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1 IJAPR Available Online through Research Paper ISSN: METHOD DEVELOPMENT AND VALIDATION FOR SYMULTANEOUS ESTIMATION OF CITICOLINE AND METHYLCOBALAMIN BY RP- HPLC METHOD China Babu. Dara*, Dr.Sreenivasulu. M, Vasanth P M, Anusha M Narayana Pharmacy College, Chinthareddypalem, Nellore Received on Revised on Accepted on ABSTRACT A simple, rapid, and accurate reversed phase high-performance liquid chromatographic (RP-HPLC) method has been developed and subsequently validated for the simultaneous determination of citicoline and methylcobalamine in combination. The separation was carried out using a mobile phase consisting of phosphate buffer and acetonitrile in the ratio of 50: 50. The ph of the mobile phase was adjusted to 3.0 with orthophosphoric acid. The column used was Inertsil C18 YMC ( mm, 5 μm) with flow rate of 0.8 ml/min using UV detection at 295nm. The total run time was 8 min and the retention time of citicoline and methylcobalamine was 3.2 min and 4.1min, respectively. The described method was linear for the assay of citicoline and methylcobalamine over a concentration range of 1000 μg/ml and 1.5/mL respectively. Results of the analysis have been validated statistically and by recovery studies. The limit of quantitation for citicoline and was methylcobalamine found to be μg/mL and μg/mL respectively. The results of the studies showed that the proposed RP-HPLC method is simple, rapid, precise, and accurate, which is useful for the routine determination of citicoline and methylcobalamine bulk drug and its pharmaceutical dosage form. Key Word: Citicoline, HPLC, INTRODUCTION (1,2) Citicoline Sodium Citicoline is a complex organic molecule that functions as an intermediate in the biosynthesis of cell membrane phospholipids. Citicoline is also known as CDP-choline and cytidine diphosphate choline (cytidine 5 -diphosphocholine). CDP-choline belongs to the group of biomolecules in living systems known as nucleotides that play important roles in cellular metabolism. CDP-choline is composed of ribose, pyrophosphate, cytosine (a nitrogenous base), and choline.1 Exogenous Citicoline research in animal experiments and human clinical trials provides evidence of its cholinergic and neuro protective actions. As a dietary supplement, Citicoline appears useful for improving both the structural integrity and functionality of the neuronal membrane that may assist in membrane repair. Animal and clinical studies indicate the potential of Citicoline to improve cognitive deficits, stroke rehabilitation, brain and spinal cord injuries, neurological diseases, and eye conditions. sodium Structure of Citicoline is one of the two coenzyme forms of vitamin B12 (the other being adenosylcobalamin). It is a cofactor in the enzyme methionine synthase which functions to transfer methyl groups for the regeneration of methionine from homocysteine. IJAPR /Oct. 2015/ Vol. 6/Issue.10/

2 Evidence indicates methylcobalamin is utilized more efficiently than Cyanocobalamin to increase levels of one of the coenzyme forms of vitamin B12. Experiments have demonstrated similar absorption of methylcobalamin following oral administration. The quantity of cobalamin detected following a small oral dose of methylcobalamin is similar to the amount following administration of Cyanocobalamin; but significantly more cobalamin accumulates in liver tissue following administration of methylcobalamin. Human urinary excretion of methylcobalamin is about one-third that of a similar dose of Cyanocobalamin, indicating substantially greater tissue retention. solution containing 1000μg/mL of Citicoline and 1.5μg/mL. Preparation of working standard solutions Different volumes of stock solutions of each were accurately transferred in to 10mL volumetric flasks and diluted to mark to yield a concentration range of μg/ml solutions of Citicoline and μg/mL solutions of Methylcobalain. Preparation of Sample Solution A powder quantity equivalent to 500 mg Citicoline and 0.75 mg was accurately weighed and transferred to volumetric flask of 100 ml capacity. 