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METHODICAL INSTRUCTION FOR STUDENTS OF THE 3rd COURSE

June 22, 2024

METHODICAL INSTRUCTION FOR STUDENTS OF THE 3rd COURSE

Pharmaceutical Faculty

LESSON № 15 (PRACTICAL – 6 HOURS)

Theme: Pharmaceutical analysis of amide of p-aminobenzenesulfonic acid derivatives (sulfanilamides) as drug substances: synthesis, properties, analysis, storage, action and use.

Aim: to acquire theoretical knowledge and practical skills for quality analysis, tests and quantitative definition of sulfanilamides.

Professional orientation of students:

Sulfanilamides are chemotherapeutic antibacterial drugs. The structural formula of sulfanilamides:

R – radical (aliphatic or heterocyclic) in sulfamide group – SO₂NH₂

R₁– radical (aliphatic, aromatic or heterocyclic) in aromatic amino group– NH₂.

Sulfanilamides are used for treatment of infections, caused by many gram-positive and gram-negative bacterias, some Protozoa (agents of malaria and toxoplasmosis), chlamydia (trachoma, paratrachoma). Between structure of sulfanilamides and pharmacological activity are relationships.

Action of sulfanilamides is connected with infringement of formation by microorganisms of factors of growth necessary for their development – folic and dihydrofolic acids, which structure of molecules includes p-aminobenzoic acid (the theory of “competitive antagonism”). Sulfanilamides have bacteriostatic action.

Considering the theoretical material, concerning methods of synthesis, physical and chemical properties, techniques of identification, tests and quantitative definition of drug substances, their storage conditions and application in the medical practice and pharmaceutical analysis, students acquire knowledge which are necessary in future for professional work.

Performing practical work, students get new practical skills and improve earlier ones from the quality control of drugs and pharmaceuticals.

Methodology of Practical Class

Sulfanilamide

General Notices

(Ph Eur monograph 1571)

Sulfanilamidum

Streptocidum (SPU, suppl. 2, N )

C₆H₈N₂O₂S

172.2

DEFINITION

Sulfanilamide contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of 4-aminobenzenesulphonamide, calculated with reference to the dried substance.

CHARACTERS

White or yellowish-white crystals or fine powder, slightly soluble in water, freely soluble in acetone, sparingly soluble in alcohol, practically insoluble in methylene chloride. It dissolves in solutions of alkali hydroxides and in dilute mineral acids.

IDENTIFICATION

D. (BrPh, SPU, suppl. 2). Reaction of diazotization with the next azocoupling

Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid. Dilute 1 ml of the solution to 10 ml with water R. The solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1).

Amines, Primary Aromatic

Acidify the prescribed solution with dilute hydrochloric acid R and add 0.2 ml of sodium nitrite solution R. After 1 min to 2 min, add 1 ml of -naphthol solution R. An intense orange or red colour and usually a precipitate of the same colour are produced.

orange or red colour of azo dye

ASSAY

(BrPH, SPU, suppl. 2). Nitritometry, direct titration

Carry out the determination of primary aromatic amino-nitrogen (2.5.8), using 0.140 g and determining the end-point electrometrically.

Determination of Primary Aromatic Amino-nitrogen:

(Ph. Eur method 2.5.8)

Dissolve the prescribed quantity of the substance (0.140 g) to be examined in 50 ml of dilute hydrochloric acid R or in another prescribed solvent and add 3 g of potassium bromide R. Cool in ice-water and titrate by slowly adding 0.1 M sodium nitrite with constant stirring.

Determine the end-point electrometrically (BrPh) or by the use of the prescribed indicator (mix tropeolin 00 (4 drops) and methylene dark blue (2 drops). Then titrate from violet to blue colour. – SPU, suppl. 2).

1 ml of 0.1 M sodium nitrite is equivalent to 17.22 mg of C₆H₈N₂O₂S.

Е_m = М m.

STORAGE

Store protected from light.

Ph Eur

Action and use

Antibacterial.

Ph Eur

Sulfacetamide Sodium

General Notices

Soluble Sulfacetamide

(Ph Eur monograph 0107)

Sulfacetamidum natricum

C₈H₉N₂NaO₃S,H₂O

254.2

Chemical name: sodium derivative of N-[(4-aminophenyl)sulphonyl]acetamide (BrPh, 2007).

DEFINITION

Sodium acetyl[(4-aminophenyl)sulphonyl]azanide.

