Quality analysis of medical drugs from the vitamins group of heterocyclic row
Derivatives of pyrimidine and thiazole
Molecule of pyrimidine-thiazol vitamins (vitamins B1 – thiamines) consists of two heterocycles – pyrimidine (A) and thiazole (B) connected by a methylene group:
In medical practice used thiamine hydrobromide, thiamine hydrochloride, diphosphate ester of thiamine hydrochloride (cocarboxylase).
Preparations of vitamin В1
Thiamine hydrobromide (Thiamini hydrobromidum) (SPhU)
3-[(4-Amino-2-methyl–pyrimidine-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazole bromide hydrobromide
Thiamine hydrochloride (Thiamini hydrochloridum) (SPhU)
3-[(4-Amino-2-methyl–pyrimidine-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazole chloride hydrochloride
Vitamin В1 is the first vitamin, which pioneered the opening of vitaminology science .
For the first time it was identified from the rice bran (Funk in 1912 y.). This substance in small doses cured polyneuritis of pigeons and because its structure contains sulfur atom, called thiamine.
Most contained in the cereal (grain) plants, yeast, egg yolks, ox liver. Extraction from natural sources is difficult with low output (from 1 t of yeast – 0,25 g of vitamin В1).
For the medical purpose it is received synthetically, at the first pyrimidine and thiazole compounds should be extracted separately, then they are condensed .
Thiamine belongs to the aminoalcohol of heterocyclic series (availability of carbonyl and amino-groups ).
Properties of thiamines
Thiamine hydrobromide
The crystalline powder of white or white with yellowish tint with a specific smell. Easily soluble in water, little soluble in 96% alcohol, practically insoluble in ether.
Thiamine hydrochloride
The crystalline powder of white or almost white or colorless crystals. Easily soluble in water, soluble in glycerin, slightly soluble in 96% alcohol. Hygroscopic.
This compounds are stable only in acidic environment In the neutral and alkaline environments decompose with rupture of thiazole cycle and formation of mercapto group.
Identification of thiamines
Thiamine hydrobromide
1. IR–spectrophotometry.
2. Thiochrome test.
3. Reactions on bromides.
Thiamine hydrochloride
1. IR–spectrophotometry.
2. Thiochrome test.
3. Reactions on chlorides.
Unpharmacopoeial reactions
а) With solutions of FeCl3 and K3[Fe(CN)6] a blue coloration of Berlin blue formed.
b) At the melting with crystalline NaOH or metallic sodium sulfide ions formed, which can be detected by the reaction with sodium nitroprusside (red-purple coloration).
c) Thiamine can be quantitatively precipitated from the solutions by the usage of some general alkaloid reagents (Dragendorff, Bertran, Sheibler, picric acid, p–aminoacetophenone, Reinecke salt).
Thiochrome test
Thiamine is oxidized by the alkali solution of potassium ferricyanide with the thiochrome formation (bright yellow substance), which is extracted by isoamyl or butyl alcohol – alcohol layer gives blue fluorescence in UV light (λ = 365 nm):
Quantitative determination of thiamine hydrochloride and hydrobromide
Thiamine h/br. (SPhU). Acidimetry in non-aqueous environment in the presence of mercury (II) acetate . Equivalent point is determined by potentiometric method:
Е = ½ М.m.(C12H18Br2N4OS)
2. Thiamine h/chl. (SPhU). Alkalimetry in the mixture of 0,01 М chloride acid solution and 96 % alcohol. Titrant – 0,1 М NaOH solution. Equivalent point is determined by potentiometric method. Volume of titrant between two potential jumps on the titration curve is taken in the calculation. Е = ½ М.m.
3. Thiamine h/chl.(SPhU addition 1). Acidimetry in non-aqueous environment in the mixture of formic acid and acetic anhydride. Equivalent point is determined by potentiometric method. Е = ½ М.m.
4. Gravimetry after precipitation of the drug by silicon tungsten acid. Sediment composition: SiO2•12WO3• 2C12H17BrN4OS або SiO2•12WO3• 2C12H17ClN4OS.
5. Alkalimetry, direct titration, indicator – bromothymol blue orphenolphthalein. Е=М.m.
6. Argentometry by Fayans method, indicator – bromophenol blue. Е=1/2 М.m.
4. Fluorimetry (by the intensity of thiochrome fluorescence).
5. Argentometry after the neutralization of the substance solution with alkali. Analytical weight of thiamine bromide is titrated by sodium hydroxide solution to the blue-green color, indicator – bromothymol blue .
Then solution acidified by nitrate acid, add indicator – iron (III) ammonium sulfate and 0,1 ml of 0,1М ammonium thiocyanate solution – red color appears due to the formation of iron (III) thiocyanate:
Reaction mixture is titrated by 0,1 М silver nitrate solution:
In the equivalent point excess of silver nitrate reacts with iron (III) thiocyanate – solution becomes colorless:
The content of thiamine bromide is calculated by the difference of volumes of silver nitrate, ammonium thiocyanate and sodium hydroxide. Е = М.m.
Storage of the thiamine preparations
In airtight containers, that protect from the action of light. Not allowed contact with metals to prevent the reduction to dihydrothiamine:
Application of the thiamine preparations
В1 plays an important role in human body. Is part of coenzyme – cocarboxylase. While lack of vitamin B1 carbohydrate metabolism disorder arising, lactic and pyruvic acid accumulate in the tissues, and therefore may be neuritis and disorders of the heart. Thiamine affects on protein and lipid metabolism, participates in the regulation of water exchange.
Daily demand 2 mg (1 vg of the drug – 330 IU).
Avitaminosis –a beriberi disease, hypovitaminosis – disorders of the nervous system.
Used for treatment of hypo- and avitaminosis of this vitamin and diseases associated with dysfunction of the nervous system.
