Corticosteroids (Figure 1) containing the 1,3-dihydroxyacetone side chain on 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)    . Based on the formulation composition, excipient, pH and thermal stability, the degradation of corticosteroids in varied formulations can be induced via different mechanisms    .
There were several articles reported for the corticosteroids degradation by various analytical techniques  - . 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   - .
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    . 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
Figure 1. Chemical structures of corticosteroids.
Figure 2. Hydrocortisone impurities.
predominate oxidative degradant as well as metabolite of the many corticosteroids   . 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      . However there was no detailed degradation pathways of Clobetasol not been published to the best of our knowledge. In this article, degradation pathways of Clobetasol propionate evaluated in ointment, lotion and topical solution. During the stability of the topical solution unknown impurity was observed. UPLC-MS/MS and NMR performed for the Characterization of the unknown impurities in the stability samples of the Pharmaceutical Dosage forms.
Desonide (Figure 4) is a glucocorticoid with anti-inflammatory and antipruritic activities that is used in the treatment of corticosteroid-responsive dermatoses. These drugs are widely used for the treatment of skin diseases of inflammatory, proliferative, or immunological origin. In this article Desonide stability in the pharmaceutical dosage forms of ointment and lotion is discussed. During the stability sample analysis of Desonide lotion there was one unknown impurity observed which was characterized by UPLC-MS/MS analysis.
Figure 3. Clobetasol propionate impurities.
Figure 4. Desonide impurities.
2.1. 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 MilliQ Plus water purification system. Drug product samples were supplied by Lupin Somerset, New Jersey.
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).
3. 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 (min)/% solution B: 0.0/26, 18/26, 32/45, 48.00/70, 58/70, 60/26, 75/26. The column temperature was maintained at 40˚C and the injection volume was 20 μL. Acetonitrile and a mixture of Milli-Q water in the proportion of 60:40 (v/v); respectively used as diluent. The mobile phase was pumped at 0.8 mL/min−1. The eluted compounds were monitored at 254 nm. The run time was 75 min.
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 Clobetasol propionate. The chromatographic column used was X Bridge C 18, 4.6 × 250 mm 5 µm. The separation was achieved on a gradient method. 0.05 M of sodium phosphate buffer adjusted to pH 5.5 was used as a buffer and ratio of methanol: acetonitrile (10:90 v/v) was used as a mobile phase B. The HPLC gradient program was set as Time (min)/% solution B: 0.0/40, 12/40, 35/65, 40/65, 45/40, 55/40. The column temperature was maintained at 40˚C and the injection volume was 20 μL. Methanol and a mixture of Milli-Q water in the proportion of 70:30 (v/v); respectively used as Diluent. The mobile phase pumped at 1.00 mL/min−1. The eluted compounds were monitored at 240 nm. The run time was 55 min.
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 Desonide. The chromatographic column used was Inertsil ODS-3V, 4.6 × 250 mm 5.0 µm particle size, Manufacturer: GL Sciences. 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 (min)/% solution B: 0.00/5, 5.00/25, 30.00/40, 35.00/40, 45.00/80, 50.00/80, 52.00/5, 65.00/5.00. The column temperature was maintained at 40˚C and the injection volume was 20 μL. Acetonitrile used as Diluent. The mobile phase pumped at 1.00 mL/min−1. The eluted compounds were monitored at 245 nm. The run time was 65 min.
4. 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.
