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

 

 

Sulfanilic acid (p-aminobenzenesulphoacid)

– is substitution product –ОН group in molecule of sulphatic acid H₂SO₄  on rest of aniline   –C₆H₅–NH₂ . Sulfanilic acid is not drug.   

 

–      

amide of sulfanilic acid (sulfanilamide) – is sours for synthesis many drugs.

–       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 with antibacterial action

 

The term sulfanilamide is commonly used to antibacterials that are aniline-substituted sulfonamides, the “sulanilamides”; prodrugs that produce sulfanilamides (sulfasalazune); and nonanilane sulfonamides (mafenide).

Mechanism of action

Folinic acid, N⁵, N¹⁰-methylenetetrahydrofolic acid, and N¹⁰– formyltetrahydrofolic acid are indispensable for several biosynthetic pathways in humans, bacteria, animals and plants. Without these folate coenzymes will not available to produce nucleic acids needed for cell division.  The result of any drug blocking the biosynthesis of folate coenzymes in bacteria, for example, is that growth and cell division are stopped. Such drugs – including the sulfonamides and trimethoprim – are thus bacteriostatic.

Folate coenzymes are biosynthesized from dietary folic acid in humans (and other animals). However, bacteria must make them from p-aminobensoic acid (PABA). The microbes cannot use dietary folic acid from the host, for reasons not yet completely understood. It may be that folic acid cannot penetrate the cell wall.

The sulfonamides act as competitive inhibitors for the incorporation of PABA to form dihydroptiroic acid.

 

 

 

General methods of synthesis sulfanilamides

For synthesis of sulfanilamides use diferents organic compounds with general formula                    , that is aniline derivatives C₆H₅–NH₂, with protected amino-group –NH_2.        

 

Method I. 3 stages:

  1) Sulphonation of acetanilide by means of sulphuric chlorohydrin (sulphate-acid chloride HO–SO₂–Cl) with formation acetyl sulfanilic-acid chloride:

 

 

 

                                                                                                                                    

                                                                                                 acetylsulfanilchloride     

2) Condensation obtaining product with ammoniac NH₃ or other amine R–NH₂ with formationс образованием acetyl sulfanilamides:

 

 

 

 

Medium HCl.

3) Saponification acyl group by means of boiling with mineral acids (HCl, H₂SO₄ – acid hydrolis) or alkali (obtained Na–salt of sulfanilamide).

 

 

 

 

 

 

Method 2. Initial substance – sulfanilic acid.

 

 

 

 

 

sulfanilic acid                         Na p-aminobenzene sulphonate    acylation H₂ N-                                                                                                                                    group

 

 

 

 

                         Na p-acetylaminobenzene sulphonate     Na p-acetylaminobenzene sulphchloride

 

 

 

 

 

                alkyl-amide of p-N-acetylsulfanilic acid                              alkylsulfanilamide

 

 

Method 3 (most rational and economic). Initial substance – phosgene Cl–CO–Cl (toxic gas) and aniline C₆H₅NH_2, by means of which synthesis carbmethoxyanilide.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chemical properties and reaction of identification

 

І. Amphoteric characters of sulfanilamides and its solubility in cids and alkalis

General formula of many sulfanilamides                                           , its consists in molecule amino–group–NH₂ with basic properties and substituted sulphamide–group                  with acid properties. Therefore many sulfanilamides 

are amphoteric compounds.

 

а) as base its dissolves in acids with formation salts:

 

HCl·                   .

But this salts in water very hydrolyzes and practical not exists.

 

b) in acid sulfanilamide-group                  Hydrogene-atom can substitutes on metal with formation salts. Therefore sulfanilamides  can dissolutions in alkalis and carbonates of alkalis metals:

 

 

 

 

 

 

 

 

 

 

Sulfacetamide (N-[(4-Aminophenyl)sulfonyl]-acetamide; N–sulfanylacetamide). Its plasma half-life is seven hours. This compound is a white crystalline powder, soluble in water and in alcohol. It is very soluble in hot water, and its water solution is acidic.

 

 

 

 

 

Sulfacetamide sodium (Sodium sulamyd)

 is obtained as a monohydrate and is the white, odorless, bitter, crystalline powder that is very soluble in water. Because the sodium salt is highly soluble at the physiologic pH of 7.4, it is especially suited, as a solution, for repeated topical applications in the local management of ophthalmic infections susceptible to sulfonamide therapy.

 

 

        

 

 

Silver sulfadiasine (Silvedene). The silver salt of sulfadiasine applied in a water-miscible cream base has proved to be an effective topical antimicrobial agent, especially against Pseudomonas species. This is of particular significance in burn therapy because pseudomonad is often responsible for failures in therapy.  The salt is only very slightly soluble and does not penetrate the cell structure. Studies using radioactive silver have shown essentially no adsorption into body fluids. Sulfadiazine levels in serum were about 0.5 to 2 mg/100 ml.

The preparation is reported to be simpler and easier to use than other standard burn treatments, such as application of freshly prepared dilute silver nitrate solutions or mafenide ointment.

 

 

Sulfanilamides are white, and odorless compounds. Only sulfapyridazine is a yellow powder and salasopyridasine is a orange powder. Sulfanilamides are slightly soluble or insoluble in water, alcohol, CHCl₃, ether. Some of sulfanilamides are soluble in acetone. In practice we often use sodium salts of sulfanilamides: sulfacetamide sodium, norsulfasole sodium, aethazole sodium, sulfapyridazine sodium, which have general formula:

 

Sodium salts of sulfanilamides are very soluble in water and slightly soluble or insoluble in organic solvents.

Because of amphoteric properties sulfanilamides dissolve in acids and bases to form a salts. 

 

Identification of sulfanilamides

For identification of sulfanilamides carry out general reactions characteristic of functional groups and specific reaction for each substance.

1.     Diazo coupling reaction.

It is general reaction for all substances containing primary aromatic amino group. For sulfanilamides with replaced amino group (streptocide, phthazine, phthalasole) previously make hydrolysis by boiling with diluted hydrochloric acid.

2.     Lignin test.

At drawing on a paper or wood of drops of a diluted hydrochloric acid and sulfanilamide there is a formation of orange-yellow coloring (lignin hydrolyzes to form aromatic aldehydes, which cooperate with primary aromatic amines).

3.     Reactions of halogenation.

4.                Pyrolysis of  sulfanilamide. At thermal decomposition of a preparation in dry tube melts get various coloring. For example, at pyrolysis of streptocide a violet melt with odor of aniline and ammonia forms; norsulfazole gets a brown melt with odor of H₂S; urosulfan – violet melt with odor of NH₃.

5. Revealing Sulphur in molecule sulfanilamide after mineralization drug in conc. Nitric acid HNO₃ or fusion (melting) with 10-multiple quantity potassium nitrate KNO₃.

 

 

 

         Forming sulphate-ions SO₄^2– revealing by means of barium chloride in the medium diluted HCl.  

SO₄^2– + Ba²⁺ ® BaSO₄¯

                     white

Not dissolve in mineral acids and alkalis.

 

6.     Reaction with solution of heavy metals.

Sulfanilamides form color complexes with solutions of cuprum, silver, cobalt and iron. For example, sulfacetamide sodium forms a blue-green precipitate with solutions of cuprum salts; norsulfazole – violet precipitate; aethazole – dirty green precipitate that goes to black; sulfadimethoxine – dirty yellow; sulfalene – dirty green precipitate that goes to green- blue; sulfadimezine – green-yellow precipitate that goes to brown; phtazine – green-blue precipitate; and salopiridazine – green precipitate.

General formula of complexes is:

 

 

 

 

 

 

 

 

 

 

 

7.                Reaction with sodium nitroprusside (Legalya reagent).

Solutions of sulfanilamides react with 1% solution of sodium nitroprusside Na₂[Fe(CN)₅NO]  in presence of bases (NaOH) and following acidification to form red or red-brown solutions (streptocide, streptocide soluble, sulgine, urosulfane, sulfacetamide sodium), or precipitates (aethazole, norsulfasole, sulfadimezine).

8.     UV-spectroscopy.

9.     IR- spectroscopy.

 

 

ASSAY of sulfanilamides

         Many methods:

         1. Nitritometry, direct titration

For many sulfanilamides, if in molecules is free aromatic amino-group: стрептоцида, сульфацил–натрия, сульгина, уросульфана, норсульфазола, норсульфазол–натрия, сульфадимезина, сульфадиметоксина, сульфапиридазина и др.

