Quality analysis of medical drugs from the vitamins group of aliphatic and alicyclic rows.
Vitamins – are organic compounds of different chemical structures, which in small amounts required for the normal organism living. They are a part of enzyme systems, which are biological catalysts of the chemical reactions in living cells and are involved in metabolism. The human and animals caot synthesize vitamins or synthesize them in insufficient quantities (nicotinic acid) and therefore must obtain them from food. In some cases vitamins are formed in the tissues as a result of chemical transformation of substances that are their precursors (provitamins).
In 1912 y. polish scientist K. Funk proposed term “vitamins” that means “amines necessary for life”.
Classification of vitamins
1. By the solubility:
• fat soluble (A, D, E, F, K )
• Water-soluble (group В, С, synthetic analogs of К, РР, Р)
2. By letters:
• by the disease that occurs at the insufficient amount of this vitamin (С – scurvy (antiscorbutic), А –antixerophthalmic, В – beri-beri, Е – one that ensures procreation.
• additional figures at the expanding of the group of vitamins (В1 – В15, Вс)
3. Chemical classification.
Chemical classification of vitamins
I. Vitamins of the aliphatic row:
а) Derivatives of the unsaturated polyoxy–g– lactones (ascorbic acid (vitamin C));
b) Derivatives of gluconic acid esters (pangamic acid (vitamin В15));
c) Derivatives of b– aminoacids (pantothenic acid (vitamin В3)).
II. Vitamins of the alicyclic row:
а) cyclohexyl isoprenoids(retinols (vitamins of A group));
b) Cyclohexanolethylenehydrindanoic (calciferols (vitamins of group D)).
III. Vitamins of the aromatic row (naphthoquinone derivatives):
c) natural vitamins of К group (phylloquinone– К1, menaquinone – К2);
d) synthetic analogues of vitamins of К group (menadione, vikasol).
IV. Vitamins of the heterocyclic row:
1) Chromane derivatives
а) tocopherols (vitamins of Е group);
b) bioflavonoids (vitamins of Р group).
2) Pyridine derivatives
а) nicotinic acid and its amide (vitamine of РР group);
b) oxymethylpyridine vitamines (vitamins of В6 group).
3) pyrimidine derivatives (thiamin (vitamin В1).
4) Pterine derivatives (folic acid (vitamin Вс).
5) Isoalloxazine derivatives or flavin vitamins (riboflavin (vitamin В2))
6) Corrine derivatives – cobalamins (vitamins of В12 group).
Obtaining of vitamins
• Synthetic and semi-synthetic methods (С, А, Е, D, В1, В2, РР and others).
• From plant and animal raw materials , microorganisms (С – fruit-bloom, Р –waste of the tea industry, D –natural sterines, А –fish oil, Е –vegetable fats, В12 –product of microbiological synthesis of antibiotics, effluents).
Properties
White crystals or crystalline powder (rutin – a green- yellow, folic acid – a yellow or yellow-orange, riboflavin – yellow-orange, cyanocobalamin – dark red). Tocopherol acetate-light yellow, transparent, dense, oily liquid with slight odor.
Vitamins are unstable under the action of light, air and heat (some of them).
Identification of vitamins
• There are not general reactions, except nitrogen determination by the Kjeldahl method.
• Often the group reaction(on the pyridine cycle etc.)
• More often – reactions that depend on the individual characteristics of the chemical structure.
• According to the SPhU requirements – 10 preparations (D, С, РР, nicotinamide В6, В2, В1 (hydrobromide), В1 (hydrochloride), Вс, В12:
ІR-, UV– spectroscopy, chromatography.
Assay of vitamins
• Biological methods – determining of the biological activity of vitamins. Studies conducted on rats, pigeons, guinea pigs, which are moved on a diet (exclude the study vitamin from the food). Then determines which of the study vitamin can cure or save the animal from avitaminosis. Parallel conduct similar studies with the standard drug. Activity in IU (international unit) – the notional amount of the standard drug in mg or mcg (g). For one unit is considered to be the minimum amount of vitamin that cures or prevents the animal from avitaminosis. 1 IU is different in different vitamins (1 IU of vitamin A corresponds to 0,344 g of tocopherol acetate, vitamin D – 0,25 g of ergocalciferol).
• Currently, the most frequently for quantification use physical, physico-chemical and chemical methods.
USAGE
• Parts of biocatalysts (enzymes) that cause different functions in the body metabolism. Hypo-and avitaminosis of the some vitamin. In addition, vitamins of В group –CNS diseases, heart diseases; В12 – anemia; РР – a violation of the peripheral circulation; Е – for the pregnancy keeping; D – rachitis; А – skin and eye disease. Multivitamins–support during the cold season, reduce the risk of heart diseases and others.
• Side effects – allergic reactions (Multi-tabs, Kinder-biovital), dyspeptic disorders. Supervitaminosis – vitamines А, D, К; essentiale Н (vitamins of Вgroup) – at the expense of long reception.
Vitamins of the aliphatic row
• Ascorbic acid belongs to the derivatives of polyoxy-γ-lactones of the unsaturated carboxylic acids, On the other hand it can be attributed to the vitamins of heterocyclic series as furan derivative. It is widely distributed iature. Especially rich in it is flora: fresh vegetables, fruits, etc. In industry, ascorbic acid synthesized from D-glucose.
