Methods for the Analysis of Clobetasol Propionate Gel

To initiate the standard development process and engage stakeholders early, USP is piloting a new, agile approach for developing and sharing information with stakeholders. USP’s science departments and laboratories are collaborating to develop and validate methods for the testing of drug substances, drug products, and excipients. Through this iterative process, USP introduces Emerging Standards – potential standards in early development – to stimulate dialogue and participation, which may contribute to these documents potentially achieving official status in the future by going through USP’s standards-setting processes.  

Clobetasol propionate gel has been evaluated and shown to be a suitable candidate for development under this new program. The methods in this document are being published to help analyze the drug product. Additional method development and validation information is provided to justify the use of method parameters.  

This document may identify certain commercial software, instruments, or materials to adequately specify the experimental procedure. Such identification does not imply USP approval, endorsement, or certification of a particular brand or product, nor does it imply that the software, instrument, or material is necessarily the best available for the purpose or that any other brand or product was judged to be unsatisfactory or inadequate.

This document is not a USP compendial standard and is intended to serve as a resource for informational purposes only. It does not reflect USP or USP’s Expert Body opinions of future revisions to the official text of the USP–NF. Parties relying on the information in this document bear independent responsibility for awareness of and compliance with any applicable federal, state, or local laws and requirements.

Clobetasol propionate is used to treat the itching, redness, dryness, crusting, scaling, inflammation, and discomfort of various scalp and skin conditions, including psoriasis and eczema.¹ 

USP–NF contains monographs for clobetasol propionate,² clobetasol propionate topical solution,³ clobetasol propionate cream,⁴ and clobetasol propionate ointment.⁵ Clobetasol propionate is the active ingredient in clobetasol propionate gel. However, there is no monograph for clobetasol propionate gel. USP received submissions for the clobetasol propionate family of monographs including impurity profiles and specifications from several manufacturers. As part of the Emerging Standards initiative, USP developed and validated a method for clobetasol propionate gel.

The recently published USP General Chapter <1220> Analytical Procedure Life Cycle⁶ provides a framework for the implementation of the analytical procedure life cycle approach consistent with the quality by design (QbD) concepts described in International Council for Harmonization (ICH) guidelines. Stage 1 of the life cycle includes systematic procedure development experiments that result in an understanding of the effect of procedure parameters on the procedure performance. Design of experiment (DOE), which includes statistical multivariate analysis and modeling, is an important tool in Stage 1. The knowledge acquired through DOE studies also enables the determination of a robust method operable design region (MODR) for procedure parameters.

This document summarizes method development efforts assisted by the Fusion QbD^® software, as well as robustness and forced degradation study results. It also describes final procedures that may be suitable for identification, strength, and purity determination of clobetasol propionate in the presence of various impurities and excipients in clobetasol propionate gel. A summary of validation data and representative chromatographic results are included. 

3.1. Clobetasol Propionate and Impurities

USP Reference Standards for clobetasol propionate, clobetasol propionate related compound A, betamethasone, betamethasone dipropionate related compound B, and betamethasone dipropionate related compound C were used. The other impurities were procured from commercial sources. The chemical structures of clobetasol propionate and related impurities are shown in Figures 1 and 2.

Figure 1. Clobetasol propionate (CloPro)

Figure 2a. Betamethasone dipropionate related compound B (IMP A) 

Figure 2b. IMP B

Figure 2c. IMP C

Figure 2d. IMP D

Figure 2e. IMP E

Figure 2f. IMP F

Figure 2g. IMP G

Figure 2h. IMP H

Figure 2i. IMP I

Figure 2j. Clobetasol propionate related compound A (IMP J)

Figure 2k. Betamethasone dipropionate related compound C (IMP K)

Figure 2l. Betamethasone (Beta)

Figure 2m. Clobetasone 17-propionate (Cl17)

Figure 2n. Epoxybetamethasone (Epox)

Figure 2o. Desmethyl clobetasone 17-propionate (Desm)

Figure 2. Impurities

3.2. Samples

Clobetasol propionate gel samples (0.05%) from three commercial sources were used to evaluate methods described in this document.

3.3. Reagents

Formic acid (ACS grade) and acetonitrile (HPLC grade) were obtained from Fisher Chemical. Ultrapure water was obtained from a Milli-Q water purification system.

The goal was to develop stability-indicating assay and organic impurities (OI) methods for known degradation products and other potential degradants in clobetasol propionate gel. The Phenomenex Kinetex XB-C18 column (150 × 4.6 mm, 2.6-µm) was evaluated for method development. Initially, mobile phases of 0.1% phosphoric acid and acetonitrile at different ratios were tested. Based on the ultraviolet (UV) spectra, 240 nm was set as the detection wavelength. When the mobile phase was delivered in a gradient mode (scouting gradient from 5% to 95% of acetonitrile within 0 – 9 minutes at 0.9 mL/min flow rate), most of the 16 peaks eluted at the middle of the gradient. During another set of studies, isocratic mobile phases of 0.1% phosphoric acid and acetonitrile (50:50) showed potential capability to separate all 16 peaks. Most of the peaks were separated with a resolution of not less than (NLT) 1.5. However, the resolution between IMP I and Desm was less than 1.5, and IMP E eluted after the isocratic stage. Fusion QbD® software was used for the DOE in an optimization study to explore the MODR and to select chromatographic conditions.