Thiamine hydrobromide due to its higher molecular weight are taken in large doses (1 mg of thiamine hydrochloride is responsible by the activity to 1,29 mg of thiamine hydrobromide).
Issue: Thiamine hydrochloride – dragee by 0,002 g, amp. 5%-1,0 №10; thiamine hydrobromide – dragee by 0,0026 g №50.
Included in multivitamin preparations and vitamin of B group (neurobex, neuron, neurovitan, neurorubine, multi-tabs B-complex).
It is not recommended to enter at once parenterally with B6 and B1 vitamins (delay esterification of thiamine by phosphate acid) and B12 (enhances allergic effect of thiamine) and mixed in one syringe vitamin B1 with penicillin or streptomitsin (decomposition of antibiotics) and nicotinic acid (thiamine decomposition).
Cocarboxylase
(Cocarboxylasum) CCB
Hydrochloride of thiamine diphosphate ester
CCB – freeze (lyophylization) dry white porous mass with low specific smell and bitter-sour taste. Easily soluble in water, sparingly soluble in ethanol.
Decomposes at the heated above 35°С.
Store in a dark place at a temperature less than 5°С.
CCB –coenzyme of enzymes involved in carbohydrate metabolism. In the compound with proteins and magnesium ions is a part of the carboxylase enzyme that catalyzes carboxylation and decarboxylation of a-ketoacids.
In connection with the lack of CCB at the beri-beri diseases a-ketoacids (especially pyruvic and СН3СОСООН acids) accumulate in the tissues.
Assign it foe the treatment of arrhythmia, coronary circulation failure and other cardiovascular diseases, diabetes and various pathological processes associated with deterioration of carbohydrate metabolism.
Enter by i/m, i/v, s/c way by 0,05-0,1 g 1 time per day.
Issue: sealed ampoules by 0,05 g of sterile powder, which before usage (ex tempore) is dissolved in 0,5% novocaine solution or 0,9% sodium chloride solution.
Derivatives of pterin
Pterin vitamins, which include folic acid (vitamin Вс), contained in green leaves of spinach, parsley, lettuce, in legume and cereal crops (wheat, rye, corn), as well as in yeast, liver.
The basis of chemical structure of the data vitamins is a pteridine kernel which is a condensed system of pyrimidine (A) and pyrazine (B) cycles. Derivative of pteridine 2-amino-4-oxypteridine is called pterin.
Folic acid
(Acidum folicum), vitamin Вс (SPhU)
(2S)-2-[[4-[[(2-Amino-4-oxy–pteridine-6-yl)methyl]amino]benzoyl]amino]pentadioc acid
Name of the vitamin is derived from the Latin word folium – leaf. Folic acid is iature both in free state and as polyglutaminates in which there are additional glutamic acid residues.
Extraction of folic acid
(method of А.V. Truchanova and B.А. Kirsanova)
Condensed equimolar quantity of 2,5,6-tri-amino-4-oxypirimidine hydrochloride, p–aminobenzoyl–L(+)-glutamic acid and 2,3-dibromopropionic aldehyde:
Storage of folic acid
In airtight containers, in the place protected from light.
Properties of folic acid
Crystalline yellowish or orange powder. Practically not soluble in water and most organic solvents (different from other vitamins). Soluble in dilute acids (the presence of nitrogen atoms of amine nature) and alkali (availability of free carboxyl groups). Decomposing under the action of light, hygroscopic.
Folic acid molecule consists of three main parts: 2-amino-4-oxypteridine (pterin), p–aminobenzoic acid and associated with this acid residue of glutamic acid.
Folic acid is an amphoteric compounds: basic properties is caused by the nitrogen atoms of the pteridine molecule, acidic – the carboxyl groups and hydroxyl group in position 4.
Identification of folic acid
By the physico-chemical constants: specific rotation, by liquid chromatography method, TLC.
Unpharmacopeial reactions:
a) at the adding of potassium permanganate to the drug solution in hydrochloric acid (with following selection of its excess by hydrogen peroxide) p-aminobenzoylglutamic and pterine-6-carboxylic (pteric) acid are produced. The last has a much stronger blue fluorescence than folic acid, and its use for fluorimetric quantification of the drug.
b) determination of UV–spectral characteristics of folic acid.
c) due to its acidic properties folic acid with salts of heavy metals forms insoluble colored complexes; with CuSO4 – green precipitate, with Co(NO3)3 – dark yellow precipitate, with FeCl3 – red-yellow precipitate. The general formula of these salts:
Assay of folic acid
1. Liquid chromatography.
2. Polarographic method. Is used the folic acid ability to recover in the medium of sodium carbonate to 7,8-dyhydrofolic acid which is easily oxidized to folic even by atmospheric oxygen, polarographic cell is constantly blowing by nitrogen.
3. Photocolorimetric method. In the core of method is folic acid oxidation by potassium permanganate with the following formation of p-aminobenzoylglutamic acid, its diazotation and azoconnection with N-(1-naphthyl)-ethylenediamine dyhydrochloride. As a result formed colored in purple color azo dye, the intensity of its coloration in a solution is determined by photocolorimeter at l=550 nm.
Application of folic acid
Folic acid is involved in the process of blood formation, so it is called antianemic factor. Together with vitamin В12 it stimulates erythropoiesis, is involved in the synthesis of amino–acids (methionine, serine, etc.), nucleic acids, purine and pyrimidine metabolism and in choline metabolism. In the body is redused to tetrahydrofolic acid – coenzyme that participates in various metabolic processes.
Daily demand of the folic acid for a healthy human is 0,2 – 1 mg. Its deficiency leads to anemia.
To prevent a lack of folic acid at the unbalanced or poor nutrition take 20-50 mcg inside daily, during pregnancy – 400 micrograms per day and lactation – 300 mcg.
