Bioanalytical Method Development and Validation for

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Citation Patel P Patel P Giram P 2015 Bioanalytical Method Development and Validation for Latanoprost Quantification in Pharmaceutical. Opthalmic Microemulsion Formulation by RP HPLC J Anal Bioanal Tech 6 284 doi 10 4172 2155 9872 1000284. Page 2 of 5, The known quantity of the drug substance corresponding to 80. 100 120 of standard LAT and the mixture were analyzed by the. proposed method At each level of the amounts six determinations. were performed This was done to check the accuracy of the drug at. different levels in the formulations 13, Six injections of three different concentrations 1 3 5 g ml were. given on the same day and the values of the RSD were calculated. to determine intra day precision Three injections of three different. concentrations 1 3 5 g ml were given on three different days to. determine inter day precision,Figure 2 Materials of analytical method. Limit of quantitation LOQ and limit of detection LOD. an ultrasonic bath prior to use 7 Data acquisition and integration was The LOQ and LOD were determined based on a signal to noise. performed using LC solution software Spincho Biotech Vadodara ratios and were based on analytical responses of 10 and 3 times the. background noise respectively LOD and LOQ were experimentally. Preparation of mobile phase verified by diluting known concentration of LAT until the average. responses were approximately 3 or 10 times the standard deviation of. Three mg Latanoprost was accurately weighed in 25 ml volumetric. the responses for six replicate determinations, flask Approximate 15 ml acetonitrile was added and vortex mixed to. dissolve drug into acetonitrile The final volume was adjusted upto 25 Stability in sample solutions. ml mark with acetonitrile to prepare stock solution of concentration. 120 g ml Three different concentrations of LAT 1 3 5 g ml were prepared. from sample solution and stored at room temperature for 2 days They. Preparation of standard solution were then injected into HPLC system 14. 2 5 ml of above stock solution was added to 10 ml volumetric flask Analysis of marketed and developed formulations of LAT. and diluted to 10 ml with acetonitrile to produce 30 g ml From this. solution 1 ml of 30 g ml was taken in 10 ml volumetric flask and To determine the content of LAT in formulations an accurately. diluted with mobile phase to prepare 10 g ml of working standard weighed amount equivalent to 50 g of LAT was transferred in to 50 ml. solution volumetric flask dissolved in 25 ml of acetonitrile water and volume. was made up to the mark with the same solvent The volumetric flask. Sample preparation for calibration curve of Latanoprost was sonicated for 2 min for complete dissolution and then filtered The. concentration of LAT in sample stock solution was 1 g ml Then 20. Aliquots ranging from 1 ml to 10 ml were taken from working l of this solution was injected in to column and chromatogram was. standard solution in 10 ml volumetric flask and diluted to 10 ml recorded The analysis was repeated in triplicate 15. with mobile phase to give final concentration of 1 2 3 4 5 6 7 8. 9 10 g ml Injections of 20 l were made for each concentration Results. and chromatographed under the condition described in Table 1. Calibration graph was constructed by plotting peak area versus Validation of HPLC method. concentration of Latanoprost and the regression equation was The method was validated with respect to parameters including. calculated 8 10 Linearity Limit of Quantitation LOQ Limit of Detection LOD. Robustness Precision Recovery and Selectivity, To evaluate LC method robustness a few parameters were Validation of developed HPLC method was carried out as per ICH.
deliberately varied 11 The parameters included variation of flow guidelines Q2 R1 Table 1 Optimized parameters of HPLC method. rate percentage of acetonitrile in the mobile phase and acetonitrile of Robustness. different lots Robustness of the method was done at three different. concentration levels 2 5 5 7 5 g ml respectively Each factor selected Results presented in Table 2 indicate that the selected factors. except solvents of different lots to examine were changed at three remained unaffected by small variation of these parameters It was also. levels 1 0 1 One factor at the time was changed to estimate the found that acetonitrile of different lots from the same manufacture has. effect Thus replicate injections n 3 of standard solution at three no significant influence on the determination. concentration levels were performed under small changes of three Insignificant difference in assay and less variability in retention. chromatographic parameters factors time were observed Table 2 a Robustness Evaluation. Linearity Linearity, The calibration curves n 3 constructed for LAT were checked for Three correlation coefficient of 1 r2 0 9983 2 r2 0 9987 3. linearity over the concentration range of 1 10 g ml Peak areas of LAT r 0 9987 with RSD values less than 2 across the concentration range. were plotted versus LAT concentration and linear regression analysis studied were obtained following linear regression analysis Table 3. was performed on the resultant curves 12 Typically the regression equation for the calibration curve was found. J Anal Bioanal Tech, ISSN 2155 9872 JABT an open access journal Volume 6 Issue 6 1000284. Citation Patel P Patel P Giram P 2015 Bioanalytical Method Development and Validation for Latanoprost Quantification in Pharmaceutical. Opthalmic Microemulsion Formulation by RP HPLC J Anal Bioanal Tech 6 284 doi 10 4172 2155 9872 1000284. Page 3 of 5, Kromasil C18 250 mm 4 6 mm i d 5 m System suitability. particle size,Mobile Phase Acetonitrile Water 60 40. The experiment was performed by injecting six consecutive. Flow rate 1 0 ml min, injections of solution having concentration of 10 g ml during the start.
