Analysis of the quality of drugs from the alkaloids group derivatives of indole and purine
Indole – condensed system of pyrrole and benzene cycles which have two share atoms:
Indole is a structural basis of physostigmine, strychnine, reserpine alkaloids.
Group reaction on indole derivatives – Van-Urk’s reaction
In the base of reaction is the process of electrophyllic substitution. Reagent p-dimethylaminobenzaldehyde. Reaction conducts at the presence of conc. H2SO4 and FeCl3 as oxidant.
Derivatives of indole which have free 2 and 3 positions give this reaction. Reserpine gives this reaction by the opening of ring C in the presence of acids, as a result position 2 becomes free.
Product of reaction can exist in 2 forms. Color of the reaction product depends on the chemical structure of primer compounds conditions of the reaction. Van-Urk’s reaction can be hold with another aldehyde. So, for reserpine solution of vanillin in chloride acid is used.
Physostigmine salicylate (Physostigmini salicylas), Eserini salicylas
Salicylate of ester of methylcarbaminic acid and eseroline or 5-methylcarbaminoiloxy-1,3,1’-trimethyl-2,3,2’,3’-tetrahydropyrrole indole
Physostigmine– the main alkaloid of calabaric beans (Faba calabarica) – poisonous seeds of West African plant Physostigma venenosus , Fabaceae
Physical properties
Physostigmine salicylate – brilliant colorless or almost colourless prismatic crystals. Soluble in water, easily soluble in alcohol, practically insoluble in ether. Aqueous solutions are unstable. It melts at about 182 °C, with decomposition Optic active compound. When heated with water easy hydrolyze and therefore its solutions for parenteral usage produce in aseptic introductions. On the air and light product paints in the red color – pharmacological inactive rubrezerine formed.
Pharmacological action cased by the methylurethane group.
Proserine – white crystalline powder with bitter taste. Very easily soluble in water, easily soluble in alcohol and chloroform, ether. Hygroscopic. Becomes pink on the light.
Identification of Physostigmine salicylate
1. Melting point, the specific rotation.
2. Substance gives reaction to salicylates (2 reactions in SPU).
3. Total Pharmacopeial reaction on alkaloids (with Dragendorff’s reagent)
4. After evaporation of the preparation with ammonium forms blue residue (physostigmine base), which is dissolved in ethanol with formation of a blue solution which after the acidification by СН3СООН becomes red.
5. Drug solution in H2SO4 conc. gradually becomes yellow.
6. Erdman and Frede reagents with medication give reddish-yellow color, with HNO3 conc. – yellow color.
7. When heated with alkalis (and gradually when heated with water) physostigmine salycilate hydrolyzed and appears character odor methylamine:
8. At the heating with 0,1 % ninhydrine solution in conc. H2SO4 on the water bath at 60 0С during 10 min. And than after cooling solution have green fluorescence. Proserine gives blue fluorescence at this conditions.
9. At the gradually adding to the solution boric acid, 0,1 М solution of nitrate acid and sodium nitrite, after 1 min. add sodium hydroxide solution, violet color appears.
Assay
Physostigmine salicylate
1. Alkalimetriya, direct titration . The drug is dissolved in a mixture of ethanol and chloroform and titrated by 0,1 М NаОН to the pink color (indicator – phenolphthalein). Е = М.m.
2. Acidimetry ion-aqueous medium. The drug is dissolved in a mixture of chloroform and conc. CH3COOH, titrated by 0,1 М НClО4. For determination of the end-point use potentiometry. Equivalent point is fixed by potentiometric method. Е = М.m./2.
3. Complexonometry, reverse titration. As a stable titrant acetic-acidic solution of bismuth nitrate is used in the presence of potassium iodide. Scheme of reaction:
Product of the interaction is filtrated and an excess of reagent is titrated by 0,1 М sodium EDTA solution. Е = М.m.
STORAGE and USAGE of Physostigmine salicylate
In an airtight containers of dark glass, protected from light. Poison compound.
Cholinesterase inhibitor, myotic mean (atropine antagonist). Used for the treatment of glaucoma as 0,25-1% eye drops. Introduce subcutaneous 0,1% -1,0 solution the neuromuscular diseases (Alzheimer’s disease). H. d. – 0,0005 g, H. d. d. – 0,001 g.
Synthetic substitute of physostigmine
Proserine (Proserinum) Neostigmine methylsulfate*
N-(m–dimethylcarbamoiloxiphenyl)-N,N,N–trimethylammonium methylsulfate
Identification of proserine
1. Reaction to methylsulfate-ion. If after heating the drug with HNO3 conc. add solution of BaCl2, white precipitate falls (BaSO4).
2. With a solution of iodine preparation forms brown sediment of periodide.
At the heating of drug with alkali dissolution of urethane group takes place (m–dimethylaminophenol formed, which is detected by the condensation with diazotative sulfanylic acid – cherry-red color (azo-dyes)):
Assay
The modified K’yeldal method. The drug is boiled in a K’yeldal flask with NaOH. Dimethylamine, which evaporates, distilled with water vapor in the receiver with a solution of boric acid. Metaborate and tetraborate of dimethylamine formed, which are titrated by 0,1 М solution of HCl (mixed indicator). Е = М.m.
STORAGE and USAGE of proserine
In an airtight containers of dark glass, protected from light. Poison compound.
Substitute of physostigmine. Anticholinesterase, antimyasthenic means. Curare antagonist drugs. Used for treatment of myasthenia, paralysis, neuritis, atony of intestine and urinary bladder, glaucoma, for stimulating labor activity as 0,25-1% as eye drops.
Issue – tablets 0,015 g, amp. 0,05%-1,0.
H. d., internally – 0,015 g, H. d. d., internally – 0,05 g; H. d. subcutaneous – 0,002 g, H. d. d. s/c – 0,006 g.
Strychnine nitrate (Strychnini nitras)
Cycles АВ, BD, ED – derivatives of indole.
Cycle А – aromatic and strychnine can be nitrated and halogenated.
N19 – tertiary atom, has a base character and gives salts with acids.
N9 – is in the lactam group, which may be disclosed by the interaction with alcohol solution of KOH with formation of carboxyl and secondary amino-groups.
Strychnine can be found with brucine in the seeds of tropical plant Strychnos Nux Vomica (emetic nut)
Reserpine (Reserpinum)
11,17-dimethoxy-16-carbmethoxy-18(3’,4’,5’,–trimethoxibenzoyloxy)-alloyohimban
Reserpine molecule contains indole (АВ), dihydroquinolysidine (СD),
partially hydrogenated 3-carbolynic (АВС), hydrogenated isoquinoline (ED) cycles.
Reserpine contains in the roots of the plant Rauwolfia serpentina Benth
Physical properties
Strychnine nitrate – a colorless brilliant crystals with very bitter taste. Difficultly soluble in water and alcohol, easily soluble in boiling water, practically insoluble in ether.
Reserpine – colorless, white or slightly yellow, small crystals or crystalline powder with melting point 261-265°С. Insoluble in water, soluble in chloroform, acetone, pyridine and ether, darkening slowly on the exposure to
Light. Optic active compound. At the heating with acids or alkalis hydrolysis takes place (reserpinic acid, methanol, trimethoxybenzoic acid form).
Identification of strychnine nitrate
1. Pharmacopeial reaction on alkaloids.
2. Solution of the drug in H2SO4 conc. + crystal of K2Cr2O7 – formed the blue-violet strips which pass into the red and lilac-green.
3. Vitali-Moren’s reaction. At the interaction with HNO3 conc. drug becomes yellow (as opposed to brucine, which becomes blood-red) by nitration of benzene cycle А; after the evaporation of reaction product the residue gives with alcohol solution of КОН formed red-violet color.
4. Van-Urk’s reaction (on indole cycles). With 1% vanillin in glycerol in the presence of H2SO4 dil. Pink-violet color appears.