• To the vitamins of aliphatic row, derivatives of gluconic acid esters, pangamic acid belongs (vitamin В15). In medical practice is used its calcium salt. Pangamic acid is a part of the rice bran, yeast, blood, liver.
• To the vitamins of aliphatic row, derivatives of β-aminoacids, pantothenic acid belongs. Yeast, eggs, liver and egg yolk are rich on pentothenic acid. In medical practice is used its calcium salt.
Ascorbic acid (Acidum ascorbicum) Vitamin С (SPhU)
(R)-5-[(S)-1,2- Dihydroxyethyl]-3,4-dihydroxy-5H–furan-2-on
g -lactone-2,3-dehydro-L–gulonic acid
Extraction of ascorbic acid
Available in fresh vegetables – cabbage, beets, lettuce, tomatoes, potatoes, berries – strawberries, currents, fruits – lemon, orange. t is also found in milk, eggs, rose hips, fennel, etc.. For the extraction of ascorbic acid from the rose hips produced water extract, which is thickened in vacuum to the thicken of syrup. From the remainder precipitated ballast substances with alcohol and ether, and the filtrate evaporated to the dry state. Residue is clearing by crystallization or chromatographic method.
The basic amount of ascorbic acid is now extracted synthetically, based on D-glucose, that can be transformed by the reduction in the D-sorbitol and D-sorbitol by enzymatic oxidation – in the L–sorbose
To protect the alcohol group of sorbose, at first it is condensed with two molecules of acetone, and then oxidize diacetone sorbose by КMnO4. Obtained diacetone ketoglutaric acid is saponificated to ketogulonic acid.
Properties of ascorbic acid
• White or almost white crystalline powder of or colorless crystals that change color under the action of air and moisture. Easily soluble in water, soluble in 96% alcohol, practically indissoluble in ether. Melts at about 190 ° C with decomposition.
• By endiol group ascorbic acid shows both acidic and restorative properties.
• Acidic nature is cased by the mobility of hydrogen atom in hydroxyl group in position 3; at the alkali titration ascorbic acid behaves like a monobasic acid.
Ascorbic acid oxidizes in two stages:
1. reversible oxidation to dehydroascorbic acid (keto-form);
2. irreversible process of oxidation, which leads to the formation of furfural:
Identification of ascorbic acid
1. By the physico-chemical constants : UV– and IR- spectroscopy, determination of рН and the specific optical rotation.
2. Add to the solution of ascorbic acid diluted nitrate acid and silver nitrate solution – silver gray metallic precipitate falls:
3. Unpharmacopoeial reactions:
a) To 1 ml of obtained solution add 2 drops of iron (III) chloride R2 solution and after1 min. 1 drop of of potassium ferricyanide R; blue color appeares:
3FeCl2 + 2K3[Fe(CN)6] → Fe3[Fe(CN)6]2 + 6KCl.
b) To 1 ml of obtained solution add 5-7 drops of 0,05 М iodine solution; solution becomes transparent:
c) To 1 ml of obtained solution add 1 drop of CuSO4 solution and 2-3 drops of 1 % NH4CNS solution; white precipitate forms:
d) To the aqueous solution of the preparation add NaHCO3 and FeSO4, shake and leave to stand; a dark purple color appears which disappears at the adding of H2SO4 dil.:
e) at the adding by drops 2,6- dichlorophenolinedophenolate solution to the solution of ascorbic acid blue color disappears:
f) Ascorbic acid is easily oxidized at the interaction with :
• Phosphoric-molybdenum acid (the products of blue color form);
• methylene blue (discoloration occurs due to formation of leucobase);
• Potassium permanganate(discoloration;
• Fehling reagent (red color)
Assay of ascorbic acid
Iodometry, direct titration (SPhU). Indicator – starch. E m .= М. m./2
Alkalimetry, direct titration. Indicator – phenolphthalein).
E.m. = М. m.
• Iodatometry, direct titration in acidic medium in the presence of potassium iodide, indicator – starch. At the point of equivalence excess of potassium iodate causes the blue color of solution. E.m. = М. m./2.
• Titration by sodium 2,6-dichlorophenolindophenolate solution (for the determination of ascorbic acid in row material). E.m. = М. m./2
• Other redox methods (Iodochlorometry, cerimetry and others).
Ascorbic acid storage
• Ionmetallic airtight containers, in the dark place.
Usage of ascorbic acid
• Takes part in oxidative-reduction processes, in carbohydrates metabolism and tissue regeneration, the formation of steroid hormones. Daily demand of a healthy person – 80-100 mg.
• In preventive and therapeutic purposes at the scorbutus (scurvy), bleeding of various etiologies, infectious diseases and poisonings, liver and kidneys diseases.
• Issue: tablets, dragee by 0,025; 0,05; 0,1; 0,5 g; 5% and 10% solutions for injections. Soluble tablets by 1,0 g.
Calcium pangamate
(Calcii pangamas), vit. В15, Kalham
By the chemical structure pangamic acid is an ester of D–gluconic and dimethylaminoacetic acids (dimethylglycine).
Substance, except for calcium pangamate contains calcium gluconate (25%) and calcium chloride (6%).
Extraction of pangamic acid
• Isolated from rice bran, yeast, blood, liver.