Liquid Chromatography and Mass Spectrometric Conditions
LC-MS/MS system (Acquity UPLC coupled with TQD mass spectrometer with empower software, Waters Corporation, Milford, USA) was used for the identification of unknown compounds formed during forced degradation and stability testing studies for Hydrocortisone. The chromatographic column used was an Acquity UPLC BEH C18 1.7 µm, 3 × 100 mm. The separation was achieved on a gradient method. Ammonium formate buffer (0.01 M) adjusted to pH 4.5 was used as a buffer and acetonitrile was used as mobile phase B. The HPLC gradient program was set as Time (min)/% solution B: 0.0/26, 6.83/26, 12.43/45, 18.83/70, 25/70, 28/26, 35/26. The column temperature was maintained at 30˚C and the injection volume was 5 μL. Acetonitrile and a mixture of milli-Q water in the proportion of 60:40 (v/v); respectively was used as diluent. The mobile phase pumped at 0.3 mL/min−1. The eluted compounds were monitored at 254 nm. The run time was 35 min. Mass spectrometric conditions optimized as cone gas 30 V, cone value 30 V, collision flow 50 L/Hr., ion energy 2 entrance and exit Potentials 1 V, source temperature 130˚C, desolovation gas 300 L/hr., desolvation temperature 300˚C.
LC-MS/MS system (Acquity UPLC coupled with TQD mass spectrometer with empower software, Waters Corporation, Milford, USA) was used for the identification of unknown compounds formed during forced degradation and stability testing studies of clobetasol propionate. The chromatographic column used was an Acquity UPLC BEH C18 1.7 µm, 3 × 100 mm. The separation was achieved on a gradient method. Ammonium formate buffer (0.01M) adjusted to pH 5.25 with 10% formic acid was used as a buffer and ratio of methanol:acetonitrile (10:90 (v/v) was used as a mobile phase B. The UPLC gradient program was set as Time (min)/% solution B: 0.0/40, 2.00/40, 4.00/45, 14.00/65, 18.00/65, 20.00/40, 25.00/40. The column temperature was maintained at 40˚C and the injection volume was 5 μL. Acetonitrile and a mixture of Milli-Q water in the proportion of 60:40 (v/v); respectively used as diluent. The mobile phase pumped at 0.4 mL/min−1. The eluted compounds were monitored at 240 nm. The run time was 25 min. Mass spectrometric conditions optimized as cone gas 30 V, cone value 30 V, collision flow 50 L/Hr., ion energy 2 entrance and exit Potentials 1 V, source temperature 130˚C, desolovation gas 300 L/hr., desolvation temperature 300˚C.
LC-MS/MS system (Acquity UPLC coupled with TQD mass spectrometer with empower software, Waters Corporation, Milford, USA) was used for the identification of unknown compounds formed during forced degradation and stability testing studies of Desonide. The chromatographic column used was an Acquity UPLC BEH C18 1.7 µm, 3 × 100 mm. The separation was achieved on a gradient method. Ammonium formate buffer (0.01 M) adjusted to pH 4.48 with 10% formic acid was used as a buffer and ratio of methanol:acetonitrile (20:80 (v/v)) was used as a mobile phase B. The UPLC gradient program was set as Time (min)/% solution B: 0.0/5.00, 2.00/25, 12.00/35, 15.00/35, 20.00/80, 25.00/80, 27.00/5, 33.00/5.00. The column temperature was maintained at 40˚C and the injection volume was 5 μL. Acetonitrile used as diluent. The mobile phase was pumped at 0.4 mL/min−1. The eluted compounds were monitored at 240 nm. The run time was 33 min. Mass spectrometric conditions optimized as cone gas 30 V, cone value 30 V, collision flow 50 L/Hr., ion energy 2 entrance and exit Potentials 1 V, source temperature 130˚C, desolovation gas 300 L/hr., desolvation temperature 300˚C.
5. Degradation of Corticosteroids
There were several degradation mechanisms reported for corticosteroids especially with the D ring and 1,3-dihydroxyacetone side chains      . 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.
5.1. 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    . The formation of enol aldehydes from these corticosteroids is via acid-catalyzed β-elimination of water from the side chain, a process known as Mattox rearrangement (Figure 5). Bin Chen et al. reported that enol aldehydes could also be formed directly from the corresponding 17,21-diesters of these corticosteroids but only under alkaline condition, which was proposed to follow a variation pathway of the original Mattox rearrangement. Bin Chen et al. proposed the mattox rearrangement in both acidic conditions as well as in basic conditions. The characteristic UV Spectra (Figure 6) is the informal identification for the formation of these impurities due to mattox rearrangement. This impurity was also observed in the degradation of Clobetasol propionate and Desonide in the transdermal dosage forms.
