Page3639 Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: 2231-6876 RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS DETERMINATION OF CITICOLINE AND PIRACETAM IN PHARMACEUTICAL DOSAGE FORM Manutosh Acharya 1, A.K Jain 2, Naveen Sharma 3, Suman Jain 4, Gurdeep Singh Saluja 5, Ashish Dixit 6 1 OmniActive Health Technologies, Thane (W)-400 064. 2 Pharmaceutical Chemistry, Department of Pharmacology, Gajraraja Medical College, Gwalior (M.P. 3 Department of Pharmacology, Shri Ram Pharmacy College, Gwalior (M.P.) 4 SOS Pharmaceutical Sciences, Jiwaji University, Gwalior (M.P) 5 Department of Pharmaceutical Chemistry, Shri Ram Pharmacy College, Gwalior (M.P.) 6 Department of Pharmaceutical Analysis, Shri Ramnath Singh Institute of Pharmaceutical Science and Technology, Gwalior (M.P.)- 474011. ARTICLE INFO Article history Received 17/08/2015 Available online 10/09/2015 Keywords, Citicoline, Assay, RP-HPLC. Corresponding author Manutosh Acharya Vice President-R&D OmniActive Health Technologies New Technology Centre, A-10 Road No 1, Wagle Industrial Estate, Thane (W)-400 064, India m.acharya@omniactives.com ABSTRACT A new simple, selective, rapid, precise reversed phase high performance liquid chromatography method has been developed and validated for the simultaneous estimation of and Citicoline in a pharmaceutical dosage form. The separation was made using Inertsil C18, (250 x 4.6) mm, 5µm column. Mobile phase used contained Phosphate buffer and acetonitrile in gradient mode at wavelength of 210nm. The mobile-phase flow rate and the sample volume injected were 1 ml/min and 10 μl, respectively. Retention time of Citicoline and were found to be 4.2 ±0.2mins, 12.3±0.2minsrespectively. A good linear relationship (Citicoline r=0.999& r=0.999) was observed over a concentration range of 80.50 to 603.73 µg/ml of and 50.66 to 379.94 µg/ml of Citicoline. The limit of detection (LOD) and limit of quantification (LOQ) for Citicoline was found to be 0.08 ppm&0.26ppm and for it was found to be0.12 ppm & 0.42ppm. It was concluded that in the present developed RP- HPLC method, the standard and sample preparation required less time and without tedious extraction. The developed method is simple, rapid, and accurate, hence can be used for routine quality control analysis in Pharmaceutical industry. The method was optimized on gradient mode; offering better resolution of peak of interest as well as impurities in contrast to all existing methods which are on isocratic mode. Please cite this article in press as Manutosh Acharya et al. RP-HPLC Method Development and Validation For Simultaneous Determination of Citicoline and in Pharmaceutical Dosage Form. Indo American Journal of Pharmaceutical Research.2015:5(08). Copy right 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Page3640 INTRODUCTION Citicoline (Cytidine-5 -diphosphocholine or CDP) is an intermediate in the biosynthesis of phosphatidylcholine.it play important roles in cellular metabolism. Citicoline is readily absorbed in the GIT and widely distributed throughout the body, crosses the Central Nervous system, where it is incorporated into the membrane and microsoma fraction. It activates biosynthesis of structural phospholipids, increases brain metabolism, and acts on the levels of different neurotransmitters. It has a variety of applications in CNS injury models neurological disorders of the brain such as stroke, brain, trauma, Alzheimer's and Parkinson s disease. (1,2,3) is a nootropic drug in the racetams group, with chemical name 2-oxo-1-pyrrolidine acetamide. is a cyclic derivative of GABA. Both drugs are psychotherapeutic agents, used as psycho stimulant, nontropic and neurotonics.both drugs are freely soluble in water. These drugswill increase cerebral metabolism and increase level of various neurotransmitters, including acetylcholine and dopamine, exerting its action by activating the biosynthesis of structural phospholipids in neuronal membrane. This drug will increase the blood flow and oxygen consumption in brain. (1, 2, 3) The review of literature regarding quantitative analysis of Citicoline and revealed that the attempts were made to develop analytical methods for estimaton of Citicoline and in serum. Some spectrometric methods and LC methods have been reported for the estimation of the individual drugs. The focus of the present study was to develop, optimize and validate a rapid, specific, and economic and stability indicating RP-HPLC method for the simultaneous estimation of Citicoline and in tablet dosage form adaptable by pharmaceutical industry. (4,5,6) The method was optimized on gradient mode ;offering better resolution of peak of interest as well as impurities so it is useful even for studies degradation impurities. MATERIALS AND METHODS Materials HPLC:Waters HPLC 2 2695 series consisting pump, Auto sampler, UV- Visible detector, Thermostat