Description
[7-(Dimethylamino)phenothiazin-3-ylidene]dimethylazanium chloride (common name: methylene blue, methylthioninium chloride), crystalline powder, 1000 mg pack. Material intended exclusively for laboratory and analytical in vitro research. This product is not intended for human or animal consumption; it is not a medicinal product, food supplement, foodstuff or cosmetic.
Three distinct entities that must not be confused
Methylene blue is a special case in this catalogue: the same molecule functions simultaneously as the active substance of registered medicinal products administered intravenously and as a common staining reagent and analytical standard. Separating the layers is just as important here as for substances with a clearly “pharmaceutical” lineage:
| Entity | What it is | What it concerns |
|---|---|---|
| Chemical substance methylene blue, CAS 61-73-4 |
A chemical concept — a phenothiazinium cation with chloride as the counter-ion. In itself it is neither a medicine nor a reagent; its status is conferred only by the form in which it has been manufactured and authorised. | chemistry, substance registers |
| Medicinal product e.g. solutions for injection based on methylthioninium chloride, registered under trade names such as Proveblue |
A pharmaceutical preparation: a strictly defined concentration of solution for intravenous administration, manufacture under GMP, registration dossier, safety monitoring. Used in the treatment of acquired methaemoglobinaemia and studied as an adjunctive drug in the therapy of Plasmodium falciparum infections. It is this entity that the clinical studies and toxicological reviews cited below concern. | clinical pharmacology, toxicology |
| Chemical reagent the material offered here |
Material for laboratory and analytical work — a dye, a spectrophotometric standard, a redox label. It does not have, and cannot have, any authorisation for use in humans or animals — it has no pharmaceutical form and no medicinal product documentation. | analytics, chemistry, reference standards |
The consequence is unambiguous. The research results referred to later on this page were obtained either with a registered medicinal product administered intravenously under medical supervision, or with the substance used under laboratory experimental conditions or in preclinical animal studies. None of these studies was conducted with an analytical reagent, and none of these results transfers to the material offered. The reagent is not a form in which the substance has ever been administered to humans, and it cannot be used in that way.
Reagent identification sheet
| Systematic name (IUPAC) | [7-(dimethylamino)phenothiazin-3-ylidene]dimethylazanium chloride |
|---|---|
| Common name | methylene blue |
| INN / pharmacopoeial name | methylthioninium chloride |
| CAS number | 61-73-4 |
| Molecular formula | C16H18ClN3S |
| Molar mass | 319.9 g·mol−1 |
| Monoisotopic mass | 319.0910 Da |
| InChIKey | CXKWCBBOMKCUKX-UHFFFAOYSA-M |
| SMILES | CN(C)C1=CC2=C(C=C1)N=C3C=CC(=[N+](C)C)C=C3S2.[Cl-] |
| PubChem CID | 6099 |
| ChEMBL | CHEMBL3942629 |
| Form | crystalline powder (basic dye); aqueous solution intensely blue |
| Purity | ≥ 99.5% |
| Intended use | research reagent — not for human or animal consumption |
Origins of the molecule: from aniline dye to the phenothiazine core
Methylene blue is not a product of medicinal chemistry — it is a by-product of the aniline dye industry of the second half of the nineteenth century, which only after the fact found applications in biology and pharmacology. This distinguishes it from most substances in this catalogue: its career began on the production floor of a chemical works, not in a pharmacological laboratory.
Born in a dye factory (1876)
The compound was synthesised by Heinrich Caro in 1876 at BASF — a fact documented in the historical paper by Krafts and co-workers [1]. Six years later Robert Koch used methylene blue in the discovery of the tubercle bacillus, and in 1880 Paul Ehrlich described the concept of “neutral” dyes — mixtures of an acidic and a basic dye for differentiating peripheral blood cells [1].
The dye that taught biology to see
In 1886 Bernthsen obtained from the decomposition of methylene blue a derivative named methylene azure. In 1891 Malachowski developed a method combining eosin with “ripened” methylene blue which for the first time revealed the nuclei of malaria parasites in a blood smear — Romanowsky reproduced this effect by an independent method. The following decades brought a series of refinements to the staining technique: Nocht (1899), Jenner (1899, methanol as fixing solvent), Wright (1902) and finally Giemsa (1902), who perfected the demethylation of the dye and added glycerol as a stabiliser. Malachowski–Wright–Giemsa staining remains to this day the worldwide standard for the microscopic diagnosis of malaria [1]. The context of this diagnostic revolution — dyes as a tool that laid the foundations of modern cell biology (Virchow, Papanicolaou) and of the neuron theory (Golgi, Cajal) — is described in Chast’s historical review [2].