5.2. 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 . 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  
Figure 5. Enol-Aldehyde: Mattox Rearrangement.
Figure 6. Characteristic UV spectra of Enol-Aldehyde Impurity.
. 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.
5.3. 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 . 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    . 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.
5.4. Desonide Degradation
HPLC and UPLC analysis was performed for Desonide finished product samples (lotion, ointment and cream). Based on the excipients and the sample preparation 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.
6. Results and Discussion
Literature studies indicated that 21-dehydrohydrocortisone was the principal degradation product of hydrocortisone formed at 50˚C and room temperature   . 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).
Figure 7. UPLC-MS/MS (PDA) Chromatogram of the Hydrocortisone stability sample.
Figure 8. MS/MS Chromatogram of the keto impurity of the Hydrocortisone.
Figure 9. UPLC-MS/MS Chromatogram of the formoxyl acid impurity.
6.2. 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
Figure 10. MS/MS Chromatogram of the Hydrocortisone.
Figure 11. H NMR spectrum of the formoxyl acid impurity.
Figure 12. Mechanism of Hydrocortisone via Baeyer-Villiger oxidation.
reaction to form a cyclic compound (Figure 14 & Figure 15). Clobetasol propionate topical solution generates the same impurity at a very minimal level. The nominal degradants of the topical solution are delta chloro impurity and Clobetasol beta chloro diene impurity which will form due to the loss of hydroxy group at 17 position. Another major unknown degradant observed during the stability sample analysis of the topical solution was later identified as an acetic acid impurity. LC-MS/MS analysis was performed and the molecular ion of the peak was found as 374 (Figure 16). Several possibilities of degradation pattern were drawn with the identified mass number and through which all the possible impurities were injected to identify the unknown impurity (Figure 17). Unknown impurity characterization was performed by the UPLC-MS/MS and H NMR analysis (Figure 18 & Figure 19).
Structural confirmation and chemical shift assignments of the acetic acid impurity was achieved from the analyses of H NMR, in conjunction with mass spectral information. Analysis of NMR data clearly showed the absence the resonances due to the chloromethyl ketone and propionate ester moieties in the degradant as compared to the API. An olefin proton resonance was observed at 5.42 ppm (H-21). In addition, the singlet at 5.42 ppm also showed a C correlation to the carbonyl carbon at 170.9 ppm (C-22), consistent with the carboxyl carbonyl carbon. The stereochemistry around C17 - C21 double bond could not
Figure 13. Stability sample results of Clobetasol Propionate formulations.
Figure 14. MS/MS spectral Pattern of Clobetasol related compound-A.
Figure 15. Mechanism of Impurity-A formation from clobetasol propionate.
Figure 16. MS/MS spectral pattern of the unknown impurity.
Figure 17. Spiking of the possible MW 374 degradants and related impurities in the Clobetasol spiking solution.
Figure 18. H NMR spectrum of acetic acid impurity.
Figure 19. C13 NMR Spectrum of the acetic acid impurity.
be confirmed by NMR experiments Which indicates because of the steric hindrance, the bulky −COOH group is expected to be away from the C-18 methyl group and shown as drawn. Analysis of the NMR data also revealed that there are no structural modifications in A, B, and C ring moieties of the degradant. The only structural changes observed are with respect to the substituents attached to C17 position of the molecule. Mass spectral characterization was performed for this impurity and observed that the molecular ions of 355 (M-HF), 337 (M-HF+H2O), 319 (M-HF-2H2O), 171 (Cleavage in the B ring) and 121 (Cleavage at the C ring) confirm the structure of acetic acid impurity. Mechanism involves formation of enol aldehyde through intra molecular cannizaro reaction (Figure 20).