Issue: tabl. by 0,001 g and 0,005 g № 50.
Used for increased erythropoiesis, in certain types of anemia, including anemia and leukopenia caused by drugs and ionizing radiation, chronic gastroenteritis and tuberculosis of intestine.
Derivatives of isoalloxazine
Isoalloxazine –heterocyclic system, which consists of condensed pyrazine, pyrimidine and benzene cycles, that is derived from benzopteridine. Pyrimidine nucleus of isoalloxazine has character of lactam cycle because it contains two keto-groups :
Benzopteridine Isoalloxazine
Detection of vitamin properties of flavin associated with the presence in the molecule extremely labile group with two conjugated double bonds in isoalloxazine nucleus. This group of atoms causes the redox properties of riboflavin.
Riboflavin (Riboflavinum)
vitamin В2 (SPhU)
7,8-Dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxy–pentyl] benzo[g]pteridine-2,4(ЗH,10H)-dion,
or 6,7-dimethyl-9-(1’–D-рibityl)-isoalloxazine
At the first isoalloxazine vitamins (vitamin B2) was allocated from whey (because it is also called “laktoflavin). Name of vitamin B2 “Riboflavin” comes from the fact that it has a residue of polyhydric alcohols of the ribose original, and its solutions are yellow (Latin flavus – yellow).
Riboflavin is very common in plant and in animal products: contained in the milk serum, liver, kidney, brewer’s and baker yeast; in grains – millet, barley; in vegetables – spinach and tomatoes.
For extraction of 1 g of the drug should be processed 5,4 tons of whey, so now vitamin B2 is synthesizeed from D-ribose, о-4-xylidine and barbituric acid.
Characters. Yellow or orange-yellow crystalline powder. Very few soluble in water, hardly soluble in 96% alcohol, ether, acetone, chloroform. Easily soluble in chloride (forms salts) and acetic acid. Solutions decompose under the influence of light, especially in the presence of alkali. Detects polymorphism.
Identification of riboflavin
1. By the physico-chemical constants: the specific rotation, infrared spectroscopy, TLC.
2. Solution of the substance in the light that passes through it, has a pale greenish-yellow color, and in reflected light – an intense yellow-green fluorescence, which disappears at the adding of mineral acid or alkali (in acidic medium on the light forms riboflavin forms lumichrome (6,7-dymethylalloxazine) – a colorless substance that breaks down into ethanol and chloroform with the appearance of blue fluorescence, in alkaline medium on light riboflavin forms lumiflavin (6,7,9-trimethylalloxazine), which solution has the same color and fluorescence as riboflavin, but soluble in chloroform).
3. At the riboflavin reduction by sodium hydrosulfite and fluorescence and coloration of aqueous solution disappear (a colorless compound formed – leukoriboflavin):
3. Unpharmacopoeial reaction:
a) at the adding of sulfate concentrated acid to riboflavin red coloration appears, which becomes yellow after the adding of water.
b) with a silver nitrate solution orange-red coloration forms (presence of imide group).
Test on purity
Determined lyumiflavin that formed as a result of changes in the chemical structure of matter under the influence of light and alkaline environment.
Determination of the lyumiflavin impurity based on its solubility in chloroform (in chloroform riboflavin is insoluble) – color of the filtrate should not exceed the standard(SPhU).
Determination of the lyumiflavin impurity conducted by thin layer chromatography(SPhU, additions).
Storage
In airtight containers, in the place protected from light.
Assay of riboflavin
1. UV spectrophotometry (SPhU). Determination of the optical density is conducted at the weakened transmitting light in aqueous solution, acidified by acetic acid at l=444 nm. Riboflavin content is calculated using the specific absorption rate, which equals 328.
2. Photocolorimetry.
3. Fluorimetry.
4. Alkalimetry by substituent after the interaction with silver nitrate solution. Е = М.m.
5. Periodate oxidation (Malaprad’s reaction). Methodic is based on the ribityl fragment of riboflavin molecule oxidation with formation of formic acid.
Formic acid, which eliminated as a result of reaction, is titrated by alkalimetry method :
НСООН + NaOH → НСООNa + Н2О
According to another method after the action of periodate to the solution is added sodium iodide and sulphate acid:
5 NaI + NaIO3 + 3 H2SO4 → 3 I2 + 3 Na2SO4 + 3 H2O
Iodine that allocated by the reaction is titrated by the standard solution of sodium thiosulfate.
6. Estarification byconcentrated sulfate acid. Due to the hydroxyl groups are formed by mono-, di-, tri- and tetrasulfoxyl esters are formed. Then by potentiometric titration with KOH solution determine an excess of sulfate acid. The reaction proceeds in a stechiometric ratio 1 : 3.
Application
Plays an important physiological role, because is a part of the oxidativ cell enzymes as riboflavin-phosphate ester. Daily demand – 2 mg. Hyporiboflavinose is characterized by deterioration of appetite, weight loss, sores in the corners of mouth; ariboflavinose – conjunctivitis, clouding of the cornea and lens its..
Vitamin В2 is used orally in doses of 5-10 mg per day (depending on the degree of disease) at ariboflavinose, conjunctivitis, irytah, keratitis, radiation sickness and others.
Issue: tabl. by 0,002 g; 0,005 g; 0,01 g; 0,01% eye drops; is a part of riboflavin mononucleotide (riboflavin-5′-monophosphate sodium) – amp. 1%-1,0 №10.
Corrine derivatives
Corrine vitamins (group В12) were found iatural products of the animal origin, mainly in the internal organs. So, the richest source of vitamin B12 are fish entrails, high content of this vitamin is in the whale liver and the highest content meat is in the meat of molluscs. In human and animal B12 is synthesized by microflora of intestine and accumulates in the liver, kidney, intestine walls.