The phenothiazine core: from methylene blue to chlorpromazine
Methylene blue is the first member of the family of phenothiazine dyes — a tricyclic system with nitrogen and sulfur atoms in the central ring. Attempts to modify this core in search of an antimalarial drug (a continuation of Ehrlich’s work) did not yield an effective antimalarial compound, but led organic chemistry to a new class of antihistamine and sedative drugs: promethazine, promazine and finally chlorpromazine — the first effective antipsychotic drug, as described by Frankenburg and Baldessarini [3]. The history of this line of development “from methylene blue via chlorpromazine to modern neuroleptics”, in the context of Ehrlich’s receptor concept, was traced by Kristiansen [4]. For the laboratory this has a classification consequence: methylene blue is sometimes cited as the parent compound of the entire phenothiazine family, not as a side branch of it.
Timeline
| 1876 | synthesis of the compound by Heinrich Caro at the BASF works [1] |
|---|---|
| 1880 | Ehrlich describes the concept of “neutral” dyes for differentiating blood cells [1] |
| 1882 | Koch uses methylene blue in the discovery of the tubercle bacillus [1] |
| 1886–1891 | Bernthsen obtains methylene azure; Malachowski and Romanowsky reveal malaria parasites in a blood smear [1] |
| 1899–1902 | Nocht, Jenner, Wright and Giemsa refine the staining technique — Malachowski–Wright–Giemsa staining is born [1] |
| 1st half of the twentieth century | modifications of the phenothiazine core in search of an antimalarial drug lead to promethazine, promazine and chlorpromazine [3][4] |
| twentieth century | methylene blue establishes its position as the standard antidote in acquired methaemoglobinaemia [6][7] |
| 2017 | ex vivo study of the activity of a modern methylthioninium chloride preparation against multidrug-resistant strains of Plasmodium falciparum — a possible return of the compound to the antimalarial role it held over a century ago [5] |
| 2012–2023 | development of LC-MS/MS and UPLC-MS/MS methods for determining residues of the compound in food of aquatic origin, and of electrochemical methods using it as a redox label [10][9][14] |
Four research streams that shaped knowledge of this molecule
Unlike substances with a single research centre, the literature on methylene blue divides into four largely independent streams. For a laboratory using this reagent this matters in practice — when looking for a specific piece of information, it helps to know in which stream to look for it.
- The tradition of staining and microscopic diagnostics. The oldest stream, going back to Ehrlich and Koch, leading via Giemsa to today’s standard of malaria diagnostics [1][2].
- Clinical toxicology and antidotes. A stream focused on the mechanism of methaemoglobin reduction and the use of the compound as an antidote in emergency medicine [6][7].
- Analytical chemistry and food safety. The quantitatively youngest stream: HPLC and LC-MS/MS methods for determining residues of the compound in fish and seafood tissues, where it was at times used as an antifungal agent in aquaculture and is now subject to residue control [9][10], as well as bioanalytical methods for determining the compound and its metabolite in plasma [11].
- Electrochemistry and DNA biosensing. Use of the methylene blue redox couple as a label in electrochemical DNA sensors [14][15].
Chemistry: charge, redox couple and the chlorine signature
Why the compound is a salt rather than a neutral molecule
Unlike modafinil, which is a neutral organic molecule with a stereogenic centre on the sulfur atom, methylene blue is a permanent cation — the positive charge is delocalised over the phenothiazine system, and chloride acts as the counter-ion. This has a concrete computational consequence: no XLogP partition coefficient value appears in the source data, because classical logP is defined for neutral molecules, and a permanently charged cation does not lend itself to such a description in a simple way. The missing value is not a gap in the data but a consequence of the structure of the compound.
The redox couple: methylene blue / leucomethylene blue
The most important chemical feature of this molecule is its reversible redox couple: the oxidised cationic form (blue) and the reduced leucomethylene blue form (colourless). This same redox property underlies three seemingly separate applications: as an antidote it reduces the ferric iron of methaemoglobin back to the ferrous iron of haemoglobin [7]; as an electrochemical label it generates a measurable redox signal on intercalation with DNA in electrochemical sensors [14][15]; and as a colorimetric reagent its decolourisation in a redox reaction is used for the spectrophotometric determination of other analytes. One chemical property, three independent laboratory applications.
