Estimation of Tranexamic Acid Andethamsylate Using RP-HPLC

Estimation of Tranexamic Acid Andethamsylate Using RP-HPLC Chapter-3 Experimental work 3. EXPERIMENTAL WORK 3.1 MATERIALS AND METHODS Table 2. List of Chemical and standers used S.No Chemicals Manufacturer Name Grade 1 Water Processed in Bright Labs HPLC grade 2 Acetonitrile Fisher scientific HPLC grade 3 Orthophosphoric acid Merck GR grade 4 Tranexamic acid Sun pharma ltd BP 5 Ethamsylate Sun pharma ltd USP 6 KH2PO4 Merck GR grade 7 K2HPO4 Merck GR grade 8 Methanol Merck HPLC grade Table 3. List of instruments used S.No Instrumentname Model Number Soft Ware Manufacturers name 1 HPLC-auto sampler-UV detector ACME9000 Auto crome 3000 Youngline 2 Electronic balance Lab India 3 Sonicator CWUC9L 201402822 Spectrum tek 4 Vacuum Pump 28965405-289717 Vacuubrand 5 0.45 µ filter paper HPLC grade Rankem 3.2. Method development for the simultaneous estimation of Tranexamic acid andethamsylate by using RP-HPLC. Selection of mobile phase Selection of detectionwavelength Selection of column Selection of solvent delivery system Selection of flow rate Selection of column temperature Selection of diluent Selection of test concentration and injection volume 3.2.1. Selection of mobile phase Phosphate Buffer: Methanol (30:70) 3.2.2. Selection of wavelength 10mg Tranexamic acid and Ethamsylate were dissolved in mobile phase. The overlay spectrum was used for selection of wavelength for Tranexamic acid and Ethamsylate The iso-bestic point was taken as detection wavelength 286nm. 3.2.3. Selection of column Heart of HPLC made of 316 grade stainless steel packed with stationary phase. Silica based columns with different cross linking’s in the increasing order of polarity are as follows: à ¯Ã†â€™Ã… ¸- Non-polar-moderately polarPolar-à ¯Ã†â€™Ã‚   C1886 In reverse phase chromatography, hydrophobic interaction between drug molecule and the alkyl chains on the column packing material. Column is selected based on solubility, polarity chemical differences among analytes and Column selected: i.e. X-Bridge C18 (150 Ãâ€" 4.6 mm, packed with 5  µm), particle size Reasons: Better separation, Good tailing factor. 3.2.4. Selection of solvent delivery system Always preferable solvent delivery system. More chance of getting reproducible result on retention time of analytes. More economic than gradient technique. 3.2.5. Selection of flow rate Acceptable limit: Not more than 2.5 ml/min Flow rate selected was 1.0ml/min Flow rate is selected based on 1. Retention time 2. Column back pressure 3. Peak symmetry. 4. Separation of impurities. Reasons: For earlier elution of analyte and elution of all impurities within 10 min Information from the reference method in literature. 3.2.6. Selection of diluent Selection of diluents is based on the solubility of the analyte Diluent selected: Phosphate Buffer: Methanol (30:70 % v/v) Reason: Analyte is soluble in acetonitrile and water. 3.2.7. Selection of column temperature Preferable temperature is ambient or room temperature. Reasons: To elute all impurities along with analyte with in 10 min of run time. Less retention time Good peak shape Higher theoretical plates. Good resolution. 3.2.8. Selection of test concentration and injection volume Test concentration is finalized after it is proved that API is completely extractable at the selected test concentration. Test concentration is fixed based upon the response of API peak at selected detector wavelength. Tranexamic Acid and Ethamsylate label claimed 25mg and 50 mg And the test concentration selected is 100ppm Injection volume selected is 20 µL. Reason: good peak area, retention time, peak symmetry Chromatographic trails for simultaneous estimation Tranexamic acid Ethamsylate TRIAL 1 Parameters Method Stationary phase (column) : Kromosil C18 (150 Ãâ€" 4.6 mm, packed with 5  µm) Mobile Phase : 100% of Methanol Ph : 3.0  ± 0.02 Flow rate (ml/min) : 1.0 Run time (minutes) : 8.0 Column hotness ( °C) : Ambient Volume of injection loop (à ¯Ã‚ Ã‚ ­l) : 20 Detection wavelength (nm) : 242 Drugs