In the Laboratory

Quantitative Determination of Caffeine in Beverages

Using a Combined SPME-GC/MS Method
Min J. Yang, Maureen L. Orton, and Janusz Pawliszyn*
The Guelph-Waterloo Center for Graduate Work in Chemistry, University of Waterloo, Waterloo, ON N2L 3G1, Canada

Caffeine occurs naturally in tea, coffee, and cola nuts.

Caffeine analysis is performed for quality control purposes,
for example to insure proper caffeine levels in decaffeinated
coffee to meet regulatory standards. Current methods for
determining caffeine in beverages require pH adjustments
and often involve the use of toxic organic solvents. These
methods are labor-intensive, generate large quantities of
organic waste, and have relatively poor precision. Alterna-
tively, solid-phase microextraction (SPME) can be used. The
SPME technique has many advantages including simplic-
ity, portability, time efficiency, sensitivity, and compatibil-
ity with a GC system. Initial work on SPME caffeine analy-
sis has been reported (1). The SPME principle is based on
an equilibrium process in which the analyte partitions be-
tween the fiber and the aqueous phase (2, 3). Many appli-
cations of SPME have been investigated (4–8), and a recent
review provides a good overview (9). The main objective of
this paper is to describe a simple undergraduate experi-
ment for determination of caffeine in common beverages
using the combined SPME-GC/MS methods.

Experimental Procedure
Materials and Equipment
The SPME device was purchased from Supelco
(Supelco Canada, Mississauga, ON). For caffeine analysis,
an uncoated fused silica fiber was used for extraction. Com-
mercial fibers are often supplied with a coating such as
poly(dimethylsiloxane) (PDMS) and polyacrylate. Uncoated
fiber can be prepared by dissolving the fiber coating in hot
sulfuric acid.
Two 5-mg/mL stock solutions of regular 12C caffeine
(Aldrich Chemical Co, Inc., Milwaukee, WI) and isotopically
labeled (trimethyl 13C) caffeine (Cambridge Isotope Labo-
ratories, Woburn, MA) were separately prepared in metha-
nol. Regular coffee, decaffeinated coffee, and teas were
brewed as for normal consumption. Soft drinks were taken
from beverage containers.
A Varian 3400 gas chromatograph equipped with a Sat-
urn II ion trap MS (Varian, Mississauga, ON) detector was
used. The column was a 30 m × 0.25 mm SPB-5 with a sta-
tionary phase thickness of 0.25 µm (Supelco Canada,
Mississauga, ON).
Extraction Procedures
Many different versions of SPME procedures are de-
scribed; which one to use depends on the application (4–8).
For this experiment, the adsorption period was set for 5
minutes. The sample was magnetically stirred during ex-
traction (Fig. 1). After the adsorption step, the fiber was di-
rectly transferred into the GC injector in which the ana-
lytes were thermally desorbed at 250 °C, and the GC/MS
run was started. To obtain a complete desorption, the fiber
*Corresponding author.
Figure 1. Sample extraction setup for the manual SPME method.
was allowed to stay in the hot GC injection port for 1 min
before withdrawal.
GC/MS Methods
The GC oven was maintained at 200 °C. for 1.5 min,
then ramped at 30 °C/min to 275 °C. The temperatures of
the injector and transfer line were set at 250 and 220 °C,
respectively. Helium was used as the carrier gas at a flow
rate of 1.0 mL/min. MS conditions were electron impact,
ionization, positive ion, mass range 50–300 amu, and 60
scans/min. The integrated areas from the extracted mass
chromatogram of the molecular ions (194 and 197 for 12C
caffeine and 13C 3 caffeine) were used for all quantitation.
The entire caffeine analysis run took 4 minutes to complete.
Determination of the Calibration Curve
For quantitative caffeine determination, a calibration
curve was obtained by analyzing a set of standard aqueous
samples with 12C caffeine concentrations of 50, 100, 200,
300 and 500 µg/mL spiked with 240 µL of the 13C 3 caffeine
stock solution, which served as an internal standard. The
ratio of the integrated peak areas for 12C caffeine (m/z 194)
and 13C3 caffeine (m/z 197) was plotted versus 12C caffeine
concentration in the standard solutions to provide external
calibration for caffeine determination in various beverage
samples. This method of isotopic dilution has been previ-
ously described (10).
Analysis of Caffeine Content in Selected Beverage
All beverages were allowed to reach room temperature
before the analysis. A 12-mL aliquot of each beverage was

