Warfarin Sodium C19H15NaO4 330.31
2H-1-Benzopyran-2-one,4-hydroxy-3-(3-oxo-1-phenylbutyl)-,sodium salt.
3-(a-Acetonylbenzyl)-4-hydroxycoumarin sodium salt [129-06-6]. »Warfarin Sodium is an amorphous solid or a crystalline clathrate.The clathrate form consists principally of warfarin sodium and isopropyl alcohol,in a 2:1molecular ratio;it contains not less than 8.0percent and not more than 8.5percent of isopropyl alcohol.Warfarin Sodium contains not less than 97.0percent and not more than 102.0percent of C19H15NaO4,calculated on the anhydrous basis for the amorphous form or on the anhydrous and isopropyl alcohol-free basis for the crystalline form. Packaging and storage— Preserve in well-closed,light-resistant containers. Labeling— Label it to indicate whether it is the amorphous or the crystalline form. USP Reference standards á11ñ USP Warfarin RS.USP Warfarin Related Compound A RS. Identification— A: Infrared Absorption á197Kñ—The test specimen is the residue of warfarin obtained in Identificationtest B. B: Dissolve about 100mg in 25mLof water,and adjust with hydrochloric acid to a pHof less than 3,using short-range pHindicator paper.Stir the mixture and allow the precipitate to coagulate.Filter the mixture,wash the precipitate with four 5-mLportions of water,and dry in vacuum over phosphorus pentoxide for 4hours:the warfarin so obtained melts between 157and 167,but the range between beginning and end of melting does not exceed 4. C: Asolution of it responds to the tests for Sodium á191ñ.The filtrate obtained in Identificationtest Bresponds to the flame test for Sodium á191ñ. pHá791ñ: between 7.2and 8.3,in a solution (1in 100). Water,Method Iá921ñ: not more than 4.5%for the amorphous form;not more than 0.3%for the crystalline clathrate form. Heavy metals á231ñ Dissolve 4.0g in 45mLof water,add 5mLof glacial acetic acid,stir until the precipitate agglomerates,filter,and use 25mLof the filtrate,employing glacial acetic acid,if necessary,to make the pHadjustment:the limit is 0.001%. Absorbance in alkaline solution— Dissolve 1.25g,accurately weighed,in 10mLof sodium hydroxide solution (1in 20),filter through a membrane filter,and within 15minutes determine the absorbance of the solution in a 1-cm cell at 385nm,with a suitable spectrophotometer,using sodium hydroxide solution (1in 20)as the blank:the absorbance does not exceed 0.1. Chromatographic purity— Solvent mixture— Prepare a mixture of water and methanol (75:25). Mobile phase— Prepare a filtered and degassed mixture of water,acetonitrile,and glacial acetic acid (68:32:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ). Standard solution— Transfer an accurately weighed quantity of about 24mg of USP Warfarin RSand 24mg of USP Warfarin Related Compound A RSto a 200-mLvolumetric flask,add 4.0mLof 0.1Nsodium hydroxide,50mLof methanol,and dissolve.Dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 200-mLvolumetric flask,dilute with Solvent mixtureto volume,and mix.Transfer 20.0mLof this solution to a 50-mLvolumetric flask,dilute with Solvent mixtureto volume,and mix. Test solution— Transfer an accurately weighed quantity of about 80mg of Warfarin Sodium to a 100-mLvolumetric flask,dissolve in and dilute with Solvent mixtureto volume,and mix. Chromatographic system(see Chromatography á621ñ) The liquid chromatograph is equipped with a 260-nm detector and a 4.6-mm ×25-cm column that contains packing L10.The flow rate is about 1.5mLper minute.Chromatograph the Standard solution,and record the chromatogram as directed for Procedure:the resolution,R,between warfarin and warfarin related compound Ais not less than 3;and the relative standard deviation for replicate injections is not more than 5.0%. Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for all of the peaks.The relative retention times of warfarin and warfarin related compound Aare 1.0and about 1.2,respectively.Calculate the percentage of each impurity in the portion of Warfarin Sodium taken by the formula: 10,000(C/M)(ri/rS) in which Cis the concentration,in mg per mL,of warfarin sodium in the Standard solution;Mis the quantity,in mg,of warfarin sodium taken to prepare the Test solution;riis the peak response of the individual impurity;and rSis the peak response due to warfarin in the Standard solution:not more than 0.3%of any individual impurity and not more than 1.0%of total impurities is found. Isopropyl alcohol content (crystalline clathrate form) Internal standard solution— Dilute 2mLof n-propyl alcohol with water to 100.0mLin a volumetric flask. Standard preparation— Transfer an accurately weighed quantity of about 1.6g of isopropyl alcohol to a 100-mLvolumetric flask,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,add 10.0mLof the Internal standard solution,dilute with water to volume,and mix to obtain a Standard preparationhaving a known concentration of about 1.6mg of isopropyl alcohol per mL. Test preparation— Dissolve an accurately weighed quantity of about 1.85g of Warfarin Sodium in about 50mLof water in a 100-mLvolumetric flask.Add 10.0mLof the Internal standard solution,dilute with water to volume,and mix. Chromatographic system(see Chromatography á621ñ) The gas chromatograph is equipped with a flame-ionization detector and a 4-mm ×1.8-m column packed with 80-to 100-mesh support S2.The temperatures of the column,injector,and the detector are maintained at about 140,200,and 250,respectively.The carrier gas is nitrogen,flowing at the rate of about 40mLper minute.The column temperature may be varied so that the following system suitability criteria are met:the resolution,R,between n-propyl alcohol and isopropyl alcohol is not less than 2.0;the tailing factor,T,for the isopropyl alcohol peak is not more than 1.5;and the relative standard deviation of the ratio of isopropyl alcohol area to n-propyl alcohol area for five replicate injections of the Standard preparationis not more than 2.0%. Procedure— Separately inject equal volumes (about 5µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and measure the areas of the major peaks.Calculate the weight,in mg,of the major peaks.Calculate the weight,in mg,of isopropyl alcohol in the portion of Warfarin Sodium taken by the formula: 100C(RU/RS), in which Cis the concentration,in mg per mL,of isopropyl alcohol in the Standard preparation;and RUand RSare the peak area ratios of isopropyl alcohol to n-propyl alcohol obtained from the Test preparationand the Standard preparation,respectively. Organic volatile impurities,Method Iá467ñ: meets the requirements. Assay— pH7.4Buffer— Transfer 1.36g of monobasic potassium phosphate to a 200-mLvolumetric flask,and dissolve in 50mLof water.Add 39.1mLof 0.2Nsodium hydroxide,and dilute with water to volume.Adjust with sodium hydroxide or phosphoric acid to a pHof 7.4±0.1. Mobile phase— Prepare a degassed solution containing a mixture of methanol,water,and glacial acetic acid (64:36:1).Adjust the ratio as necessary. Internal standard solution— Dissolve propylparaben in a mixed solvent consisting of acetonitrile and glacial acetic acid (988:12),to obtain a solution having a concentration of about 0.2mg per mL. Standard preparation— Transfer about 94mg of USP Warfarin RS,accurately weighed,to a 250-mLvolumetric flask,and dissolve in 97.8mLof 0.1Nsodium hydroxide.Add 62.5mLof 0.2Mmonobasic potassium phosphate,dilute with water to volume,and mix.Pipet 5mLof this solution,5mLof pH7.4Buffer,and 10mLof Internal standard solutioninto a conical flask,and mix. Assay preparation— Using about 100mg of Warfarin Sodium,accurately weighed,prepare as directed under Standard preparation. Chromatographic system (see Chromatography á621ñ) The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains packing L7.The flow rate is about 1.4mLper minute.Chromatograph five replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times of propylparaben and warfarin are about 0.75and 1.0,respectively;the resolution of the two peaks is not less than 2.0;and the relative standard deviation of the warfarin responses is not more than 2.0%. Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C19H15NaO4in the portion of Warfarin Sodium taken by the formula: (330.32/308.34)C(RU/RS), in which 330.32and 308.34are the molecular weights of warfarin sodium and warfarin,respectively;Cis the concentration,in µg per mL,of USP Warfarin RSin the Standard preparation;and RUand RSare the peak response ratios of warfarin to propylparaben obtained from the Assay preparationand the Standard preparation,respectively. Auxiliary Information— Staff Liaison:Lawrence Evans,III,Ph.D.,Scientist Expert Committee:(PA6)Pharmaceutical Analysis 6 USP28–NF23Page 2031 Phone Number:1-301-816-8389