A: Transfer a portion of powdered Tablets,equivalent to about 50mg of baclofen,to a glass-stoppered 40-mLcentrifuge tube.Add 10.0mLof a mixture of dehydrated alcohol and glacial acetic acid (4:1),shake by mechanical means for 30minutes,and centrifuge.Apply 20µLof this solution and 20µLof a Standard solution containing 5mg of USP Baclofen RSper mLin a mixture of dehydrated alcohol and glacial acetic acid (4:1)to a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel.Place the plate in a chromatographic chamber (see Chromatography á621ñ)containing a solvent system consisting of a mixture of butyl alcohol,glacial acetic acid,and water (4:1:1),and develop the chromatogram until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the chamber,and dry in a current of warm air.Spray with a detecting reagent consisting of 0.4g of ninhydrin in 95mLof butyl alcohol and 5mLof dilute glacial acetic acid (1in 10)until the plate is slightly wet.Place the plate in an oven maintained at 100for 10minutes:the RFvalue of the principal orange-red spot obtained from the solution from the Tablets corresponds to that obtained from the Standard solution.

B: 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.

Medium: 0.01Nhydrochloric acid;500mLfor Tablets containing 10mg or less of drug and 1000mLfor Tablets containing more than 10mg of drug.

Apparatus 2: 50rpm.

Time: 30minutes.

Mobile phase and Chromatographic system—Proceed as directed in the Assay.

Procedure— Inject an accurately measured volume (about 190µL)of a filtered portion of the solution under test into the chromatograph by means of a microsyringe or a sampling valve,record the chromatogram,and measure the response for the major peak.Calculate the quantity of C10H12ClNO2dissolved in comparison with a Standard solution having a known concentration of USP Baclofen RSin the same Mediumand similarly chromatographed.

Tolerances— Not less than 75%(Q)of the labeled amount of C10H12ClNO2is dissolved in 30minutes.

Diluting solution ,Mobile phase,and Chromatographic system—Proceed as directed in the Assay.

Standard preparation— Transfer about 10mg of USP Baclofen Related Compound A RS,accurately weighed,to a 10-mLvolumetric flask,dissolve in and dilute with methanol to volume,and mix.Transfer 4.0mLof this solution to a 25-mLvolumetric flask,dilute with Diluting solutionto volume,and mix.

Test preparation— Use the Assay preparation.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 10µL)of the Standard preparationand the Test preparationinto the chromatograph,and proceed as directed in the Procedureunder Assay.Calculate the percentage of baclofen related compound Ain the portion of Tablets taken by the formula: in which Cis the concentration of USP Baclofen Related Compound A RS,in mg per mL,in the Standard preparation;Wis the weight of baclofen in the Test preparation;and rUand rSare the peak responses due to baclofen related compound Ain the chromatograms of the Test preparationand the Standard preparation,respectively.Not more than 4.0%is found.

Diluting solution— Transfer 75mLof methanol and 10mLof glacial acetic acid to a 250-mLvolumetric flask.Dilute with water to volume,and mix.

Mobile phase— Prepare a filtered and degassed mixture of 0.3Nacetic acid,methanol,and 0.36Nsodium 1-pentanesulfonate (550:440:20).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Baclofen RSin Diluting solutionto obtain a solution having a known concentration of about 4mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 40mg of baclofen,to a 50-mLflask.Transfer 10.0mLof Diluting solutionto the flask,sonicate to disperse,and shake by mechanical means for 30minutes.Centrifuge a portion of this solution for 5minutes,and filter.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 265-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 0.6mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,and allow the Assay preparationto elute for not less than three times the retention time of baclofen.Record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C10H12ClNO2in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Baclofen RSin the Standard preparation;and rUand rSare the baclofen peak responses obtained from the Assay preparationand the Standard preparation,respectively.