ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF MELATONIN AND PYRIDOXINE IN TABLET DOSAGE FORM BY HPLC

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Rathod et al. SJIF Impact Factor 2.786 Volume 3, Issue 6, 676-686. Research Article ISSN 2278 4357 ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF MELATONIN AND PYRIDOXINE IN TABLET DOSAGE FORM BY HPLC Sangeeta Rathod *, Ankita Bhavsar, Bhagirath Patel Department of Quality Assurance, Sat Kaival College of Pharmacy, Sarsa-388365, Gujarat, India. Article Received on 14 April 2014, Revised on 09 May 2014, Accepted on 28 May 2014 *Author for Correspondence Sangeeta Rathod Department of Quality Assurance, Sat Kaival College of Pharmacy, Sarsa-388365, Gujrat, India ABSTRACT A simple, rapid and accurate High performance Liquid Chromatography method was developed and validated for simultaneous estimation of Melatonin and Pyridoxine in tablet dosage form. The High performance Liquid Chromatography method has shown adequate separation of Melatonin and Pyridoxine in its tablet Dosage form. The separation was achieved BDS hypersil C 18, with an isocratic system of 0.2M KH 2 PO4 buffer (ph - 4): methanol: TEA in the ratio of 70:30:0.1 v/v. The mobile phase at a flow rate of 1.0 ml/min, Injection volume 20µl and wavelength of detection used was 244 nm. The retention time for Melatonin and Pyridoxine were obtained as 3.760 min and 6.107 min, respectively. The linearity of the proposed method was investigated in the range of 3-9 µg/ml and 10-30µg/ml for Melatonin and Pyridoxine respectively. Correlation coefficient was 0.9939 and 0.9995 for Melatonin and Pyridoxine respectively. The developed method was validated as per International conference Harmonization (ICH) guideline for its accuracy, precision, Limit of detection and Limit of quantitation. Key Words: Melatonin, Pyridoxine, HPLC method development, Validation. 1. INTRODUCTION Analytical method development [1,2] Analytical methods are intended to establish the identity, purity, physical characteristics and potency of the drugs and to support drug testing against specifications during manufacturing and quality release operations as well as during long term stability studies. www.wjpps.com Vol 3, Issue 6, 2014. 676

Method validation [3,4] Validation of an analytical method is the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications. Melatonin [6,7,8] It is a biogenic amine that is found in animals, plants and microbes. Melatonin regulates the sleep-wake cycle by chemically causing drowsiness and lowering the body temperature. Melatonin is also implicated in the regulation of mood, learning and memory, immune activity, dreaming, fertility and reproduction. Melatonin is also an effective antioxidant. Pyridoxine [9,10] Pyridoxine is one of the compounds that can be called vitamin B6, along with pyridoxal and pyridoxamine. It is often used as 'pyridoxine hydrochloride.' The Chemical structures of Melatonin (A) and Pyridoxine (B) are shown in fig.1. Melatonin (A) Pyridoxine (B) Fig. 1 Chemical structures of Melatonin (A) and Pyridoxine (B) 2. MATERIALS AND METHOD 2.1 Chemical and solvents Melatonin and Pyridoxine are procured from Merck Pharmaceutical pvt. Ltd. as a gift sample. HPLC grade solvents: Water, Methanol, Acetonitrile, KH 2 PO 4 buffer, Tri ethyl amine and Ortho phosphoric acid were obtained from Gitar laboratory, Ahmedabad. Zytonin TAB (Melatonin 3 mg, Pyridoxine 10 mg ) was gifted from Indon Zydus Cadila Health Care Ltd. Ahmedabad. 2.2 HPLC instrumentation and chromatographic conditions Shimadzu SPD 20AT system equipped with, isocratic pump LC-20AT, and UV detector www.wjpps.com Vol 3, Issue 6, 2014. 677

