Oxytetracycline hydrochloric acid

To a stirred solution of 4.6 g (0.01 mol) of anhydrous oxytetracycline in 40 ml of dry tetrahydrofuran is added 3.5 g (0.021 mol) of pyridine-sulfur trioxide complex. After 16 hours of stirring at room temperature, the resulting suspension is filtered, and the solid is slurried with 25 ml of 2% hydrochloric acid for 10 minutes, filtered and thoroughly washed with methanol followed by ether. The pale yellow crystalline 5-oxytetracycline-6, 12-hemiketal-12-sulfuric acid ester melts at 210°C. 

The light-absorbing impurities of oxytetracycline hydrochloride and oxytetracycline dihydrate [2,4,6] are detected using a UV-spectrophotometric method. In all the compendia, the absorbance of a solution of 2.0 mg/mL in a mixture of 1 volume of hydrochloric acid solution (0.1 mol/L) and 99 volumes of methanol at 430 nm not greater than 0.50 is required the absorbance of a solution of 10 mg/mL in the same solvent at 490 nm is not greater than 0.20. The measurements are carried out within 1 h of the preparation of the solutions. 

A microbiological assay is the method of choice (see p. 813) but spectro-photometric methods are also applicable. One spectrophotometric method is based on the formation of a yellow colour with an absorption maximum at 380 m//, when tetracycline is dissolved in 0-25N sodium hydroxide, another on the orange-brown colour (maximum absorption 490 m//) formed on mixing a dilute hydrochloric acid solution of the sample with ferric chloride solution. The former method which is described below is also applicable to oxytetracycline but not to chlortetracycline while the ferric chloride reaction, which is given in detail under oxytetracycline, applies to all three. 

Calculate the content of oxytetracycline free base by reference to a standard curve prepared by using suitable volumes of a standard solution of oxytetracycline hydrochloride in O OIN hydrochloric acid covering the range 0 to 2,000 fxg. 

Pour about 50 ml of the solution above on the dry column and allow it to percolate. Adjust the inactivated extract to pH 4-5 with N hydrochloric acid and transfer 20 ml to a 50-ml graduated flask. Add a known amount of a solution of the oxytetracycline standard equal to the amount estimated to be present in 4 g of the test sample and make up to volume with phosphate buffer solution. From this prepare further final dilutions as required in the assay. 

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