A VALIDATED RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF TIROFIBAN HYDROCHLORIDE IN PURE AND MARKETED FORMULATION

About Authors:
Sukanto Paul, Krishan R Bhadu
Department of Quality Assurance, School of Pharmaceutical Sciences,
Jaipur National University,
Jagatpura, Jaipur-302025,
Rajasthan, India.

ABSTRACT
A validated reverse phase high performance liquid chromatography method has been developed for the simultaneous determination of Tirofiban hydrochloride in pure and marketed formulation. Chromatography was carried out on a BDS Hypersil C18 (4.6 mm × 250 mm, 5 μm) using Buffer: Acetonitrile in the ratio of 80:20 (v/v) as the mobile phase at a flow rate of 1.5 mL/min and eluents were monitored at 274 nm using UV detector at ambient temperature.  The average retention time of Tirofiban was found to be 9.124 min. The method was validated for linearity, precision, accuracy, specificity, robustness and solution stability. The calibration curve was linear (R²≥0.9999) over the range of 12.5-75 μg/mL.Limit of detection (LOD) and Limit of quantitation (LOQ) were 0.11 μg/mL and 0.33 μg/mL respectively. This method can be successfully employed for the quantitative analysis of Tirofiban hydrochloride in bulk drugs and formulations.

Reference Id: PHARMATUTOR-ART-1125

INTRODUCTION
Tirofiban hydrochloride 11(S)-2-(butylsulfonamino)-3-(4-[4-(piperidin-4-yl)butoxy]phenyl)propanoic acid(fig. 1), is a white free flowing powder, freely soluble in alcohol and .slightly soluble in water. It is a specific non peptide platelet fibrinogen receptor (GPIIb/IIIa)-antagonist and this compound is an antithrombotic and used in treatment of unstable angina[7]

Literature survey indicated that estimation of Tirofiban was done by using LC-MS.in biological fluid [8] It also includes quantitative determination of Tirofiban hydrochloride by UV spectrophotometry.[9] The literature survey does not reveal any validated assay method for the estimation of Tirofiban hydrochloride in bulk and Pharmaceutical dosage form by RP-HPLC method.

The objective of this work was to develop simple and rapid RP-HPLC method which would be accurate and precise.

The methods were validated according to ICH guidelines. The linearity of response, accuracy, and intermediate precision of the described methods has been validated.[1,2,3]

2. MATERIALS AND METHODS:
Tirofiban Hydrochloride of working standard grade was kindly supplied as gift sample by Venus Remedies Ltd., Panchkula, India and was certified to contain 83.93% (w/w), on as is basis. Tirofiban Hydrochloride Active Pharmaceutical Ingredient (API) was kindly supplied as gift sample by Taj Pharmaceuticals, Valsad, India and was certified to contain 99.6% (w/w), on as is basis. Acetonitrile and water used were of HPLC grade and were purchased from Spectrochem Pvt. Ltd. Mumbai, India. The parenteral formulation (Aggrastat injection) containing 5 mg of Tirofiban Hydrochloride was procured from local market and used for analysis of marketed formulation. After several trials taken by changing mobile phase, columns, flow rate, column temperature, the following chromatographic conditions were finalised.

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Chromatographic separation was performed on a Agilant 1200 at HPLC (Double pump) having a auto-sampler with 100μl loop capacity and UV, Prominence Diode Array Detector. The wavelength of detection chosen was 274 nm. A reverse phase Hypersil BDS C-18 column (4.6 mm × 250 mm, 5 μm) was used for the analysis. The mobile phase comprising of a mixture of phosphate buffer, and acetonitrile with pH 5.0 adjusted with 1.0N sodium hydroxide solution in the ratio of 80:20 % v/v with a flow rate of 1.5 ml/min. The injection volume was 20 μl.

In addition, an electronic balance (Shimadzu AX200), a pH meter (Systronics model EQMK VI) and a sonicator (Spectra Lab, model UCB 40) were used in this study.

