LectureLecture №28 №28Vitamins of the heterocyclic row: Vitamins of the heterocyclic row:

derivatives of pyrimidinethiazol, derivatives of pyrimidinethiazol, isoalloxazine, corrine. Properties, isoalloxazine, corrine. Properties,

quality requirements, storage, quality requirements, storage, application. Multivitamin drugsapplication. Multivitamin drugs ..

prepared ass. Medvid I.I.prepared ass. Medvid I.I.

Derivatives of pyrimidine and Derivatives of pyrimidine and thiazolethiazole

Molecule Molecule of of pyrimidinepyrimidine--thiazolthiazol vitamins (vitamins B1 - vitamins (vitamins B1 - thiaminethiaminess) consists of two heterocycles - pyrimidine (A) ) consists of two heterocycles - pyrimidine (A) and thiazole (B) connected by a methylene group:and thiazole (B) connected by a methylene group:

In medical practice used thiamine hydrobromide, thiamine In medical practice used thiamine hydrobromide, thiamine hydrochloride, diphosphate ester of thiamine hydrochloride, diphosphate ester of thiamine hydrochloride (cocarboxylase).hydrochloride (cocarboxylase).

Preparations of vitaminPreparations of vitamin ВВ11Thiamine hydrobromideThiamine hydrobromide ((Thiamini Thiamini hydrobromidumhydrobromidum)) ((SPhUSPhU))

3-[(4-3-[(4-AminoAmino-2--2-methylmethyl-- pyrimidinepyrimidine-5--5-ylyl))methylmethyl]-5-(2-]-5-(2-

hydroxyethylhydroxyethyl)-4-)-4-methylthiazolemethylthiazole bromidebromide hydrobromidehydrobromide

Thiamine hydrochlorideThiamine hydrochloride ((Thiamini Thiamini hydrochloridumhydrochloridum) () (SPhUSPhU))

3-[(4-3-[(4-AminoAmino-2--2-methylmethyl-- pyrimidinepyrimidine-5--5-ylyl))methylmethyl]-5-(2-]-5-(2-

hydroxyethylhydroxyethyl)-4-)-4-methylthiazolemethylthiazole chloridechloride hydrochloridehydrochloride

VitaminVitamin ВВ11 iis the first vitamin, which pioneered the s the first vitamin, which pioneered the opening of vitaminoloopening of vitaminology gy sciencescience . . For the first timeFor the first time it was it was identified identified from thefrom the rice bran rice bran (Funk in(Funk in 1912 1912 yy.). This substance in small doses cured .). This substance in small doses cured polyneuritis of pigeons and because its structure contains polyneuritis of pigeons and because its structure contains sulfur atom, called thiamine.sulfur atom, called thiamine.Most contained in the cereal (grain) plants, yeast, egg Most contained in the cereal (grain) plants, yeast, egg yolks, ox liver.yolks, ox liver. ExtractExtractionion from natural sources is difficult from natural sources is difficult withwith low output (from 1 t low output (from 1 t of of yeast - 0,25 g yeast - 0,25 g of of vitamin Вvitamin В11).).

For the For the medical medical purpose purpose it isit is receiv receiveded synthetically, synthetically, at the at the first first pyrimidine and thiazole compounds should be pyrimidine and thiazole compounds should be extracted extracted separately, then separately, then they are they are condensedcondensed ..Thiamine Thiamine belongs tobelongs to the aminoalcohol the aminoalcohol of of heterocyclic heterocyclic series (availability series (availability of carbonyl of carbonyl and aminand amino-o-groupsgroups ). ).

Properties of thiamineProperties of thiaminess ThiamineThiamine

hydrobromidehydrobromide The crystalline powder of The crystalline powder of

white or white with white or white with yellowish tint with a yellowish tint with a specific smellspecific smell. . Easily Easily soluble in water, little soluble in water, little soluble in 96% alcohol, soluble in 96% alcohol, practically insoluble in practically insoluble in ether.ether.

ThiamineThiamine hydrochloridehydrochloride

The crystalline powder of The crystalline powder of white or almost white or white or almost white or colorless crystals. Easily colorless crystals. Easily soluble in water, soluble in soluble in water, soluble in glycerin, slightly soluble in glycerin, slightly soluble in 96% alcohol. Hygroscopic.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 thiamineIdentification of thiaminessThiamineThiamine hydrobromidehydrobromide

1.1. IRIR--spectrophotometryspectrophotometry..

2.2. ThiochromeThiochrome testtest..

3.3. Reactions on bromidesReactions on bromides..

Thiamine hydrochlorideThiamine hydrochloride

1.1. IRIR--spectrophotometryspectrophotometry..

2.2. Thiochrome testThiochrome test..

3.3. Reactions on chloridesReactions on chlorides..

Unpharmacopoeial Unpharmacopoeial reactionsreactionsа) а) With solutions ofWith solutions of FeClFeCl33 and and KK33[Fe(CN)[Fe(CN)66] ] a blue coloration a blue coloration

of Berlin blue of Berlin blue formedformed..bb) ) At the meltingAt the melting with crystallinewith crystalline NaOHNaOH oror metallic sodium metallic sodium

sulfide ions formedsulfide ions formed, , which can be detected by the reaction which can be detected by the reaction with sodium nitroprusside with sodium nitroprusside ((red-purple colorationred-purple coloration).).

cc) ) Thiamine can be quantitatively precipitated from the Thiamine can be quantitatively precipitated from the solutionssolutions by the usage of some general alkaloid reagents by the usage of some general alkaloid reagents ((DragendorffDragendorff, , BertranBertran, , SheiblerSheibler, , picric acidpicric acid, , pp--aminoacetophenoneaminoacetophenone,, Reinecke salt Reinecke salt).).

