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Journal of Chromatography A, xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Determination of caffeine, theobromine and theophylline in Mate beer

and Mate soft drinks by high-performance thin-layer chromatography
Claudia Oellig ∗ , Jacob Schunck, Wolfgang Schwack
Institute of Food Chemistry, University of Hohenheim, Garbenstrasse 28, 70599, Stuttgart, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Mate beer and Mate soft drinks are beverages produced from the dried leaves of Ilex paraguariensis (Yerba
Received 20 November 2017 Mate). In Yerba Mate, the xanthine derivatives caffeine, theobromine and theophylline, also known as
Received in revised form 8 December 2017 methylxanthines, are important active components. The presented method for the determination of
Accepted 8 December 2017
caffeine, theobromine and theophylline in Mate beer and Mate soft drinks by high-performance thin-
Available online xxx
layer chromatography with ultraviolet detection (HPTLC–UV) offers a fully automated and sensitive
determination of the three methylxanthines. Filtration of the samples was followed by degassing, dilu-
Keywords:
tion with acetonitrile in the case of Mate beers for protein precipitation, and centrifugation before the
Caffeine
Theobromine
extracts were analyzed by HPTLC–UV on LiChrospher silica gel plates with fluorescence indicator and
Theophylline acetone/toluene/chloroform (4:3:3, v/v/v) as the mobile phase. For quantitation, the absorbance was
Mate beer and Mate soft drinks scanned at 274 nm. Limits of detection and quantitation were 1 and 4 ng/zone, respectively, for caffeine,
High-performance thin-layer theobromine and theophylline. With recoveries close to 100% and low standard deviations reliable results
chromatography–ultraviolet detection were guaranteed. Experimental Mate beers as well as Mate beers and Mate soft drinks from the market
(HPTLC–UV) were analyzed for their concentrations of methylxanthines.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction sentative. Methylxanthines also affect the cardiovascular system,

Yerba Mate (Ilex paraguariensis) is a plant of the Aquifoliaceae
family that typically grows in South America [1,2] and comprises
several constituents which are known to be anti-carcinogenic and
anti-oxidative [2]. Ilex paraguariensis is widely known as the source
of Yerba Mate tea, also simply called Mate. Mate and further Mate
beverages are infusions made from green or dried leaves or other
plant parts of Ilex paraguariensis [1,2]. The infusion Mate is known
as stimulating beverage with health promoting properties that has
been traditionally consumed in South America [1]. The extract of
the infusion is used as ingredient in alcoholic and non-alcoholic soft
drinks. Recently also a flavored beer, the Mate beer, was designed,
when smoked and not smoked Yerba Mate is added during the beer
brewing.
Among others the plant alkaloids caffeine, theophylline, and
theobromine are important active components in Yerba Mate [2],
which are derivatives of xanthine and also are known as methylx-
anthines [3]. They all have in common the stimulating effect on
the central nervous system, with caffeine as the most potent repre-
∗ Corresponding author.
E-mail address: claudia.oellig@uni-hohenheim.de (C. Oellig).
with theophylline generating the strongest effect [2,3]. Thus, the
concentration of caffeine, theophylline, and theobromine in Mate
beverages is generally of great interest. In this context, for the man-
ufacturing of Mate beer, the influence of different procedures of the
Yerba Mate addition during the beer brewing on the quantity of the
active components is an aspect, which has to be evaluated.
For the determination of caffeine, theobromine, and theo-
phylline in different foods, high-performance liquid chromatog-
raphy (HPLC) and high-performance thin-layer chromatography
(HPTLC) were suggested. For coffee, tea and coca samples, HPLC
with ultraviolet (UV) detection or mass selective detection was
often reported [4–7]. HPTLC was suggested for the analysis of caf-
feine and other active components in herbal products, power drinks
[8], and energy drinks [9]. A TLC approach for the determination
of caffeine and theobromine in Mate tea was mentioned by Bojić
et al. [10]. Cimpoiu et al. [11] presented a TLC method for the anal-
ysis of caffeine, theobromine and theophylline in different types
of tea, and Badea [12] described a TLC/GC/MS method for tea and
coffee. However, a fully automated and validated HPTLC method
for the simultaneous determination of the three methylxanthines,
caffeine, theobromine and theophylline, in Mate beer and Mate soft
drinks is not available.

