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Allison Blonski-Analytical Problem-Taurine in Energy Drinks

Large numbers of college students turn to energy drinks for a "quick boost," some infrequently and others almost every day. Most energy drinks contain large amounts of caffeine, plus other assorted organic compounds which claim to boost energy. One of these compounds is taurine, a sulfonic acid commonly classified as an amino acid. Taurine is typically ingested through meat, and is important for skeletal muscle growth and functioning. When added to energy drinks, marketers claim it boosts energy, increases concentration and memory and supplements physical performance. However, the quantities added are far below levels of taurine shown to produce such effects. Additionally, taurine and caffeine are known to have adverse interactions, although these have not been thoroughly studied or quantified. One of the major interactions appears to be increased cardiac stress in the form of increased stroke volume. Accurately determining the taurine content is these energy drinks is important, because people may be consuming levels of taurine that can interact with the caffeine in these drinks to cause cardiac stress.

The hypothesis is that taurine levels in energy drinks are less than the standard effective dosage for taurine, but great enough to cause harmful interactions with caffeine. Essentially, the taurine in energy drinks is not great enough to add any benefits, but prevalent enough to cause cardiac distress. The primary analyte is taurine, and the matrix is the energy drink, including the added caffeine, sugars, vitamins, and organic compounds.

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).

Reissig, C.J.; Strain, E.C.; Griffiths, R.R. Caffeinated energy drinks--A growing problem. Drug Alcoh. Dep.[Online] 2009, 99, 1-10. http://www.sciencedirect.com.ezp1.lib.umn.edu/science/article/pii/S0376871608002858 (accessed Sept 20, 2011).

UV-Vis absorption spectrometry

Taurine does not have detectable uv-vis activity. However, numerous different compounds can be complexed with taurine to obtain either uv-vis or fluorescent spectra. One method that has shown success in complex matrices, such as energy drinks, is derivitization with 4-fluoro-7-nitrobenzofurazan (NBD-F). To create this complex, a small amount of energy drink is heated with EDTA and the NBD-F. After 10 minutes at 60 degrees Celsius, the complex is formed an can be analyzed at 470 nm.

To find information on uv-vis data for taurine, I performed a journal search for papers that determined taurine content and provided uv-vis data. This search lead me to numerous publications explaining different methods for complexing taurine.

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).

See "Andrew-Xayamongkhon-Analytical Problem- Nitric oxide and muscle growth" for similarities between our problems.

Similar Analytical Problem(s)

One problem that will be similar to mine is Andrew Xayamongkhon's on nitric oxide and muscle growth. Andrew's problem looks at the effectiveness of nitric oxide in muscle building compounds and their health effects. He will measure levels of nitrate and nitrite found in blood. His hypothesis states that nitric oxide in these supplements is not effective for muscle growth.

Another problem similar to mine is Vinh Tran's examination of triclocarban in human urine. TCC, an antimicrobial agent, is found in many soaps, but can have adverse health effects. His hypothesis states that TCC levels will increase in people who have greater exposure to TCC through soaps. He will measure levels of TCC in urine.

One similar study between my problem and these two problems is the determination of total content of the analyte in the matrix. We all have complex matrices, and we will need to determine the content of our compounds from this complexity. Additionally, we all are working with health risks associated with compounds that have intended health benefits. We will have to determine what levels of these compounds are associated with said risks.

However, it will be very different establishing controls for our analytes. While I can easily examine energy drinks that lack taurine as a negative control, it will be harder for Vinh and Andrew to determine baseline levels of their analytes in biological fluids. I will also be able to add standard taurine to energy drinks as a positive control, something that will be harder to do for my two peers.

Chemical Structure and Standards

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Standards for taurine can be purchased through Abblis Scientific. 100 grams can be purchased for $37 (catalog number AB1002120). This standard has a purity of greater than 99%.

CAS Number: 107-35-7: 2-Aminoethanesulfonic acid. http://www.abblis.com/product_AB1002120.html?&referrer=chemexper (accessed Oct 25, 2011).

