NOTE—Throughout the following procedures avoid the use of tetrahydrofuran stabilized with butylated hydroxytoluene (BHT).In the presence of peroxides,BHTmay react with clonidine,producing impurity peaks.

A:Infrared Absorption á197Kñ.

B: The retention time of the major peak in the chromatogram of theAssay preparation corresponds to that in the chromatogram of theStandard preparation,as obtained in theAssay.

Medium: 0.001Mphosphoric acid;80mLfor systems containing 5mg or less of clonidine;200mLfor systems containing more than 5mg of clonidine.

Time: 8,24,96,and 168hours.

Apparatus 7— Proceed as directed in the chapter,using the transdermal system holder-angled disk (see Figure 7a).The appropriate size of the holder,1.42or 1.98inches,should be chosen based on the size of the system to prevent overhang.Use 100-mLbeakers forMedium volumes of 80-mLand 300-mLbeakers forMedium volumes of 200mL.Gently press the transdermal system to a dry,smooth,square piece of cellulose membrane*,or equivalent,with the adhesive side against the membrane.Attach the membrane/system to a suitable inert sample holder with a Viton O-ring,or equivalent,such that the backing of the system is adjacent to,and centered on,the bottom of the sample holder.Trim the excess of cellulose membrane with scissors.Suspend each sample holder from the arm of a reciprocating shaker such that each system is continuously immersed in a beaker containing the specified volume ofMedium.The filled beakers are weighed and pre-equilibrated to 32.0±0.3prior to immersing the test sample.Agitate the sample in an up-down motion at a frequency of 30cycles per minute with an amplitude of 2.0±0.1cm.TheMediummust be added daily to the beakers during each interval to maintain sample immersion.At the end of each time interval,transfer the test sample to a fresh beaker containing the appropriate volume of Medium,weighed and pre-equilibrated to 32.0±0.3.

Mobile phase— Use a filtered and degassed 0.1%solution of triethylamine in a mixture of water and methanol (70:30),adjust with phosphoric acid to a pHof 6.0±0.2.Make adjustments if necessary (seeSystem Suitability under Chromatography á621ñ).

System suitability solution— Prepare a solution of USP Clonidine Hydrochloride RSin 0.001Mphosphoric acid having a known concentration of about 10µg per mL.

Standard solutions— Prepare a minimum of four standard solutions of USP Clonidine RSin 0.001Mphosphoric acid having known concentrations of clonidine similar to those of theTest solutions.

Test solutions— At the end of each release interval,allow the beakers to cool to room temperature and make up for evaporativeMedium losses by addingMedium to obtain the original weight.Mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm ×15-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph theSystem suitability solution,and record the peak responses as directed forProcedure:the tailing factor is not more than 2.0;the capacity factor is not less than 0.5;the column efficiency is not less than 2000theoretical plates;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)of filtered portions of theStandard solution and theTest solution into the chromatograph,record the chromatograms,and measure the responses for the major peaks.Construct a standard curve of concentration (µg per mL)of clonidine in theStandard solutions versus peak area by linear regression analysis.The correlation coefficient is not less than 0.995.Calculate the release rate of clonidine by the formula: in whichCis the concentration,in µg per mL,of clonidine in the sample obtained from the standard curve;Vis the volume,in mL,of theMedium;Tis the time,in hours;andAis the area,in cm2,of the transdermal system.

Tolerances— The release rate of C9H9Cl2N3from the Transdermal System,expressed as µg per hour per cm2at the times specified,conforms to Acceptance Table 4.

Mobile phase,Diluent,System suitability solution,and Chromatographic system— Proceed as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Clonidine Related Compound B RSin tetrahydrofuran,and dilute quantitatively,and stepwise if necessary,to obtain a solution having a known concentration of about 1mg per mL.Prepare a minimum of four Standard solutionsin Diluent that bracket the expected clonidine related compound Bconcentration in the sample.The standard concentrations should be within the range of 0.2to 10.0µg per mL.[NOTE—Standard solutionsare stable for up to 2days if stored at 4.]