Content

99.0 per cent to 101.0 per cent (anhydrous substance).

CHARACTERS

Appearance

White or yellowish-white, crystalline powder.

Solubility

Freely soluble in water, slightly soluble in anhydrous ethanol.

IDENTIFICATION

E. (BrPh, SPU, suppl. 2). Reaction of diazotization with the next azocoupling

Dissolve about 1 mg of the precipitate obtained in identification C, with heating, in 1 ml of water R . The solution gives the reaction of primary aromatic amines (2.3.1) with formation of an orange-red precipitate.

Amines, Primary Aromatic

Acidify the prescribed solution with dilute hydrochloric acid R and add 0.2 ml of sodium nitrite solution R. After 1 min to 2 min, add 1 ml of β-naphthol solution R. An intense orange or red colour and usually a precipitate of the same colour are produced.

эF. (BrPh, SPU, suppl. 2).Solution S (see Tests) gives the reactions of sodium (2.3.1) .

Sodium and Sodium Salts:

A. (BrPh, SPU). Reaction with potassium pyroantimonate solution. Dissolve 0.1 g of the substance to be examined in 2 ml of water R or use 2 ml of the prescribed solution. Add 2 ml of a 150 g/l solution of potassium carbonate R and heat to boiling. No precipitate is formed. Add 4 ml of potassium pyroantimonate solution R and heat to boiling. Allow to cool in iced water and if necessary rub the inside of the test-tube with a glass rod. A dense white precipitate is formed.

Na⁺ + [Sb(OH)₆]^– Na[Sb(OH)₆]

dense white precipitate

TESTS

Solution S

Dissolve 1.25 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent.

pH (2.2.3)

8.0 to 9.5 for solution S.

ASSAY

(BP, SPU, suppl. 2). Nitritometry, direct titration

Carry out the determination of primary aromatic amino-nitrogen:

Dissolve about 0.14 g (exact shot of the substance) in 50 ml of dilute hydrochloric acid R and add 3 g of potassium bromide R. Cool in ice-water and titrate by slowly adding 0.1 M sodium nitrite with constant stirring.

As indicator use mix of tropeolin 00 (4 drops) and methylen dark blue (2 drops) and titrate until blue colour is obtained.

1 ml of 0.1 M sodium nitrite is equivalent to 17.22 mg of C₆H₈N₂O₂S.

STORAGE

Protected from light.

Action and use

Sulfonamide antibacterial.

Ph Eur

In Ukraine is preparation Sulfacetamide sodium (30 %) (Solutio Sulfacyli-natrii 30 %) eyes drops. Use for treatment of eye infectious diseases.

Phthalylsulfathiazole

General Notices

(Ph Eur monograph 0352)

Phthalylsulfathiazolum

Phtalazolum (SPU, suppl. 2, N)

C₁₇H₁₃N₃O₅S₂

ээ403.4ээ

DEFINITION

2-[[4-(Thiazol-2-ylsulphamoyl)phenyl]carbamoyl]benzoic acid.

Content

98.5 per cent to 101.5 per cent (dried substance).

CHARACTERS

Appearance

White or yellowish-white, crystalline powder.

Solubility

Practically insoluble in water, freely soluble in dimethylformamide, slightly soluble in acetone

and in ethanol (96 per cent).

IDENTIFICATION

D. (BP, SPU, suppl. 2). Reaction of fluoresceine formation (for rest of phthalic acid)

To 0.1 g add 0.5 g of resorcinol R and 0.3 ml of sulphuric acid R and heat on a waterbath until a homogeneous mixture is obtained. Allow to cool. Add 5 ml of dilute sodium hydroxide solution R. Dilute 0.1 ml of this brownish-red mixture to 25 ml with water R. An intense green fluorescence appears which disappears on acidification.

E. (BrPh, SPU, suppl. 2). Reaction of diazotization with the next azocoupling (for primary aromatic amino group)

Dissolve about 10 mg of test substance in 200 ml of 0.1 M hydrochloric acid . To 2 ml of the solution add 0.2 ml of sodium nitrite solution R. After 1 min to 2 min, add 1 ml of -naphthol solution R. An orange precipitate is produced.

ASSAY

(BrPh, SPU, suppl. 2). Alkalimetry, non-aqueous titration (direct titration)

Dissolve 0.300 g in 40 ml of dimethylformamide R. Titrate with 0.1 M sodium hydroxide until

the colour becomes blue. using 0.2 ml of thymolphthalein solution R as indicator.

