In the Laboratory
Simultaneous Determination of Caffeine and Vitamin B6 in Energy Drinks by High-Performance Liquid Chromatography (HPLC) Rachel E. Leacock, John J. Stankus,* and Julian M. Davis Department of Chemistry, University of the Incarnate Word, San Antonio, Texas 78209, United States *
[email protected] The analysis of popular commercial products in chemistry laboratory courses is an established method of promoting student engagement, interest, and understanding (1-6). Several methods have been developed and incorporated into the classroom to analyze caffeine in products such as coffee (1, 2), soft drinks (2), tea (3), and headache medicine (4). The newest popular caffeinated beverages on the market are energy drinks such as Red Bull, Monster, Rockstar, and Full Throttle. Between July 2006 and July 2007, over 200 new energy drinks were introduced in the U.S. market (7). Energy drinks have become very popular with college students. Several studies have been conducted regarding the physiological effects of the drinks and reported side effects range from headaches and heart palpitations and, in extreme cases, even death (7, 8). To date, there is only one previous study describing the use of energy drinks in an undergraduate laboratory experiment, and this study uses NMR rather than high-performance liquid chromatography (HPLC) (5). It has been suggested in this Journal that energy drinks are a good introduction for students to analytical methods (6). We have developed a method for determining the concentration of caffeine and vitamin B6 (Figure 1) in Red Bull and other energy drinks using HPLC. Although experiments involving HPLC analysis of other caffeinated beverages have been published (1-3), we believe this is the first example of such study with energy drinks. Vitamin B6, an essential water-soluble vitamin, is commonly added to multivitamin tablets and many energy drinks in the form of pyridoxine hydrochloride (Figure 1). Vitamin B6 is a key cofactor in transamination and other biochemical reactions and plays several important roles in human health. There are several methods described in the literature for determining vitamin B6 (9-12), but to our knowledge, this work is the first application of analysis of vitamin B6 in the undergraduate laboratory. Red Bull contains about 5 mg of vitamin B6, which corresponds to 250% of the Recommended Daily Allowance (RDA) (13). We present an experiment that can be performed in one or two 3-h laboratory sessions of an undergraduate instrumental analysis course. In this experiment, students measure the concentration of two analytes simultaneously using the standard addition method. Alternately, a standard calibration curve can be established if the ability to run numerous samples is desired. The experiment has been introduced in an instrument analysis course successfully with positive feedback from students.
Figure 1. Structures of the compounds examined in the energy drinks.
and a 250 4.60 mm 5 μm Phenomenex C18 column. The injection port was equipped with a 20 μL injection–Flag deleted toal count is 0 loop and samples were analyzed under UV detection at 292 nm. Although our system is equipped with a diode array detector, this experiment can be performed on a single wavelength detector. Reagents Red Bull was obtained from the university cafeteria. The Red Bull was degassed by manual agitation (5 min). Pyridoxine hydrochloride (vitamin B6) (Sigma Aldrich), caffeine (Eastman Co.), anhydrous sodium phosphate dibasic (Mallinckrodt), HPLCgrade water (Fischer Scientific), and HPLC-grade methanol (Mallinckrodt) were used as received. Unless otherwise noted, all water and methanol used in these experiments were HPLC grade. Mobile-Phase Solutions The HPLC was eluted with an isocratic 60:40 (v/v) solution of phosphate buffer (50 mM, pH 3.0) and methanol at a flow rate of 1.0 mL/min. In our HPLC setup, one reservoir was loaded with aqueous phosphate buffer and another with HPLCgrade methanol, and the HPLC pump was used to create the isocratic mixture. A third reservoir contained HPLC-grade water; this was used in conjunction with the methanol for the column wash. Alternately, the 60:40 buffer/methanol solution could be mixed and deposited into a single HPLC reservoir. Phosphate buffer was prepared by dissolving 3.525 g Na2HPO4 in 400 mL of HPLC-grade water in a prerinsed beaker. Once dissolved, the pH was adjusted to 3.0 by the addition of concentrated hydrochloric acid. The solution was transferred to a prerinsed 500 mL volumetric flask and diluted to volume with HPLC-grade water.
