INTRODUCTION:

Seratrodast (fig-I) is a thromboxane A₂ receptor antagonist used primarily in the treatment of asthma ^[1-8]. Chemicaliy it is 7-phenyl-7-(2, 4, 5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl) heptanoic acid. It was the first TP receptor antagonist that was developed as an anti-asthmatic drug which does not affect thrombus formation thus ruling out any action on blood coagulation cascade.

Figure – I

Literature reveals different methods for its analysis in formulations. Hence our present plan is to develop a new, simple, precise and accurate method for its analysis in formulation after a detailed study, a new RP-HPLC method was decided to be developed and validated as per ICH norms^[09-11].

For the estimation of this method we used Phosphate buffer and Acetonitrile with a pH 6.8 adjusted with ortho phosphoric acid in the ratio of 50:40v/v. The column used was Thermo hypersil C 18, at a flow rate of 1.0 ml/min. and UV detector was employed in the study at 266nm.

MATERIALS AND METHODS:

Apparatus and chromatographic parameters:

A Waters HPLC with Alliance with Auto sampler with Empower 2.0 software with Thermo Hypersil C 18 (4.6 x 150mm, 5 mm, Make: Thermosil) column and UV detector was employed in the study. An Edwa pH meter Afcoset digital balance and ambient column oven were the other instruments used for this study.

Drug samples:

The Seratrodast drug used for estimation for this study was procured from tablet. The brand name seretra was used and the label claim was Seratrodast 80 mg in each tablet.

Reagents and solutions:

HPLC grade Acetonitrile, a GR grade/Merck Potassium di hydrogen phosphate, HPLC grade water and Seratrodast drug was used in the study. A mixture of Potassium di hydrogen ortho phosphate buffer, Acetonitrile and Water in the ratio of 50:40:10%v/v was used as a mobile phase at a pH 6.8 adjusted with ortho phosphoric Acid and it is also used as a diluent for preparing the working solution of drug. The mobile phase was degassed in ultrasonic water bath for 5 minutes and filtered through 0.45μm filter under vacuum filtration.

Preparation of the seratrodast standard and sample solution:

Standard solution preparation:

Accurately Weighed and transferred 40mg of Seratrodast working Standards into a 10 ml clean dry volumetric flask, add 7ml of diluent, sonicated for 5 minutes and make up to the final volume with diluents. Mixed well and filtered through 0.45µm filter.

Sample solution preparation:

5 tablets were weighed and crushed into powder, in order to calculate the average weight of each tablet. From that powder weight equivalent to 40mg of Seratrodast were transferred into a 100 mL volumetric flask, 80mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution 1ml was pipetted out into a 10 ml volumetric flask and made upto 10ml with diluent.

Method devlopment:

Three trials were performed for the method development and the best peak (fig - II) with least fronting factor was found to be the third peak with RT= 6.016 min.

Figure – II

Linearity graph

Figure – III. X-Axis = Concentration, Y-Axis = Peak area

Method validation:

Precision:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits and results were tabulated in table – I.

Acceptance Criteria:

The % RSD for the area of five standard injections results should not be more than 2%

Table – I:

S. No.	RT	Peak area	Average peak area	Standard deviation	% RSD
1.	6.130	6993040	6966748	71789.1	1.0
2.	6.133	7028808
3.	6.139	6933296
4.	6.142	7009617
5.	6.146	6999992
6	6.153	6835736

ACCURACY:

Injected the standard solutions of Accuracy -50%, 100% and 150% and calculated the Amount found, Amount added for Seratrodast and the individual recovery and mean recovery values tabulated in table – II.

Acceptance Criteria: The % Recovery for each level should be between 98.0 to 102.0%.

Table – III:

S.No	%Linearity Level	Concentration in ppm(Sera)	Area
1	50	200	3377483
2	75	300	5147445
4	100	400	6893280
5	125	500	8627527
6	150	600	10159638

RECOVERY STUDIES:

To determine the accuracy and precision of the proposed method recovery studies were carried out. A fixed amount of sample was taken and standard drug was added at 50%, 100% and 150% levels. The results were analyzed and the results were within the limits. The % recovery, Mean recovery and %Relative standard deviation value for Seratrodast drug was found to be 99.2-100.8%, 100.1% and 0.94 respectively.

