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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Yeligar et al.

World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041

Volume 5, Issue 6, 1440-1451

Research Article

ISSN 2278 – 4357

DEVELOPMENT OF SPECTROPHOTOMETRIC METHOD AND VALIDATION FOR MELATONIN IN TABLET DOSAGE FORM Veerendra C. Yeligar*, Ravindra G. Gaikwad, Kavita D. Patil, Sanjay S. Patil and Shitalkumar S. Patil Department of Quality Assurance Technique, Ashokrao Mane College of Pharmacy, Peth Vadgaon, Kolhapur-416112 Maharashtra- India. ABSTRACT

Article Received on 11 April l2016,

A simple, accurate, precise and economic spectrophotometric method

Revised on 02 May 2016, Accepted on 23 May 2016

is developed for the determination of melatonin (MLT) in tablet dosage

DOI: 10.20959/wjpps20166-6921

forms. The method is based on the formation of pink colour chromogen complex formed by oxidation of melatonin with Ferric citrate in the presence of 1,10-phenanthroline. The colour complex was

*Corresponding Author Veerendra C. Yeligar

measured at 510nm. Beers law was observed in the concentration

Department of Quality

range of 2-20µg/ml with correlation coefficient 0.988. LOQ and LOD

Assurance Technique,

was found to be 0.1229, 0.3724respectively. The method was validated

Ashokrao Mane College

for several parameters like accuracy, precision and linearity, LOQ,

of Pharmacy, Peth

LOD. The values of relative standard deviation and % recovery were

Vadgaon, Kolhapur416112 Maharashtra-

found to be satisfactory, indicating that the proposed method is precise and accurate and can be used for the determination of Melatonin in

India.

tablet dosage forms. KEYWORDS: Melatonin, ferric citrate, Spectrophotometry, 1,10-phenanthroline. INTRODUCTION The Melatonin (MEL) chemically is an N – [2-(5-methoxy-1H-indol-3-yl) ethyl] acetamide (figure 1A)[1], clinically used in the treatment of cancer, immune disorder, cardiovascular diseases, depression and sexual dysfunction. It is available for oral administration as filmcoated

compressed

tablets

containing

3.0mg

Melatonin.

Melatonin

(N-acetyl-5-

methoxytryptamine), the chief hormone of the pineal gland, attracted much interest after its antioxidant ability was proven by both in vivo and in vitro studies.[2,3] MEL acts as a powerful antioxidant and as a free radical scavenger of hydroxyl, peroxyl radicals, and

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superoxide anions.[4] Indeed, Melatonin was shown to be twice as potent as vitamin E in removing peroxyl radicals[5], and it is more effective in scavenging hydroxyl radicals than glutathione and mannitol.[6] The direct effects of this hormone on the male reproductive system have also been examined in several animal studies. Since Melatonin binding sites were detected in the reproductive system of different species[7,8], it seems reasonable to assume thatMelatonin exerts its actions via direct interaction with the steroidogenic cells of the reproductive organs. Literature survey revealed that there are several methods available for determining Melatonin individually and in combined dosage forms such as: Liquid Chromatography[9] and LC-MS method[10], spectrofluorimetry[11], precolumn derivatisation micro RP-HPLC[12] and capillary electrophoresis.[13] Since HPLC is regarded as the most reliable method and it was successfully used for determining MEL in plasma[14,15], and plants[16], and MEL and Zolpidem estimated by HPLC method.[17] However, there are no reported methods for determination of MEL in tablet dosage forms. Therefore attempt was made for a simple, precise, accurate method development for the determination of Melatonin by visible spectroscopic method. The proposed method is based on the reducing property of Melatonin which is found to quantitatively reduce ferric (III) form of iron to ferrous (II) form, which is then made to interact with 1,10-phenanthroline to give red coloured chromogen complex, whose absorbance is measured at its max of 510nm.

