Basic Test For Pharmaceutical Substances: Basic tests describe procedures for testing pharmaceutical substances, medicinal plant materials, and pharmaceutical dosage forms. Basic tests are not, intended to replace the requirements of The International Pharmacopoeia or other pharmacopoeial monographs. These give an assurance of quality whereas, basic tests merely confirm identity.
Basic tests need not be carried out by fully qualified pharmacists or chemists, but they should be performed by people with some understanding of analytical chemistry such that acquired in courses for pharmaceutical assistants.
Basic Test For Pharmaceutical Substances
1. Amikacin Sulfate
Table of Contents
Identity tests:
Description: A white to yellowish-white, crystalline powder, almost odorless.
Melting point: About 204°C.
Procedures:
- Dissolve 10 mg in 1 ml of water, add 1 ml of sodium hydroxide (~80 gm/L) TS and mix, then add 2 ml of cobalt (II) nitrate (10 gm/L) TS. Violet color is produced.
- Dissolve 0.05 gm in 3 ml of water and add slowly 4 ml of anthrone TS. Bluish violet color is produced.
- Dissolve 20 mg in 1 ml of water and add 1 ml of barium chloride (50 gm/L) TS-A white precipitate is produced which is practically Insoluble in hydrochloric acid (-250 gm/L) TS.
Degradation test: Discoloration of the test substance.
2. Bacitracin Zinc
Composition: Bacitracin zinc is a zinc complex of bacitracin, polypeptides produced by the growth of an organism of the licheniformis group of Bacillus subtilis. The main components are the bacitracin A, B₁, and B₂.
Identity tests:
Description: A white or pale brownish-yellow powder; odorless or with a faint, characteristic odor, hygroscopic.
Melting point: About 250°C.
Procedures:
- Shake 5 mg with 1 ml of water, add 1 ml of triketohydrindene/butanol TS and 0.5 ml of pyridine, and heat to 100°C for 5 minutes. Violet color is produced. Transfer about 0.5 gm to a silica crucible and ignite. Dissolve the residue in 5 ml of sulfuric acid (-5 gm/L) TS and filter. Divide the filtrate into 2 equal volumes.
(a) To 1 volume, add 1 ml of potassium ferrocyanide (45 gm/L) TS; a white precipitate is produced which is insoluble in hydrochloric acid (~250 gm/L) TS.
(b) To 1 volume, add 1 drop of copper (II) sulfate (1 gm/L) TS and 1 ml of ammonium mercurithiocyanate TS. A violet precipitate is produced.
Degradation test:
- Discoloration of the test substance and non-compliance with the following test usually indicates gross degradation:
- Dissolve 0.10 gm in 100 ml of water, a clear, colorless, or slightly yellowish solution is produced.
3. Captopril
Identity tests:
Description: A white or almost white, crystalline powder; odor, characteristic but faint.
Melting point: About 108°C.
Procedures:
- Dissolve 10 mg in 2 ml of hydrochloric acid (0.1 mol/L) VS and add about 1 ml of iodine TS; the color of the lodine disappears immediately and a white, turbid solution is produced.
- Dissolve 10 mg in 2 ml of water and add 0.5 ml of lead acetate (80 gm/L) TS; a white precipitate is produced.
- Dissolve 10 mg in 5 ml of ethanol (-750 gm/L) TS, add 0.5 ml of tetramethylammonium hydroxide/ethanol TS and shake. Then add 0.5 ml of triphenyl tetrazolium chloride/ethanol TS and shake again. The red color is produced.
Degradation tests: Discoloration of the test substance.
4. Chloramphenicol Sodium Succinate
Identity tests:
Description: A white or almost white powder; hygroscopic.
Melting point: About 42°C
Procedures:
- Dissolve about 1.4 gm in 5 ml of water and use as the test solution for the following tests:
- To 1 drop of the test solution, add 5 ml of ethanol (~750 gm/L) TS, 0.2 gm of zinc powder, and 1 ml of sulfuric acid (-100 gm/L) TS and allow to stand for 10 minutes. Filter; to the filtrate add 0.5 ml of sodium nitrite (10 gm/L) TS and allow to stand for 2 minutes. Then add about 1 gm of urea and a solution containing 10 mg of 2-naphthol in 2 ml of sodium hydroxide (~80 gm/L) TS. The red color is produced.
