Estimation of Tranexamic Acid Andethamsylate Using RP-HPLC

Estimation of Tranexamic Acid Andethamsylate Using RP-HPLCChapter-3 experimental work3. EXPERIMENTAL WORK3.1 MATERIALS AND METHODSTable 2. List of Chemical and standers utilizeS.NoChemicalsManufacturer NameGrade1WaterProcessed in Bright LabsHPLC grade2AcetonitrileFisher scientificHPLC grade3Orthophosphoric deadlyMerckGR grade4Tranexamic sultrySun pharma ltdBP5EthamsylateSun pharma ltdUSP6KH2PO4MerckGR grade7K2HPO4MerckGR grade8 methyl alcoholMerckHPLC gradeTable 3. List of instruments useS.NoInstrumentnameModel issuingSoftWareManufacturers name1HPLC-autosampler-UV detectorACME9000Auto crome 3000Youngline2Electronic balanceLab India3SonicatorCWUC9L201402822Spectrum tek4Vacuum ticker28965405-289717Vacuubrand50.45 filter paperHPLC gradeRankem3.2. Method development for the coincidental esteem of Tranexamic caustic andethamsylate by using RP-HPLC.Selection of wide awake material bodySelection of detectionwavelengthSelection of pillarSelection of resolve delivery constitutionS election of stop sum upSelection of column temperatureSelection of dilutantSelection of render ducking and injection volume3.2.1. Selection of mobile kindPhosphate fan Methanol (3070)3.2.2. Selection of wavelength10mg Tranexamic corrosive and Ethamsylate were turn in mobile stage. The overlay spectrum was used for selection of wavelength for Tranexamic acid and Ethamsylate The iso-bestic stay was taken as detection wavelength 286nm.3.2.3. Selection of columnHeart of HPLC do of 316 grade stainless steel packed with stationary pattern.Silica base columns with different cross linkings in the increasing order of signal are as follows- Non-polar-mode arrangely polarPolar-C1886In reverse phase chromatography, hydrophobic fundamental interaction amongst dose molecule and the alkyl chains on the column packing material. tugboat is selected found on solubility, polarity chemical differences among analytes and Column selected i.e. X-Bridge C18 (150 4.6 mm, packed with 5 m) , particle size originators Better separation,Good chase after factor.3.2.4. Selection of solvent delivery musical arrangementAl shipway preferable solvent delivery system.More chance of getting reproducible result on retention prison term of analytes.More economic than gradient technique.3.2.5. Selection of flow appraiseAcceptable limit Not more than than 2.5 ml/ arcminuteFlow rate selected was 1.0ml/minFlow rate is selected based on1. Retention time2. Column back pressure3. Peak symmetry.4. time interval of impurities.ReasonsFor earlier elution of analyte and elution of all impurities within 10 minInformation from the consultation method in literature.3.2.6. Selection of diluentSelection of diluents is based on the solubility of the analyteDiluent selected Phosphate Buffer Methanol (3070 % v/v)ReasonAnalyte is soluble in acetonitrile and water.3.2.7. Selection of column temperaturePreferable temperature is close or room temperature.ReasonsTo elute all impurities along wit h analyte with in 10 min of run time.Less retention timeGood florescence shapeHigher theoretical plates.Good re termination.3.2.8. Selection of foot race concentration and injection volumeTest concentration is finalized after it is proved that API is all extractable at the selected test concentration.Test concentration is fixed based upon the response of API confidential information at selected detector wavelength.Tranexamic Acid and Ethamsylate label claimed 25mg and 50 mgAnd the test concentration selected is carbonppmInjection volume selected is 20L.Reason right(a) peak area, retention time, peak symmetry Chromatographic trails for concurrent estimation Tranexamic acid Ethamsylate visitation 1ParametersMethodStationary phase (column) Kromosil C18 (150 4.6 mm, packed with 5 m) unstable material body 100% of MethanolPh 3.0 0.02Flow rate (ml/min) 1.0Run time (minutes) 8.0Column hotness (C) ambientVolume of injection grummet (l) 20 detecting wavelength (nm) 242Drugs RT (m in) 2.91 4.42Fig. 4 audition 1S.No.NameRTminAreaV*sTPTF closing1Tranexamic Acid2.91674915837707.51.08330.00002Ethamsylate4.4227107664910124.71.01245.3676Sum1568232 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) smooth Phase 3070 (Methanol water)Ph 3.5 0.02Flow rate (ml/min) 1.0Run time (minutes) 8.0Column temperature (C) closeVolume of injection loop topology (l) 20Detection wavelength (nm) 228Drugs RT (min) 2.81 5.34Fig. 5 Trial 2S.No.NameRTminAreaV*sTPTFRe source1Tranexamic 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) 15.0Column temperature (C) AmbientVolume of injection loop (l) 20Detection wavelength (nm) 236Drugs RT (min) 2.86 10.48Fig. 6 Trial 3S.No.NameRTminAreaV*sTPTFRe root word1Tranexamic 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 not 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.NameRTminA reaV*sTPTFResolution1Tranexamic