Chemical and toxicological analysis of acidic, Chemical and toxicological analysis of acidic, neutral and weak basic drugsneutral and weak basic drugs

Lecture № 8

associate prof. M.M. Mykhalkiv

OUTLINE.1. Usage, toxicological characteristics, methods of isolation from

biological material and methods of analysis of salicylic acid derivatives.

2. Usage, toxicological characteristics, methods of isolation from biological material and methods of analysis of barbituric acid derivatives.

3. Usage, toxicological characteristics, methods of isolation from biological material and methods of analysis of pyrazolone derivatives.

1. Usage, toxicological characteristics, methods of isolation from biological material and methods of analysis of salicylic acid

derivatives. Salicylic acid derivatives

Salicylic acid (hydroxybenzoic acid)

Aspirin (acetylsalicylic acid)

Sodium salicylate

Methyl salicylate

Salicylamide

Physical and chemical propertiesAspirin and other salicylic acid derivatives (except of methyl

salicylate) are crystal substances, and methyl salicylate is a colorless or yellowish liquid with characteristic odor.

They (except sodium salicylate) are slightly soluble or practically insoluble in water; soluble in alkali, alcohol and other organic solvents.

Sodium salicylate is soluble in water and alcohol.Salicylic acid and its derivatives is sublimed.

UsageSalicylic acid is used to treat of skin diseases, as a disinfectant,

decrease sweating. Drugs - salicylic acid derivatives (salts, esters, amides) are

used as antipyretic, anti-inflammatory and analgetic in the treatment of rheumatic fever, rheumatic endocarditis and myocarditis.

Methyl salicylate is applied externally for treatment of joint and muscle rheumatism, arthritis, exudative pleurisy.

Toxic actionTherapeutic doses of salicylates: may be tinnitus, impaired

hearing, edema, heartburn, vomiting. Toxic doses: exacerbation of asthma, allergic reactions, decrease

of synthesis of mucus secretion in the stomach and the formation of gastric ulcers.

Salicylates contribute to the development of bleeding. Neuropsychopathy (discoordination, speech disorder, anxiety,

convulsions) will be at higher doses. The lethal dose salicylates: 2-4 g - for children, about 20 grams

- for adults. The behavior in the organism

Salicylic acid is rapidly absorbed in the stomach; much of it is bound with plasma proteins, excreted unchanged or as metabolites through the kidneys with urine.

Salicylic acid and its derivatives are metabolized in the liver: hydrolysis, oxidation, conjugation by Glucuronides, conjugation by glycine. Salicylamide is excreted mainly unchanged.

METABOLISMBiotransformation products of salicylic acid:

2,5-dihydroxybenzoic acid

2,3-dihydroxybenzoic acid

2,3,5-trihydroxybenzoic acidsalicyluric acid

glucuronides of salicylic acid

Directional chemical-toxicological analysis of salicylic acid

derivatives

Investigation on presence of salicylic acid in biological materials

is done at special instructions or needles form on slide at evaporation

of chloroform.

Objects of investigation: stomach, intestine, liver, kidney, blood,

urine, and foodstuffs.

Isolation. General methods are used for isolation of salicylic acid

and salicylates from biological material. "Acidic" chloroform extract

is analyzed.

Solution of NaSolution of Na22COCO33 is added to foodstuffs, jams and other object for is added to foodstuffs, jams and other object for

isolation of salicylic acid. Soluble sodium salicylate forms:isolation of salicylic acid. Soluble sodium salicylate forms:

Aqueous extract is filtered, acidified by H2SO4 solution and salicylic acid is extracted by chloroform.

TLC-screening. General solvent system is acetone-chloroform (1:9), is in (Rf for salicylic acid = 0-0,25); specific system is acetone-cyclohexane (5:1) - Rf = 0,63-0, 65.

Developers: 5 or 10 % solution of FeCl3 (blue-violet stain).

Detection (identification). "Acidic" chloroform extract is purified by extraction, distillation,

chromatographic method. After that salicylic acid is detected by chemical, physical and chemical methods.

The reaction is nonspecific, highly sensitive. Forensic chemical has a negative result.