- Repeat test 1 omitting the zinc powder. No red color is produced.
- Carefully heat 1 drop of the test solution with 10 mg of resorcinol and three drops of sulfuric acid (-1760 gm/L) TS, cool, and add 2 ml of water. Cool again and pour the solution into a mixture of 100 ml of water and about 1 ml of sodium hydroxide (-400 gm/L) TS; a yellow-green fluorescence appears which disappears with the addition of 1 ml of hydrochloric acid (-250 gm/L) TS.
- Introduce the test solution into a non-luminous flame using a magnesia stick or a nichrome or platinum wire sealed to a glass rod. The flame acquires an intense yellow color.
Degradation tests:
- Discoloration of the test substance and non-compliance with the following test usually indicates gross degradation.
- Dissolve 0.2 gm in 10 ml of water. A clear solution is produced.
5. Cisplatin
Identity tests:
Description: White to yellowish crystals or a yellow powder.
Note: This substance is very toxic and should be handled with care.
Melting point: About 270°C.
Procedures:
- Dissolve 5 mg in 5 ml of hydrochloric acid (-420 gm/L) TS and heat to boiling. To half of the solution (keep the unused portion for test 2), add a few crystals of potassium iodide; a brownish yellow color is produced which changes to reddish-brown on standing.
- To the remaining solution, from test 1 add a few crystals of tin (II) chloride; a reddish-orange color is produced which changes to reddish-brown and standing.
- Transfer 0.05 gm to a glass dish and add 2 ml of sodium hydroxide (-80 gm/L) TS. Evaporate to dryness and dissolve the residue in a mixture of 0.5 ml of nitric acid (-1000 gm/L) TS and 1.5 ml of hydrochloric acid (~420 gm/L) TS. Again evaporate to dryness; an orange-colored residue is produced. Dissolve the residue in 0.5 ml of water and add 0.5 ml of ammonium chloride (100 gm/L) TS. A yellow, crystalline precipitate is produced.
Degradation test:
- Discoloration and a change in the physical state of the test substance usually indicate gross degradation.
6. Coal Tar
Composition: Coal tar is a by-product usually obtained during the destructive distillation of coal. It is a complex and undefined mixture of a great number of chemical compounds. The product is available in various compositions.
Identity tests:
Description: Brown-black or black, viscous liquid; characteristic and strong, resembling naphthalene-like odor. On exposure to air, the liquid solidifies.
Melting point: About 180°C.
- When ignited, the liquid burns in the air with a luminous sooty flame and almost no residue remains.
Procedures:
- Shake 1 drop vigorously with 5 ml of ethanol (~750 gm/L) TS and filter. The filtrate is yellow with bluish-green fluorescence.
- Shake about 1 gm vigorously with 9 ml of water for 10 minutes and filter, the filtrate gives a neutral or only slightly acid reaction when tested with litmus paper (unlike wood tar) and an odor of naphthalene is discernible (keep the filtrate for test 3).
- To 5 ml of the filtrate from test 2, add a few drops of bromine TS. Yellow turbidity develops (phenols).
Degradation tests:
- If the substance does not pass the following test, this usually indicates that gross degradation has occurred.
- Dissolve 0.10 gm in 10 ml of nitrobenzene; a clear or almost clear solution is produced.
7. Doxorubicin Hydrochloride
Identity tests:
Description: A red-orange, crystalline powder, hygroscopic.
Melting point: About 216°C.
Procedures:
- Place a small quantity of the test substance on a white test plate and add 1 drop of formaldehyde/sulfuric acid TS. The orange-red color of the substance changes to violet.
- Dissolve about 2 mg in 2 ml of methanol and add 2 ml of water and 1 drop of sodium hydroxide (-80 gm/L) TS. The orange-red color of the solution changes to blue-violet.