https://doi.org/10.1016/j.chroma.2017.12.019
0021-9673/© 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: C. Oellig, et al., Determination of caffeine, theobromine and theophylline in Mate beer and Mate soft
drinks by high-performance thin-layer chromatography, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.12.019
G Model
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Table 1
Conditions of the Yerba Mate addition and caffeine concentrations in seven experimental Mate beers.
Sample Hops Yerba Mate Method of Yerba
[g/L] Mate addition
1 Bitter and aroma 8 Loose
2 Bitter 8 Loose
3 Bitter and aroma 8 Loose
4 Bitter 8 Tea towel
5 – 8 Tea towel
6 – 16 Tea towel
7 Bitter and aroma 10 Tea strainer
a
Standard deviation.
Thus, the aim of this work was to develop a sensitive, selective
and automated planar chromatographic method for the simulta-
neous determination of caffeine, theobromine and theophylline
in Mate beer. The method should further be applicable to inves-
tigate the methylxanthines in Mate soft drinks. With the plenty
separation possibilities in HPTLC, an efficient separation of the
methylxanthines from Mate matrix compounds should easily be
possible. The highly automated HPTLC devices guarantee well
repeatable procedures for a rapid, simple and reliable determina-
tion of the methylxanthines and additionally offer the analysis of
many samples in parallel.
2. Material and methods
2.1. Chemicals and materials
Caffeine (≥99%), theobromine (≥98%) and theophylline, anhy-
drous (≥99%) were obtained from Sigma-Aldrich (Steinheim,
Germany). Acetone (Rotisolv pestilyse) was purchased from Carl
Roth (Karlsruhe, Germany). Toluene (for pesticide residue analysis),
acetonitrile (LC–MS, Chromasolv) and chloroform (for pesticide
residue analysis, >99.8%) were obtained from Sigma-Aldrich. Ultra-
pure water (>18 M cm) was supplied by a Synergy System
(Millipore, Schwalbach, Germany). Folded paper filter (diame-
ter 185 mm) and filter tips (0.45-␮m) were purchased from
Macherey-Nagel (Düren, Germany). HPTLC silica gel LiChrospher
F254 s plates from Merck (Darmstadt, Germany) were used with-
out pre-washing. Experimental Mate beer samples were obtained
from the Department of Yeast Genetics and Fermentation Technol-
ogy, University of Hohenheim (Stuttgart, Germany). The used Yerba
Mate Pajarito Selección Especial was from Lauro Raatz S.A., Itapùa,
Paraguay. Commercially available Mate beers and Mate soft drinks
were provided from different producers.
2.2. Standard solutions
For the preparation of standard stock solutions, methylxan-
thines generally were weighted into 100-mL volumetric flasks,
dissolved in 60 mL of hot water (60 ◦ C), and filled up with water
at ambient temperature.
For HPTLC–UV method development, individual stock solu-
tions of caffeine, theobromine, and theophylline (100 mg/L) were
prepared. A standard-mix solution (33.3 ng/␮L) was obtained by
pipetting 500 ␮L of the individual standard stock solutions together
in an autosampler vial. For the determination of limits of detection
and quantitation (LOD/LOQ), a combined standard stock solution
(40 mg/L) was prepared, from which a working standard solution
(1 ng/␮L) was achieved by dilution 1:40 with methanol. As calibra-
tion standards for recovery experiments and the analysis of Mate
beer samples, individual standard solutions were prepared at a con-
centration of 60 mg/L caffeine, 40 mg/L theobromine and 40 mg/L
theophylline. For the analysis of Mate soft drink samples, individual
standard solutions were prepared for calibration at a concentration
Please cite this article in press as: C. Oellig, et al., Determination of caffeine, theobromine and theophylline in Mate beer and Mate soft
drinks by high-performance thin-layer chromatography, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.12.019
Time of Yerba Mate addition Caffeine
[mg/L] ± SDa (n = 3)
30 min after the starting of the wort boiling 125 ± 2
30 min after the starting of the wort boiling 107 ± 1
During the washes 78 ± 3
During the washes 90 ± 4
5 min after the starting of the wort boiling 78 ± 4
5 min after the starting of the wort boiling 87 ± 7
5 min after the starting of the wort boiling 110 ± 4
of 150 mg/L caffeine, 100 mg/L theobromine and 100 mg/L theo-
phylline.