1130 Journal of Chemical Education • Vol. 74 No. 9 September 1997


Table 1. Quantitative Analysis of Caffeine in Beverages
Using the SPME-GC/MS Method
Peak ratio Caffeine conc.
Beverage
(m / z 194/197) (µg/mL)
100% Colombian coffee 5.6332 635
Decaffeinated coffee 0.4468 49
Chinese green tea 1.8315 206
Camomille tea 0.6596 73
Diet Coke 2.311 260
Diet decaffeinated Coke 0.4769 52
pipetted into a 15-mL vial. To each sample vial, 240 mL of
the 13C3 caffeine stock solution was added as an internal
standard. The ratio of the integrated peak area for both 12C
caffeine (m/z 194) and 13C3 caffeine (m/z 197) was used to-
gether with the previous calibration data to quantitatively
determine the caffeine content in the beverage. Table 1
shows the calculated average caffeine concentration for the
tested beverages based on the standard calibration curve.
Results and Discussion
For caffeine extraction from beverages, an uncoated fi-
ber is preferred because caffeine contents in the beverages
of interest are in the ppm range. Also, uncoated fiber mini-
mizes potential carry-over problems and produces sharp in-
jection bands owing to rapid desorption. Alternatively, the
7-µm PDMS fiber can be used, but in this case the carry-
over may become a significant issue.
Since the internal standard and native analyte have the
same chemical and physical properties, their behavior dur-
ing extraction (equilibration time, metric effects) is identi-
cal. As a result, the use of an isotopically labeled internal
standard essentially eliminates the need to reach equilib-
rium of analyte partitioning between the fiber and the
sample. Also, any change in extraction conditions, including
the change of the fiber properties due to irreversible adsorp-
tion of some of the matrix component, is compensated for.
Quantitative information can be obtained by generat-
ing extracted mass chromatograms for the specific ions of
the native analyte and the isotopically labeled internal
Retention Time (s)
Figure 2. Typical chromatograms obtained for caffeine analysis
of a brewed coffee sample using the SPME-GC/MS method. (a)
A total ion chromatogram. (b) An extracted mass chromatogram
for m /z 194. (c) An extracted mass chromatogram for m/ z 197.
Vol. 74 No. 9 September 1997 • Journal of Chemical Education
In the Laboratory
Figure 3. A standard calibration curve for quantitative caffeine de-
termination using the SPME-GC/MS method.
standard, and comparing the two peak areas obtained. Fig-
ure 2a shows that both the isotopically labeled (trimethyl
13C) caffeine internal standard and the native 12C caffeine
coelute at the end of the GC column and form a single caf-
feine peak in the total ion chromatogram. Figures 2b and
2c show the extracted mass chromatograms for the molecu-
lar ions of the native caffeine (m/z 194) and the 13C 3 caf-
feine (m/z 197), respectively.
There is a linear relationship between the amount of
caffeine adsorbed by the bare fiber and the concentration
in the solution. The precision of the method in terms of rela-
tive standard deviation (RSD), determined by analyzing 5
vials of the same sample, is 1.9%. A calibration curve (Fig.
3) was obtained by plotting the peak area relations between
the 12C caffeine and the 13C 3 caffeine versus the 12C caffeine
concentration. Excellent linearity can be observed for the
SPME-GC/MS method within the tested concentration
range. The correlation coefficient for the linear regression
was .996.
The quantitative results are in good agreement with
caffeine contents reported by Hawthorne et al. (1). There
was concern that deposition of beverage color stain on the
fiber surface could hinder sample extraction. For replicate
sample analysis, large fluctuations in component peak ar-