- Dissolve 0.05 gm in 1 ml of water, add 5 drops of ammonia (~100 gm/L) TS and filter Acidify the filtrate with nitric acid (-130 gm/L) TS and add 1 ml of silver nitrate (40 gm/L) TS. A white, curdy precipitate is produced which is soluble in ammonia (~100 gm/L) TS but practically insoluble in nitric acid (-1000 gm/L) TS.
Degradation test:
- Discoloration of the test substance.
8. Fluphenazine Decanoate
Identity tests:
Description: A pale yellow, viscous liquid or a yellow, crystalline solid with an oily appearance; odor, faint and ester-like.
Melting point: About 32°C.
Procedures:
- Dissolve 5 mg in 2 ml of sulfuric acid (-1760 gm/L) TS and allow to stand for 5 minutes. A reddish-brown color is produced.
- Dissolve 5 mg in about 2 ml of formaldehyde/sulfuric acid TS; an orange color is produced. Heat in a water bath for 2 minutes. The color changes to dark brown.
- Dissolve 5 mg in 2 ml of water and add 3 drops of potassium dichromate (100 gm/L) TS. A yellow precipitate is produced.
- Dissolve 5 mg in about 1 ml of sucrose/hydrochloric acid TS and allow standing for 5 minutes. The red color is produced.
Degradation test:
- Discoloration and a change in the physical state of the test substance usually indicate gross degradation.
9. Gallamine Triethiodide
Identity tests:
Description: A white or almost white powder, odorless; hygroscopic.
Melting point: About 270°C.
Procedures:
- Dissolve 0.05 gm in 5 ml of water and add 1 ml of potassio-mercuric iodide TS-A yellow precipitate is produced.
- Dissolve 0.05 gm in 5 ml of water and add 1 ml of sulfuric acid (-100 gm/L) TS and 1 ml of potassium nitrite (100 gm/L) TS. A brownish coloration is produced.
- Dissolve 0.05 gm in 5 ml of water and add 1 drop of nitric acid (-1000 gm/l.) TS and 1 ml of silver nitrate (40 gm/L) TS; a yellow precipitate is produced which is insoluble in ammonia (-100 gm/L) TS and nitric acid (-1000 gm/L) TS.
Degradation test:
- Discoloration and a change in the physical state of the test substance usually indicate gross degradation.
10. Hydrocortisone Sodium Succinate
Identity tests:
Description: A white or almost white, crystalline powder or amorphous solid; odorless; hygroscopic.
Melting point: About 163°C.
Procedures:
- Dissolve about 2 mg in 1 ml of alkaline potassio-mercuric iodide TS; a dark precipitate is produced.
- Dissolve a small quantity in about 2 ml of sulfuric acid (-1760 gm/L) TS; a yellow solution with a greenish fluorescence is produced. Very cautiously pour the solution into 10 ml of water. The color of the solution changes to brownish-yellow but the fluorescence remains.
- Dissolve a small quantity in about 1 ml of phosphoric acid (-1440 gm/L) TS and heat cautiously; a yellow solution is produced with a pale greenish fluorescence.
- Dissolve about 2 mg in 1 ml of water and introduce the solution into a non-luminous flame using a magnesia stick or a nichrome or platinum wire sealed to a glass rod; the flame acquires an intense yellow color.
- Heat carefully 10 mg with 1 drop of water, 10 mg of resorcinol, and 3 drops of sulfuric acid (-1760 gm/L) TS, cool and add 2 ml of water. Cool again and pour the solution into a mixture of 100 ml of water and 1 ml of sodium hydroxide (~400 gm/L) TS. A yellowish-green appears.
Degradation tests:
- Discoloration of the substance and non-compliance with the following test usually indicates gross degradation.
- Dissolve 0.20 gm in 1.0 ml of water, a clear and colorless solution is produced.
11. Ketamine Hydrochloride
Identity tests:
Description: A white, hygroscopic, crystalline powder; characteristic odor.
Melting behavior: About 260°C.
Procedures:
- Dissolve about 0.2 gm in 4 ml of water and chill the solution in ice. Add potassium carbonate (100 gm/L) TS, drop by drop, until the solution is slightly alkaline when tested with pH-indicator paper and allow standing to crystallize. Filter and dry the crystals in a vacuum over phosphorus pentoxide; melting temperature, about 92°C.