The chlorine signature in mass spectrometry
The chloride counter-ion gives a recognisable isotope pattern in the mass spectrum: 35Cl and 37Cl occur in a ratio of about 3:1, giving a pair of peaks 2 Da apart in exactly that ratio. This signature is weaker than the bromine isotope pair in bromantane (about 1:1 there), but still useful when confirming the identity of the chloride salt in LC-MS analysis — especially since the phenothiazinium cation itself contains no atoms with an unusual isotope distribution. The iodination reaction of methylene blue and its course, studied by LC-MS with ionspray ionisation, were described by Raffaelli and co-workers [12].
Physicochemical characteristics
| Parameter | Value | What follows from it |
|---|---|---|
| Charge | cation (+1), counter-ion Cl− | no classical XLogP value; the compound is readily soluble in water despite its aromatic core |
| Topological polar surface area | 43.9 Å2 | moderate — lower than in modafinil (71.7 Å2), consistent with the absence of N–H and O–H groups |
| Hydrogen bond donors | 0 | no N–H/O–H groups — all nitrogen atoms are tertiary or iminium |
| Hydrogen bond acceptors | 4 | three nitrogen atoms and the thiazine sulfur atom |
| Rotatable bonds | 1 | highly rigid system — the planar tricyclic core limits conformational freedom |
| Heavy atoms | 21 | reference parameter for checking the integrity of the MS spectrum |
| Melting point (with decomposition) | approx. 105°C | heating above this temperature leads to decomposition, not to clean melting |
| Solubility | very good in water and methanol; weaker in non-polar solvents | the reverse of the typical organic reagents in this catalogue — a consequence of the permanent charge |
Why one molecule has more than a dozen names
The divergence in nomenclature here stems from a long industrial and pharmacopoeial history, not from the number of isomers. All of the following terms denote the same compound with CAS number 61-73-4:
- Methylene blue (błękit metylenowy) — the name most common in commercial and laboratory circulation;
- Methylthioninium chloride (chlorek metylotioniniowy) — the INN, used in pharmacopoeial documentation;
- Basic Blue 9 — the name from the Colour Index of dyes, reflecting its industrial origin;
- Bleu de méthylène — the French form, found in the classical historical literature cited above;
- Chromosmon, Swiss Blue, Methylenium ceruleum — older pharmacopoeial and trade synonyms, found in historical registers and identifier databases.
When searching the literature it is worth combining the common name with the INN — part of the toxicological and pharmacological literature indexes entries only under “methylthioninium chloride”.
Laboratory applications of the reagent
- reference material for confirming identity and purity by HPLC-UV [8];
- standard in LC-MS/MS and UPLC-MS/MS methods for determining residues in food and environmental matrices [9][10], and in bioanalytical methodology — for the simultaneous determination of the compound and its metabolite in biological matrices [11];
- derivatising reagent in the determination of trace levels of sulfide in aqueous samples by LC-MS [13];
- redox label in electrochemical DNA sensors and intercalation studies [14][15];
- object of aggregation spectroscopy studies — the dye forms dimers and higher aggregates in aqueous solution in a manner dependent on concentration and chemical environment [16];
- material for studies of the chemical reactivity of the phenothiazine core, including the iodination reaction studied by LC-MS [12].
Storage, handling and safety at work
Store in the original, tightly closed packaging, in a dry and cool place, protected from light, away from food and feed and out of the reach of children. Work only under laboratory conditions, using personal protective equipment: gloves, safety glasses and laboratory clothing; weigh the powder under conditions that minimise dusting — the intense colour makes surface contamination easy to detect, but does not exempt from the dust-limiting regime. Avoid inhaling dust and contact with skin and eyes. Waste handling — in accordance with the regulations applicable to chemical waste at the place where the research is carried out.
| Classification element | Value |
|---|---|
| Signal word | Danger |
| Pictograms | GHS05 (corrosive to eyes), GHS07 (irritant/harmful), GHS08 (health hazard) |
| H302 | Harmful if swallowed |
| H318 | Causes serious eye damage |
| H361 | Suspected of damaging fertility or the unborn child |
| H370 / H372 | Causes damage to organs / through prolonged or repeated exposure |
| Precautionary statements | P264, P203 (codes as per PubChem source data) |
Regulatory status
Methylthioninium chloride is the active substance of registered medicinal products administered intravenously under medical supervision, including preparations used in the treatment of acquired methaemoglobinaemia. Trade in medicinal preparations is governed by separate pharmaceutical legislation, and this offer does not concern it. The substance is also subject to the GHS hazard classification described above (signal word Danger) — this applies to the chemical compound as such, regardless of its commercial form. The material offered is a chemical reagent, not for consumption, and has no authorisation for any use in humans or animals. The purchaser is responsible for ensuring that the intended use complies with the law in force in the country of destination.
