RP-HPLC method development and validation for estimation of doxorubici

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Monika R et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 09-16 9 IJAMSCR |Volume 7 | Issue 1 | Jan - Mar - 2019 www.ijamscr.com Research article Medical research RP-HPLC method development and validation for estimation of doxorubicin and in bulk and pharmaceutical dosage form Monika Renikunta 1 Dr.T.Rama Mohan Reddy 2 Dr.Konde Abbulu 3 1 Department of Pharmaceutical Analysis CMR College of Pharmacy Medchal Hyderabad-501401. 2Associate Professor department of Pharmaceutical Chemistry CMR College of Pharmacy Medchal Hyderabad-501401 3 Principal Department of Pharmaceutics CMR College of Pharmacy Hyderabad 501401. Corresponding Author: Monika Renikunta Dr.T.Rama Mohan Reddy Email id: ramamohanreddy40gmail.com monika.renikuntagmail.com ABSTRACT A simple Precised Accurate method was developed for the estimation of doxorubicin by RP-HPLC technique. Chromatographic conditions used are stationary phase Discovery C18 150mm x 4.6 mm 5  Mobile phase 0.1 OPA: Acetonitrile in the ratio of 45:55 and flow rate was maintained at 0.9ml/min detection wave length was 234 nm column temperature was set to 30 o C and diluent was mobile phase Conditions were finalized as optimized method. System suitability parameters were studied by injecting the standard six times and results were well under the acceptance criteria. Linearity study was carried out between 25 to150 levels R 2 value was found to be as 0.999. Precision was found to be 0.6 for repeatability and 0.7 for intermediate precision. LOD and LOQ are 0.085µg/ml and 0.258µg/ml respectively. By using above method assay of marketed formulation was carried out 99.90 was present. Degradation studies of doxorubicin were done in all conditions purity threshold was more than purity angle and within the acceptable range. Keywords: HPLC Doxorubicin Method development. ICH Guidelines INTRODUCTION Doxorubicin is a cytotoxic anthracycline antibiotic isolated from cultures of streptomyces peucetius var. caesius. Doxorubicin binds to nucleic acids presumably by specific intercalation of the planar anthracycline nucleus with the DNA double helix.Doxorubicin is capable of undergoing 3 metabolic routes: one-electron reduction two- electron reduction and deglycosidation. However approximately half of the dose is eliminated from the body unchanged. Two electron reduction yields doxorubicinol a secondary alcohol this pathway is considered the primary metabolic pathway. Literature survey revealed that there were few analytical methods have been reported for doxorubicin in LC MS/MS for doxorubicin has been reported. However an extensive literature search didn’t reveal any estimation method for doxorubicin in API and Pharmaceutical dosage form. Therefore an attempt has been made to develop and validate simple precise accurate economical RP-HPLC method as per ICH guidelines for estimation of ISSN:2347-6567 International Journal of Allied Medical Sciences and Clinical Research IJAMSCR