2.3. Sample preparation
2.3.1. Mate beer
Ten mL of Mate beer were filtered through a folded filter paper
and were degassed in an ultrasonic bath (for 45 min). A 1-mL
aliquot of the degassed sample was transferred into a 15-mL plastic
centrifuge tube equipped with a screw cap, containing 3 mL of ace-
tonitrile. The tube was vigorously shaken for 2 min and centrifuged
at 3200 × g at ambient temperature for 10 min (Biofuge primo R,
Heraeus, Hanau, Germany). A 1-mL aliquot of the supernatant was
transferred through a 0.45-␮m filter tip into an HPTLC vial.
For the determination of recovery rates, spiked samples were
prepared by dissolving 10 mg of caffeine, theophylline and theo-
bromine in 100 mL of a degassed hop beer sample (100 mg/L) as
described in Section 2.2 for the standard stock solutions. Sample
preparation was performed as described for the Mate beer samples.
2.3.2. Mate soft drink
Ten mL of Mate soft drink was degassed in an ultrasonic bath
(for 45 min) and directly transferred through a 0.45-␮m filter tip
into an HPTLC vial.
2.4. High-performance thin-layer chromatography–ultraviolet
detection (HPTLC–UV)
Application was performed by an Automatic TLC Sampler 4 (ATS
4, CAMAG, Muttenz, Switzerland). Samples and standards were
applied as 6-mm bands with the following settings leading to 21
tracks on a 20 cm × 10 cm plate: 8 mm distance from the lower
edge, 15 mm distance from the left edge, and 8.5 mm track dis-
tance. For the application parameters, the predefined settings for
methanol were used. Methanol was used as the rinsing solvent
with 1 rinsing cycle and 1 filling cycle. The application volume for
LOD/LOQ determinations was 2.5–25 ␮L of the working standard
solution, resulting in 2.5–25 ng/zone for caffeine, theobromine and
theophylline. For recovery experiments, calibration was performed
by application of 1.3–15 ␮L of the individual standard solutions
of caffeine, theobromine and theophylline in the overspray mode.
Sample application volume for recovery experiments was 10 ␮L.
After the application, a drying step with a hair dryer followed
for 5 min, and chromatography was performed without chamber
saturation in the Automatic Developing Chamber (ADC2, CAMAG)
with a 20 cm × 10 cm twin-trough chamber (CAMAG). The plate
activity was controlled to 33% relative humidity by saturated mag-
nesium chloride solution (5 min). As mobile phase, a mixture of
acetone/toluene/chloroform (4:3:3, v/v/v) was used up to a migra-
tion distance of 60 mm (developing time 9 min), and a drying
step followed for 2 min. Plate images were captured with the TLC
Visualizer (CAMAG) under UV 254 nm illumination. The plate was
scanned in the absorption mode at UV 274 nm (deuterium lamp)
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by the TLC Scanner 4 (CAMAG) in the automatic detector mode,
with a scanning speed of 20 mm/s, a data resolution of 100 ␮m/step
and a slit dimension of 4 mm × 0.3 mm. HPTLC instruments were
controlled by the winCATS software 1.4.6.2002 (CAMAG).
2.5. Sample analysis
Seven experimental Mate beers as well as four Mate beers
and six Mate soft drinks from the German market were analyzed.
The conditions of the Yerba Mate addition for the experimentally
brewed Mate beers are listed in Table 1. Sample preparation and
HPTLC analysis were done according to the Sections 2.3 and 2.4.
Application volume was 10 ␮L for the experimental Mate beers, and
ranged between 8 and 25 ␮L for the beers from the German market,
when taking into account the labelled xanthine content of theses
samples. For calibration of beers, the calibration range was lowered
(compared to the calibration range for the recovery experiments),
because low methylxanthine concentrations were expected. The
individual standard solutions (Section 2.2) were applied in the
overspray mode with application volumes of 0.5–7.5 ␮L for caffeine
and 0.2–1.5 ␮L for both theobromine and theophylline, leading
to 30–450 ng/zone caffeine, and 8–60 ng/zone theobromine and
theophylline. For Mate soft drinks, the sample application volume
was 2.5 and 5 ␮L, respectively. For calibration of these samples,
the calibration range was extended to 1–12 ␮L for caffeine and
2.5–15 ␮L for both theobromine and theophylline, because higher
xanthine contents were expected, resulting in 150–1800 ng/zone
caffeine and 250–1500 ng/zone theobromine and theophylline,
respectively.
