Studies on the Stability of Corticosteroids: Degradation of Clobetasol Propionate, Desonide and Hydrocortisone in Topical Formulations by HPLC and UPLC-MS/MS

Corticosteroids are the most widely used class of anti-inflammatory medications in the pharmaceutical industry. There are several pharmaceutical dosage forms available using different corticosteroids. Topical steroids of varying potencies are available in creams, ointments, solutions and other vehicles. Chemical instability and drug degradation are the key quality concerns for these topical dosage forms. Nature of the dosage forms, excipient quality, product composition, and process optimization are some of the common factors which affect the stability of corticosteroids. This article describes drug degradation behavior of three different corticosteroids in different topical dosage forms. Drug degradation patterns of Hydrocortisone, Clobetasol propionate and Desonide formulations observed in stability studies of respective finished drug products under ICH recommended storage conditions were investigated. HPLC, UPLC-MS/MS methods were developed for the separation and characterization of impurities. The structural elucidation of the unknown impurities observed for these steroids and mechanistic consideration of potential degradation pathways has been discussed. Detailed discussion on the analytical methodologies is included as well.


Introduction
Corticosteroids (Figure 1) containing the 1,3-dihydroxyacetone side chain on How to cite this paper: Hotha, K.K., Ramamoorthy, S. and Patel, H. (2020) Studies on the Stability of Corticosteroids: Degradation of Clobetasol Propionate, Desonide and Hydrocortisone in Topical Formulations by HPLC and UPLC-MS/MS. American Journal of Analytical Chemistry, 11, their D-rings, such as Betamethasone, Dexamethasone, Hydrocortisone, Clobetasol propionate and related compounds are an important class of organic compounds arranged in typical configuration. They are widely used as potent anti-inflammatory and immunomodulatory agents and formulated in several dosage forms (Figure 1) [1] [2] [3] [4]. Based on the formulation composition, excipient, pH and thermal stability, the degradation of corticosteroids in varied formulations can be induced via different mechanisms [5] [6] [7] [8].
There were several articles reported for the corticosteroids degradation by various analytical techniques [5]- [13]. Enol aldehyde via β-elimination, mattox rearrangement, Bayer villager oxidation are some of the known mechanisms for these degradation pathways. There were several impurities can be formed based on the formulation composition, excipient, pH and thermal stability. In the current article steroids Hydrocortisone, Clobetasol propionate and Desonide formulations were evaluated in the finished product dosage forms and impurity profiling by UPLC-MS/MS was performed. Many authors proposed methods based on gas chromatography/mass spectrometry for the detection of corticosteroids [9] [14]- [26]. Hydrocortisone ( Figure 2) is used in treating severe allergies, arthritis, asthma, multiple sclerosis, and skin conditions. There were several formulations associated with the Hydrocortisone in terms of creams, ointments and otic solution. Hydrocortisone-Acetic acid otic solution is used to treat certain problems of the ear canal. There were several analytical methods that were developed for the Hydrocortisone estimation in creams and lotion dosage forms. Several articles are available about the degradation pathways of hydrocortisone in suspensions as well as in microbial environments [6] [9] [26] [27]. The current research article thoroughly reviewed about the degradation mechanism of the Hydrocortisone in the Hydrocortisone acetic acid otic solution in the stability conditions. During the analysis of the stability samples, two unknown peaks were identified and characterized. One unknown peak is the keto impurity of the Hydrocortisone which is a  predominate oxidative degradant as well as metabolite of the many corticosteroids [3] [18] [19]. The second impurity was the 17-formoxyl impurity where it arises due to the Bayer villager oxidation of the aldehyde group. Structural elucidation was performed using LCMSMS and NMR.
Clobetasol propionate ( Figure 3) is a class 1 corticosteroid and is a super high potency di-halogenated corticosteroid that has been commercially available since 1973. It was used in the treatment of skin conditions such as severe psoriasis, seborrheic dermatitis and extreme photo dermatitis in HIV/AIDS. There were articles described for the forced degradation of Clobetasol in bulk drug and cream formulations by HPLC [9]

Chemical and Reagents
Hydrocortisone working standard and impurity standards were supplied by Tianjin Jinjin Pharmaceutical Company, China whereas Clobetasol propionate, Desonide, Dexamethasone, Betamethasone were procured from Teva Pharmaceuticals and from sigma Aldrich, USA. The HPLC grade acetonitrile, methanol, and analytical grade ammonium formate, formic acid were purchased from Merck, Darmstadt, Germany. Water used was obtained by using Millipore Mil-liQ Plus water purification system. Drug product samples were supplied by Lupin Somerset, New Jersey.