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Monika R et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 09-16 10 doxorubicin in API and Pharmaceutical dosage form. MATERIALS AND METHODS Chemicals and Reagents Acetonitrile HPLC grade orthophosphoric acid HPLC grade water HPLC grade were purchased from Mark India Ltd Worli Mumbai India. All active pharmaceutical ingredients APIs of doxorubicin as reference standards were procured from Spectrum Pharma labs Hyderabad India. Instruments and Chromatographic Conditions Electronics Balance-Denver P H meter -BVK enterprises India Ultrasonicator-BVK enterprises WATERS HPLC Acuity system equipped with quaternary pumps UV detector and Auto sampler integrated with Empower 2 Software was used for LC peak integration and Data processing. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz cells integrated with UV-win 6 Software was used for measuring absorbance of Doxorubicin solution. The mobile phase used was 0.1 orthophosphoric acid: Acetonitrile 45:55A at a flow rate of 1ml/min samples were analyzed at 234 nm detector wave length and at an injection volume of 10 μL using discovery C 18 150 x 4.6 mm 5  with run time of 5 min. METHODS Diluent Based up on the solubility of the drugs diluent was selected Acetonitrile and Water taken in the ratio of 50:50. Buffer 0.1OPA Buffer 1ml of Perchloric acid was diluted to 1000ml with HPLC grade water. Standard Preparation Accurately Weighed and transferred 7.5mg of Doxorubicin working Standards into a 10ml clean dry volumetric flask add 3/4th volume of diluent sonicated for 5 minutes and make up to the final volume with diluents. 1ml from the above stock solution was taken into a 10ml volumetric flask and made up to 10ml. Sample Preparation 10 tablet was weighed powdered and then was transferred into a 100mL volumetric flask 10mL of diluent added and sonicated for 25 min further the volume made up with diluent and filtered. From the filtered solution 1 ml was pipeted out into a 10 ml volumetric flask and made up to 10ml with diluent. Method Validation As per ICH guidelines the method was validated and the parameters like Linearity Specificity Accuracy Precision Limit of Detection LOD and Limit of Quantitation LOQ were assessed. Specificity It is the ability of analytical method to measure the response of the analyte and have no interference from other extraneous components and well resolved peaks are obtained. Linearity Linearity solutions are prepared such that 0.25 0.5 0.75 1 1.25 1.5ml from the Stock solutions of doxorubicin is taken in to 6 different volumetric flasks and diluted to 10ml with diluents to get 18.75ppm 3.75ppm 56.25ppm 75ppm 93.75ppm 112.5ppm Accuracy Preparation of Standard stock solutions Accurately Weighed and transferred 7.5mg of doxorubicin working Standard into a 10ml clean dry volumetric flask add 3/4th volume of diluent sonicated for 5 minutes and make up to the final volume with diluents. 1ml from the above stock solution was taken into a 10ml volumetric flask and made up to 10ml Preparation of 50 Spiked Solution 0.5ml of sample stock solution was taken into a 10ml volumetric flask to that 1.0ml from each standard stock solution was pipetted out and made up to the mark with diluent. Preparation of 100 Spiked Solution 1.0ml of sample stock solution was taken into a 10ml volumetric flask to that 1.0ml from each

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Monika R et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 09-16 11 standard stock solution was pipetted out and made up to the mark with diluent. Preparation of 150 Spiked Solution 1.5ml of sample stock solution was taken into a 10ml volumetric flask to that 1.0ml from each standard stock solution was pipetted out and made up to the mark with diluent. Robustness Small deliberate changes in method like Flow rate mobile phase ratio and temperature are made but there were no recognized change in the result and are within range as per ICH Guide lines. Robustness conditions like Flow minus 0.9ml/min Flow plus 1.1ml/min mobile phase minus mobile phase plus temperature minus 25°C and temperature plus 35°C was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. RSD was within the limit. LOD sample Preparation 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flasks and made up with diluents. From the above solutions 0.1ml of doxorubicin solution was transferred to 10ml volumetric flask and made up with the same diluent. LOQ sample Preparation 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flask and made up with diluent. From the above solutions 0.3ml of doxorubicin solution was transferred to 10ml volumetric flask and made up with the same diluent. System suitability parameters The system suitability parameters were determined by preparing standard solutions of doxorubicin 75ppm solution was injected six times and the parameters like peak tailing resolution and USP plate count were determined to check whether the results complies with Recommended limits. Assay of doxorubicin An Accurately measured weight equivalent to Adriamycin 75mg of doxorubicin was used to perform assay by utilizing the method developed and under the optimized chromatographic conditions. Sample solutions were injected in to the HPLC system and scanned at 234 nm from which the of drug was estimated. RESULTS DISCUSSIONS Optimization of Chromatographic Conditions To develop and establish a suitable RP-HPLC method for estimation of doxorubicin in bulk and tablet dosage forms different preliminary tests were performed and different chromatographic conditions were tested and optimized chromatographic conditions were developed which were given in Table-1.The final analysis was performed by using 45 Ortho phosphoric acid:55 Acetonitrile at a flow rate of 1ml/min. samples were analyzed at 234 nm detector wave length and at an injection volume of 10 μL using Discovery C18 4.6 x 250mm 5µm.with run time of 5 min. The proposed method was optimized to give sharp peak with good resolution and minimum tailing effect for doxorubicin the optimized chromatogram was obtained as shown in Figure- 2. Validation Linearity was established 18.75-112.5µg/ml at six different concentrations each were injected in a duplicates and average areas were determined and linearity equations were obtained as y 15456x + 2384 correlation coefficient R 2 was determined as 0.999. The Linearity calibration curves were plotted as shown in Figure-3. Retention times of doxorubicin were 2.567min. Where no interfering peaks in blank and placebo at retention time of this drug was not found in this method. So this method holds its specificity. Three levels of Accuracy samples 50 100 150 were prepared and triplicates of injections were given for each level of accuracy and mean Recovery was obtained as 99.00 was shown in Table-2. RSD was calculated from the corresponding peaks obtained by injecting six times a known concentration of doxorubicin was obtained as 0.6 and the RSD for intermediate Precision was obtained as 0.7 Low RSD values indicates that the method developed was precise as shown in Table-3. The LOD and LOQ values were evaluated based on