Mass Spectra

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Chromatographic Techniques

For taurine analysis, gas chromatography, HILIC, and ion-exchange chromatography are all feasible methods. Reverse phase is not suitable because taurine is very polar, and it would not be retained at all by the stationary phase in a reverse phase column. Taurine is too small to be considered for size-exclusion, and lacks a suitable ligand for affinity chromatography. Finally, its lack of chiral centers makes chiral chromatography a poor choice. However, derivatization of taurine to produce a fluorescent molecule makes reverse phase chromatography a possibility as well.

Of the methods above, reverse phase chromatography of a taurine NBD-F or taurine NBD-Cl derivative is the best option. This method is simple and provides good separation from the numerous other compounds found in the matrix. Additionally, the derivatized compounds make detection simple.

A suitable column for this separation would be would be a Bondclone column with a length of 300 mm and a diameter of 3.9 mm packed with C18 chain silica particles. (catalog number WAT027324) The particles are 10 micrometers. For this separation, the pH should be kept as close to 9 as possible, and a temperature between 25 and 40 degrees is suitable. The separation should use a mobile phase of THF, acetonitrile, and phosphate buffer in a volume ratio of 4:24:72 at a pressure of 1100 psi and a flow rate of 1 mL/min.

For detection, a fluorometer should be used, since the derivatized compound exhibits activity. Such a detector provides for easy quantification, because only the taurine compounds fluoresce at the selected wavelengths, plus this method produces and better S/N ratio than uv-vis absorption. An example of such a detector is Shimadzu SPD-M10AVP diode array detector.


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=&_coverDate=30-Jun-1996&view=c&wchp=dGLzVBA-zSkzk&md5=ada4407c4aeec0327c7acd073b2c28f8/1-s2.0-073170859501697X-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 Si.[Online] 2008, 54, 661-664. http://jhs.pharm.or.jp/data/54%286%29/54_661.pdf(accessed Nov 10, 2011).

Bondclone. http://www.brechbuehler.ch/fileadmin/redacteur/pdf/columns-sampleprep/lc-columns/zhbdc.pdf (accessed Nov 10, 2011).

Capillary Electrophoresis

Suitable capillary electrophoresis techniques for determining the taurine content in energy drinks include CZE and cIEF. MEKC is not suitable because most of the compounds dissolved in water-based energy drinks are highly hydrophilic. There simply would not be great enough separation between taurine and most of the other compounds in the matrix. CGE incorporates a size-related separation that is inappropriate for this study. Most of the compounds in energy drinks are quite similar in size to taurine. Focusing on the charge/size ratio will better separate taurine from other compounds, like caffeine.

Between CZE and cIEF, CZE is a better option for taurine analysis. When using cIEF for analysis, derivatization of compounds is not very suitable for the band formation in the gradient. Since taurine is most easily detected in a derivative form, CZE, which allows for this, is the better choice.

Suitable conditions for this analysis include a sodium phosphate buffer to a pH of 11.8. An applied electric field of 22 kV will produce a suitable separation in an uncoated fused-silica capillary with an internal diameter of 75 micrometers and an effective length of 40 cm.

Since CZE allows for derivatization, a fluorometer would be a suitable detector. Fluorescence is a good technique for measuring taurine because it is a selective method that has minimal interference from other compounds in the matrix and has a better signal to noise ratio than uv-vis absorbance. Given the quantity of taurine claimed to be included in energy drinks, fluorescence should not provide any issues with limits of detection. Additionally, fluorometers have proven to have high reproducibility in detecting taurine in CZE in previous studies.

Zinuella, A.; Sotgia, S.; Scanu, B.; Chessa, R.; Gaspa, L.; Franconi, F.; Deiana, L.; Carru, C. Taurine determination by capillary electrophoresis with laser-induced fluorescence detection: from clinical field to quality food applications. Amino Acids[Online] 2009, 1, 35-41. http://www.ncbi.nlm.nih.gov/pubmed/18193477(accessed Nov 20, 2011).

Analytical Electrochemistry

Although taurine can be reduced, this process is extremely difficult to carry out and would not provide an accurate way to quantify the taurine content. Therefore, an ion selective electrode would prove much more useful for quantification.

Carbon-disk electrodes respond to taurine in a system. A 300 micrometer carbon-disk electrode can serve as the working electrode, while SCE provides a suitable reference electrode.

