1. INTRODUCTION:

The dissolution test is a simple and useful in vitro tool that can provide valuable information about drug release similarity among different batches and brands. It describes about manufacturing reproducibility, product performance similarity and biological availability of drug from its formulation. Therefore, it is considered as one of the most quality control tests of solid pharmaceutical dosage forms.¹

Deflazacort is chemically, (11b, 21- Dihydroxy-2'-methyl-5'Bh pregna-1, 4-dieno [17, 16-d] oxazole3, 20 dione 21- acetate). It is an oxazoline derivative of prednisolone with anti-inflammatoryand immunosuppressive activity. It was approved in February 2017 by the FDA for use in treatment of Duchenne muscular dystrophy (trade name emflaza).²

Figure 1: Structure of deflazacort

Analytical quality by design (AQbD) and CCD help in regulatory compliance for RP-HPLC method development, stress testing, or stability indicating methods.^3-6.UV spectrophotometric methods (simultaneous equation method/ Vierodt') and LC-MS are widely acceptable for simultaneous estimation of pharmaceutical combinations^7-9. Presence of impurities critically affects the stability and pharmacological action of pharmaceutical API and drug products.^10-14 The purpose of stability and related substance study is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors.^15-16

This action of deflazacort is due to corticosteroid prodrug, whose active metabolite 21-desDFZ, acts through the glucocorticoid receptors to exert anti-inflammatory and immunosuppressive effects. The precise mechanism by which deflazacort exerts its therapeutic effects in patients with DMD is unknown. It is soluble in DMSO, methanol, chloroform, water (< 1 mg/ml at 25°C) and ethanol (12mg/ml at 25°C).¹⁷

This drug is not official in any pharmacopeia. Several methods have been reported for the analysis ofdeflazacort in pharmaceutical dosage form as well as in the biological samples like serum and urine i.e., HPLC, LC-MS/MS with ESI, UV method. Literature survey reveals that there is no single multimedia dissolution method reported^18,19 Hence, it is worthwhile to develop multimedia dissolution method for estimation of deflazacort in tablet dosage form by UV visible spectrophotometry. The present manuscript describes simple and sensitive spectroscopic procedures for the determination of deflazacort in pharmaceutical dosage form.

2. MATERIALS AND METHODS:

2.1 Chemicals and solutions:

Pharmaceutical grade deflazacort is used. All reagents used in this study were of analytical grade. Buffer solutions were prepared in distilled water. All dilutions prepared in standard volumetric flasks. Deflazacort tablets (Defcort 6mg and Defcort 30mg) were procure from local drug store.

2.2 Instrument:

A dissolution apparatus USP type-II (paddle, lab India), A Shimadzu model 1800 double beam UV visible spectrophotometer with spectral width of 2nm, wavelength accuracy of 0.5nm and 2cm cuvette used to measure absorbance of all the solutions, spectra were automatically obtained by UV- probe system. Other instruments used sonicator, digital pH Meter, electronic weighing balance and thermomixer.

3. UV Method Development:

Deflazacort is not completely soluble in any of the dissolution media i.e., 0.1N HCl, 4.5 Acetate buffer,Water and 6.8 Phosphate buffer. Hence, for complete solubilization of deflazacort methanol was used.^20,21 Stock solution of deflazacort was prepared by accurately weighing 2mg of standard in 100ml volumetric flask. Add 10ml methanol and sonicate for 10minutes. Make up the volume up to 100ml with dissolution media (0.1N HCl, water, 4.5 acetate buffer and 6.8-phosphate buffer). Pipette out 6ml from stock solution and dilute up to 20ml with dissolution media 0.1N HCL, 4.5 acetate buffer, water, 6.8-phosphate buffer and that will give 6μg/ml concentration of solution (working standard solution).

4. Dissolution Method Development:

One tablet was placed in each of the six vessels of the dissolution apparatus USP type-II (tablet dissolution tester, lab-India) containing 1000ml of different dissolutionmedia (0.1N HCL, 4.5 acetatebuffer and water), preheated at 37±0.5°C and the dissolution medium was stirred at 50RPM. Aliquots (10ml) of the dissolution medium were withdrawn at 5, 10, 20, 30, 45, 60 and 75 minutes and filteredthrough Millipore PVDF 0.45μm filter. Collect the filtrate after discarding few ml and record absorbance by UV spectrophotometer.²²

4.1 Solubility Study:

6mg of standard deflazacort was weighed and transferred into 10ml volumetric flask and make up the volume up to mark with dissolution media. Shake it for three hrs using thermomixer. Take 1ml offiltrate into 100ml volumetric flask and make up the volume up to mark with dissolution media. (6μg/ml). Record absorbance of each solution and calculate solubility.

