INTRODUCTION:

Budesonide (BDS) is a new glucocorticoid¹ with chemical name 11β, 21-Dihydroxy-16α, 17α-[butane-1,1-diylbis(oxy)] pregna-1,4-diene-3,20-dioneused for the treatment of chronic obstructive pulmonary disease and asthma. Budesonide (Figure 1) (C₂₅H₃₄O₆_&Mo. Wt. 430.534 g/mol) is marketed in different formulations such as nasal polyps, nasal spray, inhaler, rectal forms and pills colitis treatment². Budesonide is also used for the treatment of inflammatory bowel disease³.

Different analytical methods such as LC-MSMS, UFLC and HPLC and techniques were published for the estimation of Budesonide. Raveendra Babu et al., developed a LC-MS/MS method⁴ for the estimation of Budesonide in human plasma in presence of an internal standard, levenorgestreol. A mixture of tertbutyl methyl ether and n-hexane (70:30, v/v) was used for the extraction from the human plasma. LC-MS-MS system composed an Agilent 1100 infinity combined with an AB Sciex Qtrap^® 4000 thermo Finnigan TSQ quantum discovery triple quadruple mass spectrometer was used with Agilent Zorbax Eclipse XDB-C₈ column. Mobile phase consisting of ammonium formate (pH adjusted to 4.2): Acetonitrile was used for the study. Sai Sheela et al., developed a new stability indicating RP-UFLC method⁵ for the quantification of Budesonide using Agilent C18 column with mobile phase mixture, formic acid: acetonitrile (25: 75) (UV detection at 247 nm) and linearity was shown over the concentration range 10-100 µg/ml. Demurtas et al., developed a new validated HPLC method⁶ for the quantification of Budesonide in skin layers using mobile phase, methanol and water mixture (69:31, v/v). The flow rate was 0.8 ml/min and the UV detection was carried out at 254 nm. The linearity was in a very narrow range i. e. 0.05-10 μg/ml. A mixture of acetonitrile: water was used for the extraction from stratum corneum, epidermis and dermis. Yamani et al., developed a stability indicating RP-HPLC method⁷ for the estimation of Budesonide using formic acid: methanol (30: 70) as mobile phase with Agilent C18 column (flow rate was 1.0 ml/min). Budesonide has shown linearity over the concentration range 0.1-100 μg/ml and the UV detection was carried out at 243 nm. Gupta et al., developed a HPLC method⁸ using a mobile phase mixture of acetonitrile and phosphate buffer (pH 3.2) in the ratio of 55:45 (v/v) with flow rate 1.1 mL/min. Kromasil^® C₈column was used and the linearity was reported as 1–50 µg/ml (UV detection at 244 nm). Roth et al. developed a HPLC technique⁹ for the separation and identification of Budesonide epimers and 4 impurities in presence of and internal standard, Flucloronide using Hypersil C18 column and mobile phase consisting of ethanol-acetonitrile-phosphate buffer (pH 3.4 & 25.6 mM) in the ratio 2: 30: 68 (v/v/v) with a flow rate of 1.5 mL/min. Budesonide primary standard was also synthesized and purification was done in seven steps. Shuguang Hou et al., developed HPLC method¹⁰ for the assay of Budesonide using Hypersil C18 column with mobile phase mixture ethanol: acetonitrile: phosphate buffer (pH 3.4) in 2:30:68 ratio. Budesonide has shown linearity over the concentration range 2.5-25 μg/ml and the UV detection was carried out at 240 nm (Flow rate was 1.5 ml/min). Faouzi et al., determined Budesonide in bronchoalveolar lavage of asthmatic patients using HPLC technique¹¹ with Spherisorb ODS column using the mobile phase composition, methanol-aqueous buffer (69:31, v/v) with UV detection at 250 nm and the linearity range was 0.847-7.997 µg/ml.