4.1. HPLC Conditions Optimization

The following HPLC conditions were maintained constant in the optimization study: aqueous moiety of the mobile phase (Solution A: 0.1% phosphoric acid in water), detection wavelength (240 nm), and column brand/type (Phenomenex Kinetex XB-C18). Injection volume was selected at 10 µL.

A method with isocratic elution mode was targeted. The effect of four method variables — % strong solvent (Solution B), acetonitrile to methanol ratio in Solution B, column temperature, and flow rate — on resolution between all peaks of interest was studied (Table 1). The software generated 30 runs for the design, with randomization and replicates considered. The method development solution consisted of about 0.1 mg/mL of clobetasol propionate and 1 µg/mL each of IMP A, IMP B, IMP C, IMP D, IMP E, IMP F, IMP G, IMP H, IMP I, IMP J, IMP K, Beta, Epox, Cl17 and Desm in methanol and water (1:1).

Table 1. LC Conditions Optimization Design

The chromatographic results were imported back into the software for data modeling, analysis, and visualization to guide the selection of method conditions that provide the best selectivity and robustness. An in-silico robustness study was performed using theoretical Monte Carlo simulations based on the DOE-derived models. The white areas in Figure 3 represent the MODR within which the method is predicted to be robust by meeting all response goals, including peak resolution of NLT 2.0 between clobetasol propionate and its adjacent peaks, and NLT 1.5 between the other peaks. The areas within the rectangles (strong solvent: 51.5% – 52.5%, acetonitrile to methanol ratio: 100:0, column temperature: 35 – 40°, and flow rate: 0.85 – 0.95 mL/min) include method conditions that can each be independently adjusted while still meeting response goals according to the model prediction. The proposed final method was chosen at 52% Solution B (100% acetonitrile), 40° column temperature, and 0.9 mL/min flow rate.

As 0.01% formic acid as Solution A provided the same chromatographic profile as 0.1% phosphoric acid, 0.01% formic acid was used to replace 0.1% phosphoric acid due to its mass spectrometry compatibility. Injection volume was increased from 10 µL to 15 µL to accommodate the sensitivity of all peaks of interest. A representative chromatogram consisting of the 16 peaks of interest is shown in Figure 4.

Figure 3a. MODR with original parameters

Figure 3b. MODR with extrapolated parameters for Oven Temperature and % Strong Solvent

Figure 3. Potential MODR represented by the unshaded areas

Figure 4. Representative chromatogram consisting of 16 peaks of interest

4.2. Robustness

The method development solution was analyzed under the proposed and deliberately changed HPLC conditions. The changes included percent of acetonitrile ± 2% absolute (50% and 54%), column temperature ± 3° (37° and 43°), flow rate ± 10% (0.8 mL/min and 1.0 mL/min), formic acid concentration in Solution A (0.1% and 0.005%), and different column lot. Resolution criteria of NLT 2.0 between clobetasol propionate and its adjacent peaks and NLT 1.0 between any pair of impurities, and peak tailing criteria of 0.8 — 1.8 for the clobetasol propionate peak were met across all evaluated conditions.

4.3. Forced Degradation

Forced degradation studies were performed by exposing the solution prepared from USP Clobetasol Propionate Reference Standard (RS) to acid, base, and oxidation; and by exposing the RS to heat, heat/humidity, and light (Table 2). The stressed samples were analyzed against freshly prepared Control solution. The chromatograms were processed at 240 nm to detect the degradation products.

Table 2. Forced Degradation Results

Known impurities IMP G and IMP J were formed under base stress condition, while IMP H was formed under light. No unknown degradant peaks ≥1.0% total area were observed under any stress condition. Photodiode array (PDA) purity analysis (210 ‒ 400 nm) and mass spectrometry analysis showed spectral homogeneity of the clobetasol propionate peaks from the Control solution and all stressed solutions, indicating a lack of coelution. 

4.4. Chromatography Conditions 

• Solution A: 0.01% formic acid in water
• Solution B: Acetonitrile
• Mobile phase: Isocratic
  • Solution A and Solution B (48:52)
• Chromatographic system
  • Mode: LC
  • Detector: UV 240 nm
  • Column: Phenomenex Kinetex XB, 4.6-mm × 15-cm, 2.6-μm, USP packing L1
  • Column temperature: 40°
  • Flow rate: 0.9 mL/min
  • Sample compartment temperature: Ambient
  • Injection volume: 15 µL
  • Run time: 20 min

Identification (ID) of clobetasol propionate in clobetasol propionate gel was evaluated using the final HPLC conditions (Section 4.4), with PDA spectral match and retention time match. See the Assay Validation (Section 6) for solution preparations.

