Chemical and toxicological Chemical and toxicological

analysis of basic nature drugsanalysis of basic nature drugs. .

Lecture № 10

associate prof. M.M. Mykhalkiv

OUTLINE.

1. Usages, toxicological characteristics, isolation methods from

biological material and methods of p-aminobenzoic acid derivatives

analysis.

2. Usages, toxicological characteristics, methods of selection of

biological material and methods of phenothiazine derivatives

analysis.

3. Usages, toxicological characteristics, methods of selection of

biological material and methods of 1,4-benzodiazepines derivatives

analysis.

1. USAGES, TOXICOLOGICAL CHARACTERISTICS, ISOLATION METHODS FROM BIOLOGICAL

MATERIAL AND METHODS OF P-AMINOBENZOIC ACID DERIVATIVES ANALYSIS.

Novocaine - -diethylaminoethyl ester of p-aminobenzoic acid

COO-CH2-CH2-N(C2H5)2H2N

Dicain - - dimethylaminoethyl esters of p- butilaminobenzoic acid

COO-CH2-CH2-N(CH3)2C4H9-HN

Procainamide - - diethylaminoethylamid p-aminobenzoic acid

CO-NH-CH2-CH2-N(C2H5)2H2N

Toxic effect

Drugs lead to neuropsychiatric and cardiovascular disorders.

They cause allergic complications (skin rash), dyspepsia, swelling of the skin, mucous membranes, bronchospasm at therapeutic doses.

They cause disruption and eventually paralysis of the central nervous system in toxic doses. The clinical picture is characterized by psychomotor disturbances, convulsions, loss of consciousness, decreased blood pressure, bradycardia.

Toxicity of dicain exceeds the toxicity of procaine (lethal dose is 1 g) and procainamide (lethal dose is 1.5 g).

MetabolismMetabolismNovocaine is rapidly hydrolyzed to p-aminobenzoic acid and diethylaminoethanol:

H2N COO-CH2-CH2-N(C2H5)2

COOHH2N HO-CH2-CH2-N(C2H5)2

+ H2O

+

Dicain is metabolized to p-aminobenzoic acid.Procainamide is metabolized by N-acetylation.

H2N CO-NH-CH2-CH2-N(C2H5)2

CH3-C-HN CO-NH-CH2-CH2-N(C2H5)2

O

N-acetylnovocainamide

p-aminobenzoic acid is metabolized to glucuronide

H2N COOH H2N COOC6H9O6

Directional chemical toxicological analysis on p-aminobenzoic acid derivatives

Objects of investigation. Liver, kidney, blood, urine.Isolation - at directional analysis - general methods (Vasilyeva, Stas-Otto), at nondirectional analysis - biological objects (organ tissues) is extracted by water, acidified to pH = 2-3 by hydrochloric acid (salts are soluble in water). From the aqueous phase drugs in the form of bases is extracted with chloroform at pH 11 (alkalinization of the aqueous phase by NaOH). Impurities remain in the aqueous phase.

TLC screening: in general solvent system - chloroform-

dioxane-acetone-25 % ammonia solution (45:47,5:5:2,5),

sorbent - silica gel, they will be in third zone (Rf = 0,63-

0,83).

After elution by methanol-25% ammonia solution (9:1) drugs

are analyzed in specific solvent system: chloroform-ethanol

(20:1), sorbent - aluminum oxide.

Developer: Dragendorff's reagent (appear orange, orange-

brown stains) or identify the reaction of azo dye formation

with β-naphthol (orange stains).

Chemical methods of analysis:1. 1. Precipitation reactionsPrecipitation reactions with reagents of alkaloidal group

precipitation (picric acid, Bushard, Dragendorff, Mayer, Zonnenstein, reagents etc.).

Amorphous or crystalline precipitates with uncharacteristic shapes indicating the presence of heterocyclic nitrogen atom in drugs. Reactions high sensitivity, but nonspecific.

2. 2. Microcrystaloscopic reaction.Microcrystaloscopic reaction.Reactions are high sensitive and specific for certain conditions

(additional purification, temperature):- Dragendorff’s reagent - crystals in thin needles collected in bundles

(procaine) or diamond-shaped plates (procainamide);- With solution of tetrabromoauratic acid - crystals as plates and needles

(procaine);- With solution of hexachloroplatinatic acid - dense rosettes

(procainamide);- With solution of sodium nitrite - prisms, forked at the ends (dicain).