3. Results and discussion
3.1. Method development
Experimentally brewed Mate beers and a “blank” beer (without
the addition of Yerba Mate), were applied to cover the spectrum
of the Mate beer matrix, and caffeine, theobromine, and theo-
phylline were used as standard substances. Beer samples were
prepared by degassing, the addition of acetonitrile for protein pre-
cipitation, and a subsequent centrifugation step. Silica gel and
amino-modified silica gel plates with irregular shaped material
and fluorescence indicator were evaluated first. Separation was
directly visible under UV 254 nm illumination. According to the
method of Baranowska and Zydron [13], amino-modified silica
gel plates delivered good separation of caffeine, theobromine,
and theophylline with ethanol/diethylamine (10:1, v/v) as the
mobile phase. For Mate beer, however, this chromatographic sys-
tem turned out to be unsuitable, as matrix compounds co-migrated
either with theophylline or with theobromine depending on plate
(plate manufacturer-dependent). Thus, irregular-shaped normal
silica gel HPTLC plates were investigated next. Based on the method
of Morlock and Aranda [9] for the determination of caffeine in
energy drinks, mixtures of ethyl acetate/methanol/25% ammonium
hydroxide solution were evaluated. Separation of methylxanthines
was achieved on irregular-shaped silica gel material with the pub-
lished ratio of 90:15:1 (v/v/v) [9], and sharpness of the analytes’
zones was optimized by spherical-shaped silica gel (LiChrospher
plates). However, the entire separation of Mate matrix compo-
nents and methylxanthines was not possible. Badea [12] suggested
acetone/toluene/chloroform 40:30:30 (v/v/v) on silica gel material
for the separation of xanthine derivatives in tea, coffee and soft
drinks, which also turned out to be suitable for the separation
in Mate beer. Best separation of caffeine, theobromine and theo-
phylline from the Mate beer matrix and sharpest standard zones
were obtained on LiChrospher silica gel plates for a migration dis-
Please cite this article in press as: C. Oellig, et al., Determination of caffeine, theobromine and theophylline in Mate beer and Mate soft
drinks by high-performance thin-layer chromatography, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.12.019
3
Fig. 1. Images of HPTLC LiChrospher silica gel plates developed with ace-
tone/toluene/chloroform (4:3:3, v/v/v) up to a migration distance of 60 mm under
UV 254 nm illumination: (A) caffeine (900 ng/zone), theophylline and theobromine
(both 600 ng/zone); (B) extract of a spiked hop beer sample with 100 mg methylx-
anthines/L (corresponding to 250 ng methylxanthines/zone); (C) extract of a “blank”
beer sample; (D) extract of a Mate soft drink sample with 200 mg caffeine/L (cor-
responding to 600 ng caffeine/zone). All samples were prepared according to the
developed method, the application volume was 10 ␮L for both the “blank” beer
sample and the spiked hop beer sample, and 3 ␮L for the Mate soft drink sample.
tance of 60 mm including humidity control (33% relative humidity).
hRF values were 18, 25, and 35 for theobromine, theophylline and
caffeine, respectively, as shown for the standards and a spiked
hop beer sample (Fig. 1A and B), while the “blank” beer sample
showed no interfering matrix (Fig. 1C). To assess the suitability
of the developed HPTLC separation for Mate soft drinks, samples
from the German market were tested. Matrix components like pro-
teins that are present in Mate beer were not expected in Mate soft
drinks, thus the samples were centrifuged after degassing, without
the addition of acetonitrile, and directly analyzed by HPTLC. Matrix
interferences in Mate soft drinks generally were not detected,
when detectable sample matrix compounds mainly remained at the
application position, as exemplarily shown in Fig. 1D. Quantitation
of caffeine, theobromine and theophylline was performed in the
absorption mode. Recorded absorption spectra of the methylxan-
thines showed a maximum at UV 274 nm, why 274 nm was selected
as wavelength for densitometry.
