk material actually is, 14.5 percent water of crystallization, and a gross weight quoted against the anhydrous molecular weight overstates dye content by about 17 percent. This is the commonest quantitative error on this compound and it is arithmetic, not a difference in the material. (3) AZURE B chloride, C15H16ClN3S, 305.82 / cation 270.1060 - exactly one methylene lighter, the principal related dye, present in essentially all commercial material at some level, and the attribute that pharmacopeial grade limits and dye grade does not. (4) Azure A chloride, C14H14ClN3S, 291.80 / 256.0903. (5) Azure C chloride, C13H12ClN3S, 277.77 / 242.0747. (6) Thionine chloride, C12H10ClN3S, 263.74 / 228.0590. Each successive demethylation is exactly -14.0157 Da monoisotopic, so the homolog series is a regular ladder and a mass spectrometer that reports only the parent has not looked for it. (7) LEUCOMETHYLENE blue, the reduced form C16H19N3S, 285.41 - COLORLESS. The two formulas differ by one hydrogen - C16H18N3S+ at 284.40 against C16H19N3S at 285.41 - so it is +1.01 Da from the cation, not +2 Da. It re-oxidizes in air, so a partially reduced lot changes both its assay and its appearance between the supplier's bench and the buyer's. (8) Methylene violet Bernthsen, an oxidative degradation product of the same core. (9) inorganic contaminant load, which is a variant in the only sense that matters commercially: dye-grade and technical-grade material is documented to carry heavy metals - arsenic, aluminum, cadmium, chromium, lead, mercury, zinc - at levels pharmacopeial grade limits, and no mass spectrum of the cation detects any of them.

2 Class & testing panel

Form

Single article

Testing panel

P6 — panel definition

3 Primary sources & evidence

The index reports the design and provenance of the literature, not a conclusion about effect.

Published literature exists and is very large and very old; the constraint on this record is not evidence. What exists, by document type. Chemistry and analysis: the compound was first synthesized in 1876 and has a continuous analytical literature since, including standard spectrophotometric and chromatographic methods and a well-documented demethylated-homolog impurity profile. Histology and microbiology: it is a foundational biological stain with more than a century of method literature and is a Biological Stain Commission certified dye, which is itself a documented standard rather than a marketing term. Regulatory documents: FDA-approved labeling exists for a methylene blue injection product (NDA 204630, approved 2016) together with its review record, and USP-NF publishes a monograph for the substance. Human interventional literature: randomized controlled trials and systematic reviews exist across several fields, and registered studies under the methylene blue and methylthioninium terms number in the hundreds on ClinicalTrials.gov. Analytical-survey literature: published surveys of non-pharmaceutical grade material report inorganic contaminant loads that pharmacopeial grade limits, and those surveys are the most directly relevant published work for a catalog record. Identifiers are not individually re-verified here; the point is the shape and size of the published literature, and no statement is made about what the compound does.

4 Storage & specification

Storage

Dark green to bronze crystalline solid with a metallic luster, in a screw-cap amber borosilicate or HDPE container with a PTFE-lined closure, non-sterile, with no sterility claim. Store at 15 to 30 degrees C tightly closed, protected from light, in a dry place; refrigeration is not required and is not specified, because the compound is thermally robust and the failure mode is chemical rather than thermal. Two controls are specific rather than general. First, light: the phenothiazinium chromophore is photochemically active and photodegradation drives both demethylation and oxidative products, so amber glass and an opaque secondary pack are part of the specification and not a precaution. Second, redox state: the dye is reduced to its colorless leuco form in the presence of reducing agents and re-oxidizes in air, so the container is kept closed, reducing agents are excluded from the weighing area, and any color change from dark green toward pale or colorless is treated as an out-of-specification event rather than as fading. The solid stains skin, glassware, benches and clothing indelibly; dedicated glassware and a dedicated weighing area are specified. Solutions are prepared fresh in a stated solvent, protected from light, and not stored for reuse.

Shelf life

Provisional retest interval 60 months from QA release for the solid at 15 to 30 degrees C, tightly closed and protected from light, stated as provisional pending the company's own stability data - a longer interval than any peptide in this catalog, because a phenothiazinium dye salt is genuinely more robust than a peptide, and the interval is set by the dye's own failure modes rather than by analogy. Protocol ICH Q1A(R2)-style: 12 months real time with pulls at 0, 3, 6, 9 and 12 months plus 6 months accelerated at 40 degrees C / 75 percent relative humidity, with appearance, Karl Fischer water, dye content by absorbance, and the individually named homologs Azure B, Azure A, Azure C and thionine reported at every pull. A photostability arm under ICH Q1B is mandatory and not optional on this article, because light is its principal degradation vector and a dark-storage stability study would demonstrate nothing about the way it actually fails. Retest date printed on every container, first-expiry-first-out enforced against that date.