In the Laboratory edited by
Topics in Chemical Instrumentation
David Treichel Nebraska Wesleyan University Lincoln, NE 68504
Determination of Aspartame and Caffeine in Carbonated Beverages Utilizing Electrospray Ionization–Mass Spectrometry H. Robert Bergen III,* Linda M. Benson, and Stephen Naylor Biomedical Mass Spectrometry and Functional Proteomics Facility, Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, MN 55905-0002; *
[email protected] The advent of ionization techniques such as matrixassisted laser desorption ionization (MALDI) and electrospray ionization (ESI), which are amenable to thermally labile and nonvolatile molecules, has greatly increased the utility of mass spectral analysis (1). Reviews of electrospray ionization–mass spectrometry (ESI–MS) in this Journal and elsewhere indicate the widespread application of these techniques to biomolecules including proteins and nucleic acids (2–4). However, simple undergraduate protocols that utilize ESI in an undergraduate laboratory setting have not been previously reported. In this laboratory protocol we demonstrate the use of ESI– MS for the analysis of caffeine (MW = 194) and aspartylphenylalanine methyl ester (aspartame, MW = 294) in carbonated beverages. The potential and ease of use of ESI–MS as well as caveats inherent in ESI are explored. Lab Rationale ESI has facilitated the mass spectral analysis of high molecular weight thermally labile compounds including proteins and nucleic acids. The analyte in question must be capable of forming either a positive or negative ion or an adduct between the analyte and a charged ion (e.g. [M + H]+, [M – H]᎑, [M + Na]+, etc.). Our facility was recently involved in hosting several groups of advanced high school students in an organized effort to encourage them to consider careers in the sciences and career opportunities at the Mayo Clinic. As our facility’s contribution, we allowed students to analyze the caffeine and aspartame content of carbonated beverages. These analytes are familiar to students and require little or no background explanation. Both caffeine and aspartame are amines that are readily protonated to give a strong (M + H)+ ion. Many biological compounds and metabolites are also amines and behave similarly (i.e., add H+) and would be detected in positive ion detection mode. Biological acids produce anions seen in negative ion mode, but not under the conditions of our analysis (positive ion). Compounds that don’t readily protonate or deprotonate can often be seen as adducts with metal cations (e.g. carbohydrates). These differences in charge and ionization characteristics provide a great opportunity to discuss the capabilities and limitations of ESI–MS. The protocols described would be appropriate, with proper modification, for inclusion in quantitative analysis or instrumental analysis curriculums.
Experimental Procedures
Sample Preparation Caffeine, aspartame, theophylline, or xanthine standards were purchased from Sigma (Sigma Chem. Co., St. Louis, MO). Standards were prepared in water to give a final concentration between 0.001 and 0.2 mM. Internal standards were added to a concentration of 0.1 mM. Carbonated beverage samples were prepared by allowing them to degas at room temperature and then diluting them 1:10 into water or water containing internal standard. Mass Spectrometer A Perkin-Elmer Sciex API 365 triple quadrupole MS system (Foster City, CA) was equipped with a standard electrospray ion source (without turbo pumping) and run in single quadruple positive ion mode. Ion optics were tuned to maximize the (M + H)+ ion of an infused caffeine standard. The scan parameters were 150–300 amu scan width, 0.1 amu/ step, and a 0.5-ms dwell time. Chromatographic data was smoothed prior to analysis.
Chromatography Samples were minimally purified by HPLC on a C-8 reversed-phase column (Columbus 5 µm, C-8, 1 × 250 mm; Phenomenex, Torrance, CA) using HPLC grade acetonitrile/ water/acetic acid (23:77:1) isocratically (50 µL/min) as a mobile phase. A Rheodyne 9725I injector (Rheodyne L.P., Cotati, CA) equipped with a 20-µL sample loop was used to introduce samples (0.5 µL). Flow from the column passed through a UV detector (785A UV–vis, 254 nm, Perkin Elmer–Applied Biosystems, Foster City, CA) before entering the ESI source. The HPLC system comprised Shimadzu LC-10AD pumps equipped with a Shimadzu SCL-10Avp pump controller (Shimadzu Corporation, Kyoto). Effluent from the UV detector was split before the ESI source, allowing approximately 10 µL/min into the ion source. Hazards There are no significant safety issues unless the highvoltage safety interlocks are defeated on the ion source or the injection syringe is used improperly.
JChemEd.chem.wisc.edu • Vol. 77 No. 10 October 2000 • Journal of Chemical Education
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In the Laboratory 0.25
Table 1. Caffeine and Aspartame Content of Carbonated Beverages Measured by ESI–MS
Concentration / mM
0.20
Beverage 0.15
0.10
0.05
0.00
-0.05 0
2
4
6
8
10
12
14
16
Area or area ratio Figure 1. A standard curve for the quantification of aspartame and caffeine. 䊉 Aspartame, 䊊 caffeine, 䉲 caffeine utilizing an internal standard.
