226 4EPIANHYDROTETRACYCLINE
This chromatographic procedure is provided to demonstrate that the content of 4epianhydrotetracycline, a degradation product of tetracycline, does not exceed the limit given in the individual monograph.
EDTA Buffer— Dissolve 37.2 g of edetate disodium in 800 mL of water, adjust with ammonium hydroxide to a pH of 7.8, dilute with water to 1000 mL, and mix. Support Phase— Add 5 mL of EDTA Buffer to 10 g of acidwashed chromatographic siliceous earth for column chromatography, and mix until the siliceous earth is uniformly moistened. Test Solution— Prepare as directed in the individual monograph. Procedure— Prepare a 15mm × 170mm chromatographic tube with a 4mm × 50mm outlet by packing it, in increments, with Support Phase, firmly tamping down each increment, until the tube is filled to a height of about 10 cm. In a beaker, prepare a mixture of 1 g of acidwashed chromatographic siliceous earth for column chromatography and 1 mL of Test Solution. Transfer the mixture to the top of the column. Drywash the beaker with Support Phase, and transfer to the column to provide an additional 1cm layer on top of the mixture containing the Test Solution. Within 30 minutes, pass chloroform through the column, and collect successive fractions of 5.0 mL, 5.0 mL, 10.0 mL, 10.0 mL, and 5.0 mL. Observe the column during elution, and note the appearance of two separate yellow bands. The fraction or fractions containing the first yellow band contain the anhydrotetracyclines. Discard these fractions. The fractions after the first yellow band contain the 4epianhydrotetracycline. Determine the absorbance of each 4epianhydrotetracycline fraction at the wavelength of maximum absorbance at about 438 nm, with a suitable spectrophotometer, diluting each fraction, if necessary, with chloroform, and using chloroform as the blank. Calculate the quantity, in mg, of 4epianhydrotetracycline in each fraction by the formula: AVD/20.08
in which A is the absorbance, V is the volume, in mL, of the fraction taken, D is the dilution factor, if the fraction was diluted, and 20.08 is the absorptivity of 4epianhydrotetracycline at 438 nm. From the sum of the quantities of 4epianhydrotetracycline found in the fractions, calculate the percentage of 4epianhydrotetracycline in relation to the tetracycline hydrochloride equivalent contained in the Test Solution.
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USP35–NF30 Page 142