Test solution— Use the Assay preparation,prepared as directed in the Assay.

Procedure— Separately inject equal volumes (about 25µL)of at least three Standard solutions that will bracket the expected sample concentration range and the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the clonidine related compound B.Calculate the peak response ratios of the analyte,and plot the results.Determine the linear regression equation of the standards by the mean-square method,and record the linear regression equation and the correlation coefficient;it should be not less than 0.995.Determine the concentration of the clonidine related compound B.Calculate the amount,in µg per cm2,of the clonidine related compound Bin the portion of the Transdermal System taken by the formula: in which Cis the concentration of clonidine related compound B,in µg per mL,obtained from the linear regression analysis;Vis the volume of the Test solution in mL;and Ais the area of the sample system in cm2.Not more than 10.0µg per cm2of clonidine related compound Bis found.

Mobile phase— Dissolve 4mLof triethylamine in 1.6Lof water,and adjust with phosphoric acid to a pHof 3.0.Add 2.4Lof acetonitrile,stir the solution for 30minutes,filter,and degas.Make adjustments if necessary (see System Suitability under Chromatography á621ñ).

Diluent— Prepare a mixture of tetrahydrofuran and methanol (1:1).

System suitability solution— Dissolve an accurately weighed quantity of USP Clonidine RSand USP Clonidine Related Compound B RSin Diluent to obtain a solution having known concentrations of about 250µg per mLand 10µg per mL,respectively.

Standard preparation— Dissolve a suitable amount,accurately weighed,of USP Clonidine RSin tetrahydrofuran to obtain a solution having a known concentration of about 1mg per mL.Prepare a minimum of four Standard preparationsin Diluentthat bracket the expected clonidine concentration in the sample.The standard concentrations should be within the range of 50to 300µg per mL.[NOTE—Standard preparationsare stable for up to 2days if stored at 4.]

Assay preparation— Remove each Transdermal System from its package,discard the release liner from each system,and transfer into a 50-mLcentrifuge tube with a Teflon-lined screw cap.Add the appropriate volume of tetrahydrofuran as listed in the table below. Mix vigorously on a vortex mixer until the systems are washed down and fully submerged in the tetrahydrofuran.Let the systems soak in tetrahydrofuran for about 5minutes,and mix on a vortex mixer until the systems are completely delaminated.Allow the systems to remain submerged for an additional 60minutes,mixing on a vortex mixer every 30minutes.Add methanol in a volume equal to the volume of tetrahydrofuran,and mix vigorously on a vortex mixer.The solution turns milky.Centrifuge for 10minutes at 2000rpm.Use the supernatant as the Assay preparation.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a detector capable of measuring at 210nm and 242nm,and a 4.6-mm ×15-cm column that contains packing L10.The flow rate is about 1mLper minute.The detector is programmed initially to 242nm and 210nm after the elution of the clonidine peak but prior to the elution of the clonidine related compound B.The relative retention time is 1.0for clonidine and 1.5for clonidine related compound B.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the resolution,R,between clonidine and clonidine related compound Bis not less than 2.0;the capacity factor,k ¢,is not less than 0.6for clonidine;the tailing factor for both clonidine and clonidine related compound Bis not more than 3.0;and the relative standard deviation of the clonidine peak area for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)of at least three Standard solutionsthat will bracket the expected sample concentration range and the Assay preparation into the chromatograph,record the chromatograms,and measure the responses for the clonidine.Calculate the peak response ratios of the analyte,and plot the results.Determine the linear regression equation of the standards by the mean-square method,and record the linear regression equation and the correlation coefficient;it should be not less than 0.995.Calculate the amount,in mg,of C9H9Cl2N3in the Transdermal System taken by the formula: in which Cis the concentration of clonidine,in µg per mL,obtained from the linear regression analysis;and Vis the volume of the Assay preparation in mLper sample system.USP28