Clonidine Transdermal System
724
Test 3:
If the product complies with this test, the labeling indicates that it meets USP Drug Release Test 3.
DEFINITION
Clonidine Transdermal System contains NLT 80.0% and NMT 120.0% of the labeled amount of clonidine (C9H9Cl2N3).
[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. ]
IDENTIFICATION
• A. Infrared Absorption 197K
197K
Buffer solution:
242.28 g/L of tris-(hydroxymethyl)aminomethane in water. Adjust with dilute hydrochloric acid to a pH of 9.2.
Sample:
Carefully peel the release liner from each Transdermal System, and place a number of Transdermal Systems equivalent to 25 mg of clonidine into a 50-mL screw-capped centrifuge tube. Add 5 mL of chloroform, and mix on a vortex mixer for 5 min. Allow to stand for 30 min, and mix intermittently on a vortex mixer. Transfer the chloroform solution to another 50-mL centrifuge tube, and wash the residue with an additional 3 mL of chloroform, combining the extracts. Add 2 mL of 0.5 N hydrochloric acid to the extract, mix on a vortex mixer for 1 min, and centrifuge at about 1000 rpm for 4 min. Remove and discard the bottom chloroform layer. Extract the aqueous layer with 4 mL of chloroform. Centrifuge at 1000 rpm for an additional 5 min, and again discard the bottom chloroform layer. Add 5 mL of Buffer solution and 3 mL of methylene chloride. Mix on a vortex mixer for 1 min. Centrifuge at 1000 rpm for 4 min. Transfer the bottom methylene chloride layer into a 100-mL beaker, and dry the methylene chloride with anhydrous sodium sulfate (about 1/4 liquid height). Decant, and evaporate to dryness with a stream of nitrogen. Dry at 105
for 30 min, and allow to cool in a desiccator.
for 30 min, and allow to cool in a desiccator.
Analysis:
Determine the IR spectrum of the Sample solution and USP Clonidine Hydrochloride RS in the wavelength region of 3500–600 cm
1.
1.
Acceptance criteria:
Meets the requirements
• B.
The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
ASSAY
• Procedure
Buffer solution:
2.5 mL of triethylamine in 1 L of water. Adjust with phosphoric acid to a pH of 3.0.
Mobile phase:
Acetonitrile and Buffer solution (60:40). [Note—Stir the solution for 30 min. ]
Diluent:
Tetrahydrofuran and methanol (1:1)
System suitability solution:
250 µg/mL of USP Clonidine RS and 10 µg/mL of USP Clonidine Related Compound B RS in Diluent
Standard stock solution:
1 mg/mL of USP Clonidine RS in tetrahydrofuran
Standard solutions:
Prepare a minimum of four Standard solutions from the Standard stock solution in Diluent that bracket the expected clonidine concentration in the sample. The standard concentrations should be within the range of 50–300 µg/mL. [Note—The Standard solutions are stable for up to 2 days if stored at 4. ]
. ]
Sample solution:
357 µg/mL of clonidine prepared as follows. 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 Table 1.
Table 1
| For systems containing about 2.5 mg of clonidine | 7.0 mL |
| For systems containing about 5.0 mg of clonidine | 14.0 mL |
| For systems containing about 7.5 mg of clonidine | 21.0 mL |
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 min, and mix on a vortex mixer until the systems are completely delaminated. Allow the systems to remain submerged for an additional 60 min, mixing on a vortex mixer every 30 min. 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 min at 2000 rpm. Use the supernatant as the Sample solution.
Chromatographic system
Mode:
LC
Detector:
UV 210 and 242 nm
[Note—The detector is programmed initially to 242 nm and switched to 210 nm after the elution of the clonidine peak but before the elution of the clonidine related compound B peak. ]
Column:
4.6-mm × 15-cm; packing L10
Flow rate:
1 mL/min
Injection size:
25 µL
System suitability
Sample:
System suitability solution
[Note—The relative retention times for clonidine and clonidine related compound B are 1.0 and 1.5, respectively. ]
Suitability requirements
Resolution:
NLT 2.0 between clonidine and clonidine related compound B
Capacity factor (k¢):
NLT 0.6 for clonidine
Tailing factor:
NMT 3.0 for both clonidine and clonidine related compound B
Relative standard deviation:
NMT 2.0% for the clonidine peak area
Analysis
Samples:
At least three Standard solutions that will bracket the expected sample concentration range and the Sample solution
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 NLT 0.995.