of method validation and the start of each day,Retention time 7 02 min. Detector UV detector 210 nm Different Peak parameters were observed like retention time tailing. Needle wash Methanol Water 50 50 factor theoretical plates and RSD of area These are summarized in. Table 1 Optimized parameters of HPLC method Table 8. Retention Time,In vitro drug release,Factor Level Assay. tR of LAT min Comparative diffusion studies were carried out for F2 F3 F4 F5. A Flow rate ml min and Ch F4 microemulsions and plain drug solution using the diffusion. 0 95 1 7 24 101 62 0 34 method for a period of 24 hours and results were compared after. 1 0 0 7 01 101 37 0 42 applying different kinetic models The results of in vitro drug release. 1 05 1 6 95 101 64 0 23 studies were indicating that after the end of 24 hours study 52 56 of the. Mean SD 101 61 0 33 drug was released from plain drug solution and 66 4 68 34 83 06. B of ACN in mobile phase 69 93 and 90 43 from F2 F3 F4 F5 and Ch F4 microemulsion. 55 1 5 29 101 56 0 48 respectively The results showed that the drug released from plain. 60 0 7 01 101 29 0 37,65 1 8 94 101 63 0 49,Mean SD 101 49 0 45. Table 2 Robustness Evaluation,Mean Area V s SD n 3 RSD. 1 26594 5 275 1 03429,2 61273 411 0 67164,3 83589 589 0 18949.
4 115204 1902 1 81435,5 132271 1423 1 35358,6 167439 1049 1 28635. 7 187567 5 1499 1 86496,8 211312 5 1774 0 83958,9 237610 5 3027 1 37636. 10 247109 5 3203 1 34691,Average of three determinants. Table 3 Calibration curve result, to be y 25780x 8095 6 where x is the concentration in g ml Figures. Average of three determinants, This was done to check the accuracy of the drug at different levels.
Figure 3 Calibration curve results,in the formulations Table 4. Table 5 shows that for both the cases RSD was less than 2. which complies with specified limit, Limit of quantitation LOQ and limit of detection LOD. The LOQ was found to be 0 025 g ml with resultant RSD of. 1 2 n 5 The LOD was found to be 0 07 g ml,Stability in sample solutions. No additional peak was found in chromatogram indicating the. stability of LAT in the sample solution Table 6, Analysis of marketed and developed formulations of LAT. The validated LC method was successfully applied for the assay. of LAT in marketed and developed formulations Assay results are. Figure 4 Linearity Plot,represented in Table 7,J Anal Bioanal Tech.
ISSN 2155 9872 JABT an open access journal Volume 6 Issue 6 1000284. Citation Patel P Patel P Giram P 2015 Bioanalytical Method Development and Validation for Latanoprost Quantification in Pharmaceutical. Opthalmic Microemulsion Formulation by RP HPLC J Anal Bioanal Tech 6 284 doi 10 4172 2155 9872 1000284. Page 4 of 5,In Vitro Drug Release Study,70 Plain Drug Solution. 0 5 10 15 20 25 30,Time Hours, Figure 5 Overlay chart of Calibration curve Figure 6 In vitro drug release study. Excess drug drug solution was less than half of the drug released from other drug. Theoretical a,content g ml Found n 3, RSD n 3 formulations So these formulations increased the drug permeation. analyte Ch F4 showed controlled release than other microemulsions These are. 80 4 4 02 100 5 0 54 summarized in Figure 6,100 5 5 04 100 8 1 5. Analysis of marketed and developed formulations of LAT. 120 6 6 02 100 33 0 46,mean concentration of three determinants.