Frequently Asked Questions
How does methylene blue as a reagent differ from a preparation for injection?
A preparation for injection is a medicinal product: a strictly defined solution concentration, manufacture under GMP, a registration dossier and administration only under medical supervision. The reagent offered here is material for laboratory work, with no pharmaceutical form and no authorisation for use in humans or animals.
Who synthesised methylene blue, and when?
The compound was synthesised by Heinrich Caro in 1876 at the BASF works, as an aniline dye. Biological and therapeutic applications came later, thanks to Ehrlich, Koch and subsequent researchers developing staining techniques [1].
What are the chemical formula and molar mass of methylene blue?
C16H18ClN3S; molar mass 319.9 g·mol−1, monoisotopic mass 319.0910 Da. CAS number 61-73-4, InChIKey CXKWCBBOMKCUKX-UHFFFAOYSA-M.
Why is there no XLogP value in the identification sheet?
Because methylene blue is a permanent cation, not a neutral molecule. The classical octanol/water partition coefficient is defined for neutral compounds — for a salt with a delocalised positive charge there is no simple equivalent, which is why the data source leaves this field empty.
Is the reagent suitable for applications other than laboratory ones?
No. The material is intended exclusively for laboratory and analytical research. It is not intended for human or animal consumption; it is not a medicine, food supplement, food or cosmetic and may not be used for medical, diagnostic or consumption purposes.
How can methylene blue be recognised in mass spectrometry?
By the mass of the cation and by the isotope signature of the chloride counter-ion: 35Cl and 37Cl in a ratio of about 3:1 give a pair of peaks 2 Da apart. This signature is clearly weaker than the bromine isotope pair, but recognisable in the spectrum of the chloride salt.
What is a methylene blue standard used for in an analytical laboratory?
For confirming identity and purity by HPLC-UV, as a standard in LC-MS/MS for the determination of residues in food matrices, as a derivatising reagent in the determination of sulfides and as a redox label in electrochemical DNA sensors.
How should methylene blue be stored as a laboratory reagent?
In the original, tightly closed packaging, in a dry and cool place, protected from light, away from food and feed and out of the reach of children.
What should methylene blue be dissolved in to prepare a stock solution?
The compound is very readily soluble in water and methanol — the reverse of most neutral organic reagents in this catalogue, which is a direct consequence of its ionic character.
What is the hazard classification of methylene blue?
Signal word Danger, pictograms GHS05, GHS07, GHS08; hazard statements H302, H318, H361, H370 and H372. The full summary is given in the table in the section on storage and safety at work above.
Is a safety data sheet supplied with methylene blue?
We make the safety data sheet available on request to recipients conducting research or analytical activities.
Is methylene blue legal in Poland?
As a chemical compound it is not a controlled substance. As an ingredient of registered medicinal products it is subject to separate pharmaceutical legislation, to which this offer does not relate. The material offered is a chemical reagent without authorisation for use in humans or animals. The purchaser is responsible for ensuring that the intended use complies with the law of the country of destination.
Why is methylene blue sometimes called the parent compound of antipsychotic drugs?
Because it is the first link in the phenothiazine family: attempts to turn it into an antimalarial drug led to promethazine, promazine and chlorpromazine — the first effective drug in the treatment of psychoses [3][4]. This is a historical and chemical fact, not information about the action of the reagent offered.
References
The entries come from the PubMed database and concern the history of the compound, the clinical pharmacology of registered preparations and analytical chemistry. Entries [1]–[4] are historical papers on the synthesis of the dye and its role in the development of diagnostics and pharmacology; [5]–[7] concern the registered medicinal product, not the analytical reagent; [8]–[16] are methodological papers in the fields of analytical chemistry, electrochemistry and spectroscopy. None of them describes the use of the material offered or constitutes an encouragement to any use of it outside the laboratory. This is the complete list of literature used in preparing this page as of 2026-09-06.
- Krafts K, Hempelmann E, Oleksyn BJ (2011). “In search of the malarial parasite: biographical sketches of the blood stain contributors.” Parasitol Res. PMID 21660627. doi:10.1007/s00436-011-2475-4.
- Chast F (2005). “Dyes, indispensable tools for the 19th century’s biologic and therapeutic revolution.” Rev Hist Pharm (Paris). PMID 17152864.