Vitamin В12 (cyanocobalamine) is extracted from the waste of the production of streptomycin and chlortetracycline antibiotics. Another source – sewage (waste products of actinomycetes, bacteria, blue-green algae), from this sourse vitamin B12 is allocated by adsorption or extraction.
Cyanocobalamin (Cyanocobalami–num) vitamin В12
(SPhU)
α-(5,6-dimethyl–benzimidazole-1-yl)cobamide cyanide
Corrine
Cyanocobalamin molecule consists of two main parts. First – nucleotide containing 5,6-dimethylbenzimidazole associated with D-ribofuranose, which, in turn, linked by etheric bound with phosphate acid. Nucleotide connected with macrocyclic corrine system (second part) by peptide bond. Nitrogen atom of 5,6-dimethylbenzimidazole in position 3 bounded by coordination bond with cobalt atom. Cobalt forms a chelate compound with cyano-group and with nitrogen atoms and of hydrogenated pyrrole cycles of corrine system.
Positive charge of cobalt ion is neutralized by negatively charged phosphate acid anion. Therefore, cyanocobalamin is not only a chelating compound, but an internal salt.
Properties of cyanocobalamin
Crystalline powder with dark red color, odorless, hygroscopic. Sparingly soluble in water, soluble in 95 % alcohol, practically insoluble in ether, chloroform, acetone. The anhydrous substance is very hygroscopic
At the heating above 300°С decomposes. Stable at рН=4-6. Easily decomposed in an alkaline environment.
СN-group in the molecule of vitamin В12 can be replace by the ОН-group (oxycobalamin) or NO2 (nitrocobalamin). Both products are converting to a group of vitamin В12.
At the heating of cyanocobalamin solution with HCl ammonium chloride, 5,6-dimethylbenzimidazole, aminoisopropanol, cobalt chloride, cyanic acid are formed.
Antianemic action associated with the presence of 5,6-dimethylbenzimidazole in the molecule structure.
Identification of cyanocobalamine
Determination of UV-spectral characteristics.
TLC.
Cobalt ions are determined after the mineralization by alloying with potassium hydrosulfate and interaction with sodium 1-nitroso-2-naphthol-3,6-disulfonate – a red color of internally complex salts of cobalt appeares.
Cobalt ion can be detected after evaporation and roasting of 0,25 mg of cyanocobalamin with 10 mg of potassium sulfate and 2 drops of 15 % sulfate acid. Saturated solution of ammonium thiocyanate in acetone is added to the residue; blue-green coloration appears :
Со2+ + 2 NH4SCN → Со(SCN)2 + 2 NH4+
Detection of cyano-group. Analytical weight of cyanocobalamin is heated in the test-tube with oxalic acid, under the action of this acid cyanic acid eliminates, which is detected with filtration paper soaked by the solution of benzidine and copper (ІІ) acetate, as a result blue complex compound formes.
Assay
The method of UV-spectrophotometry.
Storage
In airtight containers, in the place protected from light.
Application of cyanocobalamin
§ Vitamin В12 is a growth factor necessary for normal hematopoiesis and maturation of erythrocytes. He participates in the formation of choline, methionine, nucleic acids, shows a positive effect on liver function and nervous system.
§ Daily demand for healthy person – 10-20 mcg.v This quantity of vitamin is not produced in the intestine and should be complemented by the products of animal origin
§ Apply at the malignant anemia, various forms of anemia, liver disease, nervous system, skin diseases and others.
§ Issue: solution for injection 200 mcg (0,02%) or 500 mcg (0,05%) 1,0 №10.
Multivitamin drugs
Iature vitamins commonly found in the form of various combinations. Herbal products often contain vitamins of group B, vitamin C and others. The combination of vitamins is also found in animal products.
. In many cases, vitamins mutually reinforce physiological effects that they have. For example, the effect of vitamin P on vascular permeability amplified by ascorbic acid, mutually amplifying effect on hematopoiesis of folic acid and cyanocobalamin.
In some cases, toxicity of vitamins is reduced at their combined application. For example, vitamin D toxicity is reduceing by vitamin A. For example, vitamin D toxicity is reduceing by vitamin A. Vitamins can also detect antagonistic effect (nicotinic acid slows the lipotropic action of choline).
Vitamins participating actively in various biochemical processes, if they are associated and show a stronger complex biological action that is served for the combined usage in both cases for the preventive and therapeutic purposes.
Multivitamin drugs have different compositions (some vitamins alone or in combination with microelements) and issued in the form of various dosage forms: tablets, “effervescent” tablets (upsavit, supradine) tablets for chewing (multi-tabs, jungle), beans (sob) , syrups (multivitamol), gels (Kinder–biovital), solution for injection (nerviplex (В1, В6, В12)).
Ukrainian multivitamin drugs: revit (А, В1, В2, С), hexavit (revit+ В6, РР), undevit (hexavit+ Е, Р, Вс, В3), decamevit (А, В1, В2, В6, В12, РР, С, Е, Р, Вс, methionine), complevit (В1, В2, В6, В12, РР, С, Е, Вс), quadevit (decamevit + В3, glutamic acid, fitine, KCl. CuSO4), vitam (В1, В2, В6, РР, В3,mefenamic, Fe, Zn, Mn, Cu, Co, Cr).
Multivitamin preparations of the foreign origin: Vitrum vitamins (Kids, Junior, Beauty, Prenatal, Energy, Cardio, etc..) Centrum, multi-tabsy, Duovit, Pikovit, Jungle, Unicap, Pregnavit, Oligovit, Theravit, Supradyn, Kinder-biovital and others.
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Thiamine Hydrochloride
General Notices
(Ph Eur monograph 0303)
C12H17ClN4OS,HClıı337.3ıı 67-03-8
Action and use
Vitamin B1.