Sonali et al., developed HPLC method¹² for Budesonide using Hypersil C18 column with mobile phase mixture methanol: water in 80:20 ratio. The flow rate was 1.5 ml/min and the UV detection was carried at 244 nm. The alkali degradation product was characterized using UV, 1H -NMR, 13CNMR, DEPT-NMR, MS and IR and the linearity of this method was reported as 0.01-100 µg/ml. In the present study a new validated stability indicating RP-HPLC method has been proposed for the estimation of Budesonide in presence of an internal standard (IS), Etoricoxib (ETC).

Figure 1: Structure of Budesonide

MATERIALS AND METHODS:

Budesonide was obtained as gift sample from Cipla Ltd (India). 25 mg Budesonide was weighed carefully and transferred in to a 25 ml volumetric flask and dissolved in HPLC grade methanol (Stock solution: 1000 µg/ml). The stock solution was diluted according to the requirement for the studies of accuracy, precision, linearity, robustness and other studies. All the solutions were filtered through 0.45 μm membrane filter before use.

Instrumentation and Chromatographic conditions

Shimadzu Model HPLC system with PDA detector and Zorbox C18 column was used for the chromatographic study. Mobile phase including a mixture of formic acid: acetonitrile: methanol (25: 10: 65) was chosen for the chromatographic study with flow rate 1.0 ml/min. The PDA detection was carried at 240 nm.

Method validation¹³

Linearity, Precision, Accuracy and Robustness studies

A series of Budesonide standard solutions, 0.05-120 µg/mL were prepared from the stock solution and 10 µg/mL Etoricoxib solution (Internal standard) was added and diluted with mobile phase and injected into the HPLC system (n=3). The peak area of Budesonide and Etoricoxib was noted from the respective chromatogram and the mean peak area ratio of Budesonide to Etoricoxib (BDS / ETC) was calculated. A calibration

curve was drawn by plotting the concentration of Budesonide on the x-axis and the corresponding mean peak area ratio of Budesonide to Etoricoxib (BDS / ETC) on the y-axis. The LOQ and LOD were calculated from the S/N ratio.

The interday precision (10, 20 and 40 µg/mL) and intraday precision (20, 40 and 60 µg/mL) studies were performed on three different days and on the same day and the mean peak area ratio (BDS / ETC) was calculated from the chromatograms obtained. The percentage relative standard deviation was calculated from the mean peak area ratio (BDS / ETC).

Accuracy study was performed by spiking the Budesonide (20 µg/mL) formulation solution (50, 100, 150%) with known concentration of Budesonide API (10, 20 and 30 µg/mL) in presence of the internal standard, Etoricoxib solution (10 µg/mL). The peak area ratio of Budesonide to that of the internal standard, Etoricoxib (BDS / ETC) was noted and the percentage recovery was calculated from the linear regression equation.of the calibration curve along with % RSD.

Small deliberate changes were made in the optimized chromatographic conditions for the robustness study such as flow rate (± 0.1 mL; 1.1 and 0.9 mL/min), mobile phase composition formic acid: acetonitrile: methanol (± 5% v/v; 30: 10: 60 and 20: 10: 70) and detection wavelength (± 2 nm; 242 and 238 nm).

Stress degradation studies¹⁴

Stress degradation studies were performed for studying the specificity of the proposed method. Budesonide (20 µg/mL) was allowed to undergo acidic hydrolysis, alkaline hydrolysis, oxidation and thermal degradation reactions in presence of the internal standard, Etoricoxib (10 µg/mL).

Acidic degradation was performed by refluxing Budesonide (20 µg/mL) solution with 1 mL of 0.1 N HCl solution at 80⁰ for 30 minutes on a water bath. The stressed sample was then cooled and neutralized with 1.0 mL 0.1N sodium hydroxide solution. 10 µg/mL of the internal standard, Etoricoxib was added just before injecting the sample and the solution was made up to volume with the mobile phase so that the internal standard will not be affected by the stress reagents. 20 μl of the resulting mixture was injected in to the HPLC system the peak area of Budesonide and that of the internal standard, Etoricoxib was noted and then the peak area ratio (BDS / ETC) was calculated. The percentage degradation was calculated from the linear regression equation of the calibration curve along with the system suitability parameters.

