RP-HPLC Method Development and Validation for the Estimation of Seratrodast in Bulk and Pharmaceutical Dosage Form

 

B. Sivagami1*, D. Nagendramma1, R. Chandrasekar2, Dr. M. Niranjan babu2

1Associate Professor, Department of Pharmaceutical Analysis, Seven Hills College of Pharmacy, Venkataramapuram, Tirupati, Chitoor Dist, 517561, A. P.

2Department of Pharmacognosy, Seven Hills College of Pharmacy, Venkataramapuram, Tirupati, Chitoor Dist, 517561, A. P.

*Corresponding Author E-mail: sivagamib_27@rediffmail.com

 

ABSTRACT:

A simple, specific, accurate, and precise reverse phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the estimation of Seratrodast in Bulk and pharmaceutical dosage forms. The chromatographic separation of Seratrodast was achieved on a LC-1220 C 18 column with 100 x 4.6mm i.d. and 3.5µm particle size, using VW detection at 267ηm. The optimized mobile phase was consisted of Methanol: Phosphate buffer (pH adjusted to 4 with Orthophosphoric acid) (70:30, v/v). The flow rate was 1ml/min and effluents were monitored at 267ηm. Chromatogram showed the main peak at a retention time of 2.270min. The method was validated for linearity, accuracy, precision, and limit of detection, limit of quantification, robustness and ruggedness. The linearity was found in the concentration range of 10-150µg/ml. The Correlation coefficient was 0.999. The regression equation was found to be Y = 64890x + 18895. The limit of detection and limit of quantitation for estimation of Seratrodast was found to be 0.168µg/ml and 0.512µg/ml respectively. Recovery of Seratrodast was found to be in the range of 99.1-101.8%. Proposed method was successfully applied for the quantitative determination of Seratrodast in Bulk and Pharmaceutical dosage forms.

 

KEYWORDS: Seratrodast, RP-HPLC, Method validation, ICH guidelines.

 

 


INTRODUCTION:

Seratrodast is a thromboxane A2 (TXA2) receptor (TP) antagonist used primarily in the treatment of Asthma. TXA2 and other bronchoconstrictor prostanoids, like PGD2 and PGF2α, are generated in asthma and participate in acute and chronic inflammatory processes.

 

 

Seratrodast, being a TP receptor antagonist, inhibits the pathophysiological processes in asthma. Chemically it is known as 7-Phenly-7(2, 4, 5-trimethyl-3, 6-dioxocyclohexa-1, 4-dien-1-yl) hepatonic acid. Seratrodast is an orally active Quinone derivative and a potent TXA2 receptor antagonist used in the prophylactic management of asthma and treatment of allergic rhinitis.1-4 The survey of literature reveals that good analytical methods are not available for drugs like Seratrodast. Even though very few methods are available, many of them suffer from one disadvantage or the other such as low sensitivity, lack of selectivity, and simplicity. In the present work, an attempt was made to provide novel, simple, accurate and economic RP-HPLC methods for the effective quantitative determination of Seratrodast, in bulk as well as in pharmaceutical Dosage forms. The specific aim of the research work was to develop and validate reversed phase high performance liquid chromatography (RP-HPLC) method for the estimation of Seratrodast in bulk and pharmaceutical dosage forms. The developed methods can successfully be applied to estimate the amount of Seratrodast in formulations and bulk drugs. The proposed method to be validated as per ICH guidelines.

 

SERATRODAST:

Structure:

 

Fig 1: Chemical Structure of Seratrodast

 

MATERIALS AND METHODS:

Apparatus and chromatographic parameters:

The chromatographic separation was achieved on anAgilentLC-1220 C18 column (100 x 4.6mm i.d. and 3.5µm particle size) HPLC with VWD detector and a UV detector was employed in the study. A3 Star pH meter and Precision balance were the other instruments used for this study.

 

Drug samples:

The Seratrodast drug used for estimation for this study was procured as tablet form and brand name seretra 80 (10 mg)

 

Reagents and solutions:

Methanol Merck, (HPLC-Grade) Water Merck, (HPLC-Grade) Potassium dihydrogen phosphate Merck, (GR-Grade) Orthophosphoric acid Merck, (GR-Grade) and Seratrodast drug was used in the study. A mixture of Methanol: Phosphate buffer (pH adjusted to 4 with Orthophosphoric acid) (70:30, v/v) was used as a mobile phase.

 

Preparation of Standard stock solution of Seratrodast:

An accurately weighed quantity of Seratrodast100mg was transferred to 100ml volumetric flask, dissolved in Methanol, the final volume (100ml) was made with Methanol to obtain standard solution having concentration of 1000μg/mL. 1ml of this solution was transferred to 10ml volumetric flask, volume was made with Methanol. It gives 100µg/ml. These stock solutions were used to prepare further dilutions.