4.2 Solution Stability study:

Stability study was performed by preparing solutions of pure drug and marketed formulation. An accurately weigh of 2mg of standard deflazacort was transferred in 100ml volumetric flask, add 10ml methanol, sonicate for 10minutes and make up the volume up to 100ml with dissolution media. Pipette out 6ml and dilute up to 20ml with dissolution media (6μg/ml). Similarly prepare 6 μg/ml solution of deflazacort tablets.

Solution Stability was calculated by below equation:

% difference = (initial absorbance – final absorbance)/ initial absorbance × 100.

5. Validation:

The propose method was validate for the parameters like linearity, accuracy, specificity and precision as per ICH guidelines.^23,24

5.1 Specificity:

Specificity of an analytical method indicates that the analytical method is able to measure accurately and specifically the analyte of interest without any interference from dissolution media (Blank), placebo and degradation products. Record spectra of dissolution media, placebo, standard deflazacort (6μg/ml) and sample solution (6μg/ml) in range of 200-400nm. Check the interference from dissolution media and placebo.:

5.2 Linearity:

Linearity of the method was determined by analyzing five independent levels of calibration curve in the range of 1.2 to 7.2μg/ml for deflazacort. Plot graph of absorbance v/s concentration. The correlation coefficient and regression equation for drug was calculated. Stock solution for linearity was prepared by accurately weigh and transfer 4mg of deflazacort into 100ml volumetric flask. Add 10ml methanol and make volume up to the mark with different dissolution media (40μg/ml). Requiredvolume was pipette from the stock solution to prepare solutions in the range of 1.2 to 7.2μg/ml.

5.3 Accuracy:

The accuracy of the proposed method was determined by performing recovery studies. It was carried out at 20% (1.2μg/ml), 100% (6μg/ml) and 120% (7.2μg/ml) range for deflazacort. The placebo solution was spiked with standard deflazacort and %recovery was performed. Standard solution for accuracy was prepared by accurately weigh and transfer 4mg of deflazacort into 100ml volumetric flask, add 10ml methanol and make volume up to the mark with different dissolution media (40μg/ml). Pipette out 1.5ml from the standard solution and dilute up to 20ml media (6μg/ml). Add 1.2mg, 6mg and 7.2mg to dissolution medium (1000mL) to get the drug concentration in the range of 20–120% of the nominal dose and stirred at 50rpm for 75min. Samples (10mL) were withdrawn, analyzed for drug content, and percent recovery was calculated.

5.4 Precision:

Precision was determined as system precision, repeatability, interday and intraday precision. System Precision was performed by recording absorbance of standard solution of 6μg/mL concentration for 6 times and %RSD was calculated. Intra-day precision was performed by conducting dissolution studies on three tablets on the same day and record absorbance at 247 nm of every dissolution sample at working concentration 6μg/mL and calculating relative standard deviation of %drug release at 75minutes. For interday precision, dissolution study was performed on three tablets by different analysts on two consecutive days and record absorbance at 247nm of every dissolution sample at working concentration 6μg/mL and calculating relative standard deviation of %drug release at 75minutes.

6. RESULTS AND DISCUSSION:

6.1 UV Method Development:

Spectra of working solution (6μg/ml) prepared in different dissolution media were recorded in range of 200-400nm and from that 247nm was selected as maximum absorptive point for deflazacort.

6.2 Dissolution Method Development:

The experimental results revealed that for the selection of volume, graph of time point vs % drug release was plotted for 500ml and 1000ml media volume. From graph, (figure 2) it was concluded that % drug release was greater than 85% in 1000ml. So, 1000ml volume selected. It was found that the drug was less absorbed from Millipore PVDF 0.45μm filter and showed less % difference as well as the pressure was also minimum. So, Millipore PVDF 0.45μm filter selected. Deflazacort tablets are immediate release tablets So, USP type -II paddle selected for the study.²⁵ At 50 RPM, deflazacort tablets show 95-100 % drug release so 50 RPM selected as agitation speed. Deflazacort tablets (dose 6mg) shows more than 90% drug release within 60 minutes and % RSD is less than 2% In different dissolution media (Table 2).

6.2.1. Solubility Study:

Based on solubility study different dissolution media selected for dissolution method development are 0.1 N HCl, water, 4.5 acetate buffer and 6.8 Phosphate buffer. Results of solubility study given in Table 1.

