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

A: Infrared Absorption 197K.

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

Medium: 0.001 M phosphoric acid; 80 mL for systems containing 5 mg or less of clonidine; 200 mL for systems containing more than 5 mg of clonidine.

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

Apparatus 7— Proceed as directed in the chapter, using the transdermal system holder-angled disk (see Figure 4a). The appropriate size of the holder, 1.42 or 1.98 inches, should be chosen based on the size of the system to prevent overhang. Use 100-mL beakers for Medium volumes of 80-mL and 300-mL beakers for Medium volumes of 200 mL. 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 of Medium. The filled beakers are weighed and pre-equilibrated to 32.0 ± 0.3 prior to immersing the test sample. Agitate the sample in an up-down motion at a frequency of 30 cycles per minute with an amplitude of 2.0 ± 0.1 cm. The Medium must 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 pH of 6.0 ± 0.2. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Prepare a solution of USP Clonidine RS in 0.001 M phosphoric acid having a known concentration of about 10 µg per mL.

Standard solutions— Prepare a minimum of four standard solutions of USP Clonidine RS in 0.001 M phosphoric acid having known concentrations of clonidine similar to those of the Test solutions.

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

Chromatographic system (see Chromatography 621)— 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 1.5 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: 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 2000 theoretical plates; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25 µL) of filtered portions of each Standard solution and Test 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 the Standard 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 which C is the concentration, in µg per mL, of clonidine in the sample obtained from the standard curve; V is the volume, in mL, of the Medium; T is the time, in hours; and A is the area, in cm2, of the transdermal system.

Tolerances— The release rate of C9H9Cl2N3 from the Transdermal System, expressed as µg per hour per cm2 at the times specified, conforms to Acceptance Table 1 under Drug Release 724.

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 RS in tetrahydrofuran, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 1 mg per mL. Prepare a minimum of four Standard solutions in Diluent that bracket the expected clonidine related compound B concentration in the sample. The standard concentrations should be within the range of 0.2 to 10.0 µg per mL. [note—Standard solutions are stable for up to 2 days if stored at 4.]

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

Procedure— Separately inject equal volumes (about 25 µL) of at least three Standard solutions that will bracket the expected sample concentration range and the Test solution into 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 B in the portion of the Transdermal System taken by the formula: in which C is the concentration of clonidine related compound B, in µg per mL, obtained from the linear regression analysis; V is the volume of the Test solution in mL; and A is the area of the sample system in cm2. Not more than 10.0 µg per cm2 of clonidine related compound B is found.

Mobile phase— Dissolve 4 mL of triethylamine in 1.6 L of water, and adjust with phosphoric acid to a pH of 3.0. Add 2.4 L of acetonitrile, stir the solution for 30 minutes, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

Standard preparation— Dissolve a suitable amount, accurately weighed, of USP Clonidine RS in tetrahydrofuran to obtain a solution having a known concentration of about 1 mg per mL. Prepare a minimum of four Standard preparations in Diluent that bracket the expected clonidine concentration in the sample. The standard concentrations should be within the range of 50 to 300 µg per mL. [note—Standard preparations are stable for up to 2 days 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-mL centrifuge 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 5 minutes, and mix on a vortex mixer until the systems are completely delaminated. Allow the systems to remain submerged for an additional 60 minutes, mixing on a vortex mixer every 30 minutes. Add methanol in a volume equal to the volume of tetrahydrofuran, and mix vigorously on a vortex mixer. The solution turns milky. Centrifuge for 10 minutes at 2000 rpm. Use the supernatant as the Assay preparation.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a detector capable of measuring at 210 nm and 242 nm, and a 4.6-mm × 15-cm column that contains packing L10. The flow rate is about 1 mL per minute. The detector is programmed initially to 242 nm and 210 nm after the elution of the clonidine peak but prior to the elution of the clonidine related compound B. The relative retention time is 1.0 for clonidine and 1.5 for 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 B is not less than 2.0; the capacity factor, k ¢, is not less than 0.6 for clonidine; the tailing factor for both clonidine and clonidine related compound B is 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 25 µL) of at least three Standard solutions that 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 C9H9Cl2N3 in the Transdermal System taken by the formula: in which C is the concentration of clonidine, in µg per mL, obtained from the linear regression analysis; and V is the volume of the Assay preparation in mL per sample system.