SPD-20AT, Rheodyne injector (20 µl Capacity), Syringe: Hamilton (25µl), Data were processed using Chromatographic software Spinchrom. Analytical balance: AX 200 Shimadzu, Japan. The chromatographic saparation was carried out on HPLC system C 18 (simadzu LC 20 AT) with UV visible detector (SPD 20 AT), C (250 4.6,5 µm) column. The mobile phase consisting of 0.02M KH 2 PO 4 buffer (ph-4): Methanol: Tri Ethyl Amine (70:30:0.1, v/v). Mobile phase was filtered through a 0.45 µm membrane filter paper and sonicated before use. The flow rate of the mobile phase was maintained at 1.0 ml/min. 2.3 Preparation of Stock solution Accurately weighed 6 mg of standard Melatonin and 20 mg of standard Pyridoxine API and transferred to a 100 ml volumetric flask and dissolved in methanol. The flasks were shaken and volume was made up to the mark with mobile phase to obtain standard stock solution of 60µg/ml Melatonin and 200µg/ml Pyridoxine. Stock solution filtered through a 0.45 µm whatman filter paper. 1 ml Melatonin solution was withdrawn to 10 ml volumetric flask and diluted up to mark with mobile phase to get working standard of Melatonin. 1 ml Pyridoxine solution was withdrawn to 10 ml volumetric flask and diluted up to mark with mobile phase to get working standard of Pyridoxine. The working standard solution of Melatonin and Pyridoxine were prepared from suitable aliquots of stock solutions. 2.4 Preparation of sample solution Twenty tablets were weighed and finely powdered. Powder equivalent to 6 mg Melatonin and 20mg Pyridoxine was accurately weighed and transferred to volumetric flask of 100 ml capacity. 100 ml of methanol was transferred to this volumetric flask. The flask was shaken and volume was made up to the mark with mobile phase. The above solution was filtered through whatman filter paper (0.45µ). From this solution 10 ml was transferred to volumetric flask of 100 ml capacity. Volume was made up to the mark to give a solution containing 6 mg Melatonin and 20 mg. Pyridoxine. 2.5 Selection of analytical wavelength The standard solution of Melatonin and Pyridoxine were scanned in the UV region of 200-400 nm using methanol as a blank and the overlain spectra was recorded. At the 244nm both the drugs gave good response around this point. Therefore, 244.17 nm analytical wavelength was selected for estimation of Melatonin and Pyridoxine. www.wjpps.com Vol 3, Issue 6, 2014. 678

Pyridoxine Melatonin Fig. 2 Analytical wavelength of Melatonin and Pyridoxine 2.6 Optimization of HPLC Method The pure drug solutions of Melatonin (6µg/ml) and Pyridoxine (20 µg/ml) were injected individually into HPLC system and allow to run in different mobile phases like methanol, water: methanol, water: acetonitrile, phosphate buffer: methanol and phosphate buffer: acetonitrile were tried in order to find the optimum conditions for the separation of Melatonin and Pyridoxine It was found that mobile phase containing 0.02M KH 2 PO 4 buffer (ph-4): Methanol: Tri Ethyl Amine (70:30:0.1, v/v) at a flow rate of 1.0 ml/min with detection wavelength 244.17 nm gave satisfactory results with sharp, well defined and resolved peaks with minimum tailing as compared to other mobile phases. Under these conditions the retention times were typically 3.760 min and 6.107 min for Melatonin and Pyridoxine (Figure 3) and optimized chromatographic conditions described in (Table 1) Fig. 3 Chromatogram of standard drugs of Melatonin (6 µg/ml), Pyridoxine (20 µg/ml) www.wjpps.com Vol 3, Issue 6, 2014. 679

Table 1 Optimized chromatographic conditions for simultaneous estimation of Melatonin and Pyridoxine Parameters Conditions Mobile phase Phosphate buffer (ph 4): Methanol (65:35 v/v) Stationary phase BDS hypersil C 18, 250mm 4.6mm, 5µ (particle size) Flow rate (ml/min.) 1 Run time (min.) 10 Volume of injection (µl) 20.0 Detection wavelength (nm) 244.17 Retention time (min.) Melatonin : 3.76 Pyridoxine : 6.107 3. Validation of the method Validation of the optimized RP-HPLC method was carried out with respect to the following parameters. 3.1 Linearity and range The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. [4] Linearity responses for Melatonin and Pyridoxine were assessed in the concentration range 3-9 µg/ml and 10-30 µg/ml of standard solutions, respectively. Result is shown in table 2. 3.2 Sensitivity The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The sensitivity measurement of Melatonin and Pyridoxine by the use of proposed method was estimated in terms of Limit of Detection (LOD) and Limit of Quantitation (LOQ). The LOD and LOQ were calculated using following equations. LOD = 3.3 σ/s LOQ = 10 σ/s Where, σ = the standard deviation of the response S = slope of the calibration curve www.wjpps.com Vol 3, Issue 6, 2014. 680

3.3 Precision The precisions of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. [4] Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of the method was verified by repeatability, interday and intraday precision. Repeatability study was performed by analysis of three different concentrations of the drug in six replicates on the same day. Intraday precision was determined by analysing sample solutions at different time intervals on the same day and on different day for interday precision. Results are mentioned in table 3 and 4. 3.4 Accuracy The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. [5] To the pre analysed sample, a known amount of standard solution of pure drugs (Melatonin and Pyridoxine) was spiked at three different levels. This study was carried out at 80%, 100% and 120% level. 3.5 Robustness Robustness was performed by deliberately changing the chromatographic conditions. The important parameter to be studied was the resolution factor between two peaks. The robustness was checked by changing following parameters one by one. Change in the ratio of mobile phase by ± 0.2 ml [phosphate buffer (ph 4): methanol (68:28 v/v) and phosphate buffer (ph 4): methanol (72:32 v/v)] Change in flow rate by ± 0.2 ml/minute (0.8 ml/min. and 1.2 ml/min.) After each change, sample solution was injected and % assay with system suitability parameters were checked. Change in ph of mobile phase by ± 0.2 ph [phosphate buffer (ph 4.2): methanol (70:30v/v) and phosphate buffer (ph 3.8): methanol (70:30v/v)] Results are mentioned in table 7. 3.6 System suitability parameters System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. To check system suitability, number of theoretical plates, resolution, retention time and tailing factor were determined. Results are mentioned in table 8. www.wjpps.com Vol 3, Issue 6, 2014. 681