Preparations
Thiamine Injection
Thiamine Tablets
Vitamins B and C Injection
Ph Eur
DEFINITION
3-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium chloride
hydrochloride.
Content
98.5 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance
White or almost white, crystalline powder or colourless crystals.
Solubility
Freely soluble in water, soluble in glycerol, slightly soluble in alcohol.
IDENTIFICATION
First identificationı A, C.
Second identificationı B, C.
ıA. Infrared absorption spectrophotometry (2.2.24) .
Comparisonı thiamine hydrochloride CRS .
ıB. Dissolve about 20 mg in 10 ml of water R, add 1 ml of dilute acetic acid R and 1.6 ml of
1 M sodium hydroxide , heat on a water-bath for 30 min and allow to cool. Add 5 ml of dilute
sodium hydroxide solution R , 10 ml of potassium ferricyanide solution R and 10 ml of
butanol R and shake vigorously for 2 min. The upper alcoholic layer shows an intense lightblue
fluorescence, especially in ultraviolet light at 365 nm. Repeat the test using 0.9 ml of 1
M sodium hydroxide and 0.2 g of sodium sulphite R instead of 1.6 ml of 1 M sodium
hydroxide . Practically no fluorescence is seen.
ıC. It gives reaction (a) of chlorides (2.3.1) .
TESTS
Solution S
Dissolve 2.5 g in distilled water R and dilute to 25 ml with the same solvent.
Appearance of solution
The solution is clear (2.2.1) and not more intensely coloured than reference solution Y7 or
GY7 (2.2.2, Method II).
Dilute 2.5 ml of solution S to 5 ml with water R .
pH (2.2.3)
2.7 to 3.3.
Dilute 2.5 ml of solution S to 10 ml with water R .
Related substances
Liquid chromatography (2.2.29) .
Solution Aı Add 5 volumes of glacial acetic acid R to 95 volumes of water R and mix.
Test solutionı Dissolve 0.35 g of the substance to be examined in 15.0 ml of solution A and
dilute to 100.0 ml with water R .
Reference solution (a)ı Dissolve 5 mg of the substance to be examined and 5 mg of
thiamine impurity E CRS in 4 ml of solution A and dilute to 25.0 ml with water R . Dilute 5.0
ml of the solution to 25.0 ml with water R .
Reference solution (b)ı Dilute 1.0 ml of the test solution to 50.0 ml with water R . Dilute 5.0
ml of this solution to 25.0 ml with water R .
Column: ı
ı— size: l = 0.25 m, Ø = 4.0 mm;
ı— stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R (5
μm) with a specific surface area of 350 m2/g and a pore size of 10 nm;
ı— temperature: 45 °C.
Mobile phase:
ı— mobile phase A: 3.764 g/l solution of sodium hexanesulphonate R adjusted to pH 3.1
with phosphoric acid R;
ı— mobile phase B: methanol R2 ,
Flow rateı 1.0 ml/min.
Detectionı Spectrophotometer at 248 nm.
Injectionı 25 μl.
Relative retentionı With reference to thiamine (retention time = about 30 min): impurity A =
about 0.3; impurity B = about 0.9; impurity C = about 1.2.
System suitabilityı Reference solution (a):
ı— resolution: minimum 1.6 between the peaks due to impurity E and to thiamine.
Limits:
ı— any impurity: not more than the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.4 per cent);
ı— total: not more than 2.5 times the area of the principal peak in the chromatogram
obtained with reference solution (b) (1.0 per cent);
ı— disregard limit: 0.125 times the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.05 per cent).
Sulphates (2.4.13)
Maximum 300 ppm.
5 ml of solution S diluted to 15 ml with distilled water R complies with the limit test for
sulphates.
Heavy metals (2.4.8)
Maximum 20 ppm.
12 ml of solution S complies with limit test A. Prepare the standard using lead standard
solution (2 ppm Pb) R.
Water (2.5.12)
Maximum 5.0 per cent, determined on 0.40 g.
Sulphated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.110 g in 5 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R .
Titrate immediately with 0.1 M perchloric acid , determining the end-point potentiometrically
(2.2.20) and carrying out the titration within 2 min. Carry out a blank titration.
1 ml of 0.1 M perchloric acid is equivalent to 16.86 mg of C12H18Cl2N4OS.
STORAGE
In a non-metallic container, protected from light.
IMPURITIES
Specified impuritiesı A, B, C.
Other detectable impuritiesı (The following substances would, if present at a sufficient level,
be detected by one or other of the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or by the general monograph
Substances for pharmaceutical use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. Control of impurities in substances
for pharmaceutical use): D, E, F, G, H.
ıA. R1 = CH3, R2 = O-SO3-: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-5-[2-
(sulphonatooxy)ethyl]thiazolium (thiamine sulphate ester),
ıB. R1 = H, R2 = OH: 3-[(4-aminopyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium
(desmethylthiamine),
ıC. R1 = CH3, R2 = Cl: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-chloroethyl)-4-
methylthiazolium (chlorothiamine),
ıF. R1 = C2H5, R2 = OH: 3-[(4-amino-2-ethylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-
methylthiazolium (ethylthiamine),
ıG. R1 = CH3, R2 = O-CO-CH3: 5-[2-(acetyloxy)ethyl]-3-[(4-amino-2-methylpyrimidin-5-yl)
methyl]-4-methylthiazolium (acetylthiamine),
ıD. X = O: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazol-2
(3H)-one (oxothiamine),
ıE. X = S: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazol-2
(3H)-thione (thioxothiamine),
ıH. (3RS)-3-[[[(4-amino-2-methylpyrimidin-5-yl)methyl]thiocarbamoyl]sulphanyl]-4-oxopentyl
acetate (ketodithiocarbamate).
Ph Eur
Riboflavin
General Notices
(Ph Eur monograph 0292)
C17H20N4O6
ıı376.4ıı83-88-5
Action and use
Vitamin B2.