ThiochromeThiochrome testtest ThiamineThiamine is oxidizedis oxidized by the alkali solution of potassiumby the alkali solution of potassium

ferricyanideferricyanide with the thiochrome formationwith the thiochrome formation ( (bright bright yellow substanceyellow substance), ), which is extracted by isoamyl or which is extracted by isoamyl or butyl alcohol - alcohol layer gives blue fluorescence in butyl alcohol - alcohol layer gives blue fluorescence in UV light UV light ((λλ = 365 nm = 365 nm):):

Quantitative determination of thiamine Quantitative determination of thiamine

hydrochloride and hydrobromidehydrochloride and hydrobromide 1.1. ThiamineThiamine hh//brbr. (. (SPhUSPhU).). Acidimetry in Acidimetry in non-aqueous non-aqueous

environmentenvironment in the presence of in the presence of mercury (II) acetatemercury (II) acetate . . EEquivalenquivalentt point point is determined byis determined by potentiometric potentiometric method:method:

Е = ½ М.Е = ½ М.mm.(.(CC1212HH1818BrBr22NN44OSOS))

2.2. ThiamineThiamine hh//chlchl. (. (SPhUSPhU). ). AlkalimetryAlkalimetry in the mixture ofin the mixture of 0,01 М 0,01 М chloride acid solutionchloride acid solution andand 96 % 96 % alcohol alcohol. . Titrant –Titrant – 0,1 М 0,1 М NaOH solutionNaOH solution. . EEquivalenquivalentt point point is determined byis determined by potentiometric method. potentiometric method. Volume of titrant between Volume of titrant between two two potential jumps on the titration curvepotential jumps on the titration curve is taken in the is taken in the calculationcalculation. Е = ½ М.. Е = ½ М.mm..

3.3. ThiamineThiamine hh//chlchl.(.(SPhUSPhU additionaddition 1). 1). Acidimetry inAcidimetry in non-aqueous non-aqueous environmentenvironment in the mixture of formic acid and acetic in the mixture of formic acid and acetic anhydrideanhydride. . EEquivalenquivalentt point point is determined byis determined by potentiometric potentiometric method. Е = ½ М.method. Е = ½ М.mm..

4.4. Gravimetry after precipitation of the drug Gravimetry after precipitation of the drug by silicon tungsten acidby silicon tungsten acid. . Sediment composition: Sediment composition: SiOSiO22••12WO12WO33•• 2C 2C1212HH1717BrNBrN44OSOS або або SiOSiO22••12WO12WO33•• 2C 2C1212HH1717ClNClN44OSOS..

5.5. AlkalimetryAlkalimetry,, direct titration direct titration, , indicator – bromothymolindicator – bromothymol blueblue ororphenolphthalein. Е=М.phenolphthalein. Е=М.mm..

6.6. Argentometry by Fayans methodArgentometry by Fayans method, , indicatorindicator – – bromophenol bluebromophenol blue. . Е=1/2 М.Е=1/2 М.mm. .

7.7. FluorimetryFluorimetry ( (by the intensity of thiochrome fluorescenceby the intensity of thiochrome fluorescence).).8.8. ArgentometryArgentometry after the neutralization of the substance solution after the neutralization of the substance solution

with alkaliwith alkali. . Analytical weight of thiamine bromideAnalytical weight of thiamine bromide is titrated by is titrated by sodium hydroxide solution tosodium hydroxide solution to the blue-green colorthe blue-green color, , indicator – indicator – bromothymolbromothymol blueblue . .

Then solution acidified by nitrate acidThen solution acidified by nitrate acid, , addadd indicatorindicator – – iron iron ((IIIIII) ) ammonium sulfate andammonium sulfate and 0,1 0,1 ml ofml of 0,1М 0,1М ammonium thiocyanate ammonium thiocyanate solution solution – – red color appears red color appears due to due to the the formation of iron formation of iron ((IIIIII) ) thiocyanatethiocyanate::

Reaction mixture is titrated byReaction mixture is titrated by 0,1 0,1 М М silver nitrate solutionsilver nitrate solution::

In the equivalent point excess of silver nitrate reacts with In the equivalent point excess of silver nitrate reacts with iron iron ((IIIIII) ) thiocyanatethiocyanate – – solution becomes colorlesssolution becomes colorless: :

The content of thiamine bromide is calculated by the The content of thiamine bromide is calculated by the difference of volumes ofdifference of volumes of silver nitratesilver nitrate, , ammonium ammonium thiocyanatethiocyanate and sodium hydroxideand sodium hydroxide. Е = М.. Е = М.mm..