Diazotization of free aromatic amino-group:

E_m = M.m.

For drugs free aromatic amino-group whis method is possible after preliminary hydrolysis:

 

 

 

 

2. Alkalimetry (direct titration) in the medium organic solvent (for drugs with acid character)

T – NaOH

Medium – alcohol or water-acetone solution

Indicator – thymolphthalein.

 

 

 

E_m = M.m.

 

3. Alkalimetry, non-aqueous titration

  T – NaOH

Medium – DMFA, methanol, benzene

Indicator – thymol dark blue.

Titrate to change colour from yellow to dark blue.

 

 

 

 

 

E_m = M.m./2

5.     Acidimetry in the presence of organic solvent (for Na-salts of sulfanilamides).

T – HCl

Medium – mix of alcohol and acid

Indicator – methyl orange.

Titrate to pink colour.

 

 

 

 

 

E_m = M.m.

 5. Bromatometry, back titration, with iodometric finishing    

Aliquot of test solution add in flask, add excess of standard solution of potassium bromate КBrO₃, crystalline potassium bromide KBr, acidifies by means of HCl (or H₂SO₄),   stand  15 minutes, stir up.

KBrO₃ + 5KBr + 6HCl = 3Br₂ + 6KCl + 3H₂O

Allocated bromine Br₂ react with drug (reaction for benzene ring) with formation dibromoderivative:

 

 

 

 

 

 

Add solution of potassium iodide KI. Not reacted bromine Br₂ react with potassium iodide KI with formation iodine I₂:

Br₂ + 2KI = I₂ + 2KBr

Allocated iodine I₂ titrate with standard solution of sodium thiosulphate Na₂S₂O_3. As indicator use starch solution and titrate to dissapperance dark blue colouring (add starch solution in the end titration):

I₂ + 2Na₂S₂O₃ = 2NaI + Na₂S₄O₆

I₂ + 2е ® 2I^–

2S₂O₃^2– – 2е ® S₄O₆^2–

Carry out a control titration.

Е_m = М. m./4

6. Iodochlorometry, back titration

 To test solution add excess standard solution of iodomonochloride ICl, which react with drug (iodination of benzene ring in free о–positions rather (of) Н₂N–group):

                      

 

 

 

Not reacted ICl react with potassium iodide  KI with formation iodine I₂, which titrate with standard solution of Na₂S₂O₃ (indicator – starch solution).

ICl + KI = I₂ + KCl

I₂ + 2Na₂S₂O₃ = 2NaI + Na₂S₄O₆

Carry out a control titration.

Е_m = М.m./4

 

7. Argentometry, Morh method

 Some sulfanilamides (for example, sulfathiazole) forming salts at titration by means of standard solution of AgNO₃ (acid properties of sulfamide group –SO₂–NH–):

 

 

 

 

 

In the Morh method is necessary neutral medium. Formed nitric acid HNO₃ will be promote dissolution Ag-salt (displacement reaction to the left). Therefore titrate in the presence of borax Na₂B₄O₇×10Н₂О, which neutralised HNO₃:

Na₂B₄O₇ + 2HNO₃ + 5Н₂О = 4H₃BO₃ + 2NaNO₃

As indicator use solution of potassium chromate K₂CrО₄. Titrate to orange-red precipitate: excess drop of titrant AgNO₃ react with indicator K₂CrО₄ with formation precipitate Ag₂CrО₄:

2AgNO₃ + K₂CrО₄ ®Ag₂CrО₄¯ + 2КNO₃

                                    orange-red precipitate

Е_m = М. m.

8. Coppermetry

To solution of Na-salt sulfanilamide add solution of copper sulphate CuSO₄  (not standard solution)  and sodium hydroxide NaOH.

 

 

 

 

 

 

 

 

Add solution of sulphatic acid H₂SO₄, which destroys complex and frees equivalent quantity CuSO₄. After that add solution of potassium  iodide KI:

2CuSO₄ + 4KI ® Cu₂I₂ + I₂ + 2K₂SO₄

Allocated iodine I₂ titrate with standard solution of sodium thiosulphate Na₂S₂O_3. As indicator use starch solution and titrate to dissapperance dark blue colouring (add starch solution in the end titration):

I₂ + 2Na₂S₂O₃ = 2NaI + Na₂S₄O₆

E_m = 2М.m.

         9. Gravimetry after mineralization of preparation

At mineralization of sulfanilamide preparation by means of concentrated nitric acid HNO₃ or melts with 10-multiple quantity of potassium nitrate KNO₃ Sulphur passes into sulphate-ions SO₄^2–.

 

 

 

Their quantity precipitates by means of soluble barium salt – ВаCl₂:

SO₄^2– + Ba²⁺ → BaSO₄↓

Precipitate BaSO₄ filteres, dry up, incinerates (calcinates) to constant weight (mass) and weighs. By means of mass of BaSO₄ calculate % maintenance of sulfanilamide preparation.

         10. Keldal method (classic) – definition of Nitrogen in the preparation.

Substance of drug mineralizated by means of boiling in the special instrument at presence of K₂SO₄, CuSO₄ and concentrated H₂SO₄. Nitroge passes into ammonium hydrosulphate NH₄HSO₄, which at interaction with alkali NaOH formed ammonia  NH₃:

NH₄HSO₄ + 2NaOH ® NH₃­ + Na₂SO₄ + 2H₂O

Allocated ammonia NH₃ distilates into flask-receiver (collector) with boric acid H₃BO₃:

NH₃ + H₃BO₃ ® NH₄BO₂ + H₂O

2NH₃ + 4H₃BO₃ ® (NH₄)₂B₄O₇ + 5H₂O

Formed salts (metaborate NH₄BO₂ and tetraborate ammonium (NH₄)₂B₄O₇) titrate with standard solution of HCl in the presence of mix indicators (mix of methyl red and methylene dark blue (2:1)):

NH₄BO₂ + HCl + H₂O ® NH₄Cl + H₃BO₃

(NH₄)₂B₄O₇ + 2HCl + 5H₂O ® 2NH₄Cl + 4H₃BO₃

 Em of preparation depended of figure atoms of Nitrogen in the substance molecule.

11. Colourimetry

Method based on synthesis colored products diferents reactions (azo dyes, complex with heavy metals and other).

12. Spectrofotometry in UV- or visible spectral range. 

 

Storage. List of strong substance. In dense corked containers, at protecting from light, a dry and cool place.

 

Action and use. Chemiotherapeutic antibacterial agents.

 

 

 

 

Table 1

Classification, structure and chemical names of sulfanilamides preparations

 