Fig. 1: Chemical Structure of Melatonin METHODS AND MATERIALS Instrument Agilent carry 60 UV /Visible spectrophotometer with spectral bandwidth of 1.00 nm and a pair of matched quartz cells was used for measuring the absorbance. Materials

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All the chemicals and reagents used in the spectrophotometric analysis were of AR grade. Standard Melatonin BP was procured from SWAPNROOP DRUGS PVT LTD. Aurangabad, Maharashtra India. Methanol, 0.005M Ferric citrate, 0.02% w/v 1,10-phenanthroline was of analytical grade. Tablets – Meloset, manufactured by Aristo Pharma LTD. containing 3.0 mg per tablet was purchased from the market. Preparation of standard stock solution of MEL Accurately weighed 50 mg of pure MEL BP was transferred in to a 50 ml volumetric flask and dissolved in methanol solvent was diluted with Methanol to make final 50 ml. This solution was kept for 10 minutes in bath Ultra sonicator for sonication after sonication volume was made upto the mark with the same solvent the concentration of stock solution (1000µ/ml). Preparation of 0.005M Ferric citrate solution Accurately weighed 1.22gm of ferric citrate was added in 100 ml volumetric flask distilled water added in same and the volumetric flask was kept in bath ultrasonicator for 20 minutes. Removed the flask and final volume was made up to the mark with the distilled water. Preparation of 0.02 M 1-10, phenathroline reagent solution Accurately weighed 0.570 gm of 1,10,phenathroline was added in a 100 ml volumetric flask .to the same volumetric flask .volumetric flask was kept in bath ultrasonicatot for 20 minutes removed the flask and final volume made up with distilled water. Preparation of standard calibration curve From the stock solution of (1000µ/ml) aliquots was transferred in a 10 ml volumetric flask to get concentration of 2-20µ/ml.to each of volumetric flask 0.2 ml of 0.005M ferric citrate was added and the contents of the flask were shacked for 5 minutes. After shaking of contents 3 ml of 1-10, phenathroline was added to each volumetric flask .the flask kept in water bath for 5 minutes .the final volume up to the mark was maintained by adding methanol in it. Blank solution was prepared in the same manner as above but omitting standard MEL. The absorbance of the resulting orange coloured solution was measured at 510 nm by using spectrophotometer. Calibration curve of the drug was then plotted by taking the absorbance obtained on y-axis and the concentration of the solution on x-axis. (Fig.2).The curve showed linearity in the concentration range of 2-20 μg/ml with correlation coefficient 0.988. Determination of MEL in marketed tablet dosage formulation www.wjpps.com

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The method was extended for the determination of MEL from tablets which were purchased from the local market. Tablets are weighed individually and triturated in mortar and pestle. Powder equivalent to 50 mg of MEL was weighed accurately and dissolved in 50 ml methanol. The resulting solution was filtered through Whatman no.42 paper. Then the filtrate was diluted to 10.0 ml with water. The procedure given for standard calibration curve was then followed for development of colour. Optimization of reagent volumes and conditions The volume of reagent concentrations required for obtaining maximum absorbance for the solutions has been optimized. METHOD VALIDATION The method was validated according to ICH Q2 (R1) guidelines for parameters like linearity, accuracy, precision, LOD, LOQ and specificity of the analyte[33] Table 1: validation parametres Parameters Absorption Maximum Linearity range Correlation coefficient Regression equation Slope Intercept Accuracy Precision (%RSD) LOD LOQ

Observations 510 nm 2-20 µg/ml 0.988 0.0537x 0.0537 0.024291 98-101% 0.7206% 0.37243 0.12290

Selection of solvent The selection of solvent was made after assessing the solubility of the drug in differentSolvent like wiz water, ethanol, acetone, methanol, According to the solubility characteristics of the drug, methanol was selected as solvent for analysis from the scanning of the drug by UV spectra; wavelength were selected for estimation of MEL at and 510 nm for colorimetric method development.

