June 3, 2024
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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

Physostigminethe 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.

 Proserinewhite 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 (indicatorphenolphthalein). 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-(mdimethylcarbamoiloxiphenyl)-N,N,Ntrimethylammonium 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 (mdimethylaminophenol 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, EDderivatives of indole.

   Cycle А – aromatic and strychnine  can be nitrated and halogenated.

    N19tertiary atom, has a base character and gives salts with acids.

    N9is 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), dihydroquinolysidineD),

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.

*    Reserpinecolorless, 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 Н2О2yellow-lilac color.

4.     Water solutions of reserpine in UV-light blue fluorescence.

5.     Alcohol solution of the preparation + H2SO4 + NaNO2green fluorescence.

6.     With Frede reagentred 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.

ReserpineAcidimetry 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), Adelphanesidrex (Triresid) (Adelfane + 10 mg of dichlorothiazide), Adelphaneesidrex -К (Triresid К)(0,6 g of КСl), Crystepin, Neocrystepin. Raunatinethe 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

350px-Caffeine_metabolites

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– 1Hpurine-2,6-dione

1,3,7-trimethylxantine

         Caffeinesodium 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, indicatorstarch (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-1Нpurine-2,6-dione, or 3,7- dimethylxantine    

 

Theophylline monohydrate Theophyllinum monohydricum (SPhU)

1,3-Dimethyl-3,7- dihydro-1Нpurine-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 solutionintense 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).
Indicatorphenolphthalein  (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-(5oxohexyl)-3,7-dihydro-7Нpurine-2,6-dion or 1-(5oxohexyl)-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. Issuetablets 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

 

Theophyllineethylenediamine (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, indicatormethyl 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. Issuetablets by 0,2 g; amp. 10%-5,0; candles by 0,5 g.

 

*    Xantinole nicotinate (Xantinoli nicotinas) Complamine, Theonicol

7-[2oxi-3-(N‘-methyl-β-oxiethylamino)-propyl]-theophylline nicotinate

Used to improve peripheral and cerebral circulation

Issuetablets by 0,15 g; amp. 15%-2,0 і 10,0.

 

 

   

 

 

 

 

 

 

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 182 °C, with decomposition.

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 0.900 g, without heating, in 95 ml of carbon dioxide-free water R prepared from

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 0.2 g of the substance to be examined in alcohol R and dilute to 10

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 15 cm using a 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

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 1.00 g by drying in an oven at 105 °C.

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 0.350 g in 50 ml of a mixture of equal volumes of anhydrous acetic acid R and

chloroform 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 41.35 mg of C22H27N3O5.

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 145 °C, with decomposition.

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 0.500 g without heating in carbon dioxide-free water R and dilute to 50.0 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 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 0.15 g of the substance to be examined in alcohol R and dilute to 5

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 15 cm using a

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 1.000 g by drying in an oven at 105 °C.

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 0.5000 g in a mixture of 20 ml of anhydrous acetic acid R and 40 ml of acetic

anhydride R. Titrate with 0.1 M perchloric acid determining the end-point potentiometrically

(2.2.20) at the first inflexion point.

1 ml of 0.1 M perchloric acid is equivalent to 64.88 mg of C30H44N6O8S.

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

ıreserpine: 98.0 per cent to 102.0 per cent (dried substance);

ıtotal 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 0.250 g in chloroform R and dilute to 25.0 ml with the same solvent. Examine

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 0.500 g by drying at 60 °C over diphosphorus

pentoxide R at a pressure not exceeding 667 Pa for 3 h.

Sulphated ash (2.4.14)

Maximum 0.1 per cent, determined on 0.5 g.

ASSAY

Total alkaloids

Dissolve 0.500 g in a mixture of 6 ml of acetic anhydride R and 40 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 60.9 mg of total alkaloids.

Reserpine

Protect the solutions from light. Moisten 25.0 mg with 2 ml of ethanol (96 per cent) R, add 2

ml of 0.25 M sulphuric acid and 10 ml of ethanol (96 per cent) R, and warm gently to dissolve.