2.1 Preparation of Mobile Phase: 13.6 gm of monobasic potassium phosphate was dissolved in 1000 ml water and the pH of the solution was adjusted to 5.0 by 1.0N sodium hydroxide solution.Mixture of above buffer and acetonitrile in the ratio of 80:20 % v/v for each 1000 ml of mobile phase was prepared.

2.2 Preparation of Stock Solution: 50 mg of Tirofiban hydrochloride was accurately transferred into 100 ml of volumetric flask and 30 ml mobile phase was added by pipette, sonicated to dissolve and the volume was made upto 100ml with mobile phase. Concentration of solution was 500µg/ml.
The solution was further diluted with the same solvent to obtain final concentrations of 50μg/ml. The standard solution was sonicated and injected.
The HPLC analysis was performed on reversed-phase high-performance liquid chromatographic system with isocratic elution mode using a mobile phase of Buffer: Acetonitrile (80:20, %v/v), pH 5.0 adjusted with 1.0N sodium hydroxide solution with flow rate 1.5 ml/min at 274 nm using UV detector then retention time was observed.

2.3 Preparation of Sample Solution: 50 mg of Tirofiban hydrochloride (Tirofiban infusion) was accurately transferred into 100 ml of volumetric flask and 30 ml mobile phase was added by pipette, the mixture was sonicated for 20 min in an ultrasonic bath and the volume was made upto 100ml with mobile phase. Concentration of solution was 500µg/ml. Above solution was filtered using membrane filter (0.22μ). Appropriate volume of the aliquot was transferred to a 50 ml volumetric flask and the volume was made up to the mark with mobile phase to obtain a solution containing 50 μg/ml of Tirofiban HCl. A 20 μl volume of above sample solution was injected into HPLC and peak areas were measured under optimized chromatographic conditions.

2.4 Method Validation: The method of analysis was validated as per the recommendations of ICH [7] for the parameters like accuracy, linearity, intermediate precision, specificity, detection limit, quantitation limit ruggedness, robustness and solution stability.
2.4.1 Linearity: Linearity study was carried out with sample solution containing Tirofiban infusion solution in six different concentrations (25-150% of the target concentration. A calibration curve was plotted between concentration and area response and statistical analysis of the calibration curve was done (fig. 2).
2.4.2 Accuracy: Accuracy was done by adding known amount of standard solution in the range 75-125% of the target concentration. The area observed at each level was compared with expected theoretical value calculated by injecting standard solution at one concentration.

2.4.3 Precision: Method precision was done by determining the assay in six different preparations of a homogeneous batch of Tirofiban infusion solution."
Intermediate precision was done by studying the variation in assay of homogeneous sample analyzed by 2 different equipment and analyst.

2.4.4 LOD and LOQ: The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using following formulae: LOD= 3.3(SD)/S and LOQ= 10 (SD)/S, where SD=standard deviation of response (peak area) and S= average of the slope of the calibration curve.

2.4.5 System Suitability: System suitability tests are an integral part of chromatographic method which are used to verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain system suitability test parameters were checked by repetitively injecting the drug solution at the concentration level 50 μg/ml Tirofiban HCl to check the reproducibility of the system.

2.4.6 Specificity: To carryout specificity test injection of placebo solution was injected to check whether there is no carry over in blank solution as well as the placebo does not interferes with the drug peak. The drug peak was also checked for its peak purity in presence of excipients.

2.4.7 Robustness: For robustness evaluation of HPLC method a few parameters like flow rate, columns and mobile phase composition were deliberately changed. One factor was changed at one time to estimate the effect. Each factor selected was changed at three levels (-1, 0, +1) with respect to optimized parameters. Robustness of the method was done at the concentration level 50 μg/ml for Tirofiban HCl.

2.4.8 Solution Stability: The standard and sample solutions were injected at different time intervals for 24 hrs to observe % difference in chromatographic responses relative to freshly prepared solutions.

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3. RESULTS AND DISCUSSION:
The mobile phase consisting of Buffer: ACN (80:20, %v/v), pH 5.0 adjusted with 1N sodium hydroxide, at 1.5ml/min flow rate was optimised which gave sharp peak with minimum tailing factor for Tirofiban HCl (fig. 3). The retention time for Tirofiban HCl was 9.124 min. UV spectra of Tirofiban HCl showed the λ_max at 274 nm, so this wavelength was selected as the detection wavelength.
*  The calibration curve for Tirofiban HCl was found to be linear over the range of 12.5-75 μg/ml. The graph gave a regression coefficient of 0.9999. The data of regression analysis of the calibration curves is shown in Table 1 & 2.