• Synthesized from D-glucose:
• Properties. White, sometimes yellowish crystalline powder with characteristic odor. Hygroscopic. Easily soluble in water and hardly soluble in organic solvents.
Identification of calcium pangamate
• By the physico-chemical constants: IR spectroscopy.
• Substance gives reactions on calcium (4 pharmacopoeial reaction).
• Residue of gluconic is confirmed by the reaction with iron (III) salts by the formation of light green color.
• Alkaline hydrolysis. After the heating of substance with solution of sodium hydroxide smell of amines appears.
• The reaction of formation of colored iron hydroxamate (esteric group):
Assay of calcium pengamate
• Given that the substance except calcium pangamate, also includes calcium gluconate and calcium chloride, quantitatively determine the content of:
1. Nitrogen (3,6 – 4,2%) – acidimetry ion-aqueous medium;
2. Calcium (5,8 – 7,4%) – complexonometry method;
3. Chlorides (not more than 2,2%) – a method of reverse argentometry by Folgard;
4. The amount of carboxyl groups (11 – 15%) – by ion exchange chromatography.
STORAGE
• In glass airtight containers, in a dry place.
Usage of calcium pangamate
• Activates the transfer of oxygen to the cells of tissues and takes part in the methylation of biosubstrates, improves lipid metabolism.
• Similar to vitamins preparation. Calcium pangamate is used for the treatment of various forms of atherosclerosis, cirrhosis, alcoholism and other diseases.
• Issue : tablets by 0,05 g.
Calcium pantothenate
(Calcii pantothenas), vit. В3
Calcium salt of D-(+)-α,γ-dioxy-β,β-dimethylbutyryl–N–amide-β’-aminopropionic acid (β-alanine).
Properties. White fine crystalline powder without smell. Easily soluble in water, very little soluble in organic solvents.
Extraction of calcium pantothenate
• In large quantities contained in caviar, yeast, liver, egg yolk, rice.
• Synthetically is extracted by the following scheme:
Identification of calcium pantothenate
1. By the physico-chemical constants: the specific rotation from +25 to +28° (5 % aqueous solution)
2. Substance gives reactions on calcium.
3. With the solution of copper (II) sulfate in an alkaline medium substance forms a blue complex (β-alanine):
4. Residue of α,γ –dioxy– β,β –dimethylbutyric acid determine after alkaline hydrolysis. . Substance is boiled with sodium hydroxide solution, after cooling acidified by acid chloride solution and added iron (III) chloride – yellow color appears:
The reaction of colored iron hydroxamate formation : γ-butyrolactone
Assay of calcium pantothenate
Determine the content of:
l Nitrogen (5,7 – 6,0%) – Kjeldahl method;
l Calcium (8,2 – 8,6%) – complexonometry method.
Test on purity
Specific impurity. Pantoyl lactone –is determined spectrophotometricaly in the visible part of the spectrum by the reaction of iron hydroxammate formation.
Storage
• In glass airtight containers, in a dry place at the room temperature.
Usage of calcium pantothenate
• Participates in hydrocarbon and fat metabolism, stimulates the formation of corticosteroids and is part of coenzyme A.
• The daily demand – 10-12 mg. Since it is formed during the vital functions of Escherichia coli, its avitaminosis is not observed.
• To treat neuralgia, eczema, allergies, polyneuritis, and other diseases related with violation of metabolism and at the inflammatory processes.
• Issue: tablets by 0,1 g.
Vitamins of the alicyclic row
• Vitamins of the alyciclic row include retinols (vitamins of А group) and calciferols (vitamins of D group).
• In the core of the retinol molecule is trimethylcyclohexanoic cycle associated with tetraenol conjugate chain, in the end of which is hydroxyl or aldehyde group. Retinol was obtained from the liver of fish in 1909 year. In 1928 y., Euler found that some plants are substances that have provitamin activity, scilicet are the precursors of vitamins. Provitamins of vitamin A are α-, β-and γ-carotene.
• . To vitamins of D group, which is called calciferols or antirachitis include derivatives of cyclohexanol ethylene-hydrindane. Is opened few vitamins of D group: D1-D7 similar by the chemical structure, physico-chemical properties and pharmacological action. The practical usage hame the following vitamins: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol).
• Vitamins D2 and D3 are in egg yolks, caviar, butter, milk. A significant number of them accompanied by retinol in liver and adipose tissue of fish and marine animals.
• In medicine is used retinol acetate and ergocalciferol.
Retinol acetate
(Retinoli acetas) vit. А
Trans–9,13-Dimethyl-7-( 1,1,5-trimethylcyclohexene5-yl-6)-nonatatraene-7,9,11,13-ol-15 acetate
Extraction of retinol
Hydrolysis of fish liver by 15 % КОН solution in the atmosphere of inert gas is used for the vitamin A obtaining.
Main quantity of vitamin A is obtained synthetically from citral by the following scheme: β-ionone
Properties of the retinol acetate
White or pale yellow crystals with a weak odor. Extremely unstable under the action of air oxygen and light (easily oxidizes by the atmospheric oxygen, especially at elevated temperatures and light, with the formation of heronic acid). Practically insoluble in water, soluble in 95% alcohol, chloroform, ether and oils.
Identification of retinol acetate
1. The reaction with antimony (III) chloride in chloroform environment – blue color appears.
2. The reaction with aluminum chloride – blue color appears.
3. Reactions on the unsaturated bonds (discoloration of iodine, bromine water, reduction of phosphorwolframic acid, AuCl3).