Ph Eur
DEFINITION
7,8-Dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4(3H,10H)-dione.
This monograph applies to riboflavin produced by fermentation.
Content
97.0 per cent to 103.0 per cent (dried substance).
CHARACTERS
Appearance
Yellow or orange-yellow, crystalline powder.
Solubility
Very slightly soluble in water, practically insoluble in ethanol (96 per cent).
Solutions deteriorate on exposure to light, especially in the presence of alkali.
It shows polymorphism (5.9).
IDENTIFICATION
ıA. Specific optical rotation (see Tests).
ıB. Thin-layer chromatography (2.2.27).
Test solutionıSuspend 25 mg of the substance to be examined in 10 ml of water R, shake for
5 min and filter the suspension to remove the undissolved material.
Reference solutionıSuspend 25 mg of riboflavin CRS in 10 ml of water R, shake for 5 min
and filter the suspension to remove the undissolved material.
PlateıTLC silica gel plate R (2-10 μm).
Mobile phaseıwater R.
ApplicationıAs follows, drying in a current of cold air after each individual application:
ı
ı— 1st application: 2 μl of methylene chloride R then 2 μl of the test solution;
ı— 2nd application: 2 μl of methylene chloride R then 2 μl of the reference solution.
DevelopmentıOver a path of 6 cm.
DryingıIn a current of cold air.
DetectionıExamine in ultraviolet light at 365 nm.
ResultsıThe principal spot in the chromatogram obtained with the test solution is similar in
position and size to the principal spot in the chromatogram obtained with the reference
solution.
ıC. Dissolve about 1 mg in 100 ml of water R. The solution has, by transmitted light, a pale
greenish-yellow colour, and, by reflected light, an intense yellowish-green fluorescence
which disappears on the addition of mineral acids or alkalis.
TESTS
Specific optical rotation (2.2.7)
– 115 to – 135 (dried substance).
Dissolve 50.0 mg in 0.05 M sodium hydroxide free from carbonate and dilute to 10.0 ml with
the same alkaline solution. Measure the optical rotation within 30 min of dissolution.
Absorbance (2.2.25)
Test solutionıDilute the final solution prepared for the assay with an equal volume of water R.
Absorption maximaıAt 223 nm, 267 nm, 373 nm and 444 nm.
Absorbance ratios: ı
ı— A373/A267 = 0.31 to 0.33;
ı— A444/A267 = 0.36 to 0.39.
Related substances
Liquid chromatography (2.2.29). Prepare the solutions immediately before use and protect
from light.
Solution Aı13.6 g/l solution of sodium acetate R.
Test solutionıWith the aid of ultrasound, dissolve 0.120 g of the substance to be examined in
10 ml of 0.1 M sodium hydroxide and dilute to 100 ml with solution A.
Reference solution (a)ıDilute 1.0 ml of the test solution to 10.0 ml with solution A. Dilute 1.0
ml of this solution to 100.0 ml with solution A.
Reference solution (b)ıWith the aid of ultrasound, dissolve the contents of a vial of riboflavin
for peak identification CRS (containing impurities C and D) in 1.0 ml of a mixture of 1 volume
of mobile phase B and 9 volumes of mobile phase A.
Reference solution (c)ıIn order to prepare in situ impurities A and B, dissolve 10 mg of the
substance to be examined in 1 ml of 0.5 M sodium hydroxide. Expose to daylight for 1.5 h.
Add 0.5 ml of acetic acid R and dilute to 100 ml with water R.
Column:ı
ı— size: l = 0.25 m, Ø = 4.6 mm;
ı— stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 μm).
Mobile phase:
ı— mobile phase A: phosphoric acid R, water R (1:1000 V/V);
ı— mobile phase B: acetonitrile R;
Flow rateı1.0 ml/min.
DetectionıSpectrophotometer at 267 nm.
Injectionı10 μl.
Identification of impuritiesıUse the chromatogram supplied with riboflavin for peak
identification CRS and the chromatogram obtained with reference solution (b) to identify the
peaks due to impurities C and D.
Relative retentionıWith reference to riboflavin (retention time = about 16 min): impurity C =
about 0.2; impurity D = about 0.5; impurity A = about 1.4; impurity B = about 1.9.
System suitability:
ı— resolution: minimum 5 between the peaks due to impurities A and B in the chromatogram
obtained with reference solution (c);
ı— the chromatogram obtained with reference solution (b) is similar to the chromatogram
supplied with riboflavin for peak identification CRS.
Limits:
ı— correction factors: for the calculation of content, multiply the peak areas of the following
impurities by the corresponding correction factor: impurity A = 0.7; impurity B = 1.4; impurity
C = 2.3; impurity D = 1.4;
ı— impurity A: not more than 0.25 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.025 per cent);
ı— impurities B, C, D: for each impurity, not more than twice the area of the principal peak in
the chromatogram obtained with reference solution (a) (0.2 per cent);
ı— unspecified impurities: for each impurity, not more than the area of the principal peak in
the chromatogram obtained with reference solution (a) (0.10 per cent);
ı— total: not more than 5 times the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.5 per cent);
ı— disregard limit for peaks other than those due to impurity A: 0.5 times the area of the
principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).
Loss on drying (2.2.32)
Maximum 1.5 per cent, determined on 1.000 g by drying in an oven at 105 °C.
Sulphated ash (2.4.14)
Maximum 0.1 per cent, determined on the residue obtained in the test for loss on drying.
ASSAY
Carry out the assay protected from light.