5. Reaction oitrate ions NO3–-:
6. а) SPU. The interaction with nitrobenzene in the presence of sulfate acid
7. Quantity of substance, listed in a separate article, add to the mixture of 0,1 ml of nitrobenzol R and 0,2 ml of sulfate acid R and after 5 min. cooled in ice water. Continuing to cool slowly and while stirring add 5 ml of water R, 5 ml of sodium hydroxide concentrated solution R NaOH, 5 ml of acetone R, shake and put for standing; the apper layer becomes dark purple.
8. b) SPU, N. Not discolors potassium permanganate
9. The solution of the substance, acidified by acid sulfate diluted R H2SO4, not discolors solution of 1 g/l potassium permanganate R (difference of nitrite ).
10. с) Not pharmacopeial reaction. Interaction with iron (ІІ) sulfate FeSO4 in the medium of conc. H2SO4; brown ring is formed (FeSO4×NO) (on the clock glass ):
11. 2 Strychnine•НNO3 + 6FeSO4 + 4H2SO4 = 2NO + 3Fe2(SO4)3 + (Strychnine)2•Н2SO4 + 4H2O
12. NO + Fe2+ + SO42– ® [Fe(NO)]SO4
Unpharmacopeial reaction. Interaction with diphenylamine in acidic medium. (conc. H2SO4), formed an bright blue organic dye:
diphenylbenzidine
Sulfimmonium salt of diphenylbenzidine (blue dye)
Identification of reserpine
1. Specific optical rotation (6 assymetric carbon atoms).
2. UV-spectroscopy (chromophor groups – indole and trimethoxybenzoatic acid – 2 maximum of absorption on UV-spectrum).
3. Reactions of indole cycle. With chloric water –purple, with KMnO4 – dark lilac, with vanillin in the presence of HCl – pink, with Н2О2 – yellow-lilac color.
4. Water solutions of reserpine in UV-light – blue fluorescence.
5. Alcohol solution of the preparation + H2SO4 + NaNO2 – green fluorescence.
6. With Frede reagent – red color, which goes to the blue.
7. On ester groups:
а) alkali hydrolysis;
б) hydroxame sample.
8. Van-Urk’s reaction. With p- dimethylaminobenzaldehyde + H2SO4 + СН3СООН – green coloring that goes into the red. With vanillin in chloride acid – pink color.
Assay
Strychnine nitrate – Alkalimetry, direct titration. Titration of the drug in alcohol-chloroform solution of 0,1 М NaOH (phenolphthalein indicator).
Е = М.m.
Specific additive – brucine.
Reserpine – Acidimetry ion-aqueous medium. Hatch is titrated in the medium of anhydrous СН3СООН by 0,1 М solution HClO4 (indicator crystal violet) to the appearance of green color. Е = М.m.
Storage, usage
Strychnine nitrate
In airtight containers. Poison compound.
CNS stimulant, tonic mean.
Issue – amp. 0,1%-1,0.
H. d. s/c – 0,002 g; H. d. d. s/c – 0,005 g.
Reserpine
In airtight containers, in a dark place. Powder – poisonous substance.
Neuroleptic , treatment of hypertension. Included in tablets: Adelphane (0.1 mg of reserpine,10 mg of dihydralasine), Adelphan – esidrex (Triresid) (Adelfane + 10 mg of dichlorothiazide), Adelphane – esidrex -К (Triresid К)(0,6 g of КСl), Crystepin, Neocrystepin. Raunatine–the amount of Rauwolfia alkaloids.
Alkaloids, purine derivatives
Purine –condensed system of pyrimidine and imidazol
If in the core of purine hydrogen atoms in the pyrimidine nucleus replaced by hydroxyl groups, we will get xantine :
Caffeine is contained in coffee beans (Coffea arabica), tea leaves (Thea sinensis), cola (Cola acuminata)
Pharmacology
Caffeine stimulates the central nervous system first at the higher levels, resulting in increased alertness and wakefulness, faster and clearer flow of thought, increased focus, and better general body coordination, and later at the spinal cord level at higher doses. Once inside the body, it has a complex chemistry, and acts through several mechanisms as described below.
Metabolism and half-life
Theophylline first was isolated from tea leaves (Thea sinensis)
Theobromine is extracted from cocoa beans (Theobroma cacao)
Three natural alkaloids, derivatives xantine: caffeine, theophylline, theobromine:
Extracted from semi-synthetic uric acid, guanine, and urea.
Very convenient is the method of extraction of caffeine and theobromine from xantine, which can be extracted from uric acid (waste poultry farms) and guanine (fish flakes, waste of paper):
Synthesis of caffeine and theophyllin by method Hmelevskiy – Abramova( firs was synthesed by Traube in1900 year).
Caffeine Medications
Caffeine (Соffеіnuт) , Caffeine monohydrate (Соffеіnuт monohydricum) (SPhU
1,3,7-Trimethyl-3,7-dihydro– 1H–purine-2,6-dione
1,3,7-trimethylxantine
• Caffeine–sodium benzoate (Coffeinum-natrii benzoas)
Physical properties
Caffeine – White or almost white, crystalline powder or silky crystals, sublimes readily. Moderately soluble in water, freely soluble in boiling water, slightly soluble in ethanol and ether. Soluble in the concentrated solutions of alkali benzoate or salicylates. Very weak base, forms unstable salts with acids by nitrogen in position 9.
Caffeine-sodiume benzoate – white powder, odorless, bitter taste.
Easily soluble in water, slightly soluble in ethanol. Contains 38-40% caffeine.
Extracted by the mixing and evaporation to the dry state of aqueous solutions containing equimolar quantity of caffeine and sodium benzoate.
General Pharmacopeial reaction – a reaction on xantines (Murexide reaction or reaction on purine alkaloids):
Identification of caffeine
By the physico-chemical constants: melting point, IR-spectroscopy.
With potassium iodide in iodine in the presence of HCl dil.- brown precipitate formed(periodide С8Н10N4О2•J2•HJ), which dissolves in NaOH dil. solution at the neutralization.
Murexide sample.
Unpharmacopoeial reaction – with 1% solution of tannin – white precipitate dissolved in excess of reagent.
With HgCl2 – white crystalline sediment, which is a complex compound with the following content C5H10N4O2· HgCl2.
With acetylacetone and dimethylaminobenzaldehyde. Solution of the substance in a mixture of acetylacetone and dil. NaOH heated in a water bath, cooled, than add solution of dimethylaminobenzaldehyde and heat again, cool and add water – appears an intense blue color:
Caffeine monohydrate gives all reactions on caffeine after a preliminary drying at 100-105 ° C.
Identification of caffeine-sodium benzoate
1. Caffeine identify by:
a) melting temperature (234-237 ° C) after extraction by chloroform from alkaline solution;
b) Murexide sample;
c) reaction with 1% solution of tannin;
d) reaction with iodine solution;
2. Sodium benzoate identify by:
e) the reaction with solution of iron (III) chloride – pink-yellow sediment;
f) the sodium cation paints the flame in yellow color.
Assay
Caffeine
1. Acidimetry ion-aqueous medium in a mixture of acetic acid anhydrous, acetic anhydride and toluene, a direct titration. Potentiometric indication, the control experiment (Е=М.m).
2. Iodometry, reverse titration, indicator – starch (Е=М.m/4).
3. Cerimetry, reverce titration, with iodometric ending. An excess of cerium is neutralized by potassium iodide solution. Sodium thiosulfate used as titrant. (Е=М.m/4).
Caffeine-sodium benzoate
1. Caffeine content is determined by iodometric method (Е=М.m/4). ). In the dry matter it should be 38,0 – 40,0 %.
2. Sodium is determined in the presence of mixed indicator (methyl orange solution and methylene blue at a ratio of 1:1) and ether (for the extraction of benzoic acid, available in the titration) (Е=М.m). Sodium benzoate in the dry matter must be not less than 58,0 % and not more than 62,0 %.