1. Precipitation reaction - the formation of 2,4,6-tribromphenol (white precipitate).

2. Colored reaction - with Iron (III) chloride. Color may change 2. Colored reaction - with Iron (III) chloride. Color may change depending on the pH of the solution:depending on the pH of the solution:

Nonspecific reaction, sensitive. 3. Formation of methyl salicylate - with methanol in the presence of concentrated H2SO4. Characteristic smell of methyl salicylate appears:

Nonspecific reaction, sensitive.

Blue-violet color

red-brown color

yellowcolor

4. Detection of salicylic acid in the UV spectra:4. Detection of salicylic acid in the UV spectra:

a) 0.5 N solution of NaOH - λmax = 300 nm;a) 0.5 N solution of NaOH - λmax = 300 nm;

b) 0.1 N H2SO4 solution - λmax = 302 nm.b) 0.1 N H2SO4 solution - λmax = 302 nm.

5. Identification by chromatographic methods: TLC, HPLC, GLC.5. Identification by chromatographic methods: TLC, HPLC, GLC.

Quantitative analysis:Quantitative analysis:

photometric methods (UV spectrophotometry, photocolorimetry, photometric methods (UV spectrophotometry, photocolorimetry,

extraction photometry), chromatographic methods (GLC, HPLC, extraction photometry), chromatographic methods (GLC, HPLC,

TLC).TLC).

Neutralization, bromatometry is used at drug analysis.Neutralization, bromatometry is used at drug analysis.

Acetylsalicylic acid in aspirin was determined by the Acetylsalicylic acid in aspirin was determined by the

fluorescence spectroscopy.fluorescence spectroscopy.

2. Usage, toxicological characteristics, methods of isolation

from biological material and methods of analysis of

barbituric acid derivatives.

barbiturates

ONH

NH

O

OC2H5

R

Physical and chemical propertiesBarbiturates - white or yellow crystalline amorphous powders, odorless, taste bitter.These substances are poorly soluble in water, soluble in ethanol, chloroform, ether, fats, aqueous solutions of alkalis, due to imide-imido-tautomerism:

Sodium salts of barbiturates are better soluble in water. All barbiturates Sodium salts of barbiturates are better soluble in water. All barbiturates are weak acids.are weak acids.

Barbiturates have the ability to sublimate.Barbiturates have the ability to sublimate.

UsageUsage

Derivatives of barbituric acid are used as hypnotic (soporific), sedative, Derivatives of barbituric acid are used as hypnotic (soporific), sedative, anticonvulsive. Barbiturates are used at treatment of epilepsy, anticonvulsive. Barbiturates are used at treatment of epilepsy, atherosclerosis, for local anesthesia, general anesthesia. Barbiturates atherosclerosis, for local anesthesia, general anesthesia. Barbiturates as sedatives are part of some drugs.as sedatives are part of some drugs.

Toxic actionToxic action

Barbiturates depress the central nervous system, mainly with the Barbiturates depress the central nervous system, mainly with the inhibition in the cerebral cortex, depress respiratory center, causing a inhibition in the cerebral cortex, depress respiratory center, causing a toxic lesion of brain capillaries.toxic lesion of brain capillaries.

Barbiturates accumulate in organism at continued usage of therapeutic Barbiturates accumulate in organism at continued usage of therapeutic doses. Increased sweating, decreased respiratory center, sluggishness, doses. Increased sweating, decreased respiratory center, sluggishness, hesitant gait, speech and vision disorder will be hesitant gait, speech and vision disorder will be at mild poisoningat mild poisoning of of barbiturates.barbiturates.

Anesthesia, which quickly turns into a coma, will be Anesthesia, which quickly turns into a coma, will be at heavy poisoningat heavy poisoning of barbiturates with breathlessness (respiratory impairment), of barbiturates with breathlessness (respiratory impairment), neurological disorders, decrease tendon reflexes and reaction of pupils neurological disorders, decrease tendon reflexes and reaction of pupils to light. to light.

Death Death occurs due to pulmonary edema and respiratory paralysis. occurs due to pulmonary edema and respiratory paralysis.

Some substances (drugs, alcohol, tranquilizers) increase the toxicity of Some substances (drugs, alcohol, tranquilizers) increase the toxicity of barbiturates. barbiturates.