Equipment
LC-MS/MS system (Acquity UPLC coupled with TQD mass spectrometer with empower software, HPLC with empower software Waters Corporation, Milford, USA) was used for the identification of unknown compounds formed during forced degradation and stability testing studies. Cintex digital water bath was used for hydrolysis studies. Thermal stability studies were performed in a dry air oven (Cintex, Mumbai, India).

Chromatographic Conditions
HPLC and UPLC-MS/MS analysis was performed for stability analysis and for the impurity identification. All the chromatographic conditions were described as follows.

HPLC Chromatographic Conditions
HPLC (PDA Detector with empower software, Waters Corporation, Milford, USA) was used for the analysis of finished product samples and forced degradation and stability testing studies for Hydrocortisone. The chromatographic column used was Luna C18 (2), 100A, 4.6 × 250 mm, 5.0 µm particle size, Manufacturer: Phenomenex. The separation was achieved on a gradient method. 0.05 M of potassium phosphate monobasic buffer adjusted to pH 4.5 was used as a buffer and acetonitrile was used as a mobile phase B. The HPLC gradient program was set as Time

UPLC-MS/MS Method Development and Optimization
In order to run the samples in UPLC-MS/MS method, it was decided to develop a method compatible for mass spectrometry. Sodium phosphate buffer used in the HPLC method is not compatible with UPLC-MS/MS. So that UPLC method was developed using volatile buffer ammonium formate and chromatographic conditions were optimized for the separation. Electrospray Ionization of positive ion mode was selected for the mass spectrometric analysis. Identical Pattern was reproduced using these conditions. To confirm the unknown peak of interest, elution pattern and % area of the unknown peak in HPLC were compared against UPLC-MS/MS method and the respective retention times of unknown peaks were identified and confirmed.

Degradation of Corticosteroids
There were several degradation mechanisms reported for corticosteroids especially with the D ring and 1,3-dihydroxyacetone side chains [3] [27] [28] [29] [30] [31]. Betamethasone and Dexamethasone form corresponding enol aldehydes and carboxylic acid impurities during the stability in the transdermal dosage and in solutions. The introduction of the phosphoric group at the 21-hydroxyl of the steroid core structures might impart additional degradation pathways to the resulting phosphonosteroids.

Enol-Aldehyde Degradation-Mattox Rearrangement
Enol aldehydes are one type of key degradants and metabolic intermediates formed from a group of corticosteroids containing the 1,3-dihydroxyacetone side chain on their D-rings, such as Betamethasone, Dexamethasone, Beclomethasone, and related compounds [27]

Hydrocortisone Degradation
Jen Hansen et al. reported the degradation pattern of Hydrocortisone and its major degradation products in aqueous solution was investigated utilizing an HPLC method [19]. The product distribution was characterized qualitatively and quantitatively as a function of pH in the range 0 -11, nature of buffers and trace metal impurities. Two major decomposition pathways were reported, an oxidative degradation leading to the formation of 21-dehydrohydrocortisone which subsequently degraded to a 17-carboxylic acid and a 17,20-dihydroxy-21-carboxylic acid derivative, and a non-oxidative reaction giving a 17-oxo, 17-deoxy-21-aldehyde and 17-deoxy-20-hydroxy-21-carboxylic acid derivative. HPLC and UPLC-MS/MS methods were developed and performed the forced degradation study and stability sample analysis of Hydrocortisone in otic solution [10] [24]  . Characteristic UV spectra of Enol-Aldehyde Impurity. [25]. During the stability studies testing of the otic solution, two unknown impurities was observed, one unknown impurity was identified as a keto-hydrocortisone impurity whereas the other impurity was identified as Hydrocortisone acid impurity. These two impurities were identified by UPLC-MS/MS fragmentation pattern and by NMR evaluation.

