Development and Validation of New Analytical Method for Estimation of Anti-histamine in Tablet Dosage Form

 

Kalyani Gudadhe1*, Mukund Tawar2

1Department of Quality Assurance, P.R. Pote Patil College of Pharmacy, Amravati 444604.

2Department of Pharmaceutics, P.R. Pote Patil College of Pharmacy, Amravati 444604.

*Corresponding Author E-mail: kalyanigudadhe9@gmail.com

 

ABSTRACT:

A rapid, sensitive and accurate method for Estimation of Anti- Histamine in Tablet Dosage Form by RP-HPLC. Mobile phase was Methanol: 0.1%. Ortho phosphoric acid (80:20% v/v). Flow rate was 0.9ml/min. The detection Wavelength was 227nm. The injection volume was 20l.The proposed method was validated for selectivity, precision, linearity, and accuracy. All validation parameters were within the acceptable range. Linearity studies for Rupatadine Fumarate were performed (10-60g/ml). The %RSD for accuracy found to be less than 2%. Assay was found to be 99.17 to 101.65 respectively. The range of 10-60g /ml was selected for the linearity of a standard Rupatadine Fumarate. The 80%, 100% and 120% levels of recovery study were selected to perform the recovery study. Linearity studies for Rupatadine Fumarate was performed on (10-60g/ml).

 

KEYWORDS: RP-HPLC, Rupatadine Fumarate, Methanol, Ortho phosphoric acid.

 

 


INTRODUCTION:

The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing one. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs.1-2

 

Rupatadine Fumarate C30H30CIN3O4 is a second generation, non-sedating, long-acting histamine antagonist with selective peripheral H1 receptor antagonist activity. It further blocks the receptors of the platelet-activating factor (PAF).3

 

Figure 1: Structure of Rupatadine Fumarate

 

An attempt has been made to develop and validate a simple, economic, rapid and accurate method. The proposed method was validated according to ICH guidelines4-6.

 

The reported simple RP-HPLC method used methanol: Methanol and 0.05% Ortho phosphoric acid (60:40v/v) as a mobile phase. The goal of this study was to develop a method without using buffer in mobile phase, has less run time, and more sensitive compare to developed method for analysis of Rupatadine fumarate, with extremely low LOD and LOQ values.5

 

MATERIALS AND METHODS:

Rupatadine Fumarate was procured from Yarrow-chem, Mumbai, India. HPLC Grade methanol, Ortho-phosphoric acid obtained from S.D. Fine Chem, Pvt. Ltd. and Laboratory grade Hydrochloric acid, hydrogen peroxide and sodium hydroxide obtained from Loba Chem, Mumbai.

 

Instrumentation:

Agilent technology HPLC instrument was used for the analysis purpose. Column Agilent C18 (250mm 4.6 mm). UV-2080 UV-Visible Spectrophotometer Analytical Technologies Pvt. Ltd.

 

RESULT AND DISCUSSION:

Chromatographic Conditions:

The chromatographic separation was carried out at room temperature on Agilent C18(250mm4.6mm) analytical column under RP-HPLC conditions. Mobile phase was Methanol: 0.1% Ortho phosphoric acid (80:20%v/v). The mobile phase was filtered through 0.45micron membrane filter which was degassed by ultrasonic bath. Flow rate was 0.9ml/min. The detection Wavelength was 227nm. The injection volume was 20l.

 

Selection and Preparation of Mobile Phase:

0.1% ortho phosphoric acid is prepare by take a 0.1ml ortho phosphoric acid and dissolved in 100ml water. An accurately measured quantity of 320ml methanol and 80 ml 0.1% ortho phosphoric acid were transferred in a 500 ml volumetric flask followed by proper mixing of resultant solution, then filtered through 0.45 membrane filter and sonicated with intermittent shaking for the 15 min in a bath sonicator. And inject the 50g/ml standard solution with injection volume 20l flow rate 0.9 ml/min and Wavelength 227nm.