10 therapeutic single doses of each drug or their mixture are 10 therapeutic single doses of each drug or their mixture are lethal lethal dosesdoses: for barbital - 3-4, phenobarbital - 1.4-2, barbamyl - 4-6, sodium : for barbital - 3-4, phenobarbital - 1.4-2, barbamyl - 4-6, sodium ethaminal (pentobarbital sodium) - 1 gram.ethaminal (pentobarbital sodium) - 1 gram.

The behavior of the organismThe behavior of the organism

Barbiturates are rapidly absorbed in the stomach by passive diffusion, Barbiturates are rapidly absorbed in the stomach by passive diffusion, this process increase in the presence of alcohol. this process increase in the presence of alcohol.

Unchanged or as metabolites are entered from organism with the urine. Unchanged or as metabolites are entered from organism with the urine. The strength and effective barbiturates duration depends on their The strength and effective barbiturates duration depends on their metabolism. metabolism.

Half-life: barbital - 4, phenobarbital - 3, barbamyl - 8 ethaminal sodium - Half-life: barbital - 4, phenobarbital - 3, barbamyl - 8 ethaminal sodium - 15 days.15 days.

Barbiturates are distributed in all tissues and liquids. The highest Barbiturates are distributed in all tissues and liquids. The highest concentration of barbiturates will be in the liver, kidney, spleen and concentration of barbiturates will be in the liver, kidney, spleen and brain. brain.

METABOLISM- Oxidation of the radicals in position 5 to alcohols, glucuronides formation:

Barbamyl

- Destruction of the pyrimidine cycle

Directional chemical-toxicological analysis of barbituric acid Directional chemical-toxicological analysis of barbituric acid derivativesderivatives

Objects of analysis:Objects of analysis: liver, kidney, brain, spleen, stomach with contents, liver, kidney, brain, spleen, stomach with contents, blood, urine.blood, urine.

Isolation.Isolation. Specific methods are used for isolation barbiturates - Specific methods are used for isolation barbiturates - extraction of aqueous solution of sodium hydroxide (Valov’s method) extraction of aqueous solution of sodium hydroxide (Valov’s method) and extraction with water, acidified by sulphuric acid (Popova’s and extraction with water, acidified by sulphuric acid (Popova’s method).method).

TLC-screening.TLC-screening. General solvent system: acetone-chloroform (1:9), General solvent system: acetone-chloroform (1:9), barbiturates are in zone 2 ( Rf = 0,31-0,41). Developer of barbiturates: barbiturates are in zone 2 ( Rf = 0,31-0,41). Developer of barbiturates: 5 % solution of mercury sulfate and 0.1 % 5 % solution of mercury sulfate and 0.1 % diphenylcarbazonediphenylcarbazone solution solution in chloroform (appear blue-violet or red-purple stains). Then drugs in chloroform (appear blue-violet or red-purple stains). Then drugs were eluted from the sorbent layer by acetone, eluates are investigated were eluted from the sorbent layer by acetone, eluates are investigated in specific solvent system chloroform-n-butanol-25 % solution of in specific solvent system chloroform-n-butanol-25 % solution of ammonia (70:40:5), sorbent - silica gel, buffered by 0.1 N boric acid ammonia (70:40:5), sorbent - silica gel, buffered by 0.1 N boric acid solution. solution.

IDENTIFICATION1. Colored reaction:a) with cobalt salts in the presence of ammonia or isopropylamine:

b) murexide reaction.2. Microcrystaloscopic reaction.A) Isolation of acidic form of barbiturates:

barbital phenobarbital barbamyl

B) With Zinc chloride-iodide

barbital barbamyl

phenobarbital barbamyl

C) with a mixture of iron (III) chloride and potassium iodide solution

D) withPotassium diiodocopprate in iodine solutionE) With acidified solution of potassium iodide

butobarbital

F) with Copper salts and pyridine:

barbital

where C - concentration of the substance,%;ΔA - the difference in optical density at 260 nm, which are measured at pH 2 and pH 10 (pH = 2 - absorb impurities, pH = 10 - imidol form of barbiturates and impurities); - specific absorptivity;

l - width of cuvette, sm.

