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Taurine in Energy Drinks Allison Blonski CHEM 4101: Modern Instrumental Methods of Analysis December 9, 2011 What is Taurine? Sulfonic acid, often considered a free amino acid1 Obtained in diet through meat consumption Important for muscle functioning Interacts with caffeine, distressing cardiac output Exact Mass = 125.01466 Da2 Energy Drinks and Taurine Taurine added to energy drinks to boost energy, concentration, memory, and physical performance Effective dosages of taurine range from 2,000 to 6,000 mg1 Potential health risks arise from interactions with other compounds, including caffeine Energy drinks containing both caffeine and taurine have been connected to athlete deaths in Europe1 Central Problem How much taurine is found in a serving of energy drinks? Does this amount of taurine provide any benefits? Does this amount of taurine cause any health issues? Hypothesis The taurine content in a typical energy is below the effective dosage required for any benefits, but high enough to interact with caffeine, making taurinecontaining drinks more dangerous than those without taurine. Separation Techniques Technique Advantages Disadvantages Reverse Phase High Performance Liquid Chromatography (HPLC) Relatively low cost, Somewhat slow commonly used procedure technique, low sample volume, good separation Capillary Isoelectric Focusing (cIEF) Sharp separation, relatively fast, low sample volume Compound cannot be derivatized, may be difficult to fully separate all compounds in matrix Capillary Zone Electrophoresis (CZE) Sharp separation, relatively fast, low sample volume Difficult to combine with optical detection methods Detection Methods Technique Advantages Disadvantages Fluorescence Spectroscopy Excellent S/N ratio, high sensitivity Compound requires derivatization Ultraviolet-Visible Low cost, rapid Absorption procedure Spectroscopy (UV-Vis) Time of Flight Mass Spectrometry (MSTOF) 2 Good resolution, provides qualitative confirmation Relatively high limit of detection (LOD) and signal to noise ratio (S/N), compound requires derivatization Expensive, weak sensitivity, relatively difficult to pair with some separation techniques Procedure Sample Preparation3 Derivitazation4 Measurement5 Deproteinate with acetonitrile Heat to 60° C Inject 20 μL Add 1 mL of sample with 2 mL of 0.1 M phosphate buffer Excite sample at 473 nm Centrifuge Dilute to concentration of 0.015 mg/mL Add 1 mL 4-fluoro-7nitrobenzofurazan (NBD-F) and stir 10 min Measure emission at 550 nm Reverse Phase HPLC Fluorescence Separation based on polarity of compounds Detection based on compound excitation and wavelength emission Compatible methods of separation and detection Relatively low cost Suitable selectivity and sensitivity for the matrix 6 Instrumentation Bondclone μBondapak C18 column WAT0273247 300 mm x 3.9 mm Stationary phase = C18 chain silica (10 μm diameter) Mobile phase = 4:24:72 (THF: acetonitrile: phosphate buffer), pH = 9 1100 psi at 1 mL/min Hitachi HTA HPLC Detector - L-2485 Fluorescence9 8 S/N > 900 Excitation wavelength = 200 -850 nm Emission wavelength = 250 – 900 nm Band width = 15 nm Accuracy within 3 nm Limit of detection (LOD) = 0.015 ng/mL Limit of quantification (LOQ) = 0.0625 ng/mL Possible Results 9 Calibration curve would be created via standard addition Standard taurine can be purchased through Abblis Scientific10 Addition of standard taurine to energy drinks without taurine will serve as a positive control Conclusions Reverse phase HPLC with detection using fluorescence will provide a suitable quantification of the taurine found in energy drinks. Further clinical research should be done to examine the effects of taurine, caffeine, and the combination of the two on the human body. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Clauson, K.A.; Shields, K.M.; McQueen, C.E.; Persad, N. Safety issues associated with commercially available energy drinks. J. Amer. Pharm. Assoc.[Online] 2008, 48, e55-e67. http://www.pharmacytoday.org/ pdf/2008/May_CE_exam.pdf (accessed Sept 20, 2011). Chen, Z.; Chen, B.; Yao, S. High-performance liquid chromatography/electrospray ionization-mass spectrometry for simultaneous determination of taurine and 10 water-soluble vitamins in multivitamin tablets. A. Chem. Acta.[Online] 2006, 569, 169-175. http://hgxy.hunnu.edu.cn/UploadFile/20061116102343585.pdf (accessed Oct 26, 2011). McMahon, G.P.; O'Kennedy, R.; Kelly, M.T. High-performance liquid chromatographic determination of taurine in human plasma using pre-column extraction and derivatization. J. Pharm. Biomed. Anal.[Online] 1996, 14, 1287-1294. http://www.sciencedirect.com.ezp1.lib.umn.edu/science? _ob=MiamiImageURL&_cid=271442&_user=616288&_pii=073170859501697X&_check=y&_origin=&_c overDate=30-Jun-1996&view=c&wchp=dGLzVBA-zSkzk&md5=ada4407c4aeec0327c7acd073b2c28f8/1s2.0-073170859501697X-main.pdf(accessed Nov 3, 2011). Sawabe, Y.; Tagami, T.; Yamasaki, K. Determination of Taurine in Energy Drinks by HPLC Using a Pre-Column Derivative. J. Health Sci.[Online] 2008, 54, 661-664. http://jhs.pharm.or.jp/data/54%286%29/54_661.pdf (accessed Sept 29, 2011). Eidelman, O.; Zangvill, M.; Razin, M.; Ginsburg, H.; Cabantchik, Z.I. The anion-transfer system of erythrocyte membranes. Biochem. 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