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Monika R et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 09-16 12 Relative standard deviation of response and slope of the calibration curve doxorubicin. The detection limit values were obtained as 0.085 and Quantitation limit were fund to be 0.258 as given in Table-4.Robustness conditions like Flow minus 0.9ml/min Flow plus 1.1ml/min mobile phase minus 50:50 mobile phase plus 40:60 temperature minus 25°C and temperature plus 35°C was maintained and samples were injected in duplicate manner Table -5. System suitability parameters were not much affected and all the parameters were passed. RSD was within the limit Table -6. Doxorubicin pure drugs API was obtained from spectrum Pharma research solutions and pfizer Ltd Adriamycin bearing the label claim 75mg. Assay was performed with the above formulation. Average Assay obtained was 98.82 the result was shown in Table-7 and the chromatogram of standard drugs and pharmaceutical dosage forms were shown in Figure-4 5 respectively. Degradation Studies Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples was calculated and all the samples passed the limits of degradation Table 8. Figure-1: Chemical Structure of doxorubicin Figure-2: Optimized Chromatogram of Doxorubicin

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Monika R et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 09-16 13 Figure-3: Linearity Curve of Doxorubicin Figure-4: Standard Chromatogram of Doxorubicin Figure-5: A Sample Chromatogram of Doxorubicin in Pharmaceutical Dosage FormTable-1: Optimized Chromatographic Conditions Parameter Condition RP-HPLC WATERS HPLC SYSTEM equipped with quaternary pumps with PDA detector Mobile phase 45 OPA 0.1 : 55 Acetonitrile y 15456x + 2384.2 R² 0.9996 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 0 20 40 60 80 100 120 Concentration A U C

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Monika R et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 09-16 14 Flow rate 1ml/min Column Discovery 250x4.6mm 5µ Detector wave length 234nm Column temperature 30°C Injection volume 10 L Run time 5 min Diluent Water and Acetonitrile in the ratio 50:50 Retention Time Doxorubicin 2.588 min Theoretical Plates Doxorubicin 2820 Table-2: Accuracy results of Doxorubicin Sample Amount added µg/ml Recovery RSD Doxorubicin 37.5 99.16 0.44 75 98.99 0.46 122.5 98.84 0.040 Table-3: Precision Result of Doxorubicin S.no Repeatability Intermediate precision 1. 1145785 1080737 2. 1147001 1070565 3. 1153316 1068333 4. 1165564 1060334 5. 1157097 1078539 6. 1156369 1078539 Mean 1154189 1072841 S.D 7289.1 7863.9 RSD 0.6 0.7 Table-4: LOD and LOQ values of Doxorubicin and Elbasvir Molecule LOD LOQ Doxorubicin 0.085 0.258 Table-5 Robustness Data of Doxorubicin S.no. Condition RSD of Doxorubicin 1 Flow rate - 0.9ml/min 0.2 2 Flow rate + 1.1 ml/min 0.2 3 Mobile phase - 50B:50A 0.5 4 Mobile phase + 60B:40A 0.6 5 Temperature - 25°C 0.5 6 Temperature + 35°C 0.3