Carry out a blank titration.

1 ml of 0.1 M sodium hydroxide is equivalent to 20.17 mg of C₁₇H₁₃N₃O₅S₂.

Е_m = М m./2

STORAGE

Protected from light.

Ph Eur

Action and use

Antibacterial.

Ph Eur

Individual Students Program

1. Latiames and synonyms, structural formulas and chemical names of Sulfanilamide, Streptocide soluble, Sulfacetamide Sodium, Sulfaguanidine, Sulfacarbamide, Sulfathiazole, Phthalylsulfathiazole, Sulfadimethoxine, Salazopyridazine, Sulfadiazine, Silver Sulfadiazine, Sulfaethidole, Sulfaethidole Sodium, Sulfalene, Sulfapyridazine, Sulfapyridazine Sodium, Sulfamonomethoxine, Phthazine, Biseptol (combined drug, which consists of Sulfamethoxazole and Trimethoprim), etc.

2. The synthesis methods.

3. Physical properties of the above-named drugs.

4. The reactions of identification of Sodium-cations, sulphide-, and sulphate-ions, primary aromatic amino group, sulfamide group in molecules of drugs; thermal decomposition (streptocide, norsulfazole, sulgine).

5. The specific impurities of test substances (streptocide soluble and phthalazole).

6. The methods of ASSAY of drugs.

7. Storage of sulphanyl amides .

8. Action and use of drugs.

Seminar discussion of theoretical issues

1. What are latiames of Sulfanilamide, Streptocide soluble, Sulfacetamide Sodium, Sulfaguanidine, Sulfacarbamide, Sulfathiazole, Phthalylsulfathiazole, Sulfadimethoxine, Salazopyridazine, Sulfadiazine, Silver Sulfadiazine, Sulfaethidole, Sulfaethidole Sodium, Sulfalene, Sulfapyridazine, Sulfapyridazine Sodium, Sulfamonomethoxine, Phthazine, Biseptol (combined drug, which consists of Sulfamethoxazole and Trimethoprim), etc.?

2. What are structural formulas of Sulfanilamide, Streptocide soluble, Sulfacetamide Sodium, Sulfaguanidine, Sulfacarbamide, Sulfathiazole, Phthalylsulfathiazole, Sulfadimethoxine, Salazopyridazine, Sulfadiazine, Silver Sulfadiazine, Sulfaethidole, Sulfaethidole Sodium, Sulfalene, Sulfapyridazine, Sulfapyridazine Sodium, Sulfamonomethoxine, Phthazine, Biseptol (combined drug, which consists of Sulfamethoxazole and Trimethoprim), etc?

3. What are the general methods of synthesis of sulfanilamides?

4. What functional groups are in the formulas of the given drugs?

5. What are general reactions of sulfanilamides identification?

6. What are general methods of sulfanilamides quantitative definition?

7. What storage conditions of sulfanilamides?

8. What are actions and use in the medical practice of given drugs?

Test evaluation and situational tasks

1. Characters of sulfanilamide is:

White amorphous powder, soluble in ether
White amorphous powder, soluble in acetone
White amorphous powder, soluble in alkalis
White or yellowish-white crystals or fine powder, slightly soluble in water
Yellow crystalline powder, soluble in water

Write the structural formula of preparation.

2. What analytical effect of reaction we can be see after addition to sulfacetamide sodium solution copper sulphate solution and sodium hydroxide?

A. White smoke

B. Precipitate of blue-green colour

C. Brown gas

D. White precipitate

E. Red gelatinous precipitate

Write the equations of corresponding reactions.

3. The specific impurity in preparation of streptocide soluble is:

A. Sodium sulphite

B. Sulfate ashes

C. Hlorides

D. Heavy metals

E. Potassium sulphate

Write the reaction of its revealing.

4. What reagents can be used for identification of thiazole cycle in the molecule of sulfathiazole?

A. Barium chloride in the medium of HCl

B. Silver nitrate in the medium of HNO₃ after a preparation mineralization

C. Barium chloride in the medium of HCl after mineralization

D. Silver nitrate in the medium of HNO₃

E. Sodim nitrite in the medium of HCl

Write the equations of corresponding reactions.

5. What method of assay we can be used for quantitative analysis of Trimethoprim (part of Co-Trimoxazolle)?

A. Acidimetry

B. Argentometry

C. Permanganatometry

D. Ceriumetry

E. Keldal’s

Write the equations of reactions.