- Frankenburg FR, Baldessarini RJ (2008). “Neurosyphilis, malaria, and the discovery of antipsychotic agents.” Harv Rev Psychiatry. PMID 18803105. doi:10.1080/10673220802432350.
- Kristiansen JE (1989). “Dyes, antipsychotic drugs, and antimicrobial activity. Fragments of a development, with special reference to the influence of Paul Ehrlich.” Dan Med Bull. PMID 2651032.
- Fall B et al. (2017). “Ex vivo activity of Proveblue, a methylene blue, against field isolates of Plasmodium falciparum in Dakar, Senegal from 2013-2015.” Int J Antimicrob Agents. PMID 28689867. doi:10.1016/j.ijantimicag.2017.03.019.
- Clifton J 2nd, Leikin JB (2003). “Methylene blue.” Am J Ther. PMID 12845393.
- Pushparajah Mak RS, Liebelt EL (2021). “Methylene Blue: An Antidote for Methemoglobinemia and Beyond.” Pediatr Emerg Care. PMID 34463662. doi:10.1097/PEC.0000000000002526.
- de Carvalho Matos L, Calixto LA, Junqueira Garcia MT (2021). “Developing an analytical method by HPLC for simultaneous quantification of methylene blue and metformin applied to in vitro skin permeation and retention studies.” Biomed Chromatogr. PMID 33675106. doi:10.1002/bmc.5112.
- Mitrowska K, Kijewska L, Giannetti L, Neri B (2023). “A simple and sensitive method for the determination of methylene blue and its analogues in fish muscle using UPLC-MS/MS.” Food Addit Contam Part A. PMID 37011028. doi:10.1080/19440049.2023.2195948.
- Xu YJ et al. (2012). “Simultaneous determination of malachite green, crystal violet, methylene blue and the metabolite residues in aquatic products by ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry.” J Chromatogr Sci. PMID 22542891. doi:10.1093/chromsci/bms054.
- Kim SJ, Ha DJ, Koo TS (2014). “Simultaneous quantification of methylene blue and its major metabolite, azure B, in plasma by LC-MS/MS and its application for a pharmacokinetic study.” Biomed Chromatogr. PMID 24122875. doi:10.1002/bmc.3063.
- Raffaelli A, Pucci S, Desideri I et al. (1999). “Investigation on the iodination reaction of methylene blue by liquid chromatography-mass spectrometry with ionspray ionisation.” J Chromatogr A. PMID 10497928.
- Small JM, Hintelmann H (2007). “Methylene blue derivatization then LC-MS analysis for measurement of trace levels of sulfide in aquatic samples.” Anal Bioanal Chem. PMID 17323053.
- Pheeney CG, Barton JK (2012). “DNA electrochemistry with tethered methylene blue.” Langmuir. PMID 22512327. doi:10.1021/la300566x.
- Lin X, Ni Y, Kokot S (2015). “An electrochemical DNA-sensor developed with the use of methylene blue as a redox indicator for the detection of DNA damage induced by endocrine-disrupting compounds.” Anal Chim Acta. PMID 25813025. doi:10.1016/j.aca.2015.02.050.
- Jalili-Jahani N, Abdollahi H, Rabbani F (2023). “Spectrophotometric thermodynamic analysis of methylene blue aggregation in water by chemometrics; what comes in the presence of different cyclodextrins?” Spectrochim Acta A Mol Biomol Spectrosc. PMID 37506453. doi:10.1016/j.saa.2023.123126.
Identification data and computed descriptors from PubChem (CID 6099) and the ChEMBL register.
Related reagents in our catalogue
- Modafinil (CAS 68693-11-8) — structural contrast: there a neutral molecule with a stereogenic centre on sulfur, here a permanent cation without chirality
- Bromantane (CAS 87913-26-6) — isotopic contrast: bromine gives a pattern of about 1:1, chlorine (the counter-ion of methylene blue) gives a pattern of about 3:1
Statement of intended use
The material offered is a chemical reagent intended exclusively for research, analytical and laboratory purposes. It is not intended for human or animal consumption. It is not a medicinal product, food supplement, foodstuff, medical device or cosmetic. It may not be used for medical, diagnostic, therapeutic, prophylactic or consumption purposes, administered to humans or animals, applied to the skin, or added to food, beverages or feed. Sale exclusively to recipients conducting research, scientific or analytical activities, with laboratory facilities and the knowledge required for the safe handling of chemical reagents. The purchaser bears sole responsibility for the lawful and safe use of the reagent and for compliance with the regulations in force in the country of destination.