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Monika R et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 09-16 15 Table-6: System Suitability Parameters Result of Doxorubicin S no Doxorubicin Inj RTmin USP Plate Count Tailing 1 2.585 2513 1.13 2 2.585 2640 1.12 3 2.588 2922 1.15 4 2.590 2291 1.12 5 2.597 2900 1.13 6 2.603 2234 1.12 Table -7: Assay Results of Doxorubicin S. No. Doxorubicin Assay 1 98.10 2 98.20 3 98.74 4 99.79 5 99.07 6 99.00 AVG 98.82 STDEV 0.6241 RSD 0.63 Table 8. Degradation Data of Doxorubicin S.NO Degradation Condition Drug Degraded Purity Angle Purity Threshold 1 Acid 4.98 0.368 0.665 2 Alkali 2.79 0.402 0.712 3 Oxidation 1.98 0.381 0.517 4 Thermal 0.51 0.358 0.526 5 UV 0.54 0.416 0.571 6 Water 0.54 0.316 0.532 CONCLUSION A simple Accurate precise method was developed for the simultaneous estimation of the doxorubicin in injection dosage form. Retention time of doxorubicin was found to be 2.58min. RSD was found to be 0.6. Recover was obtained as 99.90. LOD LOQ values were obtained from regression equations of doxorubicin was 0.085µg/ml and 0.258µg/ml respectively. Regression equation of doxorubicin is y 15456x + 2384. Retention times are decreased and that run time was decreased so the method developed was simple and economical that can be adopted in regular Quality control test in Industries.

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Monika R et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-71 2019 09-16 16 REFERENCES 1. https://www.drugbank.ca/drugs/DB00997. 2. https://www.rxlist.com/adriamycin-pfs-side-effects-drug-center.htm. 3. https://en.wikipedia.org/wiki/Doxorubicin. 4. Hajare ashok a powar trupti a bhatia neela m More harinath n. Development and validation of rp-hplc method for determination of doxorubicin hydrochloride from vacuum foam dried formulation. Research j. pharm. and tech 99 2016 1352-1356. 5. B 1 meenhorst pl van gijn r fromme m rosing h underberg wj. Hplc determination of doxorubicin doxorubicinol and four aglycone metabolites in plasma of aids patients. J pharm biomed anal. 910-12 1991 995-1002. 6. Khairallahalharethab1christinevauthier ab clairegueutin ab gilles.Hplc quantification of doxorubicin in plasma and tissues of rats treated with doxorubicin loaded poly alkylcyanoacrylate nanoparticles. Journal of chromatography 887–888 2012 128-132. 7. Drugs Cosmetics Act 1940 Rules 2 1945 Susmit publishers Mumbai India 2000. 8. Indian Pharmacopoeia Ministry of Health Family Welfare Government of India New Delhi 1996. 9. The United States Pharmacopoeia- the National Formulary United States Pharmacopoeial convention Rockville 2007. 10. British Pharmacopoeia the Stationary Office London 2005. 11. ICH Harmonised Tripartite Guideline 2015. How to cite this article: Monika Renikunta Dr.T.Rama Mohan Reddy Dr.Konde Abbulu. RP-HPLC method development and validation for estimation of doxorubicin and in bulk and pharmaceutical dosage form. Int J of Allied Med Sci and Clin Res 2019 71: 09-16. Source of Support: Nil. Conflict of Interest: None declared.

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