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Tetracycline
Appearance: yellow, crystalline powder.
2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethane sulphonic Solubility: very slightly soluble in water, soluble in ethanol (96 per cent) and in methanol, sparingly soluble in acetone.
Dissolve the above ingredients in about 950 ml of water R, It dissolves in dilute acid and alkaline solutions.
adjust to pH 7.4 with 1 M sodium hydroxide and add 60 mg IDENTIFICATION of benzylpenicillin sodium R and a quantity of streptomycin sulphate R equivalent to 100 mg of streptomycin. Dilute to A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 5 mg of the substance to be examined in methanol R and dilute to 10 ml with the Add 2 g/l of glucose R to solution A.
Reference solution (a). Dissolve 5 mg of tetracycline hydrochloride CRS in methanol R and dilute to 10 ml To solution B add 1 g/l of a preparation of collagenase obtained from Clostridium histolyticum and of a grade Reference solution (b). Dissolve 5 mg of tetracycline suitable for the preparation of dispersed cells.
hydrochloride CRS, 5 mg of demeclocycline hydrochloride R and 5 mg of oxytetracycline hydrochloride R in methanol R and dilute to 10 ml with Add 5 g/l of bovine albumin R to solution B.
Plate: TLC octadecylsilyl silica gel F A 9 g/l sterile solution of sodium chloride R containing Mobile phase: mix 20 volumes of acetonitrile R, 1 g/l of bovine albumin R and adjusted to pH 2.0 with 1 M 20 volumes of methanol R and 60 volumes of a 63 g/l solution of oxalic acid R previously adjusted to pH 2 with Store under nitrogen, protected from light, at a temperature Development: over 3/4 of the plate.
Drying: in air.
Detection: examine in ultraviolet light at 254 nm.
System suitability: the chromatogram obtained with — the potency in International Units per milligram, reference solution (b) shows 3 clearly separated spots.
Results: the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with B. To about 2 mg add 5 ml of sulphuric acid R. A violet-red colour develops. Add the solution to 2.5 ml of water R.
01/2008:0211
corrected 6.0 C. Dissolve about 10 mg in a mixture of 1 ml of dilute nitric
acid R and 5 ml of water R. Shake and add 1 ml of silver TETRACYCLINE
nitrate solution R2. Any opalescence in the solution is not more intense than that in a mixture of 1 ml of dilute nitric acid R, 5 ml of water R and 1 ml of silver nitrate TESTS
pH (2.2.3): 3.5 to 6.0.
Suspend 0.1 g in 10 ml of carbon dioxide-free water R.
Specific optical rotation (2.2.7): − 260 to − 280 (dried
substance).
Dissolve 0.250 g in 0.1 M hydrochloric acid and dilute to 50.0 ml with the same acid.
Related substances. Liquid chromatography (2.2.29).
Prepare the solutions immediately before use. Test solution. Dissolve 25.0 mg of the substance to be examined in 0.01 M hydrochloric acid and dilute to 25.0 ml (4S,4aS,5aS,6S,12aS)-4-(Dimethylamino)-3,6,10,12,12a- Reference solution (a). Dissolve 25.0 mg of tetracycline pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a- hydrochloride CRS in 0.01 M hydrochloric acid and dilute Substance produced by certain strains of Streptomyces Reference solution (b). Dissolve 12.5 mg of 4-epitetracycline aerofaciens or obtained by any other means.
hydrochloride CRS in 0.01 M hydrochloric acid and dilute Content: 88.0 per cent to 102.0 per cent (dried substance).
See the information section on general monographs (cover pages) Tetracycline hydrochloride
Reference solution (c). Dissolve 10.0 mg of Heavy metals (2.4.8): maximum 50 ppm.
anhydrotetracycline hydrochloride CRS in 0.01 M 0.5 g complies with test C. Prepare the reference solution hydrochloric acid and dilute to 100.0 ml with the same acid. using 2.5 ml of lead standard solution (10 ppm Pb) R.
