A: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.

B: Shake a portion of finely powdered Tablets,equivalent to about 2mg of melphalan,with 20mLof alcohol,and filter:a 1-mLportion of the solution so obtained responds to Identificationtest Bunder Melphalan.

Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 2: 50rpm.

Time: 30minutes.

Mobile phase— Prepare a filtered and degassed mixture of water,acetonitrile,ammonium acetate,glacial acetic acid,and triethylamine (1500:500:2:2:0.4).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Chromatographic system (seeChromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×5-cm column that contains packing L7.The flow rate is about 1.5mLper minute.Chromatograph replicate injections of the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 3.0%.

Procedure— Inject a volume (about 50µL)of a filtered portion of the solution under test into the chromatograph,record the chromatogram,and measure the response for the major peak.Calculate the quantity of C13H18Cl2N2O2dissolved in comparison with a Standard solution having a known concentration of USP Melphalan Hydrochloride RSin the same Mediumand similarly chromatographed.

Tolerances— Not less than 80%(Q)of the labeled amount of C13H18Cl2N2O2is dissolved in 30minutes.

Procedure for content uniformity— Place 1Tablet in a 200-mLvolumetric flask,add 10mLof water and 10mLof alcohol,sonicate to dissolve the soluble components in the mixture,dilute with alcohol to volume,mix,and filter to obtain a clear solution.Dissolve an accurately weighed quantity of USP Melphalan Hydrochloride RSin alcohol to obtain a Standard solution having a known concentration of about 10µg per mL.Concomitantly determine the absorbances of the solution from the Tablet and the Standard solution in 1-cm cells at the wavelength of maximum absorbance at about 260nm,with a suitable spectrophotometer,using alcohol as the blank.Calculate the quantity,in mg,of melphalan (C13H18Cl2N2O2)in the Tablet taken by the formula: in which 305.20and 341.66are the molecular weights of melphalan and melphalan hydrochloride,respectively;Tis the labeled quantity,in mg,of melphalan in the Tablet;Dis the concentration,in µg per mL,of melphalan in the solution from the Tablet,on the basis of the labeled quantity per Tablet and the extent of dilution;Cis the concentration,in µg per mL,of USP Melphalan Hydrochloride RSin the Standard solution;and AUand ASare the absorbances of the solution from the Tablet and the Standard solution,respectively.

Mobile phase— Prepare a solution of 0.025Mdiethylamine in a mixture of methanol and water (1:1),adjust with 3.5Nhydrochloric acid to a pHof 5.5,filter,and degas.Make adjustments if necessary (seeSystem Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Melphalan Hydrochloride RSin alcohol,and quantitatively dilute with alcohol to obtain a solution having a known concentration of about 0.9mg of melphalan hydrochloride per mL.Pipet 10mLof this solution into a 100-mLvolumetric flask containing 75mLof alcohol and 2.0mLof glacial acetic acid,dilute with alcohol to volume,and mix to obtain a Standard preparationhaving a known concentration of about 90µg of USP Melphalan Hydrochloride RSper mL(equivalent to about 80µg of melphalan per mL).

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to 8mg of anhydrous melphalan,to a 100-mLvolumetric flask.Add about 75mLof alcohol and 2.0mLof glacial acetic acid to the flask,and sonicate for 15minutes.Cool,dilute with alcohol to volume,and mix.Filter through a medium-porosity,sintered-glass funnel,discarding the first few mLof the filtrate,and use the remainder of the filtrate as the Assay preparation.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.2-mm ×25-cm column that contains packing L7.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor for the analyte peak is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (between 10and 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 melphalan (C13H18Cl2N2O2)in the portion of Tablets taken by the formula: in which 305.20and 341.67are the molecular weights of melphalan and melphalan hydrochloride,respectively;Cis the concentration,in µg per mL,of melphalan hydrochloride in the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.