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METHODICAL INSTRUCTION FOR STUDENTS OF THE 3rd COURSE

June 15, 2024

METHODICAL INSTRUCTION FOR STUDENTS OF THE 3rd COURSE

Pharmaceutical Faculty

LESSON № 18 (PRACTICAL – 6 HOURS)

Theme: Pharmaceutical analysis of derivatives of pyridine (pyridine-3-carboxylic acid, pyridine-4-carboxylic acid) and pyrimidine (barbituric acid) 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 six-membered heterocycles.

Professional orientation of students:

The six-membered heterocycles with one heteroatom of Nitrogen are pyridine derivatives:

Pyridine is used as a precursor to pharmaceuticals and is also an important solvent and reagent. Pyridine has strong bactericidal action, however because of toxicity in medicine it is not applied, but many its derivatives are not only medical products, but also are used for synthesis of some synthetic drugs (nicotinic acid and its amide, Nikethamide injection). Isonicotinic acid (pyridin-4-carboxylic acid or γ-pyridincarboxylic acid)) is used for synthesis of drugs with antitubercular action (Isoniazid, Ftivazide, Flurenizide, etc.).

The six-membered heterocycles with two heteroatoms of Nitrogen at position 1 and 3 are pyrimidine derivatives:

Pyrimidine cycle is a part of molecules of many drugs, both natural, and synthetic origin (some alkaloids, vitamins, sulfanilamides), and also in structure of nucleinic acids. The derivatives of barbituric acid are synthetic compounds of pyrimidine, in the medical practice they apply as hypnagogue and anticonvulsant, and also for intravenous narcosis.

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

Isoniazid

Izoniazidum

C₆H₇N₃O

137.1

DEFINITION

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

CHARACTERS

A white, crystalline powder or colourless crystals, freely soluble in water, sparingly soluble in alcohol.

IDENTIFICATION

C. (BP, SPU, suppl. 2). Reaction with solution of vanillin

Dissolve 0.1 g in 2 ml of water R and add 10 ml of a warm 10 g/l solution of vanillin R. Allow to stand and scratch the wall of the test tube with a glass rod. A yellow precipitate is formed, which, after recrystallisation from 5 ml of alcohol (70 per cent V/V) R and drying at 100 °C to 105 °C, melts at 226 °C to 231 °C.

yellow precipitate

Other reactions:

Reaction with copper(ІІ) sulphate solution

To 0,1 g of drug substance add 5 ml of water and 4–5 drops of copper(ІІ) sulphate solution; a blue precipitate is formed; shake and solution becomes blue. At heating the solution and precipitate change colour to pale green, after that – yellow-green and gas vials are allocated.

Reaction with 2,4-dinitrochlorobenzene and glutaconic aldehyde formation (for pyridine cycle)

To several crystals of drug substance add 0,05 g of 2,4-dinitrochlorobenzene, 3 ml of 95 % ethanol and boil during 1–1,5 minutes. After that to cool solution, add 2 drops of sodium hydroxide solution; a red-brownish colour is formed, which quickly changes to red-brown.

Definition of hydrazine rest

To 0,1 g of drug substance add 2 ml of water and 1 ml of ammoniac solution of silver nitrate; a yellowish precipitate is formed, which at heating by a water heater darkens and on test tube walls the “silver mirror” is formed.

TESTS

Solution S

Dissolve 2.5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent.

pH (2.2.3)

The pH of solution S is 6.0 to 8.0.

ASSAY

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

Dissolve 0.250 g in water R and dilute to 100.0 ml with the same solvent. To 20.0 ml of the solution add 100 ml of water R, 20 ml of hydrochloric acid R, 0.2 g of potassium bromide R

and 0.05 ml of methyl red solution R. Titrate dropwise with 0.0167 M potassium bromate, shaking continuously, until the red colour disappears.

1 ml of 0.0167 M potassium bromate is equivalent to 3.429 mg of C₆H₇N₃O.

KBrO₃ + 5KBr + 6HCl → 3Br₂ + 6KBr + 3H₂O

1 mol 4 equivalents

E_m (С₆Н₇N₃O) = М. m./4

STORAGE

Protected from light, in the well-closed containers. At temperature not more than +10°C.

Action and use

Antituberculosis drug.

Preparations

Isoniazid Injection

Isoniazid Tablets

Ph Eur

Flurenizide

Flurenizidum

C₁₉H₁₃N₃O

299.33

DEFINITION

Flurenizidе contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of N-(fluorenylidene)-N-pyridine-4-carboxylic acid hydrazide, calculated with reference to the dried substance.