Calculate the percentage of the labeled amount of clonidine (C9H9Cl2N3) in the Transdermal System taken:
Result = (CS/CU) × 100
| CS | = | = concentration of clonidine from the linear regression analysis (µg/mL) |
| CU | = | = nominal concentration of clonidine in the Sample solution (µg/mL) |
Acceptance criteria:
80.0%–120.0%
PERFORMANCE TESTS
Change to read:
• Drug Release 724
Test 1
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 h
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, so that the backing of the system is adjacent to and centered on the bottom of the sample holder. Trim the excess cellulose membrane with scissors. Suspend each sample holder from the arm of a reciprocating shaker so 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 before immersing the test sample. Agitate the sample in an up-down motion at a frequency of 30 cycles/min 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.
before immersing the test sample. Agitate the sample in an up-down motion at a frequency of 30 cycles/min 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:
0.1% solution of triethylamine in a mixture of methanol and water (30:70). Adjust with phosphoric acid to a pH of 6.0 ± 0.2.
System suitability solution:
10 µg/mL of USP Clonidine RS in 0.001 M phosphoric acid
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 Sample solutions.
Sample 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, then mix.
Chromatographic system
Mode:
LC
Detector:
UV 220 nm
Column:
4.6-mm × 15-cm; packing L1
Flow rate:
1.5 mL/min
Injection size:
25 µL
System suitability
Sample:
System suitability solution
Suitability requirements
Column efficiency:
NLT 2000 theoretical plates
Tailing factor:
NMT 2.0
Capacity factor (k¢):
NLT 0.5
Relative standard deviation:
NMT 2.0%
Analysis
Samples:
Standard solutions and Sample solutions
Construct a standard curve of concentration (µg/mL) of clonidine in the Standard solutions versus peak area by linear regression analysis. The correlation coefficient is NLT 0.995.
Calculate the release rate of clonidine:
Result = CV/TA
| C | = | = concentration of clonidine in the sample of the standard curve (µg/mL) |
| V | = | = volume of the Medium (mL) |
| T | = | = time (h) |
| A | = | = area of the Transdermal System (cm2) |
Tolerances:
See Table 2.
Table 2
| Time (h) |
Time for Sampling (h) |
Release Rate (µg/h/cm2) |
|---|---|---|
| 0–8 | 8 | 7.5–16.0 |
| 8–24 | 24 | 1.5–4.6 |
| 24–96 | 96 | 1.5–4.6 |
| 96–168 | 168 | 1.5–3.3 |
The release rate of clonidine (C9H9Cl2N3) from the Transdermal System, expressed as µg/h/cm2 at the times specified, conforms to Acceptance Table 1 in 724.
724.
Test 2:
If the product complies with this test, the labeling indicates that it meets USP Drug Release Test 2.
Medium:
0.01 N hydrochloric acid; 500 mL for systems labeled as 0.1 mg/day, 900 mL for systems labeled as 0.2 or 0.3 mg/day
Apparatus 6:
100 rpm. Apply double-sided tape around the lower-most circumference of the cylinder, overlapping the ends to prevent peeling of the tape end from the cylinder. Remove the outer layer of the tape. Attach the Transdermal System to the cylinder with the backing side against the double-sided tape and the longitudinal axis parallel to the bottom of the cylinder. Carefully smooth the system to remove any air bubbles, and remove the release liner from the system. For systems requiring 500 mL of Medium, apply the double-sided tape to the system such that the bottom edge of each is NMT 2 mm from the bottom of the cylinder to prevent evaporation during the test from exposure to air. After setting the cylinder in the vessel, cover the vessel to minimize evaporation.
Times:
6, 48, 96, and 168 h
Buffer:
0.3% triethylamine in 0.025 M monobasic potassium phosphate. Adjust with phosphoric acid to a pH of 6.20 ± 0.10.
Mobile phase:
Buffer and tetrahydrofuran (94:6)
Standard solutions:
Solutions containing 0.7, 3.0, 5.3, 7.5, and 9.8 µg/mL of USP Clonidine RS in Medium. A small amount of methanol (not exceeding 10% of the final volume) can be used to solubilize clonidine.