Cerimetric determination of caffeine
Storage, Usage
Caffeine
In a dry, dark place.
Central nervous system stimulant, cardiotonic mean, at the angiospasms; enuresis in children; stimulant of mental and physical disability, poisoning with drugs. Produced as powder.
Caffeine monohydrate is part of the tablets: Theophedrine, Cytramone, Cytropak, Askofene, Cofficyll, Cophetamine, Benalgin, Coldrex, Solpadein, Panadol-екстра. Apply in doses by 0,05-0,1 g as CNS stimulant.
Caffeine-sodium benzoate
In a dry, dark place.
Central nervous system stimulant, cardiotonic mean. Thanks to the solubility in water used in the form of injection solutions. Issue – powder, tablets 0,1 and 0,2 g, 0,075 g (for children); 10% і 20% solutions in amp. by 1,0-2,0 ml. Included in the tablets: Anaprylline, Pentalgin.
Theobromine Theobrominum (SPhU)
3,7-Dimethyl-3,7- dihydro-1Н–purine-2,6-dione, or 3,7- dimethylxantine
Theophylline monohydrate Theophyllinum monohydricum (SPhU)
1,3-Dimethyl-3,7- dihydro-1Н–purine-2,6-dione monohydrate, or monohydrate of 1,3- dimethylxantine
Properties
Theobromine
White crystalline powder, with bitter taste. very slightly soluble in water, ethanol ,ether and chloroform; slightly soluble in hot water; easily in dil. Solutions of alkalis and acids.
Theobromine and theophylline – amphoteric compounds with a predominance of acidic properties (by moving the hydrogen atom at the nitrogen atom in position 1 or 7).
Theophylline
White crystalline powder. Sightly soluble in water, ethanol and chloroform; easily soluble in hot water; soluble in dil. Solutions of acids and alkalis.
Identification of theobromine
1. IR-spectrophotometry.
2. Dissolve the substance in ammonium solution at the heating. After cooling add silver nitrate solution – solution must still be colorless. After the boiling during few minutes white precipitate formed.
3. Reaction on xantines (murexide sample). At the oxidation of theobromine 3-methylalloxane and methylurea formed. Ammonium salt of dimethylpuepuric acid formed as a result of murexide reaction:
Unpharmacopoeial reactions
4. Reaction of the sodium salt of theobromine, obtained by the interaction of alkali with an excess of preparation, with cobalt (II)chloride solution – intense violet color appears, which quickly disappears, grey-blue precipitate:
5. The reaction of theobromine sodium salt with silver nitrate – formed a dense gelatin mass (silver salt), which is thinning by heating to 80° С and solidifies again at the cooling.
6. With HgCl2 – white crystalline precipitate.
Identification of theophylline
Determination of the melting temperature alter the drying.
IR spectrophotometry.
Theophylline in the alkali medium decomposes to teophyllidine, which can be identified by the reaction of azojoining with diazonium salts, red color azo-dye formed.
4. Determination of the water by semimicromethod (К. Fisher) (8-9,5 %).
5. Reaction on xantines (murexide sample). At the oxidation of theophylline 1,3-dimethylalloxane and urea. Ammonium salt of tetramethylpuepuric acid forme as a result of murexide reaction:
2. The reaction of theobromine sodium salt obtained by the interaction of alkali with an excess of drug, with a solution of cobalt (II) chloride – formed white with pink tinge precipitate of cobalt salt.
2. With HgCl2 –white crystalline precipitate.
3. With alkali solution of sodium nitroprusside green color formed dissappears at the adding an excess of acid.
4. The reaction of theobromine sodium salt with silver nirate – formed a dense gelatin mass.
5. Theophylline with 2,6-dichloroqiunonechloroimide in borate buffer solution (рН 8,5) gives intense blue merocyanic dye:
Assay
Theophylline and Theobromine
Alkalimetry by the substituent (indirect alkalimetry).
Indicator – phenolphthalein (theobromine) or Bromothymol blue (theophylline). Е = М.m.
Storage, usage
Theobromine
In airtight containers, place protected from light.
Stimulates the activity of the heart, somewhat expands the coronary vessels and bronchi, shows diuretic effect. Issue – powder and tablets by 0,25 g.
Included in tablets: Theminal (with amidopyrine and Phenobarbital), Theodibaverine (with papaverine and dibazole), Theoephedrine.
Theophylline
In airtight containers, place protected from light.
Non-selective phosphodiesterase inhibitor (xanthine); treatment of reversible airways obstruction. Broncholitic, cardiotonic and diuretic mean with moderate influence on the stagnation phenomena in the cardiac and renal origin organs. Issue – powder and tablets by 0,1 and 0,3 g; amp. 2%-5,0; candles by 0,2 g. Teopek, Theotard, Neophylline, Euphylline. Included in tablets: Theoephedrine.
Pentoxifylline (Pentoxifyllinium)
Agapurine, Pentyline, Trental
A synthetic analogue of theobromine
3,7-dimethyl-1-(5’–oxohexyl)-3,7-dihydro-7Н–purine-2,6-dion or 1-(5’–oxohexyl)-theobromine
Identification of pentoxifylline
Temperature of melting
IR-spectroscopy
TLC
Reaction on xantines
Formation of azo-dye (look theophylline)
Assay
Pentoxifylline
Acidimetry ion-aqueous media in a mixture of anhydrous acetic acid and acetic anhydride, direct titration. Potentiometric indication. (Е=М.m).
Usage of pentoxifylline
Has a vasodilator effect, improves tissue oxygen supply, decreases thrombosis aggregation and reduces blood viscosity. Apply at the peripheral circulatory disorders, atherosclerotic disorders, ischemic condition after heart attack, in ophthalmology, at the hearing disorders. Issue – tablets of 0,1 g, ampoules of 2%-5,0. Adopt inside by 0,2 g 3 times daily after meals. In the acute disorders of peripheral or cerebral circulation injected 0,1 g intravenous in 250-500 ml of NaCl or 5% glucose solution.
Synthetic analogues of theophylline
Theophylline–ethylenediamine (Theophyllinum et ethylenediaminum) (SPhU),
Euphylline (Euphyllinum), Aminophilline
Theophylline with 1,2-ethylenediamine
White, sometimes with yellowish crystalline powder with slight ammonia odor. On the air absorbs carbon dioxide, thus decreasing its solubility. Soluble in water, aqueous solutions have an alkaline reaction.
Identification of euphylline
Theophylline is identified after the separation by HCl of ethylenediamine according to SPhU:
а) By the melting temperature of theophylline (269 – 274°С) after HCl acidification to рН 4-5;
b) IR-spectroscopy;
c) Reaction of azojoining (look theophylline);
d) murexide sample.
Ethylenediamine can be confirmed by the following reactions:
а) determination of the melting temperature of the product of reaction with benzoyl in alkali medium (dibenzoylethylenediamine) (SPhU):
b) with a solution of copper (II) sulfate a bright purple color forms :
c) with 2,4-dinitrochlorobenzene –yellow precipitate falls.
Assay
Euphylline
Ethylenediamine can be determined by acidimetry, indicator –methyl orange. Е = М.m./2.
Ethylenediamine in euphylline should be 13,5—15 % in the dry matter.
2.Theophylline can be determined by alkalimetry by substituent 1 after drying in a drying cabinet at 25-130 °С to the disappearance of amine odor.
The content of waterless theophylline in euphylline should be 84- 87,4 %.
Storage, usage of euphylline
Given the ability to absorb carbon dioxide, stored in a well corked filled to the end container, protected from the effects of light and moisture.
Antispasmodic, bronchodilatin, diuretic mean. At the bronchial asthma and bronchospasm, hypertension, cardiac asthma, to improve blood circulation to the brain, decreasing the intraperitoneal pressure and brain edema in ischemic stroke.