3.7 Quantitative estimation of pharmaceutical dosage form Twenty tablets were weighed; their average weight was determined and finally powdered. An accurately weighed tablet powder equivalent to 6 mg of Melatonin and 20 mg Pyridoxine were then transferred to 10 ml volumetric flask containing 5 ml methanol and sonicated for 20 min. The solution was filtered through 0.45µm filter and the volume was adjusted up to mark with methanol. From the above solution 1 ml was taken into a 10 ml volumetric flask and the volume was adjusted up to mark with methanol to get a final concentration of 6 µg/ml of Melatonin and 20 µg/ml Pyridoxine. 20 µl of the test solution was injected and chromatogram was recorded for the same and the amount of the drug was calculated. 4. RESULTS AND DISCUSSION The results of method development and validation studies on simultaneous estimation of Melatonin and Pyridoxine in the current study involving phosphate buffer (ph-4): methanol: TEA (70:30:0.1v/v) as the mobile phase for RP-HPLC are given below. 4.1 Method development Melatonin and Pyridoxine were completely separated on C 18 column by RP-HPLC using the isocratic elution of phosphate buffer and methanol as mobile phase. When the methanol percentage was reduced starting from 80% by a decrement of every 5%, broadening, fronting and tailing of peaks were observed. As a result of decrease in the percentage of methanol and using phosphate buffer (ph-4) a sharp pointed and well separated peak was observed. As methanol concentration gradually decreases the peak broadening, fronting and tailing were remarkably reduced. Eventually proper resolution was achieved at flow rate of 1ml/min and using phosphate buffer (ph-4): methanol: TEA (70:30:0.1 v/v) as the mobile phase for RP- HPLC. (Figure 3) (Table 1) Table 2 Linearity data of Melatonin and Pyridoxine by proposed method Melatonin Pyridoxine Conc. Mean Peak Area Conc. Mean Peak Area 3 866.287 10 628.293 4.5 1308.802 15 927.815 6 1789.682 20 1268.224 7.5 2103.893 25 1562.288 9 2681.647 30 1900.924. Correlation coefficient : 0.9993 Correlation coefficient : 0.9995 Regression Equation : y = 292.3x - 0.262 Regression Equation : y = 63.59x - 14.30 LOD : 0.70 LOD : 0.69 LOQ : 2.14 LOQ : 2.10 www.wjpps.com Vol 3, Issue 6, 2014. 682

4.2 Linearity The drug response was linear (R2 = 0.993 for Melatonin and 0.9995 for Pyridoxine) over the concentration range between 3-9 µg/ml for Melatonin and 10-30 µg/ml for Pyridoxine. The result is shown in (Table 2). 4.3 Sensitivity The LOD and LOQ were calculated by respective equations. The LOD values were found to be 0.70 and 0.69 µg/ml for Melatonin and Pyridoxine respectively. The LOQ values were found to be and 2.14 and 2.10 µg/ml for Melatonin and Pyridoxine respectively. (Table 2) 4.4 Precision The results of the repeatability, intra-day and inter-day precision experiments are shown respectively as given in (Table 3) and (Table 4). The developed method was found to be precise as the RSD values for repeatability of intra-day and interday precision studies were < 2 %. Table 3 Repeatability study of Melatonin and Pyridoxine Concentration Melatonin (6µg/ml) Pyridoxine (5µg/ml) Area 1782.51 1263.62 1786.08 1242.96 1749.44 1268.70 1793.23 1271.24 1786.09 1266.15 1789.66 1268.76 ± SD 15.96432 10.42526 %RSD 0.896281 0.825060 Table 4: Intra-day and inter-day study of Melatonin and Pyridoxine Drug Melatonin Pyridoxine 4.5 Accuracy Concentration Intra-day area mean (n=3) ± SD %RSD Inter-day area mean (n=3) ± SD %RSD 3 880.10 ±8.73 0.994 880.991 1.39 6 1777.14 ± 14.63 0.824 1778.961 1.05 9 2662.88 ± 26.81 1.011 2665.537 0.74 10 611.84 ± 8.29 1.334 617.262 0.76 20 1244.13 ± 11.79 0.938 1243.146 0.91 30 1866.41 ± 15.06 0.799 1860.676 1.06 As shown in (Table 5) and (Table 6), good recoveries of the Melatonin and Pyridoxine in the range from 98 to 102 % were obtained at various added concentrations. www.wjpps.com Vol 3, Issue 6, 2014. 683