CHARACTERS

A fine-crystalline powder with acicular crystals or yellow or greenish-yellow lamellar crystals, without a smell, freely soluble in acetic acid, sparingly soluble in chloroform, slightly soluble in 96 % alcohol, practically insoluble in water.

IDENTIFICATION

A. Determination of fluorene fragment (non-pharmacopoeial reaction)

Place about 0,1 g of the substance to be examined on hour glass and add 0,03 ml of concentrated nitric acid. A orange-red colour is produced. The red colour disappears at addition 0,04 ml of water.

orange-red colour

B. Reaction with alkaline solution of copper(ІІ) sulphate

To about 0,1 g of the substance to be examined add 1,0 ml of 96 % ethanol, 0.5 ml of water, 0,08 ml of sodium hydroxide solution and 0,15 ml of copper(ІІ) sulphate solution. A bluish-green precipitate is formed, which is turning to brown-green and gas vials are allocated.

C. Reaction with 2,4-dinitrochlorobenzene and glutaconic aldehyde formation (for pyridine cycle)

To about 0,1 g of the substance to be examined add some crystals of 2,4-dinitrochlorobenzene, 3 ml of 95 % alcohol and boil during 1,5 minutes. After cooling add 0,08 ml of sodium hydroxide solution. A brown-red colour is produced, which is quickly turning to reddish-brown.

STORAGE

In the well-closed containers, in the place protected from light.

Action and use

Antitubercular, antimicrobial, antichlamidial agent.

Corvalol

Corvalolum

Corvalol is a barbiturate-based heart medication and a mild tranquilizer, popular in Eastern Europe and the former Soviet Union.

The composition of this medication may vary to some degree. In a typical preparation, a single dose of Corvalol ( 1 ml ) contains:

· Phenobarbital – 16mg

· Bromisovalum (derivative of bromine and valeric acid) – 20mg or Ethylbromisovalerinate ( ethyl ester of α-Bromoisovaleric acid ) – 20 mg

· Peppermint oil – 1.5 mg

· Hop oil – 0.2 mg (not always present)

as well as inactive ingredients:

· Sodium hydroxide (to convert poorly soluble phenobarbital into phenobarbital sodium)

· Ethanol and water as solutes.

Phenobarbital

Phenobarbitalum

C₁₂H₁₂N₂O₃

232.2

DEFINITION

Corvalol contains as the main active ingredient 5-ethyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trione.

CHARACTERS

It is a transparent liquid with a characteristic strong smell.

IDENTIFICATION

D. (BP, SPU, suppl. 2). It gives the reaction of non-nitrogen substituted barbiturates.

Barbiturates, Non-nitrogen Substituted:

(BP, SPU). Reaction with salts of heavy metals (colored complexes formation)

Dissolve about 5 mg of the substance to be examined in 3 ml of methanol R, add 0.1 ml of a solution containing 100 g/l of cobalt nitrate R and 100 g/l of calcium chloride R. Mix and add, with shaking, 0.1 ml of dilute sodium hydroxide solution R. A violet-blue colour and precipitate are formed.

STORAGE

In the well-closed containers, in the place protected from light.

Action and use

Barbiturate.

Sedative; anticonvulsant.

Ph Eur

Individual Students Program

1. Latin names and synonyms, structural formulas and chemical names of Nikethamide, Nikethamide Injection, Hydroxymethylnicotinamide, Isoniazid, Ftivazide, Flurenizide; Barbital, Barbital sodium, Phenobarbital, Hexobarbital, Thiopental Sodium, Amobarbital, Benzobarbital.

2. Synthesis of afore-mentioned preparations.

3. Physical properties of investigated medical products.

4. Identification of given drugs.

5. Tests of above-mentioned substances.

6. The methods of quantitative definition of drug substances.

7. Storage conditions of above-mentioned drugs, according to their physical and chemical properties.

8. Application of drugs in the medical practice and pharmaceutical analysis.

Seminar discussion of theoretical issues

1. What are latiames of Nikethamide, Nikethamide Injection, Hydroxymethylnicotinamide, Isoniazid, Ftivazide, Flurenizide; Barbital, Barbital sodium, Phenobarbital, Hexobarbital, Thiopental Sodium, Amobarbital, Benzobarbital?

2. What are structures of Nikethamide, Nikethamide Injection, Hydroxymethylnicotinamide, Isoniazid, Ftivazide, Flurenizide; Barbital, Barbital sodium, Phenobarbital, Hexobarbital, Thiopental Sodium, Amobarbital, Benzobarbital?