- Dissolve 10 mg in 4 ml of water and add 0.5 ml of nitric acid (-130 gm/L) TS and 0.5 ml of silver nitrate (40 gm/L) TS; a white, curdy precipitate is produced. Separate the precipitate, wash it with water and add an excess of ammonia (-100 gm/L) TS. The precipitate dissolves.
- Dissolve 10 mg in 4 ml of sulfuric acid (-5 gm/L) TS and add 1 drop of potassium iodobismuthate/acetic acid TS. A reddish-brown precipitate is produced.
Degradation test:
- Discoloration and a change in the physical state of the test substance usually indicate gross degradation.
12. Levamisole
Identity tests:
Description: A white, hygroscopic, crystalline powder, characteristic odor.
Melting point: About 59°C.
Procedure:
- Dissolve 0.05 gm in 20 ml of water. Add 1 ml of sodium hydroxide (-80 gm/L) TS, boil for 10 minutes, and cool. Add a few drops of sodium nitroprusside (45 gm/L) TS; the red color is produced which fades with time.
Degradation test: Discoloration of the test substance.
13. Levamisole Hydrochloride
Identity tests:
Description: A white to pale cream-colored, crystalline powder; odorless or almost odorless. Melting point: About 228°C.
Procedures:
- Dissolve 0.25 gm in 20 ml of water and add 1.5 ml of sodium hydroxide (-80 gm/L) TS. Extract with 20 ml of dichloromethane, discard the aqueous layer and wash the dichloromethane layer with 10 ml of water. Discard the aqueous layer, shake the dichloromethane layer with about 1 gm of anhydrous sodium sulfate, and filter. Evaporate the filtrate at room temperature and dry in a vacuum at a temperature not exceeding 40°C; the residue melts at about 59°C.
- Dissolve 0,10 gm in 40 ml of water. To 20 ml of this solution (keep the unused portion for test 3), add 1. ml of sodium hydroxide (-80 gm/L) TS, boil for 10 minutes and cool Add a few drops of sodium nitroprusside (45 gm/L) TS. The red color is produced which fades with time.
- To 20 ml of the solution prepared for test-2, add 1 ml of nitric acid (-130 gm/L) TS and a few drops of silver nitrate (40 gm/L) TS; a white precipitate is produced. Separate the precipitate, wash with water and add an excess of ammonia (-100 gm/L) TS; the precipitate dissolves.
Degradation test: Discoloration of the test substance.
14. Magnesium Sulfate
Identity tests:
Description: Brilliant, colorless crystals or a white, crystalline powder, odorless; effloresces in warm and dry air.
Melting point: About 1124°C.
Procedures:
- Dissolve 10 mg in 2 ml of water and add 1 ml of ammonia (-100 gm/L) TS; a white precipitate is produced. Add 1 ml of ammonium chloride (100 gm/L) TS; the precipitate dissolves. Add 1 ml of disodium hydrogen phosphate (100 gm/L) TS; a white precipitate is produced.
- Dissolve 10 mg in 2 ml of water and add 3 drops of titan yellow TS and 2 ml of sodium hydroxide (-80 gm/L) TS; a distinct pink color is produced.
- Dissolve 0.05 g in 5 ml of water. Add 1 ml of hydrochloric acid (~70 gm/L) TS and 1 ml of barium chloride (50 gm/L) TS; a white precipitate is produced.
Degradation test: Discoloration of the test substance.
15. Medroxyprogesterone Acetate
Identity tests:
Description: A white or almost white, crystalline powder; odorless or almost odorless.
Melting point: About 204°C.
Procedures:
- Dissolve 5 mg in 5 ml of sulfuric acid (-1760 gm/L) TS. Incline the tube and carefully add, without mixing, 5 ml of ethanol (-750 gm/L) TS; a blue-violet color is produced at the interface of the two liquids.
- Heat 0.05 gm with 2 ml of potassium hydroxide/ethanol TS in a water bath for 5 minutes. Cool and add 1 ml of water and about 1 ml of sulfuric acid (-1760 gm/L) TS. Boil gently for 1 minute: ethyl acetate, perceptible by its odor (proceed with caution), is produced.