RT (min) : 2.91 4.42 Fig. 4: Trial 1 S.No. Name RT[min] Area[ µV*s] TP TF Resolution 1 Tranexamic Acid 2.9167 491583 7707.5 1.0833 0.0000 2 Ethamsylate 4.4227 1076649 10124.7 1.0124 5.3676 Sum 1568232 Observation: 100% Methanol used for this trial, flow rate was 1ml/min at ambient temperature. Faster elution of the analyte takes place . TRIAL 2 Parameters Method Stationary phase (column) : Inertsil C18 (250 Ãâ€" 4.6 mm, packed with 5  µm) Mobile Phase : 30:70 (Methanol : water) Ph : 3.5  ± 0.02 Flow rate (ml/min) : 1.0 Run time (minutes) : 8.0 Column temperature ( °C) : Ambient Volume of injection loop (à ¯Ã‚ Ã‚ ­l) : 20 Detection wavelength (nm) : 228 Drugs RT (min) : 2.81 5.34 Fig. 5: Trial 2 S.No. Name RT[min] Area[ µV*s] TP TF Resolution 1 Tranexamic Acid 2.8167 1272583 4707.5 1.0333 0.0000 2 Ethamsylate 5.3467 1952369 9124.7 1.0524 7.1376 Sum 3224952 Observation: Methanol and water was used in the ratio of 70:30. The flow rate was 1ml/min at ambient temperature.Couldn’t get consistent retention time TRIAL 3 Parameters Method Stationary phase (column) : Inertsil C18 (250 Ãâ€" 4.6 mm, packed with 5  µm) Mobile Phase : 30:70 (Methanol : Phosphate Buffer) Ph : 3.0  ± 0.02 Flow rate (ml/min) : 1.0 Run time (minutes) : 15.0 Column temperature ( °C) : Ambient Volume of injection loop (à ¯Ã‚ Ã‚ ­l) : 20 Detection wavelength (nm) : 236 Drugs RT (min) : 2.86 10.48 Fig. 6: Trial 3 S.No. Name RT[min] Area[ µV*s] TP TF Resolution 1 Tranexamic Acid 2.8627 407583 2307.5 1.2833 0.0000 2 Ethamsylate 10.4802 9792049 9901.7 1.3124 10.2646 Sum 10199632 Observation: Methanol and Phosphate Buffer used in the ratio of (30:70 ) Couldn’t get consistent retention time Discussion: The above trials indicating that RT for the drug was not constant and elution time was faster which not prefered for the analysis. TRAIL 4 Optimizing method Parameters Method Stationary phase (column) : X-Bridge C18 (150 Ãâ€" 4.6 mm, packed with 5  µm) Mobile Phase : 30:70 (Phosphate Buffer : Methanol) pH : 3.2  ± 0.02 Flow rate (ml/min) : 1.0 Run time (minutes) : 8.0 Column temperature ( °C) : Ambient Volume of injection loop (à ¯Ã‚ Ã‚ ­l) : 20 Detection wavelength (nm) : 286 Drugs RT (min) : 3.01 5.06 Fig. 7: Developed Chromatogram S.No. Name RT[min] Area[ µV*s] TP TF Resolution 1 Tranexamic Acid 3.0167 1574827 3707.5 1.0833 0.0000 2 Ethamsylate 5.0667 2779277 5124.7 1.0124 8.5376 Sum 4354104 Discussion: All the experiments were complete by the higher than developed method and the consequences were acceptable. Optimized chromatographic conditions for simultaneous estimation of Tranexamic Acid and Ethamsylate Trail 4: (Optimized Chromatographic Conditions) Parameters Method Stationary phase (column) : X-Bridge C18 (150 Ãâ€" 4.6 mm, packed with 5  µm) Mobile Phase : 30:70 (Phosphate Buffer : Methanol) PH : 3.2  ± 0.02 Flow rate : 1.0 Run time (min) : 8.0 Column temperature ( °C) : Ambient Volume of injection loop (à ¯Ã‚ Ã‚ ­l) : 20 Detection wavelength (nm) : 286 Drugs RT (min) : 3.01 5.06 Assay procedure Preparation of 0.2M phosphate buffer: Buffer solution prepares by dissolving 2.72g of Potassium dihydrogen ortho phosphate (KH2PO4) in 1L of water and the degassing of the solution. Diluents Preparation: 1L of diluents was prepared by mixing 300 ml of 0.02 M Phosphate Buffer and 700 ml of Methanol. Preparation of stock solution: Accurately weighed 10 mg of the both Tranexamic Acid and Ethamsylate is transferred to 10 ml fresh and dry volumetric flask. The amount was making up to the mark among the Methanol and mixed well. This yielded a stock solution with concentration 1000 ppm of Tranexamic Acid and Ethamsylate mixture. Preparation of standard solution: Accurately amount of 0.25 and 0.25 ml of the Tranexamic Acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Then compose up the amount up to the mark among the diluents and mix well. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of Tranexamic Acid and Ethamsylate respectively. Procedure 20à ¯Ã‚ Ã‚ ­Lof the standard and sample was injected into the chromatographic system and areas for the Tranexamic acid and Ethamsylate from the peaks were used for calculating the % assay by using the formulae. Assay calculation AT WS DT P Avg. Wt Assay % = x -x x -x 100 AS DS WT 100 Label Claim Where: AT = Average area counts of sample preparation. AS = Average area counts of standard preparation. WS=Weight of working standard taken in mg. P= Percentage purity of working standard LC = Label Claim of Tranexamic acid , Ethamsylate mg/ml. 3.4 METHOD VALIDATION 3.4.1 ANALYTICAL METHOD VALIDATION Validation parameters Specificity Linearity Range Accuracy Precision System precision Repeatability Intermediate Precision Detection Limit Quantitation Limit Robustness 1. Specificity The system suitability for specificity was carried out to determine whether there are any interference of any impurities in retention time of analytical peak. The study was performed by injecting blank. 2. Linearity The linearity is a systematic method its ability (within a given range) to get assessment results, which are directly relative to the absorption (amount) of analyte in sample. Preparation of standard stock solution: Accurately weighed 10 mg of the both tranexamic acid and Ethamsylate was transferred in to 10 ml fresh and dry volumetric flask. After that the amount was made up to the mark with the Methanol and mix well. This yielded a stock solution amid attention 1000 ppm of tranexamic acid with Ethamsylate mixture. Preparation of standard solution: Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock was transferred to 10 ml clean and dry volumetric flask. Then the volume was made up to the mark with the diluent and mixed well. This yielded a standard stock solution with concentrations of 25ppm and 25ppm of tranexamic acid and Ethamsylate respectively10 Procedure: Prepared a series of standard solutions not less than five during the particular concentration range along with investigate them like for each method. Acceptance criteria: The correlation coefficient should be not less than 0.9990 3. Range The range of a systematic process is the gap between the superior and lower concentration of analyte in sample for which it has been established to the investigative practice was a suitable level of accuracy, precision and linearity. Acceptance criteria: Linearity, Precision and Recovery should be shown. The logic behind this parameter was – typical concentration range was essential between which the actual concentration should fall when performing real sample analysis.10 4. Accuracy Preparation of standard stock solution: Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transfer to 10 ml fresh and dried volumetric flask. Make up the volume up to mark with the diluents and mix well. The standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively. Method procedure: Prepared solutions in triplicate at levels 80%, 100% and 120s% of test concentrations using for tranexamic acid and Ethamsylate working Standards as per the test method and injected each solution in triplicate. Sample Are 100 % Recovery = x x 100 Standared Area conc. in % Accuracy normally refers to the difference between the mean of the set of results and the true or correct value for the quantity measured. According to IUPAC accuracy relates to the difference between results (or mean) and the true value. For analytical methods, there are two possible ways of determining the accuracy, absolute method and comparative method. Accuracy is best reported as percentage bias, which is calculated from the expression Procedure: Known amount of drug substance spiked with known amount of standard drug- minimum of three levels (80%, 100% 120% of test concentration), each level was triplicate. Acceptance criteria: Assay recovery should be between 97%-103%.10 5. Precision Preparation of standard solution: Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Subsequently make up the volume up to the mark among the diluent and well mixed. