1. Chemical and toxicological analysis of basic nature drugs. Lecture № 10 associate prof. M.M. Mykhalkiv

2. 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.

3. 1. USAGES, TOXICOLOGICAL CHARACTERISTICS, ISOLATION METHODS FROM BIOLOGICAL MATERIAL AND METHODS OF P-AMINOBENZOIC ACID DERIVATIVES ANALYSIS. Novocaine -  -diethylaminoethyl ester of p-aminobenzoic acid Dicain -  - dimethylaminoethyl esters of p- butilaminobenzoic acid Procainamide -  - diethylaminoethylamid p-aminobenzoic acid

4. 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).

5. Metabolism Novocaine is rapidly hydrolyzed to p-aminobenzoic acid and diethylaminoethanol: Dicain is metabolized to p-aminobenzoic acid. Procainamide is metabolized by N-acetylation. N-acetylnovocainamide

6. p-aminobenzoic acid is metabolized to glucuronide 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.

7. 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).

8. Chemical methods of analysis: 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. 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).

9. 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.

10. 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.

11. 2. USAGES, TOXICOLOGICAL CHARACTERISTICS, METHODS OF SELECTION OF BIOLOGICAL MATERIAL AND METHODS OF PHENOTHIAZINE DERIVATIVES ANALYSIS aminazine diprazinum (isopromethazine) Tisercinum

12. 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

13. METABOLISM OF PHENOTHIAZINE a) demethylation (I phase biotransformation) 2-hydroxy-10-(3'- aminopropyl)-phenothiazine tisercin b) oxidation of hetero atom - sulfur to sulphoxide and sulphone promethazinesulphoxidesulphone

14. 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

15. TLC-screening: in general solvent system: chloroform- dioxane-acetone-25% ammonia solution (45:47,5:5:2,5), sorbent - silica gel; developers - concentrated acid (H 2 SO 4, HNO 3, HC1) and solutions of oxidizers HClO 4 and NaNO 2 ; 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.

16. Chemical methods of analysis: 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 reactions 2. Color reactions are mainly based on chemical oxidation, dehydration, condensation with aldehydes at usages of concentrated acids H 2 SO 4, HNO 3, HCl, HClO 4 ; reagents Marqui's, Mandelin's, Frohdes's; solutions of salt FeCl 3, NaNO 2. The reaction products are colored in red and purple, blue and red colors. Reactions sensitive, nonspecific.

17. 3. 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.

18. 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.

19. 3. USAGES, TOXICOLOGICAL CHARACTERISTICS, METHODS OF SELECTION OF BIOLOGICAL MATERIAL AND METHODS OF 1,4-BENZODIAZEPINES DERIVATIVES ANALYSIS. DIAZEPAMUMNITRAZEPAMUM OXAZEPAM CHLORDIASEPOXIDUM

20. 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.

21. 1. N-dealkylation - chlordiazepoxide Metabolism 2. Reduction - nitrazepam dezmethylchlordiazepoxide

22. 3. oxidation and N-demethylation - diazepam, oxazepam  - oxyglycine 2-amino-5- chlorobenzophenon diazepam oxazepam glucuronides

23. 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).

24. Schemes of acidic hydrolysis of 1, 4-benzodiazepine derivatives: chlordiazepoxide 2-amino-5- glycine methyl amine chlorobenzophenone oxazepam 2-amino-5-  - oxyglycine chlorobenzophenone

25. nitrazepam 2-amino-5-glycine nitrobenzophenone diazepam 2-methylamino-5-glycine chlorobenzophenone

26. 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 (R f = 0,63-0,70) is eluated from sorbent by benzene. Stage 2 – analysis in specific solvent system: chloroform- ethanol (20:1); sorbent - aluminum oxide; R f = 0,60-0,63; eluent - benzene.

27. Identification of benzophenones by reaction: color reactions - formation of azo dyes: pink-lilac color orange color

28. 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.

29. 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 R f = 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.

30. 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 reactions 2. 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 reactions 3. 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).

31. 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.

32. Thanks for your attention!