A: The RFvalues of the principal fluorescent spot and the principal blue spot in the chromatogram of the Test preparationcorrespond to those in the chromatogram of the Standard preparation,as obtained in the test for Related alkaloidsunder Ergonovine Maleate,using the Injection instead of Ergonovine Maleate.

B: Dilute a volume of Injection with water to obtain a solution having a concentration of about 0.67mg per mL:the solution exhibits a bluish fluorescence under UVlight.To this solution,add 2mLof a solution of glacial acetic acid in ethyl acetate (1in 2),and stratify 2mLof sulfuric acid,by pipetting,under the solution:a bluish purple ring appears at the interface of the two liquids.

Solvent mixture— Mix 9volumes of alcohol with 1volume of ammonium hydroxide.

Test preparation— Transfer a volume of Injection,equivalent to about 5mg of methylergonovine maleate,to a separator,and extract with three 5-mLportions of chloroform.Discard the chloroform extracts.Render alkaline to litmus with 6Nammonium hydroxide,and extract with three 5-mLportions of chloroform.Evaporate the combined extracts with the aid of a current of air,but without heat,to dryness.Dissolve the residue so obtained in 0.5mLof Solvent mixture.

Standard preparation andStandard dilutions— Prepare a solution of USP Methylergonovine Maleate RSin Solvent mixtureto contain 10mg per mL(Standard preparation).Prepare a series of dilutions of the Standard preparationin Solvent mixtureto contain 0.50mg,0.20mg,0.10mg,and 0.05mg per mL(Standard dilutions).

Procedure— In a suitable chromatographic chamber arranged for thin-layer chromatography place a volume of a solvent system consisting of a mixture of chloroform,methanol,and water (75:25:3)sufficient to develop the chromatogram,cover,and allow to equilibrate for 30minutes.Apply 5-µLportions of the Test preparation,the Standard preparation,and each of the three Standard dilutionsto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Allow the spots to dry,and develop the chromatogram until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the spots on the plate by spraying thoroughly and evenly with a solution prepared by dissolving 1g of p-dimethylaminobenzaldehyde in a cooled mixture of 50mLof alcohol and 50mLof hydrochloric acid:the RFvalue of the principal spot obtained from the Test preparationcorresponds to that obtained from the Standard preparation.Estimate the concentration of any other spots observed in the lane for the Test preparationby comparison with the Standard dilutions:the spots from the 0.50-,0.20-,0.10-,and 0.05-mg-per-mLdilutions are equivalent to 5.0%,2.0%,1.0%,and 0.50%of impurities,respectively.The sum of the impurities is not greater than 5.0%.

Mobile phase— Prepare a filtered and degassed mixture of 800mLof monobasic potassium phosphate solution (1in 500)and 200mLof acetonitrile.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Extraction solvent— Dissolve 5g of tartaric acid in 500mLof water.Add 500mLof methanol,and mix.

Standard preparation— Transfer about 20mg of USP Methylergonovine Maleate RS,accurately weighed,to a 200-mLvolumetric flask.Add 150mLof Extraction solvent,and shake by mechanical means for 15minutes.Dilute with the Extraction solventto volume,and mix to obtain the Standard preparationhaving a known concentration of about 100µg of USP Methylergonovine Maleate RSper mL.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 10mg of methylergonovine maleate to a 100-mLvolumetric flask.Dilute with Extraction solventto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 240-nm detector,and a 4-mm ×25-cm column that contains packing L7maintained at 30.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency determined from the analyte peak is not less than 1000theoretical plates,the tailing factor 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 (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 methylergonovine maleate (C20H25N3O2·C4H4O4)in each mLof the Injection taken by the formula: in which Cis the concentration,in µg per mL,of USP Methylergonovine Maleate RSin the Standard preparation;Vis the volume,in mL,of Injection taken;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.