Storage of the thiamine preparationsStorage of the thiamine preparations

InIn airtight containersairtight containers, , that protect from the action of lightthat protect from the action of light. . Not Not allowed contact with metals to prevent the reduction to allowed contact with metals to prevent the reduction to dihydrothiaminedihydrothiamine::

Application of the thiamine Application of the thiamine preparationspreparations

ВВ11 plays an important role in human body. Is part of plays an important role in human body. Is part of coenzymecoenzyme – – cocarboxylasecocarboxylase. While lack of vitamin B1 . While lack of vitamin B1 carbohydrate carbohydrate metabolismmetabolism disorder arising, lactic and disorder arising, lactic and pyruvic acid accumulate in the tissues, and therefore may pyruvic acid accumulate in the tissues, and therefore may bebe neuritis and disorders of the heart. neuritis and disorders of the heart. Thiamine aThiamine affects ffects on on protein and lipid metabolism, participates in the regulation protein and lipid metabolism, participates in the regulation of water exchange. of water exchange. Daily demand Daily demand 2 2 mgmg (1 (1 vgvg of the drugof the drug – 330 – 330 IUIU).).AvitaminosisAvitaminosis –a beriberi disease, hypovitaminosis - –a beriberi disease, hypovitaminosis - disorders of the nervous system.disorders of the nervous system.Used for treatment of hypo- and avitaminosis Used for treatment of hypo- and avitaminosis of of this this vitamin and diseases associated with dysfunction of the vitamin and diseases associated with dysfunction of the nervous system.nervous system.

Thiamine hydrobromideThiamine hydrobromide due to its higher molecular due to its higher molecular weight are taken in large doses (1 mg weight are taken in large doses (1 mg of of thiamine thiamine hydrochloride is responsible hydrochloride is responsible byby the activity the activity toto 1 1,,29 mg 29 mg of of thiamine hydrobromide). thiamine hydrobromide). Issue: Issue: Thiamine hydrochlorideThiamine hydrochloride – – dragee by dragee by 0,002 0,002 gg, , ampamp. 5%-1,0 №10; . 5%-1,0 №10; thiamine hydrobromidethiamine hydrobromide – – dragee bydragee by 0,0026 0,0026 gg №50. №50.Included in multivitamin preparations and vitamin Included in multivitamin preparations and vitamin of of B B group group (neurobe(neurobexx, neuron, , neuron, nneurovitan, neurorubine, eurovitan, neurorubine, multi-tabs B-complex). multi-tabs B-complex). It is not recommended to enter at once parenterally with It is not recommended to enter at once parenterally with B6 B6 and and B1 vitaminB1 vitamins s (delay esterification (delay esterification of of thiaminethiamine by by phosphate acid) and B12 (enhances allergic effectphosphate acid) and B12 (enhances allergic effect of of tthiamine) and mixed in one syringe vitamin B1 hiamine) and mixed in one syringe vitamin B1 with with penicillin or streptomitsin (decomposition of antibiotics) penicillin or streptomitsin (decomposition of antibiotics) and nicotinic acid (thiamine decomposition).and nicotinic acid (thiamine decomposition).

CocarboxylaseCocarboxylase((CocarboxylasumCocarboxylasum) ) CCBCCB

Hydrochloride of thiamine diphosphate esterHydrochloride of thiamine diphosphate ester

CCBCCB – – freeze freeze ((lyophylizationlyophylization)) dry white porous mass with dry white porous mass with low specific smell and bitter-sour tastelow specific smell and bitter-sour taste.. Easily soluble in Easily soluble in water, sparingly soluble in ethanolwater, sparingly soluble in ethanol..

Decomposes at the heated above Decomposes at the heated above 3535°°С.С.

Store in a dark place at a temperature less than Store in a dark place at a temperature less than 55°°С.С.

CCBCCB –coenzyme –coenzyme of of enzymes involved in carbohydrate enzymes involved in carbohydrate metabolism. In the compound metabolism. In the compound withwith proteins and magnesium proteins and magnesium ions isions is a a part of the carboxylase enzyme that catalyzes part of the carboxylase enzyme that catalyzes carboxylation andcarboxylation and decarboxylation decarboxylation of of a-a-ketoacidsketoacids..

In connection with the lack of CCB at the beri-beri diseases In connection with the lack of CCB at the beri-beri diseases a-a-ketoacids (especially pyruvic and ketoacids (especially pyruvic and СНСН33СОСООНСОСООН acids) acids) accumulate in the tissuesaccumulate in the tissues..Assign it Assign it foefoe the the treatment of treatment of arrhythmia, coronary circulation arrhythmia, coronary circulation failure and other cardiovascular diseases, diabetes and various failure and other cardiovascular diseases, diabetes and various pathological processes associated with deterioration of pathological processes associated with deterioration of carbohydrate metabolism.carbohydrate metabolism.Enter Enter by iby i/m/m, i/v, s/c way by, i/v, s/c way by 0,05-0,1 g 1 0,05-0,1 g 1 time time per day.per day.

IssueIssue:: sealed ampoules sealed ampoules by by 0,05 g 0,05 g of of sterile powder, which sterile powder, which before usagebefore usage (ex tempore) (ex tempore) is is dissolved in 0dissolved in 0,,5% novocaine 5% novocaine solution or 0,9% sodium chloride solution.solution or 0,9% sodium chloride solution.

Derivatives of Derivatives of pterinpterinPterPterinin vitamins, which include folic acid (vitamin Вс), vitamins, which include folic acid (vitamin Вс), contained in green leaves of spinach, parsley, lettuce, in contained in green leaves of spinach, parsley, lettuce, in legume and cereal crops legume and cereal crops ((wheat, rye, corn), as well as in wheat, rye, corn), as well as in yeast, liver.yeast, liver.

The basis of chemical structure The basis of chemical structure of the of the data vitamins is a data vitamins is a pteridine pteridine kernel which is kernel which is a a condensed systemcondensed system of of pyrimidine pyrimidine (A) and p(A) and pyrazineyrazine (B) cycles. (B) cycles. Derivative of pteridineDerivative of pteridine 2- 2-aminoamino-4--4-oxypteridineoxypteridine is called pterinis called pterin..

