Food Chemistry 141 (2013) 4087–4093

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Simultaneous determination of caffeine, theophylline and theobromine

in food samples by a kinetic spectrophotometric method
Zhenzhen Xia a,b, Yongnian Ni a,b,⇑, Serge Kokot c
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
b
Department of Chemistry, Nanchang University, Nanchang 330031, China
c
School of Chemistry, Physics and Mechanical Engineering, Faculty of Science and Engineering, Queensland University of Technology, Brisbane 4001, Australia

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

Article history: A novel kinetic spectrophotometric method was developed for the simultaneous determination of caf-
Received 5 July 2012 feine, theobromine and theophylline in food samples. This method was based on the different kinetic
Received in revised form 4 June 2013 characteristics between the reactions of analytes with cerium sulphate in sulphuric acid and the associ-
Accepted 26 June 2013
ated change in absorbance at 320 nm. Experimental conditions, the effects of sulphuric acid, cerium sul-
Available online 4 July 2013
phate and temperature, were optimised. Linear ranges (0.4–8.4 lg mL1) for all three analytes were
established, and the limits of detection were: 0.30 lg mL1 (caffeine), 0.33 lg mL1 (theobromine) and
Keywords:
0.16 lg mL1 (theophylline). The recorded data were processed by partial least squares and artificial neu-
Spectrophotometry
Methylxanthines in food
ral network, and the developed mathematical models were then used for prediction. The proposed, novel
Kinetic reaction method was applied to determine the analytes in commercial food samples, and there were no significant
Cerium sulphate differences between the results from the proposed method and those obtained by high-performance
liquid chromatography.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction in human diet, such as cocoa, teas, and chocolate products

(Buyuktuncel, 2010). It is therefore, important to assay caffeine,
Some stimulants are derived from methylxanthines, and they theobromine and theophylline, because their levels depend on
include caffeine, theobromine and theophylline (Spiller, 1998). Caf- the genotype and affect the flavour of food and drinks (Brunetto
feine as an alkaloid is found in varying quantities in the seeds, et al., 2007).
leaves, and fruit of some plants, where it acts as a natural pesticide Different analytical techniques and sample preparation proto-
that paralyses and kills certain insects feeding on the plants. It is cols have been proposed for the simultaneous determination of
most commonly consumed as infusions extracted from the beans the methylxanthines in foods and biological fluids. Several differ-
of the coffee plant and the leaves of a tea bush, as well as from var- ent pretreatments have been employed to minimise the deleteri-
ious foods and drinks containing products derived from the kola ous effects of complex biofluid matrices with liquid–liquid
nut. Theobromine is a bitter alkaloid of the cacao plant, and it is extraction (Begas, Kouvaras, Tsakalof, Papakosta, & Asprodini,
found in chocolate as well as in a number of other foods. Theoph- 2007), solid-phase extraction (Fenske, 2007) and microwaves-as-
ylline, also known as dimethylxanthine, is naturally found in tea sisted extraction (Gonzalez-Nunez & Canizares-Macias, 2011).
and cocoa beans. As a member of the methylxanthine family, it Once isolated from the matrix, numerous analytical techniques
bears structural and pharmacological similarity to caffeine and have been used for the analysis of these components, including
theobromine. The differences in the structures between these com- high-performance liquid chromatography (HPLC) (Ptolemy, Tzio-
pounds are that the NH group in theobromine and theophylline umis, Thomke, Rifai, & Kellogg, 2010; Aresta, Palmisano, & Zambo-
molecules is replaced in caffeine by an N–CH3 group, and also that nim, 2005), gas chromatography–mass spectrometry (GC–MS)
theobromine is the isomer of theophylline. These substances show (Shrivas & Wu, 2007), spectrophotometry (Khanchi et al., 2007;
various physiological effects on different parts of the body, includ- Khoshayand, Abdollahi, Shariatpahahi, Saadatfard, & Mohammadi,
ing the central nervous, cardiovascular, gastrointestinal, respira- 2008), capillary electrophoresis (CE) (Meinhart et al., 2010), spec-
tory, and renal systems, and these compounds may also cause trofluorimetry (Alves & Poppi, 2009; Moreira et al., 2005), and vol-
depression and hyperactivity. They are very common chemicals tammetry (Sanghavi & Sricastava, 2010; Yang et al., 2010).
Nevertheless, some of these methods, such as chromatography,
⇑ Corresponding author at: Department of Chemistry, Nanchang University, are time-consuming, expensive, and need complicated pre-concen-
Nanchang 330031, China. Tel./fax: +86 791 83969500. tration or multi-solvent extraction. Electrochemical methods,
E-mail addresses: [email protected] (Y. Ni), [email protected] (S. Kokot).

