Analytical Method Development and Validation of Beclomethasone Dipropionate and Fusidic Acid in pure and Pharmaceutical Dosage Form (Cream) by RP-HPLC

 

Anuradha Tiwari, Dimpy Patel, Ishvarchandra Parmar*, Vishalkumar Modi

Department of Pharmaceutical Chemistry, Department of Quality Assurance, S.S.R. College of Pharmacy,

S.S.R. Campus, Sayli Road, Silvassa - 396230, UT of Dadra and Nagar Haveli and Daman and Diu, India.

*Corresponding Author E-mail: ijparmar2266@gmail.com

 

ABSTRACT:

A simple, rapid, specific, cheap, eco-friendly, and accurate RP-HPLC method for simultaneous estimation of Beclomethasone Dipropionate and Fusidic Acid in pure and combination drugs has been developed and validated. A stationary phase of BDS Hypersil C18 ((250mm x 4.6m, 5m) and a mobile phase of Acetonitrile: Methanol: Orthophosphoric acid (60:20:20v/v) were used for the separation. The flow rate was continued at 1.0 ml/min and the effluent was observed at 230nm. The designated situations were appropriate for separating Beclomethasone Dipropionate and Fusidic acid eluted with a mean retention time of 7.2 and 5.1 minutes, respectively. The planned technique was validated as per the International Council for Harmonisation recommendation. Its linearity, specificity, system suitability, accuracy and precision, limit of detection, limit of quantitation, and robustness are some of the parameters that have been validated. The concentration range produced linear calibration curves of 1-6g/ml and 2-12g/ml for Beclomethasone Dipropionate and Fusidic Acid with R2 = 0.9961 and 0.9963. Percent RSD values for both intraday and interday were determined to be less than 2.0%. Demonstrating the accuracy of the approach. The technique was proven to be reliable. A successful application of the developed RP-HPLC method for the quantification of Beclomethasone Dipropionate and Fusidic Acid in marketed semi-solid dosage form (Cream).

 

KEYWORDS: Antibiotic, skin infections, Beclomethasone Dipropionate, Fusidic Acid, Method validation, RP HPLC.

 

 

INTRODUCTION: 

Beclomethasone Dipropionate (BD) belongs to the class of corticosteroids. It is chemically is (8S,9R,10S,11S,13S,14S,16S,17R)-9-Chloro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-[2-(propionyloxy)acetyl]-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate.It is used as an anti-inflammatory and anti-asthmatic medication, as well as a stimulant.Its hydrophilic 17-hydroxyl group forms the valerate ester with it, increasing its lipophilicity and making it more appropriate for topical application.

 

Corticosteroids are frequently utilized in topical preparations1. It is used for asthmatics, seasonal allergies, and varying stages that respond to corticosteroids. The active monoester, 17 monopropionate (17-BMP), acts as a mediator of anti- inflammatory actions.The active monoester, 17-monopropionate (17-BMP), acts as a mediator of anti-inflammatory actions. The binding affinity of 7-BMP in BD is 25 times that of dexamethasone and 13 times that of cortisol.

 

Fusidic Acid (FA)is an antistaphylococcal antibiotic recommended to treat skin infections. Chemically, it is (2Z). -2-[(3R,4S,5S,8S, 9S, 10S, 11R, 13R, 14S, 16S)] -16-acetyloxy-3,11-dihydroxy-4,8,10,14-tetramethyl-2,3,4,5,6,7,9,11,12,13,15,16-dodecahydro-1H-cyclopenta[a][phenanthren-17-ylidene] -6-methylhept-5-enoic acid.Fusidic Acid is used to treat bacterial infections and interacts with the elongation factor G (EF-G).In the 50S subunit of the ribosome, elongation factor G hydrolyses Guanosine-5'-triphosphate (GTP) and Guanosine Diphosphate (GDP) to generate energy for the translocation of peptidyl-transfer ribonucleic acid (tRNA) from the A to P site. After GTP hydrolysis, EF-G stays bound to the ribosome, preventing the next stage of protein synthesis2.It works by inhibiting peptidyl tRNA from translocating. It is employed to treat bacterial infections such as cellulitis, impetigo, and conjunctivitis in the eyes and on the epidermis (red, itchy eyes). Skin infections caused by susceptible strains of S. aureus, Streptococci species, and C. minutissimum are treated with fusidic acid, which acts as bacteriostatic antibiotic.Fusidic Acid helps prevent the growth of bacteria while the immune system clears the infection. Its working mechanism is to stop the growth of bacteria after it is applied to the skin.