 

Optimized Chromatographic Conditions:

The optimized chromatographic conditions are shown in Table .1

 

Table 1: Optimized Chromatographic Conditions

Column

Agilent C18 (250mm 4.6 mm)

Flow rate

0.9 ml/min

Wavelength

227nm

Injection volume

20 L

Mobile phase

Methanol: 0.1% Ortho phosphoric acid (80:20% v/v)

 

Figure 2: The chromatogram of method development and optimization

 

Preparation of Standard Solution:

Accurately weighed 10mg of Rupatadine Fumarate was transferred to a 10ml volumetric flask, sufficient amount of Methanol was added to dissolve it and volume was made up to 10ml. pipette out 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.6ml and diluted with mobile phase (Methanol: 0.1% Ortho phosphoric acid (80:20% v/v) up to 10 ml to get concentration of 10, 20, 30, 40, and 60g/ml for the linearity study.

 

Method Validation:

The validation is performed According to ICH guidelines.7-8

 

Linearity:

The Linearity of Analytical procedure is its ability to obtained test result which is directly proportional to the concentration of analyte in the sample. The range of 10-60g /ml was selected for the linearity of a standard Rupatadine Fumarate.

 

Figure 3: Chromatogram of Linearity

 

Figure 4: Standard calibration curve of Rupatadine Fumarate

 

Table 2: Results of linearity

Sr No.

Concen tration

Area I

Area II

Mean

SD

% RSD

1

10

335.29

336.01

335.65

0.51

0.15

2

20

739.94

743.73

741.84

2.68

0.36

3

30

1150.26

1141.85

1146.06

5.95

0.52

4

40

1562.06

1556.27

1559.17

4.09

0.26

5

60

2397.8

2397.05

2397.43

0.53

0.02

r2= 0.999

Slope = 41.23

Y-intercept= 83.38

Avg. SD=2.75

 

 

System Suitability Test:

The system suitability test is a pharmacopoeial requirement and is carried out to verify whether the analytical system is adequate or not for analysis of drug9. The 20g/ml of Rupatadine Fumarate standard solution was prepared and injected same solution into three replicates the Chromatogram of system suitability test.

 

Figure 5: System Suitability Test Chromatogram

 

Table 3: Result of system suitability

Sr. No

Concentration (g/ml)

Peak area

No. of Theoretical plates

Asymmetric factor

 

1

20

746.08

8096

0.77

2

20

745.59

7867

0.77

3

20

746.34

8065

0.76

Mean 746.00

S.D. 0.346482

%RSD 0.04644

 

Accuracy Study:

The recovery study was performed to evaluate the developed method was accurate for the analysis of Rupatadine Fumarate. The 80%, 100% and 120% levels of recovery study were selected to perform the recovery study.


 

Table 4: Result of % Recovery Test

Level of % recovery

Amount of tablet solution taken (g/ml)

Amount of standard solution add (g/ml)

Area

Amount found

Amount Recovery

% Recovery

80%

10

8

659.39

18.01

8.01

100.12

10

8

661.42

18.06

8.06

100.75

100%

10

10

742.87

20.04

10.04

100.40

10

10

740.32

19.97

9.97

99.70

120%

10

12

820.11

21.91

11.91

99.25

10

12

821.23

21.94

11.94

99.50

 


Table 5: Statistical result of accuracy

Level of % recovery

Mean

% Recovery

SD

%RSD

80%

100.44

0.45

0.44

100%

100.05

0.49

0.49

120%

99.38

0.18

0.18

Mean

99.95

 

 

Precision:

ICH defines the precision of an analytical procedure as the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.10-11 Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of the method was demonstrated by intra-day and inter-day studies. The 10g/ml, 30g/ml, 60g/ml standard Rupatadine Fumarate solution was suitably selected for method repeatability.

 

A) Intra-day Precision:

In the intra-day studies, 2 replicates of 3 standard solutions (10g/ml, 30g/ml, 60g/ml) were analyzed in a same day and percentage RSD was calculated.

 

B) Inter-day Precision:

For the interday variation studies, 2 replicates of 3 standard solutions (10g/ml, 30g/ml, 60g/ml) were analyzed on 2 consecutive days and percentage RSD was calculated.

 


 

Table 6: Result of Intra-day Precision

Sr No.

Conc.

Area-I

Area-II

Mean

Amount Found

%Amount Found

SD

% RSD

% RSD mean

1

10

333.6

332.54

333.07

10.10

101.04

0.75

0.23

0.093

 

 

2

30

1152.24

1151.71

1151.98

29.96

99.86

0.37

0.03

3

60

2400.74

2401.31

2401.03

60.26

100.43

0.40

0.02

 

Table 7: Result of Inter-day Precision

Sr No.

Conc.