Table 1: Data for Calibration Curves

The calibration curve was plotted as Area vs. Concentration

Table 2:  Regression Analysis Data for Calibration Curves

Table 3: Results of Accuracy Data of Tirofiban HCl

Accuracy was checked at three levels viz. 75%, 100% and 5%

*  The accuracy results show that the method is accurate to practically achieve added amount at three different levels within 98%-102% with RSD less than 2%. The average %recovery was found to be 99.4%. The results are shown in Table 3.
*  The method precision gave results obtained within 2% RSD suggesting the method is precise. The results are shown in Table 4.

Table 4: Results for Method Precision

Table 5: Results for Intermediate Precision

Table 6: System Suitability Data

*  The LOD for Tirofiban HCl was found to be 0.11 μg/ml while LOQ was 0.33 μg/ml.
*  The developed method was also found to be specific, since there was no interference of excipients and the purity angle<purity threshold (fig. 4).
*  The results for system suitability parameters are shown in Table 6.
*  Results for robustness evaluation for drug are presented in Table 7. Insignificant differences in peak areas and less variability in retention times were observed. The results mentioned are of area obtained.
*  The results for solution stability are shown in Table 8. The standard and sample solutions were found to be stable up to 24 hrs.
*  The results for validation and system suitability test parameters are summarized in Table 9.
*  The proposed method was successfully applied to the determination of Tirofiban HCl in its dosage form. The results for the combination were comparable with the corresponding labelled amounts.

Table 7: Results of Robustness Study

Concentrations level used for robustness evaluation was 50 μg/ml. Three factors were slightly changed at three levels (-1, 0, 1). The results shows area obtained.

Table 8: Results for Solution Stability for Sample Preparation

Table 9: Summary of Validation Parameters of RP-HPLC Method for Estimation of Tirofiban HCl

S/N stands for signal to noise ratio

4.    CONCLUSION:
In the proposed study, HPLC method was developed for the estimation of Tirofiban HCl and validated as per ICH guidelines. Statistical analysis proved that method was accurate, precise, and repeatable. The developed method was found to be simple, sensitive and selective for analysis of Tirofiban HCl without any interference from the excipients. The method was successfully used for determination of drugs in a pharmaceutical formulation. Assay results for its dosage form using proposed method showed 98.42±0.34 % of Tirofiban HCl. Hence, the method is recommended for routine quality control analysis and also stability sample analysis.

5.    ACKNOWLEDGEMENTS:
The authors thank Venus Remedies Ltd., Panckula, India for providing Tirofiban hydrochloride as gift sample for this work. They also thank the management of Jaipur National University for providing required facilities to carry out this research work.

6.    REFERENCES:
1.    ICH, Q2A, Harmonized Tripartite Guideline, Test On Validation of Analytical Procedures, IFPMA, in: Proceedings of the International Conference on Harmonization, Geneva, March, 1994.
2.    ICH, Q2B, Harmonized Tripartite Guideline, Validation of Analytical Procedure: Methodology, IFPMA, in: Proceedings of the International Conference on Harmonization, Geneva, March 1996.
3.    ICH Guidance on Analytical Method Validation, in: Proceedings of the International Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, and September, 2002.
4.    Oertel R, Köhler A, Koster A and Kirch W, (5 June 2004)J. Chromatography B, ,805(1),181-185
5.    A. Bergquist,W. A. Hunke, R.A. Reed,D. Manas,R.J. Forsyth MS,R.R. Kenney,J. Cook,M. Holahan, (April 1999) Journal. Cli. Phar. Thera, 24(2), 125–132.
6.    HUANG rong-fu,ZHAO chi,SUN Jun-feng,WEI Qing, (May 2010), China pharmacy.
7.    wikipedia.org/wiki/Tirofiban

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