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively. Method precision: Six individual preparations were prepared using single batch of tranexamic acid and Ethamsylate functioning standard as for each test process and injected each one solutions. Injection precision: Solo preparation was prepared using single batch of Tranexamic acid and Ethamsylate effective standard as for each urbanized process in addition to injected six injections10. Acceptance Criteria: 1. RSD should not be more than 2.0% for five replicate injections of standard. 6. Ruggedness Preparation of standard solution: Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Subsequently make up the quantity up to the mark among the diluents and well mix. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively. Method Procedure: The standard solution was individually prepared as per the test method and injected each solution in six times using different system, analyst, and date. Acceptance Criteria: Overall RSD should not be more than 2.0 %. 7. Limit detection and limit of quantitation LOD: Lowest amount of analyte in a sample that can be detected but not necessarily quanities, under the stated experimental conditions. Preparation of standard solution: Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Then build up the quantity up to the mark with the diluents and mix well. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of Tranexamic acid and Ethamsylate respectively. Method Procedure: The mobile phase was permissible to run equilibrate with stationary phase up to good baseline was obtained. The different concentration ranging from 0.01 to 0.1ppm of tranexamic acid and 0.01 to 0.1ppm Ethamsylate was injected and peaks were recorded. 0.03 and 0.03ppm for tranexamic acid and Ethamsylate concentrations were detected respectively. LOD can be calculated based on signal-noise ratio,by using following formula LOD = S/N Where, S = Signal Obtained From LOD Solution. N = Average Baseline Noise Obtained from Blank Acceptance criteria for LOD and LOQ RSD Criteria Concentration at which RSD Concentration at which RSD 8. Robustness Preparation of standard solution: Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. After that make up the quantity up to the mark with diluent and well mixed. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively. Method procedure: 1. Flow: The standard solution was prepared and injected for the two times with (+1) flow rate. 2. Mobile Phase: The standard solution was prepared and injected for the two times with (+5) Mobile Phase composition. Appraise of its capability to remain unchanged by minute, but conscious variations in process parameters and provides signal of its reliability during its normal usage. Procedure: samples were analyzed under the following conditions.10 3. Stability studies In the rational design and evaluation of dosage forms for the drugs, the stability of the activity components must be a major criterion in determining their stability. The medicine has to reach the patient in an active and acceptable form maintaining the criteria for acceptable equality. The quality of the product has to be retained as long as the product is offered for sale or for administration to the patient. 10 Acceptance Criteria: Overall RSD should not be more than 2.0 %. 9. System suitability Preparation of standard solution: Accurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transferred to 10 ml clean and dried volumetric flask. Subsequently make up the amount up to the mark with diluent and well mixed. Finally the standard stock solution with concentrations of 25 ppm and 25 ppm of tranexamic acid and Ethamsylate respectively. Procedure: Standard solution was prepared and injected six times to test the performance of the chromatographic instrument. Acceptance Criteria: 1. RSD should not be more than 2.0% for five replicate injections of standard 2. USP Tailing for tranexamic acid and Ethamsylate peak in not more than 2.0 3. The column efficiency as determined for tranexamic acid and ethamsylate Plate Count should not be more than 2000. Dept.of Pharmaceutical Analysis JNTUA-OTRI, Ananthapuramu Page 1