Used oral by 0,15g after food, i/v (2,4% solutions by 5,0) and i/m (24 % solution by 1 ml).
Diprophylline (Diprophyllinum)
7-(2′,3′-Dioxipropyl)-theophylline
Less toxic than theophylline. Used for treatment of coronary spasm, cardiac and bronchial asthma, hypertension. Issue – tablets by 0,2 g; amp. 10%-5,0; candles by 0,5 g.
Xantinole nicotinate (Xantinoli nicotinas) Complamine, Theonicol
7-[2’–oxi-3’-(N‘-methyl-β-oxiethylamino)-propyl]-theophylline nicotinate
Used to improve peripheral and cerebral circulation
Issue – tablets by 0,15 g; amp. 15%-2,0 і 10,0.
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Physostigmine Salicylate
General Notices
(Ph Eur monograph 0286)
C15H21N3O2,C7H6O3
ıı413.5ıı57-64-7
Action and use
Cholinesterase inhibitor.
Ph Eur
DEFINITION
Physostigmine salicylate contains not less than 98.5 per cent and not more than the
equivalent of 101.0 per cent of (3aS,8aR)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,
3-b]indol-5-yl methylcarbamate salicylate, calculated with reference to the dried substance.
CHARACTERS
Colourless or almost colourless crystals, sparingly soluble in water, soluble in alcohol. The
crystals gradually become red when exposed to air and light; the colour develops more
quickly when the crystals are also exposed to moisture. Aqueous solutions are unstable.
It melts at about
IDENTIFICATION
First identificationıA, B.
Second identificationıB, C, D.
ıA. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with physostigmine salicylate CRS.
ıB. Examine the chromatograms obtained in the test for related substances. 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. Heat about 10 mg in a porcelain dish with a few drops of dilute ammonia R1. An orange
colour develops. Evaporate the solution to dryness. The residue dissolves in alcohol R
giving a blue solution. Add 0.1 ml of glacial acetic acid R. The colour becomes violet. Dilute with water R. An intense red fluorescence appears.
ıD. Solution S (see Tests) gives reaction (a) of salicylates (2.3.1).
TESTS
Solution S
Dissolve
distilled water R and dilute to 100.0 ml with the same solvent. Prepare immediately before
use.
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 5.1 to 5.9.
Specific optical rotation (2.2.7)
– 90 to – 94, determined on solution S and calculated with reference to the dried substance.
Related substances
Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Test solution (a)ıDissolve
ml with the same solvent.
Test solution (b)ıDilute 2.5 ml of test solution (a) to 50 ml with alcohol R.
Reference solution (a)ıDissolve 10 mg of physostigmine salicylate CRS in alcohol R and
dilute to 10 ml with the same solvent.
Reference solution (b)ıDilute 2 ml of reference solution (a) to 20 ml with alcohol R.
Apply to the plate 20 μl of each solution. Develop over a path of
volumes of concentrated ammonia R, 23 volumes of 2-propanol R and 100 volumes of
cyclohexane R. Dry the plate in a current of cold air and carry out a second development in
the same direction. Allow the plate to dry in air and spray with freshly prepared potassium
iodobismuthate solution R and then with dilute hydrogen peroxide solution R. Examine the
plate within 2 min. Any spot in the chromatogram obtained with test solution (a), apart from
the principal spot, is not more intense than the spot in the chromatogram obtained with
reference solution (b) (0.5 per cent).
Eseridine
To 5 ml of solution S add a few crystals of potassium iodate R, 0.05 ml of dilute hydrochloric
acid R and 2 ml of chloroform R. Shake. No violet colour develops in the chloroform layer
within 1 min.
Sulphates (2.4.13)
15 ml of solution S complies with the limit test for sulphates (0.1 per cent).
Loss on drying (2.2.32)
Not more than 1.0 per cent, determined on
Sulphated ash (2.4.14)
Not more than 0.1 per cent, determined on the residue obtained in the test for loss on drying.
ASSAY
Dissolve
chloroform R. Titrate with
(2.2.20).
1 ml of
STORAGE
Store in an airtight container , protected from light.
Physostigmine Sulphate
General Notices
(Ph Eur monograph 0684)
(C15H21N3O2)2,H2SO4
ıı648.8ıı64-47-1
Action and use
Cholinesterase inhibitor.
Ph Eur
DEFINITION
Physostigmine sulphate contains not less than 97.0 per cent and not more than the equivalent
of 101.0 per cent of di[(3aS,8aR)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-
5-yl methylcarbamate] sulphate, calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, hygroscopic, very soluble in water, freely soluble
in alcohol. It gradually becomes red when exposed to air and light; the colour develops more
quickly when the substance is also exposed to moisture. Aqueous solutions are unstable.
It melts at about
IDENTIFICATION
First identificationıA, D.
Second identificationıB, C, D.
ıA. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with physostigmine sulphate CRS. Examine the substances prepared as discs
using potassium bromide R.
ıB. Examine the chromatograms obtained in the test for related substances. 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. Heat about 10 mg in a porcelain dish with 0.5 ml of dilute ammonia R1. An orange colour
develops. Evaporate the solution to dryness. The residue dissolves in alcohol R giving a
blue solution. Add 0.1 ml of glacial acetic acid R. The colour becomes violet. Dilute with
water. An intense red fluorescence appears.
ıD. It gives reaction (a) of sulphates (2.3.1).
TESTS
Solution S
Dissolve
same solvent. Prepare immediately before use.
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 3.5 to 5.5.
Specific optical rotation (2.2.7)
– 116 to – 120, determined on solution S and calculated with reference to the dried substance.
Related substances
Examine by thin-layer chromatography (2.2.27), using silica gel G R as the coating
substance.
Test solution (a)ıDissolve
ml with the same solvent.
Test solution (b)ıDilute 1 ml of test solution (a) to 10 ml with alcohol R.
Reference solution (a)ıDissolve 30 mg of physostigmine sulphate CRS in alcohol R and
dilute to 10 ml with the same solvent.
Reference solution (b)ıDilute 5 ml of test solution (b) to 100 ml with alcohol R.
Apply separately to the plate 10 μl of each solution. Develop over a path of
mixture of 2 volumes of concentrated ammonia R, 23 volumes of 2-propanol R and 100
volumes of cyclohexane R. Dry the plate in a current of cold air and carry out a second
development in the same direction. Allow the plate to dry in air and spray with freshly
prepared potassium iodo-bismuthate solution R and then with dilute hydrogen peroxide
solution R. Examine the plate within 2 min. Any spot in the chromatogram obtained with test
solution (a), apart from the principal spot, is not more intense than the spot in the
chromatogram obtained with reference solution (b) (0.5 per cent).
Eseridine
To 5 ml of solution S add a few crystals of potassium iodate R, 0.05 ml of dilute hydrochloric
acid R and 2 ml of chloroform R and shake. After 1 min, the chloroform layer is not more
intensely coloured than a reference solution prepared at the same time in the same manner
using 5 ml of water R instead of solution S.
Loss on drying (2.2.32)
Not more than 1.0 per cent, determined on
Sulphated ash (2.4.14)
Not more than 0.1 per cent, determined on the residue obtained in the test for loss on drying.
ASSAY
Dissolve
anhydride R. Titrate with
(2.2.20) at the first inflexion point.
1 ml of
STORAGE
Store in a well-filled, airtight glass container, protected from light.
Reserpine
General Notices
(Ph Eur monograph 0528)
C33H40N2O9
ıı609ıı50-55-5
Action and use
Rauwolfia alkaloid; treatment of hypertension.
Ph Eur
DEFINITION
Methyl 11,17-dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]-3,20-yohimban-16–
carboxylate.