Table 5 Determination of accuracy for Melatonin % Level of Recovery Conc. of sample solution Conc. Of standard Solution Total conc. Mean peak area (n=3) Conc. found (n=3) % Recovery mean(n=3) 80 3 2.4 5.4 737.939 5.33 99.95 100 3 3 6 925.218 5.94 99.42 120 3 3.6 6.6 1117.237 6.56 99.50 Table 6 Determination of accuracy for Pyridoxine % Level of Recovery Conc. of sample solution Conc. Of standard Solution Total conc. Mean peak area (n=3) Conc. found (n=3) % Recovery mean(n=3) 80 10 8 18 523.505 17.78 99.73 100 10 10 20 665.012 19.94 99.95 120 10 12 22 796.564 21.94 99.84 4.6 Robustness The standard deviation of the peak areas was calculated for each parameter and the % RSD was found to be less than 2 %. Result shows low values of % RSD as shown in (Table 7) and signifies the robustness of the method. Table 7 Robustness for Melatonin and Pyridoxine Parameters Column temperature ±2 ºC Flow rate ±0.2 ml ph ±0.2 Change in condition Drug Conc. Mean area SD % RSD 23 ºC Melatonin 3 1735.66 21.1296 1.2173 Pyridoxine 10 1233.79 13.9667 1.1320 27 ºC Melatonin 3 1824.19 20.1136 1.1026 Pyridoxine 10 1297.41 11.8128 0.9104 1.2 ml Melatonin 3 1739.71 20.2967 1.1666 Pyridoxine 10 1233.74 17.0115 1.3788 0.8 ml Melatonin 3 1844.21 22.9760 1.2458 Pyridoxine 10 1312.45 12.8602 0.9798 4.2 Melatonin 3 1701.78 20.0910 1.1805 Pyridoxine 10 1207.52 1216.669 1.7572 www.wjpps.com Vol 3, Issue 6, 2014. 684

3.8 Melatonin 3 1824.96 25.0648 1.3734 Pyridoxine 10 1297.20 17.1249 1.3201 4.7 System suitability studies The column efficiency, resolution and peak asymmetry were calculated for the standard solutions and the results are mention in (Table 8). Table 8 System suitability parameters Parameters Melatonin Pyridoxine Theoretical plates 6884 7149 Retention time (min) 3.760 6.107 Tailing factor 1.417 1.350 Resolution 9.982 Table 9 Quantitative estimation of pharmaceutical dosage form 5. CONCLUSION Zytonin Parameters Melatonin Pyridoxine Actual Concentration 3 10 Concentration Obtained 2.9682 9.8731 % Assay 99.91 99.62 %RSD 0.8832 0.7917 Limit 98-102% Development and validation of RP-HPLC method was found to be simple, accurate, precise and economical. This method can be applied for routine quantitative analysis of Melatonin and Pyridoxine in pharmaceutical dosage form. 6. ACKNOWLEDGEMENTS The authors would like to thank, Merck pharmaceutical Pvt. Ltd. India for providing a gift sample of standard Melatonin and Pyridoxine. The authors would like to thank to Department of Quality Assurance, Sat Kaival College of Pharmacy, Sarsa, Gujrat, India for providing necessary facilities to carry out the work. 7. REFERENCES 1. Singh RM. HPLC method development validation an overview. J Pharm Edu Res, 2013; 4 (1): 26-32. www.wjpps.com Vol 3, Issue 6, 2014. 685

2. Haghi AE. Analytical method validation: Razi vaccine and serum research Institute. J Pharm 2009; 3(2) 67-8. 3. Sharma BK. Instrumental Method of Chemical Analysis. 21st ed., Delhi; Goel Publishing House: 2002, pp.3-10. 4. Mayer V. Practical HPLC. 2nd ed., New York; John wiley and sons: 1990, pp. 26-27. 5. ICH Q2B (2005) Validation of Analytical Procedure: Methodology, International Conference on Harmonization, IFPMA, Geneva, Switzerland. 6. http:// www.drugs.com/melatonin.html 7. Bruno C, Bruno J, Chazat G. The basic physiology and pathophysiology of melatonin. Sleep Med Rev Elsvier, 2005; 12(9): 11-24. 8. http://www.drugbank.ca/drugs/db01065 9. http://www.drugbank.ca/drugs/db01065 10. http://www.medindia.net/doctors/drug_information/ www.wjpps.com Vol 3, Issue 6, 2014. 686