Simultaneous Determination of Caffeine and Nicotinamde in Energy Drinks by First-Order Derivative Spectrophotometry

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1 Simultaneous Determination of Caffeine and Nicotinamde in Energy Drinks by First-Order Derivative Spectrophotometry Liliek Nurhidayati, Nurdiani Faculty of Pharmacy, Pancasila University, Jakarta Abstract A simple analytical method is described for the simultaneous determination of caffeine and nicotinamide in powder form of energy drinks. The first-order derivative UV-spectrophotometry was employed using water as solvent, the zero-crossing wavelengths were found at 273 and 261 nm for caffeine and nicotinamide, respectively. The calibration graphs were rectilinear up to 25.4 µg/ml and the relative standard deviations values were lower to 1.7 % for both analytes. The limits of detection were µg/ml caffeine and µg/ml nicotinamide, respectively with recovery values of to % for caffeine and to % for nicotinamide Key words: energy drink, caffeine, nicotinamide, zero-crossing method Introduction Caffeine and nicotinamide are two of substituent that always could be found in energy drink. Numbers of reported method were already available for the individual determination of both compounds. Coffeine has been determined by titration in nonaqueous solvent (Depkes RI 1995); high performance liquid chromatography (HPLC) (BP Commision 2009; USP Convention 2009). Nicotinamide has been determined by titration in nonaqueous solvent (Depkes RI 1995; BP Commision 2009) and HPLC (USP Convention 2009). Caffeine in beverage has been estimated using derivatives spectrophotometry (Aldpogan et al 2002) and HPLC (Aditia LS 2005). No spectroscopy method has been developed for the simultaneous determination of them in mixture. Derivative spectroscopy provides a greater selectivity than common spectroscopy and offers a powerful approach for resolution of band overlapping quantitative analysis of multicomponent mixture (Hayun et al 2006). The aim of the present study is the development of a simple, accurate

2 and sensitive derivative spectroscopic method for the determination of caffeine and nicotinamide in energy drinks on the basis of zero-crossing measurement. Methodology Chemicals and Reagents Caffeine and nicotinamide were obtained from BPOM. Commercial energy drinks was purchased from the local supermarket in South Jakarta. Instrumentation Spectroscopic analysis was carried out on Shimadzu UV 1700 double beam UV/Visible spectrophotometer. The zero order absorption spectra were recorded over the wavelength range of 200 to 400 nm, against solvent blank, in quartz cuvetts with 1 cm diameters. For all solutions, the derivative spectra were obtained over nm range at 2,4 nm. Standard and calibration solutions Standard stock solutions of a concentration of 250 µg/ml of caffeine and nicotinamide were prepared separately using water as solvent. Appropriate volume of standard stock solution was diluted with water to get concentration of 10 µg/ml of both analytes. Further dilutions were made from stock solutions in same solvent to get linearity concentrations 5-25 µg/ml for caffeine and nicotinamide. Linearity and Range Calibration curves were constructed by analysis of working standard solutions of caffeine and nicotinamide with at least 5 different concentrations in the range between 5-25 µg/ml. Each concentration was analysed in triplicate. First derivative values (D1) of caffeine and nicotinamide were measured at 261 and 273 nm respectively. Calibration curve was plotted by taking first derivative values (D1) on Y-axis and concentrations on X- axis. The relation between drug concentration (x) and its corresponding D1 value (y) i.e., expressed by the equation y = mx + b, where m is slope and b is intercept. Limit of Detection and Quantitation The limit of detection (LOD) and limit of Quantification (LOQ) was estimated from the standard calibration curve. The residual standard deviation of regression line or standard deviation of y intercepts of regression lines used to calculate LOD and LOQ. Here, LOD=3.3* D/S and LOQ=10*D/S. Where, D is the standard deviation of y intercept of regression line and S is the slope of calibration curves.

3 Precision and Accuracy Precision of procedure was calculated from analysis of the powder of energy drinks that made five replications of synthetic mixture and two commercial brand name. Quantities of the energy drinks powder equivalent to 50 mg of caffeine and 20 mg of nicotinamide were accurately weighed and transferred in a 100 ml volumetric flask. Weighed powder was dissolved in 50 ml of water, shake well. Then the volume made up to 100 ml with water. Solution obtained was filtered and diluted with water solvent to get the concentration within linearity and used for the measurement of caffeine and nicotinamide derivative spectra. The concentration of caffeine and nicotinamide in energy drinks were calculated using the corresponding calibrated curve. Accuracy of method was measured as percentage of deviation between added and measured concentrations (recovery study). Quantities of the energy drinks powder equivalent to 37.5 mg of caffeine and 15 mg of nicotinamide plus 12.5 mg of caffeine and 5 mg of nicotinamide were accurately weighed, transferred in a 100 ml volumetric flask. Weighed powder was dissolved in 50 ml of water, shake well. Then the volume made up to 100 ml with water. Solution obtained was filtered and diluted with water solvent to get the concentration within linearity and used for the measurement of caffeine and nicotinamide derivative spectra. The concentration of caffeine and nicotinamide in energy drinks blets were calculated using the corresponding calibrated curve. The other one, quantities of the energy drinks powder equivalent to 25 mg of caffeine and 10 mg of nicotinamide plus 25 mg of caffeine and 10 mg of nicotinamide were accurately weighed, transferred in a 100 ml volumetric flask. The next step were done as above. Results and Discussions The zero-order spectra of caffeine and nicotinamide were found in Fig. 1 and 2. As a result, the simultaneous determination of the two compounds cannot be possible by direct absorbance measurements. The first order spectra of them were shown in Fig 3 and Fig 4.