 

Different method are used like HPLC,UV spectrophotometry, HPTLC, they developed to determination of Beclomethasone Dipropionate alone or in combination with other drugs and for same inFusidic Acid method used like RH LC, HPLC, HPTLC. Some methods for determination of Beclomethasone Dipropionate and Fusidic Acid were reported, but for the best results, and ecofriendly chemical are used in this research article and retention time is less, a new method has been developed for Beclomethasone Dipropionate and Fusidic Acid3,4.

 

 

Fig.1: Structure of Beclomethasone Dipropionate

 

Fig.2: Structure of Fusidic Acid

 

MATERIALS AND METHODS:

Chemicals and reagents:

All chemicals and substances used for method development are HPLC grade. Beclomethasone Dipropionate and Fusidic Acid were kindly supplied as gift samples for KLM, Lab. Pvt. Ltd., Vadodara, Gujrat. The commercial fixed dose combination product and bulk powder were obtained from a local market. The HPLC grade acetonitrile, methanol, and water were purchased from Fisher Scientific, Mumbai, India. Orthophosphoric Acid (Molychem Pvt. Ltd., Mumbai, India. All the remaining chemicals and reagents are pure analytical grade.

 
Instruments and Apparatus:

A high-performance liquid chromatographic system (Jasco LC-4000 quaternary pump) with an auto sampler and PDA detector, a digital ultra-sonication cleaner (Equiptron-CD-4820), The Lab Solutions application was used to record the data. BDS Hypersil C18 (250 mm x 4.6mm, 5m) Column was used at room temperature (25°). An analytical balance (Precise) for weighing of samples, conical flasks, beakers, volumetric flasks, and borosilicate glass was used in the study.

Chromatographic conditions:

1.    Column: BDS Hypersil C18 (250*4.6)mm; 5µm

2.    Mobile Phase: Acetonitrile: Methanol: Orthophosphoric acid 60: 20:20v/v

3.    Flow Rate:1.0ml/min

4.    Detection Wavelength: 230nm

5.    Injection volume: 20.0μl

 
Preparation of stock solution:5

Standards solutions (stock solutions) are prepared by dissolving 5mg of Beclomethasone Dipropionate and Fusidic Acid (weighed accurately) each in methanol as the mobile phase in 2 separate 50-ml volumetric flasks, and sonicating for 10minutes. The final make with solution is made up to 50ml with methanol to get a stock solution containing 100ppm of Beclomethasone Dipropionate and Fusidic acid solution, respectively.

 

Preparation of Sample Solution:

A samplecombinationis prepared by addition or mixingthe sample accurately weighed and prepared by dissolving 1g of cream in 100ml volumetric flask with Methanol, and sonicated for 20-25minutes and the remaining methanol add to final volume up to 50ml to getstandard solution containing 100ppm of Beclomethasone Dipropionate and Fusidic Acid respectively.

 

Preparation of Level (Dilution):

The standard solutions of Beclomethasone Dipropionate and Fusidic Acid are in the range of 1-6 and 2-10ppm. From 100ppm standard stock solution, 6 solutions of different concentrations were prepared from 100ppm standard stock solution by pipetting out 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6ml and 0.2, 0.4, 0.6, 0.8, 1, and 1.2 from stock solution and added in a 10ml volumetric flask, and the final volume was made up with mobile phase.