1. What volume of 0,0167 M potassium bromate (f = 0,9690) for titration of 2,0172 g of sulfanilamide (streptocide) ointment (M = 172,21 g/mol) will be used, if mass fraction of sulfanilamide in preparation is 5 %?

2. Calculate the mass of phthalylsulfathiazole (M = 403,4 g/mol) in one tablet, if for titration of 0,2505 g of powdered tablets were used: 11,4 ml of 0,1 M sodium hydroxide (f = 0,9879). The average mass of 1 tablet is 0,55 g.

3. Calculate the percentage content of sulfathiazole (M = 255,32 g/mol), if for titration of 0,1553 g of sulfathiazole by means of Volhard method were used: 4,1 ml of 0,1 M ammonium thiocyanate (f = 1,0015). For control test was used 10,2 ml of 0,1 M ammonium thiocyanate.

4. Calculate mass of sulfaguanidine (M = 232,26 g/mol), if for titration was used: 5,1 ml of 0,1 M sodium nitrite (f = 1,0000). The percentage content of sulfaguanidine in preparation is 99,5 %, volume of volumetric flask is 100 ml, volume of aliquot is 10 ml.

5. What volume of 0,1 M sodium thiosulphate (f = 1,0000) will be used for titration of 0,1010 g of sulfadiazine (M = 250,3 g/mol), if mass fraction of sulfadiazine in preparation is 0,2 %. Volume of volumetric flask is 100 ml, volume of aliquot is 50 ml. For control test 25,0 ml of sodium thiosulphate was used.

Initial level of knowledge and skills are checked by solving situational tasks for each topic, answers in test evaluations and constructive questions.

(the instructor has tests & situational tasks)

Student should know:

1. Latin and chemical names, synonyms, structural formulas of drugs from group of sulphanilamides: streptocide, streptocide soluble, sulfacetamide sodium, norsulfazole (sulfathiazole), phthalazole (sulphothalidine, phthalylsulfathiazole), sulgine (sulfaguanidine), sulfacarbamide (urosulfanum), sulfadimethoxine, salazodimethoxine, salazopyridazine (salazodine), sulfadiazine (sulfanilamidopyrimidine), silver salt of sulfadiazine, sulfadimezine sodium, etazole, sulfalene, sulfapyridazine, sulfamethoxypyrydazine, sulfamonomethoxine, phthazine (phthazinum), Co–Trimoxazolle, etc.

2. The methods of synthesis of sulphanilamides, its physical and chemical characteristics, methods of identification and assay.

3. Storage, action and use of drugs from specified group.

Student should be able to:

1. To write reactions of synthesis, identification and quantitative definition of aforementioned preparations.

2. Calculate the molar mass of equivalent at definition of mass fraction of active ingredient of investigated preparations.

3. To do conclusion about conformity of quality of aforementioned preparations to requirements of the AND.

Correct answers of test evaluations and situational tasks:

1. D; 2. B; 3. A; 4. C; 5. E.

1. 24,10 ml; 2. 0,499 g; 3. 100,4 %; 4. 1,1905 g; 5. 17,70 ml.

References:

А – Basic:

1. The International Pharmacopoeia / World Health Organization. – 3^rd Edition, Volume 5. Tests and general requirements for dosage forms; Quality specifications for pharmaceutical substances and dosage forms. – Geneva, 2003. – 371 p.

2. European Pharmacopoeia /Directorate for the Quality of Medicines & HealthCare of the Council of Europe (EDQM). – Sixth Edition, Volume 1. – Strasbourg, 2007. – 1083 p.

3. British Pharmacopoeia /published by The Stationery Office on behalf of the Medicines and Healthcare products Regulatory Agency (MHRA). – Volumes I–IV. – London, 2009. – 10952 p.

4. Pedersen O. Pharmaceutical chemical analysis: methods for identification and limit tests. – USA: Taylor and Francis Group, 2006. – 150 p.

5. Lecture materials.

В – Additional:

1. David G. Watson. Pharmaceutical analysis. – New York: Churchill Livingstone, 2000. – 400 p.

2. Cairns D. Essentials of Pharmaceutical Chemistry. – Third edition. – London: Pharmaceutical Press, 2008. – 280 p.

Methodical instruction has been worked out by: associate professor L.M. Mosula

Methodical instruction was discussed and adopted at the Department sitting

on the 7-th of June 2012. Minute № 17