4 crossing wavelength of the derivative spectra of individual components, which should be only the function a the concentration of the other component. Fig 1. UV Absorption spectra for caffeine in water (10 µg/ml) Fig.3. First-order spectra of caffeine Fig 2. UV Absorption spectra for nicotinamide in water (10 µg/ml) Derivative spectroscopy, based on mathematical transformation of spectra zero-order curves in to derivative spectra, allows a fast sensitive and precise resolution of a multi-component mixture and overcomes the problem of overlapping of a multicomponent system. Derivative spectroscopy on the basis of zero crossing measurements involves measurement of absolute value of total derivative spectrum at an abscissa value corresponding to the zero Fig.4. First-order spectra of nicotinamide Zero-crossing points of caffeine and nicotinamide were found to be 273 and 261 nm respectively (Fig. 3 and Fig. 4). The measurements exhibited the best linear response and have given a near zero intercept on the coordinate of the calibration graph, and is less affected by the concentration of any other component. Caffeine was determined by measurement of its D1 amplitude at the zero-crossing point of nicotinamide (at 261 nm). Nicotinamide was determined by measurement of its D1 at the zerocrossing point of coffeine (at 273 nm).

5 Linearity, Range, LOD and LOQ The developed UV derivative method has shown the linearity in range of 5 to 25 µg/ml for caffeine and nicotinamide. The calibration curves were constructed by plotting the D1 value against caffeine (at 261 nm) or nicotinamide (at 273 nm) concentration at zero-crossing wavelength of caffeine and nicotinamide, respectively. The results obtained are summarized in Table 1. The linearity of the calibration curves and the adherence of the method to Beer s law are validated by the high value of correlation coefficient. The LOD and LOQ values are also summarized in Table 1. less than 1.7 % for both analytes in all three kind sample. The percentage recovery in each case was calculated. The results obtained from the recoveries of both analytes showed good accuracy. (Table 2). No interference was observed from the presence of the other constituent in energy drink. Applicability of the proposed methods for the simultaneous estimation of caffeine and nicotinamide was studied by assay of commercial powder of energy drinks (Brand A and Brand B).The results obtained are given in Table 2. The results obtained are in good agreement with the labled content. Accuracy and Precision Data of these tables showed a good accuracy and precision over the entire concentration range. The relative standard deviation (RSD%) values were Table 1: Regression analysis data for the determination of caffeine and nicotinamide using first order derivative spectra. Statistical Parameter Caffeine Nicotinamide Wavelength (nm) Regression Equation a y = x y= x Correlation coefficient (r2) Limit of Detection(µg/mL) µg/ml µg/ml Limit of Quantitation (µg/ml) µg/ml µg/ml a Y = mx + b where, y is the D1 amplitude at specified wavelength, x is the concentration of drug in µg/ml, m is slope and b is intercept.

6 Table 2. Accuracy and precision data for determination of caffeine and nicotinamide Name of sample analyte Amount (mg/sachet) Precision % recovery in addition of analyte (n=5) % Lable claim %RSD 25% 50% (%Found ± SD,n=5) Synthetic Caffeine ± mixture Nicotinamide ± Brand A Caffeine ± Nicotinamide ± Brand B Caffeine ± Nicotinamide ± Conclusion A rapid, simple and specific UV first derivative spectroscopic method has been developed for the simultaneous determination of caffeine and nicotinamide. Method is also successfully applied for determination of both compounds in energy drinks. Acknowledgement The authors are grateful to Kopertis Wilayah III for research funding and also Faculty of Pharmacy, Pancasila University for the facilities provision. References Aberásturi F, Jiménez AI, Jiménez F, and Arias JJ UV-visible first derivative spectrophotometry applied to an analysis of vitamin mixture. J Chem Educ 78 (6): Aditia LS Penetapan Kadar Kofein dalam minuman ringan secara Kromatografi Cair Kinerja Tinggi [skripsi]. Jakarta: Fakultas Farmasi Universitas Pancasila. 34. Alpdoğan G, Karabіna K, Sungur S Derivative spectrophotometric determination of caffeine in some beverages. Turk J Chem 26: [BP] British Pharmacopoeia Commision British Pharmacopoeia. Volume ke-1,2. London: Hers Majesty s Stationary Office. 309, Nurhidayati L Spektrofotometri Derivatif dan Aplikasinya dalam Bidang Farmasi. Jurnal Ilmu Kefarmasian Indonesia vol 5 No. 2 September ISSN [Depkes RI] Departemen Kesehatan Republik Indonesia Farmakope Indonesia. Edisi IV. Jakarta : Direktorat Jendral Pengawasan Obat dan Makanan , , 925-6, 1032, 1043, [USP] United States Pharmacopeia Convention The United States Pharmacopeia 32. The National Formulary 27. Rockville: United States Pharmacopeia Convention Inc. 1741, 3079.

7 Hayun, Harianto dan Yenti Penetapan kadar triprolidina hidroklorida dan pseudoefedrina hidroklorida dalam tablet influenza secara spektrofotometri derivatif. Majalah Ilmu Kefarmasian 3 (2): Ismail NE, Suheryanto R, Kustomo S, JB Wudangadi Efektivitas extra joss dalam memperbaiki ketahanan kerja. [30 Januari 2007] Sulistia GG, Rianto S, Frans DS, Purwantyastuti, Nafrialdi, editor Farmakologi dan terapi. Edisi ke-4. Jakarta: Bagian Farmakologi Fakultas Kedokteran Universitas Indonesia , 719. Tan Hoat Tjay, Raharja K Obat-obat penting. Edisi ke-4. Jakarta : Direktorat Jendral Pengawasan Obat dan Makanan ,

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