 

System Suitability Solutions:6,7
System suitability analysis is an important stage in many analytical methods. The tests are based on the theory that a system can be evaluated as a whole if its components—tools, electronics, analytical processes, and test samples—are all used together. The parameters for a system suitability test that must be created for a specific operation depend on the kind of procedure that is being verified. It is integral part of liquid chromatography in this way. System suitability was performed by six injections of standard solution (100 ppm) and two injections of sample solution (100 ppm) to HPLC under optimised chromatographic conditions These tests include tests for the number of theoretical plates, resolution, and tailing factor. The data for system suitability parameter are shown in the table 5.

 

Method development and optimization of chromatographic parameters:7,8

a.    Selection of wavelength:

The appropriate choice of wavelength determines the sensitivity of the HPLC process that uses UV detection. An optimum wavelength is one that responds well and allows for the detection of all drugs. A UV spectrum of Beclomethasone Dipropionate and Fusidic Acid was recorded between 200-400nm.

 

b.    Selection of chromatographic methods:

The right choice of chromatographic parameters depends on the sample's type (ionic, ionisable, or neutral molecule), molecular weight, and stability. Reversed phase chromatography can be employed since it is straightforward and appropriate for the polar, ionic drugs that were selected.
 
Validation of Developed RP-HPLC Method:8

Validation of the developed method was carried out according to the ICH recommendation Q2 (R1). Performance attributes are defined in terms of analytical metrics. The validation of the RP-HPLC technique for the measurement of beclomethasone dipropionate and fusidic acid was completed in compliance with ICH guidelines.

 

1.    Linearity:

Linearity is a series of solutions made by mixing the standard solution with mobile phase were analysed to determine linearity. Calibration curve of Beclomethasone Dipropionate and Fusidic Acidwas constructed by plotting peak area vs. applied concentrations. The obtained data demonstrated a strong relationship between peak area and pure drug  concentration range and Calculated the correlation coefficient between the average area at each level and the concentration of analysis show in table no 1.

 

2.    Accuracy (%Recovery):9-11

The degree of agreement between a value considered to be either the value discovered is expressed as the correctness of the analytical technique and is compared to a conventional true value or a recognised reference value. The incorporation of conventional techniques was used to conduct recovery studies. The percent recovery is determined after examining three concentration levels (50%, 100%, and 150%) with a specified amount of analyte added and three replications of the test shown in table No 2.

 

3.    Precision (Intraday and Interday):12-14

The precision of an analytical technique is defined as the degree of agreement between a set of measurements made using several samples of a single homogeneous sample under the given conditions. Within-day precision (repeatability), between-day precision, and precision between research labs are the three levels of accuracy (reproducibility) Six injections of 100% of the test concentration readings are used to establish repeatability. The accuracy of both intraday and interday precision is being investigated.

 

4.    Limitofdetection and Limit of Quantification (LOQ):15-18

An analytical process must be validated using the limits of detection (LOD) and limit of quantification (LOQ). The smallest amount of analyte in a sample that may be detected is known as LOD. It is not essential to quantify this sum. The lowest quantitative level of the analyte is known as LOQ. There are three techniques to calculate LOD or LOQ that are based on visual inspection, signal to noise ratio, standard deviation (SD) of the response, and slope, there are three techniques to calculate LOD or LOQ

LOD  = 3.3* σ /S

LOQ  = 10* σ /S

Where,

Sigma (σ) = Standard deviation of intercept

S = Mean of the slope

 

5.    Robustness:19,20

Robustness means theability of an analytical procedure to be unaffected by minor but intentional changes to method parameters, and it gives an indication as to how reliable the process will be under typical conditions. A method's robustness was determined by intentionally altering the flow rate, the composition of the mobile phase, and the wavelength variation to assess the impact on the process shown in table No 3.