Area-I

Area-II

Mean

Amount Found

%Amount Found

SD

% RSD

% RSD Mean

1

10

339.83

336.18

338.01

10.22

102.2

2.58

0.76

 

0.35

2

30

1156.52

1152.91

1154.72

30.03

100.1

2.55

0.22

3

60

2402.09

2404.5

2403.30

60.31

100.5

1.70

0.07

 


Robustness: ICH defines the robustness of an analytical method was carried out to confirm the analytical method remains unaffected by small variations in the optimized method parameters, the 20g/ml of standard solution were injected for a each varied conditions like change in flow rate 1 ml/min, change in wavelength 1 nm and change in composition of mobile phase 1 ml and the chromatograms were recorded, then results were obtained by calculating the %RSD of a peak area for each varied conditions.

 

Table 8: Result of robustness

Flow Rate

Sr. no

Conc.

(g/ml)

Area

SD

% RSD

% RSD Mean

(0.8ml/min)

1

20

847.72

 

4.75

 

0.56

 

 

 

0.78

2

20

854.44

(1.0ml/min)

1

20

682.67

 

6.78

 

1.00

2

20

673.08

Mobile Phase

Sr. no

Conc.

(g/ml)

Area

SD

SD

% RSD

Mean

(79:81%v/v)

1

20

739.38

 

8.34

 

1.12

 

 

 

0.29

2

20

751.15

(81:19% v/v)

1

20

747.04

0.84

0.11

2

20

748.23

Wavelength

(nm)

Sr. no

Conc.

(g/ml)

Area

SD

%RSD

% RSD

(226nm)

1

20

745.39

 

3.50

 

0.47

 

 

0.28

2

20

740.44

(228nm)

1

20

747.96

 

5.07

 

0.11

2

20

740.79

 

Ruggedness:

ICH defines the ruggedness or Intermediate precision of a method was assessed to check the effect of change in analyst on analysis, different laboratories, instruments and environmental conditions of Rupatadine Fumarate, the linearity as a method validation parameter was repeated by second/other analyst to verify the ruggedness, and %RSD was calculated. The %RSD was found to be 0.63 which is under accepted criteria; hence the developed method was rugged.12

 

Table 9: Result of Ruggedness

Sr No.

Concen tration

Area-I

Area-II

Mean

SD

% RSD

1

30

1963.64

1945.15

1954.40

13.07

0.67

2

40

1986.66

1989.14

1987.90

1.75

0.09

3

60

2227.84

2192.09

2209.97

25.28

1.14

AVG SD = 13.36

AVG %RSD = 0.63

 

LOD and LOQ:

ICH defines the detection limit of an individual analytical procedure as the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The limit of detection (LOD) is the point at which a measured value is larger than the uncertainty associated with it. It is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified.13

 

ICH defines the limit of quantitation (LOQ) of an individual analytical procedure as the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities or degradation products.14

 

The limit of detection and limit of quantitation were calculated on the basis of standard deviation of the accuracy response and slope of the linearity calibration curve. The result of LOD and LOQ was shown in Table 12.

 

Table 10: Results of LOD and LOQ

Drug

LOD

LOQ

Rupatadine Fumarate

0.2201

0.6669

 

Forced Degradation Study:

In order to establish the forced degradation study, the standard API was subjected in to the various stress conditions as follows15-18:

 

Alkaline Degradation:

An accurately weighed quantity of 10mg Rupatadine Fumarate was transferred to the round bottom flask, then 10ml of 0.1N NaOH was added into it, afterwards; refluxed at 60oC for 1hr by using reflux assembly, after 1hr the flask was removed to cool the solution, 500l (0.5ml) solution was withdrawn and transferred into 10 ml of volumetric flask and the final volume was made up to mark with selected mobile phase, the resultant sample was filtered through 0.45 membrane syringe filter. Finally, the sample was injected into the system to record the chromatogram. The percent degradation of drug in alkaline condition was found to be 5.06%.

 

Figure 6: Chromatogram of base degradation sample

 

Acid degradation:

An accurately weighed quantity of 10mg Rupatadine Fumarate was transferred to the round bottom flask, then 10ml of 0.1N HCl was added into it, afterwards; refluxed at 60oC for 1hr by using reflux assembly, after 1hr the flask was removed to cool the solution, 500l (0.5ml) solution was withdrawn and transferred into 10ml of volumetric flask and the final volume was made up to mark with selected mobile phase, the resultant sample was filtered through 0.45 membrane syringe filter. Finally, the sample was injected into the system to record the chromatogram. The degradation of drug in acidic condition was found to be 4.71%.