column compartment connected with Waters (alliance) Empower2 software Balance Sartorius Cpa225d Semi Micro Balance. Sonicator Bio-Technics India, Mumbai Reagents buffer reagents such as potassium dihydrogen phosphate, ion pairing reagents such as hexane sulphonic acid sodium salt. Organic solvents such as methanol and acetonitrile Methodology-Assay and Validation of Simultaneous determination of and Citicoline in tablet Chemicals and Reagents Potassium dihydrogen orthophosphate, hexane sulphonic acid sodium salt, ortho phosphoric acid Chromatographic conditions Column : Inertsil C18, (250 x 4.6) mm, 5µm or equivalent Detector : UV-Visible (Use HPLC with PDA, MWD or DWD) Detection Wavelength : 210 nm Flow rate : 1.0 ml/min Injection volume : 10 µl Column oven temperature : 30 C Sample cooler temperature : NA Runtime : 25minutes Diluent : 0.001 N HCl (ph 3.0 HCl) Table 1:Gradient Program. Time (Minutes) Mobile Phase A Mobile Phase B 0 100 0 8 100 0 18 90 10 20 100 0 25 100 0 i)blank: 0.001 N HCl (ph 3.0 HCl) ii)mobile Phase Mobile Phase - A: 6.8 gm of potassium dihydrogen orthophosphate & 1.0 gm of hexane sulphonic acid sodium salt were dissolved in 1000 ml Mili-Q water, ph of above solution was adjusted to 3.0 ± 0.05 with diluted ortho phosphoric acid.themobile phase buffer was filtered through 0.45 µm membrane filter. Mobile Phase - B: Acetonitrile Preparation of Diluent: 0.001 N HCl
Page3641 iii) Standard Preparation: 40 mg of working standard and 27.2 mg of Citicoline sodium working standard (eq.to 25 mg of Citicoline) was weighed accurately into a clean and dry 100 ml volumetric flask, 50 ml of diluent was added and sonicated to dissolve. Volume was make-up to the mark with diluent.(concentration of Citicoline was about 250µg/ml and was about 400µg/ml). iv) Sample Preparation: 20 tablets were weighed accurately and the average weight was determined. The tablets were crushed to fine powder. The powder equivalent to 500 mg of Citicoline were weighed into a 200 ml volumetric flask, 100 ml of Diluent was added to it and sonicated for 15 minutes with intermittent shaking. The solution was allowed to cool at room temperature. Volume was make-up to the mark with diluent, mixed & Filtered. 5 ml of this stock solution was pipetted out in 50 ml volumetric flask and diluted up to the mark with diluent.(concentration of Citicoline was about 250µg/ml and was about 400µg/ml). The order of injection followed in HPLC was: Table 2:Order Of Injection. Sr.No. Sample Name No. of Injections 1 Blank 1 2 Standard preparation 5 3 Test preparation 2 Validation of quantitative HPLC method (7, 8): The optimized RP-HPLC method for assay was validated according to the procedures described in ICH guidelines Q2 (R1) for the validation of analytical method (ICH 2005). Specificity Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc Linearity & Range It is the ability of the method to elicit test result that is directly proportional to analyte concentration within a given range. Acceptance criteria: The correlation coefficient should be NLT 0.999. Calibration curve was plotted over a concentration range of 50.66 to 379.94 µg/ml of Citicoline and 80.50 to 603.73 µg/ml of. Samples are prepared, filtered and injected into HPLC system. Aliquots (10 µl) of each solution were injected from auto sampler under the operating chromatographic conditions described above. Calibration Curve was constructed by plotting peak area verses concentration and the regression equation was calculated. Accuracy& Recovery The absolute recovery of analytical method was measured as the response of a processed spiked matrix standard expressed as a percentage of the response of pure standard which has not been subjected to sample pretreatment and indicates whether the method provides a response for the entire amount of analyte that was present in the sample. The accuracy of the method was determined by calculating recoveries of Citicoline and by the standard addition method. Known amounts of standard solutions of Citicoline and were spiked to placebo preparation at different levels (40%, 80%, 100% and 120% levels). The area under curve obtained was checked and analyzed for the recovery percentage. Limit of Detection (LOD) and Limit of Quantification (LOQ) The limit of detection (LOD) and the limit of quantification (LOQ) of Citicoline and were derived by Calculating the signal-to-noise ratio (S/N) using the following equations designated by International Conference on Harmonization (ICH) guidelines. LOD = 3.3 s/s LOQ = 10 s/s Where, s = the standard deviation of the response and S = slope of the calibration curve. The LOD and LOQ were estimated by injecting lower concentration solutions of Citicoline and and determining % RSD of area responses of 6 replicate injections. The LOD and LOQ were confirmed and recorded separately on the basis of signal (S) to noise (N) ratio.