- Dissolve 5 mg in 0.5 ml of methanol in a small test tube, add about 3 mg of sodium nitroprusside 0.05 gm of sodium carbonate, and 0.5 gm of ammonium acetate and shake. Allow to stand for 10-30 minutes, a violet-red color is produced (distinction from progesterone).
- Dissolve a few crystals in about 1 ml of sulfuric acid (-1760 gm/L) TS. Incline the tube and carefully add, without mixing, 1 ml of water, a green color is produced at the interface of the two liquids. Allow to stand; the color changes to bluish violet (distinction from hydroxyprogesterone caproate).
Degradation test:
- Discoloration and a change in the physical state of the test substance usually indicate gross degradation.
16. Methionine
Identity tests:
Description: White crystals or a white, crystalline powder, characteristic odor.
Melting point: About 281°C.
Procedures:
- Dissolve about 0.1 gm in 5 ml of hydrochloric acid (0.1 mol/L) VS, add 0.2 ml of triketohydrindene/ethanol TS and heat; a violet color is produced.
- Dissolve 10 mg in 1 ml of water and add 1-2 drops of hydrochloric acid (-250 gm/L) TS, 0.5 ml of copper (II) sulfate (1 gm/L) TS, and 1-2 ml of sodium hydroxide (-150) gm/L) TS; a blue-violet color is produced.
- Dissolve about 0.1 gm in 5 ml of potassium hydroxide (-110 gm/L) TS and add about. 0.3 ml of sodium nitroprusside (45 gm/L) TS with shaking. Heat the solution in a water bath at a temperature of 35-40°C for 10 minutes. Cool in ice for 2 minutes, add about 2 ml of hydrochloric acid (-250 gm/L) TS, and shake well; the red color is produced.
Degradation test:
- Discoloration and a change in the physical state of the test substance usually indicate gross degradation.
17. Pentamidine Mesilate
Identity tests:
Description: A white or light pink, granular powder; almost odorless.
Melting point: About 265°C,
Procedures:
- To about 1 gm, add 10 ml of water and heat to 80°C to dissolve. Add 15 ml of sodium hydroxide (-80 gm/L) TS, cool in ice, and filter. To 2 ml of the filtrate (keep the unused portion for tests 2 and 3) add about 0.2 ml of nitric acid (-1000 gm/1) TS and about 0.2 ml of ceric ammonium nitrate TS; a yellow color is produced.
- Neutralize 5 ml of the remaining filtrate from test 1 with hydrochloric acid (-70 gm/L) TS testing with pH-indicator paper, and add 3 ml of the same acid and a few drops of barium chloride (50 gm/L) TS; no precipitate is produced.
- Transfer a further 10 ml of the remaining filtrate from test 1 to a crucible, add 2.5 ml of hydrogen peroxide (-60 gm/L) TS, mix and evaporate to dryness over a water bath. Dissolve the residue in 1 ml of water, add about 1 ml of glacial acetic acid, evaporate again and ignite until free from carbon. After cooling, add 5 ml of water and filter. If necessary, neutralize the filtrate with hydrochloric acid (-70 gm/L) TS, testing with pH-indicator paper, add 3 ml of the same acid, heat to boiling for 30 seconds, cool, and add a few drops of barium chloride (~50 gm/L) TS; a white precipitate is produced which is practically insoluble in hydrochloric acid (-250 gm/L) TS.
Degradation test:
- Discoloration and a change in the physical state of the test substance usually indicate gross degradation.
18. Prednisolone Sodium Phosphate
Identity tests:
Description: A white or almost white powder, odorless; hygroscopic.
Melting point: About 220°C.
Procedures:
- Dissolve about 2 mg in about 2 ml of sulfuric acid (~1760 gm/L) TS and allow to stand for 5 minutes; a wine-red solution is produced. Dilute the solution very cautiously with 10 ml of water, the color fades and a greyish brown, flocculent precipitate is produced.
- Dissolve 5 mg in about 1 ml of phosphoric acid (-1440 gm/L) TS and heat cautiously; the solution changes from colorless to yellow, then to orange, and later to reddish-brown.