 

Preparation of sample solution:

Twenty tablets of Seratrodast were taken and into a fine powder of the tablets and the powder equivalent to 100mg of Seratrodast was weighed accurately and transferred into a 100ml volumetric flask. The contents were dissolved in Methanol and sonicated for 30 Minutes. This entire solution was filtered through 0.45 micron Whatmann filter paper (No. 41) and the final solution was made with Methanol to get the solution of 1000μg/ ml. 1ml of this solution was transferred to 10ml volumetric flask, volume was made with Methanol. It gives 100µg/ml.1ml of the solutions were pipetted out separately into 10 ml volumetric flask to give 10μg/ml concentration.

 

Method development:

Five trials were performed for the method development and the trail 5 was selected for the further work in HPLC as the result was satisfactory with a sharp peak with the retention time of 2.270mins.

 

Various standards in the range 10-150μg/ml of Seratrodast were injected onto the chromatographic system and the peak area was measured. A graph of peak area (on Y-axis) versus concentration (on X-axis) is plotted and the correlation coefficient was calculated.Table1gives information about the linearity data. From the results, the correlation coefficient for Seratrodast is 0.999 and, it can be concluded that the linearity is well within the limit. Hence the method is linear.

 

Table1: Summary of Linearity

Concentration (μg/ml)

Peak Area

10

7356988

20

13015418

30

19523127

40

26030836

50

32538546

60

39046255

80

52061673

100

65077092

120

78092510

150

97615638

 

 

Fig.2: Linearity graph of Seratrodast

Precision:

Precision of an analytical method is usually expressed as standard deviation and relative standard deviation. The standard deviation and % relative standard deviation are calculated from statistical formula. The standard deviation and relative standard deviation for the peak area were calculated from statistical formula.

 


 

 

 

Table 2: Summary of system precision & Summary of method Precision

Concentration

[µg/ml]

Peak area

Concentration

[µg/ml]

%Assay

50

33066181

50

101.1

50

33520677

50

98.98

50

33877386

50

100.2

50

34146354

50

99.83

50

33115940

50

101.42

50

34355975

50

99.5

Area Mean

33680419

Area Mean

100.1717

S.D

535482.9

S.D

0.93903

%R.S.D

1.5

%R.S.D

0.92

 

 

 


Accuracy studies:

The accuracy of the method, recovery studies were carried out by adding different amounts (50%, 100% and 150%) of bulk samples of Seratrodast within the linearity range were taken and added to the pre-analyzed formulation of concentration 20µg/ml. From that percentage recovery values were calculated. The results were within the range and were found to be highly accurate which was shown in table 4.


 

 

 

Table 3: Summary of Accuracy studies

Spiked level (%)

Formulation

Conc (µg/ml)

PureDrug Conc (µg/ml)

Amount

Found

% Recovery

% Mean recovery

SD

%RSD

 

50

50

25

79.55

99.45

99.66

0.599

0.601

50

25

79.41

99.2

50

25

80.27

100.34

 

100

50

50

81.14

101.4

101.3

0.602

0.594

50

50

81.57

101.9

50

50

80.62

100.7

 

150

 

50

75

79.94

99.92

100.17

0.296

0.296

50

75

80.1

100.1

50

75

80.42

100.5

 


 

Fig.3: Chromatogram of 50%

 

 

Fig.4: Chromatogram of 100%

 

Fig.5: Chromatogram of 150%

 

 

Limit of Detection and Limit of Quantification

The LOD and LOQ were calculated based on the standard deviation of the response and the slope of the constructed calibration curve, as described in International Conference on Harmonization guidelines Q2 (R1) 5-7was shown in table 5.

 

Table 4: Summaries of LOD and LOQ

Parameter

Concentration (μg/ml)

LOD

0.168

LOQ

0.512

 

Robustness:

The Robustness of the method was determined by making slight changes in the experimental conditions such as change in the flow rate, mobile phase.


 

Table 5: Summary of Robustness

Parameters

Mean peak Area (n=3)

S.D

%R.S.D

RT

Theoretical plates

Flow rate 0.9ml/min

33556661

127201.4

0.379

2.290

7421

Actual flow rate 1ml/min

32592992

76997.56

0.236

2.270

6831

Flow rate 1.1ml/min

31487991

71480.72

0.22

2.170

5961

10% less organic (60:40 v/v)

32786011

71496.27

0.218

2.273

6521

Actual mobile Phase ((70:30 v/v) )

32592992

76997.56

0.236

2.270

6831

10% more organic (80:20 v/v)

27085281

69940.64

0.258

2.610

7573

 


 

Fig.6: Chromatogram of Flow rate: 0.9ml/min

 

 

Fig.7: Chromatogram of Flow rate: 1.1ml/min

 

 

Fig.8: Chromatogram of Mobile phase: 10%less organic

 

 

Fig.9: Chromatogram of Mobile phase: 10%more organic


Table 6: Summary of Accuracy studies

Spiked level (%)

Formulation

Conc (µg/ml)

PureDrug Conc (µg/ml)

Amount

Found

% Recovery

% Mean recovery

SD

% RSD

 

50

50

25

79.55

99.45

99.66

0.599

0.601

50

25

79.41

99.2

50

25

80.27

100.34

 

100

50

50

81.14

101.4

101.3

0.602

0.594

50

50

81.57

101.9

50

50

80.62

100.7

 

150

 

50

75

79.94

99.92

100.17

0.296

0.296

50

75

80.1

100.1

50

75

80.42

100.5

 


Accuracy studies:

The accuracy of the method, recovery studies were carried out by adding different amounts (50%, 100% and 150%) of bulk samples of Seratrodast within the linearity range were taken and added to the pre-analyzed formulation of concentration 20µg/ml. From that percentage recovery values were calculated. The results were within the range and were found to be highly accurate which was shown in table 4.