In a brown-glass 500 ml volumetric flask, suspend 65.0 mg in 5 ml of water R ensuring that it
is completely wetted and dissolve in 5 ml of dilute sodium hydroxide solution R. As soon as
dissolution is complete, add 100 ml of water R and 2.5 ml of glacial acetic acid R and dilute to
500.0 ml with water R. Place 20.0 ml of this solution in a 200 ml brown-glass volumetric flask,
add 3.5 ml of a 14 g/l solution of sodium acetate R and dilute to 200.0 ml with water R.
Measure the absorbance (2.2.25) at the absorption maximum at 444 nm.
Calculate the content of C17H20N4O6 taking the specific absorbance to be 328.
STORAGE
In an airtight container , protected from light.
IMPURITIES
Specified impuritiesıA, B, C, D.
ıA. 7,8,10-trimethylbenzo[g]pteridine-2,4(3H,10H)-dione (lumiflavine),
ıB. 7,8-dimethylbenzo[g]pteridine-2,4(1H,3H)-dione,
ıC. 6,7-dimethyl-8-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]pteridine-2,4(3H,8H)-dione,
ıD. 8-(hydroxymethyl)-7-methyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-
2,4(3H,10H)-dione.
Ph Eur
Folic Acid
General Notices
(Ph Eur monograph 0067)
C19H19N7O6
ıı441.4ıı59-30-3
Action and use
Vitamin B component.
Preparations
Folic Acid Tablets
Ferrous Fumarate and Folic Acid Tablets
Ph Eur
DEFINITION
(2S)-2-[[4-[[(2-Amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino]benzoyl]amino]pentanedioic
acid.
Content
96.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance
Yellowish or orange, crystalline powder.
Solubility
Practically insoluble in water and in most organic solvents. It dissolves in dilute acids and in
alkaline solutions.
IDENTIFICATION
First identificationıA, B.
Second identificationıA, C.
ıA. Specific optical rotation (2.2.7): + 18 to + 22 (anhydrous substance).
Dissolve 0.25 g in 0.1 M sodium hydroxide and dilute to 25.0 ml with the same solvent.
ıB. Examine the chromatograms obtained in the assay.
ResultsıThe principal peak in the chromatogram obtained with the test solution is similar in
retention time to the principal peak in the chromatogram obtained with reference solution (a).
ıC. Thin-layer chromatography (2.2.27).
Test solutionıDissolve 50 mg of the substance to be examined in a mixture of 2 volumes of
concentrated ammonia R and 9 volumes of methanol R and dilute to 100 ml with the same
mixture of solvents.
Reference solutionıDissolve 50 mg of folic acid CRS in a mixture of 2 volumes of
concentrated ammonia R and 9 volumes of methanol R and dilute to 100 ml with the same
mixture of solvents.
PlateıTLC silica gel G plate R.
Mobile phaseıconcentrated ammonia R, propanol R, ethanol (96 per cent) R (20:20:60 V/V/
V).
Applicationı2 μl.
DevelopmentıOver 3/4 of the plate.
DryingıIn air.
DetectionıExamine in ultraviolet light at 365 nm.
ResultsıThe principal spot in the chromatogram obtained with the test solution is similar in
position, fluorescence and size to the principal spot in the chromatogram obtained with the
reference solution.
TESTS
Related substances
Liquid chromatography (2.2.29).
Test solutionıDissolve 0.100 g of the substance to be examined in 5 ml of a 28.6 g/l solution
of sodium carbonate R and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (a)ıDissolve 0.100 g of folic acid CRS in 5 ml of a 28.6 g/l solution of
sodium carbonate R and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (b)ıTo 20 mg of pteroic acid R, add 5 ml of a 28.6 g/l solution of sodium
carbonate R, dilute to 100.0 ml with the mobile phase and mix until completely dissolved. Mix
1.0 ml of this solution with 1.0 ml of reference solution (a) and dilute to 100.0 ml with the
mobile phase.
Reference solution (c)ıDilute 2.0 ml of the test solution to 20.0 ml with the mobile phase.
Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase.
Reference solution (d)ıDissolve 10.0 mg of N-(4-aminobenzoyl)-L-glutamic acid R in 1 ml of a
28.6 g/l solution of sodium carbonate R and dilute to 100.0 ml with the mobile phase. Dilute
1.0 ml of this solution to 100.0 ml with the mobile phase.
Reference solution (e)ıTo 12.0 mg of pteroic acid R , add 1 ml of a 28.6 g/l solution of
sodium carbonate R, dilute to 100.0 ml with the mobile phase and mix until completely
dissolved. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase.
Column:ı
ı— size: l = 0.25 m, Ø = 4.0 mm;
ı— stationary phase: spherical octylsilyl silica gel for chromatography R (5 μm) with a carbon
loading of 12.5 per cent, a specific surface of 350 m2/g and a pore size of 10 nm.
Mobile phaseıMix 12 volumes of methanol R and 88 volumes of a solution containing 11.16
g/l of potassium dihydrogen phosphate R and 5.50 g/l of dipotassium hydrogen phosphate R.
Flow rateı0.6 ml/min.
DetectionıSpectrophotometer at 280 nm.
Injectionı5 μl of the test solution and reference solutions (b), (c) (d) and (e).
Run timeı3 times the retention time of folic acid.
Relative retentionıWith reference to folic acid (retention time = about 8.5 min): impurity A =
about 0.5; impurity B = about 0.6; impurity C = about 0.9; impurity E = about 1.27; impurity D =
about 1.33; impurity F = about 2.2.
System suitabilityıReference solution (b):
ı— resolution: minimum 4.0 between the peaks due to folic acid and impurity D.
Limits:
ı— impurity A: not more than the area of the principal peak in the chromatogram obtained
with reference solution (d) (0.5 per cent);
ı— impurity D: not more than the area of the principal peak in the chromatogram obtained
with reference solution (e) (0.6 per cent);
ı— any other impurity: not more than the area of the principal peak in the chromatogram
obtained with reference solution (c) (0.5 per cent);
ı— Total of other impurities: not more than twice the area of the principal peak in the
chromatogram obtained with reference solution (c) (1.0 per cent);
ı— disregard limit: 0.1 times the area of the principal peak in the chromatogram obtained
with reference solution (c) (0.05 per cent).