Page3642 Method Precision The precision of method was checked and verified by repeatability,inter-day precision and variability due to analyst.repeatability was checked by injecting six test preparations into the HPLC system as per the test method.ruggedness of the proposed analytical method was evaluated for variability studies like, Variability due to Analyst and variability due to different day. Assay of Citicoline sodium & tablets was performed in six replicates as per the proposed test method and analysedfor proposed variability. Results were compared with the method precision data obtained under precision studies. The % Assay was calculated for each of the sample. Solution Stability The solution stability of Citicoline and sample and standard solution was carried out by leaving both the test and standard solution in tightly capped volumetric flask at room temperature for 15 hours. The same sample solution was analyzed initially and at various time intervals up to the 15 hours throughout the study period. Robustness To determine the robustness of the developed method, minute changes were made in the flow rate, percentage of organic phase, mobile phase ph and column compartment temperature. The deviations in all parameters from optimized method were studied. System Precision System suitability experiments can be defined as tests to ensure that the system can generate results of acceptable accuracy and precision. The requirements for system suitability are usually developed after method development and validation have been completed. To perform the system suitability test the standard solution were freshly prepared and injected under the condition of optimized method. Table 3: Validation Parameters &their Acceptance criteria. S.No. Parameters Acceptance criteria 1 Specificity No interference observed for response due to analyte or impurities of interest 2 Linearity & Range correlation coefficient NLT 0.999 3 Accuracy & Recovery NLT 98.0% and NMT 102.0% at various recovery levels 4 Limit of Detection (LOD) To be determined by test 5 Limit of Quantification (LOQ) To be determined by test 6 Method Precision RSD NMT 2.0% 7 Solution Stability To be determined by test 9 System Precision RSD NMT 2.0% RESULTS AND DISCUSSION Optimised method obtained was using Phosphate buffer (6.8 gm of potassium dihydrogen orthophosphate & 1.0 gm of hexane sulphonic acid sodium salt in 1000 ml Mili-Q water, ph 3.0±0.1 adjusted with diluted ortho phosphoric acid) and acetonitrile under gradient mode as Mobile phase and a flow rate of 1.0 ml/min. Peaks were well defined, symmetrical and resolved. Retention time of Citicoline and was found to be 4.2 ±0.2, 12.3±0.2 mins respectively and the optimum wavelength was determined to be 210 nm. Specificity Blank (diluent), placebo and samples were injected into HPLC and peak Purity of analyte Peaks and impurity peaks was checked. The impurity peaks were found to be well resolved from each other and from analytes peaks. No interference was observed at the retention time of analyte peak from the blank & placebo solution. Linearity & Range: The linearity data for and Citicoline has been given in Table 4.
Page3643 Table 4: Linearity data for. Citicoline S. No. Conc. Conc. (µg/ml ) (µg/ml ) 1 80.50 1054996 50.66 627429 2 160.99 2099812 101.32 1246557 3 321.99 4136773 202.63 2482310 4 362.24 4627756 227.96 2782466 5 402.48 5138647 253.29 3102798 6 7 442.73 482.98 5562852 6110723 278.62 303.95 3361595 3709260 8 603.73 7576585 379.94 4618302 Slope 12450 12108 12108 Intercept 91335 19472 19472 CC 0.99988 0.99994 0.99994 The correlation coefficient was observed to be 0.99988 and 0.99994 for and Citicoline respectively that meets the acceptance criteria (NLT 0.999). This concludes that the method is linear throughout the range selected. Limit of Detection (LOD) and Limit of Quantification (LOQ): The LOD and LOQ were estimated by injecting lower concentration solutions of Citicoline and and determining % RSD of area responses of 6 replicate injections. Data obtained is shown in Table-5. Table-5: LOD and LOQ data of. Concentration ppm % w/w LOD 0.12 0.01 LOQ 0.42 0.04 Injection no. Counts -LOD S/N Value Counts - LOQ S/N Value 1 5185 4 9848 12 2 3266 3 9458 10 3 4149 5 8183 10 4 2964 4 8246 8 5 3998 3 8845 9 6 5349 5 7586 8 Mean 4152 4 8694 10 SD 971.6-851.8 - RSD (%) 23.40-9.80 - Table-6: LOD and LOQ data of Citicoline. Injection no. Counts -LOD S/N Value Counts - LOQ S/N Value 1 2714 5 7089 16 2 2948 5 6616 12 3 4002 8 6543 14 4 4071 8 6439 11 5 2942 5 7464 13 6 4017 7 6841 13 Mean 3449 6 6832 13 SD 642.4-387.3 - RSD (%) 18.63-5.67 - The % RSD for both the analytes was found well within the accepted criteria (NMT 10.0% at LOQ level and NMT 33.0% at LOD level). Hence the method has suitable level of LOD/LOQ. Accuracy: Data obtained shown in Table 7.