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.06.121
4088 Z. Xia et al. / Food Chemistry 141 (2013) 4087–4093

which use modified electrodes, are generally highly sensitivity pro- acid (0.5 mol L1) was prepared by diluting a suitable amount of
cedures but often have poor stability and repeatability. Santos and concentrated sulphuric acid.
Rangel (2012) developed an HPLC method to determine two meth-
ylxanthines. The coupling of a commercial monolithic column to a 2.2. Apparatus and software
traditional low pressure flow injection system, facilitated the anal-
ysis with the use of ultraviolet (UV) detection; a substantial An Agilent 8453 UV–visible photodiode-array spectrophotome-
improvement in analytical results was achieved. The limits of ter (Aglient Instruments, USA) equipped with a 1.0 cm quartz cuv-
detection (LODs) were 106 mol L1 for theobromine and theoph- ette was used for measuring the absorbance. All measurements
ylline, and 105 mol L1 for caffeine. In another application (Llo- were performed in a thermostat cell compartment at 70 ± 0.5 °C
rent-Martinez, Garcia-Reyes, Ortega-Barrales, & Molina-Diaz, with the aid of a model ZC-10 temperature control accessory
2005), a solid-phase ultraviolet sensing system was used to deter- (Ningbo Tianheng Instruments Factory, China). An Orion SA 720
mine methylxanthines in different kinds of food, beverages and digital pH-meter equipped with a combined glass/Ag–AgCl elec-
pharmaceutical samples; the concentration ranges were 1– trode was used for pH adjustment. Micropipettes (Finnpipette,
16 mg L1 for caffeine, and 1–12 mg L1 for theophylline and Labsystems, Finland) were used to deliver solutions less than
theobromine. 1.0 mL, and a stop watch was used to record the cell heating time.
In order to enhance the sensitivity of measurements and sim- HPLC measurements were performed on an Agilent 1100 series
plify the analytical procedures, an alternative method, kinetic HPLC–DAD system (Aglient Instruments, USA) equipped with a
spectrophotometry, was researched and developed for quantita- vacuum degasser, a quaternary pump, an autosampler, an injector
tive, simultaneous analysis of complex systems, such as the ternary with a 30 lL loop, an Agilent Zorbax eclipse XDB-C18 column
mixtures of carbidopa, levodopa and methyldopa (Chamsaz, Safavi, (250 mm  4.6 mm, 5 lm) with an Agilent Zorbax high pressure
& Fadaee, 2007), and the determination of ascorbic acid, uric acid reliance cartridge guard-column (C18, 12.5 mm  4.6 mm, 5 lm)
and dopamine (Moghadam, Dadlfarnia, Shabani, & Dhahbazikhah, and a DAD detector. The HPLC system was operated at 25 ± 0.5 °C.
2011), with the aid of chemometrics methods. The kinetics meth- All the programs used in the computing process were written in
od, based on the difference in reaction rates, especially when the MATLAB 6.5 for microsoft windows.
analytes react with a common reagent, is an efficient way to ana-
lyse simultaneously several analytes without any separation or 2.3. UV and HPLC procedure
masking processes; this approach has been significantly improved
with the use of chemometrics modelling (Chamsaz et al., 2007). Appropriate amounts of the solutions of caffeine, theobromine
In recent years, chemometrics has been applied for the analysis and theopylline or their mixture were transferred into a 1.0 mL
of multicomponent systems because, for example, of the develop- cuvette. Then, taking into account that the total volume required,
ment of fast data collection with the use of rapid scanning spectro- was 2.30 mL, 0.3 mL of sulphuric acid (0.5 mol L1) and appropri-
photometers (Moghadam et al., 2011). Chemometrics methods ate amounts of double distilled water were transferred into the