Water (2.5.12)
5.0 per cent to 8.5 per cent, determined on 0.150 g.
Sulphated ash (2.4.14)
Maximum 0.2 per cent, determined on 1.0 g.
ASSAY
Liquid chromatography (2.2.29) as described in the test for related substances with the
following modification.
InjectionıTest solution and reference solution (a).
STORAGE
Protected from light.
IMPURITIES
Specified impuritiesıA, B, C, D, E, F.
ıA. (2S)-2-[(4-aminobenzoyl)amino]pentanedioic acid (N-(4-aminobenzoyl)-L-glutamic acid),
ıB. 2,5,6-triaminopyrimidin-4(1H)-one,
ıC. (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-7-yl)methyl]amino]benzoyl]amino]
pentanedioic acid (isofolic acid),
ıD. 4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino]benzoic acid (pteroic acid),
ıE. (2S)-2-[[4-[bis[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino]benzoyl]amino]
pentanedioic acid (6-pterinylfolic acid),
ıF. 2-amino-7-(chloromethyl)pteridin-4(1H)-one.
Ph Eur
Cyanocobalamin
General Notices
(Ph Eur monograph 0547)
C63H88CoN14O14Pıı1355ıı68-19-9
Action and use
Vitamin B12 analogue.
Preparation
Cyanocobalamin Tablets
Ph Eur
DEFINITION
-(5,6-Dimethylbenzimidazol-1-yl)cobamide cyanide.
Content
96.0 per cent to 102.0 per cent (dried substance).
This monograph applies to cyanocobalamin produced by fermentation.
CHARACTERS
Appearance
Dark red, crystalline powder or dark red crystals.
Solubility
Sparingly soluble in water and in ethanol (96 per cent), practically insoluble in acetone.
The anhydrous substance is very hygroscopic.
IDENTIFICATION
ıA. Ultraviolet and visible absorption spectrophotometry (2.2.25).
Test solutionıDissolve 2.5 mg in water R and dilute to 100.0 ml with the same solvent.
Spectral rangeı260-610 nm.
Absorption maximaıAt 278 nm, 361 nm and from 547 nm to 559 nm.
Absorbance ratioı
ı— A361 / A547-559 = 3.15 to 3.45;
ı— A361 / A278 = 1.70 to 1.90.
ıB. Thin-layer chromatography (2.2.27). Carry out the test protected from light.
Test solutionıDissolve 2 mg of the substance to be examined in 1 ml of a mixture of equal
volumes of ethanol (96 per cent) R and water R.
Reference solutionıDissolve 2 mg of cyanocobalamin CRS in 1 ml of a mixture of equal
volumes of ethanol (96 per cent) R and water R.
PlateıTLC silica gel G plate R.
Mobile phaseıdilute ammonia R1, methanol R, methylene chloride R (9:30:45 V/V/V).
Applicationı10 μl.
DevelopmentıIn an unsaturated tank, over a path of 12 cm.
DryingıIn air.
DetectionıExamine in daylight.
ResultsıThe principal spot in the chromatogram obtained with the test solution is similar in
position, colour and size to the principal spot in the chromatogram obtained with the reference
solution.
TESTS
Related substances
Liquid chromatography (2.2.29).
Test solutionıDissolve 10.0 mg of the substance to be examined in the mobile phase and
dilute to 10.0 ml with the mobile phase. Use within 1 h.
Reference solution (a)ıDilute 3.0 ml of the test solution to 100.0 ml with the mobile phase.
Use within 1 h.
Reference solution (b)ıDilute 5.0 ml of the test solution to 50.0 ml with the mobile phase.
Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Use within 1 h.
Reference solution (c)ıDissolve 25 mg of the substance to be examined in 10 ml of water R,
warming if necessary. Allow to cool and add 5 ml of a 1.0 g/l solution of chloramine R and 0.5
ml of 0.05 M hydrochloric acid , then dilute to 25 ml with water R. Shake and allow to stand for
5 min. Dilute 1 ml of this solution to 10 ml with the mobile phase and inject immediately.
Column:ı
ı— size: l = 0.25 m, Ø = 4 mm;
ı— stationary phase: octylsilyl silica gel for chromatography R (5 μm).
Mobile phaseıMix 26.5 volumes of methanol R and 73.5 volumes of a 10 g/l solution of
disodium hydrogen phosphate R adjusted to pH 3.5 with phosphoric acid R and use within 2
days.
Flow rateı0.8 ml/min.
DetectionıSpectrophotometer at 361 nm.
Injectionı20 μl.
Run timeı3 times the retention time of cyanocobalamin.
System suitability:
ı— the chromatogram obtained with reference solution (c) shows 2 principal peaks;
ı— resolution: minimum 2.5 between the 2 principal peaks in the chromatogram obtained
with reference solution (c);
ı— signal-to-noise ratio: minimum 5 for the principal peak in the chromatogram obtained with
reference solution (b).
Limits:
ı— total: not more than the area of the principal peak in the chromatogram obtained with
reference solution (a) (3 per cent);
ı— disregard limit: the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent).
Loss on drying (2.2.32)
Maximum 12.0 per cent, determined on 20.00 mg by drying in vacuo at 105 °C for 2 h.
ASSAY
Dissolve 25.00 mg in water R and dilute to 1000.0 ml with the same solvent. Measure the
absorbance (2.2.25) at the absorption maximum at 361 nm.
Calculate the content of C63H88CoN14O14P taking the specific absorbance to be 207.
STORAGE
In an airtight container , protected from light.
Ph Eur