Page3644 Table 7: Accuracy (% Recovery) data for and Citicoline. Recovery Level % Recovery for % Recovery for Citicoline 40% level 101.0 98.7 80 % Level 99.5 100.0 100% Level 98.9 100.9 120% Level 99.3 99.2 The % Recovery was found well within the acceptance criteria at various recovery levels (NLT 98.0% and NMT 102.0%). Hence the method is accurate throughout the selected range. Solution Stability: The data shown in Table 8 indicate that the sample solution is stable for 13 hours at room temperature. Timepoint Table-8: Solution Stability data of Citicoline& at room temperature. Standard Preparation Sample Preparation Citicoline Citicoline Absolute % Absolute % Absolute % Time- Difference Difference Difference Counts Counts point Counts Counts w.r.t. Initial w.r.t. Initial w.r.t. Initial Initial 3397524-5809667 - Initial 3509894-5582976 - 5 hr 3393275 0.13 5780806 0.50 3 hr 3481310 0.81 5543938 0.70 8 hr 3372739 0.73 5767386 0.73 6 hr 3512760 0.08 5611669 0.51 11 hr 3359139 1.13 5740500 1.19 9 hr 3458630 1.46 5526455 1.01 12 hr 3361566 1.06 5737012 1.25 10 hr 3466733 1.23 5536896 0.83 13 hr 3351778 1.35 5728847 1.39 11 hr 3483952 0.74 5570250 0.23 14 hr 3351824 1.35 5726390 1.43 12 hr 3484869 0.71 5566785 0.29 15 hr 3345934 1.52 5734372 1.30 13 hr 3509239 0.02 5596031 0.23 Absolute % Difference w.r.t. Initial From the results it is concluded that the standard solution is stable for about 15 hours and sample solution is stable for about 13 hours at room temperature. Robustness: System suitability parameters were met under all robustness conditions except change in organic content in gradient program where significant retention time shift was observed for both the analyses peaks. Hence it is recommended that organic content should be strictly adhered to as per the method. The system suitability data under robustness conditions has been summarized in the Table 9.
Page3645 Table 9: Robustness Data for Standard solution. Condition Standard Preparation Citicoline RT USP Plate Count USP Tailing RT USP Plate Count USP Tailing Control 4.22 5408 1.16 13.31 12048 1.05 Flow Minus (0.9 ml/min) Flow Plus (1.1 ml/min) 4.69 5728 1.16 14.78 12602 1.06 3.85 5020 1.15 12.07 11156 1.04 Temperature Minus (25 C) 4.50 5269 1.14 14.16 11249 1.04 Temperature Plus (35 C) 4.01 5468 1.17 12.55 12244 1.05 Control 4.869 6101 1.07 16.012 15594 1.15 (-) Organic content in Mobile Phase B in gradient program* 4.876 6089 1.07 15.653 15354 1.13 (+) Organic content in Mobile Phase B in gradient program* 3.614 4425 1.09 9.096 13240 1.05 ph Minus (ph=2.80) 5.174 6438 1.07 14.593 14737 1.13 ph Plus (ph=3.20) 5.134 6329 1.07 14.606 14718 1.13 Wavelength minus (λ=205) 4.823 6075 1.07 15.705 15697 1.11 Wavelength plus (λ=215) 4.823 6073 1.07 15.705 15790 1.11 The retention time (RT) and system suitability data under various robustness conditions in comparison to control sample has been summarized in the Table 10. Table 10: Robustness Data for Control Sample. Control Sample Condition Citicoline RT USP Plate Count USP Tailing RT USP Plate Count USP Tailing Control 4.23 5343 1.18 13.21 10824 1.27 Flow Minus (0.9 ml/min) 4.68 5683 1.18 14.70 11266 1.29 Flow Plus (1.1 ml/min) 3.84 5011 1.17 12.01 10247 1.24 Temperature Minus (25 C) 4.51 5226 1.17 14.05 10368 1.27 Temperature Plus (35 C) 4.00 5434 1.19 12.44 11100 1.25 Control 4.864 6090 1.07 16.037 16229 1.08 (-) Organic content in Mobile Phase B in gradient program (+) Organic content in Mobile Phase B in gradient program 4.885 6136 1.06 15.688 15895 1.04 3.613 4432 1.09 9.100 13385 1.03 ph Minus (ph=2.80) 5.173 6409 1.07 14.622 15290 1.05 ph Plus (ph=3.20) 5.175 6431 1.06 14.649 15271 1.05 Wavelength minus (λ=205) 4.818 6181 1.06 15.623 15686 1.11 Wavelength plus (λ=215) 4.818 6180 1.06 15.623 15764 1.11