based on principal component regression (PCR), partial least cuvette. This was placed into the cuvette compartment, stirred
squares (PLS) and artificial neural networks (ANN), have found and thermostated at 70 °C for 1.5 min. Then, 0.40 mL cerium sul-
increasing applications for multicomponent kinetic determinations phate solution (2.5  103 mol L1) was added to give the final vol-
(Chu & Fan, 2009; Ni, Xia, & Kokot, 2009, 2011). ume of 2.5 mL, and absorbance measurement was started
In this work, a novel kinetic spectrophotometric method, based immediately. All samples were measured with respect to a distilled
on kinetic reactions, was researched and developed for the simul- water blank every 4 s between 0 and 600 s at 320 nm (total 150
taneous determination of caffeine, theobromine and theophylline points). Each reagent was added so as to keep the experimental re-
in food samples. To interpret the overlapping kinetics data, two sults consistent and reproducible within the precision of the mic-
chemometrics methods of data analysis – partial least squares ropipettes, i.e. ±0.1–1.5 lL; thus, the relative error introduced
(PLS) and radial basis function artificial neural networks (RBF- was <1%.
ANN), were applied for data resolution. Reaction kinetics and the An HPLC procedure was used as a reference method (Buyuktun-
experimental conditions were investigated in detail, and the devel- cel, 2010). All analyte samples and the standard solutions (caffeine,
oped calibrations were applied for the analysis of caffeine, theoph- theobromine and theophylline, Section 2.1) were injected into the
ylline and theobromine in commercial samples, such as tea, cocoa, monolithic column via the autosampler of the HPLC instrument
coffee and cola drinks; finally, to verify the proposed method, the (volume – 30 lL for all samples). A mixture of methanol and phos-
results obtained were compared with those obtained with the phoric acid (0.1%) was used as the mobile phase, while the flow
use of a reference method – high performance liquid chromatogra- rate was set at 1.0 mL min1 at ambient temperature. The gradient
phy method (HPLC). elution was applied as follows: 0–3 min – methanol was at 25%; 3–
5 min, – methanol was from 25% to 40%; 5–10 min – methanol was
at 40%. All analytes were detected at 271 nm, and the retention
2. Experimental times for caffeine, thophylline and thobromine were 7.8 min,
3.9 min, and 5.8 min, respectively. Peak area was used for signals
2.1. Reagents and solutions evaluation, while each sample was filtered through 0.45 lm mem-
brane nylon filters before injection.
All reagents used were of analytical reagent grade, and double
distilled water was used throughout the experiments. A solution 2.4. General procedure for the preparation of food samples
of Ce4+, 2.5  103 mol L1, was prepared by dissolving 0.101 g of
cerium sulphate tetrahydrate (Xilong Chemical Ind., Co., Ltd, Four kinds of commercial sample – cola drinks, caffeine powder,
Guangzhou, China) in 5.0 mL 1.0 mol L1 warm sulphuric acid, cocoa powder and tea samples, were purchased from the local
and then diluted to 100 mL with water. Stock solutions of analytes markets in Nanchang city, and were quantitatively analysed for
(caffeine, theobromine and theophylline, 100 lg mL1) were pre- caffeine, theobromine and theophylline.
pared by dissolving 10.0 mg of each analyte (Sigma–Aldrich, St. Five grams of a tea sample was weighed into a 250 mL beaker,
Louis, MO, USA) in a 3.0 mL sodium hydrate (4.0 mol L1), respec- and 150 mL double distilled water was added (Santos & Rangel,
tively and then diluted to 100 mL with water. Solution of sulphuric 2012). This beaker was heated on a water bath at 80 °C for 4 h.
 Z. Xia et al. / Food Chemistry 141 (2013) 4087–4093 4089