50mL of distilled water was transferred to this volumetric flask and sonicated for 15 min. The flask was shaken and volume was made up to the mark with distilled water. The above solution was filtered through Whatmann filter paper (0.45μ). From this solution 5mL was transferred to volumetric flask of 25mL capacity. Volume was made up to the mark to give a solution containing 1000μg/mL of Citicoline and 1.5μg/mL of. Structure of methylcobalamine MATERIAL AND METHODS Materials and reagents Analytically pure Citicoline and were kindly provided by Lara Laboratory, Hyderabad as gift samples. HPLC grade acetonitrile was purchased from Merck & Co. Glass wares used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in hot air oven. Triple distilled water is used for all purpose. The commercial combined dosage form was purchased from local pharmacy. Instrumentation HPLC system (Waters with Empower2 Software) containing C 18 (YMC, 250 x 4.6mm, 5µ) column with UV- PDA detection. Preparation of standard solutions Accurately weighed 500mg of Citicoline and 0.75mg standard were transferred to separate 100 ml volumetric flask and dissolved in 10 ml methanol. The flasks were shaken and volume was made up to the mark with distilled water to give solutions containing 5000 μg/ml Citicoline and 7.5 μg/ml. From this solution 5mL was transferred to volumetric flask of 25 ml capacity. Volume was made up to the mark to give a METHOD DEVELOPMENT (3,4) Chromatographic Conditions The mobile phase consisted of Potassium dihydrogen phosphate: Acetonitrile (adjusted to ph 3.0 using orthophosphoric acid) in the ratio of 50:50 v/v. The contents of the mobile phase were filtered before use through a 0.45μ membrane and degassed for 10 min. The mobile phase was pumped from the solvent reservoir to the column at a flow rate of 0.8 ml/min and the injection volume was 20μL. The column temperature was maintained at ambient temperature. The eluents were monitored at 295 nm. Method Validation (5) The proposed method has been extensively validated in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility as per ICH guidelines. The accuracy was expressed in terms of percent recovery of the known amount of the standard drugs added to the known amount of the pharmaceutical dosage forms. The precision was expressed with respect to the repeatability, intra and inter-day variation in the expected drug concentrations. After validation, the developed methods have been applied to pharmaceutical dosage form. Experimental Proceedings Experimental Approach Towards Method Development The proposed method has been extensively validated in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility as per ICH guidelines. The accuracy was expressed in terms of percent IJAPR /Oct. 2015/ Vol. 6/Issue.10/

3 recovery of the known amount of the standard drugs added to the known amount of the pharmaceutical dosage forms. The precision was expressed with respect to the repeatability, intra and inter-day variation in the expected drug concentrations. After validation, the developed methods have been applied to pharmaceutical dosage form. RESULTS System Suitability Criteria It is defined as tests to measure the method that can generate result of acceptable accuracy and precision. Table No: 1 The system suitability was carried out after the method development and validation have been completed. The system suitability was assessed by five replicate analyses of the drugs at concentrations of 1000μg ml 1 of Citicoline and 1.5μg ml 1 of and for this, parameters like plate number (n), tailing factor, retention time, resolution, similarity of samples were measured. System suitability parameters for Citicoline and Parameter Value obtained (n=6) Acceptance Citicoline criteria Plate count >2000 Tailing factor Resolution >2 Similarity Rt (min) %RSD Fig: 1 Standard chromatogram for Citicoline and Linearity Data Linearity Appropriate volume of aliquot from Citicoline and standard stock solution was transferred to volumetric flask of 10mL capacity. The volume was adjusted to the mark with methanol to give a solution containing μg/mL Citicoline and μg/mL. The slope, Y-intercept and correlation coefficient were calculated Calibration Cruves IJAPR /Oct. 2015/ Vol. 6/Issue.10/