3. Color reactions (chromogenic):3. Color reactions (chromogenic):- Vitali - Morena reaction: orange-yellow color (procaine), yellow-brown color (procainamide), a blood-red color (dicain). Nonspecific reaction;- Reaction of azo dye formation: with β-naphthol (red-orange), or N-α-naphtylethylendiamine (lilac color) - for novocaine and procainamide.

NH2

R

R

N N-

N

R

N

Cl-

OH

NH-CH2-CH2-NH2

NH-CH2-CH2-NH2

N

R

N-

OH

+ HCl, NaNO2

-NaCl, H2O

+

4. Physical-chemical methods of identification: UV and IR

spectroscopy, TLC, GLH, HPLC

Quantitative analysis

spectroscopic (UV spectrophotometry, photometry, extraction

photometry) and chromatographic (TLC - planimetric and

densitometric methods, gas-liquid and liquid

chromatography) methods.

2. USAGES, TOXICOLOGICAL CHARACTERISTICS, METHODS OF SELECTION OF BIOLOGICAL MATERIAL

AND METHODS OF PHENOTHIAZINE DERIVATIVES ANALYSIS

S

N

R1

R21

23

456

78

9

10

aminazine S

N

CH2-CH2-CH2-N(CH3)2

Cl1

23

456

78

9

10

diprazinum (isopromethazine)

S

N H

CH2-CH(CH3)-N(CH3)2

123

456

78

9

10

Tisercinum

S

N OCH3

CH2-CH(CH3)-CH2-N(CH3)2

12

345

6

78

9

10

Toxic effect

Products are characterized by the neurotoxic effect, mental disorders, decrease of the cardio-vascular system activity, dyspepsia, hemodynamic instability.

Complications are possible at therapeutic doses: fall in blood pressure, palpitations, dryness in oral cavity, photophobia, drowsiness.

Comatose condition occurs at acute poisoning of phenothiazine derivatives. It is characterized expansion of pupils, decrease of temperature, depression of the respiratory center, tachycardia, thready pulse. Death occurs during cardiopulmonary diseases.

The lethal dose of chlorpromazine (for adults) is 5-10 g

METABOLISM OF PHENOTHIAZINEa) demethylation (I phase biotransformation)

S

N OCH3

CH2-CH(CH3)-CH2-N(CH3)2

S

N

CH2-CH2-CH2-NH2

OH

2-hydroxy-10-(3'-aminopropyl)-phenothiazine

tisercin

b) oxidation of hetero atom - sulfur to sulphoxide and sulphone

S

N H

CH2-CH(CH3)-N(CH3)2

S

N H

CH2-CH(CH3)-N(CH3)2

O

S

N H

CH2-CH(CH3)-N(CH3)2

OO

promethazine sulphoxide sulphone

Directional chemical-toxicological analysis on the phenothiazines

Objects of investigation: brain, liver, kidney, stomach with contents, lungs, urine.

Isolation: is used general methods at nondirectional analysis (Vasilyeva’s or Stas-Otto’s), the directional - Salomatin’s method.

c) aromatic hydroxylation at 3 and 6 positions, with further conjugation with glucuronic acid

S

N

CH2-(CH2)2-N(CH3)2

Cl

S

N

CH2-(CH2)2-N(CH3)2

OH

Cl

S

N

CH2-(CH2)2-N(CH3)2

Cl

OC6H9O6

TLC-screening: in general solvent system: chloroform-

dioxane-acetone-25% ammonia solution (45:47,5:5:2,5),

sorbent - silica gel; developers - concentrated acid (H2SO4,

HNO3, HC1) and solutions of oxidizers HClO4 and NaNO2;

Marqui's or Mandelin's reagents.

The presence of pink and purple stains in the 3rd zone (Rf =

0,63-0,83) indicates the possible presence of phenothiazine

derivatives.

Mixture of methanol-25% ammonia solution (9:1) is used for

elyation drugs and analyzed in specific solvent system:

chloroform-ethanol (20:1) and cyclohexane-acetone (5:1);

sorbent - aluminum oxide.