Cao, Y.; Zhang, X.; Chu, Q.; Fang, Y.; Ye, J. Determination of Taurine in Lycium Barbarum L. and Other Foods by Capillary Electrophoresis with Electrochemical Detection. Electroanalysis[Online] 2003, 15, 898-902. http://onlinelibrary.wiley.com.ezp2.lib.umn.edu/doi/10.1002/elan.200390112/pdf (accessed Dec 6, 2011).


Blog 13. Good answers

Blogs 9, 10, 11. Good answers

For my analytical problem, I will measure taurine content by derivitization with NBD-F, followed by reverse phase HPLC for separation with quantifiable detection using fluorescence. This method is simple, selective, and provides a suitable limit of detection.

Other problems using HPLC-fluorescence include Abdihakim Abdullahi's examination of mercury in skin supplies, Ziyue Yi's look at alfatoxin in oil, and Rajvi Mehta's exploration of DEHP leaching from PVC into the contents of medical devices.

Good answers.

Sample preparation:

1) Deproteinization (to removed proteins added to the energy drinks) through the addition of pure acetonitrile in a ratio of 3 parts acetonitrile for every 2 parts sample

2) Centrifuge the mixture for 10 minutes at 5800 g

3) Removal of the supernatant via pipette

4) Storage at room temperature until needed, though it should be noted that any desired derivitizations should be performed immediately following the removal of the supernatant.

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=&_coverDate=30-Jun-1996&view=c&wchp=dGLzVBA-zSkzk&md5=ada4407c4aeec0327c7acd073b2c28f8/1-s2.0-073170859501697X-main.pdf(accessed Nov 3, 2011).

Good answers for Blogs 6 and 7

Although atomic spectrometry will not work for taurine, mass spectrometry can be used to quantify taurine. Taurine has a nominal mass of 125 Daltons and an exact mass of 125.01466 Daltons.

One type of mass spectrometry that would work well for taurine is ESI-MS. The ionization source is electrospray ionization, and the mass analyzer is a quadrupole. An example of the mass spectrum obtained using this equipment with an injection volume of 5 microliters and and flow rate of 1 milliliter per minute can be found in my original post.

Another mass spectrometry method is EI-TOF. This equipment also uses electorspray ionization as an ion source, but it uses time of flight for the mass analyzer. An example of a taurine spectrum for this method can also be found in the original post.

Ara, T.; Morishita, Y.; Shibata, D. Taurine Mass Spectrum. http://www.massbank.jp/jsp/Dispatcher.jsp?type=disp&id=KZ000076&site=5 (accessed Oct 26, 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).

Good answers for BLOGS 4 and 5.

Although taurine is not fluorescent, it can be complexed to form a fluorescent compound. Taurine can be complexed with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in ethanol to produce N-(7-nitrobenzofurazan-
4-yl)taurine (NBD-Tau). NBD-Tau exhibits fluorescence, with an excitation maximum at 483 nm and an emission maximum at 550 nm.

Given the volume of the samples likely to be used and the number of samples needed to be measured, a spectrofluorimeter will work well for making these measurements.

Eidelman, O.; Zangvill, M.; Razin, M.; Ginsburg, H.; Cabantchik, Z.I. The anion-transfer system of erythrocyte membranes. Biochem. J.[Online] 1981, 195, 503-513. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1162915/pdf/biochemj00401-0160.pdf(accessed Oct 18, 2011).

Studies needed to analyze my hypothesis include 1) the content, per serving and per container, of taurine in energy drinks that both advertise taurine inclusion and those that claim not to include taurine, 2) determining the median effective dosage (ED-50) of taurine in children and adults, males and females, through observing physical and mental performance after consuming controlled quantities of taurine, and 3) comparisons of cardiac output and heart rate after consuming energy drinks with taurine and caffeine to those with just taurine. The results from study 1 will need to be compared with results from studies 2 and 3. Depending on the differences displayed in study 3, it may be useful to quantify the caffeine in various energy drinks.

In energy drinks that contain taurine, there is usually around 1 gram of taurine, according to manufacturers.