METHOD DEVELOPMENT www.wjpps.com

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Preparation of standard stock solution of MEL The standard stock solution of MEL was prepared by dissolving 50mg of MEL in 50ml of AR grade methanol to produce concentration of 1000μm/ml. Methanol solvent was added to volumetric flask and it was kept in ultrasonicator for 10 minutes. After sonication volume was made up to the mark with the same solvent i.e. methanol .the concentration of stock solution (1000µ/ml). 1ml of this stock solution was taken and then diluted up to 10 ml by using AR grade methanol to produce concentration of 100μg/ml. 1. Determination of λmax The standard stock solution of MEL was prepared by dissolving 50mg of MEL in 50ml of AR grade methanol to produce concentration of 1000μm/ml.1ml of this stock solution was taken and then diluted up to 10 ml by using AR grade methanol to produce concentration of 100μg/ml which is the standard stock solution scanned at different concentration in the range 200-800nm and the λmax was determined against reagent blank. The maximum absorbance for the colored solution was found to be 510 nm. Preparation of standard calibration curve: Into a series of 10ml volumetric flasks appropriate aliquots of the standard solution was taken to Finally produce a concentration range of 2-20μg/ml. To each volumetric flask 0.2ml of 0.005M Ferric citrate was added. The contents in the flasks were mixed for 5 min and further 3ml of 0.02M, 1, 10-Phenanthroline was added. The solutions were kept in a water bath at temp of 60-70ºc for 5 minutes to stand at room temperature .after that the volume was made up to the mark with methanol. Absorbance of the resulting red colour chromogen was measured at 510nm against reagent blank prepared in the same manner as described above, but omitting the standard substance. Calibration curve of the drug was then plotted by taking the absorbance obtained on y-axis and the concentration of the solution on x-axis. The curve showed linearity in the concentration range of 2-20μg/ml with correlation coefficient 0.988. METHOD VALIDATION The method was validated according to ICH Q2B guidelines for validation of analytical Procedure in other to determine linearity, specificity, precision, limit of detection (LOD), Limit of quantification (LOQ), ruggedness and accuracy for the analyte.

Linearity

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The linearity of the analytical method was its ability to elicit test results which are directly proportional to analyte concentration in samples within a given range. To establish the linearity of the proposed method, various aliquots 2-20µg/ml of the standard solution of the drug were prepared from stock solution and analysed. The drug showed linearity in the range of 2-20μg/ml with correlation coefficient 0.988. Accuracy (Recovery studies) The accuracy of the method was determined by calculating recovery of MEL by the standard addition method. To the sample solutions, known concentration of was added in different level viz., 80,100 and 120% level. The amounts of MEL was recorded and calculated as per the ICH guidelines. This concentration was repeated for three times. The results are shown in (Table 2). Specificity The specificity of the method was determined by calculating the recovery of melatonin. The 20 tablets of melatonin was triturated and amount equivalent to get concentration of 1000 (µg/ml) was added in volumetric flask. From this stock solution of 1000(µg/ml) the solution was pipet out 4,6,8 (µg/ml) to make the concentration added in 10 ml volumetric flask and the final volume was made up to the mark with the methanol and the absorbance was consideredas 510 nm. Precision Precision of the method was carried out by repeatability, intradayand interday variation studies. For the repeatability study six samples of same concentration, 10μg/ml, were taken and the absorbance’s were observed and the %RSD was calculated. The acceptable limit should be within 2%.The results are shown in (Table 3). Intraday precision for intraday precision study nine different solutions of same concentration 10μg/ml were analysed three times in a day i.e. from morning, afternoon and evening and the absorbances were noted. From the absorbance result mean, standard deviation and %RSD were calculated. The acceptable limit for intraday variation should be within 1%. Results were shown in (Table 4). Inter DayPrecision

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for inter day precision studies, solution of same concentration 10μg/ml were analysed three times for the three consecutive days and the absorbance result was observed. Mean, standard deviation and %RSD were calculated. The acceptable limit for interday variation should be within 2%. Results are shown in (Table 4). LOD Based on the standard deviation of the blank: Measurement of the magnitude of analytical background response was performed by analysing the six replicates of blank samples and calculating the standard deviation of these responses by using formula, LOD = 3.3 σ/S Where, σ = Standard deviation of the response and S = Slope of the corresponding calibration curve. LOQ Based on the standard deviation of the blank: Measurement of the magnitude of analytical background response was performed by analysing the six replicates of blank samples and calculating the standard deviation of these responses by using formula, LOQ = 10 σ/S Where, σ = Standard deviation of the response and S = Slope of the corresponding calibration curve. Ruggedness Ruggedness of the proposed method was evaluated by applying developed procedure the concentration of 10 µg/ml of MEL by using same instrument by two different analyst under optimised conditions for two days. The obtained result was found to be reproducible since there was no difference showing in the result by different analyst so method could be considered as rugged. RESULTS Linearity Standard calibration curve for MEL, covering the range 2-20 µg/ml, prepared by serial dilution with methanol and its oxidation reaction with ferric citrate and 1, 10-phenathroline for pure drug and tablet formulation were developed and validated. The procedure was adopted as per desired protocol, based on ICH Q2B guidelines. The calibration curve was