To determine the content of LAT in formulations an accurately. Recovery mean measured concentration nominal concentration 100 weighed amount equivalent to 50 g of LAT was transferred in to 50 ml. Table 4 Recovery Study volumetric flask dissolved in 25 ml of acetonitrile water and volume. was made up to the mark 50 ml with the same solvent The volumetric. Precision flask was sonicated for 2 min for complete dissolution and then filtered. RSD n 3 The concentration of LAT in sample stock solution was 1 g ml Then. Interday Intraday 20 l of this solution was injected in to column and chromatogram was. 1 1 23 1 06 recorded The analysis was repeated in triplicate. 2 0 94 0 99,Conclusion,3 1 04 0 96, The developed HPLC method is very simple and results confirm. Table 5 Data for Precision, suitable accuracy linearity and precision Therefore the method. could be useful for both routine analytical and quality control assay of. Actual conc g ml a,Measured conc g ml RSD, Latanoprost in ophthalmic microemulsion and stability studies. 1 0 98 1 22,References,10 10 03 1 03, 1 Nash RA Watcher AH 2003 Pharmaceutical Process Validation Marcel. 30 30 32 1 17,Dekker Inc New York pp 159 190,mean concentration of three determinants.
2 United States Pharmacopoeia 2012 US Pharmaceutical Convention Inc. Table 6 Stability of LAT in sample solution Rockville Volume I II and III. 3 British Pharmacopoeia 2012 Her Majesty s Stationary Office London Volume. Actual Amount,Formulations a,Amount found SD Assay I II and III. Market formulation 50 49 89 0 35 99 78 4 Indian Pharmacopoeia 2010 Controller of Publication Delhi Volume I II and III. Developed formulation 50 49 77 0 28 99 54 5 European Pharmacopoeia 2012 Council of Europe 67075 Strasbourg cedex. France Volume I II and III,Drug solutiion 50 50 19 0 21 100 38. 6 http www ich org fileadmin Public Web Site ICH Products Guidelines. mean concentration of three determinants Quality Q2 R1 Step4 Q2 R1 Guideline pdf. Table 7 Assay of different formulations, 7 Mehta J Patel V Kshatri N Vyas N 2010 A versatile LC method for the. simultaneous quantification of Latanoprost Timolol and Benzalkonium chloride. Parameters Value Standard value and related substances in the presence of their degradation products in. Re t Time min 7 01 5 10 ophthalmic solution Analytical Methods 11 1737 1744. LOQ ppm 0 019 0 1 8 Skoog FA Holler FJ Nieman DJ 2009 Introduction to UV Spectroscopy. LOD ppm 0 07 0 05 Principle of instrumental analysis Brooks Cole Publications p 301. T Plates 14253 2000 9 Jeffrey GH Bassett J Mendham J Denney RC 1997 Introduction Vogel. Tailing Factor 1 089 2 Textbook of Quantitative Chemical Analysis ELBS Longman pp 3 8. Resolution 4 23 2 10 Snyder LR Kirkland JJ Glajch JL 1997 Practical HPLC method development. A Wiley Interscience Publication pp 697 709,Capacity factor 2 33 2. 11 Lorenz LJ 2000 Modern Methods of Pharmaceutical Analysis CRC Press. Table 8 Summary of validation parameters,Florida pp 241 244.
J Anal Bioanal Tech, ISSN 2155 9872 JABT an open access journal Volume 6 Issue 6 1000284. Citation Patel P Patel P Giram P 2015 Bioanalytical Method Development and Validation for Latanoprost Quantification in Pharmaceutical. Opthalmic Microemulsion Formulation by RP HPLC J Anal Bioanal Tech 6 284 doi 10 4172 2155 9872 1000284. Page 5 of 5, 12 Mermet JM Otto M Valcarcel M Widmer HM Kellner R 2004 Analytical 14 Morgan PV Proniuk S Blanchard J Noecker RJ 1999 Effect of temperature. Chemistry Wiley VCH UK pp 533 534 and light on the stability of latanoprost and its clinical relevance J Glaucoma. 10 401 405, 13 Rele RV Mhatre VV Parab JM Warkar CB 2011 Simultaneous RP HPLC. determination of Latanoprost and Timolol Maleate in combined pharmaceutical 15 Redulescu V Doneanu C Mandruta C Cocu F 1997 HPLC analysis of some. dosage form J Chem Pharm Res 3 138 144 synthetic prostaglandin compounds of therapeutic interest Rev Roum Chim. 45 1129 1135,J Anal Bioanal Tech, ISSN 2155 9872 JABT an open access journal Volume 6 Issue 6 1000284. Bioanalytical Method Development and Validation for Latanoprost Quantification in Pharmaceutical Opthalmic Microemulsion Formulation by RP HPLC Pratikeshkumar Patel1 Priti Patel2 and Prabhanjan Giram1 1Polymer Science and Engineering CSIR NCL Pune Maharashtra India 2Sayajirao University of Baroda Gujarat India HO HO O O HO Figure 1 Chemical structure of Latanoprost Journal of

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