- Dissolve 10 mg in 1 ml of methanol, add 1 ml of potassio-cupric tartrate TS and heat in a water bath; an orange precipitate is gradually produced.
- Carefully heat 0.04 gm with about 2 ml of sulfuric acid (~1760 gm/L) TS until white fumes are evolved. Add, drop by drop, nitric acid (-1000 gm/L) TS until the oxidation is complete. Allow to cool, add 10 ml of water, and heat again until white fumes are evolved. Cool, add 10 ml of water, and neutralize with ammonia (-100 gm/L) TS, using pH-indicator paper. Introduce an aliquot of this solution into a non-luminous flame using a magnesia stick or a nichrome or platinum wire sealed to a glass rod; the flame acquires an intense yellow color. To the remaining solution, add 5 ml of ammonium molybdate (95 gm/L) TS, acidify with nitric acid (-130 gm/L) TS, and heat a bright yellow precipitate is produced.
Degradation test: Discoloration of the test substance.
19. Pyrimethamine
Identity tests:
Description: A white, crystalline powder, hygroscopic; odorless.
Melting point: About 240°C.
Procedures:
- Dissolve 0.05 gm in 5 ml of sulfuric acid (-100 gm/L) TS and add about 0.2 ml of freshly prepared potassio-mercuric iodide TS; a creamy white precipitate is produced.
- To 1 ml of methyl orange/ethanol TS add 5 ml of water and 2 ml of ethyl acetate and shake; the ethyl acetate layer remains colorless. Add a solution of 5 mg of the test substance dissolved in 5 ml of sulfuric acid (-5 gm/L) TS, shake and allow to separate (about 30 minutes); a yellow color is produced in the ethyl acetate layer.
- Ignite about 0.1 gm with 0.5 gm of anhydrous sodium carbonate, extract the residue with water, and filter. Neutralize the filtrate with nitric acid (-130 gm/L) TS and add 0.5 ml of silver nitrate (-40 gm/L) TS; a white precipitate is produced. Add ammonia (~100 gm/L) TS; the precipitate dissolves.
Degradation test: Discoloration of the test substance.
20. Tamoxifen Citrate
Identity tests:
Description: A white or almost white, crystalline powder.
Melting point: About 142°C with decomposition.
Procedures:
- Shake 10 mg with 5 ml of dehydrated ethanol. Add 3 ml of water, 0.5 ml of ammonia (-100 gm/L) TS, and 2 ml of phosphomolybdic acid/ethanol TS. Within a few minutes, the color of the solution changes to light blue.
- To 10 mg, add 4 ml of pyridine and about 2 ml of acetic anhydride and shake; a yellow color is immediately produced. Heat in a water bath for 2 minutes. A light pink to red color is produced.
Degradation test: Discoloration of the test substance.
21. Vinblastine Sulfate
Identity tests:
Description: A white to slightly yellow, amorphous or crystalline powder, hygroscopic.
Note: This substance is very toxic and should be handled with care.
Melting point: About 200°C.
Procedures:
- To about 0.5 mg, add 2 drops of a 10 mg/ml solution of ceric ammonium sulfate in phosphoric acid (-1440 gm/L) TS; a purplish red color is produced which darkens with time.
- To about 1 mg, add 0.2 ml of a freshly prepared 10 mg/ml solution of vanillin in hydrochloric acid (-420 gm/L) TS; after about 1 minute, pink color is produced (distinction from vincristine sulfate).
- Mix about 0.5 mg with 5 mg of 4-dimethylaminobenzaldehyde, about 0.2 ml of glacial acetic acid, and about 0.2 ml of sulfuric acid (~1760 gm/L) TS. A reddish-brown color is produced which changes to pink after the addition of about 1 ml of glacial acetic acid.
- Dissolve 10 mg in 2 ml of water. Add 1 ml of hydrochloric acid (~70 gm/L) TS and 1 ml of barium chloride (50 gm/L) TS. A white precipitate is produced which is practically insoluble in hydrochloric acid (~250 gm/L) TS.
Degradation test: Discoloration of the test substance.
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