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obtained by plotting absorbance Vs analyte concentration. The slope and intercept of the calibration line was determined by linear regression. Table 2: Evaluation data of Linearity Concentration Absorbance (µg/ml) (nm) 0 0 2 0.1121 4 0.2406 6 0.3096 8 0.4528 10 0.5997 12 0.6395 14 0.8051 16 0.8798 18 0.9499 20 0.9991

Figure: 2 Standard calibration curve of MEL. Accuracy (Recovery studies) Table 3: Recovery study of MEL from tablet samples.

Sr.No.

1

2

Level of % recovery 80 80 80 100 100 100

Initial amount present μg/ml 2.4 2.4 2.4 3 3 3

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Amount of standard added μg/ml 3 3 3 3 3 3

Statistical Total Total analysis amount amount % Mean present recovered recovery % S.D μg/ml μg/ml RSD 5.4 5.43072 100.5689 5.4 5.41566 100.29 100.959 0.928342 0.91952 5.4 5.5090 102.01 6 5.95481 99.246 6 6.01506 100.251 100.681 1.698934 1.687358 6 6.01506 102.5602

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3

120 120 120


3.6 3.6 3.6

3 3 3

6.6 6.6 6.6

6.68674 6.66566 6.73795

101.3143 0.563268 0.555128 100.994 101.048 101.4665

Specificity Table 4: Result of specificity study Precision Conc ( µg/ml) 4

6

8

Conc. estimated (μg/ml) 4.029964 3.981547 4.043 6.134248 6.082106 6.063484 7.925681 7.934992 7.921957

% conc estimated 100.74911 99.5386829 101.074996 102.237458 101.368433 101.058067 99.0710174 99.1874048 99.0244625

(%RSD) 0.657953

0.491524

0.069153

(repeatability). Table5: Repeatability study of MEL. Sr.no. 1 2 3 4 5 6

Concentration Absorbance (µg/ml) (nm) 10 0.6185 10 0.6087 10 0.6134 10 0.6007 10 0.6173 10 0.6114

Mean SD

%RSD

0.6116 ±0.0058

0.96097

Intraday precision Table6: Intradayprecision for MEL Time Concentration (µg/ml) 10:00 a.m 2:00p.m 4:30 p.m 10 0.6108 0.6126 0.6100 10 0.6134 0.6234 0.5967 10 0.6160 0.6182 0.5989 10 0.6018 0.6001 0.6015 10 0.6109 0.6063 0.6104 10 0.6127 0.5976 0.6042 Mean 0.6108 0.6097 0.60363 SD 0.004401 0.00929 0.005207 RSD 0.7206209 1.524664 0.862662

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Inter-day precision Table 7: Interday precision for MEL Day Concentration (µg/ml) 1 2 3 10 0.6108 0.6011 0.5987 10 0.6134 0.5935 0.6107 10 0.616 0.6107 0.6147 10 0.6018 0.6014 0.6044 10 0.6109 0.5935 0.5904 10 0.612 0.6014 0.6023 Mean 0.6108 0.6013 0.6035 SD 0.00440 0.00503 0.007897 RSD 0.72062 0.836561 1.308457 LOD Based on the standard deviation of the blank: Measurement of the magnitude of analytical background response was performed by analysing the six replicates of blank samples and calculating the standard deviation of these responses by using formula, LOD = 3.3 σ/S LOD=3.3*0.002/0.0537 =0.1229 LOQ Based on the standard deviation of the blank: Measurement of the magnitude of analytical background response was performed by analysing the six replicates of blank samples and calculating the standard deviation of these responses by using formula, LOQ = 10 σ/S =10*0.002/0.0537 =0.3724 Table 8: Ruggedness study Test concentration 10 10 10 Mean SD RSD DISCUSSION