Sample solution:
1.5 mL aliquots of the solution under test. After sampling the last time point, measure the volume of Medium remaining in the vessel.
Chromatographic system
Mode:
LC
Detector:
UV 210 nm
Columns
Guard:
3.0-mm × 4-mm; packing L1
Analytical:
4.6-mm × 15-cm; packing L1
Flow rate:
1.0 mL/min
Injection size:
50 µL
System suitability
Sample:
5.3 µg/mL of the Standard solution
Suitability requirements
Tailing factor:
NMT 2.0
Relative standard deviation:
NMT 3.0%
Analysis
Samples:
Standard solutions and Sample solutions
Construct a standard curve of concentration (µg/mL) of clonidine in the Standard solutions versus peak area by linear regression analysis. The correlation coefficient is NLT 0.997. Calculate the release rate of clonidine.
Calculate the volume loss rate in mL/h (L):
L = [V F + (N × 1.5)]/T
F + (N × 1.5)]/T
| V | = | = initial volume of Medium (mL) |
| F | = | = final volume of Medium (mL) |
| N | = | = number of sampling time points |
| T | = | = total elapsed time between start of run and final volume measurement (h) |
Calculate the volume (mL) at each sampling time adjusted for evaporation (Vadj):
Vadj = V (L × tC) [(n 1) × 1.5]
(L × tC) [(n 1) × 1.5]| tC | = | = cumulative time for the sample withdrawal (6, 48, 96, or 168 h) |
| n | = | = sampling number (1, 2, 3, or 4 for the 6-, 48-, 96-, and 168-h sampling times, respectively) |
Calculate the release rate of clonidine (µg/h/cm2):
Result = [(rU b) × Vadj]/(m × A × ti)
b) × Vadj]/(m × A × ti)| rU | = | = peak response from the Sample solution |
| b | = | = y-intercept of the standard curve |
| m | = | = slope of the standard curve |
| A | = | = area of the system (cm2) |
| ti | = | = interval time (h) |
Tolerances:
See Table 3.
Table 3
| Time (h) |
Time for Sampling (h) |
Interval Time (h) |
Release Rate (µg/h/cm2) |
|---|---|---|---|
| 0–6 | 6 | 6 | 7.6–12.0 |
| 6–48 | 48 | 42 | 1.7–2.5 |
| 48–96 | 96 | 48 | 2.0–2.9 |
| 96–168 | 168 | 72 | 1.7–2.6 |
The release rate of clonidine (C9H9Cl2N3) from the Transdermal System, expressed as µg/h/cm2 at the times specified, conforms to Acceptance Table 1 in 724.
724.Test 3:
If the product complies with this test, the labeling indicates that it meets USP Drug Release Test 3.
Medium:
100 mM acetate buffer, pH 5.0, with 0.01% of cetyltrimethylammonium bromide (13.6 g/L of sodium acetate monohydrate in water, adjust with glacial acetic acid to a pH of 5.0, and add 0.1 g/L of cetyltrimethylammonium bromide); 900 mL
Apparatus 5:
100 rpm, with the 76-mm disk
Times:
8, 24, 96, and 168 h
Solution A:
2.4 g/L of octanesulfonic acid sodium salt and 2 mL/L of phosphoric acid in water
Mobile phase:
Methanol and Solution A (45:55). Adjust with 10 N sodium hydroxide to a pH of 3.0.
Standard stock solution:
1 mg/mL of USP Clonidine RS in methanol
Standard solution:
Dilute the Standard stock solution with Medium to obtain a final concentration similar to the expected concentration in the Sample solution, considering complete drug release.
Sample solution:
Apply double-sided adhesive tape to the stainless steel disk to cover enough of the disk area so that the entire patch is secured by the tape. Apply a Transdermal System with the release liner intact to the adhesive layer on the stainless steel disk. Press the backing film of the patch to the adhesive tape with the clear release liner film of the system facing up. Peel the release liner from the affixed system on the disk assembly, and place the disk assembly flat on the bottom of the vessel with the exposed transdermal adhesive side up and parallel to the bottom edge of the paddle blade. Lower the paddle, and start the equipment. At each sampling time withdraw an appropriate volume of the solution under test.