4 Fig:2 Calibration curve of Citicoline Fig:3 Calibration curve of Recovery Report of Citicoline and Accuracy Accuracy of the method was studied by recovery experiment.the recovery experiment were performed by adding kown amount of the pure. The recover was done at three levels 50%, 100% and 150% of the lable claim three samples were prepared for each recovery level. The recovery values for citicoline and methycobalamin ranged from and , respectively (Table:3) average recovery of three levels for citicoline and methycobalamin were and Table No: 3 Drug Amount taken (µg/ml) Citicoline Recovery level (%) Amount of drug added Amount of drug found % RSD % Recovery Precision The repeatability was evaluated by assaying 6 times of sample solution prepared for assay determination. The intra and inter-day precision study of Citicoline and was carried out at 100% concentrations of Citicoline and, 3 times on the same day and on 3 different days (first, second, third). Intraday Precision Data for Citicoline and Table No: 4 S.No Rt of citicoline (min) Peak area of citicoline Rt of methylcobalamin (min) Peak area of methylcobalamin Citicoline %RSD Methylcobalami n IJAPR /Oct. 2015/ Vol. 6/Issue.10/

5 Average Inter-day Precision Data of Citicoline and Table No: 5 S.No Rt Of Peak Rt Of Peak Area Of %RSD Citicoline Area Of Citicoline (min) Citicoline (min) Average Robustness The robustness of the method was evaluated by analyzing the system suitability standards and evaluating system suitability parameter data after varying the HPLC pump flow rate (±0.1mL) and temperature (±1 o C). None of the alterations caused a significant change in peak area, retention time, USP tailing factor. Robustness studies of Citicoline and Table No : 6 S.No Parameter Modification Retention Time Tailing Factor Citicoline Methylcobalami Citicoline Methylcobala n min Flow Rate (ml/min) 2 Temperature c c LOD and LOQ LOD and LOQ were calculated from the formula 3.3 x (σ/s) and 10 x (σ/s), respectively where, σ is standard deviation of intercept and S is the mean of slope. The LOD and LOQ can also be determined by S/N. The limit of detection of citicoline and methylcobalamine were found to be and 0.05µg/mL. The limit of Quantification of citicoline and methylcobalamine were found to be and 0.15 µg/ml. Assay of formulation Assay was performed by taking standard solution and sample solutions were injected.the percentage purity of citicoline and methylcobalamine were found to be 99% and 101%. Table No:7 S.No Brand name Content Peak area Assay %RSD 1 Tablet (Nervijen-CT ) 500mg- Citicoline % µg % 0.56 IJAPR /Oct. 2015/ Vol. 6/Issue.10/

6 RESULTS AND DISCUSSION A RP-HPLC method was developed and validated for the determination of citicoline and methylcobalamin in tablet dosage forms on Itersil C 18 YMC (250 X 4.6 mm 5μm) with wavelength detection at 295 nm. The retention time of citicoline and methylcobalamin was 3.2 min and 4.1 min respectively. Linear correlation was obtained between area and concentration of citicoline and methylcobalamine in the concentration range of μg/ml and μg/ml respectively. The low RSD value of inter-day and intraday at 295 nm, reveal that proposed method is precise. The limit of detection (LOD) and limit of quantification (LOQ) for citicoline and methylcobalamin were found to be μg/ml and 0.05μg/ml and 90.54μg/ml and 0.15μg/ml respectively. These data show that method is sensitive for the determination of citicoline and methylcobalamine. The recovery experiment was performed by the standard addition method. The mean recoveries of citicoline and methylcobalamin were % and % respectively. The results of recovery studies indicate that the proposed method is highly accurate. The proposed method was found