Estimation of Tranexamic Acid Andethamsylate Using RP-HPLC

Estimation of Tranexamic sexually transmitted disease Andethamsylate Using RP-HPLCChapter-3 Experimental work3. EXPERIMENTAL WORK3.1 MATERIALS AND METHODS dodge 2. List of Chemical and standers utiliseS.NoChemicalsManufacturer NameGrade1WaterProcessed in Bright LabsHPLC position2AcetonitrileFisher scientificHPLC grade3Orthophosphoric paneMerckGR grade4Tranexamic hotSun pharma ltdBP5EthamsylateSun pharma ltdUSP6KH2PO4MerckGR grade7K2HPO4MerckGR grade8 methanolMerckHPLC gradeTable 3. List of instruments usedS.NoInstrumentnameModelNumberSoftW areManufacturers name1HPLC-autosampler-UV detectorACME9000Auto crome 3000Youngline2Electronic balanceLab India3SonicatorCWUC9L201402822Spectrum tek4Vacuum Pump28965405-289717Vacuubrand50.45 filter paperHPLC gradeRankem3.2. Method development for the simultaneous affection of Tranexamic harsh andethamsylate by utilise RP-HPLC. choice of mobile stagecoachSelection of detectionwavelengthSelection of columnSelection of solvent delivery systemS election of advert rankSelection of column temperatureSelection of diluentSelection of establish concentration and pellet volume3.2.1. Selection of mobile phasePhosphate cowcatcher wood spirit (3070)3.2.2. Selection of wavelength10mg Tranexamic pane 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 columnHeart of HPLC made of 316 grade stainless steel jammed with stationary phase.Silica establish columns with different cross linkings in the change magnitude order of polarity are as follows- Non-polar-moderately polarPolar-C1886In 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 4.6 mm, packed with 5 m), particle sizeReasons Better separation,Good shadowing factor.3.2.4. Selection of solvent delivery systemAlways preferable solvent delivery system.More chance of getting reproducible result on remembering period of analytes.More economic than gradient technique.3.2.5. Selection of flow rateAcceptable limit Not more than 2.5 ml/minFlow rate selected was 1.0ml/minFlow rate is selected based on1. Retention clock time2. Column back pressure3. Peak symmetry.4. Separation of impurities.ReasonsFor earlier elution of analyte and elution of all impurities inwardly 10 minInformation from the reference method in literature.3.2.6. Selection of diluentSelection of diluents is based on the solubility of the analyteDiluent selected Phosphate mince Methanol (3070 % v/v)ReasonAnalyte is soluble in acetonitrile and water.3.2.7. Selection of column temperaturePreferable temperature is ambient or room temperature.ReasonsTo elute all impurities along with analyte with in 10 min of run time.L ess retention timeGood peak shapeHigher theoretical plates.Good re ascendent.3.2.8. Selection of test concentration and guesswork volumeTest concentration is finalized after it is proved that API is make lovely 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 mgAnd the test concentration selected is 100ppmInjection volume selected is 20L.Reason just peak area, retention time, peak symmetry Chromatographic trails for simultaneous estimation Tranexamic acid Ethamsylate TRIAL 1ParametersMethodStationary phase (column) Kromosil C18 (150 4.6 mm, packed with 5 m)Mobile Phase 100% of MethanolPh 3.0 0.02Flow rate (ml/min) 1.0Run time (minutes) 8.0Column hotness (C) AmbientVolume of injection draw in (l) 20Detection wavelength (nm) 242Drugs RT (min) 2.91 4.42Fig. 4 Trial 1S.No.NameRTminAreaV*sTPTFRe closure1Tranexamic Acid2.91674915837 707.51.08330.00002Ethamsylate4.4227107664910124.71.01245.3676Sum1568232 Observation 100% Methanol used for this trial, flow rate was 1ml/min at ambient temperature. Faster elution of the analyte takes place . TRIAL 2ParametersMethodStationary phase (column) Inertsil C18 (250 4.6 mm, packed with 5 m)Mobile Phase 3070 (Methanol water)Ph 3.5 0.02Flow rate (ml/min) 1.0Run time (minutes) 8.0Column temperature (C) AmbientVolume of injection loop (l) 20Detection wavelength (nm) 228Drugs RT (min) 2.81 5.34Fig. 5 Trial 2S.No.NameRTminAreaV*sTPTFReroot1Tranexamic Acid2.816712725834707.51.03330.00002Ethamsylate5.346719523699124.71.05247.1376Sum3224952 Observation Methanol and water was used in the ratio of 7030. The flow rate was 1ml/min at ambient temperature.Couldnt get consistent retention time TRIAL 3ParametersMethodStationary phase (column) Inertsil C18 (250 4.6 mm, packed with 5 m)Mobile Phase 3070 (Methanol Phosphate Buffer)Ph 3.0 0.02Flow rate (ml/min) 1.0Run time (minutes) 15.0 Column temperature (C) AmbientVolume of injection loop (l) 20Detection wavelength (nm) 236Drugs RT (min) 2.86 10.48Fig. 6 Trial 3S.No.NameRTminAreaV*sTPTFResolution1Tranexamic Acid2.86274075832307.51.28330.00002Ethamsylate10.480297920499901.71.312410.2646Sum10199632 Observation Methanol and Phosphate Buffer used in the ratio of (3070 ) Couldnt get consistent retention time Discussion The above trials indicating that RT for the drug was non constant and elution time was faster which not prefered for the analysis.TRAIL 4Optimizing methodParametersMethodStationary phase (column) X-Bridge C18 (150 4.6 mm, packed with 5 m)Mobile Phase 3070 (Phosphate Buffer Methanol)pH 3.2 0.02Flow rate (ml/min) 1.0Run time (minutes) 8.0Column temperature (C) AmbientVolume of injection loop (l) 20Detection wavelength (nm) 286Drugs RT (min) 3.01 5.06Fig. 7 Developed ChromatogramS.No.NameRTminAreaV*sTPTFResolution1Tranexamic Acid3.016715748273707.51.08330.00002Ethamsylate5.066727792775124.71.01248.53 76Sum4354104 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 EthamsylateTrail 4 (Optimized Chromatographic Conditions)ParametersMethodStationary phase (column) X-Bridge C18 (150 4.6 mm, packed with 5 m)Mobile Phase 3070 (Phosphate Buffer Methanol)PH 3.2 0.02Flow rate 1.0Run time (min) 8.0Column temperature (C) AmbientVolume of injection loop (l) 20Detection wavelength (nm) 286Drugs RT (min) 3.01 5.06Assay usePreparation of 0.2M phosphate bufferBuffer solution prepares by dissolving 2.72g of Potassium dihydrogen ortho phosphate (KH2PO4) in 1L of water and the degassing of the solution.Diluents Preparation1L of diluents was prepared by mixing 300 ml of 0.02 M Phosphate Buffer and 700 ml of Methanol.Preparation of product line solutionaccurately weighed 10 mg of the both Tranexamic Acid and Ethamsylate is transferred to 10 ml fresh and dry volumetrical flask. The amount was making up to the mark among the Methanol and mix well. This yielded a clove pink solution with concentration 1000 ppm of Tranexamic Acid and Ethamsylate mixture.Preparation of regulation solutionAccurately amount of 0.25 and 0.25 ml of the Tranexamic Acid and Ethamsylate line of descent solution transferred to 10 ml clean and alter volumetric flask. Then compose up the amount up to the mark among the diluents and mix well. eventually the standard stock solution with concentrations of 25 ppm and 25 ppm of Tranexamic Acid and Ethamsylate respectively.Procedure20Lof 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 calculationAT WS DT P Avg. WtAssay % = x -x x -x 100AS DS WT 100 Label ClaimWhereAT = Average area counts of sample preparation.AS = Average area counts of standard pr eparation.WS=Weight of working standard taken in mg.P= Percentage worth of working standardLC = Label Claim of Tranexamic acid , Ethamsylate mg/ml.3.4 METHOD VALIDATION3.4.1 ANALYTICAL METHOD VALIDATIONValidation parametersSpecificityLinearityRange true statementPrecision strategy precisionRepeat top executiveIntermediate PrecisionDetection LimitQuantitation LimitRobustness1. SpecificityThe 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. LinearityThe 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 solutionAccurately 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 solutionAccurately 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 respectively10Procedure Prepared a series of standard solutions not less than five dollar bill during the particular concentration range along with ask them like for each method. betrothal criteria The correlation coefficient should be not less than 0.99903. RangeThe range of a systematic process is the gap between the super 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 criter ia 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. verityPreparation of standard stock solutionAccurately amount of 0.25 and 0.25 ml of the tranexamic acid and Ethamsylate stock solution transfer to 10 ml fresh and arid 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 100Standared Area conc. in %Accuracy averagely refers to the difference between the specify of the set of results and the true or correct val ue 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 2 possible ways of determining the accuracy, absolute method and comparative method. Accuracy is best reported as percentage bias, which is calculated from the expressionProcedure cognize 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. PrecisionPreparation of standard solutionAccurately 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 in dividual preparations were prepared using single kettle of fish of tranexamic acid and Ethamsylate surgical process standard as for each test process and injected each one solutions.Injection precisionSolo 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 Criteria1. RSD should not be more than 2.0% for five recur injections of standard.6. RuggednessPreparation of standard solutionAccurately 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 quantitationLOD Lowest amount of analyte in a sample that canister be detected but not necessarily quanities, under the stated experimental conditions. Preparation of standard solutionAccurately 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 ProcedureThe 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 concentr ations were detected respectively.LOD can be calculated based on signal-noise ratio,by using pursual formulaLOD = S/NWhere,S = Signal Obtained From LOD Solution.N = Average Baseline Noise Obtained from Blank Acceptance criteria for LOD and LOQRSD CriteriaConcentration at which RSD Concentration at whichRSD 8. RobustnessPreparation of standard solutionAccurately 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 procedure1. Flow The standard solution was prepared and injected for the 2 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 conscio us variations in process parameters and provides signal of its reliability during its normal usage.Procedure samples were analyzed under the following conditions.103. Stability studiesIn 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. 10Acceptance Criteria Overall RSD should not be more than 2.0 %.9. System suitabilityPreparation of standard solutionAccurately 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 Criteria1. RSD should not be more than 2.0% for five replicate injections of standard2. USP Tailing for tranexamic acid and Ethamsylate peak in not more than 2.03. The column efficiency as determined for tranexamic acid and ethamsylatePlate Count should not be more than 2000.Dept.of Pharmaceutical Analysis JNTUA-OTRI, Ananthapuramu Page 1