 

Figure.7 Chromatogram of acid degradation sample

 

Peroxide degradation

An accurately weighed quantity of 10mg Rupatadine Fumarate was transferred to the round bottom flask, 10 ml of 3% H2O2 was added, afterwards; refluxed at 60oC for 1hr. after 1hr flask was removed to cool the solution, 500l (0.5ml) of solution was withdraw and transferred to a 10ml volumetric flask and the final volume was adjusted up to mark with mobile phase, the resultant sample was filtered through 0.45 membrane syringe filter, and inject it. The degradation of drug in oxidative condition was found to be 4.04%.

 

Figure 8: Chromatogram of peroxide degradation sample

 

Thermal degradation:

The sample was heated in loss on drying oven at 60C in Petri plate for 24 Hour. An accurately weighed quantity of 10mg sample was transferred into 10ml of volumetric flask and 5ml of mobile phase was added to it, the resultant solution was sonicated for 25min with intermittent shaking and diluted up to the mark with mobile phase, then it was allowed to settle for 15 min, the 100l of supernatant solution was diluted up to 10ml by mobile phase, then filtered through 0.45 membrane syringe filter-media and injected into the system to obtain the chromatogram Figure. 9. The degradation of drug in thermal condition was found to be 5.45%.

 

Figure 9: Chromatogram of thermal degradation sample

 

Table 11: Result of forced degradation study

Sr. No.

Stress Condition

% Degradation

1

Alkaline condition

5.06%

2

Acidic condition

4.71%

3

Oxidative condition

4.04%

4

Thermal condition

5.45%

 

The validation of the developed method was carried out as per ICH guidelines and the results were found to be complying with the acceptable limit.

 

Table 12: Results of validation

Sr. No.

Parameter

Result

Acceptance criteria

1

Linearity

10-60 g/ml,

R2 = 0.999

R2 >0.999

2

System suitability test

T.plates=8009,

A. factor=0.76

T.Plates > 2000, A. factor<1.75

3

Accuracy

99.95

98-102%

4

Intraday precision

0.09%

%RSD < 2%

5

Intra-day precision

0.35%

% RSD < 2%

6

Specificity

No Interference

No Interference

7

Robustness

Change in wavelength: 0.28%, Change in flow rate:0.78%, Change in composition: 0.29%

 

% RSD < 2%

8

Ruggedness

0.63

% RSD < 2%

9

LOD and LOQ

0.22 and 0.66

LOQ is three times of LOD

 

The forced degradation study was carried out in acid, base, peroxide and thermal stress condition. In alkaline condition 5.06% drug was degraded. In acidic condition 4.71% drug was degraded and in oxidative drug was 4.04% degraded. In thermal degradation, 5.45% of drug was degraded at 600C.

 

CONCLUSION:

The present research work based on Stability Indicating RP-HPLC Method Development and Validation for the Estimation of Rupatadine Fumarate in bulk and pharmaceutical dosage form, was successfully completed. The parent drug and degradation products were well resolved under optimized chromatographic condition indicating the selective nature of developed method. Based on the test results, it was concluded that the method is simple, accurate, sensitive, precise, rapid, and free from any kind of interference of the excipients from the formulation; therefore the proposed method can be used for routine analysis of estimation of Rupatadine Fumarate in its tablet formulation.

 

ACKNOWLEDGEMENT:

The Authors are thankful to the management of P.R. Pote Patil College of Pharmacy for providing us with the facilities to carry out this work without which the research could not have been performed so well and good.

 

REFERENCES:

1.      Malviya VR, Pande SD, Bobade NN. Preparation and Evaluation of Sustained Release Beads of Zolmitriptan Hydrochloride. Research Journal of Pharmacy and Technology. 2019;12(12):5972-6.

2.      Shaiba M, Devi K, Prashanthi R, Raghavi K, Sindhura M. High Performance Thin Layer Chromatographic Estimation of Rupatadine Fumerate. Research Journal of Pharmacy and Technology. 2011;4(3):420-2.

3.      Fidan V, Fidan T. Fixed drug eruption against rupatadine fumarate. Journal of Craniofacial Surgery. 2011 Sep 1;22(5):1682-3.