Folic acidFolic acid ((Acidum folicumAcidum folicum), ), vitaminvitamin В Вс с ((SPhUSPhU))

(2(2SS)-2-[[4-[[(2-)-2-[[4-[[(2-AminoAmino-4--4-oxyoxy--pteridinepteridine-6--6-ylyl))methylmethyl]]aminoamino]]benzoylbenzoyl]]aminoamino]]pentadiocpentadioc acidacid

Name of the vitamin is derived from the Latin word folium – leafName of the vitamin is derived from the Latin word folium – leaf.. Folic acid is in nature both in free state and as polyglutaminates Folic acid is in nature both in free state and as polyglutaminates in which there are additional glutamic acid residuesin which there are additional glutamic acid residues..

Extraction of folic acidExtraction of folic acid (method of(method of А. А.VV. . TruchanovaTruchanova andand BB.А. .А. KirsanovaKirsanova))

CondensedCondensed equimolar quantity ofequimolar quantity of 2,5,6- 2,5,6-tri-aminotri-amino-4--4-oxypirimidine hydrochlorideoxypirimidine hydrochloride, , pp--aminobenzoylaminobenzoyl--LL(+)-(+)-glutamic acidglutamic acid andand 2,3- 2,3-dibromopropionic aldehydedibromopropionic aldehyde::

Storage of folic acidStorage of folic acidInIn airtight containersairtight containers, , in the place protected from lightin the place protected from light..

Properties of folic acidProperties of folic acidCrystalline yellowishCrystalline yellowish or or orange powder. orange powder. Practically not soluble in water and most organic Practically not soluble in water and most organic solvents (different from other vitamins). Soluble in solvents (different from other vitamins). Soluble in dilute acids (the presence of nitrogen atoms of dilute acids (the presence of nitrogen atoms of amine nature) and alkali (availabilityamine nature) and alkali (availability of of free free carboxyl groups). Decomposing under the action carboxyl groups). Decomposing under the action of light, hygroscopic.of light, hygroscopic.Folic acid molecule consists of three main parts: 2-Folic acid molecule consists of three main parts: 2-aminoamino-4--4-oxypteridineoxypteridine ( (pterinpterin), ), pp--aminobenzoic aminobenzoic acidacid andand associated with this acid residue of associated with this acid residue of glutamic acidglutamic acid..Folic acid isFolic acid is an amphoteric an amphoteric compounds: basic compounds: basic properties properties is is caused by the nitrogen atoms of the caused by the nitrogen atoms of the pteridine pteridine molecule, molecule, acidicacidic - the carboxyl groups - the carboxyl groups and hydroxyl group in position 4.and hydroxyl group in position 4.

Identification of folic acidIdentification of folic acid

By theBy the physic physicoo-chemical constants: -chemical constants: sspecific rotationpecific rotation,, b byy liquid chromatographyliquid chromatography method method, TLC., TLC.

Unpharmacopeial reactionsUnpharmacopeial reactions: :

aa)) at the at the addadding of ing of potassium permanganate to the drug potassium permanganate to the drug solution in hydrochloric acid (solution in hydrochloric acid (with with following following selection selection of of its excess its excess by by hydrogen peroxide) hydrogen peroxide) p-aminobenzoylglutamic p-aminobenzoylglutamic and pterine-6-carboxylic (pteric) acid and pterine-6-carboxylic (pteric) acid are produced.are produced. The The last has a much stronger blue fluorescence than folic acid, last has a much stronger blue fluorescence than folic acid, and its use for fluorimetric quantification of the drugand its use for fluorimetric quantification of the drug..

bb)) determination ofdetermination of UVUV--spectral characteristics of folic acidspectral characteristics of folic acid..

cc)) due to its acidic properties folic acid with salts of heavy due to its acidic properties folic acid with salts of heavy metals forms insoluble colored complexes; with CuSOmetals forms insoluble colored complexes; with CuSO44 – –

green precipitategreen precipitate, , withwith Co(NOCo(NO33))33 – – dark yellow precipitate dark yellow precipitate, ,

withwith FeClFeCl33 – – red-yellow precipitatered-yellow precipitate. . The general formula of The general formula of

these saltsthese salts::

Assay of folic acid Assay of folic acid

1.1. LLiquid chromatography.iquid chromatography.

2.2. Polarographic method.Polarographic method. Is used the Is used the folic acid ability to folic acid ability to recover in the medium of sodium carbonate to 7,8-recover in the medium of sodium carbonate to 7,8-dyhdyhyydrofoldrofolic acidic acid which is easily oxidized to folic even which is easily oxidized to folic even by by atmospheric oxygen, polarographic cell is constantly atmospheric oxygen, polarographic cell is constantly blowblowing bying by nitrogen. nitrogen.

3.3. Photocolorimetric method.Photocolorimetric method. In In the core the core of method of method is folic is folic acid oxidation acid oxidation byby potassium perman potassium permanganate with the ganate with the following formation of pfollowing formation of p-aminobenzoy-aminobenzoylglutamiclglutamic aci acid,d, its its diazotationdiazotation and and azo azoconnection withconnection with N-(1-napht N-(1-naphthhyl)-yl)-ethylenediamine dyhethylenediamine dyhydrochlorideydrochloride. . As aAs a result formed result formed colored in purplecolored in purple color color azo dye, the intensity of azo dye, the intensity of its its colorationcoloration in a solution in a solution is is determinedetermined by photocolorimeterd by photocolorimeter at at =550 =550 nmnm..