Page3646 Parameters Linearity And Range Regression Line Equation Correlation Coefficient (R 2 ) Precision (%RSD) Method Precision (n=6) Intermediate Table 11: Summary Of Validation Parameters For Assay. Acceptance Criteria Correlation Coefficient(R 2 ) NLT 0.999 NMT 2.0 NMT 2.0 Citicoline 50.66-379.94 µg/ml y=12108x+19472 0.99994 1.83 1.24 80.50-603.73 µg/ml y=12450x+91335 0.99988 1.75 1.28 Ruggedness (n=12) RSD 2.0% 1.93 1.65 LOD (n=6) 0.08µg/mL 0.12µg/mL (0.01%w/w) (0.01%w/w) LOQ (n=6) 0.26µg/ml 0.42µg/ml (0.04%w/w) (0.04%w/w) Accuracy (98.0%- 102.0%) 1. 40% level 2. 80% Level 3. 100% Level 4. 120% Level Solution Stability (At Room Temperature) Specificity Robustness 2% RSD 98.7 100.0 100.9 99.2 Complies 2% RSD for 13 Hours 101.0 99.5 98.9 99.3 Complies 2% RSD for 13 Hours Specific (Passes peak purity data analysis) Robust except Organic Plus condition CONCLUSION A new simple, selective, rapid, precise reversed phase high performance liquid chromatography method has been developed and validated for the simultaneous estimation of and Citicoline in a pharmaceutical dosage form. The method is suitable for routine QC analysis in Pharmaceutical industry. The method was optimized on gradient mode and is capable of resolving peaks between main peaks and any impurities &/or degradants formed during stability and hence can be used as stability indicating method (work to be published subsequently).the similar chromatography method may be applied to related substances also. Future research is recommended in any other pharmaceutical dosage form as the method is optimized to elute the maximum numbers of impurities. CONFLICT OF INTEREST The authors confirm that there is no conflict of interest. ACKNOWLEDMENT The author is thankful to the management and colleagues of Plethico Pharmaceuticals Limited and Jiwaji University for providing the research facilities, drugs and initiatives to carry out the work. The author is thankful to Ratna Upadhyay for supporting in research publication.
Page3647 REFERENCES 1. Wurtman RJ et al., Effect of oral CDP- choline on plasma choline and uridine levels in humans,biochempharmacol, 2000, 60 (7), 989-92. 2. Rao AM et al., CDP- choline: neuroprotection in transient forebrain ischemia of gerbils." Journal ofneuroscience Res, 1999, 58(5), 697-705. 3. Neil MJ, editor. The Merck Index- and Encyclopedia of chemicals. 13 th Ed. New Jercydrugs and biological; Merck and Co; 2001; 388 and 1290. 4. RaveendraB.Ganduri, et al.,stability indicating LC method for the determination of citicoline sodium in injection formulation, International Journal of Pharm Tech Research, Vol.2, No.1, Page no. 427-433, (Jan-Mar 2010). 5. Sonali O. Uttarwar, et al., Stability indicating LC Method for Citicoline Sustained Release Tablet,International Journal of PharmTech Research, Vol.2, No.4, pp 2482-2486, (Oct-Dec 2010) 6. Robert N. Nalbandlan, et al., Liquid-Chromatographic Quantification of, CLINICAL CHEM., Vol. 29, No. 4,page no.664-666 (1983) 7. ICH guidelines, Q1 A stability testing of new drug substances and products, 1993. 8. ICH guidelines, Q2 B Analytical procedure, Methodology, 1996. 54878478451150824