Chemical methods of analysis:

1. 1. Precipitation reactionsPrecipitation reactions with reagents of alkaloidal group precipitation (picric acid, Bushard, Dragendorff, Mayer, Zonnenstein, reagents etc.).

Amorphous or crystalline precipitates with uncharacteristic shapes indicating the presence of heterocyclic nitrogen atom in drugs. Reactions high sensitivity, but nonspecific.

2. Color reactions2. Color reactions are mainly based on chemical oxidation, dehydration, condensation with aldehydes at usages of concentrated acids H2SO4, HNO3, HCl, HClO4; reagents

Marqui's, Mandelin's, Frohdes's; solutions of salt FeCl3,

NaNO2.

The reaction products are colored in red and purple, blue and red colors. Reactions sensitive, nonspecific.

3. 3. Microcrystaloscopic reaction.Microcrystaloscopic reaction.

Most of phenothiazines form characteristic crystalline precipitation of Reinecke salt, but differentiation of individual drugs of the group on the form of crystals is difficult.

4. Physical-chemical methods of analysis:

1. UV spectra of phenothiazine derivatives and their products of oxidation.

Absorption of phenothiazine in the UV region of the spectrum is characterized by two peaks:

a) max = 250-260 nm, b) max = 300-315 nm.

Sulphoxides of phenothiazines have four maximums in the UV region: max= 230, 265, 285 and 400 nm.

Quantitative analysis

spectroscopic (UV spectrophotometry, photometry, extraction

photometry) and chromatographic (TLC - planimetric and

densitometric methods, gas-liquid and liquid chromatography)

methods.

Extraction photometry is based on chloroform extraction of ion associates phenothiazines with acidic indicator (methyl orange) with subsequent measurement of the optical density of colored organic layer.

The method is sensitive, does not require a high degree of purification from impurities.

S

N

CH2-(CH2)2-N(CH3)2

Cl

H

-N=N- -SO3-

+ * (CH3)2-N-

3. USAGES, TOXICOLOGICAL

CHARACTERISTICS, METHODS OF SELECTION

OF BIOLOGICAL MATERIAL AND METHODS OF

1,4-BENZODIAZEPINES DERIVATIVES ANALYSIS.

N

NCl

CH3

C6H5

H

H

O

DIAZEPAMUM

N

NO2N

C6H5

H

H

H

O

NITRAZEPAMUM

N

N

C6H5

OH

H

H

Cl

O

OXAZEPAM

CHLORDIASEPOXIDUM

Cl

N

NH-CH3

N

H

C6H5

H

O

Toxic action

Inhibition of CNS, neurons in the spinal cord and thalamus (muscle relaxation).

Complications: digestive disorders, cardiovascular disease, neuropsychiatric disorders, allergic reactions are possible at the usages of therapeutic doses.

Mild poisoning is characterized by weakness, lethargy, drowsiness, expansion of the pupils, decreased muscle tone.

Medium poisoning is characterized more severe symptoms, and there are skin hyperemia, dry skin, tachycardia. In some cases there is euphoria, hypotonia.

Heavy poisoning is characterized by mental confusion, the appearance of convulsion, hallucinations. Death occurs due to respiratory and circulatory failure - circulatory collapse, respiratory arrest, pulmonary edema.

The lethal dose is 1-2 g for chlordiazepoxide, toxic concentration in the blood - 5-20 mg/L, the deadly - over 50 mg/L.

1. N-dealkylation - chlordiazepoxide

Metabolism

N

NH-CH3

H

NCl

C6H5 O

HCl

N

C6H5

N

NH2

O

H

H

2. Reduction - nitrazepamdezmethylchlordiazepoxide

N

NO2N

C6H5

H

H

H

ON

NH2N

C6H5

H

H

H

O

3. oxidation and N-demethylation - diazepam, oxazepam- oxyglycine

N

CH3O

H [O]

NCl

C6H5

H Cl-CH3

N

H

C6H5

N

O

H

OH +H2O

Cl

C6H5

NH2

O

NH2

COOH

CH-OH+

2-amino-5-chlorobenzophenon

diazepam oxazepam

H NHC6H9O6O

N OC6H9O6 OClHNCl C6H5

C6H5

glucuronides

Directional chemical-toxicological analysis on derivatives of 1, 4-benzodiazepine

Objects of investigation. Stomach and small intestine with contents, brain, liver, kidneys, blood and urine.