Assay of retinol acetate
• UV–spectrophotometry
• Photocolorimetry by the reaction with SbCl3
Storige of retinol acetate
Due to the fact that the substance is easily oxidizes, store it in sealed ampoules in a stream of nitrogen, which keep from the action of light, at the temperatures no more than +5°. Oil solutions of retinol acetate are stored in chock-filled, well corked cups of dark glasses at the temperatures no more than +10 °С.
Usage of retinol acetate
• In the treatment of avitaminosis, diseases and lesions of the skin, eye diseases. Prescribed as pills, granules, oil solutions orally, intramuscularly and topically. During the treatment is necessary to consider the possibility of hypervitaminosis.
• Daily dose for a healthy person – 1 mg treatment dose – up to 10 mg (33000 UA), but not more than 30 mg (100 000 UA).
• 1 IU = 0,344 microgram or 100000 IU = 0,0344 g of retinol acetate.
• There are two forms of retinol: acetate and palmytate (more stable then acetate).
• Issue. VitaminА gel. caps. By 2500, 8000, 12000, 50000, 100000 IU.
• Caps. Aevit – vit. А + Е – caps. №24 (retinol 30000 IU, tocopherol 70 IU).
Vit. A are carotenes, which contained in fruits, carrots, red peppers and others. b–Carotene in the human body decomposes under the influence of liver enzymes into two molecules of vitamin А, a– and g–carotenes form only one molecule of vitamin А.β α γ
Ergocalciferol (Ergocalciferolum), vit. D2 (SPhU)
(5Z,7E,22E)-9,10-Secoergosta-5,7,10(19),22-tetraene-3β-ol
Cyclopentanophenantrene General formula of calciferols
Extraction of ergocalciferol
Vitamin D2 by its structure is similar to steroids. This vitamin is obtained by the UV-radiation of ergosterine, which contains in yeast, the uterine horn etc.. Process of ergosterine transformation in ergocalciferol conducts through the formation of lumisterine and tachysterine:
Properties of ergocalciferol
• White or slightly yellowish crystalline powder or white or almost white crystals. Practically insoluble in water, easily soluble in 96%alcohol, soluble in fatty oils. Sensitive to air, heat and light. Solutions in volatile solvents are volatile and must been used immediately after preparation. In the solutions is possible the depending on temperature and time reversible isomerization in pre-ergocalciferol. Activity of the substance is caused by both components.
• Solution of ergocalciferol in oil – is a transparent oily liquid from light yellow to dark yellow.
Identification of ergocalciferol
1. By the physico-chemical constants: IR–spectroscopy.
2. Steroid part of molecule cases Liberman reaction (chloroform solution of the drug at the shaking with acetic anhydride and sulfate acid becomes red, color transforms to purple, then to blue and finally to green).
3. At the interaction with antimony (III) chloride solution in the presence of acetylchloride formed orange-pink color. This reaction is also used for determination of impurities by TLC and assay by the photocolorimetry method.
4. Sobel-Meyer’s reaction (with glycerin-1 ,3-dichlohydrine) – green coloration.
5. Shaltegger’s reaction (drug is boiled with benzene solutions of aldehydes (vanillin, furfural, anisic, etc.),then add perchlorate acid) – red color appears.
Assay fo ergocalciferol
• Liquid chromatography (SPhU).
• Photocolorimetry
Storage of ergocalciferol
Ergocalciferol is stored in an airtight container under nitrogen in a dark place at a temperature from 2 °С to 8 °С. The contents of opened containers should be used immediately. Medical forms of ergocalciferol are stored in chock-filled, well corked cups of dark glasses, because it easily oxidizes by the air oxygen, under the action of light gradually decomposes and forms toxic products.
Cholecalciferol (Cholecalciferolum), vit. D3
(5Z,7E)-9,10-Secocholesta-5,7,10(19)-triene-3β-ol
At first was obtained from fish oil. Provitamin of vitamin D3 is 7-dihydrocholesterol
Because of the presence of cholesterol and 7-dehydrocholesterol in the humane skin lipids content it is possible to synthesize vitamin D3 under the action of sun radiation or UV-radiation on the surface of the humane body.
Usage of calciferols
• In the medical practice use alcoholic (0,5 %) and oil (0,125 %) solutions of vitamin D2 to prevent and treat rachitis, but also at the bone diseases associated with violations of calcium metabolism. Vitamins of D group are effective for treatment of all forms of erythematosus and other skin diseases.
• Daily demand for a healthy man– 1000 IU.
• Vitamin D3 – cholecalciferol. Tablets Videine (2000 іand5000 IU). Vit. А+D3 – aqueous solution bottles by 10 ml.
• Fish Oil.
Ascorbic Acid
General Notices
(Ph Eur monograph 0253)
C6H8O6
ıı176.1ıı50-81-7
Action and use
Vitamin C.
Preparations
Ascorbic Acid Injection
Ascorbic Acid Tablets
Paediatric Vitamins A, C and D Oral Drops
Vitamins B and C Injection
When Vitamin C is prescribed or demanded, Ascorbic Acid shall be dispensed or supplied.
Ph Eur
DEFINITION
Ascorbic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5
per cent of (5R)-5-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, becoming discoloured on
exposure to air and moisture, freely soluble in water, soluble in ethanol (96 per cent).
