Specifications of Calcium Levulinate USP Grade
C10H14CaO6.2H2O -- 306.32
Pentanoic acid, 4-oxo-, calcium salt (2:1), dihydrate.
Calcium levulinate (1:2) dihydrate [5743-49-7].
Anhydrous 270.30 [591-64-0].
Calcium Levulinate contains not less than 97.5 percent and not more than 100.5 percent of C10H14CaO6, calculated on the dried basis.

Identification:
A: A solution (1 in 10) responds to the tests for Calcium.
B: To a solution of 0.5 g in 5 mL of water add 5 mL of 1 N sodium hydroxide, and filter. To the filtrate add 5 mL of iodine: a precipitate of iodoform is produced.

IDENTIFICATION
First identification A, D, E.
Second identification B, C, D, E.
A. Infrared absorption spectrophotometry comparison calcium levulinate dihydrate CRS.
B. Thin-layer chromatography.
Test solution: Dissolve 60 mg of the substance to be examined in water and dilute to 1 ml with the same solvent.
Reference solution: Dissolve 60 mg of calcium levulinate dihydrate CRS in water and dilute to 1 ml with the same solvent.
Plate: TLC silica gel plate.
Mobile phase: concentrated ammonia, ethyl acetate, water, ethanol (96 per cent) (10:10:30:50 V/V/V/V).
Application: 10 μl.
Development: Over a path of 10 cm.
Drying: At 100-105C for 20 min and allow to cool.
Detection: Spray with a 30 g/l solution of potassium permanganate. Dry in a current of warm air for about 5 min or until the spots become yellow. Examine in daylight.
Results: The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution.
C. To 1 ml of solution S (see Tests), add 20 ml of a 2.5 g/l solution of dinitrophenylhydrazine in dilute hydrochloric acid. Allow to stand for 15 min. Filter, wash the precipitate with water. Dry the precipitate in an oven at 100-105C. The melting point is 203C to 210C.
D. It gives reaction of calcium.
E. Loss on drying (see Tests).

TESTS
Solution S: Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water and dilute to 100.0 ml with the same solvent.
Appearance of solution: Solution S is clear and not more intensely coloured than reference solution.
pH: 6.8 to 7.8 for solution S.
Oxidisable substances: To 1 ml of solution S, add 10 ml of water, 1 ml of dilute sulphuric acid and 0.25 ml of a 3.0 g/l solution of potassium permanganate. Mix. After 5 min, the violet colour of the mixture is still visible.
Sucrose and reducing sugars: To 5 ml of solution S add 2 ml of hydrochloric acid and dilute to 10 ml with water. Heat to boiling for 5 min and allow to cool. Add 10 ml of sodium carbonate solution. Allow to stand for 5 min, dilute to 25 ml with water and filter. To 5 ml of the filtrate add 2 ml of cupri-tartaric solution and heat to boiling for 1 min. No red precipitate is formed.
Chlorides: Maximum 50 ppm.
Sulphates: Maximum 200 ppm.
Magnesium and alkali metals: Maximum 1.0 per cent.
To 10 ml of solution S, add 80 ml of water, 10 ml of ammonium chloride solution R and 1 ml of ammonia. Heat to boiling. To the boiling solution, add drop-wise 50 ml of warm ammonium oxalate solution. Allow to stand for 4 h, then dilute to 200 ml with water and filter. To 100 ml of the filtrate, add 0.5 ml of sulphuric acid. Evaporate to dryness on a water-bath and ignite to constant mass at 600 ± 50C. The residue weighs a maximum of 5.0 mg.
Heavy metals: Maximum 10 ppm.
Loss on drying: 11.0 per cent to 12.5 per cent, determined on 0.200 g by drying at 105C.
Pyrogens: If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of the rabbit's mass 4 ml of a solution containing per millilitre 50 mg of the substance to be examined.