Fig. 1. Effects of (a) concentration of sulphuric acid (cerium sulphate = 3.0  104 mol L1, temperature = 70 °C and time = 600 s), (b) concentration of cerium (sulphuric
acid = 6.0  102 mol L1, temperature = 70 °C and time = 600 s), and (c) temperature (sulphuric acid = 6.0  102 mol L1, cerium sulphate = 4.0  104 mol L1 and
time = 600 s) on absorbance of the reaction between cerium (IV) and caffeine, theobromine and theophylline, (d) absorbance plot of the kinetics reactions with increasing
temperature (sulphuric acid = 6.0  102 mol L1 and cerium sulphate = 4.0  104 mol L1). All analytes = 4.0 lg mL1.

One millilitre of subacetate solution, prepared by dissolving 50.0 g 2.5.2. Radial basis function-artificial neural network
lead subacetate in 100 mL water and filtering after 24 h standing, RBF-ANN modelling has been often applied to analytical
was added into the tea sample and thoroughly mixed. The solution problems and offers many advantages when dealing with the
was then centrifuged at 8000 r min1 for 2 min. The supernatant
layer was transferred into a separating funnel and extracted with
50 mL chloroform, and then the organic phase was evaporated
with the use of a rotary evaporator. The residue was dissolved in
100 mL double distilled water.
Fifty millilitres of cola drink sample was poured into a beaker,
and the dissolved CO2 was expelled by heating (85 °C) for 15 min
(Jafari, Rezaei, & Javaheri, 2011). The solution was then transferred
into a separating funnel and extracted with 50 mL chloroform; the
organic phase was then treated as above.
For the preparation of coffee and cocoa samples (Wang, Jia, &
Xia, 2011), 5.0 g of their powders and 2.0 g MgO were each trans-
ferred into separate 250 mL flasks and 150 mL double distilled
water was added. Each sample was shaken and kept refluxing at
100 °C for 45 min. After cooling to room temperature, each sample
was filtered and the filtrate was transferred into a separating fun-
nel and extracted according to the same method as the tea samples
above.

2.5. Chemometrics methods

2.5.1. Partial least squares

PLS is a well known multivariate regression method (Martens &
Naes, 1989), and PLS calibration modelling is becoming a very
widely used regression technique for spectroscopic data analysis.
It relates the calibration spectral data matrix to the analyte con-
centration data so as to predict simultaneously several response
variables. Compared with the classical multivariate linear regres-
sion (MLR), PLS is more powerful for dealing with multi-collinear
data, such as the successive data created by spectroscopy. In this
study, the PLS model was constructed by relating the kinetic spec-
trophotometric data to the concentrations of the methylxanthines, Fig. 2. Plot of kinetic behaviour versus time (a) and the absorbance spectra (b) of
the aim being to predict simultaneously caffeine, theobromine and caffeine, theobromine and theophylline against water. sulphuric acid = 6.0  102 -
theophylline in food samples. mol L1, cerium sulphate = 4.0  104 mol L1, and all analytes were 4.0 lg mL1.
4090 Z. Xia et al. / Food Chemistry 141 (2013) 4087–4093