3. Physical-chemical methods of identification: detection on ultraviolet and 3. Physical-chemical methods of identification: detection on ultraviolet and

infrared spectra, methods of TLC, GLC, HPLC.infrared spectra, methods of TLC, GLC, HPLC.

Quantitative determination of barbiturates in chemical-toxicological analysisQuantitative determination of barbiturates in chemical-toxicological analysis

physical and chemical methods: spectral (UV-spectrophotometery, physical and chemical methods: spectral (UV-spectrophotometery,

photocolorimetery, differential spectrophotometry, extraction photometry), photocolorimetery, differential spectrophotometry, extraction photometry),

chromatographic (thin layer chromatography, gas-liquid and high-chromatographic (thin layer chromatography, gas-liquid and high-

performance liquid chromatography).performance liquid chromatography).

The most perspective method is differential spectrophotometry, based on The most perspective method is differential spectrophotometry, based on

imino-imido tautomerism of barbiturates. Optical density (imino-imido tautomerism of barbiturates. Optical density (AbsorbanceAbsorbance) is ) is

measured at different pH values:measured at different pH values:

lE

AC

см

%1

1

%11смE

3. Usage, toxicological characteristics, methods of isolation from

biological material and methods of analysis of pyrazolone

derivatives.

Amidopyrine - 1-phenyl-2,3-dimethyl-4-dimethylamino- pyrazolone-5

Antipyrine - 1-phenyl-2 ,3-dimethyl- pyrazolone-5

CH3 C

C

C-N-CH3

CH3

ON

N

CH3

C6H5

CH3

CH3 N

N

C

C6H5

C

CH

O

Analgin (sodium metamizol) - sodium 1-phenyl-2 ,3-dimethyl-4-methylamino pyrazolone-5

Phenylbutazone - 1,2-diphenyl-4-butilpyrazolidindion-3, 5

CH3

CH3 N

N

C

C6H5

C

CH3

O

C-N-CH2-SO3NaC6H5 N

N

C6H5

C

CH-C4H9

O

CO

UsagePyrazolone derivatives are used in neuralgia, rheumatism, chorea and myositis. They are antipyretic, anti-inflammatory and analgesic medication.

Toxic actionIntoxication of drug will be at overdoses, increased sensitivity to these drugs. Danger of chronic poisoning will be at long-term using of drugs.Analgin causes the symptoms of anemia. It has nephrotoxic and hepatotoxic action. Prolonged usage of pyrazolone derivatives causes oppression of blood formation (leukopenia, agranulocytosis), causing disruption of the central nervous system, decrease body temperature, kidney damage, allergic reactions.The lethal dose of pyrazolone derivatives is 5-15 g.

The behavior in organismPyrazolone derivatives rapidly absorbed into the body, trace quantity of them appears in the urine within 10-20 minutes after entering.

Metabolism:a) N-demethylation of Amidopyrine:

4- monometylaminoantipyrine

CH3 C

C

C-NH2

ON

N

CH3

C6H5

4-aminoantipyrine

CH3 C

C

C-NH

CH3

ON

N

CH3

C6H5

b) hydrolysis to 4-monometylaminoantipyrine (analgin);c) acetylation to N-acetyl-4-aminoantipyrine (amidopyrine, Analgin):

CH3 C

C

C-NH

COCH3

ON

N

CH3

C6H5

N-acetyl-4-aminoantipyrine

d) oxidation (analgin, Amidopyrine, antipyrine):CH3

CH3 N

N

C

C6H5

C

C-OH

O

4-hydroxy antipyrine

e) conjugation of 4-hydroxy antipyrine with glucuronic acid:

CH3 C

C

C-N OC6H9O6

ON

N

CH3

C6H5

H

f) hydroxylation in the para position of phenyl radicals of Phenylbutazone.

C6H5 N

N

C

CH-C4H9

O

CO

OH

Objects of analysis.Objects of analysis. Stomach, liver, kidneys, blood, urine. Stomach, liver, kidneys, blood, urine.

Isolation.Isolation. General methods (Vasilieva’s, Stas-Otto’s methods), the drugs General methods (Vasilieva’s, Stas-Otto’s methods), the drugs can be detect in "acidic" and "alkaline" chloroform extract. can be detect in "acidic" and "alkaline" chloroform extract.