Results and Discussion Electrospray ionization results when a continuous stream of solvent is nebulized as a fine aerosol in the presence of a strong electric field (2). The aerosol desolvates, leaving charged ions in the gas phase. The ions are focused into the ion optics and then into the mass spectrometer, where separation is based on differences in the ratio of mass to charge (m/z). The (M + H)+ ions for caffeine (m/z 195.2) and aspartame (m/z 295.4) are readily detected, since both molecules contain easily protonated amine groups. A weak acid (e.g. acetic, formic, trifluoroacetic) is usually added to the solvent to assist in protonating the analytes. We originally performed this experiment without chromatography, utilizing a simple loop injection into a stream of solvent flowing into the ion source. We found this to be adequate for qualitative determinations, but quantitation requires removal of components that ablate the ion signal (presumably phosphates) and result in erroneous quantitation even in the presence of an internal xanthine or theophylline standard. HPLC was used as a means to remove interfering components that hinder ion production, but was not optimized to chromatographically resolve analytes. It is informative for students to note that under our conditions aspartame and caffeine are not resolved chromatographically. The MS neatly resolves both caffeine and aspartame, however, because of their mass differences. Standard curves generated for caffeine and aspartame with and without internal standard can be seen in Figure 1. Standard curves were generated by calculating the area under the peak corresponding to the (M + H)+ ion of caffeine and aspartame standards. As shown in Figure 1, linearity can be improved (caffeine shown) by the addition of an internal xanthine or theophylline standard, illustrating the caveats inherent in not utilizing an internal standard. Analysis is rapid: approximately one sample is analyzed every seven minutes. Quantitation gives results comparable to published levels of aspartame (5, 6 ) (180 mg/12 oz) and caffeine (7) (35–50 mg/12 oz) in common carbonated beverages (Table 1). Pepsi ONE utilizes a combination of artificial sweeteners, aspartame and potassium acesulfame (personal communication with the Pepsi-Cola Co., Purchase, NY, Jul 2000), and therefore has lower
1326
Aspartame, mg/12 oz Measured c
Publisheda
Caffeine, mg/12 oz Measured
Publishedb
Coke
N.D.
—
34
46
Diet Coke
197
180
50
46
Pepsi
N.D.
—
31
37
Diet Pepsi
161
180
34
35
Caffeine Free Diet Pepsi
170
180
N.D.
N.A.d
105.e
Pepsi ONE
86
56
55.e
Dr. Pepper
N.D.
—
45
40
Diet Mountain Dew
162
180
51
55
a Ref
6, 7. 5–7. c N.D. indicates not detected. d N.A. indicates not available. ePersonal communication with the Pepsi-Cola Co., Purchase, NY, Jul 2000. b Refs
aspartame concentrations than the other beverages (Table 1). Although the assay was not optimized for sensitivity, we were easily able to measure 0.5 pmol of either caffeine or aspartame. Conclusions Students with little to no laboratory experience participated in an analysis of beverage components, using state-of-the-art ESI–MS equipment. The analysis is quick (seven minutes), allowing many samples to be analyzed in a routine laboratory period. The analytes proved interesting to students, who are personally familiar with carbonated beverages (in contrast to biological samples, which often require extensive knowledge to comprehend). Many aspects of this analysis would lend themselves to a quantitative analysis laboratory (generation of standard curve, use of internal standards) or instrumental analysis laboratory (electrospray ionization, ion production, mass spectrometry, etc.). The analysis is comparable to the analyses of many biological compounds performed in our laboratory (peptides, proteins, nucleic acids, etc., which can become positively or negatively charged) and demonstrates the capabilities of this exciting analytical technology. Literature Cited 1. Mass Spectrometry in the Biological Sciences; Burlingame, A. L.; Carr, S. A., Eds.; Humana: Totowa, NJ, 1996. 2. Hofstadler, S. A.; Bakhtiar, R.; Smith, R. D. J. Chem. Educ. 1996, 73, A82–A87. 3. Bakhtiar, R.; Hofsadler, S. A.; Smith, R. D. J. Chem. Educ. 1996, 73, A118–A123. 4. Hop, E. C. A.; Bakhtiar, R. J. Chem Educ. 1996, 73, A118–A123. 5. The National Soft Drink Association. Soft Drinks; http://www. nsda.org/SoftDrinks/History/whatsin.html (accessed Jun 2000). 6. The NutraSweet Company. All about NutraSweet®; http://www. nutrasweet.com/html/his_about.html (accessed Jun 2000). 7. Bowes, A. de P. In Bowes and Church’s Food Values of Portions Commonly Used, 17th ed.; revised by Pennington, J. A. T.; Lippincott-Raven: Philadelphia, PA, 1998; p 383.
Journal of Chemical Education • Vol. 77 No. 10 October 2000 • JChemEd.chem.wisc.edu