3.2. Method validation
Limits of detection (LOD) and quantitation (LOQ) for caffeine,
theobromine and theophylline were determined according to
the DIN 32645 calibration method [14]. Calibrations were per-
formed in the range 2.5–25 ng/zone for caffeine, theobromine
and theophylline, calculated to 1–10 mg methylxanthines/L Mate
beer (n = 5), taking into account the sample dilution of 1:4 and
a sample application volume of 10 ␮L. Calibration graphs of
good linearity with high calibration correlation coefficients were
obtained (R2 > 0.9974). LOD and LOQ were determined to 1 and
4 ng/zone, respectively, for caffeine, theobromine and theophylline,
corresponding to 0.4 and 1.6 mg methylxanthines/L Mate beer,
respectively. With %RSDs <6% the determination was well repeat-
able. Thus, the HPTLC–UV screening allows the reliable quantitation
of low concentrations of methylxanthines in Mate beer and Mate
soft drinks. To obtain even lower LOQ and LOD values, higher sam-
ple volumes can be applied.
Recovery experiments for caffeine, theobromine and theo-
phylline were carried out with hop beer at a level of 100 mg/L
(n = 4). Quantitation of caffeine by HPTLC−UV exemplarily is dis-
played in Fig. 2. For the three methylxanthines, the recovery rates
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Fig. 2. (A) HPTLC chromatogram on LiChrospher silica gel plates with acetone/toluene/chloroform (4:3:3, v/v/v) as the mobile phase under UV 254 nm illumination of caffeine,
theophylline, and theobromine and extracts of spiked hop beer samples, spiking level 100 mg methylxanthines/L (n = 4); (B) resulting 3D densitogram of the absorbance scan
at UV 274 nm, and (C) corresponding calibration graph of caffeine (75–900 ng/zone).

Table 2
Recoveries of caffeine, theobromine and theophylline from hop beer (spiking level
100 mg/L).
Caffeine Theobromine Theophylline
Mean recovery [%] ± SDa (n = 4) 104 ± 4 95 ± 5 89 ± 4
a
Standard deviation.
Fig. 3. Separation of four Mate beer samples from the German market (sam-
ples 1–4, n = 3) and calibration standards (30–450 ng/zone caffeine, 8–60 ng/zone
theobromine and theophylline) on LiChrospher silica gel plates with ace-
tone/toluene/chloroform (4:3:3, v/v/v) under UV 254 nm illumination. Application
volumes were: 5 ␮L sample 1, 8 ␮L sample 2 and 3, and 20 ␮L sample 4.
were close to 100% (Table 2) and the results were well repeatable
with %RSD values less than 5%. Consequently, the analysis of the
hop beer before spiking (blank sample) revealed no caffeine, theo-
bromine and theophylline and matrix interferences by the hop beer
were not detected.
3.3. Caffeine concentrations in experimental Mate beers
The manufacturing of Mate beer was studied at the Yeast Genet-
ics and Fermentation Technology Department of the University of
Hohenheim. Seven different procedures of the Yerba Mate addition
were investigated, when the manner, time and amount of the addi-
tion were varied (Table 1), while the brewing process itself was the
same for all samples. Furthermore, the addition of the hops was var-
ied. For the evaluation of the different procedures, the quantity of
caffeine in the resulting beers was of great interest. The determined
caffeine concentrations in the seven Mate beers ranged between
78 and 125 mg/L (Table 1), while theobromine and theophylline
were not detected. To assess the influence of the quantity of Yerba
Mate on the caffeine concentration in the beer, the analyzed con-
centrations were compared with calculated concentrations. Taking
into account the Yerba Mate quantity used for the beer brewing, an
average caffeine content in Yerba Mate of 1.3% [1], and assuming a
total extraction during the brewing, 104, 208 and 130 mg caffeine/L
beer would be expected for the samples 1–5, 6, and 7, respectively.
For the samples 1 and 2, and 7, the calculated concentrations corre-
lated well with the analyzed concentrations (125 and 107 mg/L, and
110 mg/L). For the samples 3–6, however, the concentrations were
noticeably lower. The analyzed concentrations of 78 and 87 mg/L
in sample 5 and 6 (8 and 16 g Yerba Mate/L beer) did not indicate
a direct correlation between the quantity of used Yerba Mate and
the caffeine concentration in the beer. Likewise, the addition of dif-
ferent kinds (bitter or aroma hops) and amounts of hops did not
show an effect on the caffeine concentration in the beer. The man-
ner of the Yerba Mate addition did not significantly influence the
caffeine concentration (sample 3 and 4), but an interference-free
production was only possible when the Yerba Mate was added in a
packaged manner, while a loose addition led to pipe blockages. The
addition in a tea towel at the beginning of the wort boiling (sam-
ple 5 and 6) showed lower caffeine concentrations than the loose
addition (sample 1 and 2), due to an incomplete extraction through
the tea towel. When the Yerba Mate was added in a tea strainer

Please cite this article in press as: C. Oellig, et al., Determination of caffeine, theobromine and theophylline in Mate beer and Mate soft
drinks by high-performance thin-layer chromatography, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.12.019
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Table 3
Determined caffeine concentrations in four Mate beer and six Mate soft drink sam-
ples from the German market.
