INTRODUCTION:

Clobetasol propionate (Fig.1) belongs to the class of medications known as corticosteroids. Clobetasol propionate foam has been extensively used for the treatment of certain scalp and skin conditions like plaque psoriasis, rash and dermatitis ^1-7. It is branded under the trade name OLUX® Foam and approved by the United States Food and Drug Administration (USFDA) in 2000 ^8.

Fig.1 Chemical structure of Clobetasol Propionate.

Clobetasol propionate foam, 0.05% is a mixture of aqueous and organic phases and pressurized with hydrocarbon propellants like Butane or Propane. Aqueous phase contains 0.04 g of Citric acid anhydrous and 0.065 g of Potassium citrate monohydrate in 17.575 g of purified water and organic phase contains 0.025 g of Clobetasol propionate, 0.57 g of Cetyl alcohol, 0.255 g of Stearyl alcohol, 0.205 g of Polysorbate 60, 1.045 g of Propylene glycol in 30.220 g of Ethanol ⁸. Overall, Ethanol is presented 60% (w/w) in the formulation composition of the OLUX foam drug product. Monitor of Ethanol content in pharmaceutical aerosol products is very important during manufacturing and stability process because aerosol rate, droplet shape and particle size are closely linked to the formulation vapor pressure, which is regulated by the ethanol concentration ^9-13. To address this challenge, a simple, accurate and quantitative analysis method for the determination of Ethanol content is needed as the standard method of quality control for aerosol manufacturers and as a guide for government related sanitary agencies.

Official compendial analytical methods are not available for the determination of Ethanol content in topical aerosol foam products. In the public domain, Gas chromatography (GC) methods were reported for the determination of Ethanol in various samples using flame ionization detection (GC-FID) ^14-18 and mass spectrometry detection (GC-MS)^19,20. However, the reported methods are not suitable for complex mixture of aerosol formulation and so far, there is no stability indicating chromatographic method for assay determination for Ethanol in topical aerosol foam formulation. This paper narrates the development and validation of stability indicating method for the determination of assay of Ethanol in clobetasol propionate foam, 0.05% using a thermal gradient GC method with FID detection. The method is validated as per ICH Q2 guidelines ²¹. The reported GC method is sensitive enough to quantify the Ethanol content in Clobetasol propionate foam at a level of 50% to 150% with respect to analyte concentration (10 mg/mLEthanol).

MATERIALS AND METHODS:

Chemicals and reagents:

Dehydrated alcohol standard was procured from USP (Lot no. R119J0, potency: 100.0%). Isopropyl alcohol (99.9%, HPLC grade, catalogue no. 1010402500) and Methanol (99.9%, HPLC grade, catalogue no. 1060072500) were procured from Merck, India. OLUX (clobetasol propionate foam, 0.05%; Lot no. MHDK, expiry date: February 2020, United states) cans were procured from US pharmacy.

Instrumentation and chromatographic conditions:

All chromatography experiments were carried out on an Agilent 7890 GC system with a flame ionization detector (Agilent, USA) equipped with Empower software (Waters, USA). The selected column for chromatographic separations was JandW HP-5 GC column, 30m, 0.53 mm ID, 1.5 µm film thickness column (part no. 19095J-323, Agilent USA). The injector temperature and FID detector temperature were maintained at 300°C ± 2°C. The carrier gas is Helium and the selected flow rate was 2.3 mL/min. Hydrogen and Air flow rate were set at 40 mL/minand 400 mL/min respectively (may be adjusted to obtain a suitable flame). The make-up flow was set as 10 mL/min and viscosity delay was set as zero. The injection volume was 1 µL and split ratio was set at 50:1. A thermal gradient elution with the following settings was used: 50°C at 4 min; increase at a rate of 40°C/min to 300°C, hold at 300°C for 5 mins. Total chromatographic run time was set at 15.25mins. Methanol (HPLC grade, catalogue no. 1060072500, Merck, India) was selected as diluent for the preparations of standard and sample solutions. The samples were filled in 1 mL glass screw top vials (Agilent, USA) before injection into the GC system.

Internal standard solution:

An internal standard solution was prepared in methanol by dissolving an appropriate amount of Isopropyl alcohol to get the concentration of 200 mg/mL.

Dehydrated alcohol standard solution:

One gram of Dehydrated alcohol standard and 5 mL of Internal standard solution were dispensed into a 100 mL volumetric flask and then methanol was added to 100 mL mark. This was the Standard solution contains 10 mg/mL dehydrated alcohol and 10 mg/mL internal (Isopropyl alcohol). This solution was used to assess the peak response reliability of the GC instrument by determining the % relative standard deviation (RSD) from five replicate injections.