Alkaline degradation was performed by refluxing Budesonide (20 µg/mL) solution with 1 mL of 0.1 N NaOH solution at 80⁰ for 30 minutes on a water bath. The stressed sample was then cooled and neutralized with 1.0 mL 0.1N HCl solution. 10 µg/mL of the internal standard, Etoricoxib was added just before injecting the sample and the solution was made up to volume with the mobile phase so that the internal standard will not be affected by the stress reagents. 20 μl of the resulting mixture was injected in to the HPLC system and the peak area of Budesonide and that of the internal standard, Etoricoxib was noted and then the peak area ratio (BDS / ETC) was calculated. The percentage degradation was calculated from the linear regression equation of the calibration curve along with the system suitability parameters.

Oxidation reaction was performed by refluxing Budesonide (20 µg/mL) solution with 1 mL 30% H₂O₂ solution at 80⁰ for 30 minutes on a water bath. The stressed sample was then cooled and 10 µg/mL of the internal standard, Etoricoxib was added just before injecting the sample and the solution was made up to volume with the mobile phase so that the internal standard will not be affected by the stress reagents. 20 μl of the resulting mixture was injected in to the HPLC system and the peak area of Budesonide and that of the internal standard, Etoricoxib was noted and then the peak area ratio (BDS / ETC) was calculated. The percentage degradation was calculated from the linear regression equation of the calibration curve along with the system suitability parameters.

Thermal degradation was performed by refluxing Budesonide (20 µg/mL) solution at 80⁰ for 30 minutes on a water bath. The stressed sample was then cooled and 10 µg/mL of the internal standard, Etoricoxib was added just before injecting the sample and the solution was made up to volume with the mobile phase so that the internal standard will not be affected by the stress reagents. 20 μl of the resulting mixture was injected in to the HPLC system and the peak area of Budesonide and that of the internal standard, Etoricoxib was noted and then the peak area ratio (BDS / ETC) was calculated. The percentage degradation was calculated from the linear regression equation of the calibration curve along with the system suitability parameters.

Assay of Budesonide formulations

Budesonide is available with brand name Rotacaps (Inhaler suspension), Budamate Transcaps (Lupin Ltd), Budecort CR capsules (Label claim 3.0 mg) (Natco Pharma), Budenase AQ nasal spray (Cipla Ltd), (Label claim: 0.5 mg/2ml & 1 mg/2 ml), Budecort inhaler (200 μg/dose), Breemax (Intra Labs), Budez CR capsules (Label claim 3.0 mg) and Budamate inhaler etc. Twenty capsules of Budesonide of two different brands were collected and the contents inside the capsules were transferred in to two different volumetric flasks and extracted with HPLC grade methanol. Each of the extracted solutions were added 10 µg/ml of IS and then diluted with the mobile phase. 20 µl of each resulting solution was injected in to the HPLC system and the peak area ratio of BDS to that of ETC was calculated from the individual peak areas and the mean peak area ratio was calculated from the resultant chromatograms. The percentage of purity was calculated from the the linear regression equation obtained from the calibration curve.

RESULTS AND DISCUSSION:

A new stability indicating RP-HPLC method has been developed for the quantification of Budesonide in pharmaceutical fomulations in presence of an internal standard, Etoricoxib. Mobile phase mixture consisting of formic acid: acetonitrile: methanol (25: 10: 65) was chosen with flow rate 1.0 ml/min for the chromatographic study (Isocratic mode) using Shimadzu Model HPLC system with Zorbox C18 column was used and the UV detection was carried at 240 nm. A detailed survey of the analytical techniques so far published for the quantification of Budesonide were reviewed and some of the parameters were highlighted in Table 1.