№	Drug		Radical R (in sulfamide group)	Radical R1 (in aromatic amino group)	The formula and the chemical name of drug	Use and preparations
Aliphatic (R) derivatives
1.	Sulfanilamide* Streptocide		–	–	p-Aminobenzene sulfonamide	Various infectious diseases. Powder, tablets (0,3 g and 0,5 g), 10 % ointment, 5 % linimentum.
2.	Streptocide soluble Streptocidum solubile		–	–CH2SO3Na	Sodium p-sulfamidobenzolamino methylenesulphonate	Powder, 1–1,5 % intramuscular and subcutaneous injections; 2–5–10 % intravenous injections; 5 % linimentum;  The combined drug – Inhalyptum.
3.	Sulfacetamide sodium Sulfacetamidum natricum* Albucid-sodium		The rest sodium-acetamide hydrate	–	Sodium p-aminobenzolsulphonylacetamide hydrate	Various eyes diseases. Powder, 10–20–30 % solutions (eyes drops); 30 % injection solution; 10–20–30 % eyes ointments.
4.	Sulfaguanidine* Sulgine		The rest guanidine hydrate	–	(4-aminophenylsulphonyl)guanidine or   p-Aminobenzolsulphonylguanidine hydrate	Intestinal infections. Powder, tablets (0,5 g).
5.	Sulfacarbamide Urosulfanum		The rest of urea hydrate	–	p-Aminobenzolsulphonyurea hydrate	At infections of urethras (uric ways) such as cystitis, pyelitis, pyelonephritises (tablets – 0,5 g).
Heterocyclic (R) derivatives
6.	Sulfathiazole* Norsulfazole	Thiazole cycle		–	2 (p-Aminobenzolsulfamide)-thiazole	Pneumonia, meningitis, sepsis, dysentery, etc. Powder, tablets (0,25 g and 0,5 g).
7.	Norsulfazole sodium Sulfathiazolum natricum*	Thiazole          cycle		–	Sodium 2 (p-aminobenzolsulfamide)-thiazole	At blephatitis, conjuctives; the combined drug – Inhalyptum. 5 % and 10 % intravenous injections, 10 % eyes drops.
8.	Sulfadiazine, sulfanilamidopyrimidine	Pyrimidine		–	2 (p-Aminobenzolsulfamide)-pyrimidine	Dysentery, malaria. Powder, tablets (0,5 g).
9.	Silver salt of Sulfadiazine	Pyrimidine cycle		–	Silver salt 2 (p-aminobenzolsulfamide)-pyrimidine	Bactericidal action. 1 % ointment for treatment wound and burn tissues.
10.	Sulfadimezine, Sulfadimidine*	Pyrimidine cycle		–	2 (p-Aminobenzolsulfamide)-4,6-dimethylpyrimidine	At various coccal infections,  sepsis,  gonorrhoea,  dysentery. Powder, tablets (0,25 g and 0,5 g).
11.	Etazole, Sulfaethidole*, Etazolum	1,3,4-Thiadiazole cycle		–	2 (p-Aminobenzolsulfamide)-5-ethyl-1,3,4-thiadiazole	Pneumonia,  dysentery, pyelites, cystitis, wound infections, etc. Powder, tablets (0,25 g and 0,5 g), 5 %ointment.
12.	Etazole sodium, Sulfaethidolum natricum* Aethazolum solubile	1,3,4     Thiadiazole cycle		–	Sodium 2 (p-Aminobenzolsulfamide)-5-ethyl-1,3,4-thiadiazole	Pneumonia, dysentery, pyelites, cystitis, wound infections,  etc.10 % and 20 % intravenous injections (slowly); granules for children.
13.	Sulfalene, Sulfalenum	Pyrazine cycle		–	2 (p-Aminobenzolsulfamide)-3-metoxypyrazine	Infections of respiratory organs, biliferous and urinary ways. Tablets (0,2 g 1 time in 7–10 days – very long action).
14.	Sulfapyridazine Sulfamethoxypyridazine*	Pyridazine cycle		–	6 (p-Aminobenzolsulfamide)-3-methoxypyridazine	For treatment and preventive maintenance (prophylactics) of purulent surgical infections, etc.(long action). Powder, tablets (0,5 g).
15.	Sulfapyridazine sodium, Sulfapyridazinum-natrium	Pyridazine cycle		–	Sodium 6 (p-aminobenzolsulfamide)-3-methoxypyridazine tetrahydrate	At local purulent infection for spraying of wounds in the form of bandages from 3–5–10 % solutions; 10 % and 20 % eyes drops for trachoma treatment, eyes membranulas.
16.	Sulfamonomethoxine, Sulfamonomethoxinum	Pyrimidine cycle		–	6 (p-Aminobenzolsulfamide)-4-methoxypyrimidine
17.	Sulfadimethoxine, Madribonum	Pyrimidine cycle		–	6 (p-Aminobenzolsulfamide)-2,4-dimethoxypyrimidine	Sulfanilamide a long action; used at many infections.
Heterocyclic (R) derivative and aromatic, heterocyclic (R1) derivatives
18.	Phthalylsulfathiazole* Phthalazole, Sulphothalidine,	Thiazole cycle		The rest of phthalic acid (phthalyl-)	2 (p-Phthalylaminobenzolsulfamide)-thiazole	At intestinal infections (dysentery, colitises, coloenterites), at operations on intestines. Powder, tablets (0,5 g).
19.	Phthazine, Phthazinum	Pyridazine cycle		The rest of phthalic acid (phthalyl-)	6(p-Phthalylaminobenzolsulfamide)-3-methoxypyridazine	At heavy forms of  dysentery, colitises coloenterites. Powder, tablets (0,5 g).
20.	Salazopyridazine Salazodine*	Pyridazine cycle		The rest of 5-aminosalicylicacid	5 (p – [N – (3-methoxypyridazinyl-6)-sulfamide]-phenylazo)-salicylic acid	Decays in intestines to sulfapyridazine and 5-aminosalicylic acid. For treatment nonspecific ulcer colitis and Crone illness in the form of 5 % suspension   and tablets ( 0,5 g).
21.	Salazodimethoxine, Salazodimethoxinum	Pyrimidine cycle		The rest of 5-aminosalicylic acid	5 {p – [(2,4-dimethoxypyrimidinyl-6)-sulfamide]-phenylazo}-salicylic acid	At intestinal infections. Decays in intestines to Sulfadimethoxine and 5-aminosalitcylic acid. Tablets (after meal) (0,5 g).
22.	Bactrimum Biseptol Co-Trimoxazolle*	The combined drug           Sulfamethoxazole (bacteriostatic action) 3 (p-Aminobenzolsulfamide)-5-methylizoxazole                           Trimethoprimum (bacteriostatic action) 2,4-diamino-5 (3,4,5-trimethoxybenzil)-pyrimidine				Double blocking of metabolism of bacteria: Sulfamethoxazole breaks biosynthesis dihydrofolic acid, and Trimethoprimum blocks the next stage – reduction of dihydrofolic acids in tetrahydrofolic acid. Tablets Biseptol-480 (Poland).

 