Chromatographic system
Mode:
LC
Detector:
UV 220 nm
Column:
4.6-mm × 15-cm; packing L7
Column temperature:
30
Flow rate:
1.5 mL/min
Injection size:
30 µL
System suitability
Sample:
Standard solution
Suitability requirements
Tailing factor:
NMT 1.8
Relative standard deviation:
NMT 2.0%
Analysis:
Samples:
Standard solution and Sample solution
Calculate the concentration (Ci) of clonidine (C9H9Cl2N3) in the Medium (mg/mL) at each time point:
Ci = (rU/rS) × CS
| rU | = | = peak response from the Sample solution |
| rS | = | = peak response from the Standard solution |
| CS | = | = concentration of the Standard solution (mg/mL) |
| i | = | = interval, where i = 1 at 8 h, i = 2 at 24 h, i = 3 at 96 h, i = 4 at 168 h |
Calculate the rate of clonidine (C9H9Cl2N3) released in µg/h/cm2 at each time point:
Result = [(Ci Ci1) × Vi × 1000]/[S × (Ti Ti1)]
Ci1) × Vi × 1000]/[S × (Ti Ti1)]
Vi = V0 [(i 1) × VA]
[(i 1) × VA]| Vi | = | = volume of Medium at a given time point |
| V0 | = | = initial volume of Medium, 900 mL |
| VA | = | = volume of Medium withdrawn at each time point |
| 1000 | = | = conversion factor from mg to µg |
| S | = | = system size in cm2 |
| Ti | = | = current time point |
| Ti-1 | = | = previous time point |
Tolerances:
See Table 4.
Table 4
| Time (h) |
Release Rate (µg/h/cm2) |
|---|---|
| 8 | 6.5–11.0 |
| 24 | 2.5–5.5 |
| 96 | 2.5–5.0 |
| 168 | 2.0–3.8 |
The release rate of clonidine (C9H9Cl2N3) from the Transdermal System, expressed as µg/h/cm2 at the times specified, conforms to Acceptance Table 1 in 724.(RB 1-Jul-2011)
724.(RB 1-Jul-2011)
• Uniformity of Dosage Units 905:
Meets the requirements
905:
Meets the requirements
IMPURITIES
• Organic Impurities
Mobile phase, Diluent, System suitability solution, Sample solution, and Chromatographic system:
Proceed as directed in the Assay.
Standard stock solution:
1 mg/mL of USP Clonidine Related Compound B RS in tetrahydrofuran
Standard solutions:
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 0.2–10.0 µg/mL.
[Note—The Standard solutions are stable for up to 2 days if stored at 4. ]
. ]
Analysis
Samples:
At least three Standard solutions that will bracket the expected sample concentration range and the Sample solution
Measure the responses for 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 NLT 0.995. Determine the concentration of clonidine related compound B.
Calculate the amount, in µg/cm2, of clonidine related compound B in the portion of the Transdermal System taken:
Result = CV/A
| C | = | = concentration of clonidine related compound B from the linear regression analysis (µg/mL) |
| V | = | = volume of the Sample solution (mL) |
| A | = | = area of the sample system (cm2) |
Acceptance criteria:
NMT 10.0 µg/cm2
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in sealed, single-dose containers at a temperature not exceeding 30.
.
• Labeling:
The label states the total amount of clonidine in the Transdermal System and the release rate, in mg/day, for the duration of the application of one system. When more than one Drug Release test is given, the labeling states the Drug Release test used only if Test 1 is not used.
• USP Reference Standards 11
11
USP Clonidine RS 
') + '&img=../../images/v35300/cas-4205-90-7.gif',600,500);)
USP Clonidine Related Compound B RS
2-[(E)-2,6-Dichlorophenylimino]-1-(1-{2-[(E)-2,6-dichlorophenylimino]-imidazolidin-1-yl}-ethyl) imidazolidine.
C20H20Cl4N6 486.23
2-[(E)-2,6-Dichlorophenylimino]-1-(1-{2-[(E)-2,6-dichlorophenylimino]-imidazolidin-1-yl}-ethyl) imidazolidine.
C20H20Cl4N6 486.23
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Sujatha Ramakrishna, Ph.D.
Senior Scientific Liaison 1-301-816-8349 |
(SM22010) Monographs - Small Molecules 2 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 2730
Pharmacopeial Forum: Volume No. 31(1) Page 146