Clobetasol Propionate Degradation
Clobetasol propionate degradation behavior was studied in the lotion, ointment and topical solution. Ayesha et al. reported forced degradation of Clobetasol propionate in various solvents but there was no detailed identification of the degradation reported [32]. Forced degradation study and stability analysis of Clobetasol propionate in different dosage forms were performed using HPLC and UPLC-MS/MS and possible major degradants were identified. Possible mechanisms for the degradation behavior were drafted and one unknown impurity in the topical solution identified as Clobetasol acetic acid impurity was characterized by LCMSMS and NMR studies [11] [12] [13] [33]. Extensive studies were conducted evaluating different residual solvents and excipients' interaction with the drug in order to identify the root cause of the generation of these impurities.

Desonide Degradation
HPLC and UPLC analysis was performed for Desonide finished product samples (lotion, ointment and cream). Based on the excipients and the sample prepara-tion involved, three different HPLC methods were developed for three different formulations. Forced degradation study and stability analysis of Desonide in different dosage forms performed using HPLC and UPLC-MS/MS and possible major degradants identified. Possible mechanisms for the degradation identified. One unknown impurity identified in the lotion as methoxy impurity of the Desonide. All the other impurities were separated using the developed HPLC method.

Hydrocortisone
Literature studies indicated that 21-dehydrohydrocortisone was the principal degradation product of hydrocortisone formed at 50˚C and room temperature [31] [32] [33]. Significant amounts of the 17-ketosteroid (11) and small amounts of the acid (VI) were detected in samples stored at 50˚C in Hydrocortisone lotion. Forced degradation study and drug stability studies in the accelerated conditions of Hydrocortisone otic solution formed major impurities as hydrocortisone acetate, cortisone 17 keto steroid impurity and one unknown impurity (Figure 7). 17-Keto steroid impurity was identified by LC-MS/MS spectral pattern ( Figure 8). The mass of unknown impurity was identified as 377 in the positive mode (Figure 9). The retention behavior and spectral pattern indicate that it could be a polar impurity when compared to Hydrocortisone. Mass spectrum of the unknown was compared against Hydrocortisone ( Figure 10). Molecular ion fragments of 267, 285, 295 and 313 confirm the presence of the side chain. H NMR spectrum confirms the presence of aldehyde and carboxylic acid proton at singlet 8.9 and broad hump at 9.10 respectively ( Figure 11). The formation of the impurity is due to the oxidation of the Hydrocortisone which involves the Baeyer-Villiger oxidation which forms the hydroxyl group into aldehyde group ( Figure 12).

Clobetasol Propionate
Clobetasol propionate formed several impurities during the forced degradation and in the finished product stability studies. Impurity profiling of the ointment, lotion and topical solution were studied in detail ( Figure 13). During the stability analysis of these dosage forms, Clobetasol tend to form USP Related Compound A due to the keto enol tautomeric rearrangement followed by the dehydration           chloro impurity were evaluated during the impurity profiling of the topical solution. All the impurities were separated using HPLC and characterized using LC-MS/MS and NMR.

Desonide
Desonide-21-dehydro is found to be the major known acid degradant and 16-Alpha-Hydroxy prednisolone is found to be major known base degradant generated during the forced degradation studies. Based on the degradation data, it was found Desonide to be sensitive to both acid and base degradation (       pH), Hydrocortisone has proved to be unstable only on the basic side.

Conclusion
The degradants generated during the forced degradation and stability indicating studies of three corticosteroids in different topical formulations were investigated. The impurities were separated using a validated HPLC method and further elucidated using mass analysis by UPLC/MS-MS and NMR spectroscopic analyses. The mechanism of the formation of these impurities in corticosteroids was discussed in detail. The findings in the present study show that the choice of excipient grade, particle size and morphological characteristics of drug substance, emulsifying agent, solubilizing agent, presence of residual solvent in trace amount, water content in non-aqueous formulations are some of the critical parameters which could affect the stability of the drug product. The implementation of Quality By Design in every stage of the product life is encouraged to ensure both technical and regulatory success for the generic drug approval in the area of corticosteroids.