Content:
ı
ı— reserpine: 98.0 per cent to 102.0 per cent (dried substance);
ı— total alkaloids: 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
White or slightly yellow, small crystals or crystalline powder, darkening slowly on exposure to
light.
Solubility
Practically insoluble in water, very slightly soluble in ethanol (96 per cent).
IDENTIFICATION
First identificationıB.
Second identificationıA, C, D, E.
ıA. Ultraviolet and visible absorption spectrophotometry (2.2.25).
Test solutionıDissolve 20.0 mg in chloroform R and dilute to 10.0 ml with the same solvent.
Dilute 1.0 ml of this solution to 100.0 ml with ethanol (96 per cent) R. Examine immediately.
Spectral rangeı230-350 nm.
Absorption maximumıAt 268 nm.
Specific absorbance at the absorption maximumı265 to 285.
Over the range 288-295 nm, the curve shows a slight absorption minimum followed by a
shoulder or a slight absorption maximum; over this range, the specific absorbance is about
170.
ıB. Infrared absorption spectrophotometry (2.2.24).
PreparationıDiscs.
Comparisonıreserpine CRS.
ıC. To about 1 mg add 0.1 ml of a 1 g/l solution of sodium molybdate R in sulphuric acid R. A
yellow colour is produced which becomes blue within 2 min.
ıD. To about 1 mg add 0.2 ml of a freshly prepared 10 g/l solution of vanillin R in hydrochloric
acid R. A pink colour develops within 2 min.
ıE. Mix about 0.5 mg with 5 mg of dimethylaminobenzaldehyde R and 0.2 ml of glacial acetic
acid R and add 0.2 ml of sulphuric acid R. A green colour is produced. Add 1 ml of glacial
acetic acid R. The colour becomes red.
TESTS
Specific optical rotation (2.2.7)
– 116 to – 128 (dried substance).
Dissolve
immediately.
Oxidation products
Dissolve 20 mg in glacial acetic acid R and dilute to 100.0 ml with the same acid. The
absorbance (2.2.25) measured immediately at the absorption maximum at 388 nm is not
greater than 0.10.
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on
pentoxide R at a pressure not exceeding 667 Pa for 3 h.
Sulphated ash (2.4.14)
Maximum 0.1 per cent, determined on
ASSAY
Total alkaloids
Dissolve
R. Titrate with
1 ml of
Reserpine
Protect the solutions from light. Moisten 25.0 mg with 2 ml of ethanol (96 per cent) R, add 2
ml of
Cool and dilute to 100.0 ml with ethanol (96 per cent) R. Dilute 5.0 ml of this solution to 50.0
ml with ethanol (96 per cent) R. Prepare a reference solution in the same manner using 25.0
mg of reserpine CRS. Place 10.0 ml of each solution separately in 2 boiling-tubes, add 2.0 ml
of
and heat in a water-bath at
solution of sulphamic acid R and dilute to 25.0 ml with ethanol (96 per cent) R. Measure the
absorbance (2.2.25) of each solution at the absorption maximum at 388 nm, using as the
compensation liquid 10.0 ml of the same solution prepared at the same time in the same
manner, but omitting the sodium nitrite.
Calculate the content of C33H40N2O9 from the absorbances measured and the concentrations
of the solutions.
STORAGE
Protected from light.
Caffeine Hydrate
General Notices
(Caffeine Monohydrate, Ph Eur monograph 0268)
C8H10N4O2,H2Oıı212.2ıı5743-12-4
Action and use
Central nervous system stimulant.
Ph Eur
DEFINITION
Caffeine monohydrate contains not less than 98.5 per cent and not more than the equivalent
of 101.5 per cent of 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated with reference
to the dried substance.
CHARACTERS
White or almost white, crystalline powder or silky crystals, sublimes readily, sparingly soluble
in water, freely soluble in boiling water, slightly soluble in ethanol. It dissolves in concentrated
solutions of alkali benzoates or salicylates.
IDENTIFICATION
First identificationı A, B, E.
Second identificationı A, C, D, E, F.
ıA. Melting point (2.2.14)
ıB. Dry the substance to be examined at 100-
spectrophotometry (2.2.24) , comparing with the spectrum obtained with caffeine CRS .
ıC. To 2 ml of a saturated solution add 0.05 ml of iodinated potassium iodide solution R .
The solution remains clear. Add 0.1 ml of dilute hydrochloric acid R . A brown precipitate is
formed. Neutralise with dilute sodium hydroxide solution R . The precipitate dissolves.
ıD. In a glass-stoppered tube, dissolve about 10 mg in 0.25 ml of a mixture of 0.5 ml of
acetylacetone R and 5 ml of dilute sodium hydroxide solution R . Heat in a water-bath at 80
°C for 7 min. Cool and add 0.5 ml of dimethylaminobenzaldehyde solution R2 . Heat again
in a water-bath at
colour develops.
ıE. It complies with the test for loss on drying (see Tests).
ıF. It gives the reaction of xanthines (2.3.1) .
TESTS
Solution S.
Dissolve
water R, cool and dilute to 50 ml with the same solvent.
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
Acidity
To 10 ml of solution S add 0.05 ml of bromothymol blue solution R1 . The solution is green or
yellow. Not more than 0.2 ml of
the indicator to blue.
Related substances
Examine by thin-layer chromatography (2.2.27) using silica gel GF 254 R as the coating
substance.
Test solutionı Dissolve
methanol R and 6 volumes of methylene chloride R and dilute to 10 ml with the same
mixture of solvents.
Reference solutionı Dilute 0.5 ml of the test solution to 100 ml with a mixture of 4 volumes of
methanol R and 6 volumes of methylene chloride R .
Apply to the plate 10 μl of each solution. Develop over a path of
volumes of concentrated ammonia R , 30 volumes of acetone R , 30 volumes of methylene
chloride R and 40 volumes of butanol R . Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart
from the principal spot, is not more intense than the spot in the chromatogram obtained with
the reference solution (0.5 per cent).
Sulphates (2.4.13)
15 ml of solution S complies with the limit test for sulphates (500 ppm). Prepare the standard
using a mixture of 7.5 ml of sulphate standard solution (10 ppm SO 4 ) R and 7.5 ml of
distilled water R.
Heavy metals (2.4.8)
lead standard solution (10 ppm Pb) R .
Loss on drying (2.2.32)
5.0 per cent to 9.0 per cent, determined on
Sulphated ash (2.4.14)
Not more than 0.1 per cent, determined on
ASSAY
Dissolve
acid R . Allow to cool, add 10 ml of acetic anhydride R and 20 ml of toluene R . Titrate with
1 ml of
IMPURITIES
Specified impuritiesı A.
Other detectable impuritiesı B, C.
ıA. theophylline,
ıB. N-[6-amino-1,3-dimethyl-2,4(1H, 3H)-dioxopyrimidin-5-yl]formamide,
ıC. 1,3,9-trimethyl-3,9-dihydro-1H-purine-2,6-dione (isocaffeine).
Ph Eur
Caffeine
General Notices
Anhydrous Caffeine
(Ph Eur monograph 0267)
C8H10N4O2 ıı194.2ıı 58-08-2
Action and use
Central nervous system stimulant.
Preparations
Aspirin and Caffeine Tablets
Caffeine Citrate Injection
Caffeine Citrate Oral Solution
Paracetamol, Codeine Phosphate and Caffeine Capsules
Paracetamol, Codeine Phosphate and Caffeine Tablets
Ph Eur
DEFINITION
1,3,7-Trimethyl-3,7-dihydro-1H-purine-2,6-dione.
Content
98.5 per cent to 101.5 per cent (dried substance).
CHARACTERS
Appearance
White or almost white, crystalline powder or silky, white or almost white, crystals.
Solubility
Sparingly soluble in water, freely soluble in boiling water, slightly soluble in ethanol (96 per
cent). It dissolves in concentrated solutions of alkali benzoates or salicylates.
It sublimes readily.