obtain maximum differences between the measured absorbance

curves of the analytes. In this work, the influence of three impor-
tant factors on the reaction rates of caffeine, theobromine and the-
ophylline with Ce4+ were investigated; they were the
concentrations of sulphuric acid and cerium sulphate as well as
temperature.
The effects of sulphuric acid and cerium sulphate were exam-
ined over the range of 2.0  102–1.2  101 mol L1 and
2.6  104–4.4  104 mol L1, respectively (Fig. 1a and b). Almost
no obvious changes of absorbance for caffeine, theobromine and
theophylline were observed with the increasing concentrations of
both cerium sulphate and sulphuric acid. The optimum concentra-
tions selected were 4.0  104 mol L1 cerium sulphate and
6.0  102 mol L1 sulphuric acid; reaction kinetics for the three
analytes were quite stable at these concentrations.
The effect of temperature between 30 and 80 °C was investi-
gated, and in general, largest absorbance differences for the reac-
tions of caffeine, theobromine and theophylline were observed
with increasing temperature (Fig. 1c and d). It was found that
the absorbance of theophylline was almost constant between 40
and 80 °C, while that of caffeine increased almost linearly between
30 and 80 °C. The plot of absorbance changes for theobromine
effectively overlays the caffeine one between 30 and 60 °C and
then levels off to 80 °C. The absorbance versus time plots at differ-
ent temperatures suggested that there was little difference in reac-
tion kinetics behaviour of caffeine and theobromine between 30
and 60 °C (Fig. 1d). Consequently, 70 °C was chosen as the opti-
mum temperature at which clearly discernable differences in the
kinetic rate curves were noted for the reactions involving caffeine,
theobromine and theophylline.

3.2. Reaction kinetics mechanism of the methylxanthines reaction with

Ce4+

The three methylxanthines can reduce Ce4+ in an acidic med-

ium, and this reaction has a maximum absorbance at 320 nm; this
absorbance decreased as the Ce4+ was reduced to Ce3+. However,
the reduction was fastest with the theophylline (Fig. 2a), and the
reaction curves of caffeine and theobromine were also different.
It is these differences that facilitate the simultaneous determina-
tion of the three analytes in their reaction mixtures. However,
the absorbance spectra of the three methylxanthines have very
similar shapes with a maximum absorption at 271 nm (Fig. 2b),
and generally, it is difficult to discriminate the three analytes
quantitatively.
Since Ce4+ was present in excess, the kinetic reactions of these
three compounds ran according to the pseudo-first order reaction
Fig. 3. Kinetic curves of caffeine, theobromine and theophylline with different model. The rate constants for the three analytes were estimated
concentrations at optimum conditions (experimental conditions – Fig. 2).
by fitting the kinetics data obtained from single component sam-
ples of known concentrations, to the equation, A = A0 + a1exp(kt),
non-linearity inherent in such applications. In fact, ANN methods by a suitable regression method (Draper & Smith, 1981).
were designed to work on non-linear, multi-modal, and poorly
understood, improperly or ill-posed problems (Kolmogorov conti- Table 1
nuity theorem) (Castellanos, Martinez Blanco, & Palencia, 2007). Predicted results for synthetic samples by PLS and RBF-ANN methods.
Also, these methods have simple structures, well-established theo-
Methods %RPESa %RPETa
retical basis and fast learning speed. These factors make RBF-ANN
attractive for calibration modelling involving kinetic mixtures. Caffeine Theobromine Theophylline
PLS (3)b 6.0 (106)c 10.3 (110) 2.0 (102) 7.0
RBF-ANN (6,300)d 5.9 (104) 10.1 (109) 2.1 (99) 6.8
3. Results and discussion hP
a m 2
Relative prediction error: RPES ¼ 100  i¼1 ðC ijðfoundÞ  C ijðaddedÞ Þ
.P Pn Pm
n 2 0:5
3.1. Effects of temperature and concentrations of sulphuric acid and i¼1 ðC ijðaddedÞ Þ  (for single component), and RPE T ¼ 100  i¼1 i¼1
.P P
cerium sulphate on reaction kinetics ðC ijðfoundÞ  C ijðaddedÞ Þ2 n
i¼1
n 2 0:5
i¼1 ðC ijðaddedÞ Þ  (for total component).
b
Number of factors used.
P
In general, to perform simultaneous analysis of kinetic c
Mean recovery (%). %Recovery ¼ 100  ni¼1 ½C ijðfoundÞ =C ijðaddedÞ =n.
d
reactions, the optimal reaction conditions should be such so as to Number of iterations and the spread coefficient, respectively.
 Z. Xia et al. / Food Chemistry 141 (2013) 4087–4093 4091

Table 2
Analysis of the three methylxanthines in commercial samples with the use of the RBF-ANN calibration and the HPLC reference method.