Application of folic acidApplication of folic acidFolic acid is involved in the process Folic acid is involved in the process of blood formationof blood formation, so it is , so it is called antianemic factor. Together with vitamin Вcalled antianemic factor. Together with vitamin В1212 it stimulates it stimulates erythropoiesis, is involved in the synthesis of aminoerythropoiesis, is involved in the synthesis of amino--acids acids (methionine, serine, etc.), nucleic acids, purine and pyrimidine (methionine, serine, etc.), nucleic acids, purine and pyrimidine metabolism and metabolism and in in cholinecholine metabolism metabolism. In the body is re. In the body is reduseddused to to tetrahtetrahydrofolicydrofolic acid - coenzyme that participates in various acid - coenzyme that participates in various metabolic processes.metabolic processes.Daily demand of theDaily demand of the folic acidfolic acid for a healthy for a healthy humanhuman is 0,2 - 1 is 0,2 - 1 mgmg. . Its deficiency leads to anemia. Its deficiency leads to anemia. To prevent a lack of folic acid To prevent a lack of folic acid at the at the unbalanced or poor nutrition unbalanced or poor nutrition take take 20-50 20-50 mcgmcg inside daily, during pregnancy - 400 micrograms inside daily, during pregnancy - 400 micrograms perper day and lactation - 300 mcg. day and lactation - 300 mcg. IssueIssue: : tabltabl. . byby 0,001 0,001 g andg and 0,005 0,005 gg № 50. № 50.Used for increased erythropoiesis, in certain types of anemia, Used for increased erythropoiesis, in certain types of anemia, including anemia and leukopenia caused by drugs and ionizing including anemia and leukopenia caused by drugs and ionizing radiation, chronic gastroenteritis and tuberculosis radiation, chronic gastroenteritis and tuberculosis of of intestine.intestine.

Derivatives ofDerivatives of isoalloxazineisoalloxazine IsoalloxazineIsoalloxazine ––heterocyclic system, which consists of heterocyclic system, which consists of

condensed pyrazine, pyrimidine and benzene cycles, that is condensed pyrazine, pyrimidine and benzene cycles, that is derived from benzopteridinederived from benzopteridine. . Pyrimidine nucleus of Pyrimidine nucleus of isoalloxazine has character of lactam cycle because it isoalloxazine has character of lactam cycle because it contains two keto-groups contains two keto-groups ::

BenzopteridineBenzopteridine IsoalloxazineIsoalloxazineDetection of vitamin properties of flavin associated with the Detection of vitamin properties of flavin associated with the

presence in the molecule extremely labile group with two presence in the molecule extremely labile group with two conjugated double bonds in isoalloxazine nucleusconjugated double bonds in isoalloxazine nucleus.. This This group of atoms causes the redox properties of riboflavingroup of atoms causes the redox properties of riboflavin..

RiboflavinRiboflavin ( (RiboflavinumRiboflavinum))vitaminvitamin В В2 2 ((SPhUSPhU))

7,8-7,8-DimethylDimethyl-10-[(2-10-[(2SS,3,3SS,4,4RR)-)-2,3,4,5-2,3,4,5-tetrahydroxytetrahydroxy--pentylpentyl] ]

benzobenzo[[gg]]pteridinepteridine--2,4(З2,4(ЗHH,10,10HH)-)-diondion, ,

oror 6,7- 6,7-dimethyldimethyl-9-(1-9-(1’’--DD--ррibitylibityl)-)-isoalloxazineisoalloxazine

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 Riboflavin is very common in plant and in animal products: contained in the milk serum, liver, kidney, products: contained in the milk serum, liver, kidney, brewer's and baker yeastbrewer's and baker yeast;; in grains - millet, barley; in in grains - millet, barley; in vegetables - spinach and tomatoes.vegetables - spinach and tomatoes.

For extraction of 1 g of the drug For extraction of 1 g of the drug shouldshould be processed 5 be processed 5,,4 4 tons of whey, so now vitamin B2 tons of whey, so now vitamin B2 is is synthesizesynthesizeed fromed from D- D-ribose, ribose, оо-4--4-xylidine andxylidine and barbituric acidbarbituric acid..

CharactersCharacters.. Y Yellow or orange-yellow ellow or orange-yellow ccrystalline powder. rystalline powder. Very Very fewfew soluble in water, hardly soluble in 96% alcohol, soluble in water, hardly soluble in 96% alcohol, ether, acetone, chloroform. Easily soluble in chloride ether, acetone, chloroform. Easily soluble in chloride (forms(forms salts salts) and acet) and aceticic acid. Solutions decompose under acid. Solutions decompose under the influence of light, especially in the presence of alkali.the influence of light, especially in the presence of alkali. Detects polymorphismDetects polymorphism..

Identification of riboflavinIdentification of riboflavin

1.1. By theBy the physic physicoo-chemical constants: the specific rotation, -chemical constants: the specific rotation, infrared spectroscopy, TLC.infrared spectroscopy, TLC.