Table 2

Properties, reactions of identification and methods of assay of some sulfanilamides
№	Drug	The chemical formula and analytic-functional groups	Properties (the appearance and solubility), specific impurities	Reactions of identification	Methods of assay
1.	Streptocide		White, crystalline powder.     Slightly soluble in cool water, freely soluble in hot water.	1.     Reaction of formations azo dye. 2.     Pyrolysis п fusion (melt) blue-violet colour, a smell of ammonia NH3 and aniline.	1.     Nitritometry Еm = M. m.  2. Photocolorimetry3.Bromatometry Еm = M.m./4 4.Iodo–chlormetry Еm = M.m./4
2.	Streptocide soluble		White, crystalline powder.     Soluble in  water, practically insoluble in ether and chloroform.	1.     Reaction of formations azo dye only after alkaline hydrolysis. 2.     Hydrolysis with the next revealing SO2 and HCHO 3.     Reactions for Na +-ions.	1.     Acidimetry in the presence of organic dissolvent Еm = M. m. 2.     Nitritometry after alkaline hydrolysis Еm = m. M.
3.	Sulfatsil-sodium		White, crystalline powder.    Freely soluble in water, practically insoluble in organic dissolvents. Specific impurity – sodium sulphite Na2SO3 (Define by means of iodometry)	1.     Reaction of formations azo dye. 2.     With CuSO4 – precipitate of dark blue-green colour is formed. 3.     Reactions for Na +-ions. 4.     Acid hydrolysis of drug with the next revealing СН3СООН .	1.     Nitritometry Еm = M. m. 2.     Acidimetry in the presence of organic dissolvent Еm = M. m.
4.	Sulgine		White, fine-crystalline powder.    Very slightly soluble in water, organic dissolvents and alkalis (unlike urosulfanum (sulfacarbamide))	1.     Reaction of formations azo dye. 2.     Pyrolysis п fusion (melt) violet-red colour, a smell of ammonia NH3. 3.     Reaction Sakaguchy: With a-naphthol in the medium of NaOH, Br2 or NaBrO ®naphthoquinonimine of red-violet colour is formed.  4. Pyrolysis of drug NNH3	1.     Nitritometry and other metods. Еm = M. m.
5.	Urosulfanum (sulfacarbamide)	The urea rest	White, crystalline powder.    Slightly soluble in water, alcohols, freely soluble in acetone, diluted acids and alkalis.	1.     Pyrolysis п fusion (melt) violet-red colour, a smell of ammonia NH3. 2.     Reaction with NaOH ® NH3 (urea decomposition) 3.     With solution of NaNO2 at heating rubinovo-krasn red colour (Difference from sulgine and other sulfanilamides).	1.     Nitritometry and other metods. Еm = M. m.
6.	Norsulfazole		White, sometimes with yellowish shade powder.   Very slightly soluble in water and organic dissolvents, soluble in acids and alkalis.	1.     Reaction of formations azo dye. 2.     Pyrolysis п fusion (melt) of dark-brown colour, smell H2S (heterocyclic Sulphur) (PbS¯). 3.     With CuSO4 – precipitate of violet colour is formed. 4.     With CоCl2 – precipitate of lilac colour untidily-fiol violet colour is formed.	1. Nitritometry Еm = M. m. 2.Argentometry Еm = M. m. 3. Alkalimetry in the water-acetone solution or in the alcohol. Еm = M. m.
7.	Sulphathiazole sodium (Norsulfazole-sodium)		Lamellar, brilliant, colourless crystals. Freely soluble in water.	1.     Reaction of formations azo dye. 2.     Pyrolysis п fusion (melt) of dark-brown colour, smell H2S (heterocyclic Sulphur) (PbS¯) 3.     With CuSO4 – precipitate of violet colour is formed. 4.     With CоCl2 – precipitate of lilac colour untidily-fiol violet colour is formed. 5.     Reactions for Na +-ions.	1. Nitritometry Еm = M. m. 2. Acidimetry in the presence of organic dissolvent Еm = M. m.
8.	Sulfadimezine		White, or with yellowish shade powder.   Practically insoluble in water, slightly soluble in alcohol, practically insoluble in other organic dissolvents, freely soluble in acids and alkalis.	1.     Reaction of formations azo dye. 2.     With CuSO4 – precipitate of yellowish-green colours the brown brown colour is formed. 3.     With Legalya reagent (solution oxidised sodium nitroprusside Na2 [Fe (CN)5NO]) фviolet colouring.	1.Nitritometry and other metods. Еm = M. m.
9.	Sulfadimethoxine		White, sometimes with yellowish shade powder.    Practically insoluble in water, slightly soluble in alcohol, freely soluble diluted HCl and alkalis.	1.     Reaction of formations azo dye. 2.     With CuSO4 – amorphous precipitate of yellow-green colour is formed. 3.     With CоCl2 – precipitate of bright-pink colour with a lilac shade is formed.	1.Nitritometry and other metods. Еm = M. m.
10.	Phthalazole, sulphothalidine		White, sometimes with yellowish shade powder.    Practically insoluble in water, ether and chloroform, very slightly soluble in alcohol, soluble in solutions of alkalis and carbonates of alkaline metals.   Specific impurity – phthalic acid (define by means of alcalimetry); – norsulfazole (define by means of nitritometry).	1.     It is not dissolved in acids. 2.     Reaction of formations azo dye only after acid hydrolysis. 3.     Reaction of formations  fluoresceine after acid hydrolysis (for phthalic acid) green  green fluorescence.	1.     Alkalimetry, non-aqueous titration       Еm = M.m./2
11.	Salazodimethoxinum		Brownish-orange fine-crystalline powder.   Practically insoluble in water, ether and chloroform, very slightly soluble in alcohol, soluble in solutions of alkalis and carbonates of alkaline metals.	1.     Reaction of formations azo dye only after hydrolysis. 2.     Reaction with solution of  FeCl3 (for phenolic hydroxyl) sine-fiol blue-violet Colouring. 3.     Reaction with salts of heavy metals (for groups  –СООН и –SO2–NH–).	1. Nitritometry after hydrolysis     Еm = M.m./2 2. Alkalimetry in the presence of organic dissolvent    Еm = M.m./ 2 3. Method Keldalya Еm = M.m./5
12.	Bactrimum                 Co-trimoksazol   Biseptol			1.     Melting point 169–172 °C. 2.     IR-spectroscopy 3.     Thin layer chromatography 4.     Formation azo dye.     1.     Melting point 199–203 °C. 2.     IR-spectroscopy 3.     Uf-spectroscopy 4.     With solution of KMnO4 + H2SO4 at heating + HCHO + H2SO4 heating + methylene chloride green green fluorescence	1.Nitritometry and other metods. Еm = M. m. 2. Method Keldalya Еm = M.m./4 3. Alkalimetry in the presence of organic dissolvent Еm = M. m.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sulfanilamide

General Notices

  

 

 (Ph Eur monograph 1571)

 

Pantosil album

 

 

 

 C₆H₈N₂O₂S  172.2  63-74-1

  

 Action and use

  

 Antibacterial.

  

 Ph Eur

  

  

 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

  

 First identification B.

 

 Second identification A, C, D.

 

  A. Melting point (2.2.14): 164.5 °C to 166.0 °C.

 

  B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with sulfanilamide CRS. Examine the substances prepared as discs.

 

  C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (a) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a).

 

  D. 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).

  

 TESTS

  

 Solution S

  

 To 2.5 g add 50 ml of carbon dioxide-free water R. Heat at about 70 °C for about 5  min. Cool in iced water for about 15 min and filter.

  

 Acidity

  

 To 20 ml of solution S add 0.1 ml of bromothymol blue solution R1. Not more than  0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator.

  

 Related substances

  

 Examine by thin-layer chromatography (2.2.27), using a TLC silica gel F ₂₅₄plate R.

 

 Test solution (a) Dissolve 20 mg of the substance to be examined in 3 ml of a  mixture of 2 volumes of concentrated ammonia R and 48 volumes of methanol R and  dilute to 5 ml with the same mixture of solvents.

 

 Test solution (b) Dissolve 0.10 g of the substance to be examined in 0.5 ml of  concentrated ammonia R and dilute to 5 ml with methanol R. If the solution is not  clear, heat gently until dissolution is complete.

 

 Reference solution (a) Dissolve 20 mg of sulfanilamide CRS in 3 ml of a mixture of 2  volumes of concentrated ammonia R and 48 volumes of methanol R and dilute to 5  ml with the same mixture of solvents.

 

 Reference solution (b) Dilute 1.25 ml of test solution (a) to 50 ml with a mixture of 2  volumes of concentrated ammonia R and 48 volumes of methanol R.

 

 Reference solution (c) Dissolve 20 mg of the substance to be examined and 20 mg  of sulfamerazine CRS in 3 ml of a mixture of 2 volumes of concentrated ammonia R  and 48 volumes of methanol R and dilute to 5 ml with the same mixture of solvents.

 

 Apply to the plate 5 µl of each solution. Develop over a path corresponding to two-thirds of the plate height using a mixture of 3 volumes of dilute ammonia R1, 5  volumes of water R, 40 volumes of nitromethane R and 50 volumes of dioxan R. Dry  the plate at 100 °C to 105 °C and examine in ultraviolet light at 254 nm. Any spot in  the chromatogram obtained with test solution (b), apart from the principal spot, is not  more intense than the spot in the chromatogram obtained with reference solution (b)  (0.5 per cent). The test is not valid unless the chromatogram obtained with reference  solution (c) shows two clearly separated principal spots.

  

 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 100 °C to  105 °C.

  

 Sulphated ash (2.4.14)

  

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

  

 ASSAY

  

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

 

(2.5.8): Dissolve the prescribed quantity of the substance 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 or by the use of the prescribed indicator.

 

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

  

 STORAGE

  

 Store protected from light.

  

 

  Ph Eur

 

 

 

 

 

 

 

Sulfacetamide sodium

General Notices

  

 

 Soluble Sulfacetamide

 

 (Ph Eur monograph 0107)

 

 

 

 

 

 C₈H₉N₂NaO₃S,H₂O  254.2   6209-17-2

  

 Action and use

  

 Antibacterial.

  

 Ph Eur

  

  

 DEFINITION

  

 Sulfacetamide sodium contains not less than 99.0 per cent and not more than the  equivalent of 101.0 per cent of the sodium derivative of N-[(4-aminophenyl)sulphonyl]acetamide, calculated with reference to the anhydrous  substance.

  

 CHARACTERS

  

 A white or yellowish-white, crystalline powder, freely soluble in water, slightly soluble  in ethanol.

  

 IDENTIFICATION

  

 First identification B, F.  

 

 Second identification A, C, D, E, F.

 

  A. Dissolve 0.1 g in phosphate buffer solution pH 7.0 R and dilute to 100.0 ml with the same buffer solution. Dilute 1.0 ml of the solution to 100.0 ml with phosphate buffer solution pH 7.0 R. Examined between 230 nm and 350 nm (2.2.25), the solution shows an absorption maximum at 255 nm. The specific absorbance at the maximum is 660 to 720, calculated with reference to the anhydrous substance.

 

  B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with sulfacetamide sodium CRS.

 

  C. Dissolve 1 g in 10 ml of water R, add 6 ml of dilute acetic acid R and filter. The precipitate, washed with a small quantity of water R and dried at 100 °C to 105 °C for 4 h, melts (2.2.14) at 181 °C to 185 °C.

 

  D. Dissolve 0.1 g of the precipitate obtained in identification test C in 5 ml of alcohol R. Add 0.2 ml of sulphuric acid R and heat. The odour of ethyl acetate is perceptible.

 

  E. Dissolve about 1 mg of the precipitate obtained in identification test 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.