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 0.25 M sulphuric acid and 2.0 ml of a freshly prepared 3 g/l solution of sodium nitrite R. Mix

and heat in a water-bath at 55 °C for 35 min. Cool, add 1.0 ml of a freshly prepared 50 g/l

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) 234 °C to 239 °C, determined after drying at 100-105 °C.

ıB. Dry the substance to be examined at 100-105 °C. Examine by infrared absorption

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 80 °C for 7 min. Allow to cool and add 10 ml of water R . An intense blue

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 0.5 g with heating in 50 ml of carbon dioxide-free water R prepared from distilled

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 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 GF 254 R as the coating

substance.

Test solutionı Dissolve 0.2 g of the substance to be examined in a mixture of 4 volumes of

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 15 cm using a mixture of 10

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)

1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of

lead standard solution (10 ppm Pb) R .

Loss on drying (2.2.32)

5.0 per cent to 9.0 per cent, determined on 1.000 g by drying in an oven at 105 °C for 1 h.

Sulphated ash (2.4.14)

Not more than 0.1 per cent, determined on 1.0 g.

ASSAY

Dissolve 0.170 g, previously dried at 100-105 °C, with heating in 5 ml of anhydrous acetic

acid R . Allow to cool, add 10 ml of acetic anhydride R and 20 ml of toluene 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 19.42 mg of C8H10N4O2.

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): 234 °C to 239 °C.

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

80 °C for 7 min. Cool and add 0.5 ml of dimethylaminobenzaldehyde solution R2 . Heat

again in a water-bath at 80 °C for 7 min. Allow to cool and add 10 ml of water R ; an

intense blue colour develops.

ıE. Loss on drying (see Tests).

ıF. It gives the reaction of xanthines (2.3.1).

TESTS

Solution S

Dissolve 0.5 g with heating in 50 ml of carbon dioxide-free water R prepared from distilled

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 0.01 M sodium hydroxide is required to change the colour of the indicator to blue.

Related substances

Liquid chromatography (2.2.29).

Test solutionı Dissolve 0.100 g of the substance to be examined in the mobile phase and

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

ısize: l = 0.15 m, Ø = 4.6 mm;

ıstationary phase: base-deactivated end-capped octadecylsilyl silica gel for

chromatography R (5 μm).

Mobile phaseı Dissolve 1.64 g of anhydrous sodium acetate R in water R and dilute to

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):

ıresolution: 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:

ıunspecified 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);

ıtotal: not more than 0.5 times the area of the principal peak in the chromatogram

obtained with reference solution (a) (0.1 per cent);

ıdisregard 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.

1.0 g complies with 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 in an oven at 105 °C for 1 h.

Sulphated ash (2.4.14)

Maximum 0.1 per cent, determined on 1.0 g.

ASSAY

Dissolve 0.170 g with heating in 5 ml of anhydrous acetic acid R . Allow to cool, add 10 ml of

acetic anhydride R and 20 ml of toluene 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 19.42 mg of C8H10N4O2.

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 0.4 g add 20 ml of boiling water R and boil for 1 min. Allow to cool and filter. Add 0.05 ml

of bromothymol blue solution R1 . The solution is yellow or yellowish-green. Not more than

0.2 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to blue.

Related substance

Examine by thin-layer chromatography (2.2.27) , using silica gel GF 254 R as the coating

substance.

Test solutionı To 0.2 g of the finely powdered substance to be examined add 10 ml of a

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 15 cm using a

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)

1.0 g complies with limit test C for heavy metals (20 ppm). Prepare the standard using 2 ml of

lead standard solution (10 ppm Pb) R .

Loss on drying (2.2.32)

Not more than 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C.

Sulphated ash (2.4.14)

Not more than 0.1 per cent, determined on 1.0 g.

ASSAY

Dissolve 0.150 g in 125 ml of boiling water R , cool to 50 °C to 60 °C and add 25 ml of 0.1 M

silver nitrate . Using 1 ml of phenolphthalein solution R as indicator, titrate with 0.1 M

sodium hydroxide until a pink colour is obtained.

1 ml of 0.1 M sodium hydroxide is equivalent to 18.02 mg of C7H8N4O2.

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) : 270 °C to 274 °C, determined after drying at 100-105 °C.

