Standard solution— Transfer 5.0mLof dehydrated alcohol to a 250-mLvolumetric flask,dilute with water to volume,and mix.Transfer 5.0mLof this solution to a 500-mLvolumetric flask,dilute with water to volume,and mix.

Internal standard solution— Transfer 5.0mLof n-propyl alcohol to a 250-mLvolumetric flask,dilute with water to volume,and mix.Transfer 5.0mLof this solution to a 500-mLvolumetric flask,dilute with water to volume,and mix.

Standard preparation— Transfer 10.0mLof the Standard solutionto a 25-mLvolumetric flask,add 10.0mLof the Internal standard solution,dilute with water to volume,and mix.This solution contains 0.063mg of alcohol (C2H5OH)per mL.

Test preparation— Transfer about 100mg of Mitoxantrone Hydrochloride,accurately weighed,to a 5-mLvolumetric flask,add 2.0mLof the Internal standard solution,dilute with water to volume,and mix.Sonicate for 2minutes and shake for 2minutes,repeating these actions until the specimen is completely dissolved.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector and a 2-mm ×3-m column that contains 20%phase G1and 0.1%phase G39on silanized support S1A.Maintain the column at 50for 5minutes,then increase the temperature at a rate of 30per minute.When 140is reached,maintain that temperature for 20minutes.Maintain the injection port at 200and the detection block at 250.Use helium as the carrier gas at a flow rate of about 15mLper minute.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.5for alcohol and 1.0for n-propyl alcohol,the resolution,R,between the alcohol and the n-propyl alcohol peaks is not less than 6.0,and the tailing factors for the two peaks are not more than 2.0.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 1µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of alcohol (C2H5OH)in the portion of Mitoxantrone Hydrochloride taken by the formula: in which Cis the concentration,in mg per mL,of alcohol (C2H5OH)in the Standard preparation;Wis the weight,in mg,of Mitoxantrone Hydrochloride taken;and RUand RSare the ratios of the response of the alcohol peak to that of the n-propyl alcohol peak obtained from the Test preparationand the Standard preparation,respectively:not more than 1.5%is found.

100(ri/rs),

Sodium 1-heptanesulfonate solution— Dissolve 22.0g of sodium 1-heptanesulfonate in about 150mLof water,pass through a suitable filter having a 0.5-µm or finer porosity,and transfer the filtrate to a 250-mLvolumetric flask.Wash the filter with about 50mLof water,adding the filtrate to the 250-mLvolumetric flask.Add 32.0mLof glacial acetic acid to the volumetric flask,dilute with water to volume,and mix.

Mobile phase— Prepare a suitable degassed mixture of water,acetonitrile,and Sodium 1-heptanesulfonate solution(750:250:25).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Prepare a solution of USP Mitoxantrone System Suitability Mixture RSin a suitable volume of Mobile phaseto obtain a solution containing about 0.2mg of 8-amino-1,4-dihydroxy-5[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione hydrochloride (mitoxantrone related compound A)and 0.1mg of mitoxantrone hydrochloride per mL.

Standard preparation— Transfer about 20mg of USP Mitoxantrone Hydrochloride RS,accurately weighed,to a 50-mLvolumetric flask,add 40mLof Mobile phase,and dissolve by sonicating for about 5minutes.Cool to room temperature,dilute with Mobile phaseto volume,and mix.

Assay preparation— Transfer about 20mg of Mitoxantrone Hydrochloride,accurately weighed,to a 50-mLvolumetric flask,add 40mLof Mobile phase,and dissolve by sonicating for about 5minutes.Cool to room temperature,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L11.The flow rate is about 3mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.7for mitoxantrone and 1.0for mitoxantrone related compound A;the resolution,R,between mitoxantrone and mitoxantrone related compound Ais not less than 3.0;and the tailing factor for the mitoxantrone peak is not more than 2.0.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the capacity factor,k¢,for mitoxantrone is not less than 3.5;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.[NOTE—After use,wash the column with a mixture of acetonitrile and water (50:50),and store in this mixture.]Calculate the quantity,in mg,of C22H28N4O6·2HCl in the portion of Mitoxantrone Hydrochloride taken by the formula: in which Cis the concentration,in mg per mL,of anhydrous mitoxantrone hydrochloride in the Standard preparation,as determined from the content of USP Mitoxantrone Hydrochloride RScorrected for the water content determined by a titrimetric water determination;and rUand rSare the mitoxantrone peak areas obtained from the Assay preparationand the Standard preparation,respectively.