5.1. PDA Spectral Match

The ID procedure, solutions, and results are summarized in Table 3, and representative UV spectra of clobetasol propionate peak from the Standard solution and Sample solutions are shown in Figures 5 and 6, respectively.

Table 3. Summary of Procedure, Solutions, and Results for the Identification Test by PDA Spectral Match

Figure 5. UV spectrum of clobetasol propionate from the Standard solution

Figure 6. UV spectrum of clobetasol propionate from a representative Sample solution 

5.2. Retention Time Match

The ID test, solutions, and results are summarized in Table 4, and representative chromatograms of the Standard and Sample solutions are shown in Figures 7 and 8, respectively. 

Table 4. Summary of Procedure, Solutions, and Results for the Identification Test by Retention Time Match

Figure 7.Chromatogram of Standard solution using the HPLC Assay procedure

Figure 8. Chromatogram of a representative Sample solution using the HPLC Assay procedure

The final HPLC method (Section 4.4) was validated for the assay of clobetasol propionate gel following the USP General Chapter <1225> Validation of Compendial Procedures.⁷ The assay procedure was found to be specific, linear, accurate, precise, and rugged for the samples evaluated.

6.1. Solutions

• Diluent: Acetonitrile and water (4:1, v/v)
• System Suitability solution: 0.1 mg/mL of USP Clobetasol Propionate RS and 10 µg/mL of USP Clobetasol Propionate Related Compound A RS (IMP J) in Diluent
• Standard solution: 0.1 mg/mL of USP Clobetasol Propionate RS in Diluent
• Sample solution: nominally 0.1 mg/mL of clobetasol propionate from gel sample
  • Accurately weigh 1 g of clobetasol propionate gel sample into a 5-mL volumetric flask, add 2 mL of acetonitrile and sonicate for 10 min. Dilute to volume with acetonitrile. Mix well. Centrifuge the solution at NLT 3750 rpm for 15 minutes and use the supernatant for analysis.

6.2. System Suitability Parameters, Validation Performance Characteristics, and Results

The system suitability parameters and results are summarized in Table 5. The validation performance characteristics and results are summarized in Table 6. Representative chromatograms of the Standard solution and Sample solution are shown in Figures 7 and 8, respectively.

Table 5. Summary of System Suitability Parameters, Solutions, and Results for the Assay

Table 6. Summary of Validation Performance Characteristics, Solutions, and Results for the Assay

The HPLC method (Section 4.4) was validated for the OI, except that the detection of the peaks was at UV 240 nm without a need for PDA spectra. The validation was performed following the USP General Chapter <1225>.⁷ The OI procedure was found to be specific, linear, accurate, precise, and rugged for the samples evaluated.

7.1. Solutions

• Sensitivity solution: 0.1 µg/mL each of USP Clobetasol Propionate RS, IMP G, IMP H and IMP J in Diluent.
• Standard Solution: 0.2 µg/mL each of USP Clobetasol Propionate RS, IMP G, IMP H and IMP J in Diluent.

For Diluent, System suitability solution, and Sample solution preparation, refer to Assay Validation (Section 6.1).

7.2. System Suitability Parameters, Validation Performance Characteristics, and Results

The system suitability parameters and results are summarized in Table 7. The validation performance characteristics and results are summarized in Table 8. Representative chromatograms of Diluent, Sensitivity solution, Sample solution, and Sample solution spiked with impurities at 0.1% level, are presented in Figures 9–12, respectively. Linearity was established for clobetasol propionate and related impurities, whereas accuracy and repeatability were established for related impurities.

The limit of quantitation estimated from the USP signal-to-noise ratio was 0.1% for three impurities, IMP G, IMP H, and IMP J, with respect to the nominal concentration of clobetasol propionate (0.1 mg/mL). The method was validated in the range of 0.1%‒5% for IMP G, IMP H, and IMP J.

Table 7. Summary of System Suitability Parameters, Solutions, and Results for the Organic Impurities

Table 8. Summary of Validation Performance Characteristics, Solutions, and Results for the Organic Impurities

Table 9. Relative Response Factor

RRF values of the impurities were calculated by dividing the slope of the linearity curve for each impurity by the slope of the linearity curve for clobetasol propionate.

Figure 9. Chromatogram of Diluent (blank).

Figure 10. Chromatogram of Sensitivity solution.

Figure 11a. Chromatogram (at expanded scale) of Sample solution # 1

Figure 11b. Chromatogram (at expanded scale) of Sample solution # 2

Figure 11c. Chromatogram (at expanded scale) of Sample solution # 3

Figure 11. Chromatogram (at expanded scale) of Sample solutions.

Figure 12. Chromatogram (at expanded scale) of Sample solution # 1 spiked with specified impurities at the 0.1% level.