Mechanistic pathways of acetic acid impurity and USP related compound A is explained in (Figure 15 & Figure 20). Excipients composition, pH are the two important considerations in identifying the probable degradants in the Clobetasol transdermal dosage forms. Trace level residual solvents like DMF, methanol, THF which were during the synthetic manufacturing process could degrade Clobetasol when it is in the solution form. Clobetasol ointment predominantly forms related compound A whereas topical solution forms Clobetasol acetic acid as a major impurity. Water absorption, basic nature of the excipients and solution pH range are the clear-cut root causes for the formation of the Clobetasol related degradants in finished product. Clobetasol related compound B (Delta Chloro Impurity), Clobetasol related compound A (3, 3 Dione impurity), delta hydroxy impurity, 21 dehydro 17 deoxy betamethasone, dexamethasone oxetanone, enol aldehyde isomer 17 deoxy dexamethasone, Clobetasol and delta
Figure 20. Mechanism of acetic acid impurity formation in Clobetasol Propionate.
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-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 (Figures 21-23). Presence of methanol in the diluent or in the pharmaceutical dosage form degrades Desonide to methoxy degradant of Desonide (Figure 24). This impurity could be generated from the 21 dehydro compound which reacts with methanol group to form the methoxy impurity. Mass spectral fragmentation was performed and compared against Desonide Mass spectrum (Figure 25). Molecular ions of 432, 415 confirms the loss of methyl group and the presence of the methoxy group on the C-17 Sidechain (Figure 26).
The degradants generated during the forced degradation and stability indicating studies of three corticosteroids in different topical formulations were investigated. Generally, the excipients used in the RLD are presumed compatible with the drug substance. However, the formulator should be aware that different vendors or grades may contain different impurities, which in turn may trigger the drug degradation. Development of the Corticosteroid topical formulations require extensive knowledge in the area of excipients, rheological and process optimization. Excipient compatibility data, stability profile of a R&D batch and characterization of the RLD in terms of drug product stability profile are needed to increase the chances of success in pharmaceutical development and to substantiate the stability data generated in the formal stability program.
In overview, during the development of the Clobetasol topical solution and ointment several critical factors were identified which caused the chemical degradation. Presence of trace level of organic residual solvents like DMF, THF caused the formation of acetic acid impurity which was above the identification threshold. Trace level of absorption of water in the non-aqueous formulation of Clobetasol Propionate resulted in the formation of the 3, 3 20 Dione impurity which is major degradation product in the base hydrolysis and water hydrolysis of force degradation studies. Desonide is stable in the semisolid dosage forms. The selection of the petrolatum is the major challenge for the ointments which are dispersed in the petrolatum bases. It should be noted that Desonide 21 dehydro impurity the major degradant observed in Desonide formulation is a common impurity in majority of the corticosteroids. Also, presence of methanol as a residual solvent or in diluent can form methoxy impurity in Desonide formulations. The Hydrocortisone was very unstable in water and water-washable ointment base. The addition of alcohol and glycerin to water had a stabilizing effect. When subject to drastic degradations conditions (very acidic or very basic
Figure 21. Desonide Ointment sample chromatogram.
Figure 22. Desonide Lotion sample chromatogram.
Figure 23. Desonide Cream sample chromatogram.
Figure 24. Degradation of Desonide 21 dehydro impurity chromatogram.
Figure 25. MS/MS Spectra of Desonide.
Figure 26. MS/MS spectra of Methoxy Desonide Impurity.
pH), Hydrocortisone has proved to be unstable only on the basic side.
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.
The authors wish to thank the management of Lupin Somerset for providing the infrastructure for the supporting of this research work. Cooperation from colleagues Product Development, Quality Control and Analytical Research & Development of Lupin Somerset is appreciated.
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