It melts at about 190 °C, with decomposition.
IDENTIFICATION
First identificationıB, C.
Second identificationıA, C, D.
ıA. Dissolve 0.10 g in water R and dilute immediately to 100.0 ml with the same solvent. To
10 ml of 0.1 M hydrochloric acid add 1.0 ml of the solution and dilute to 100.0 ml with water
R. Measure the absorbance (2.2.25) at the maximum at 243 nm immediately after
dissolution. The specific absorbance at the maximum is 545 to 585.
ıB. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with ascorbic acid CRS. Examine the substance prepared as discs containing 1
mg.
ıC. The pH (2.2.3) of solution S (see Tests) is 2.1 to 2.6.
ıD. To 1 ml of solution S add 0.2 ml of dilute nitric acid R and 0.2 ml of silver nitrate solution
R2. A grey precipitate is formed.
TESTS
Solution S
Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent.
Appearance of solution
Solution S is clear (2.2.1) and not more intensely coloured than reference solution BY7 (2.2.2,
Method II).
Specific optical rotation (2.2.7)
Dissolve 2.50 g in water R and dilute to 25.0 ml with the same solvent. The specific optical
rotation is + 20.5 to + 21.5.
Oxalic acid
Dissolve 0.25 g in 5 ml of water R. Neutralise to red litmus paper R using dilute sodium
hydroxide solution R and add 1 ml of dilute acetic acid R and 0.5 ml of calcium chloride
solution R (test solution). Prepare a reference solution as follows: dissolve 70 mg of oxalic
acid R in water R and dilute to 500 ml with the same solvent; to 5 ml of this solution add 1 ml
of dilute acetic acid R and 0.5 ml of calcium chloride solution R (reference solution). Allow the
solutions to stand for 1 h. Any opalescence in the test solution is not more intense than that in
the reference solution (0.2 per cent).
Related substances
The thresholds indicated under Related substances (Table 2034.-1) in the general
monograph Substances for pharmaceutical use (2034) do not apply.
Copper
Not more than 5.0 ppm of Cu, determined by atomic absorption spectrometry (2.2.23, Method
I).
Test solutionıDissolve 2.0 g of the substance to be examined in 0.1 M nitric acid and dilute
to 25.0 ml with the same acid.
Reference solutionsıPrepare reference solutions containing 0.2 ppm, 0.4 ppm and 0.6 ppm
of Cu by diluting copper standard solution (10 ppm Cu) R with 0.1 M nitric acid .
Measure the absorbance at 324.8 nm using a copper hollow-cathode lamp as a source of
radiation and an air-acetylene flame. Adjust the zero of the apparatus using 0.1 M nitric acid .
Iron
Not more than 2.0 ppm of Fe, determined by atomic absorption spectrometry (2.2.23, Method
I).
Test solutionıDissolve 5.0 g of the substance to be examined in 0.1 M nitric acid and dilute
to 25.0 ml with the same acid.
Reference solutionsıPrepare reference solutions containing 0.2 ppm, 0.4 ppm and 0.6 ppm
of Fe by diluting iron standard solution (20 ppm Fe) R with 0.1 M nitric acid .
Measure the absorbance at 248.3 nm using an iron hollow-cathode lamp as a source of
radiation and an air-acetylene flame. Adjust the zero of the apparatus using 0.1 M nitric acid .
Heavy metals (2.4.8)
Dissolve 2.0 g in water R and dilute to 20 ml with the same solvent. 12 ml of the solution
complies with test A (10 ppm). Prepare the reference solution using lead standard solution (1
ppm Pb) R.
Sulphated ash (2.4.14)
Not more than 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.150 g in a mixture of 10 ml of dilute sulphuric acid R and 80 ml of carbon
dioxide–free water R. Add 1 ml of starch solution R. Titrate with 0.05 M iodine until a
persistent violet-blue colour is obtained.
1 ml of 0.05 M iodine is equivalent to 8.81 mg of C6H8O6.
STORAGE
Store in a non-metallic container, protected from light.
Ph Eur
Calcium Pantothenate
General Notices
(Ph Eur monograph 0470)
C18H32CaN2O10 ıı476.5ıı 137-08-6
Action and use
Component of vitamin B.
Ph Eur
DEFINITION
Calcium pantothenate contains not less than 98.0 per cent and not more than the equivalent
of 101.0 per cent of calcium bis[3-[[(2R)-2,4-dihydroxy-3,3-dimethylbutanoyl]amino]
propanoate], calculated with reference to the dried substance.
CHARACTERS
ıA. white or almost white powder, slightly hygroscopic, freely soluble in water, slightly soluble
in alcohol.
IDENTIFICATION
ıA. It complies with the test for specific optical rotation (see Tests).
ıB. Examine the chromatograms obtained in the test for 3-aminopropionic acid. The principal
spot in the chromatogram obtained with test solution (b) is similar in position, colour and
size to the principal spot in the chromatogram obtained with reference solution (a).
ıC. To 1 ml of solution S (see Tests) add 1 ml of dilute sodium hydroxide solution R and 0.1
ml of copper sulphate solution R . A blue colour develops.
ıD. It gives reaction (a) of calcium (2.3.1) .
TESTS
Solution S
Dissolve 2.50 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent.