IDENTIFICATION
First identificationı A, B, E.
Second identificationı A, C, D, E, F.
ıA. Melting point (2.2.14):
ıB. Infrared absorption spectrophotometry (2.2.24).
Comparisonı caffeine CRS.
ıC. To 2 ml of a saturated solution add 0.05 ml of iodinated potassium iodide solution R .
The solution remains clear. Add 0.1 ml of dilute hydrochloric acid R ; a brown precipitate is
formed. Neutralise with dilute sodium hydroxide solution R ; the precipitate dissolves.
ıD. In a ground-glass-stoppered tube, dissolve about 10 mg in 0.25 ml of a mixture of 0.5 ml
of acetylacetone R and 5 ml of dilute sodium hydroxide solution R . Heat in a water-bath at
again in a water-bath at
intense blue colour develops.
ıE. Loss on drying (see Tests).
ıF. It gives the reaction of xanthines (2.3.1).
TESTS
Solution S
Dissolve
water R , cool and dilute to 50 ml with the same solvent.
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
Acidity
To 10 ml of solution S add 0.05 ml of bromothymol blue solution R1 ; the solution is green or
yellow. Not more than 0.2 ml of
Related substances
Liquid chromatography (2.2.29).
Test solutionı Dissolve
dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile
phase.
Reference solution (a)ı Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase.
Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.
Reference solution (b)ı Dissolve 5 mg of caffeine for system suitability CRS (containing
impurities A, C, D and F) in the mobile phase and dilute to 5 ml with the mobile phase. Dilute
2 ml of this solution to 10 ml with the mobile phase.
Column: ı
ı— size: l =
ı— stationary phase: base-deactivated end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phaseı Dissolve
2000 ml with the same solvent. Adjust 1910 ml of this solution to pH 4.5 with glacial acetic
acid R and add 50 ml of acetonitrile R and 40 ml of tetrahydrofuran R.
Flow rateı 1.0 ml/min.
Detectionı Spectrophotometer at 275 nm.
Injectionı 10 μl.
Run timeı 1.5 times the retention time of caffeine.
Identification of impuritiesı Use the chromatogram supplied with caffeine for system
suitability CRS and the chromatogram obtained with reference solution (b) to identify the
peaks due to impurities A, C, D and F.
Retention timeı Caffeine = about 8 min.
System suitabilityı Reference solution (b):
ı— resolution: minimum 2.5 between the peaks due to impurities C and D and minimum 2.5
between the peaks due to impurities F and A.
Limits:
ı— unspecified impurities: for each impurity, not more than 0.5 times the area of the principal
peak in the chromatogram obtained with reference solution (a) (0.10 per cent);
ı— total: not more than 0.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.1 per cent);
ı— disregard limit: 0.25 times the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.05 per cent).
Sulphates (2.4.13)
Maximum 500 ppm, determined on 15 ml of solution S.
Prepare the standard using a mixture of 7.5 ml of sulphate standard solution (10 ppm SO 4 ) R
and 7.5 ml of distilled water R.
Heavy metals (2.4.8)
Maximum 20 ppm.
solution (10 ppm Pb) R.
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on
Sulphated ash (2.4.14)
Maximum 0.1 per cent, determined on
ASSAY
Dissolve
acetic anhydride R and 20 ml of toluene R . Titrate with
the end-point potentiometrically (2.2.20).
1 ml of
IMPURITIES
Other detectable impurities ı (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): A, B, C, D, E, F.
ıA. theophylline,
ıB. N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)formamide,
ıC. 1,3,9-trimethyl-3,9-dihydro-1H-purine-2,6-dione (isocaffeine),
ıD. R = H, R= CH3: theobromine,
ıF. R = CH3, R= H: 1,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione,
ıE. N,1-dimethyl-4-(methylamino)-1H-imidazole-5-carboxamide (caffeidine).
Ph Eur
Theobromine
General Notices
(Ph Eur monograph 0298)
C7H8N4O2 ıı180.2ıı 83-67-0
Action and use
Non-selective phosphodiesterase inhibitor (xanthine); treatment of reversible airways
obstruction.
Ph Eur
DEFINITION
Theobromine contains not less than 99.0 per cent and not more than the equivalent of 101.0
per cent of 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione, calculated with reference to the
dried substance.
CHARACTERS
A white or almost white powder, very slightly soluble in water and in ethanol, slightly soluble in
ammonia. It dissolves in dilute solutions of alkali hydroxides and in mineral acids.
IDENTIFICATION
First identificationı A, C.
Second identificationı B, C.
ıA. Examine by infrared absorption spectrophotometry (2.2.24) , comparing with the
spectrum obtained with theobromine CRS .
ıB. Dissolve about 20 mg in 2 ml of dilute ammonia R1 , warming slightly, and cool. Add 2 ml
of silver nitrate solution R2 . The solution remains clear. Boil the solution for a few minutes.
A white, crystalline precipitate is formed.
ıC. It gives the reaction of xanthines (2.3.1) .
TESTS
Acidity
To
of bromothymol blue solution R1 . The solution is yellow or yellowish-green. Not more than
0.2 ml of
Related substance
Examine by thin-layer chromatography (2.2.27) , using silica gel GF 254 R as the coating
substance.
Test solutionı To
mixture of 4 volumes of methanol R and 6 volumes of chloroform R . Heat under a reflux
condenser on a water-bath for 15 min, shaking occasionally. Cool and filter.
Reference solutionı Dissolve 5 mg of theobromine CRS in a mixture of 4 volumes of
methanol R and 6 volumes of chloroform R and dilute to 50 ml with the same mixture of
solvents.
Apply separately to the plate 10 μl of each solution. Develop over a path of
mixture of 10 volumes of concentrated ammonia R , 30 volumes of acetone R, 30 volumes of
chloroform R and 40 volumes of butanol R . Allow the plate to dry in air and examine in
ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart
from the principal spot, is not more intense than the spot in the chromatogram obtained with
the reference solution (0.5 per cent).
Heavy metals (2.4.8)
lead standard solution (10 ppm Pb) R .
Loss on drying (2.2.32)
Not more than 0.5 per cent, determined on
Sulphated ash (2.4.14)
Not more than 0.1 per cent, determined on
ASSAY
Dissolve
silver nitrate . Using 1 ml of phenolphthalein solution R as indicator, titrate with
sodium hydroxide until a pink colour is obtained.
1 ml of
Ph Eur
Theophylline Hydrate
General Notices
(Theophylline Monohydrate, Ph Eur monograph 0302)
C7H8N4O2,H2Oıı198.2ıı 5967-84-0
Action and use
Non-selective phosphodiesterase inhibitor (xanthine); treatment of reversible airways
obstruction.
Preparations
Aminophylline Injection
Prolonged-release Theophylline Tablets
Ph Eur
DEFINITION
1,3-Dimethyl-3,7-dihydro-1H-purine-2,6-dione monohydrate.
Content
99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance
White or almost white, crystalline powder.
Solubility
Slightly soluble in water, sparingly soluble in ethanol. It dissolves in solutions of alkali
hydroxides, in ammonia and in mineral acids.
IDENTIFICATION
First identificationı B, D.
Second identificationı A, C, D, E.
ıA. Melting point (2.2.14) :
ıB. Infrared absorption spectrophotometry (2.2.24) .
Preparationı Dry the substance to be examined at 100-
Comparisonı Ph. Eur. reference spectrum of theophylline.
ıC. Heat 10 mg with 1.0 ml of a 360 g/l solution of potassium hydroxide R in a water-bath at
slowly develops. Carry out a blank test.
ıD. It complies with the test for water (see Tests).
ıE. It gives the reaction of xanthines (2.3.1) .
TESTS
Solution S
Dissolve
same solvent.
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
Acidity
To 50 ml of solution S add 0.1 ml of methyl red solution R . The solution is red. Not more than
1.0 ml of
Related substances
Liquid chromatography (2.2.29) .