Analysis of biological material on derivatives of 1, 4-benzodiazepines and their metabolites is done in two ways:

direction I - by hydrolysis products - 2-aminobenzophenones (Izotov’s method);

direction II - analysis of native drugs and with their metabolites (Vasilyeva’s or Stas-Otto methods).

Schemes of acidic hydrolysis of 1, 4-benzodiazepine derivatives:

N

NH-CH3

H

NCl

C6H5 O

H

NH2

O

C6H5

Cl

NH2

CH2

COOH

+ + CH3NH2

chlordiazepoxide 2-amino-5- glycine methyl amine chlorobenzophenone

N

HO

OH +H2O

N

C6H5

HCl Cl

C6H5

NH2

O

NH2

COOH

CH-OH+

oxazepam 2-amino-5- - oxyglycine chlorobenzophenone

N

NO2N

C6H5

H

H

H

O NH2

O

C6H5

NH2

CH2

COOH

+O2N

nitrazepam 2-amino-5-glycine

nitrobenzophenone

N

CH3

OH

NCl

C6H5

H Cl

C6H5

O

NH-CH3

CH2

NH2

COOH

+

diazepam 2-methylamino-5- glycinechlorobenzophenone

Analysis of benzophenones (I direction)

TLC- screening. 1 stage – in general solvent system: chloroform-acetone-dioxane-25% ammonia solution (45:47,5:5:2,5), sorbent - silica gel. Detection of benzophenones is done by own yellow color of stain, on the reaction of azo dye formation (2-aminobenzophenones) with -naphthol (orange stains) or N--naphtylethylendiammine (pink-purple stains). Benzophenones (Rf = 0,63-0,70) is eluated from sorbent by

benzene.

Stage 2 – analysis in specific solvent system: chloroform-ethanol (20:1); sorbent - aluminum oxide; Rf = 0,60-0,63;

eluent - benzene.

Identification of benzophenones by reaction:color reactions - formation of azo dyes:

N N-

Cl-

OH

NH-CH2-CH2-NH2

NH-CH2-CH2-NH2

N N-

OH

+ HCl, NaNO2

-NaCl, H2O

NH2

O

C6H5

R

N N+

O

C6H5

R

O

C6H5

RO

C6H5

R

pink-lilac color orange color

Quantitative analysis: photometry of benzophenones in the visible region of the spectrum, in the UV region of the spectrum, the extraction-photometry with acid dyes.

Chromatographic methods: GLC and HPLC are used for identification and quantification of the hydrolysis products of 1, 4-benzodiazepines derivatives in biological extracts after purification from impurities.

Analysis by II direction

Isolation by general methods.

TLC screening in the general system (for substances with basic properties), the developer - Dragendorff's reagent (orange-brown stains with Rf = 0,63-0,77).

Substances are eluated by solvent: methanol-25%- ammonia (9:1) and chromatographed in specific solvent system chloroform-ethanol (20:1). The developer - Dragendorff's reagent.

Chemical methods:

1. Precipitation reactions.1. Precipitation reactions. with reagents of alkaloidal group precipitation (picric acid, Bushard, Dragendorff, Mayer, Zonnenstein, reagents etc.).

Amorphous or crystalline precipitates with uncharacteristic shapes indicating the presence of heterocyclic nitrogen atom in drugs. Reactions high sensitivity, but nonspecific.

2. Color reactions2. Color reactions on native substances:

Chlordiazepoxide: Marquis's reagent (yellow color)

Frohdes's reagent (orange color)

Vitali-Morena reaction (yellow color)

Diazepam and chlordiazepoxide with ninhydrin - yellow-brown color.

3. Color reactions3. Color reactions on metabolites (products of hydrolysis) – 2-aminobenzophenones, which formed at hydrolysis of chlordiazepoxide, nitrazepam, oxazepam, give reaction of azo dye formation (except for the 2-methylaminobenzophenone).

4. Physical-chemical methods. UV- and IR-

spectrometry, TLC, HPLC, GLC.

Quantitative analysis

spectroscopic (UV spectrophotometry, photometry,

extraction photometry) and chromatographic (TLC -

planimetric and densitometric methods, gas-liquid

and liquid chromatography) methods.

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