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
pH (2.2.3)
The pH of solution S is 6.8 to 8.0.
Specific optical rotation (2.2.7)
+ 25.5 to + 27.5, determined on solution S and calculated with reference to the dried
substance.
3-Aminopropionic acid
Examine by thin-layer chromatography (2.2.27) , using silica gel G R as the coating
substance.
Test solution (a)ı Dissolve 0.2 g of the substance to be examined in water R and dilute to 5
ml with the same solvent.
Test solution (b)ı Dilute 1 ml of test solution (a) to 10 ml with water R .
Reference solution (a)ı Dissolve 20 mg of calcium pantothenate CRS in water R and dilute
to 5 ml with the same solvent.
Reference solution (b)ı Dissolve 10 mg of 3-aminopropionic acid R in water R and dilute to
50 ml with the same solvent.
Apply separately to the plate 5 μl of each solution. Develop over a path of 12 cm using a
mixture of 35 volumes of water R and 65 volumes of ethanol R . Dry the plate in a current of
air and spray with ninhydrin solution R1 . Heat at 110 °C for 10 min. Any spot corresponding
to 3-aminopropionic acid in the chromatogram obtained with test solution (a) is not more
intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent).
Chlorides (2.4.4)
5 ml of solution S diluted to 15 ml with water R complies with the limit test for chlorides (200
ppm).
Heavy metals (2.4.8)
12 ml of solution S complies with limit test A for heavy metals (20 ppm). Prepare the standard
using lead standard solution (1 ppm Pb) R .
Loss on drying (2.2.32)
Not more than 3.0 per cent, determined on 1.000 g by drying in an oven at 105 °C.
ASSAY
Dissolve 0.180 g in 50 ml of anhydrous acetic acid R . Titrate with 0.1 M perchloric acid
determining the end-point potentiometrically (2.2.20) .
1 ml of 0.1 M perchloric acid is equivalent to 23.83 mg of C18H32CaN2O10.
STORAGE
Store in an airtight container .
Ph Eur
Vitamin A
General Notices
(Ph Eur monograph 0217)
In the British Pharmacopoeia, the term ‘Retinol’ is used within titles for preparations
containing synthetic ester(s) and the term ‘Vitamin A’ within the title for the preparation
containing material of natural origin.
Preparation
Paediatric Vitamins A, C and D Oral Drops
Ph Eur
DEFINITION
Vitamin A refers to a number of substances of very similar structure (including (Z)-isomers)
found in animal tissues and possessing similar activity. The principal and biologically most
active substance is all-(E)-retinol (all-(E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-
2,4,6,8-tetraen-1-ol; C20H30O). Vitamin A is generally used in the form of esters such as the
acetate, propionate and palmitate.
Synthetic retinol ester refers to an ester (acetate, propionate or palmitate) or a mixture of
synthetic retinol esters.
The activity of vitamin A is expressed in retinol equivalents (R.E.). 1 mg R.E. corresponds to
the activity of 1 mg of all-(E)-retinol. The activity of the other retinol esters is calculated
stoichiometrically, so that 1 mg R.E. of vitamin A corresponds to the activity of:
ıı
ı— 1.147 mg of all-(E)-retinol acetate;
ı— 1.195 mg of all-(E)-retinol propionate;
ı— 1.832 mg of all-(E)-retinol palmitate.
International Units (IU) are also used to express the activity of vitamin A. 1 IU of vitamin A is
equivalent to the activity of 0.300 μg of all-(E)-retinol. The activity of the other retinol esters is
calculated stoichiometrically, so that 1 IU of vitamin A is equivalent to the activity of:
ı— 0.344 μg of all-(E)-retinol acetate;
ı— 0.359 μg of all-(E)-retinol propionate;
ı— 0.550 μg of all-(E)-retinol palmitate;
1 mg of retinol equivalent is equivalent to 3333 IU.
CHARACTERS
Appearance
Retinol acetate: pale-yellow crystals (mp: about 60 °C). Once melted retinol acetate tends to
yield a supercooled melt.
Retinol propionate: reddish-brown oily liquid.
Retinol palmitate: a fat-like, light yellow solid or a yellow oily liquid, if melted (mp: about 26 °C)
.
Solubility
All retinol esters are practically insoluble in water, soluble or partly soluble in anhydrous
ethanol and miscible with organic solvents.
Vitamin A and its esters are very sensitive to the action of air, oxidising agents, acids, light
and heat.
Carry out the assay and all tests as rapidly as possible, avoiding exposure to actinic light and
air, oxidising agents, oxidation catalysts (e.g. copper , iron), acids and heat; use freshly
prepared solutions.
IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solutionıPrepare a solution containing about 3.3 IU of vitamin A per microlitre in
cyclohexane R containing 1 g/l of butylhydroxytoluene R.
Reference solutionıPrepare a 10 mg/ml solution of retinol esters CRS (i.e. 3.3 IU of each
ester per microlitre) in cyclohexane R containing 1 g/l of butylhydroxytoluene R.
PlateıTLC silica gel F254 plate R.
Mobile phaseıether R, cyclohexane R (20:80 V/V).
Applicationı3 μl.
DevelopmentıOver 2/3 of the plate.
DryingıIn air.
DetectionıExamine in ultraviolet light at 254 nm.