2.2. Solution of the substance in the light that passes throughSolution of the substance in the light that passes through itit, has a pale greenish-yellow color, and in reflected , has a pale greenish-yellow color, and in reflected light - an intense yellow-green fluorescence, which light - an intense yellow-green fluorescence, which disappears disappears at theat the adding adding ofof mineral acid or alkali (in mineral acid or alkali (in acidic medium acidic medium on theon the light forms riboflavin forms light forms riboflavin forms lumichrome lumichrome (6,7-dym(6,7-dymethylalloxazineethylalloxazine) - a colorless ) - a colorless substance that breaks down into ethanol and chloroform substance that breaks down into ethanol and chloroform with the appearance of blue fluorescence, in alkaline with the appearance of blue fluorescence, in alkaline medium on light riboflavin forms lumiflavin (6,7,9-medium on light riboflavin forms lumiflavin (6,7,9-trtrimethylalloxazineimethylalloxazine), which solution has the same color ), which solution has the same color and fluorescence and fluorescence asas riboflavin, but soluble in riboflavin, but soluble in chloroform). chloroform).

3.3. At the riboflavinAt the riboflavin reduction reduction byby sodium h sodium hydrosulfite ydrosulfite and and fluorescence fluorescence and colorationand coloration of aqueous of aqueous solution solution disappear (a disappear (a colorless compound formed - lecolorless compound formed - leukoriboflavinukoriboflavin):):

4.4. Unpharmacopoeial reactionUnpharmacopoeial reaction::

aa)) at the at the addadding of ing of sulfate concentrated sulfate concentrated acid to acid to riboflavin red riboflavin red coloration appears, which becomes yellow coloration appears, which becomes yellow after the after the adding adding of of water.water.

bb) with a ) with a silver nitrate silver nitrate solution orange-red coloration forms solution orange-red coloration forms (presence (presence of of imiimide de group)group)..

DeterminedDetermined lyumiflavin lyumiflavin that that formed as a result of formed as a result of changes in the chemical structure of matter under the changes in the chemical structure of matter under the influence of light and alkaline environmentinfluence of light and alkaline environment.

Determination of Determination of the the lyumiflavin impuritlyumiflavin impurityy based on its based on its solubility in chloroform (in chloroform riboflavinsolubility in chloroform (in chloroform riboflavin is is insoluble) - color of the filtrate should not exceed the insoluble) - color of the filtrate should not exceed the standardstandard(SPhU).

Determination of Determination of the the lyumiflavin impuritlyumiflavin impurityy conducted by conducted by thin layer chromatographythin layer chromatography(SPhU, additionsadditions).

StorageInIn airtight containersairtight containers, , in the place protected from lightin the place protected from light.

Test on purity

Assay of riboflavinAssay of riboflavin1.1. UV spectrophotometry (UV spectrophotometry (SPhUSPhU). Determination of ). Determination of the the

optical density optical density is conducted at theis conducted at the weakened weakened transmitting light in aqueous solution, acidified transmitting light in aqueous solution, acidified by by acetaceticic acid at acid at =444 =444 nmnm. Riboflavin content is . Riboflavin content is calculated using the specific absorption rate, which calculated using the specific absorption rate, which equals 328.equals 328.

2.2. Photocolorimetry.Photocolorimetry.3.3. FluorimetryFluorimetry..4.4. Alkalimetry by Alkalimetry by substituent substituent afterafter the interaction with the interaction with

silver silver nitrate solution.nitrate solution. Е = М.Е = М.mm..5.5. Periodate oxidation Periodate oxidation ((Malaprad’s reactionMalaprad’s reaction). ). Methodic Methodic

is based on the ribityl fragment of riboflavin molecule is based on the ribityl fragment of riboflavin molecule oxidation with formation of formic acidoxidation 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.

ApplicationApplicationPlays an important physiological role, Plays an important physiological role, because is abecause is a part of part of the oxidativ cell enzymethe oxidativ cell enzymess as riboflavin-phosphate ester. as riboflavin-phosphate ester. Daily demandDaily demand – 2 – 2 mgmg.. Hyporiboflavinose is Hyporiboflavinose is characterized characterized by deterioration of appetite, weight loss, sores in the corners by deterioration of appetite, weight loss, sores in the corners of mouth; arof mouth; ariboflavinoseiboflavinose - conjunctivitis, clouding of the - conjunctivitis, clouding of the cornea and lenscornea and lens its. its...

VitaminVitamin В В22 is used orally in doses of 5-10 mg per day is used orally in doses of 5-10 mg per day

(depending on the degree of disease) at ar(depending on the degree of disease) at ariboflavinoseiboflavinose, , conjunctivitis, irytah, keratitis, radiation sicknessconjunctivitis, irytah, keratitis, radiation sickness and others and others..

IssueIssue: : tabltabl. . byby 0,002 0,002 gg; 0,005 ; 0,005 gg; 0,01 ; 0,01 gg; 0,01% eye drops;; 0,01% eye drops; is is a a part of rpart of riboflavin mononucleotideiboflavin mononucleotide (riboflavin-5'- (riboflavin-5'-monophosphate sodium)monophosphate sodium) – – ampamp. 1%-1,0 №10.. 1%-1,0 №10.

Corrine derivativesCorrine derivativesCorrine vitaminsCorrine vitamins ( (groupgroup В В1212) were found in natural ) were found in natural products of products of the the animal origin, mainly in the internal animal origin, mainly in the internal organs.organs. So So, the richest source of vitamin B12 are fish , the richest source of vitamin B12 are fish entrails, high content entrails, high content of this vitamin is of this vitamin is in the whale liver in the whale liver and and the highest content the highest content meat is inmeat is in the meat of the meat of molluscs. In molluscs. In human and animal B12 is synthesized human and animal B12 is synthesized by microflora of by microflora of intestine and accumulates in the liver, kidney, intestine intestine and accumulates in the liver, kidney, intestine walls.walls.