Test solution:

Placed plastic tubing on the nozzle of Olux sample can (Lot no. MHDK, expiry date: February 2020) and then accurately dispensed approximately 1.6 g of sample into a 100 mL volumetric flask. Added 5 mL of Internal standard solution, dissolved and diluted to volume with Methanol and mixed well. This was the Test solution contains 10 mg/mL dehydrated alcohol and 10 mg/mL internal (Isopropyl alcohol).

Placebo solution:

Placebo stock solution is a mixture of aqueous phase and organic phase. Aqueous phase was prepared by dissolving 0.04 g of Citric acid anhydrous (Catalogue no. 1002415000, Merck, India) and 0.065 g of Potassium citrate monohydrate (Catalogue no. 1049560001, Merck, India) in 17.575 g of purified water and organic phase was prepared by dissolving 0.025 g of Clobetasol propionate (Catalogue no. PHR1921, Supelco, India), 0.57 g of Cetyl alcohol (Catalogue no. 1009891000, Merck, India), 0.255 g of Stearyl alcohol (Catalogue no. 8076800100, Sigma-Aldrich, India), 0.205 g of Polysorbate 60 (Catalogue no. 8170762500, Merck, India), 1.045 g of Propylene glycol (Catalogue no. W294004, Sigma-Aldrich, India) in 30.220 g of Methanol (Catalogue no. 1060072500, Merck, India). To prepare organic phase as a homogeneous mixture, alcoholic phase is required. The selected diluent for Ethanol determination is Methanol. Hence, Methanol is used as a co-solvent to dissolve all excipients and Clobetasol propionate during in organic phase preparation. However, the proportion of either excipient or diluent will not change during sample preparation.

Placebo solution was prepared by dispensing 1.6 g of placebo stock solution into a 100 mL volumetric flask. Dissolved and diluted to volume with methanol and mixed well.

Method validation:

The GC method was validated for the following parameters: system suitability and selectivity, specificity, precision (repeatability and intermediate precision), linearity, accuracy, robustness and solution stability were assessed. The system suitability and selectivity were evaluated by injecting blank (diluent) and five replicates injections of standard solution (10 mg/mL Ethanol and 10 mg/mL Isopropyl alcohol). The specificity of the method was evaluated by injecting placebo solution and the test solution of Clobetasol propionate foam sample (10 mg/mL Ethanol and 10 mg/mL Isopropyl alcohol). The precision (repeatability) of the method was verified by injecting six individual units of test solution of Clobetasol propionate foam (10 mg/mL Ethanol and 10 mg/mL Isopropyl alcohol). The method precision was performed on different days using different GC and column lots to understand the method reproducibility. The linearity response of the GC method with respect to Ethanol concentration was evaluated in the range between 50% to 150% (five concentrations: 50%, 75%, 100%, 125%, 150%) of target standard concentration for Ethanol (i.e. 5 mg/mLto 15 mg/ mL). The accuracy of the analytical method was demonstrated by spiking Ethanol to placebo at three concentration levels. i.e. 50%, 100% and 150% of the nominal concentration of Ethanol (10 mg/mL). At each concentration level, triplicate samples were prepared. The robustness of the method was assessed by deliberately changing the nominal experimental GC conditions such as carrier gas flow rate, ramp temperature rate and oven starting temperature. The effect of the carrier gas flow rate was studied at 2.1 mL/min and 2.5 mL/ min, the ramp temperature rate was studied at 38°C/min to 42°C/min and the Oven starting temperature was studied at 48°C to 52°C. In each varied condition, the system suitability (tailing factor of Ethanol, resolution of Ethanol and Isopropyl alcohol as well as % RSD for the peak area ratios (area of ethanol / area of Isopropyl alcohol) for the five replicates injections) and assay recovery differences between control and each robustness condition were evaluated. As a part of solution stability, the standard and sample solution’s stability were monitored for seven days at room temperature (25°C ± 5°C) conditions. The aged solutions were analyzed against freshly prepared standard solutions at each interval.

RESULTS AND DISCUSSIONS:

Establishment of Stability indicating method:

The main objective of the chromatographic method was to separate placebo peaks observed in samples to achieve Ethanol and Isopropyl alcohol peaks as symmetrical peaks. During the selection of GC column, we had compared the polar, mid polar and non-polar capillary columns during determination of Ethanol content in Clobetasol propionate foam. The selected diluent to dissolve the Clobetasol propionate foam sample is Methanol and the internal standard is Isopropyl alcohol. The relative polarity of Methanol (0.762), Ethanol (0.654) and Isopropyl alcohol (0.546) is almost similar. Although WAX column is a more appropriate column for the determination of alcohols, the maximum operational temperature limit is 260°C which is not apt for complex mixtures of aerosol drug products. The column should be operated at higher temperature (>260°C) to elute all other volatile compounds in aerosol drug product. Hence HP-5 GC column (maximum operational temperature limit 360°C) was a more appropriate column for the determination of Ethanol content in Clobetasol propionate foam drug product.