 

RESULTS AND DISCUSSION:

This RP-HPLC method was developed for finding and separating 2 drugs mostly prescribed to treat bacterial skin infections, such as eczema and dermatitis. Beclomethasone individual prescript for reduces the swelling, itching, and redness and Fusidic acid individual prescript for it works by stopping bacteria from growing.  In the present study, numerous mobile phase compositions were tried. A mixture had excellent peak symmetry and satisfactory separation. Acetonitrile: Methanol: Orthophosphoric acid 60:20:20v/v at a flow rate of 1ml/min. The ideal wavelength for detection was established at 230nm, with much better results found for two combination drugs.

 

 

(Peak no.1 is indicating Fusidic Acid and, Peck no.2 indicatingBeclomethasone Dipropionate)

Fig.3: Chromatogram of standard Beclomethasone Dipropionate and Fusidic Acid Solution

 

 

(Peak no.1 is indicating Beclomethasone Dipropionateand, Peck no.2 indicatingFusidic Acid)

Fig.4: Chromatogram of sample Beclomethasone Dipropionate and Fusidic Acid Solution

 
Linearity:

The calibration curves have a linear rangeSix concentrations were chosen, ranging between 1-6ug/ml for Beclomethasone Dipropionate and 20-10ug/ml for Fusidic Acid. As the developed method can detect and quantify the drug up to ug/ml level, thus the developed method was found to be sensitiveEach concentration was injected in triplicate, and the correlation coefficients for all of the analytes were determined to be greater than 0.999. The outcomes are displayed in table No.1

 

Table: 1 Linearity Data for Beclomethasone Dipropionate and Fusidic Acid

Parameters

Beclomethasone Dipropionate

Fusidic Acid

Concentration, (µg/ml)

1-5ug/ml

2-10ug/ml

Correlation coefficients (R2)

0.9961

0.9963

SD of intercept

937.9

4051.3
 
Accuracy (% Recovery):

For each of the mentioned concentrations, there were three levels of recovery: 50%, 100%, and 150%.The results of the recovery experiments were reported after the analysis of three samples for each level of recovery shown in table No.2.

 

Table: 2 Different Recovery Studies' Results

Drug

Recovery level

Drug taken (µg/ml)

Standard drug added (µg/ml)

Total conc. of drug (µg/ml)

%Recovery

Mean±SD

% RSD

Beclomethasone Dipropionate

50

2

2

4

101.94

101.35±0.88694

0.8751

50

2

2

4

101.78

 

 

50

2

2

4

100.33

 

 

100

2

4

6

100.10

100.16±0.07505

0.0749

100

2

4

6

100.20

 

 

100

2

4

6

100.20

 

 

150

2

6

8

102.10

102.12±0.02516

0.0246

150

2

6

8

102.15

 

 

150

2

6

8

102.12

 

 

Fusidic Acid

50

4

4

8

102.17

101.03±1.02101

1.0106

50

4

4

8

100.34

 

 

50

4

4

8

100.47

 

 

100

4

8

12

101.83

100.6±0.556506

0.553186

100

4

8

12

102.92

 

 

100

4

8

12

102.18

 

 

150

4

12

16

100.55

100.65±0.21455

0.213167

150

4

12

16

100.90

 

 

150

4

12

16

100.51

 

 
 
Precision (Intraday and Interday):

The precision value for this method was determined by determining the %RSD for the 2 separated drugs, and the %RSD was calculated from nine samples of the standard solutions of Beclomethasone Dipropionate and Fusidic Acid of 0.0855% and 0.0381% of Interday, and the %RSD of Beclomethasone Dipropionate and Fusidic Acid was 0.0786% and 0.0386% of Intraday, respectively. The method's accuracy was confirmed because every result met the requirements.

 

LOD and LOQ:

The LOD and LOQ were calculated using the signal to noise ratio. Considering the minimum concentration that can be identified with a certain level of confidence, the LOD for Beclomethasone Dipropionate and Fusidic Acid was 0.056 and 0.521μg/ml, respectively. Upon some repetitive dilutions, LOQ was assessed as the lowest determined concentration. Beclomethasone Dipropionate and Fusidic Acid were 0.1721 and 1.5790 μg/ml, respectively.