LINEARITY AND CALIBRATION CURVE:

Working dilutions of Seratrodast in the range of 200-600ppm was prepared by taking suitable aliquots of working standard solutions of drug in different 10ml volumetric flask and diluting up to the mark with mobile phase. 20μl quantity of each dilution was injected in to the column at a flow rate of 0.8ml/min. the drug in the elute was monitored at 266nm, tabulated in table – III and the corresponding chromatogram were recorded. From these the mean peak areas were calculated and a plot of concentration vs peak areas was constructed (fig-III). The regression of the plot was computed by least square regression method. The slope and intercept value for calibration curve was y=17078x+8327 (R2=0.999) founded.

LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION:

Limit of Detection (LOD) is the lowest concentration of an analyte in a sample that can be detected but not quantified. LOD is expressed as a concentration at a specified signal to noise ratio. The LOD will not only depend on the procedure of analysis but also on the type of instrument. In chromatography, detection limit is the injected amount that results in a peak with a height at least twice or thrice as high as baseline noise level.

The LOD for Seratrodast was found to be 2.5

Limit of Quantification (LOQ) is defined as lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy and reliability by a given method under stated experimental conditions. LOQ is expressed as a concentration at a specified signal to noise ratio. In chromatography, limit of quantification is the injected amount that results in a peak with a height, ten times as high as base line noise level.

The LOQ for Seratrodast was found to be 9.6

ROBUSTNESS:

Robustness is determined by making deliberate changes in the chromatographic conditions like change in flow rate, mobile phase composition and temperature and evaluated for the impact on the method. It was observed from the chromatograms that the results were within the limits. This indicates that the method developed is robust.

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 Phosphate buffer and Acetonitrile with a pH 6.8 adjusted with ortho phosphoric acid in the ratio of 50:40:10%v/v. Precision of the methods was studied by making repeated injections of the samples and system precision values were determined table – IV. The retention time was 6.016 min. The calibration curve was linear over the concentration range of 200-600 ppm. The LOD and LOQ values were found to be 2.5 and 9.6. 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.

Table – IV:

S. No.	Parameter	Acceptance criteria	Observed value
1	Accuracy	95-105%	99.77%
2	Precision	RSD within 2%	0.94%
3	Linearity	R² not less than 0.99	R²=0.999
4	LOD	S/N=3	2.5
5	LOQ	S/N=10	9.6

CONCLUSION:

The proposed study describes a new and simple RP-HPLC method for the estimation of Seratrodast. The method validated was found to be simple, accurate and precise. Therefore the proposed study method can be used for quantification of Seratrodast in bulk and pharmaceutical dosage form.

REFERENCES:

1. Anon. Chronic Respiratory Diseases. In J. Bousquet and N. Khaltaev (Eds). Global surveillance, prevention and control of chronic respiratory diseases, A comprehensive approach. WHO library Publication, 2007.

2. Endo S and Akiyama K; Nippon Rinsho 54 (11): 3045–8. PMID 8950952.

3. Hada S, Hashizume M, Nishii S, Yoshioka F and Yasunaga K; Arerugi 42 (1): 18–25. PMID 8457165.

4. Dogné JM, de Leval X, Benoit P, Delarge J and Masereel B; Am J Respir Med 1 (1): 11–7. PMID 14720071.

5. Bronica tablets, Takeda Pharmaceutical Co. Ltd., Japan

6. Seretra tablets, Zuventus Healthcare Ltd., India

7. Dogné JM, Hanson J, de Leval X, Kolh P, Tchana-Sato V, de Leval L, Rolin S, Ghuysen A, Segers P, Lambermont B, Masereel B and Pirotte B; J Pharmacol Exp Ther 309 (2): 498–505. PMID 14742735.

8. Samara E, Cao G, Locke C. Population analysis of the pharmacokinetics and pharmacodynamics of Seratrodast in patients with mild to moderate asthma. Clin Pharmacol Ther 1997a; 62: 426-435.

9. Synder K.L, Krikland J.J and Glajch. J.L: Practical HPLC Method Development 2nd Edn, Wiley-Interscience Publication, USA, 1983; 1-10.

10. International conference on harmonization “Validation of analytical procedures Methodology”, 14, Federal Register Nov.1996; 1-8.

11. M A Haneef, B Rajkamal, G Tharun Goud, V. Mohan Goud. Development and Validation by RP-HPLC Method for Estimation of Zidovudine in Bulk and Its Pharmaceutical Dosage Form. Asian J. Research Chem. 6(4): April 2013.

Received on 13.05.2013 Modified on 18.06.2013

Asian J. Research Chem. 6(8): August 2013; Page 749-751

%Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	1093514	4.90	4.88	99.2%	100.1%
100%	2246802	10.0	10.0	100.3%
150%	3407885	14.8	15.2	100.8%