Table 1: Review of Literature

Mobile phase (v/v)	λ (nm)	Linearity (µg/mL)	Comment	Ref
Ammonium formate (pH adjusted to 4.2 with 0.1% formic acid): Acetonitrile	-	0.0001 - 0.003	LC-MS/MS	4
Formic acid: Acetonitrile (25: 75)	247	10-100	UFLC (Stability indicating)	5
Methanol: water (69:31)	254	0.05-10	HPLC (Skin layers)	6
Formic acid: Methanol (30: 70)	243	0.1-100	HPLC (Stability indicating)	7
Acetonitrile: Phosphate buffer (pH 3.2-0.025 M) (55:45)	244	1–50	HPLC	8
Ethanol: Acetonitrile: Phosphate buffer (pH 3.4 and 25.6 mM) (2: 30: 68) Flucloronide (Internal standard)	-	-	HPLC Separation of epimers, 4 Impurities and Synthesis of primary standard, Budesonide	9
Ethanol: Acetonitrile: Phosphate buffer (pH 3.4; 25.6 mM) (2:30:68)	240	2.5-25	HPLC	10
Methanol: Aqueous buffer	250	0.847-7.997	HPLC	11
Methanol: water (80:20)	244	0.01-100	HPLC	12
Formic acid: Acetonitrile: Methanol (25: 10: 65) Etoricoxib (Internal standard)	240	0.05-150	HPLC (Stability indicating)	Present method

Method validation:

Beer-Lambert’s law was obeyed over a concentration range 0.05-120 µg/ml (% RSD 0.27-0.97) and the linear regression equation was y = 0.0489x + 0.003 with correlation coefficient 0.9997) (Figure 3). The LOQ and LOD was found to be and 0.0393 and 0.1192 µg/ml respectively. The % RSD was found to be 0.1768-1.0139 (Intraday) (Table 3) and 0.3016-0.6558 (Inter-day) (Table 3) in precision studies which is less than 2.0 indicating that the method is precise. The % recovery in accuracy studies was found to be 97.43-99.22% (Table 4) and % RSD was (0.81-1.23) less than 2% indicating that the method is accurate. The % RSD in robustness study was found to be 0.2104-1.1102 which was less than 2% indicating that the method is robust (Table 5).

Table 2: Linearity of Budesonide

Conc. (µg/mL)	*Mean peak area		Mean peak area ratio	% RSD
Conc. (µg/mL)	BDS	ETC	BDS / ETC	% RSD
0	0	0	0	-
0.05	2613	1003911	0.0026	0.36
0.1	6019	1003865	0.006	0.42
0.5	25392	1004152	0.0253	0.61
1	51652	1003829	0.0515	0.42
2	103541	1003651	0.1032	0.52
5	244572	1002657	0.2439	0.64
10	478549	1003897	0.4767	0.39
20	959418	1003948	0.9557	0.27
40	1996521	1004028	1.9885	0.81
50	2454632	1003888	2.4451	0.97
60	2952148	1003914	2.9406	0.57
80	4026514	1003741	4.0115	0.28
100	4925836	1003843	4.907	0.62
120	5826514	1003679	5.8052	0.75

*Mean of three replicates

Figure 3: Calibration curve of Budesonide in presence of IS

Table 3: Precision study of Budesonide in presence of IS

Intraday precision study
Conc. (µg/ml)	Mean peak area ratio (BDS / ETC)			Standard deviation (% RSD)
20	0.9572			0.0061 (0.6325)
20
20
40	1.9901			00035 (0.1768)
40
40
60	2.9399			0.0298 (1.0139)
60
60
Interday precision study
Conc. (µg/ml)	Mean peak area ratio			Mean peak area ratio ± SD (% RSD)*
Conc. (µg/ml)	Day 1	Day 2	Day 3	Mean peak area ratio ± SD (% RSD)*
10	0.4767	0.4796	0.4826	0.4796 ± 0.0029 (0.4368)
20	0.9561	0.9679	0.9582	0.9607 ± 0.0063 (0.6558)
40	1.9965	1.9863	1.9859	1.9896 ± 0.0060 (0.3016)