System suitabilityıReference solution:
ı
ı— the chromatogram shows the individual spots of the corresponding esters. The elution
order from bottom to top is: retinol acetate, retinol propionate and retinol palmitate.
ResultsıThe composition of esters is confirmed by the correspondence of the principal spot
or spots of the test solution with those obtained with the reference solution.
ıB. It complies with the test for related substances.
TESTS
Retinol
Thin-layer chromatography (2.2.27).
Test solutionıPrepare a solution in cyclohexane R, stabilised with a solution containing 1 g/l
of butylhydroxytoluene R, containing about 330 IU of vitamin A per microlitre.
Reference solutionıShake 1 ml of the test solution with 20 ml of 0.1 M tetrabutylammonium
hydroxide in 2-propanol for 2 min and dilute to 100 ml with cyclohexane R, stabilised with a
solution containg 1 g/l of butylhydroxytoluene R.
PlateıTLC silica gel F254 plate R.
Mobile phaseıether R, cyclohexane R (20:80 V/V).
Applicationı3 μl.
DevelopmentıOver a path of 15 cm.
DryingıIn air.
DetectionıExamine in ultraviolet light at 254 nm.
System suitabilityıReference solution:
ı
ı— in the chromatogram obtained no or only traces of the retinol esters are seen.
LimitıAny spot corresponding to retinol in the chromatogram obtained with the test solution is
not more intense than the spot in the chromatogram obtained with the reference solution (1.0
per cent).
Related substances
Ultraviolet and visible absorption spectrophotometry (2.2.25).
Test solutionıThe solution described under Activity.
Absorption maximumıAt 325 nm to 327 nm.
Absorbance ratios:ı
ı— A300/A326 = maximum 0.60;
ı— A350/A326 = maximum 0.54;
ı— A370/A326 = maximum 0.14.
The thresholds indicated under Related substances (Table 2034.-1) in the general
monograph Substances for pharmaceutical use (2034) do not apply.
ACTIVITY
The activity of the substance is determined in order to be taken into account for the
production of concentrates.
Dissolve 25-100 mg, weighed with an accuracy of 0.1 per cent, in 5 ml of pentane R and
dilute with 2-propanol R1 to a presumed concentration of 10 IU/ml to 15 IU/ml. Measure the
absorbance (2.2.25) at the absorption maximum at 326 nm. Calculate the activity of vitamin A
in International Units per gram from the expression:
A326
=
absorbance at 326 nm
m
=
mass of the substance to be examined, in grams
V
=
total volume to which the substance to be examined is diluted to give 10 IU/ml to
15 IU/ml
1900
=
factor to convert the specific absorbance of esters of retinol into International Units
per gram
STORAGE
In an airtight container , protected from light.
Once the container has been opened, its contents are to be used as soon as possible; any
part of the contents not used at once should be protected by an atmosphere of inert gas.
LABELLING
The label states:
ı
ı— the number of International Units per gram;
ı— the name of the ester or esters.
IMPURITIES
ıA. R = H, CO-CH3: kitols (Diels-Alder dimers of vitamin A),
ıB. (3E,5E,7E)-3,7-dimethyl-9-[(1Z)-2,6,6-trimethylcyclohex-2-enylidene]nona-1,3,5,7-
tetraene (anhydro-vitamin A),
ıC. (3E,5E,7E)-3,7-dimethyl-9-[(1Z)-2,6,6-trimethylcyclohex-2-enylidene]nona-3,5,7-trien-1-ol
(retro-vitamin A),
ıD. oxidation products of vitamin A.
Ph Eur
Ergocalciferol
General Notices
(Ph Eur monograph 0082)
C28H44Oıı396.7ıı50-14-6
Action and use
Vitamin D analogue (Vitamin D2).
Preparations
Calcium and Ergocalciferol Tablets
Ergocalciferol Injection
Ergocalciferol Tablets
When vitamin D2 is prescribed or demanded, Ergocalciferol shall be dispensed or supplied.
When calciferol or vitamin D is prescribed or demanded, Ergocalciferol or Colecalciferol shall
be dispensed or supplied.
Ph Eur
DEFINITION
Ergocalciferol contains not less than 97.0 per cent and not more than the equivalent of 103.0
per cent of (5Z,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3-ol.
1 mg of ergocalciferol is equivalent to 40 000 IU of antirachitic activity (vitamin D) in rats.
CHARACTERS
A white or slightly yellowish, crystalline powder or white or almost white crystals, practically
insoluble in water, freely soluble in alcohol, soluble in fatty oils. It is sensitive to air, heat and
light. Solutions in volatile solvents are unstable and are to be used immediately.
A reversible isomerisation to pre-ergocalciferol takes place in solution, depending on
temperature and time. The activity is due to both compounds.
IDENTIFICATION
Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with ergocalciferol CRS. Examine the substances prepared as discs.
TESTS
Specific optical rotation (2.2.7)
Dissolve 0.200 g rapidly and without heating in aldehyde-free alcohol R and dilute to 25.0 ml
with the same solvent. The specific optical rotation, determined within 30 min of preparing the
solution, is + 103 to + 107.