In general, any other material chromatographically adequately separated from all other components and absent in the sample can be used as an internal standard. Isopropyl alcohol was selected as internal standard for the determination of ethanol content in Clobetasol propionate foam drug product because it is not present in the drug product composition, clearly separated from the Ethanol, Methanol and all other ingredient peaks.

To get optimal separation among Methanol, Ethanol and Isopropyl alcohol, the initial step of oven program was selected with lower temperature, 50°C for 4 min and then increased rapidly to 300°C at the rate of 40°C/ min and hold at 300°C for 5 minutes. When applying the GC conditions described in Instrumentation and Chromatographic conditions, the retention time of the Methanol, Ethanol and Isopropyl alcohol in standard solution is 3.346 min, 3.755 min and 4.126 min respectively (Fig. 3). The resolution between Ethanol and Isopropyl alcohol peaks were considered as method critical quality attribute to ensure optimal and consistent separation on a day to day analysis. The Ethanol content present in the Clobetasol propionate foam was calculated by using the following formula.

As ×Wr×Pr ×100 ×100

Ethanol content (%w/w) = –––––––––––––––––––––––

Ar ×100 × 100 ×Ws)

Where ‘As’ is the peak area ratio (area of ethanol/ area of Isopropyl alcohol) in test solution, ‘Ar’ is the peak area ratio (area of ethanol/ area of Isopropyl alcohol) in standard solution, ‘Wr’ is the weight of Ethanol standard taken in ‘g’, ‘Pr’ is the Potency of Ethanol standard in “%”, Ws’ is the weight of Clobetasol propionate foam sample taken in ‘g’.

Ethanol contnet in %w/w

% Assay of Ethanol = ––––––––––––––––––––– ×100

Label claim in %w/w

Where label claim of Ethanol in Bulk solution is 93.50% w/w and label claim of Ethanol in finished product is 60.44%w/w.

Method validation:

System suitability and selectivity:

The system suitability of the method was tested to confirm reliable performance of the GC system as well as the quality of the method for accurate measurements. In this study, the resolution between ethanol and Isopropyl alcohol peaks, the USP tailing factor of Ethanol and % RSD for the peak area ratios (area of ethanol / area of Isopropyl alcohol) for the five replicate injections of standard solution were assessed as part of system suitability. Ethanol and Isopropyl alcohol peaks were separated with a resolution 4.2, the USP tailing factor of Ethanol peak was 1.0 and the relative standard deviation of peak ratios was lower than 2.0%, which indicates the system is reliable and precise (Table 1). Typical chromatograms of the Blank and Standard solution are shown in Fig.2 and Fig.3 respectively.

Table 1. Summarized data of system suitability

Injection#	Peak area of Ethanol	Peak area of Isopropyl alcohol	Peak area ratio^a
1	649	682	0.9516
2	645	675	0.9556
3	617	648	0.9522
4	632	661	0.9561
5	601	630	0.9540
Mean			0.9539
%RSD			0.2

^aPeak area ratio : area of ethanol/ area of Isopropyl alcohol

Specificity:

From the Specificity chromatograms, it was observed that there was no peak found in placebo chromatograms at the same retention time of Ethanol and Isopropyl alcohol peaks demonstrates no interference from placebo. These results confirmed the method is specific. Typical chromatograms of the Placebo and Sample solution are shown in Fig.4 and Fig.5 respectively.

Precision:

The method precision (repeatability and reproducibility) was estimated by the relative standard deviation (RSD) of the results. % RSD of assay of Ethanol was calculated and was found within 2.0%. The percent of recovery for Ethanol was calculated and found in the range of 99.4-100.1%. These results confirmed the method is precise and reproducible. The method precision results are summarized in Table 2.

Table 2. Summarized data of Precision (Method precision and Intermediate precision)

Preparation #	% Assay of Ethanol
Preparation #	Method precision	Intermediate precision
1	99.9	99..8
2	99.8	99.5
3	99.4	99.4
4	100.0	99.5
5	99.4	99.7
6	100.1	100.0
Mean	99.8	99.6
%RSD ^a	0.3	0.2

^a % RSD: % relative standard deviation

Linearity:

The correlation coefficient (r²) obtained from the calibration plots was >0.998 and % Y intercept at 100% bias was < ±2.0% showed there was an excellent correlation between the peak response ratio and concentration of Ethanol (5 mg/mlto 15 mg/ml) and the method was found to be linear. The results of linearity test are reported in Table 3. Linearity graph of Ethanol is shown in Fig.6.