*Mean of three replicates

Table 4: Accuracy study of Budesonide in presence of IS

Conc. (µg/mL)	Formulation (µg/mL)	Total Conc. (µg/mL)	Conc. obtained (µg/mL) ± SD (%RSD)*	% Recovery
10 (50%)	20 20 20	30 30 30	29.51 ± 0.2390 (0.81)	98.37
20 (100%)	20 20 20	40 40 40	38.97 ± 0.3585 (0.92)	97.43
30 (150%)	20 20 20	50 50 50	49.61 ± 0.6102 (1.23)	99.22

*Mean of three replicates

Table 5: Robustness study (20 µg/ml) of Budesonide in presence of IS

Parameter	Condition	*Mean peak area ratio ± SD (% RSD)
Flow rate (± 0.1ml/min)	0.9	0.9623 ± 0.0107 (1.1102)
	1.0
	1.1
Detection wavelength (± 2 nm)	238	0.9591 ± 0.0020 (0.2104)
	240
	242
Mobile phase composition (± 5 %) Formic acid: Acetonitrile: Methanol (25: 10: 65, v/v)	20: 10: 70	0.9541 ± 0.0025 (0.2658)
	25: 10: 65
	30: 10: 60

*Mean of three replicates

Stress degradation studies:

Budesonide eluted at 4.155 min with theoretical plates 5150.185 (> 2000) and tailing factor 1.192 (Resolution 10.342) in presence of IS (Rt 2.211 min) with theoretical plates 3761.986 and tailing factor 1.328. During the acidic degradation Budesonide was eluted at 4.204 min with theoretical plates 5094.784 (> 2000) and tailing factor 1.138 (Resolution 7.272) in presence of IS (Rt 2.274 min) with theoretical plates 5289.926 and tailing factor 1.135 with an extra degradant peak at 2.730 min (Resolution 3.096). During the alkaline degradation Budesonide was eluted at 4.283 min with theoretical plates 5001.261 (> 2000) and tailing factor 1.128 (Resolution 7.062) in presence of IS (Rt 2.268 min) with theoretical plates 5640.058 and tailing factor 1.150 with an extra degradant peak at 2.729 min (Resolution 2.895). During oxidative degradation Budesonide was eluted at 4.196 min with theoretical plates 5249.481 (> 2000) and tailing factor 1.199 (Resolution 10.082) in presence of IS (Rt 2.244 min) with theoretical plates 3328.254 with an extra degradant peak at 1.834 min. During the thermal degradation Budesonide was eluted at 4.244 min with theoretical plates 5370.675 (> 2000) and tailing factor 1.184 (Resolution 10.524) in presence of IS (Rt 2.264 min) with theoretical plates 3923.760 and tailing factor 1.359. In all the stress degradation studies the system suitability parameters were within the acceptable criteria i. e. theoretical plates were greater than 2000, tailing factor was less than 1.50 and the resolution was greater 2.0. The method is specific as Budesonide drug peak was not interfering with any other peaks. The observations recorded during the stress degradation studies were shown in Table 6 and the respective chromatograms were shown in Figure 4.

Table 6: Stress degradation studies of Budesonide

Stress condition	R_t(min)	% Recovery	% Drug degradation
Standard drug (IS)	4.155 (2.211)	100	-
Acidic degradation 0.1N HCl/80ºC/30 min	4.204	7.82	92.18
Alkaline degradation 0.1N NaOH/80ºC/30 min	4.283	2.65	97.35
Oxidation degradation H₂O₂/80ºC/30min	4.196	97.12	2.88
Thermal degradation H₂O₂/80ºC/30min	4.244	95.89	4.11

*Mean of three replicates

CONCLUSION:

A new stability indicating RP-HPLC method has been developed for the estimation of Budesonide and validated as per ICH guidelines. The proposed method is simple, precise, accurate and robust.

ACKNOWLEDGEMENT:

The authors are grateful to Cipla Ltd (India) for providing the gift samples of Budesonide. The authors declare no conflict of interest.

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Received on 08.02.2022 Modified on 19.03.2022

Research J. Pharm. and Tech. 2022; 15(5):2103-2109.

DOI: 10.52711/0974-360X.2022.00349