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ÈܼÁÈç¹ûÊÇÁ÷¶¯Ï࣬ÔÚ365nmÓ¦¸ÃûÓÐÎüÊÕ°¡ Èç¹ûÓз壬Ӧ¸ÃºÜС£¬³ö·åʱ¼äÒ²Ó¦¸ÃºÜÔçµÄ USP33 ASSAY • Procedure Mobile phase: Transfer 2.72 g of monobasic potassium phosphate, 0.74 g of sodium hydroxide, 0.50 g of tetrabutylammonium hydrogen sulfate, and 0.40 g of edetate disodium to a 1000-mL volumetric flask. Add 850 mL of water, and stir to dissolve. Add 60 g of tertiary butyl alcohol with the aid of water, dilute with water to volume, and adjust with 1 N sodium hydroxide to a pH of 8.0 ¡À 0.1. Pass this solution through a filter having a porosity of 0.5 µm or finer, and degas before using. Decreasing the proportion of tertiary butyl alcohol results in a longer retention time of doxycycline and improved separation of doxycycline from the related compounds. Diluent: 0.01 N hydrochloric acid System suitability solution: Prepare a solution of USP Doxycycline Hyclate RS in Diluent containing about 6 mg of doxycycline/mL. Transfer 5 mL of this solution to a 25-mL volumetric flask, heat on a steam bath for 60 min, and evaporate to dryness on a hot plate, taking care not to char the residue. Dissolve the residue in 0.01 N hydrochloric acid, dilute with Diluent to volume, and mix. Pass a portion of this solution through a filter having a porosity of 0.5 µm or finer, and use the filtrate. This solution contains a mixture of 4-epidoxycycline, 6-epidoxycycline, and doxycycline. When stored in a refrigerator, this solution may be used for 14 days. [Note¡ªThroughout the following procedures, protect the Standard solution and the Sample solution from light. ] Standard solution: Transfer 12 mg of USP Doxycycline Hyclate RS, to a 10-mL volumetric flask, add 6 mL of Diluent, sonicate for about 5 min or until dissolved, and dilute with Diluent to volume. Sample solution: 55 mg of Doxycycline in a 50-mL volumetric flask. Add 12 mL of 0.1 N hydrochloric acid, swirl to dissolve, and dilute with Diluent to volume. Filter through a filter of 0.5 µm or finer porosity. Chromatographic system (See Chromatography 621, System Suitability.) Mode: LC Detector: UV 270 nm Column: 4.6-mm ¡Á 25-cm; packing L21 Column temperature: 60 ¡À 1 Flow rate: 1 mL/min Injection size: 20 µL System suitability Sample: System suitability solution and Standard solution [Note¡ªThe relative retention times for 4-epidoxycycline (the main degradation product), 6-epidoxycycline, and doxycycline are 0.4, 0.7, and 1.0, respectively. ] Suitability requirements Resolution: NLT 3.0 between the 4-epidoxycycline peak and the doxycycline peak, System suitability solution Tailing factor: NMT 2.0, System suitability solution Relative standard deviation: NMT 2.0%, Standard solution Analysis Samples: Standard solution and Sample solution Calculate the quantity of C22H24N2O8, in µg, in each mg of the Doxycycline taken: Result = (rU/rS) ¡Á (CS/CU) ¡Á P rU = = peak response from the Sample solution rS = = peak response from the Standard solution CS = = concentration of USP Doxycycline Hyclate RS in the Standard solution (mg/mL) CU = = concentration of the Sample solution (mg/mL) P = = designated potency of doxycycline (C22H24N2O8), of USP Doxycycline Hyclate RS (µg/mg) Acceptance criteria: NLT 880 µg/mg and NMT 980 µg/mg |
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