 

  F. Solution S (see Tests) gives the reactions of sodium (2.3.1).

  

 TESTS

  

 Solution S

  

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

  

 Appearance of solution

  

 Solution S is clear (2.2.1) and not more intensely coloured than reference solution  GY₄ (2.2.2, Method II).

  

 pH (2.2.3)

  

 The pH of solution S is 8.0 to 9.5.

  

 Related substances

  

 Examine by thin-layer chromatography (2.2.27), using silica gel HF₂₅₄ R as the  coating substance.

 

 Test solution Dissolve 1.5 g of the substance to be examined in water R and dilute  to 15 ml with the same solvent.

 

 Reference solution (a) Dissolve 5 mg of sulfanilamide R in water R and dilute to 10  ml with the same solvent.

 

 Reference solution (b) Dilute 5 ml of reference solution (a) to 10 ml with water R.

 

 Reference solution (c) Dissolve 5 mg of sulfanilamide R in 10 ml of the test solution.

 

 Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture  of 10 volumes of concentrated ammonia R, 25 volumes of ethanol R, 25 volumes of  water R and 50 volumes of butanol R. Allow the plate to dry in air and spray with  dimethylaminobenzaldehyde solution R2. 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) (0.5 per cent), and not more  than one such spot is more intense than the spot in the chromatogram obtained with  reference solution (b) (0.25 per cent). The test is not valid unless the chromatogram  obtained with reference solution (c) shows two clearly separated spots.

  

 Sulphates (2.4.13)

  

 Dissolve 2.5 g in distilled water R and dilute to 25 ml with the same solvent. Add 25  ml of dilute acetic acid R, shake for 30 min and filter. 15 ml of the filtrate complies  with the limit test for sulphates (200 ppm).

  

 Heavy metals (2.4.8)

  

 12 ml of the filtrate obtained in the test for sulphates complies with limit test A for  heavy metals (20 ppm). Prepare the standard using lead standard solution (1 ppm  Pb) R.

  

 Water (2.5.12)

  

 6.0 per cent to 8.0 per cent, determined on 0.200 g by the semi-micro determination  of water.

  

 ASSAY

  

 Dissolve 0.500 g in a mixture of 50 ml of water R and 20 ml of dilute hydrochloric acid  R. Cool the solution in iced water and carry out the determination of primary aromatic  amino-nitrogen (2.5.8), determining the end-point electrometrically.

 

 1 ml of 0.1 M sodium nitrite is equivalent to 23.62 mg of C₈H₉N₂NaO₃S.

  

 STORAGE

  

 Store protected from light.

  

 

  Ph Eur

 

 

 

 

 

 

Sulfathiazole

norsulfazole (sulfathiazole)

General Notices

  

 

 (Ph Eur monograph 0742)

 

 

 

 

 

 C₉H₉N₃O₂S₂   255.3  72-14-0

  

 Action and use

  

 Antibacterial.

  

 Ph Eur

  

  

 DEFINITION

  

 Sulfathiazole contains not less than 99.0 per cent and not more than the equivalent  of 101.0 per cent of 4-amino-N-(thiazol-2-yl)benzenesulphonamide, calculated with  reference to the dried substance.

  

 CHARACTERS

  

 A white or slightly yellowish, crystalline powder, practically insoluble in water,  slightly soluble in alcohol, practically insoluble in methylene chloride. It dissolves in  dilute solutions of alkali hydroxides and in dilute mineral acids.

  

 IDENTIFICATION

  

 First identification A, B.

 

 Second identification A, C, D, E.

 

  A. Melting point (2.2.14): 200 °C to 203 °C. Melting may occur at about 175 °C, followed by solidification and a second melting between 200 °C and 203 °C.

 

  B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with sulfathiazole CRS. Examine the substances prepared as discs. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in alcohol R, evaporate to dryness in vacuo and record the spectra again using the residues.

 

  C. 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).

 

  D. Dissolve about 10 mg in a mixture of 10 ml of water R and 2 ml of 0.1 M sodium hydroxide and add 0.5 ml of copper sulphate solution R. A greyish-blue or purple precipitate is formed.

 

  E. 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 addition of acid, gives the reaction of primary aromatic amines (2.3.1).

  

 TESTS

  

 Appearance of solution

  

 Dissolve 1.0 g in 10 ml of 1 M sodium hydroxide. The solution is clear (2.2.1) and not  more intensely coloured than reference solution GY₄ (2.2.2, Method II).

  

 Acidity

  

 To 1.0 g add 50 ml of carbon dioxide-free water R. Heat to 70 °C for 5 min. Cool  rapidly to 20 °C and filter. To 25 ml of the filtrate add 0.1 ml of bromothymol blue  solution R1. Not more than 0.1 ml of 0.1 M sodium hydroxide is required to change  the colour of the indicator.

  

 Related substances

  

 Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating  substance.

 

 Test solution (a) Dissolve 0.10 g of the substance to be examined in a mixture of 1  volume of concentrated ammonia R and 9 volumes of alcohol R and dilute to 10 ml  with the same mixture of solvents.

 

 Test solution (b) Dilute 1 ml of test solution (a) to 5 ml with a mixture of 1 volume of  concentrated ammonia R and 9 volumes of alcohol R.

 

 Reference solution (a) Dissolve 20 mg of sulfathiazole CRS in a mixture of 1 volume  of concentrated ammonia R and 9 volumes of alcohol R and dilute to 10 ml with the  same mixture of solvents.

 

 Reference solution (b) Dissolve 50 mg of sulfanilamide R in a mixture of 1 volume of  concentrated ammonia R and 9 volumes of alcohol R and dilute to 100 ml with the  same mixture of solvents. Dilute 1 ml of this solution to 10 ml with the same mixture  of solvents.

 

 Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a  mixture of 18 volumes of ammonia R and 90 volumes of butanol R. Dry the plate at  100 °C to 105 °C for 10 min and spray with a 1 g/l solution of  dimethylaminobenzaldehyde R in alcohol R containing 1 per cent V/V of hydrochloric  acid R. 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).

  

 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 100 °C to  105 °C.

  

 Sulphated ash (2.4.14)

  

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

  

 ASSAY

  

 Carry out the determination of primary aromatic amino- nitrogen (2.5.8), using 0.200  g, determining the end-point electrometrically.

 

 1 ml of 0.1 M sodium nitrite is equivalent to 25.53 mg of C₉H₉N₃O₂S₂.

  

 STORAGE

  

 Store protected from light.

  

 

  Ph Eur

 

 

 

 

 

 

 

 

 

Sulfathiazole Sodium

 

General Notices

  

 

 

 C₉H₈N₃NaO₂S₂,1ЅH₂O  304.3  144-74-1(anhydrous

 

 C₉H₈N₃NaO₂S₂,5H₂O  367.4  6791-71-5

  

 Action and use

  

 Antibacterial.

  

 Preparation

  

 Tylosin Tartrate and Sulfathiazole Sodium Veterinary Oral Powder

  

 DEFINITION

  

 Sulfathiazole Sodium is the hydrated sodium salt of N¹-thiazol-2-yl sulphanilamide,  either the sesquihydrate or the pentahydrate. It contains not less than 99.0% and not  more than 101.0% of C₉H₈N₃NaO₂S₂, calculated with reference to the dried  substance.

  

 CHARACTERISTICS

  

 A white or yellowish white, crystalline powder or granules.

 

 Freely soluble in water ; soluble in ethanol (96%).

  

 IDENTIFICATION

  

  A. The infrared absorption spectrum of the dried substance, Appendix II A, is concordant with the reference spectrum of sulfathiazole sodium (RSV 42).

 

  B. Dissolve 1 g in 25 ml of water  and add 2 ml of 6M acetic acid. The melting point of the precipitate, after washing with water  and drying for 4 hours at 105°, is about 201°, Appendix V A.

 

  C. The precipitate obtained in test B yields the reaction characteristic of primary aromatic amines, Appendix VI, giving an orange-red precipitate.

  

 TESTS

  

 Alkalinity

  

 pH of a solution containing the equivalent of 1.0% w/v of the anhydrous substance,  9.0 to 10.0, Appendix V L.

  

 Heavy metals

  

 Dissolve a quantity containing the equivalent of 2.5 g of the anhydrous substance in  10 ml of water , add 15 ml of 2M acetic acid, shake for 30 minutes and filter. 12 ml of  the resulting solution complies with limit test A for heavy metals, Appendix VII. Use  lead standard solution (2 ppm Pb) to prepare the standard (20 ppm).