Sample Caffeine Labelled caffeine
[mg/L] ± SDa (n = 3) concentration [mg/L]
Mate beer samples
1 <LOD 80
2 175 ± 3 80
3 179 ± 1 80
4 30 ± 1 80
Mate soft drink samples
1 220 ± 2 200
2 221 ± 6 200
3 325 ± 2 250
4 285 ± 3 290
5 263 ± 3 240
6 241 ± 2 220
a
Standard deviation.
(sample 7) the efficiency of the extraction was optimized and the
caffeine concentration corresponded to the loose addition. In con-
trast to the amount and the manner of the Yerba Mate addition,
which had no influence on the caffeine concentration, the timing
of the addition did have a significant effect. Highest concentrations
were obtained, when the Yerba Mate was added after the starting
of the wort boiling (samples 1, 2 and 7). Earlier addition, after the
lautering and before the wort boiling, during the washes, delivered
lower caffeine concentrations in the beer.
3.4. Methylxanthines in commercially available Mate beers and
Mate soft drinks
To demonstrate the suitability of the developed HPTLC method,
four Mate beers and six Mate soft drinks from the German market
were analyzed and the results were compared to the labelled con-
centrations. The separation is exemplarily shown for the studied
Mate beers including the calibration in Fig. 3. The analyzed caf-
feine concentrations in the beers differed clearly from the labelled
concentration (Table 3). However, taking into account the free
accessible recipe for the production of the beer sample 3 with a
specified Yerba Mate quantity of 7 kg per 600 L beer, and an average
caffeine content in Yerba Mate of 1.3% [1], the caffeine concentra-
tion was calculated to 160 mg/L. This value clearly differed from
the labelled concentration but correlated very well with the ana-
lyzed concentration of 179 mg/L. The calculation of the caffeine
concentrations for the samples 1, 2 and 4, based on Yerba Mate
quantities, was not possible, because the Yerba Mate quantities
were not labelled. The analyzed caffeine concentration of 30 mg/L in
sample 4 and the lack of caffeine in sample 1 could not be clarified,
maybe an error occurred during the beer production. Theobromine
was detected in concentrations <LOQ in all analyzed commercially
available beer samples, which matched to the labelled concentra-
tion of 0.7 mg/L. For the Mate soft drinks, the analyzed caffeine
concentrations correlated very well with the labelled concentra-
tions (Table 3). This again showed the suitability of the developed
HPTLC-UV method. Theobromine was also not detected in the soft
drinks.
In conclusion, the caffeine concentrations in the Mate soft drinks
from the German market were consistently higher than the concen-
trations in the Mate beers, and both theobromine and theophylline
were not detected in remarkable amounts in the studied samples.
Please cite this article in press as: C. Oellig, et al., Determination of caffeine, theobromine and theophylline in Mate beer and Mate soft
drinks by high-performance thin-layer chromatography, J. Chromatogr. A (2017), https://doi.org/10.1016/j.chroma.2017.12.019
5
4. Conclusions
HPTLC–UV was shown as an efficient and reliable method to
simply determine the methylxanthines caffeine, theobromine, and
theophylline in Mate beer and Mate soft drinks. The simultane-
ous analysis of up to 21 samples including calibration standards
by HPTLC–UV was realized in less than 50 min. After a fast sam-
ple preparation, the methylxanthines were analyzed by HPTLC–UV
without matrix interferences, when quantitation was done by
absorption measurements at UV 274 nm. Well repeatable recov-
eries at relevant spiking levels were obtained and sufficient
sensitivity was guaranteed, allowing the determination of methylx-
anthines down to 1.6 mg/L in Mate beer.
Acknowledgements
The authors express many thanks to Merck (Darmstadt,
Germany) for support with plate material and to the Depart-
ment of Yeast Genetics and Fermentation Technology, University
of Hohenheim (Stuttgart, Germany), especially Dr. Thomas Senn,
for providing Mate beer samples.
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