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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