Analyst 1 0.6549 0.6345 0.6385 0.6426 0.008826 1.3764

Analyst 2 0.6597 0.6397 0.6505 0.6483 0.010324 1.5924

The proposed method provides a simple, accurate, economical and convenient method for the analysis of MEL using UV-Visible spectrophotometry. The use of ferric citrate instead of www.wjpps.com

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other ferric salts like chloride or sulphate is recommended as the solubility of citrate salts is generally satisfactory in aqueous medium and resulting solutions are stable for longer duration of time. The Beers law was obeyed in the concentration range of 2-20μg/ml, with correlation coefficient 0.988. Accuracy of the proposed method was determined by the recovery studies, and good %recovery (99.89– 101.09%) of the drug obtained indicates that the method is accurate. The method was found to be precise as %RSD value was found to be less than 2. CONCLUSION The present work describes simple, precise, accurate and economic method for selective determination of MEL in formulation based on the oxidation of MEL by Ferric citrate in the presence of 1,10-phenanthroline. The colored complex was measured at 510nm. Beers law was observed in the concentration range of 2-20µg/ml with correlation coefficient 0.970. Ferric citrate can suitably replace the commonly used ferric chloride and ferric sulphate salts used in such methods of analysis. REFERENCES 1. “The Merck Index”, 14th Edi., The Merck Research Laboratories Publishers, USA, 2006; 5811. 2. Reiter R.J., Antioxidant actions of MEL, AdvPharmacol, 1997; 38: 103–17. 3. Narayana K., D'Souza U.J., Rao K.P., Effect of ribavirin on epididymalspermcount in rat Indian J PhysiolPharmacol., 2002; 46: 97-101. 4. Reiter R., Tang L., Garcia J.J., Munoz-HoyosA.Pharmacological actions of MEL in oxygen radical pathophysiology, Life Sci., 1997; 60: 2255-2271 5. Pieri C., Marra M., Moroni F., Recchioni R., Marcheselli F., MEL: a peroxyl radical scavenger more effective than vitamin E, Life Sci., 1994; 55: L271- L276 6. Hardeland R., Reiter R.J., Poeggeler B., Tan D.X., The significance of the metabolism of theneurohormone MEL: antioxidative protection and formation of bioactive substances, NeurosciBiobehav Rev., 1993; 17: 347-357. A. and Volpe A., Influence of MEL and photoperiod on animal and human reproduction, J Endocrinol Invest., 1996; 19: 382–411. 7. Yu Z.H., Chow P.H., Pang S.F., Identification and characterization of 2 [125I] IodoMEL binding sites in the rat epididymis, J Pineal Res., 1994; 17: 195–201

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8. Jian Lin, Chunchan Zhang, YaoxiangGao, Xiaojun Zhao, Xican Li. A validated HPLC method for determining MEL in capsule dosageform. Spatula DD, 2012; 2(3): 147-151 9. Cao J, Murch SJ, O'Brien R, Saxena PK., Rapid method for accurate analysis of MEL, serotonin and auxin in plant samples using liquid chromatography-tandem mass spectrometry. JChromatogr A, Nov 17; 2006; 1134(1-2): 333-7. 10. Hany W Darwish and Mohamed I Attia., New spectrofluorimetric methods for determination of MEL in the presence of N-{2-[1-({3-[2-(acetylamino)ethyl]-5-ethoxy-Hindol-2-yl}methyl)-5-methoxy-1H-indol-3-yl]-ethyl}acetamide:

a

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chromatography with electrospray tandem mass spectrometry method for the determination of indoleamine neurotransmitters and their metabolites in sea lamprey plasma. Anal ChimActa, 2012; 721: 147-53. 15. Van Tassel DL, Roberts N, Lewy A, O'Neill SD. MEL in plant organs. J. Pineal Res., 2001; 31(1): 8-15. 16. Lalitha, KG and Venkatachalam, T “Simultaneous estimation of MEL and zolpidem in tablets dosage forms by rp-hplc method”Pharmacophore, 2014; 5(2): 252-257. 3(4): 1-5.

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