VitaminVitamin В В1212 ( (cyanocobalaminecyanocobalamine) is extracted from the ) is extracted from the waste waste ofof the production of streptomycin and the production of streptomycin and chlortetracycline chlortetracycline antibiotics. Another source - sewage antibiotics. Another source - sewage (waste products of actinomycetes, bacteria, blue-green (waste products of actinomycetes, bacteria, blue-green algae), algae), from this soursefrom this sourse vitamin B12 vitamin B12 is allocated by is allocated by adsorption or extraction.adsorption or extraction.

CyanocobalaminCyanocobalamin ((CyanocobalamiCyanocobalami--numnum) ) vitaminvitamin В В1212

((SPhUSPhU))αα-(5,6--(5,6-dimethyldimethyl--benzimidazolebenzimidazole-1--1-

ylyl))cobamidecobamide cyanidecyanide

CorrineCorrine

Cyanocobalamin moleculeCyanocobalamin molecule consists of two main parts. consists of two main parts. First - nucleotide containing 5First - nucleotide containing 5,,6-6-ddimethylbenzimidazole imethylbenzimidazole associated with D-associated with D-rribofuranose,ibofuranose, which, in turn, linked which, in turn, linked by etheric boundby etheric bound with phosphate acid. Nucleotide connected with phosphate acid. Nucleotide connected with with macrocyclic corrinemacrocyclic corrine system (second part) system (second part) by pby peptide eptide bond.bond. Nitrogen a Nitrogen atom tom of of 55,,6-d6-dimethylbenzimidazoleimethylbenzimidazole in in position 3 boundposition 3 bounded byed by coordination bond with cobalt coordination bond with cobalt atom. Cobalt forms a chelate compound with atom. Cobalt forms a chelate compound with cyano-cyano-group and withgroup and with nitrogen atoms and nitrogen atoms and of of hydrogenated hydrogenated ppyrroleyrrole cycles cycles of corrineof corrine system. system.PPositive charge ositive charge of cof cobalt ion obalt ion is is neutralized neutralized by by negatively charged phosphate acid anion. Therefore, negatively charged phosphate acid anion. Therefore, cyanocobalamin is not only a chelating compound, but cyanocobalamin is not only a chelating compound, but an internal salt.an internal salt.

Properties of cyanocobalaminProperties of cyanocobalaminCrystalline powder with dark redCrystalline powder with dark red color color, odorless, , odorless, hygroscopic. hygroscopic. Sparingly soluble in waterSparingly soluble in water,, soluble in soluble in 95 % 95 % alcoholalcohol, , practically insoluble in etherpractically insoluble in ether, , chloroformchloroform, , acetoneacetone.. The anhydrous substance is very hygroscopic The anhydrous substance is very hygroscopicAt theAt the heat heatinging above 300 above 300°°С decomposes. С decomposes. Stable atStable at рН=4- рН=4-6. Easily 6. Easily decomposeddecomposed in an alkaline environment. in an alkaline environment.ССN-group in the molecule of vitamin N-group in the molecule of vitamin ВВ1212 can can be be replace replace by by the ОНthe ОН-group-group ( (oxycobalaminoxycobalamin) or ) or NONO22 (n(nitrocobalaminitrocobalamin). Both products are converting to a group ). Both products are converting to a group of vitamin Вof vitamin В1212..At theAt the heat heating ofing of cyanocobalamin solution cyanocobalamin solution withwith HCl HCl ammonium chloride, 5,6-dammonium chloride, 5,6-dimethylbenzimidazoleimethylbenzimidazole, , aminoiaminoissopropanol, cobalt chloride, opropanol, cobalt chloride, cyanic cyanic acidacid are are formed.formed.Antianemic action associated with the presence of 5,6-Antianemic action associated with the presence of 5,6-ddimethylbenzimidazole imethylbenzimidazole in the molecule structure.in the molecule structure.

Identification of cyanocobalamineIdentification of cyanocobalamineDetermination of UV-spectral characteristics.Determination of UV-spectral characteristics.

TLCTLC..

Cobalt ions Cobalt ions are are determined after determined after the the mineralization by mineralization by alloying with potassium halloying with potassium hydrosulfateydrosulfate and interaction and interaction with sodium 1-nitroso-2-naphthol-3,6-dwith sodium 1-nitroso-2-naphthol-3,6-disulfonateisulfonate - a red - a red color color of of internally complex salts of cobaltinternally complex salts of cobalt appeares appeares..

Cobalt ion can be detected afterCobalt ion can be detected after evaporation and evaporation and roasting of roasting of 0,25 0,25 mg ofmg of cyanocobalamin withcyanocobalamin with 10 10 mg ofmg of potassium sulfate andpotassium sulfate and 2 2 drops ofdrops of 15 % 15 % sulfate acidsulfate acid. . Saturated solution of ammonium thiocyanate in Saturated solution of ammonium thiocyanate in acetone is added to the residueacetone is added to the residue; ; blue-green coloration blue-green coloration appears appears ::

СоСо2+2+ + 2 + 2 NHNH44SCNSCN → → Со(Со(SCNSCN))22 + 2 NH + 2 NH44++

Detection of cyano-groupDetection of cyano-group. . Analytical weight of Analytical weight of cyanocobalamin is heated in the test-tube with oxalic cyanocobalamin is heated in the test-tube with oxalic acidacid, , under the action of this acid cyanic acid under the action of this acid cyanic acid eliminateseliminates, , which is detected with filtration paper which is detected with filtration paper soaked by the solution of benzidine and coppersoaked by the solution of benzidine and copper (ІІ) (ІІ) acetateacetate, , as a result blue complex compound formesas a result blue complex compound formes..