TESTS
Solution S: Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water and dilute to 100.0 ml with the same solvent.
Appearance of solution: Solution S is clear and not more intensely coloured than reference solution.
pH: 6.8 to 7.8 for solution S.
Oxidisable substances: To 1 ml of solution S, add 10 ml of water, 1 ml of dilute sulphuric acid and 0.25 ml of a 3.0 g/l solution of potassium permanganate. Mix. After 5 min, the violet colour of the mixture is still visible.
Sucrose and reducing sugars: To 5 ml of solution S add 2 ml of hydrochloric acid and dilute to 10 ml with water. Heat to boiling for 5 min and allow to cool. Add 10 ml of sodium carbonate solution. Allow to stand for 5 min, dilute to 25 ml with water and filter. To 5 ml of the filtrate add 2 ml of cupri-tartaric solution and heat to boiling for 1 min. No red precipitate is formed.
Chlorides: Maximum 50 ppm.
Sulphates: Maximum 200 ppm.
Magnesium and alkali metals: Maximum 1.0 per cent.
To 10 ml of solution S, add 80 ml of water, 10 ml of ammonium chloride solution R and 1 ml of ammonia. Heat to boiling. To the boiling solution, add drop-wise 50 ml of warm ammonium oxalate solution. Allow to stand for 4 h, then dilute to 200 ml with water and filter. To 100 ml of the filtrate, add 0.5 ml of sulphuric acid. Evaporate to dryness on a water-bath and ignite to constant mass at 600 ± 50C. The residue weighs a maximum of 5.0 mg.
Heavy metals: Maximum 10 ppm.
Loss on drying: 11.0 per cent to 12.5 per cent, determined on 0.200 g by drying at 105C.
Pyrogens: If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of the rabbit's mass 4 ml of a solution containing per millilitre 50 mg of the substance to be examined.

DEFINITION
Calcium di(4-oxopentanoate) dihydrate.
Content: 98.0 per cent to 101.0 per cent (dried substance).

CHARACTERS
Appearance: White or almost white, crystalline powder.
Solubility: Freely soluble in water, very slightly soluble in ethanol (96 per cent), practically insoluble in methylene chloride.

IDENTIFICATION
First identification A, D, E.
Second identification B, C, D, E.
A. Infrared absorption spectrophotometry comparison calcium levulinate dihydrate CRS.
B. Thin-layer chromatography.
Test solution: Dissolve 60 mg of the substance to be examined in water and dilute to 1 ml with the same solvent.
Reference solution: Dissolve 60 mg of calcium levulinate dihydrate CRS in water and dilute to 1 ml with the same solvent.
Plate: TLC silica gel plate.
Mobile phase: concentrated ammonia, ethyl acetate, water, ethanol (96 per cent) (10:10:30:50 V/V/V/V).
Application: 10 μl.
Development: Over a path of 10 cm.
Drying: At 100-105C for 20 min and allow to cool.
Detection: Spray with a 30 g/l solution of potassium permanganate. Dry in a current of warm air for about 5 min or until the spots become yellow. Examine in daylight.
Results: The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution.
C. To 1 ml of solution S (see Tests), add 20 ml of a 2.5 g/l solution of dinitrophenylhydrazine in dilute hydrochloric acid. Allow to stand for 15 min. Filter, wash the precipitate with water. Dry the precipitate in an oven at 100-105C. The melting point is 203C to 210C.
D. It gives reaction of calcium.
E. Loss on drying (see Tests).

TESTS
Solution S: Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water and dilute to 100.0 ml with the same solvent.
Appearance of solution: Solution S is clear and not more intensely coloured than reference solution.
pH: 6.8 to 7.8 for solution S.
Oxidisable substances: To 1 ml of solution S, add 10 ml of water, 1 ml of dilute sulphuric acid and 0.25 ml of a 3.0 g/l solution of potassium permanganate. Mix. After 5 min, the violet colour of the mixture is still visible.
Sucrose and reducing sugars: To 5 ml of solution S add 2 ml of hydrochloric acid and dilute to 10 ml with water. Heat to boiling for 5 min and allow to cool. Add 10 ml of sodium carbonate solution. Allow to stand for 5 min, dilute to 25 ml with water and filter. To 5 ml of the filtrate add 2 ml of cupri-tartaric solution and heat to boiling for 1 min. No red precipitate is formed.
Chlorides: Maximum 50 ppm.
Sulphates: Maximum 200 ppm.
Magnesium and alkali metals: Maximum 1.0 per cent.
To 10 ml of solution S, add 80 ml of water, 10 ml of ammonium chloride solution R and 1 ml of ammonia. Heat to boiling. To the boiling solution, add drop-wise 50 ml of warm ammonium oxalate solution. Allow to stand for 4 h, then dilute to 200 ml with water and filter. To 100 ml of the filtrate, add 0.5 ml of sulphuric acid. Evaporate to dryness on a water-bath and ignite to constant mass at 600 ± 50C. The residue weighs a maximum of 5.0 mg.
Heavy metals: Maximum 10 ppm.
Loss on drying: 11.0 per cent to 12.5 per cent, determined on 0.200 g by drying at 105C.
Pyrogens: If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of the rabbit's mass 4 ml of a solution containing per millilitre 50 mg of the substance to be examined.