 

 

Fig.3: Chromatogram of 50%

 

 

Fig.4: Chromatogram of 100%

 

 

Fig.5: Chromatogram of 150%

 

Limit of Detection and Limit of Quantification:

The LOD and LOQ were calculated based on the standard deviation of the response and the slope of the constructed calibration curve, as described in International Conference on Harmonization guidelines Q2 (R1) 6-8 was shown in table 5.

 

 

Table 7: Summaries of LOD and LOQ

Parameter

Concentration (μg/ml)

LOD

0.168

LOQ

0.512

 

Robustness:

The Robustness of the method was determined by making slight changes in the experimental conditions such as change in the flow rate, mobile phase and wave length.

 

 

Fig.6: Chromatogram of Flow rate: 0.9ml/min

 

 

Fig.7: Chromatogram of Flow rate: 1.1ml/min

 

 

Fig.8: Chromatogram of Mobile phase: 10%less organic

 

 

Fig.9: Chromatogram of Mobile phase: 10%more organic

 


Table 8: Summary of Robustness

Parameters

Mean peak Area(n=3)

S.D

%R.S.D

RT

Theoretical plates

Flow rate 0.9ml/min

33556661

127201.4

0.379

2.290

7421

Actual flow rate 1ml/min

32592992

76997.56

0.236

2.270

6831

Flow rate 1.1ml/min

31487991

71480.72

0.22

2.170

5961

10% less organic (60:40 v/v)

32786011

71496.27

0.218

2.273

6521

Actual mobile Phase ((70:30 v/v) )

32592992

76997.56

0.236

2.270

6831

10% more organic

(80:25 v/v)

27085281

69940.64

0.258

2.610

7573

 


RESULTS AND DISCUSSION:

A simple, rapid and precise method has been developed and validated for the drug Seratrodast. The estimation was carried out with A mixture of Methanol: Phosphate buffer (pH adjusted to 4 with Orthophosphoric acid) (70:30, v/v) was used as a mobile phase. Precision of the methods was studied by making repeated injections of the samples and system precision values were determined table – 2. The retention time was 2.270 mins. The calibration curve was linear over the concentration range of 10-150mg/ml. The LOD and LOQ values were found to be 0.168 and 0.512. The high percentage of recovery and low percentage coefficient of variance confirm the suitability of the method. Hence it was concluded that the RP-HPLC method developed was very much suit for routine analysis.

CONCLUSION:

A new RP-HPLC method was developed for the estimation of Seratrodast. The method gave good results within a short analysis time. The developed methods were validated in accordance with ICH guidelines and all of the results were within the limits. The HPLC method for the Estimation of Seratrodast in tablet dosage form was also found to be simple, rapid, precise, accurate and sensitive. The validated HPLC method can be used for the routine analysis of quality control samples. Since the developed method has been applied only to a single brand (SERETRA 80), the same methods are applicable to different brands.

 

 

 

REFRENCES:

1.       Terao S, Shiraishi M, Matsumoto T, Ashida Y. Thromboxane A2 antagonist--discovery of seratrodast Yakugaku Zasshi. 1999; 119(5):377-90.

2.       J. Raghuram, Development and Validation of RP-HPLC Method for the Estimation of Seratrodast in Bulk and Tablet Dosage Form, International Journal of Pharmacy, 2015; 5(2): 526-530.

3.       Mohan Goud, Srinivas Rao Avanapu, Development and validation of RP-HPLC method for the Assay of Seratrodast in pharmaceutical dosage form. Asian Journal of Research Chem, 2013; 6(8): 749-751.

4.       Jufang Xu, Yihua Zhang, Yuzhu Hu,Isolation and structural Elusidation of major photodegradation products of Seratrodast, Journal of Pharmaceutical and Biomedical Analysis, 2008; 48:78-84.

5.       ICH harmonized triplicate guide line text of validation of analytical procedures recommended for adaptation at September 4th of the ICH process on 27th October 1994.

6.       ICH: Validation of analytical procedure: Methodology Q2B; 1996.

7.       ICH Stability Testing of New Drug Substances and Products Q1A (R2), International Conference on Harmonization, 2003.

 

 

 

 

 

Received on 18.04.2018       Accepted on 15.05.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(2): 96-102.

DOI:   10.5958/2231-5675.2018.00019.4