 

Robustness:

The robustness of the developed method was assessed by changing three parameters: flow rate (1.1ml/min and 0.9ml/min), mobile phase composition (Acetonitrile: Methanol: orthophoric acid (58:22:20) v/v and Acetonitrile: Methanol: orthophoric acid (62:18:20) v/v) and wavelength (228nm and 232  1nm ). Small deliberate adjustments in system parameters, including flow rate, mobile phase composition, and wavelength, have been used to evaluate the suggested technique. The result of %RSD was less than 2, indicating that the suggested approach was judged to be reliable. For both altered and unaltered conditions, the %w/w was determined to be between 98 and 102% shown in table no. 3.

 

 

Table: 3 Result of Robustness Studies

Beclomethasone Dipropionate

Fusidic Acid

Parameter

% RSD

Parameter

%RSD

Flow Rate Ml/min

Flow Rate Ml/min

0.9

0.2471

0.9

0.5815

1.1

0.2507

1.1

0.4862

 

Mobile Phase

Mobile Phase

Acetonitrile: Orthophosphoric acid: Methanol (58:20:22V/V)

0.1765

Acetonitrile: Orthophosphoric acid: Methanol (58:20:22V/V)

0.1778

Acetonitrile: Orthophosphoric acid: Methanol (62:20:18V/V)

0.0369

Acetonitrile: Orthophosphoric acid: Methanol (62:20:18V/V)

0.0650

Wavelength

Wavelength

228nm

0.4818

228nm

0.6875

232nm

0.3027

232nm

0.4523

 

Application to Pharmaceutical Dosage Form:

The analysis of commercial semisolid dosage forms was performed to determine the %w/w of the proposed method. The %w/w for Beclomethasone Dipropionate and Fusidic Acid marketed formulations was found to be 99.2850% and 98.1741%, as shown in table No. 4.

 

Table: 4 Analysis of Marketed Creamformulation

Drug

Concentration

Area

% RSD

% w/w

Acceptance criteria

Beclomethasone

Dipropionate

4µg/ml

9694

0.0465

 

99.2850%

98-102%

9698

9689

Fusidic Acid

8µg/ml

47982

0.5939

98.1741%

98-102%

47432

47747

 

Table: 5 System Suitability Parametersand Validationparameter of Beclomethasone dipropionate and Fusidic Acid

Parameter

Beclomethasone dipropionate

Fusidic Acid

Retention time

7.2 min

5.1 min

Theoretical plates

8386

6683

Tailing factor

0.946

0.971

Resolution

7.379

7.289

Correlation

Coefficient (r2)

0.9963

0.9961

LOD (ug/ml)

0.056 ug/ml

0.521 ug/ml

LOQ (ug/ml)

0.1721 ug/ml

1.5790 ug/ml

Percent recovery (n=3)

100.20-102.55%

102.97-100.51%

Precision

(% RSD)

Intraday

Interday

Intraday

Interday

0.0786%

0.0855%

0.0386%

0.038%

Specificity

Specific,no interference

 

Specific,no interference

 

 

CONCLUSION:

An accurate, sensitive, and precise HPLC method for the simultaneous determination of Beclomethasone Dipropionate and Fusidic Acid in pure and pharmaceutical dosage form was developed. This method's benefit is that it allows for the simultaneous determination of both drugs. The method could be used to conduct regular quality control checks on synthetic mixtures and bulk medications.

 

ACKNOWLEDGEMENT:

The authors are thankful to SSR College Of Pharmacy Silvassa, SAYLI Management for providing the entirenecessary facilities and infrastructure.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 
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Received on 21.10.2022           Modified on 17.02.2023

Accepted on 22.04.2023          © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):327-332.

DOI: 10.52711/0974-360X.2024.00051