eas were observed between experimental runs under the
same conditions. These fluctuations were effectively com-
pensated by the internal standard method and the peak
area ratio for the replicate samples remained relatively un-
changed. In other words, use of the internal standard has
avoided this uncertainty and leads to reproducible quanti-
tative results.
The initial acquisition of the GC/MS system and its
maintenance would be the most expensive part of the ex-
periment. The other costs can be considered minimal. The
SPME device and extraction fiber can be purchased directly
from Supelco for ~$200 U.S. and can be reused many times.
Only a small amount of (trimethyl 13C) caffeine is needed
for the experiment; for instance, 1 g of 13C-labeled caffeine
costing ~$200 U.S. is sufficient to spike more than 800
samples. If the instruments are set up and stock solutions
are prepared for the students, the entire experiment can be
completed within three hours. The instructor may choose
to prepare and run one set of calibration standards each day
for all groups of students so that the students will have
more time to comprehend the operation of the instrument
and to analyze their data. A group of no more than four stu-
dents should be allowed to run the experiment at one time
to ensure sufficient exposure to the use of the instruments.
Each student should be given an unknown sample to run.
1131
In the Laboratory
The instructor may also choose to give students real or
simulated beverage samples as unknowns. A simulated bev-
erage sample can be prepared by making an aqueous caf-
feine solution of any concentration, with or without dye ad-
dition.
Conclusions
SPME is a fast, inexpensive, and solvent-free alterna-
tive for extracting organic compounds from sample matri-
ces. Recent publications (4–8) have revealed its wide accept-
ability and applicability in many environmental and indus-
trial applications. The technique certainly deserves a place
in the classroom. The SPME-GC/MS method for analysis of
caffeine in beverages is an appropriate hands-on experi-
ment post-secondary level instrumental laboratories. The
experiment will intrigue students learning the principles of
analytical instrumentation and sample preparation tech-
niques because the experiment demonstrates the use of
today’s technology in a simple fun-to-do real life application.
Acknowledgments
This work has been financially supported by the Natu-
ral Sciences and Engineering Research Council of Canada,
Supelco Canada, and Varian Canada. The combined SPME-
1132 Journal of Chemical Education • Vol. 74 No. 9 September 1997
GC/MS method for quantitative determination of caffeine
in beverages has been implemented as a second-year un-
dergraduate laboratory experiment at the University of
Waterloo, Waterloo, Ontario. We would like to thank the un-
dergraduate laboratory TA, Lin Pan, for her cooperation in
sharing the instrument.
Literature Cited
1. Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J.; Arthur, C.
L. J. Chromatogr. 1992, 603, 185–191.
2. Louch, D.; Motlagh, S.; Pawliszyn, J. Anal. Chem. 1992, 64,
1187–1199.
3. Zhang, Z.; Pawliszyn, J. Anal. Chem. 1993, 65, 1843–1852.
4. Zhang, Z.; Yang, M; Pawliszyn, J. Anal. Chem. 1994, 66,
844A–853A.
5. Boyd-Boland, A; Chia, M; Luo, Y.; Zhang, Z.; Yang, M;
Pawliszyn, J. Environ. Sci. Technol. 1994, 28, 569A–574A.
6. Pawliszyn, J. TRAC 1995, 14, 113–122.
7. Graham. K. N.; Sarna, L. P.; Webster, G. R. B.; Gaynor, J.
D.; Ng, H. Y. F. J. Chromatogr. A 1996, 725, 129–136.
8. Langenfeld, J. J.; Hawthorne, S. B.; Miller, D. J. Anal.
Chem. 1996, 68, 144–155.
9. Eisert, R.; Levsen, K. J. Chromatogr. A 1996, 733, 143–157.
10. Hill, D. W.; McSharry, B.; Turzupet, L. S. J. Chem. Educ.
1988, 65, 907–910.