4.      Malviya VR, Pande SD. Road CKN. Preparation ad Evaluation of Zolmitriptan Hydrochloride Lozenge. J Pharma Res. 2019;8(8):624-9.

5.      Pulla RP, Sastry BS, Prasad YR, Raju NA. Simultaneous Estimation of Metformin HCl and Sitagliptin Phosphate in Tablet Dosage Forms by RP-HPLC. Research Journal of Pharmacy and Technology. 2011;4(4):646-9.

6.      Malviya V, Thakur Y, Gudadhe SS, Tawar M. Formulation and evaluation of natural gum based fast dissolving tablet of Meclizine hydrochloride by using 3 factorial design 2. Asian Journal of Pharmacy and Pharmacology. 2020;6(2):94-100.

7.      Nagappan KV, Meyyanathan SN, Raja RB, Reddy S, Jeyaprakash MR, Birajdar AS, Bhojraj S. A RP-HPLC method for Simultaneous Estimation of Ambroxol Hydrochloride and Loratidine in pharmaceutical formulation. Research Journal of Pharmacy and Technology. 2008;1(4):366-9.

8.      Malviya V, Manekar S. Design, Development and Evaluation of Aceclofenac and Curcumin Agglomerates by Crystallo Co-Agglomeration Technique. Research Journal of Pharmacy and Technology. 2021 Mar 18;14(3):1535-41.

9.      Valarmathy J, Samueljoshua L, Rathinavel G, Thanuja CS, Sivakumar T. RP-HPLC Method development and validation for assay of Levetiracetam in tablet dosage Form. Research Journal of Pharmacy and Technology. 2008;1(4):395-7.

10.   Malviya V. Preparation and Evaluation of Emulsomes as a Drug Delivery System for Bifonazole. Indian Journal of Pharmaceutical Education and Research. 2021 Jan 1;55(1):86-94.

11.   Dhongle PS, Sahare SJ, Dhongle SS, Mundhey AS, Wate SP. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Cetirizine Hydrochloride and Phenylpropanolamine Hydrochloride in Tablet Dosage Form. Research Journal of Pharmacy and Technology. 2011;4(9):1471-4.

12.   Malviya V, Ladhake V, Gajbiye K, Satao J, Tawar M. Design and Characterization of Phase Transition System of Zolmitriptan Hydrochloride for Nasal Drug Delivery System. International Journal of Pharmaceutical Sciences and Nanotechnology. 2020 May 31;13(3):4942-51.

13.   Dinda SC, Desireddy RB, Kumar PJ, Chand AS, Rao PS, Kumar BA. Development and validation of RP-HPLC method for qunatitative analysis of Amlodipine besylate in pure and pharmaceutical formulations. Research Journal of Pharmacy and Technology. 2013;6(2):204-7.

14.   Malviya VR, Tawar MG. Preparation and Evaluation of Oral Dispersible Strips of Teneligliptin Hydrobromide for Treatment of Diabetes Mellitus. International Journal of Pharmaceutical Sciences and Nanotechnology. 2020 Jan 31;13(1):4745-52.

15.   Pingale P, Singasane T. Development and validation of HPLC method for the determination of pregabalin in bulk and in pharmaceutical formulations. Research Journal of Pharmacy and Technology. 2012;5(6):829-33.

16.   Sudha T, Shanmugasundram P. Development and validation of RP-HPLC and HPTLC chromatographic methods of analysis for the quantitative estimation of raltegravir potassium in pharmaceutical dosage form. Research Journal of Pharmacy and Technology. 2011;4(11):1746-50.

17.   Nagavalli D, Sankar AS, Anandakumar K, Vetrichelvan T, Balaji M. Estimation of metaxalone in bulk and in tablet dosage form by RP-HPLC. Research Journal of Pharmacy and Technology. 2010;3(2):409-11.

18.   Burange PJ, Tawar MG, Bairagi RA, Malviya VR, Sahu VK, Shewatkar SN, Sawarkar RA, Mamurkar RR. Synthesis of silver nanoparticles by using Aloe vera and Thuja orientalis leaves extract and their biological activity: a comprehensive review. Bulletin of the National Research Centre. 2021 Dec;45(1):1-3.

 

 

 

Received on 24.06.2021 Modified on 23.12.2021

Accepted on 10.03.2022 Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech. 2022; 12(2):113-118.

DOI: 10.52711/2231-5713.2022.00019