to be robust enough (% RSD < 2.) to withstand such slight changes and allow routine analysis of the sample. The proposed validated method was successfully applied to determine citicoline and methylcobalamin in the tablet dosage form. The results obtained for citicoline and methylcobalamin were comparable with the corresponding labeled amounts. Hence the proposed method is applicable for the routine simultaneous estimation of citicolinne hydrochloride and methylcobalamin in pharmaceutical dosage form. SUMMARY AND CONCLUSION In this proposed method the linearity is observed in the concentration range of μg/ml and μg/ml with co-efficient of correlation (r 2 ) and for CITI and MCM, respectively at 295 nm. The results of the analysis of pharmaceutical formulation by the proposed method are highly reproducible and reliable and it is in good agreement with the label claim of the drug. The method can be used for the routine analysis of the CITI and MCM in combined dosage form without any interference of excipients. From the results, it was found that the developed RP-HPLC method was found to be simple, accurate, sensitive, precise, specific and rapid. The method can be applied for routine analysis of CITI and MCM in pure and its formulations. Table : 6 Summary of Validation Parameters Parameters RP-HPLC Method CITI MCM Linearity µg/ml µg/ml Regression equation y = 2108x y = 25764x Slope Intercept Correlation coefficient Precision Intraday (%RSD) Inter-day LOD (µg/ml) LOQ (µg/ml) % Recovery (Accuracy) 99.91% ± 1.04% 99% ± 1.33% % Assay 99% 101% REFERENCE 1. The Merck Index- An Encyclopedia of chemicals, Drugs and Biologicals, 14 th Edn, Merck Co. Inc: 2319, (2001). 2. Pathan AB, Doijad RC, and Gaikwrd SL. Therapeutics application of citicoline and methylcobalamin combination. Advance Res. In pharm. Bio. 2012; 2(3): Chandrul Kaushal K. and B. Srivastava; A process of method development: A chromatographic approach; J. Chem. Pharm. Res., 2010, 2(2): Synder LR, Kirland JJ, and Glajch LJ.In Practical HPLC Method Development, 2 nd edn;jhon Wiley & sons, Inc:21-57, ,(1997). 5. Q2B Validation of Analytical Procedures: Methodology; US FDA ICH Guidelines, (1996); IJAPR /Oct. 2015/ Vol. 6/Issue.10/

7 6. Neetu Sachan, Phool Chandra, Mayank Yadav, Dilipkumar Pal and Ashoke K Ghosh; Rapid analytical procedure for Citicoline in bulk and pharmaceutical dosage form by UV Spectrophotometer; Journal of Applied Pharmaceutical Sciences, (2011); 01 (6); Sanjay Surani, Ritu Kimbahune, Prachi Kabra, Urmila G. H; Spectrophotometric Determination of Citicoline Sodium in Pure Form and Pharmaceutical Formulation; Scholars Research Library, (2010); 2(5); Minakshi M. Dhoru, Sanjay Surani, Priti Mehta; UV-Spectrophotometric Methods for Determination of Citicoline sodium and Piracetam in Pharmaceutical Formulation; Scholars Research Library, (2012); 4 (5); Om Sharma and Tara Chand; Analytical Method Development and Its Validation for Estimation of Citicoline Sodium by Reversed Phase High Performance Liquid Chromatography (RP-HPLC); International Journal of Research in Pharmaceutical and Biomedical Sciences, (2013); 4 (2); Raga Pravallika E, Prashanthi D and ISMAIL Y; Method Development and Validation of RP-HPLC for Simultaneous Estimation of Citicoline and Piracetam in Tablet Dosage Form; International Journal of Chemical and Pharmaceutical Sciences, (2012); 3 (2); Sonali O. Uttarwar, R.T. Jadhav, C.G. Bonde; Stability Indicating LC Method of Citicoline Sustained Release Tablets; International Journal of PharmTech Research, (2010); 2 (4); Narmada p, Vijaya Lakshmi G, Nalini G; RP-HPLC Method Development and Validation for Determination of and Pregabalin in Combined Capsule Dosage Form; International Journal of Research in Pharmaceutical Sciences, (2013); 4 (1); Saravanan.J, Shajan A, Joshi NH, Varatharajan R.; A Simple and validated RP-HPLC method for the estimation of in bulk and capsule dosage form; International Journal of Chemical and Pharmaceutical Sciences, (2010); 1 (2); IJAPR /Oct. 2015/ Vol. 6/Issue.10/