ıB. Infrared absorption spectrophotometry (2.2.24) .

Preparationı Dry the substance to be examined at 100-105 °C before use.

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

90 °C for 3 min, then add 1.0 ml of diazotised sulphanilic acid solution R . A red colour

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 0.5 g with heating in carbon dioxide-free water R , cool and dilute to 75 ml with the

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 0.01 M sodium hydroxide is required to change the colour of the indicator to yellow.

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

ısize: l = 0.25 m, Ø = 4 mm;

ıstationary 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):

ıresolution: minimum 2.0 between the peaks due to theobromine and theophylline.

Limits:

ıimpurities 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);

ıany 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);

ıtotal: not more than 5 times the area of the principal peak in the chromatogram obtained

with reference solution (a) (0.5 per cent);

ıdisregard 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.

1.0 g complies with limit test C. Prepare the standard using 2 ml of lead standard solution (10

ppm Pb) R.

Water (2.5.12)

8.0 per cent to 9.5 per cent, determined on 0.20 g.

Sulphated ash (2.4.14)

Maximum 0.1 per cent, determined on 1.0 g.

ASSAY

Dissolve 0.160 g in 100 ml of water R , add 20 ml of 0.1 M silver nitrate and shake. Add 1 ml

of bromothymol blue solution R1 . Titrate with 0.1 M sodium hydroxide .

1 ml of 0.1 M sodium hydroxide is equivalent to 18.02 mg of C7H8N4O2.

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)

270 °C to 274 °C, determined after drying at 100-105 °C.

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

90 °C for 3 min, then add 1.0 ml of diazotised sulphanilic acid solution R . A red colour

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 0.5 g with heating in carbon dioxide-free water R , cool and dilute to 75 ml with the

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 0.01 M sodium hydroxide is required to change the colour of the indicator to yellow.

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

ısize: l = 0.25 m, Ø = 4 mm;

ıstationary 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):

ıresolution: minimum 2.0 between the peaks due to theobromine and theophylline.

Limits:

ıimpurities 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);

ıany 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);

ıtotal: not more than 5 times the area of the principal peak in the chromatogram obtained

with reference solution (a) (0.5 per cent);

ıdisregard 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.

1.0 g complies with limit test C. Prepare the standard 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 in an oven at 105 °C.

Sulphated ash (2.4.14)

Maximum 0.1 per cent, determined on 1.0 g.

ASSAY

Dissolve 0.150 g in 100 ml of water R, add 20 ml of 0.1 M silver nitrate and shake. Add 1 ml

of bromothymol blue solution R1 . Titrate with 0.1 M sodium hydroxide .

1 ml of 0.1 M sodium hydroxide is equivalent to 18.02 mg of C7H8N4O2.

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): 103 °C to 107 °C.

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 2.5 g in carbon dioxide-free water R prepared from distilled water R and dilute to 50

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 0.01 M sodium

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

ısize: l = 0.25 m, Ø = 4.0 mm;

ıstationary phase: base-deactivated octylsilyl silica gel for chromatography R (5 μm);

ıtemperature: 30 °C.

Mobile phase:

ımobile phase A: mix 30 volumes of methanol R and 70 volumes of a 5.44 g/l solution of potassium dihydrogen phosphate R;

ımobile 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):

ıretention time: impurity F = 4 min to 7 min; pentoxifylline = 9 min to 13 min; if necessary

adapt the mixing ratio of the mobile phases;

ıresolution: minimum 4 between the peaks due to impurity C and impurity F.

Limits:

ıimpurities 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);

ıimpurity J: not more than the area of the principal peak in the chromatogram obtained

with reference solution (a) (0.1 per cent);

ıany 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);

ıtotal: not more than 5 times the area of the principal peak in the chromatogram obtained

with reference solution (a) (0.5 per cent);

ıdisregard 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 1.000 g by drying in an oven at 105 °C.

Sulphated ash (2.4.14)

Not more than 0.1 per cent, determined on 1.0 g.

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 0.200 g in 3.0 ml of anhydrous formic acid R and add 50.0 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 25.42 mg of C10H14N4O4.

STORAGE

Store protected from light.

Ph Eur

 

 

 

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