Specific method of pyrazolone derivatives isolation: water acidic extract Specific method of pyrazolone derivatives isolation: water acidic extract is basified by ammonia to pH 8.5-10 and extracted by chloroform is basified by ammonia to pH 8.5-10 and extracted by chloroform from basic aqueous solution. from basic aqueous solution.

TLC-screening.TLC-screening. General solvent system: acetone-chloroform (1:9) at General solvent system: acetone-chloroform (1:9) at analysis of "acidic" chloroform extract. They will be in zone 1 ( Rf = analysis of "acidic" chloroform extract. They will be in zone 1 ( Rf = 0-0,25). Developer: 5 % solution of FeCl3 and Dragendorff's reagent. 0-0,25). Developer: 5 % solution of FeCl3 and Dragendorff's reagent.

After elution by methanol, drugs are analyzed in specific solvent system: After elution by methanol, drugs are analyzed in specific solvent system: acetone-cyclohexane (5:1), sorbent - the basic aluminum oxide. acetone-cyclohexane (5:1), sorbent - the basic aluminum oxide.

General solvent system: chloroform-dioxane-acetone-25 % ammonia General solvent system: chloroform-dioxane-acetone-25 % ammonia solution (45:47,5:5:2,5) at analysis of "alkaline" chloroform extract. solution (45:47,5:5:2,5) at analysis of "alkaline" chloroform extract. They will be in zone 2 and 3 (Rf = 0,50-0,58 and 0,63-0,83). They will be in zone 2 and 3 (Rf = 0,50-0,58 and 0,63-0,83).

After elution by a mixture of methanol-25 % ammonia solution (9:1), After elution by a mixture of methanol-25 % ammonia solution (9:1), drugs are analyzed in specific solvent systems: chloroform-acetone drugs are analyzed in specific solvent systems: chloroform-acetone (5:1) and chloroform-ethanol (20:1).(5:1) and chloroform-ethanol (20:1).

Detection. 1. Precipitation reactions with Bushard's, Zonnenstein reagent, Reinecke salt, Wagner's, Dragendorff's, Scheibler's reagent2. Colored reactions:a) Iron (III) chloride: analgin - red-violet, antipyrine - red, phenylbutazone - blue-violet color:

CH3

CH3

C

N

N

CH

C

C6H5

O3 *2FeCl3

ferripyrin

CH3

CH3

C

N

N

CH3

C

C-N-CH3

C6H5

O

[O] CH3

CH3

O

C

O

N

N

CH3

C

C-N-CH3

C6H5

O

amidopyrine dioxiamidopyrine (colorless)

Nonspecific reaction, sensitive;

b) with sodium nitrite and concentrated sulfuric acid: antipyrine - green, amidopyrine - purple (the color disappears), Analgin - greenish-blue (the color disappears), phenylbutazone - red-brown (color disappears gradually).

Nonspecific reaction, sensitive;

c) with lignin - lemon-yellow color, characteristic of Analgin;

d) with the Nessler reagent - orange precipitate is observed, characteristic of Analgin;

d) formation of azo dye - red color is observed, characteristic of antipyrine:

CH3

CH3

C

N

N

C-N=O

C

C6H5

O +H2N CH3

CH3

C

N

N

C-N=N

C

C6H5

O +H2O

2. Physical-chemical methods of identification: detection in the UV and 2. Physical-chemical methods of identification: detection in the UV and

IR spectra, chromatographic methods (GLC, HPLC, TLC).IR spectra, chromatographic methods (GLC, HPLC, TLC).

QUANTITATIVE DETERMINATIONQUANTITATIVE DETERMINATION

UV spectrophotometry, photocolorimetry (which is based on colored UV spectrophotometry, photocolorimetry (which is based on colored

reaction amidopyrine with bromophenol blue, analgin with reaction amidopyrine with bromophenol blue, analgin with

benzoquinone in acetic acid, phenylbutazone with benzidine) and benzoquinone in acetic acid, phenylbutazone with benzidine) and

chromatographic (GLC, HPLC, TLC) methods.chromatographic (GLC, HPLC, TLC) methods.

Thanks for Thanks for your your

attention!attention!