Table 3. Summarized data linearity study

Linearity level	Ethanol concentration (mg/mL)	Peak response ratio
50%	5.0	0.4814
75%	7.6	0.7265
100%	10.1	0.9712
125%	12.6	1.2128
150%	15.1	1.4637
Correlation coefficient (r²)	1.000
Slope	0.0973
Intercept	-0.0089
% Y intercept at 100% bias	-0.9

Fig. 6 Linearity graph for Ethanol

Accuracy:

The accuracy of the analytical method was demonstrated by spiking Ethanol to placebo at three concentration levels. i.e. 50%, 100% and 150% of the nominal concentration of Ethanol (10 mg/ml). At each concentration level, triplicate samples were prepared. The percent of recovery for Ethanol was calculated at each level and found in the range of 98.0-102.0%. The percent of RSD for mean recovery at each level was found within 2.0%. The results are summarized in Table 4.

Table 4. Summarized data of accuracy study

Compound	50% (5 mg/ml) (n=3)		100% (10 mg/ml) (n=3)		150% (15 mg/ml) (n=3)
Compound	Mean % Recovery	% RSD	Mean % Recovery	% RSD	Mean % Recovery	% RSD
Ethanol	99.7	0.5	100.1	0.5	100.6	0.2

Table 5. Summarized data of robustness data

Parameter	Nominal	Flow rate		Ramp temperature rate		Initial oven temperature
Parameter	Nominal	2.1 ml/min	2.5 ml/min	38°C/min	42°C/min	48°C	52°C
Resolution ^b	4.2	4.5	3.8	4.3	3.9	4.6	4.3
%RSD response ratio (n-5)^c	0.2	0.4	0.2	0.4	0.5	0.2	0.3
Avg. response ratio (n=5)^d	0.9539	0.9626	0.9429	0.9480	0.9537	0.9926	0.9907
Tailing factor ^e	1.0	1.1	1.0	1.0	1.0	1.0	1.0
% Assay ^f	99.8	99.2	99.6	100.0	100.1	99.4	99.3

^aFlow rate : Carrier gas (Helium) flow rate;

^b Resolution: Resolution between Etanol and Isopropyl alcohol peaks in the standard solution;

^c %RSD response ratio : % relative standard deviation of peak response ratio between Ethanol and Isopropyl alcohol peaks from the standard injections ;

^dAvg. response ratio: Avearge peak response ratio between Ethanol and Isopropyl alcohol peaks from the standard injections;

^e Tailing factor: The USP tailing factor of Ethanol peak in the standard injection ;

^f % Assay : % Assay of Ethanol in OLUX (Lot no. MHDK, expiry date: February 2020, United states).

Robustness:

At each varied condition, the system suitability (tailing factor of Ethanol, resolution between Ethanol and Isopropyl alcohol as well as % RSD for the peak area ratios (area of ethanol / area of Isopropyl alcohol) for the five replicates injections and Assay recovery differences between control and each robustness condition were evaluated. In all the deliberate varied chromatographic conditions the selectivity as well as the performance of the method were unchanged proves the robustness of the method. The robustness results are summarized in Table 5.

Standard and Sample solution stability:

The % Assay was calculated for stored standard and sample solutions against the freshly prepared standard solutions at each interval. The solution stability results showed that the standard and sample solutions are stable for 1 week at room temperature. The solution stability results are summarized in Table 6.

Table 6. Summarized data of solution stability study

Compound	Initial % Assay	Aged solution % Assay ^a	% Recovery difference
Standard solution	99.9	99.2	0.7
Sample solution	99.2	98.6	0.6

^aAged solution : Solution was stored at room temperature for 7 days

CONCLUSION:

Optimal chromatographic separation was achieved for Ethanol and Isopropyl alcohol peaks under selected chromatographic conditions. This newly developed rapid gradient GC method is specific, precise, linear, accurate, rugged, robust and easily implementable in quality control (QC) laboratories for the monitoring of the assay of Dehydrated alcohol (Ethanol) present in Clobetasol propionate foam formulation. The method has obvious advantages over those previously reported such as reasonable short analysis time and high separation efficiency.

ACKNOWLEDGEMENTS:

This research article is constructed potential through the aid and reinforcement from K.L. University.

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Received on 11.09.2020 Modified on 05.11.2020

Research J. Pharm. and Tech 2021; 14(11):5919-5925.

DOI: 10.52711/0974-360X.2021.01029