Experimental Section
Experimental Procedures
Apparatus and Materials
Standard Solution Preparation The combined standard solution was prepared by dissolving 0.0506 g of pyridoxine hydrochloride (vitamin B6) and 0.1253 g
The chromatograms were collected using a Shimadzu LC-10AT HPLC system equipped with a diode array detector SPD-M10A 232
Journal of Chemical Education
_
_
_
Vol. 88 No. 2 February 2011 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed100146s Published on Web 11/15/2010
In the Laboratory Table 1. HPLC Method Analytic Run and Column Wash Conditions Start Time/min
End Time/min
Eluent
Purpose
0
12
Isocratic 60:40 phosphate buffer/methanol
Analytic run
12
13
Linear ramp to 90:10 water/methanol
Column wash
13
16
Flush with 90:10 water/methanol
Column wash
16
17
Linear ramp to 90:10 methanol/water
Column wash
17
20
Flush with 90:10 methanol/water
Column wash
20
21
Linear ramp to 60:40 phosphate buffer/methanol
Column wash
21
25
Return to isocratic 60:40 phosphate buffer/methanol
Reset for analytic run
caffeine in enough 60:40 phosphate buffer/methanol solution to reach a volume of 100.00 mL. This produced a stock solution with 506 ppm vitamin B6 and 1253 ppm caffeine. Alternately, the two standards can be prepared as separate solutions. The phosphate buffer was made as described above; however, the deionized water was used to dissolve Na2HPO4 rather than HPLC-grade water (to conserve the relatively expensive HPLC-grade water), and reagent-grade methanol (Fisher Scientific) was used rather than HPLC-grade. For comparative purposes, two runs were conducted; the first with only HPLC-grade water, the second with deionized water. The results varied negligibly. Sample Preparation The samples were made by placing 10.00 mL of Red Bull in each of five 100 mL volumetric flasks. The flasks were spiked with increasing aliquots of the combined standard solution (0.00 mL, 1.00 mL, 2.00 mL, 3.00 mL, and 4.00 mL, respectively) and were each diluted to volume with the 60:40 phosphate buffer/ methanol solution. Each flask was then mixed thoroughly. These volumes and the standard solution concentrations can be adjusted to use smaller volumes of solvent. HPLC Testing Conditions and Procedures A sample, 100 μL, was carefully drawn and injected into the 20 μL loop to ensure complete filling. The mobile phase consisted of a 60:40 phosphate buffer/methanol mixture at a rate of 1 mL/min. Several alterations of the mobile phase were tested but resulted in wider peaks. Each run was followed by washes of 90:10 water/methanol and 90:10 methanol/water to ensure a clean column for the next sample run. This HPLC method, shown in Table 1, included the analytical run, water and methanol washes (to remove high-fructose corn syrup and other additives), and a return to the analytical conditions. In our experience, the washes were necessary to completely remove the sample from our column. Other authors have used this method with similar samples (specifically colas) without need of an extra wash (1, 2). It is recommended that people using this experiment first determine if a wash cycle is necessary with their system. Hazards Pyridoxine hydrochloride has no significant safety issues; however, unnecessary contact is not recommended. Caffeine ingestion can be lethal in doses of 10 g or more. Disodium hydrogen phosphate can cause irritation upon contact to skin and mucous membranes. Methanol is flammable and should be handled under the hood due to strong vapors that aggravate mucous membrane and central nervous system upon inhalation. Ingestion can
r 2010 American Chemical Society and Division of Chemical Education, Inc.
_
Figure 2. Sample chromatogram showing peaks for vitamin B6 and caffeine.
Figure 3. Sample standard addition curve showing the y intercept equation and extrapolation to the x intercept.
cause blindness and death. Concentrated hydrochloric acid and the vapors it produces are extremely corrosive. Eye protection should be worn at all times in the chemistry laboratory. Gloves are recommended when preparing samples and standards. Results and Discussion A sample chromatogram is shown in Figure 2. Vitamin B6 and caffeine appeared in the chromatogram at approximately 3 and 10 min, respectively, and both ingredients had high enough absorption at 292 nm to make reasonable concentration curves. The students were able to clearly see the quantity of added standard solution. The concentrations of caffeine and vitamin B6 were determined by the standard addition method; the caffeine data are shown in Figure 3. In the Red Bull, the concentrations of caffeine and vitamin B6 were found to be 27.2 and 4.22 ppm,
pubs.acs.org/jchemeduc
_
Vol. 88 No. 2 February 2011
_
Journal of Chemical Education
233
In the Laboratory
respectively, corresponding to 68.1 and 10.6 mg in an 8.3 oz can. The method was applied to the energy drinks Monster and Rockstar with identical retention times of caffeine and vitamin B6. This experiment was designed for upper-level undergraduate instrumental analysis course to illustrate analysis concepts and give students experience using the HPLC instrument. The common consumption of energy drinks and the possible health concerns enhance the relevance of this experiment. Owing to their familiarity with energy drinks, students were more engaged in the experiment. Because the purpose of the experiment was more clearly defined in their minds, the students reported it easier to grasp the more abstract concepts.
R.E.L. and J.M.D. would like to thank the Welch Foundation (grant number BN 0032) for financial support. We thank Elena Davila, Erica De La Rosa, Jesus Garza, and Amanda Roberts for assistance with solution preparation and data collection.
3. Friedman, M.; Levin, C. E.; Choi, S.-H.; Kozukue, E.; Kozukue, N. J. Food Sci. 2006, 71, 328–337. 4. Ferguson, G. K. J. Chem. Educ. 1998, 75 (4), 467–469. 5. Simpson, A. J.; Shirzadi, A.; Burrow, T. E.; Dicks, A. P.; Lefebvre, B.; Corrin, T. J. Chem. Educ. 2009, 86 (3), 360–362. 6. Coleman, W. F. J. Chem. Educ. 2009, 86 (3), 400. 7. Reissig, C. J.; Strain, E. C.; Griffiths, R. R. Drug Alcohol Depend. 2009, 99 (1-3), 1–10. 8. Curry, K.; Stasio, M. J. Hum. Psychopharmacol. 2009, 24 (6), 473–481. 9. Amidzic, R.; Brboric, J.; Cudina, O.; Vladimirov, S. J Serb. Chem. Soc. 2005, 70 (10), 1229–1235. 10. Otles, S. In Food Fortification and Supplementation; Ottaway, P. B., Ed.; CRC: Boca Raton, FL, 2008; pp 129-152. 11. Ollilainen, V. Agric. Food Sci. Finl. 1999, 8 (6), 515–619. 12. Grant, D. C.; Helleur, R. J. Anal. Bioanal. Chem. 2008, 391 (8), 2811–2818. 13. Dietary Supplement Fact Sheet: Vitamin B6. http://dietary-supplements.info.nih.gov/factsheets/vitaminb6.asp (accessed Oct 2010)
Literature Cited
Supporting Information Available
Acknowledgment
1. Beckers, J. L. J. Chem. Educ. 2004, 81 (1), 90–93. 2. DiNunzio, J. E. J. Chem. Educ. 1985, 62, 446–447.
234
Journal of Chemical Education
_
Vol. 88 No. 2 February 2011
Instructions for students and for instructors. This material is available via the Internet at http://pubs.acs.org.
_
pubs.acs.org/jchemeduc
_
r 2010 American Chemical Society and Division of Chemical Education, Inc.