Reference solution (d). Dissolve 10.0 mg of Loss on drying (2.2.32): maximum 13.0 per cent, determined
4-epianhydrotetracycline hydrochloride CRS in on 1.000 g by drying in an oven at 105 °C.
0.01 M hydrochloric acid and dilute to 50.0 ml with the Sulphated ash (2.4.14): maximum 0.5 per cent, determined
Reference solution (e). Mix 1.0 ml of reference solution (a), 2.0 ml of reference solution (b) and 5.0 ml of reference solution (d) and dilute to 25.0 ml with 0.01 M hydrochloric Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.
Reference solution (f). Mix 40.0 ml of reference solution (b), Injection: test solution and reference solution (a).
20.0 ml of reference solution (c) and 5.0 ml of reference Calculate the percentage content of C H N O .
solution (d) and dilute to 200.0 ml with 0.01 M hydrochloric Reference solution (g). Dilute 1.0 ml of reference solution (c) Protected from light.
to 50.0 ml with 0.01 M hydrochloric acid.
Column:— size: l = 0.25 m, Ø = 4.6 mm;— stationary phase: styrene-divinylbenzene copolymer Rtemperature: 60 °C.
Mobile phase: weigh 80.0 g of 2-methyl-2-propanol R and transfer to a 1000 ml volumetric flask with the aid A. R1 = NH , R2 = H, R3 = N(CH ) : (4R,4aS,5aS,6S,12aS)- of 200 ml of water R; add 100 ml of a 35 g/l solution of 4-(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl- dipotassium hydrogen phosphate R adjusted to pH 9.0 1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2- with dilute phosphoric acid R, 200 ml of a 10 g/l solution of tetrabutylammonium hydrogen sulphate R adjusted to pH 9.0 with dilute sodium hydroxide solution R and B. R1 = CH , R2 = N(CH ) , R3 = H: (4S,4aS,5aS,6S,12aS)- 10 ml of a 40 g/l solution of sodium edetate R adjusted to 2-acetyl-4-(dimethylamino)-3,6,10,12,12a-pentahydroxy- pH 9.0 with dilute sodium hydroxide solution R; dilute to 6-methyl-4a,5a,6,12a-tetrahydrotetracene-1,11(4H,5H)- dione (2-acetyl-2-decarbamoyltetracycline), Flow rate: 1.0 ml/min.
Detection: spectrophotometer at 254 nm.
Injection: 20 µl; inject the test solution and reference solutions (e), (f) and (g).
System suitability :— resolution: minimum 2.5 between the peaks due to C. R1 = N(CH ) , R2 = H: (4S,4aS,12aS)-4-(dimethylamino)- impurity A (1st peak) and tetracycline (2nd peak) and 3,10,11,12a-tetrahydroxy-6-methyl-1,12-dioxo-1,4, minimum 8.0 between the peaks due to tetracycline and 4a,5,12,12a-hexahydrotetracene-2-carboxamide impurity D (3rd peak) in the chromatogram obtained with reference solution (e); if necessary, adjust the concentration of 2-methyl-2-propanol in the mobile phase; D. R1 = H, R2 = N(CH ) : (4R,4aS,12aS)-4-(dimethylamino)- — signal-to-noise ratio: minimum 3 for the principal peak in 3,10,11,12a-tetrahydroxy-6-methyl-1,12-dioxo-1,4, the chromatogram obtained with reference solution (g); 4a,5,12,12a-hexahydrotetracene-2-carboxamide — symmetry factor: maximum 1.25 for the peak due to tetracycline in the chromatogram obtained with reference 01/2008:0210
corrected 6.0
Limits:— impurity A: not more than the area of the corresponding TETRACYCLINE HYDROCHLORIDE
peak in the chromatogram obtained with reference — impurity B (eluting on the tail of the principal peak): not more than 0.4 times the area of the peak due to impurity A in the chromatogram obtained with reference — impurity C: not more than the area of the corresponding peak in the chromatogram obtained with reference — impurity D: not more than the area of the corresponding peak in the chromatogram obtained with reference General Notices (1) apply to all monographs and other texts

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