  

 Related substances

  

 Carry out the method for thin-layer chromatography, Appendix III A, using silica gel H  as the coating substance and a mixture of 18 volumes of 10M ammonia and 90  volumes of butan-1-ol  as the mobile phase. Apply separately to the plate 10 µl of  each of two solutions in a mixture of 1 volume of 13.5M ammonia and 9 volumes of  ethanol (96%) containing (1) 1.0% w/v of the substance being examined and (2)  0.0050% w/v of sulfanilamide. After removal of the plate, heat it at 105° for 10  minutes and spray with a 0.1% w/v solution of 4-dimethylaminobenzaldehyde in  ethanol (96%) containing 1% v/v of hydrochloric acid. Any secondary spot in the  chromatogram obtained with solution (1) is not more intense than the spot in the  chromatogram obtained with solution (2) (0.5%).

  

 Loss on drying

  

 When dried to constant weight at 105°, loses not less than 6.0% and not more than  10.0% of its weight (sesquihydrate) or not less than 22.0% and not more than 27.0%  of its weight (pentahydrate).

  

 ASSAY

  

 Dissolve 0.5 g in a mixture of 75 ml of water  and 10 ml of hydrochloric acid, add 3 g  of potassium bromide, cool in ice and titrate slowly with 0.1M sodium nitrite VS,  stirring constantly and determining the end point electrometrically. Each ml of 0.1M  sodium nitrite VS is equivalent to 27.73 mg of C₉H₈N₃NaO₂S₂.

  

 STORAGE

  

 Sulfathiazole Sodium should be protected from light.

  

 LABELLING

  

 The label states whether the substance is the sesquihydrate or the pentahydrate.

  

 

 

Tylosin tartrate and Sulfathiazole Sodium veterinary oral powder

General Notices

  

  

 DEFINITION

  

 Tylosin Tartrate and Sulfathiazole Sodium Veterinary Oral Powder is a mixture of  Tylosin Tartrate and Sulfathiazole Sodium. The proportions of the mixture are such  that the ratio of tylosin to Sulfathiazole Sodium is about one to three.

  

 The veterinary oral powder complies with the requirements stated under Veterinary  Oral Powders and with the following requirements.

  

 Content of sulfathiazole sodium, C₉H₈NaO₂S₂,1ЅH₂O

  

 90.0 to 110.0% of the stated amount, calculated as sulfathiaziole sodium  sesquihydrate.

  

 IDENTIFICATION

  

  A. Triturate a quantity of the powder containing the equivalent of 0.25 g of tylosin with two 25 ml quantities of chloroform and filter. Reserve the chloroform-insoluble residue for test B. Wash the combined filtrates by shaking for 1 minute with 20 ml of 0.1M sodium hydroxide and filter the chloroform layer through anhydrous sodium sulphate. Evaporate the filtrate to dryness and dry the residue over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 1 hour. The infrared absorption spectrum of the dried residue, Appendix II A, is concordant with the reference spectrum of tylosin (RSV 46).

 

  B. Dry the chloroform-insoluble residue reserved in test A at 105° for 1 hour. The infrared absorption spectrum of the dried residue, Appendix II A, is concordant with the reference spectrum of sulfathiazole sodium (RSV 42).

  

 TESTS

  

 Composition

  

 Carry out the method for liquid chromatography, Appendix III D, using the following  solutions prepared immediately before use. Solution (1) contains 0.02% w/v of tylosin  BPCRS in a mixture of equal volumes of water  and acetonitrile. For solution (2)  dissolve the powder in a mixture of equal volumes of water  and acetonitrile to  produce a solution containing the equivalent of 0.02% of tylosin.

 

 The chromatographic procedure may be carried out using (a) a stainless steel  column (20 cm Ч 5 mm) packed with stationary phase C (5 µm) (Nucleosil C18 is  suitable), (b) as the mobile phase with a flow rate of 1 ml per minute 0.85M sodium  perchlorate in a 40% v/v solution of acetonitrile, the pH of the solution being adjusted  to 2.5 using 1M hydrochloric acid and (c) a detection wavelength of 290 nm.

 

 The chromatogram obtained with solution (1) shows similar resolution to the  reference chromatogram supplied with tylosin BPCRS. If necessary adjust the  molarity of the sodium perchlorate or raise the temperature of the column to a  maximum of 50°. The order of elution of the six major components of tylosin BPCRS  in the chromatogram obtained with solution (1) is desmycinosyltylosin, tylosin C,  tylosin B, tylosin D, an aldol impurity and tylosin A.

 

 The column efficiency, determined using the peak due to tylosin A in solution (1),  should be at least 22,000 theoretical plates per metre.

 

 Calculate the percentage content of components by normalisation. In the  chromatogram obtained with solution (2) the content of tylosin A is not less than  80% and the total content of tylosins A, B, C and D is not less than 95%. A peak  due to sulphathiazole will be observed eluting immediately after the solvent peak and  should be disregarded.

  

 Related substances

  

 Carry out the method for thin-layer chromatography, Appendix III A, using silica gel H  as the coating substance and a mixture of 18 volumes of 10M ammonia and 90  volumes of butan-1-ol  as the mobile phase. Apply separately to the plate 10 µl of  each of the following solutions. For solution (1) shake a quantity of the powder  containing 0.1 g of Sulfathiazole Sodium with 10 ml of a mixture of 1 volume of 13.5M  ammonia and 9 volumes of ethanol (96%), filter and use the filtrate. Solution (2)  contains 0.0050% w/v of sulfanilamide in a mixture of 9 volumes of ethanol (96%)  and 1 volume of 13.5M ammonia. After removal of the plate, heat it at 105° for 10  minutes and spray with a 0.1% w/v solution of 4-dimethylaminobenzaldehyde in  ethanol (96%) containing 1% v/v of hydrochloric acid. Any secondary spot in the  chromatogram obtained with solution (1) is not more intense than the spot in the  chromatogram obtained with solution (2) (0.5%).

  

 ASSAY

  

 For tylosin activity

  

 Transfer a quantity of the powder containing the equivalent of 0.2 g of tylosin to a 100  ml graduated flask with the aid of three 10 ml quantities of methanol , swirl to  dissolve and add sufficient sterile phosphate buffer pH 7.0 to produce 100 ml. Filter  and dilute 5 ml of the filtrate to 100 ml with sterile phosphate buffer pH 7.0. Carry out  the biological assay of antibiotics, Appendix XIV A. The precision of the assay is  such that the fiducial limits of error are not less than 95% and not more than 105% of  the estimated potency. Calculate the content of tylosin taking each 1000 IU found to  be equivalent to 1 mg of tylosin. The upper fiducial limit of error is not less than  90.0% and the lower fiducial limit of error is not more than 110.0% of the stated  amount.

  

 For sulfathiazole sodium

  

 Dissolve a quantity of the powder containing the equivalent of 0.4 g of sulphathiazole  sodium sesquihydrate in a mixture of 75 ml of water  and 10 ml of hydrochloric acid,  add 3 g of potassium bromide, cool in ice and titrate slowly with 0.1M sodium nitrite  VS, stirring constantly and determining the end point electrometrically. Each ml of  0.1M sodium nitrite VS is equivalent to 30.43 mg of C₉H₈N₃NaO₂S₂,1ЅH₂O.

  

 LABELLING

  

 The label states the quantity of Tylosin Tartrate in terms of the equivalent amount of  tylosin and the quantity of Sulfathiazole Sodium in terms of the equivalent amount of  sulfathiazole sodium sesquihydrate.

  

 

 

 

 

Phthalylsulfathiazole

(Ph Eur monograph 0352)

 

 

 C₁₇H₁₃N₃O₅S₂   403.4  85-73-4

  

 Action and use

  

 Antibacterial.

  

 Ph Eur

  

  

 DEFINITION

  

 Phthalylsulfathiazole contains not less than 98.5 per cent and not more than the  equivalent of 101.5 per cent of 2-[[4-(thiazol-2-ylsulphamoyl)phenyl]carbamoyl]benzoic acid, calculated with reference  to the dried substance.

  

 CHARACTERS

  

 A white or yellowish-white, crystalline powder, practically insoluble in water, freely  soluble in dimethylformamide, slightly soluble in acetone and in alcohol.

  

 IDENTIFICATION

  

 First identification A, B, E.

 

 Second identification B, C, D, E.

 

  A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with phthalylsulfathiazole CRS.