Test solutionı Dissolve 40.0 mg of the substance to be examined in the mobile phase and
dilute to 20.0 ml with the mobile phase.
Reference solution (a)ı Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase.
Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.
Reference solution (b)ı Dissolve 10 mg of theobromine R in the mobile phase, add 5 ml of
the test solution and dilute to 100 ml with the mobile phase. Dilute 5 ml of this solution to 50
ml with the mobile phase.
Column: ı
ı— size: l =
ı— stationary phase: octadecylsilyl silica gel for chromatography R (7 μm).
Mobile phaseı Mix 7 volumes of acetonitrile for chromatography R and 93 volumes of a
1.36 g/l solution of sodium acetate R containing 5.0 ml/l of glacial acetic acid R .
Flow rateı 2.0 ml/min.
Detectionı Spectrophotometer at 272 nm.
Injectionı 20 μl.
Run timeı 3.5 times the retention time of theophylline.
Relative retentionı With reference to theophylline (retention time = about 6 min): impurity C =
about 0.3; impurity B = about 0.4; impurity D = about 0.5; impurity A = about 2.5.
System suitabilityı Reference solution (b):
ı— resolution: minimum 2.0 between the peaks due to theobromine and theophylline.
Limits:
ı— impurities A, B, C, D: for each impurity, not more than the area of the principal peak in
the chromatogram obtained with reference solution (a) (0.1 per cent);
ı— any other impurity: for each impurity, not more than the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.1 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: 0.5 times the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.05 per cent).
Heavy metals (2.4.8)
Maximum 20 ppm.
ppm Pb) R.
Water (2.5.12)
8.0 per cent to 9.5 per cent, determined on
Sulphated ash (2.4.14)
Maximum 0.1 per cent, determined on
ASSAY
Dissolve
of bromothymol blue solution R1 . Titrate with
1 ml of
IMPURITIES
Specified impuritiesı A, B, C, D.
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): E, F.
ıA. caffeine,
ıB. 3-methyl-3,7-dihydro-1H-purine-2,6-dione,
ıC. N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)formamide,
ıD. N-methyl-5-(methylamino)-1H-imidazole-4-carboxamide,
ıE. 1,3-dimethyl-7,9-dihydro-1H-purine-2,6,8(3H)-trione,
ıF. etofylline.
Theophylline
General Notices
(Ph Eur monograph 0299)
C7H8N4O2 ıı180.2ıı 58-55-9
Action and use
Non-selective phosphodiesterase inhibitor (xanthine); treatment of reversible airways
obstruction.
Preparations
Aminophylline Injection
Prolonged-release Theophylline Tablets
Ph Eur
DEFINITION
1,3-Dimethyl-3,7-dihydro-1H-purine-2,6-dione.
Content
99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white crystalline powder.
Solubility
Slightly soluble in water, sparingly soluble in ethanol. It dissolves in solutions of alkali
hydroxides, in ammonia and in mineral acids.
IDENTIFICATION
First identificationı B, D.
Second identificationı A, C, D, E.
ıA. Melting point (2.2.14)
ıB. Infrared absorption spectrophotometry (2.2.24) .
Comparisonı Ph. Eur. reference spectrum of theophylline.
ıC. Heat 10 mg with 1.0 ml of a 360 g/l solution of potassium hydroxide R in a water-bath at
slowly develops. Carry out a blank test.
ıD. It complies with the test for loss on drying (see Tests).
ıE. It gives the reaction of xanthines (2.3.1) .
TESTS
Solution S
Dissolve
same solvent.
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
Acidity
To 50 ml of solution S add 0.1 ml of methyl red solution R . The solution is red. Not more than
1.0 ml of
Related substances
Liquid chromatography (2.2.29).
Test solutionı Dissolve 40.0 mg of the substance to be examined in the mobile phase and
dilute to 20.0 ml with the mobile phase.
Reference solution (a)ı Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase.
Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.
Reference solution (b)ı Dissolve 10 mg of theobromine R in the mobile phase, add 5 ml of
the test solution and dilute to 100 ml with the mobile phase. Dilute 5 ml of this solution to 50
ml with the mobile phase.
Column: ı
ı— size: l =
ı— stationary phase: octadecylsilyl silica gel for chromatography R (7 μm).
Mobile phaseı Mix 7 volumes of acetonitrile for chromatography R and 93 volumes of a
1.36 g/l solution of sodium acetate R containing 5.0 ml/l of glacial acetic acid R .
Flow rateı 2.0 ml/min.
Detectionı Spectrophotometer at 272 nm.
Injectionı 20 μl.
Run timeı 3.5 times the retention time of theophylline.
Relative retentionı With reference to theophylline (retention time = about 6 min): impurity C =
about 0.3; impurity B = about 0.4; impurity D = about 0.5; impurity A = about 2.5.
System suitabilityı Reference solution (b):
ı— resolution: minimum 2.0 between the peaks due to theobromine and theophylline.
Limits:
ı— impurities A, B, C, D: for each impurity, not more than the area of the principal peak in
the chromatogram obtained with reference solution (a) (0.1 per cent);
ı— any other impurity: for each impurity, not more than the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.1 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: 0.5 times the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.05 per cent).
Heavy metals (2.4.8)
Maximum 20 ppm.
ppm Pb) R.
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on
Sulphated ash (2.4.14)
Maximum 0.1 per cent, determined on
ASSAY
Dissolve
of bromothymol blue solution R1 . Titrate with
1 ml of
IMPURITIES
Specified impuritiesı A, B, C, D.
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): E, F.
ıA. caffeine,
ıB. 3-methyl-3,7-dihydro-1H-purine-2,6-dione,
ıC. N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)formamide,
ıD. N-methyl-5-(methylamino)-1H-imidazole-4-carboxamide,
ıE. 1,3-dimethyl-7,9-dihydro-1H-purine-2,6,8(3H)-trione,
ıF. etofylline.
Pentoxifylline
General Notices
(Ph Eur monograph 0851)
C13H18N4O3
ıı278.3ıı6493-05-6
Action and use
Vasodilator.
Ph Eur
DEFINITION
3,7-Dimethyl-1-(5-oxohexyl)-3,7-dihydro-1H-purine-2,6-dione.
Content
99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
White or almost white, crystalline powder.
Solubility
Soluble in water, freely soluble in methylene chloride, sparingly soluble in ethanol (96 per
cent).
IDENTIFICATION
First identificationıA, B.
Second identificationıA, C, D.
ıA. Melting point (2.2.14):
ıB. Infrared absorption spectrophotometry (2.2.24).
Comparisonıpentoxifylline CRS.
ıC. Thin-layer chromatography (2.2.27).
Test solutionıDissolve 20 mg of the substance to be examined in methanol R and dilute to 10
ml with the same solvent.
Reference solutionıDissolve 20 mg of pentoxifylline CRS in methanol R and dilute to 10 ml
with the same solvent.
PlateıTLC silica gel F254 plate R.
Mobile phaseımethanol R, ethyl acetate R (15:85 V/V).
Applicationı5 μl.
DevelopmentıOver 2/3 of the plate.
DryingıIn air.
DetectionıExamine in ultraviolet light at 254 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.
ıD. It gives the reaction of xanthines (2.3.1).
TESTS
Solution S
Dissolve
ml with the same solvent.
Appearance of solution
A 40 per cent (V/V) solution of solution S is clear (2.2.1) and not more intensely coloured than
reference solution Y7 (2.2.2, Method II).
Acidity
To 8 ml of solution S add 12 ml of carbon dioxide-free water R and 0.05 ml of bromothymol
blue solution R1. The solution is green or yellow. Not more than 0.2 ml of
hydroxide is required to change the colour of the indicator to blue.
Related substances
Liquid chromatography (2.2.29).