AssayAssayThe method of UV-spectrophotometry.The method of UV-spectrophotometry.

StorageStorageInIn airtight containersairtight containers, , in the place protected from lightin the place protected from light..

Application of cyanocobalaminApplication of cyanocobalamin VitaminVitamin В В1212 is a growth factor necessary for normal is a growth factor necessary for normal

hematopoiesis and maturation of erythrocytes. He participates in hematopoiesis and maturation of erythrocytes. He participates in the formation of choline, methionine, nucleic acids, shows a the formation of choline, methionine, nucleic acids, shows a positive effect on liver function and nervous system.positive effect on liver function and nervous system.

Daily demand for Daily demand for healthy personhealthy person – 10-20 – 10-20 mcgmcg..vv This This quantity of quantity of vitamin is vitamin is not not produced in the produced in the intestineintestine and should and should be be complementcomplemented byed by the products of animal origin the products of animal origin

Apply Apply at theat the malignant anemia, various forms of anemia, liver malignant anemia, various forms of anemia, liver disease, nervous system, skin diseases and others.disease, nervous system, skin diseases and others.

IssueIssue: : solution forsolution for injectioninjection 200 200 mcgmcg (0,02%) (0,02%) oror 500 500 mcgmcg (0,05%) 1,0 №10(0,05%) 1,0 №10..

Multivitamin drugsMultivitamin drugs In nature vitamins commonly found in the form of various In nature vitamins commonly found in the form of various combinations. Herbal products often contain vitaminscombinations. Herbal products often contain vitamins of group B of group B, , vitamin C and others. The combination of vitamins is also found vitamin C and others. The combination of vitamins is also found in animal products. in animal products. . In many cases, vitamins mutually reinforce physiological effects . In many cases, vitamins mutually reinforce physiological effects that they that they havehave. For example, the effect of vitamin P on vascular . For example, the effect of vitamin P on vascular permeability amplified permeability amplified by by ascorbic acid, mutually amplifying ascorbic acid, mutually amplifying effect on hematopoiesis of folic acid and cyanocobalamin.effect on hematopoiesis of folic acid and cyanocobalamin.In some cases, toxicity of vitamins In some cases, toxicity of vitamins is is reduced reduced atat their combined their combined application. For example, vitamin D toxicity application. For example, vitamin D toxicity is is reducereduceinging by by vitamin A. For example, vitamin D toxicity vitamin A. For example, vitamin D toxicity is is reducereduceinging by by vitamin A. Vitamins canvitamin A. Vitamins can also also detect antagonistic effect (nicotinic detect antagonistic effect (nicotinic acid slows the lipotropic action of choline).acid slows the lipotropic action of choline).VVitamins participating itamins participating aactively in various biochemical processes, ctively in various biochemical processes, if they are associated and show a stronger complex biological if they are associated and show a stronger complex biological action that action that isis served for the combined us served for the combined usage inage in both both cases for thecases for the preventive and therapeutic purposes.preventive and therapeutic purposes.

Multivitamin drugs have different compositionMultivitamin drugs have different compositionss ( (some some vitamins alone or in combination with vitamins alone or in combination with micromicroelements) and elements) and issued in the form of various dosage forms: tablets, issued in the form of various dosage forms: tablets, ““effervescenteffervescent” ” tablettabletss (upsavit, suprad (upsavit, supradineine) tablets for ) tablets for chewing (multi-tabs, jungle), beans (sob) , syrups chewing (multi-tabs, jungle), beans (sob) , syrups (mult(multiivitamol), gels (Kindervitamol), gels (Kinder--biovital), solution for injection biovital), solution for injection (nerviple(nerviplex x (В(В11, В, В66,, ВВ1212)). )).

Ukrainian multivitamin drugsUkrainian multivitamin drugs: : revitrevit (А, В (А, В11, В, В22, С), , С), hexavithexavit ((revitrevit+ В+ В66,, РР), РР), undevitundevit ( (hexavithexavit+ Е, Р, В+ Е, Р, Всс, В, В33), ), decamevitdecamevit (А, В(А, В11, В, В22, В, В66,, ВВ1212,, РР, С, Е, Р, ВРР, С, Е, Р, Всс, , methioninemethionine), ), complevitcomplevit (В(В11, В, В22, В, В66,, ВВ1212,, РР, С, Е, ВРР, С, Е, Всс), ), quadevitquadevit ( (decamevitdecamevit + В + В33,, glutamic acidglutamic acid, , fitinefitine, , KCl. CuSOKCl. CuSO44), ), vitamvitam (В (В11, В, В22, В, В66,, РР, РР, ВВ33,,mefenamicmefenamic, , Fe, Zn, Mn, Cu, Co, Cr)Fe, Zn, Mn, Cu, Co, Cr)..Multivitamin preparationsMultivitamin preparations of the of the foreign foreign origin origin: Vitrum: Vitrum vitamins vitamins (Kids, Junior, (Kids, Junior, BBeaeautyuty, , PPrenatal, Energy, renatal, Energy, CCardio, ardio, etc..) Centrum, multi-tabsy, etc..) Centrum, multi-tabsy, DDuovit, Pikovit, uovit, Pikovit, JJungle, ungle, UUniniccap, ap, Pregnavit, Pregnavit, OOliliggovit, Theravit, ovit, Theravit, SSupradyn, Kinderupradyn, Kinder--biovital biovital and and othersothers..

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