 

  B. To 1 g add 8.5 ml of dilute sodium hydroxide solution R and boil under a reflux condenser for 30 min. Cool and add 17.5 ml of dilute hydrochloric acid R. Shake vigorously and filter. Neutralise the filtrate with dilute sodium hydroxide solution R. Filter, wash the precipitate with water R, recrystallise from water R and dry the crystals at 100 °C to 105 °C. The crystals melt (2.2.14) at 200 °C to 203 °C.

 

  C. To 0.1 g in a test-tube add 3 ml of dilute sulphuric acid R and 0.5 g of zinc powder R. Fumes are evolved which produce a black stain on lead acetate paper R.

 

  D. To 0.1 g add 0.5 g of resorcinol R and 0.3 ml of sulphuric acid R and heat on a water-bath 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. Dissolve about 10 mg of the crystals obtained in identification test B in 200 ml of 0.1 M hydrochloric acid. 2 ml of the solution gives the reaction of primary aromatic amines (2.3.1) with formation of an orange precipitate.

  

 TESTS

  

 Appearance of solution

  

 Dissolve 1.0 g in 1 M sodium hydroxide and dilute to 20 ml with the same solvent.  The solution is clear (2.2.1) and not more intensely coloured than reference solution  BY₅ (2.2.2, Method II).

  

 Acidity

  

 To 2.0 g add 20 ml of water R, shake continuously for 30 min and filter. To 10 ml of  the filtrate add 0.1 ml of phenolphthalein solution R. Not more than 0.2 ml of 0.1 M  sodium hydroxide is required to change the colour of the indicator.

  

 Sulfathiazole and other primary aromatic amines

  

 Dissolve 5 mg in a mixture of 3.5 ml of water R, 6 ml of dilute hydrochloric acid R and  25 ml of alcohol R, previously cooled to 15 °C. Place immediately in iced water and  add 1 ml of a 2.5 g/l solution of sodium nitrite R. Allow to stand for 3 min, add 2.5 ml  of a 40 g/l solution of sulphamic acid R and allow to stand for 5 min. Add 1 ml of a 4  g/l solution of naphthylethylenediamine dihydrochloride R and dilute to 50 ml with  water R. Measured at 550 nm, the absorbance (2.2.25) is not greater than that of a  standard prepared at the same time and in the same manner using a mixture of 1 ml  of a solution containing in 100 ml; 10 mg of sulfathiazole R and 0.5 ml of hydrochloric  acid R, 2.5 ml of water R, 6 ml of dilute hydrochloric acid R and 25 ml of alcohol 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)

  

 Not more than 2 per cent, determined on 1.00 g by drying in an oven at 100 °C to  105 °C.

  

 Sulphated ash (2.4.14)

  

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

  

 ASSAY

  

 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.

  

 STORAGE

  

 Store protected from light.

  

 

  Ph Eur

 

 

 

 

 

 

 

Sulfaguanidine

 

General Notices

  

   (Ph Eur monograph 1476)

 

 

 

 

  C₇H₁₀N₄O₂S   214.3   56-67-0

  

 Action and use

  

 Antibacterial.

  

 Ph Eur

  

  

 DEFINITION

  

 Sulfaguanidine contains not less than 99.0 per cent and not more than the equivalent  of 101.0 per cent of (4-aminophenylsulphonyl)guanidine, calculated with reference to  the dried substance.

  

 CHARACTERS

  

 A white or almost white, fine crystalline powder, very slightly soluble in water, slightly  soluble in acetone, very slightly soluble in ethanol (96 per cent), practically insoluble  in methylene chloride. It dissolves in dilute solutions of mineral acids.

  

 IDENTIFICATION

  

 First identification A, B.

 

 Second identification A, C, D, E.

 

  A. Melting point (2.2.14): 189 °C to 193 °C, determined on the dried substance.

 

  B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with sulfaguanidine CRS.

 

  C. 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 and size to the principal spot in the chromatogram obtained with reference solution (a).

 

  D. 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).

 

  E. Suspend 0.1 g in 2 ml of water R, add 1 ml of naphthol solution R and 2 ml of a mixture of equal volumes of water R and strong sodium hypochlorite solution R. A red colour develops.

  

 TESTS

  

 Solution S

  

 To 2.5 g, add 40 ml of carbon dioxide-free water R. Heat at about 70 °C for 5 min.  Cool while stirring in iced water for about 15 min, filter and dilute to 50 ml with carbon  dioxide-free water R.

  

 Acidity

  

 To 20 ml of solution S, add 0.1 ml of bromothymol blue solution R1. Not more than  0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator.

  

 Related substances

  

 Examine by thin layer chromatography (2.2.27), using a TLC silica gel GF₂₅₄plate  R.

 

 Test solution (a) Dissolve 50 mg of the substance to be examined in acetone R and  dilute to 5 ml with the same solvent.  

 

 Test solution (b) Dilute 2 ml of test solution (a) to 10 ml with acetone R.

 

 Reference solution (a) Dissolve 10 mg of sulfaguanidine CRS in acetone R and  dilute to 5 ml with the same solvent.  

 

 Reference solution (b) Dilute 5 ml of test solution (b) to 200 ml with acetone R.

 

 Reference solution (c) Dilute 5 ml of reference solution (b) to 10 ml with acetone R.

 

 Reference solution (d) Dissolve 10 mg of sulfanilamide R in test solution (b) and  dilute to 5 ml with the same solution.  

 

 Apply to the plate 10 µl of each solution. Develop over a path of 15 cm using a  mixture of 10 volumes of anhydrous formic acid R, 20 volumes of methanol R and 70  volumes of methylene chloride R. Allow the plate to dry in air and examine in  ultraviolet light at 254 nm. 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) and at most one  such spot is more intense that the spot in the chromatogram obtained with reference  solution (c) (0.25 per cent). The test is not valid unless the chromatogram obtained  with reference solution (d) shows two clearly separated principal spots.

  

 Heavy metals (2.4.8)

  

 1.0 g complies with test F (20 ppm). Prepare the reference solution using 2 ml of  lead standard solution (10 ppm Pb) R.

  

 Loss on drying (2.2.32)

  

 Not more than 8.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C.

  

 Sulphated ash (2.4.14)

  

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

  

 ASSAY

  

 Dissolve 0.175 g in 50 ml of dilute hydrochloric acid R. Cool the solution in iced  water. Carry out the determination of primary aromatic amino-nitrogen (2.5.8),  determining the end-point electrometrically.

 

 1 ml of 0.1 M sodium nitrite is equivalent to 21.42 mg of C₇H₁₀N₄O₂S.

  

 STORAGE

  

 Store protected from light.

  

 IMPURITIES

  

 

 

 

 

 

 

 

 

  

  A. R = H: 4-aminobenzenesulphonamide (sulphanilamide),

 

  B. R = CO-NH₂: N-[(4-aminophenyl)sulphonyl]urea (sulphacarbamide).

  

 

  Ph Eur

 

 

 

Sulfadimidine

General Notices

  

 

 (Ph Eur monograph 0295)

 

 

 

 

 

 C₁₂H₁₄N₄O₂S   278.3  57-68-1

  

 Action and use

  

 Antibacterial.

  

 Preparation

  

 Sulfadimidine Injection

 

 Sulfadimidine Tablets

  

 Ph Eur

  

  

 DEFINITION

  

 Sulfadimidine contains not less than 99.0 per cent and not more than the equivalent  of 101.0 per cent of 4-amino-N-(4,6-dimethylpyrimidin-2-yl)benzenesulphonamide,  calculated with reference to the dried substance.

  

 CHARACTERS

  

 White or almost white powder or crystals, very slightly soluble in water, soluble in  acetone, slightly soluble in alcohol. It dissolves in solutions of alkali hydroxides and  in dilute mineral acids.  

 

 It melts at about 197 °C, with decomposition.

  

 IDENTIFICATION

  

 First identification A, B.

 

 Second identification B, C, D.

 

  A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with sulfadimidine CRS. Examine the substances prepared as discs.

 

  B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (a) corresponds in position and size to the principal spot in the chromatogram obtained with reference solution (a).

 

  C. Place 3 g in a dry tube. Immerse the lower part of the tube, inclined at 45°, in a silicone oil bath and heat to about 270 °C. The substance to be examined decomposes and a white or yellowish-white sublimate is formed which, after recrystallisation from toluene R and drying at 100 °C, melts (2.2.14) at 150 °C to 154 °C.

 

  D. 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).

  

 TESTS

  

 Appearance of solution

  

 Dissolve 0.5 g in a mixture of 5 ml of dilute sodium hydroxide solution R and 5 ml of  water R. The solution is not more intensely coloured than reference solution Y₅, BY₅  or GY₅ (2.2.2, Method II).