Solvent mixtureıA mixture of equal volumes of a 5.44 g/l solution of potassium dihydrogen
phosphate R and methanol R.
Test solutionıDissolve 50.0 mg of the substance to be examined in the solvent mixture and
dilute to 25.0 ml with the solvent mixture.
Reference solution (a)ıDilute 2.0 ml of the test solution to 100.0 ml with the solvent mixture.
Dilute 5.0 ml of this solution to 100.0 ml with the solvent mixture.
Reference solution (b)ıDilute 10.0 ml of reference solution (a) to 50.0 ml with the solvent
mixture.
Reference solution (c)ıDissolve 2 mg of caffeine R (impurity F) and 2 mg of theophylline R
(impurity C) in the solvent mixture, add 1 ml of the test solution and dilute to 10 ml with the
solvent mixture.
Reference solution (d)ıDissolve 5.0 mg of caffeine R (impurity F), 5.0 mg of theobromine R
(impurity A) and 5.0 mg of theophylline R (impurity C) in the solvent mixture and dilute to
100.0 ml with the solvent mixture. Dilute 1.0 ml to 25.0 ml with the solvent mixture.
Column: ı
ı— size: l = 0.25 m, Ø = 4.0 mm;
ı— stationary phase: base-deactivated octylsilyl silica gel for chromatography R (5 μm);
ı— temperature: 30 °C.
Mobile phase:
ı— mobile phase A: mix 30 volumes of methanol R and 70 volumes of a 5.44 g/l solution of potassium dihydrogen phosphate R;
ı— mobile phase B: mix 30 volumes of a 5.44 g/l solution of potassium dihydrogen
phosphate R and 70 volumes of methanol R;
Flow rateı1 ml/min.
DetectionıSpectrophotometer at 272 nm.
Injectionı10 μl.
Relative retentionıWith reference to pentoxifylline (retention time = about 12 min): impurity A
= about 0.3; impurity C = about 0.4; impurity F = about 0.5; impurity J = about 1.6.
System suitabilityıReference solution (c):
ı— retention time: impurity F = 4 min to 7 min; pentoxifylline = 9 min to 13 min; if necessary
adapt the mixing ratio of the mobile phases;
ı— resolution: minimum 4 between the peaks due to impurity C and impurity F.
Limits:
ı— impurities A, C, F: for each impurity, not more than the area of the corresponding peak in
the chromatogram obtained with reference solution (d) (0.1 per cent);
ı— impurity J: not more than the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent);
ı— any other impurity: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 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: the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.02 per cent).
Chlorides (2.4.4)
Maximum 100 ppm.
Place 20 ml of solution S in a separating funnel and shake with 2 quantities, each of 20 ml, of
2-methylpropan-1-ol R. Dilute 10 ml of the aqueous layer to 15 ml with water R.
Sulphates (2.4.13)
Maximum 200 ppm, determined on 15 ml of solution S.
Heavy metals (2.4.8)
Maximum 10 ppm.
2.0 g complies with limit test C. Prepare the reference solution using 2 ml of lead standard
solution (10 ppm Pb) R.
Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying over diphosphorus pentoxide R at 60
°C at a pressure not exceeding 700 Pa.
Sulphated ash (2.4.14)
Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.200 g in 5 ml of anhydrous acetic acid R. Add 20 ml of acetic anhydride 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 27.83 mg of C13H18N4O3.
STORAGE
Protected from light.
IMPURITIES
Specified impuritiesıA, C, F, J.
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): B, D, E, G, H, I, K.
ıA. R = H, R= CH3: theobromine,
ıB. R = R= H: 3-methyl-3,7-dihydro-1H-purine-2,6-dione,
ıC. R = CH3, R= H: theophylline,
ıD. R = CH2-CH2-CH2-OH, R= CH3: 1-(3-hydroxypropyl)-3,7-dimethyl-3,7-dihydro-1H-purine-
2,6-dione,
ıF. R = R= CH3: caffeine,
ıH. R = R= CH2-[CH2]3-CO-CH3: 3-methyl-1,7-bis(5-oxohexyl)-3,7-dihydro-1H-purine-2,6-
dione,
ıI. R = CH2-C6H5, R= CH3: 1-benzyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione,
ıE. X = CH2: 1,1-methylenebis(3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione),
ıK. X = CH2-CH2-CH2: 1,1-(propane-1,3-diyl)bis(3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione),
ıG. 3,7-dimethyl-6-(5-oxohexyloxy)-3,7-dihydro-2H-purin-2-one,
ıJ. 1-[(5E)-11-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)-5-methyl-7-oxoundec-
5-enyl]-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione.
Diprophylline
General Notices
(Ph Eur monograph 0486)
C10H14N4O4
ıı254.2ıı479-18-5
Action and use
Non-selective phosphodiesterase inhibitor (xanthine); treatment of reversible airways
obstruction.
Ph Eur
DEFINITION
Diprophylline contains not less than 98.5 per cent and not more than the equivalent of 101.0
per cent of 7-[(2RS)-2,3-dihydroxypropyl]-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione,
calculated with reference to the dried substance.
CHARACTERS
A white or almost white, crystalline powder, freely soluble in water, slightly soluble in alcohol.
IDENTIFICATION
First identificationıB, C.
Second identificationıA, C, D.
ıA. Melting point (2.2.14): 160 °C to 165 °C.
ıB. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with diprophylline CRS. Examine the substances as discs prepared using 0.5 mg
to 1 mg of the substance to be examined in 0.3 g of potassium bromide R.
ıC. Dissolve 1 g in 5 ml of acetic anhydride R and boil under a reflux condenser for 15 min.
Allow to cool and add 100 ml of a mixture of 20 volumes of ether R and 80 volumes of light
petroleum R. Cool in iced water for at least 20 min, shaking from time to time. Filter, wash
the precipitate with a mixture of 20 volumes of ether R and 80 volumes of light petroleum R,
the precipitate with a mixture of 20 volumes of ether R and 80 volumes of light petroleum R,
recrystallise from alcohol R and dry in vacuo. The crystals melt (2.2.14) at 142 °C to 148 °C.
ıD. It gives the reaction of xanthines (2.3.1).
TESTS
Solution S
Dissolve 2.5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent.
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
Acidity or alkalinity
To 10 ml of solution S add 0.25 ml of bromothymol blue solution R1. The solution is yellow or
green. Not more than 0.4 ml of 0.01 M sodium hydroxide is required to change the colour of
the indicator to blue.
Related substances
Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating
substance.
Test solutionıDissolve 0.3 g of the substance to be examined in a mixture of 20 volumes of
water R and 30 volumes of methanol R and dilute to 10 ml with the same mixture of solvents.
Prepare immediately before use.
Reference solution (a)ıDilute 1 ml of the test solution to 100 ml with methanol R.
Reference solution (b)ıDilute 0.2 ml of the test solution to 100 ml with methanol R.
Reference solution (c)ıDissolve 10 mg of theophylline R in methanol R, add 0.3 ml of the test
solution and dilute to 10 ml with methanol R.
Apply to the plate 10 μl of each solution. Develop over a path of 15 cm using a mixture of 1
volume of concentrated ammonia R, 10 volumes of ethanol R and 90 volumes of chloroform
R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with the test solution, apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent)
and at most one such spot is more intense than the spot in the chromatogram obtained with
reference solution (b) (0.2 per cent). The test is not valid unless the chromatogram obtained
with reference solution (c) shows two clearly separated spots.
Chlorides (2.4.4)
Dilute 2.5 ml of solution S to 15 ml with water R. The solution complies with the limit test for
chlorides (400 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 0.5 per cent, determined on
Sulphated ash (2.4.14)
Not more than 0.1 per cent, determined on
ASSAY
In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the
titration immediately after the end-point has been reached.
Dissolve
Titrate with
1 ml of
STORAGE
Store protected from light.
Ph Eur