Acid3.016715748273707.51.08330.00002Ethamsylate5.066727792775124.71.01248.5376Sum4354104 Discussion All the experiments were arrest 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 5.06 bank check surgery supply of 0.2M phosphate bufferBuffer solution prepares by adjournment 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 parentage solutionaccurately weighed 10 mg of the both(prenominal) Tranexamic Acid and Ethamsylate is transferred to 10 ml raw and dry volumetricalal flask. The get along was making up to the turn back off among the Methanol and mixed hygienic. This yielded a memory solution with concentration gramme ppm of Tranexamic Acid and Ethamsylate mixture.Preparation of beat solutionAccurately keep down of 0.25 and 0.25 ml of the Tranexamic Acid and Ethamsylate pack solution transferred to 10 ml clean and alter volumetric flask. Then compose up the amount up to the typeset among the diluents and mix easily. Finally the timeworn stock solution with concentrations of 25 ppm and 25 ppm of Tranexamic Acid and Ethamsylate respectively.Procedure20Lof the normal and sample was injected into the chromatographic system and areas for the Tranexamic acid and Ethamsylate from the peaks were used for calculating the % prove by using the ordinancee.Assay calculationAT WS DT P Avg. Wt Assay % = x -x x -x 100AS DS WT 100 go after citeWhereAT = second-rate area counts of sample education.AS = Average area counts of standard preparation.WS=Weight of working standard taken in mg.P= Percentage purity of working standardLC = Label Claim of Tranexamic acid , Ethamsylate mg/ml.3.4 METHOD ecesis3.4.1 ANALYTICAL METHOD VALIDATIONValidation parametersSpecificityLinearityRangetruenessPrecision form precisenessRepeatabilityIntermediate PrecisionDetection LimitQuantitation LimitRobustness1. SpecificityThe system suitability for specificity was carried out to determine whether there are any kerfuffle 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 denseness (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 newly and dry volumetric flask.After that the amount was made up to the retard 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 lively a series of standard solutions not less than five during the peculiar(a) concentration range along with investigate 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 mingled with the superior and lower concentration of analyte in sample for wh ich it has been established to the investigative practice was a fitted level of accuracy, precision and linearity. sufferance criteria Linearity, Precision and Recovery should be shown.The logical system behind this parameter was typical concentration range was essential between which the actual concentration should fall when performing real sample analysis.10 4. true statementPreparation 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 dried-out 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% Recov ery = x x 100Standared 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 beat 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 account as percentage bias, which is calculated from the expressionProcedure Known amount of drug substance spiked with known amount of standard drug- borderline 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.after 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 oneness batch of tranexamic acid and Ethamsylate functioning 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 half dozen injections10.Acceptance Criteria1. RSD should not be more than 2.0% for five replicate injections of standard.6. severityPreparation 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 res pectively.Method Procedure The standard solution was individually prepared as per the test method and injected each solution in hexad 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 washbasin be detected but not necessarily quanities, low 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 tran examic 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 formulaLOD = S/NWhere,S = Signal Obtained From LOD Solution.N = Average service line 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 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. judge 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.103. Stability studiesIn the rational design and evaluation of back breaker forms for the drugs, the stability of the activity components must be a major metre in determining their stability. The medicine has to reach the patient in an prompt 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 system 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 volumet ric 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 fixed for tranexamic acid and ethamsylatePlate Count should not be more than 2000.Dept.of pharmaceutic Analysis JNTUA-OTRI, Ananthapuramu Page 1