6.2.2. Solution Stability Study:

The solution stability study of deflazacort shows < 2% difference from initial which indicate that the drug was stable in the 0.1 N HCL, 4.5 acetate buffer, and water. In 6.8-phosphate buffer, % differencewas > 2%. Hence, it is concluded that the drug was unstable in 6.8 phosphate buffer and cannot be use in the estimation of release of deflazacort in tablet dosage form. The stability data of deflazacort in different media included in Table 1.

6.3. Validation:

The propose method was found to be specific as there was no interference of the excipients. The method was found to be linear with range of 1.2-7.2μg/ml and correlation coefficient was found to be>0.990 (Table 3). Accuracy of method is express in terms of recovery study at 20%, 100% and 120%.

Recovery of three replicate of drug was determined and the result of % recovery was found to be 98.19-101.40% (Table 3) which indicates that proposed method is accurate. The method precision was evaluated for repeatability, inter day and intraday precision and % RSD was found to be less than 2% which indicate the method developed is inter day précised. (Table 3).

Figure 2: Selection of Volume

Figure 3: % Drug Release of Deflazacort in different dissolution media

Table 1: Solubility and Stability of deflazacort in different dissolution media

Name of media	Solubility Study		Stability Study
Name of media	Absorbance	Solubility (mg/ml)	Initial absorbance	Absorbance after 24 hrs.	% Difference
0.1 NHCl	0.353	0.779	0.458	0.454	0.87
4.5Acetate buffer	0.078	0.192	0.408	0.405	0.74
6.8 Phosphate buffer	0.065	0.132	0.491	0.397	19.10
Water	0.060	0.017	0.425	0.424	0.24

Table 2: % Drug release of 6 mg deflazacort tablets in different dissolution media

Dissolution media	0.1 NHCl		4.5 Acetate buffer		Water
Time (min)	Average	% RSD	Average	% RSD	Average	% RSD
5	83.667	3.651	86.333	2.915	72.000	3.675
10	88.333	3.459	89.000	2.973	81.000	2.138
20	90.000	3.849	93.000	2.845	88.667	1.723
30	92.000	3.765	94.000	1.843	91.333	2.279
45	93.667	4.315	95.667	2.414	92.000	1.883
60	94.000	3.685	97.333	1.186	94.000	1.064
75	97.667	1.182	102.000	0.980	96.333	1.199

Table 3: Validation Parameters of Dissolution Method Developed for Deflazacort tablets (n=3)

Sr. No.	Validation Parameters		0.1 NHCl		4.5 Acetate buffer		Water
1.	Linearity
	CalibrationRange		1.2-7.2μg/ml		1.2-7.2μg/ml		1.2-7.2μg/ml
	RegressionEquation		y = 0.0772x -0.0045		y= 0.0745x-0.0115		y=0.08x-0.0093
	Correlation Co-efficient (R2)		0.9992		0.9984		0.9962
2.	Accuracy		% Recovery
	1.2 mg (20%)		97.88		100.46		96.38
	6 mg (100%)		99.92		99.76		99.92
	7.2 mg (120%)		103.08		103.72		99.76
3.	Precision		Mean	%RSD	Mean	%RSD	Mean	%RSD
	Systemprecision		0.452	0.313	0.412	0.730	0.463	0.298
	Intra-dayprecision		97.667	1.182	102.000	0.980	96.333	1.199
	Inter-dayprecision	Day1	98.000	1.020	101.667	0.568	96.667	0.597
		Day2	97.667	1.667	102.000	0.980	96.333	1.199

7. CONCLUSION:

Dissolution method for estimation of deflazacort in immediate released tablet dosage form was developed as per ICH guideline by use of double beam UV spectrophotometer. It was validated for selectivity, linearity, accuracy and precision parameters as per USP and ICH guidelines. Results of thework indicates that the method is specific as there is no interference observed. It is linear in the range of 1.2-7.2 μg/ml. Accuracy results are within acceptable limits (95-105%). RSD for precision studies is within acceptable limit (0.313-1.199%). Hence, it is concluded that the developed method can be used as dissolution method for estimation of release of deflazacort in tablet dosage form.

8. ACKNOWLEDGEMENTS:

The authors are gratefully acknowledging Piramal enterprise Ltd., Ahmedabad for providing the puredrug, equipment and guidance for the study. The authors thank the Quality Assurance department of B.K. Mody Government Pharmacy College, Rajkot to provide guidance for the study.

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Received on 27.06.2022 Modified on 29.11.2022

Research J. Pharm. and Tech 2023; 16(12):5818-5822.

DOI: 10.52711/0974-360X.2023.00942