  

 Acidity

  

 To 1.25 g, finely powdered, add 25 ml of carbon dioxide-free water R. Heat at about  70 °C for 5 min. Cool in iced water for about 15 min and filter. To 20 ml of the filtrate  add 0.1 ml of bromothymol blue solution R1. Not more than 0.2 ml of 0.1 M sodium  hydroxide is required to change the colour of the indicator.

  

 Related substances

  

 Examine by thin-layer chromatography (2.2.27), using silica gel GF₂₅₄ R as the  coating substance.

 

 Test solution (a) Dissolve 20 mg of the substance to be examined in 3 ml of a  mixture of 2 volumes of concentrated ammonia R and 48 volumes of methanol R and  dilute to 5.0 ml with the same mixture of solvents.

 

 Test solution (b) Dissolve 0.10 g of the substance to be examined in 0.5 ml of  concentrated ammonia R and dilute to 5.0 ml with methanol R. If the solution is not  clear, heat gently until dissolution is complete.

 

 Reference solution (a) Dissolve 20 mg of sulfadimidine CRS in 3 ml of a mixture of 2  volumes of concentrated ammonia R and 48 volumes of methanol R and dilute to 5.0  ml with the same mixture of solvents.

 

 Reference solution (b) Dilute 1.25 ml of test solution (a) to 50 ml with a mixture of 2  volumes of concentrated ammonia R and 48 volumes of methanol R.

 

 Apply to the plate 5 µl of each solution. Develop over a path of 15 cm using a mixture  of 3 volumes of dilute ammonia R1, 5 volumes of water R, 40 volumes of  nitromethane R and 50 volumes of dioxan R. Dry the plate at 100 °C to 105 °C and  examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with  test solution (b), apart from the principal spot, is not more intense than the spot in  the chromatogram obtained with reference solution (b) (0.5 per cent).

  

 Heavy metals (2.4.8)

  

 1.0 g complies with limit test D 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 1.00 g by drying in an oven at 100 °C to  105 °C.

  

 Sulphated ash (2.4.14)

  

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

  

 ASSAY

  

 Dissolve 0.250 g in a mixture of 20 ml of dilute hydrochloric acid R and 50 ml of water  R. Cool the solution in iced water. Carry out the determination of primary aromatic  amino– nitrogen (2.5.8), determining the end-point electrometrically.

 

 1 ml of 0.1 M sodium nitrite is equivalent to 27.83 mg of C₁₂H₁₄N₄O₂S.

  

 STORAGE

  

 Store protected from light.

  

 

  Ph Eur

 

Sulfadimidine Injection

 

 

  

 DEFINITION

  

 Sulfadimidine Injection is a sterile solution of sulfadimidine sodium in Water for  Injections free from dissolved air. It is prepared by the interaction of Sulfadimidine  and Sodium Hydroxide.

  

 The injection complies with the requirements stated under Parenteral Preparations  and with the following requirements.

  

 Content of sulfadimidine sodium, C₁₂H₁₃N₄NaO₂S

  

 95.0 to 105.0% of the stated amount.

  

 IDENTIFICATION

  

  A. Acidify a volume containing 0.1 g of sulfadimidine sodium with 6M acetic acid, filter, reserving the filtrate, wash the residue with water  and dry at 105°. The infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference spectrum of sulfadimidine (RSV 50).

 

  B. The residue obtained in test A yields the reaction characteristic of primary aromatic amines, Appendix VI, producing a bright orange-red precipitate.

  

 TESTS

  

 Alkalinity

  

 pH, 10.0 to 11.0, Appendix V L.

  

 Colour of solution

  

 An injection containing 1 g of sulfadimidine sodium in 3 ml is not more intensely  coloured than reference solution Y₄, Appendix IV B, Method I.

  

 Related substances

  

 Carry out the method for thin-layer chromatography, Appendix III A, using silica gel H  as the coating substance and a mixture of 18 volumes of 10M ammonia and 90  volumes of butan-1-ol  as the mobile phase. Apply separately to the plate 10 µl of  each of the following solutions. For solution (1) use the injection being examined  diluted with water  to contain 0.20% w/v of sulfadimidine sodium. Solution (2)  contains 0.0020% w/v of sulfanilamide in a mixture of 1 volume of 13.5M ammonia  and 9 volumes of ethanol (96%). After removal of the plate, heat it at 105° for 10  minutes and spray with a 0.1% w/v solution of 4-dimethylaminobenzaldehyde in  ethanol (96%) containing 1% v/v of hydrochloric acid. Any secondary spot in the  chromatogram obtained with solution (1) is not more intense than the spot in the  chromatogram obtained with solution (2) (1%).

  

 ASSAY

  

 Dilute a volume containing 0.5 g of sulfadimidine sodium to 75 ml with water , add 10  ml of hydrochloric acid and pass air slowly through the solution until the vapours do  not turn moistened starch iodate paper  blue. Add 3 g of potassium bromide, cool in  ice and titrate slowly with 0.1M sodium nitrite VS, stirring constantly and determining  the end point electrometrically. Each ml of 0.1M sodium nitrite VS is equivalent to  30.03 mg of C₁₂H₁₃N₄NaO₂S.

  

 STORAGE

  

 Sulfadimidine Injection should be protected from light.

  

 LABELLING

  

 The strength is stated as the amount of sulfadimidine sodium in a suitable dose-volume.

  

 

 

Sulfadimidine Tablets

DEFINITION

  

 Sulfadimidine Tablets contain Sulfadimidine.

  

 The tablets comply with the requirements stated under Tablets and with the following  requirements.

  

 Content of sulfadimidine, C₁₂H₁₄N₄O₂S

  

 95.0 to 105.0% of the stated amount.

  

 IDENTIFICATION

  

  A. Triturate a quantity of the powdered tablets containing 0.5 g of Sulfadimidine with two 5 ml quantities of chloroform and discard the chloroform. Triturate the residue with 10 ml of 5M ammonia for 5 minutes, add 10 ml of water  and filter. Warm the filtrate until most of the ammonia has been removed, cool, acidify with 6M acetic acid, wash the precipitate with water  and dry at 105°. The infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference spectrum of sulfadimidine (RSV 50).

 

  B. In the test for Related substances, the principal spot in the chromatogram obtained with solution (2) corresponds to that in the chromatogram obtained with solution (4).

 

  C. The residue obtained in test A yields the reaction characteristic of primary aromatic amines, Appendix VI, producing a bright orange-red precipitate.

  

 Related substances

  

 Carry out the method for thin-layer chromatography, Appendix III A, using silica gel  GF₂₅₄ as the coating substance and a mixture of 3 volumes of 6M ammonia, 5  volumes of water , 40 volumes of nitromethane and 50 volumes of 1,4-dioxan as the  mobile phase. Apply separately to the plate 5 µl of each of the following solutions.  For solution (1) extract a quantity of the powdered tablets containing 0.5 g of  Sulfadimidine with 25 ml of a mixture of 1 volume of 13.5M ammonia and 9 volumes of  methanol  by shaking for 10 minutes, filter and use the filtrate. For solution (2) dilute  1 volume of solution (1) to 5 volumes with a mixture of 1 volume of 13.5M ammonia  and 24 volumes of methanol . For solution (3) dilute 1 volume of solution (1) to 200  volumes with a mixture of 1 volume of 13.5M ammonia and 24 volumes of methanol .  Solution (4) contains 0.40% w/v of sulfadimidine EPCRS in a mixture of 1 volume of  13.5M ammonia and 24 volumes of methanol . After removal of the plate, dry it at 100°  to 105° and examine under ultraviolet light (254 nm). Any secondary spot in the  chromatogram obtained with solution (1) is not more intense than the spot in the  chromatogram obtained with solution (3) (0.5%).

  

 ASSAY

  

 Weigh and powder 20 tablets. Dissolve a quantity of the powder containing 0.5 g of  Sulfadimidine as completely as possible in a mixture of 50 ml of water  and 10 ml of  hydrochloric acid, add 3 g of potassium bromide, cool in ice and titrate slowly with  0.1M sodium nitrite VS, stirring constantly and determining the end point  electrometrically. Each ml of 0.1M sodium nitrite VS is equivalent to 27.83 mg of C₁₂H₁₄N₄O₂S.

  

 STORAGE

  

 Sulfadimidine Tablets should be protected from light.