3. What are methods of barbiturates synthesis?

4. What general and specific reactions of identification of afore-mentioned preparations do you know?

5. What methods of assay of afore-mentioned preparations do you know?

6. What storage conditions of drugs do you know?

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

Test evaluation and situational tasks

1. The Pharmacopoeial method of assay for Nikethamide is:

Acidimetry, non-aqueous titration

B. Argentometry, back titration

Mercurimetry, direct titration
Coppermetry, direct titration
Chelatometry, direct titration

2. The method of assay for Hydroxymethylnicotinamide is:

A. Argentometry, back titration

B. Iodometry, back titration, after alkaline hydrolysis

C. Chelatometry, direct titration

D. Permanganatometry, direct titration

E. Bromatometry, back titration, with iodometric finishing

3. In the medical practice Ftivazide can be used as:

Anaesthetic
Purgative

C. Expectorant

Antituberculous
Helminthicide

4. Initial substances for synthesis of Flurenizide are:

A. Isoniazid, vanillin

B. Isoniazid, water

C. Isoniazid, 9-fluorenone

D. Isoniazid, phenol

E. Isoniazid, formaldehyde

5. Hexobarbital in medical practice can be used as agent:

A. For intravenous narcosis

B. Sedative of long action

C. Sedative of short term action

D. Purgative

E. For an inhalatioarcosis

6. The method of assay for Phenobarbital is acidimetry, non-aqueous titration. As titrant use standard solution of:

A. H₃PO₄

B. HClO₄

C. HNO₃

D. HCl

E. H₂SO₄

7. This structure corresponds to the drug substance:

A. Niketamide injection

B. Isoniazid

C. Ftivazide

D. Flurenizide

E. Hydroxymethylnicotinamide

8. This structure corresponds to the drug substance:

A. Barbital

B. Phenobarbital

C. Hexobarbital

D. Amobarbital

E. Thiopental sodium

9. This structure corresponds to the drug substance:

A. Hexobarbital

B. Thiopental sodium

C. Benzobarbital

D. Amobarbital

E. Phenobarbital

10. This structure corresponds to the drug substance:

A. Benzobarbital

B. Barbital

C. Phenobarbital

D. Amobarbital

E. Hexobarbital

What volume of 0,05 M perchloric acid (f = 1,0000) will be used for titration 0,1534 g of Ftivazide substance (M = 271,28 g/mol)?
Calculate T_{(NaOH/Barbital),} if for titration 0,1505 g of Barbital substance was used 8,2 ml of standard solution of perchloric acid (f = 0,9958). The percentage content of Barbital in a preparation is 99,96 %.
Calculate the percentage content of Phenobarbital in preparation. For this purpose was dissolve 0,2226 g of Phenobarbital substance (M = 232,24 g/mol) in some organic solvent and was titrated with 0.1 M sodium hydroxide (f = 1,0018). For titration was used 9,5 ml of 0.1 M sodium hydroxide.

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 names and synonyms, structure formulas and chemical names of Nikethamide, Nikethamide Injection, Hydroxymethylnicotinamide, Isoniazid, Ftivazide, Flurenizide; Barbital, Barbital sodium, Phenobarbital, Hexobarbital, Thiopental Sodium, Amobarbital, Benzobarbital.

2. Methods of barbiturates synthesis.

3. Physical and chemical characteristics, methods of identification, establishment of high quality and quantitative definition of afore-mentioned drugs.

4. Storage conditions and application of drugs in medical practice and the pharmaceutical analysis.

Student should be able to:

1. To write reactions of identifications and assay of Nikethamide, Nikethamide Injection, Hydroxymethylnicotinamide, Isoniazid, Ftivazide, Flurenizide; Barbital, Barbital sodium, Phenobarbital, Hexobarbital, Thiopental Sodium, Amobarbital, Benzobarbital.

2. To calculate equivalent mass and mass fraction of active ingredients in investigated substances by different methods.

3. After analysis carrying out a conclusion about the hight quality of the drug substances.

Correct answers of test evaluations and situational tasks:

1. A; 2. B; 3. D; 4. C; 5. A; 6. B; 7. E; 8. A; 9. C; 10. D.

1. 11,3 ml; 2. 0,01842 g/ml; 3. 99,3 %.

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. Sarker S.D. Chemistry for pharmacy students: general, organic, and natural product chemistry / S.D. Sarker, L. Nahar. – UK: John Wiley & Sons, Ltd, 2007. – 383 p.

5. Kar A. Pharmaceutical drug analysis. – Revised second edition. – Addis Ababa: New Age International Publishers, 2005. – 529 p.

6. www.tdmu.edu.te.ua

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