Reducing substances
Dissolve 0.1 g in aldehyde-free alcohol R and dilute to 10.0 ml with the same solvent. Add 0.5
ml of a 5 g/l solution of tetrazolium blue R in aldehyde-free alcohol R and 0.5 ml of dilute
tetramethylammonium hydroxide solution R. Allow to stand for exactly 5 min and add 1.0 ml of
glacial acetic acid R. Prepare a reference solution at the same time and in the same manner
using 10.0 ml of a solution containing 0.2 μg/ml of hydroquinone R in aldehyde-free alcohol R.
Measure the absorbance (2.2.25) of the two solutions at 525 nm using as the compensation
liquid 10.0 ml of aldehyde-free alcohol R treated in the same manner. The absorbance of the
test solution is not greater than that of the reference solution (20 ppm).
Ergosterol
Examine by thin-layer chromatography (2.2.27), using a TLC silica gel G plate R.
Test solutionıDissolve 0.25 g of the substance to be examined in ethylene chloride R
containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 5 ml with
the same solvent. Prepare immediately before use.
Reference solution (a)ıDissolve 0.10 g of ergocalciferol CRS in ethylene chloride R
containing 10 g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 2 ml with
the same solvent. Prepare immediately before use.
Reference solution (b)ıDissolve 5 mg of ergosterol CRS in ethylene chloride R containing 10
g/l of squalane R and 0.1 g/l of butylhydroxytoluene R and dilute to 50 ml with the same
solvent. Prepare immediately before use.
Reference solution (c)ıMix equal volumes of reference solution (a) and reference solution (b)
. Prepare immediately before use.
Apply to the plate 10 μl of the test solution, 10 μl of reference solution (a), 10 μl of reference
solution (b) and 20 μl of reference solution (c). Develop immediately, protected from light,
over a path of 15 cm using a mixture of equal volumes of cyclohexane R and peroxide-free
ether R, the mixture containing 0.1 g/l of butylhydroxytoluene R. Allow the plate to dry in air
and spray three times with antimony trichloride solution R1. Examine the chromatograms for 3
min to 4 min after spraying. The principal spot in the chromatogram obtained with the test
solution is initially orange-yellow and then becomes brown. In the chromatogram obtained
with the test solution, any slowly appearing violet spot (corresponding to ergosterol)
immediately below the principal spot is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.2 per cent). There is no spot in the chromatogram
obtained with the test solution that does not correspond to one of the spots in the
chromatograms obtained with reference solutions (a) and (b). The test is not valid unless the
chromatogram obtained with reference solution (c) shows two clearly separated spots.
ASSAY
Carry out the operations as rapidly as possible, avoiding exposure to actinic light and air.
Examine by liquid chromatography (2.2.29).
Test solutionıDissolve 10.0 mg of the substance to be examined without heating in 10.0 ml of
toluene R and dilute to 100.0 ml with the mobile phase.
Reference solution (a)ıDissolve 10.0 mg of ergocalciferol CRS without heating in 10.0 ml of
toluene R and dilute to 100.0 ml with the mobile phase.
Reference solution (b)ıDilute 1.0 ml of cholecalciferol for performance test CRS to 5.0 ml
with the mobile phase. Heat in a water-bath at 90 °C under a reflux condenser for 45 min and
cool.
The chromatographic procedure may be carried out using:
ı
ı— a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with a
suitable silica gel (5 μm),
ı— as mobile phase at a flow rate of 2 ml/min a mixture of 3 volumes of pentanol R and 997
volumes of hexane R,
ı— as detector a spectrophotometer set at 254 nm.
An automatic injection device or a sample loop is recommended. Inject a suitable volume of
reference solution (b). Adjust the sensitivity of the system so that the height of the principal
peak is at least 50 per cent of the full scale of the recorder. Inject reference solution (b) 6
times. When the chromatograms are recorded in the prescribed conditions, the approximate
relative retention times with reference to cholecalciferol are 0.4 for pre-cholecalciferol and 0.5
for trans-cholecalciferol. The relative standard deviation of the response for cholecalciferol is
not greater than 1 per cent and the resolution between the peaks corresponding to precholecalciferol
and trans-cholecalciferol is not less than 1.0. If necessary adjust the
proportions of the constituents and the flow rate of the mobile phase to obtain this resolution.
Inject a suitable volume of reference solution (a). Adjust the sensitivity of the system so that
the height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject
the same volume of the test solution and record the chromatogram in the same manner.
Calculate the percentage content of ergocalciferol from the expression:
m
=
mass of the substance to be examined in the test solution, in milligrams,
m
=
mass of ergocalciferol CRS in reference solution (a), in milligrams,
SD
=
area (or height) of the peak due to ergocalciferol in the chromatogram obtained
with the test solution,
S
D
=
area (or height) of the peak due to ergocalciferol in the chromatogram obtained
with reference solution (a).
STORAGE
Store in an airtight container , under nitrogen, protected from light, at a temperature between
2 °C and 8 °C.
The contents of an opened container are to be used immediately.
IMPURITIES
ıA. (5E,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3-ol (trans-vitamin D2),
ıB. (22E)-ergosta-5,7,22-trien-3-ol (ergosterol),
ıC. (9,10,22E)-ergosta-5,7,22-trien-3-ol (lumisterol2),
ıD. (6E,22E)-9,10-secoergosta-5(10),6,8(14),22-tetraen-3-ol (iso-tachysterol2),
ıE. (6E,22E)-9,10-secoergosta-5(10),6,8,22-tetraen-3-ol (tachysterol2).
Ph Eur