Sample (mg g1) Contents detected Added (mg g1) Found (mg g1) Recovery (%)
a
CF TB TP CF TB TP CF TB TP CF TB TP
Kinetic method by PLS
Geen tea 1.07 ± 0.02b NDc ND 0.17 0.17 0.50 1.35 ± 0.02 0.13 ± 0.00 0.49 ± 0.02 108 76 98
Red tea 1.23 ± 0.02 ND ND 0.17 0.25 0.33 1.68 ± 0.03 0.26 ± 0.01 0.36 ± 0.01 120 104 109
Black tea 1.30 ± 0.01 0.09 ± 0.01 ND 0.25 0.17 0.42 1.50 ± 0.01 0.20 ± 0.01 0.47 ± 0.01 97 98 111
Olong tea 1.30 ± 0.01 0.12 ± 0.02 ND 0.17 0.33 0.50 1.17 ± 0.02 0.42 ± 0.02 0.52 ± 0.01 80 93 104
Cola 1 0.22 ± 0.01 ND ND –d – –
Cola 2 0.28 ± 0.02 ND ND – – –
Cocoa powder ND 1.56 ± 0.03 ND 5.50 0.00 2.75 4.39 ± 0.04 1.61 ± 0.02 2.63 ± 0.03 80 103 96
Coffee powder 2.67 ± 0.03 ND ND 1.50 1.50 1.50 5.22 ± 0.03 1.37 ± 0.02 1.46 ± 0.02 125 91 87
HPLC method
Geen tea 1.00 ± 0.01 ND ND 0.17 0.17 0.50 1.21 ± 0.02 0.13 ± 0.00 0.50 ± 0.01 103 76 100
Red tea 1.21 ± 0.01 ND ND 0.17 0.25 0.33 1.54 ± 0.02 0.25 ± 0.02 0.38 ± 0.00 112 100 115
Black tea 1.38 ± 0.01 0.08 ± 0.00 ND 0.25 0.17 0.42 1.58 ± 0.01 0.23 ± 0.01 0.48 ± 0.02 97 92 114
Olong tea 1.25 ± 0.02 0.10 ± 0.00 ND 0.17 0.33 0.50 1.42 ± 0.01 0.42 ± 0.02 0.50 ± 0.01 100 98 100
Cola 1 0.17 ± 0.00 ND ND – – –
Cola 2 0.21 ± 0.01 ND ND – – –
Cocoa powder ND 1.50 ± 0.02 ND 5.50 0.00 2.75 5.00 ± 0.03 1.50 ± 0.03 2.50 ± 0.03 91 100 91
Coffee powder 2.50 ± 0.03 ND ND 1.50 1.50 1.50 3.75 ± 0.02 1.25 ± 0.02 1.50 ± 0.02 94 83 100
a
CF, TB and TP represent caffeine, theobromine and theophylline, respectively.
b
Average ± standard deviation (SD) of the three replicate measurements for each test sample.
c
Not detected.
d
Not performed.

The synergistic effect of the analytes is the main cause for erro- were 0.30, 0.33 and 0.16 lg mL1 (corresponding to 1.5  106,
neous results in analysis of kinetic reactions, and this phenomenon 8.7  107 and 1.8  106 mol L1) for the same three analytes,
has attracted some attention in attempts to minimise this problem. respectively. The LODs of the proposed methods were similar or
Some years ago, it was demonstrated that this effect could be re- better than those reported in a recent HPLC study (Santos & Rangel,
duced or eliminated with the use of chemometrics methods (Ni, 2012).
Liu, & Kokot, 2000).
3.5. Simultaneous prediction of the three methylxanthine analytes in
3.3. Oxidation reactions of the three methylxanthines synthetic mixtures – PLS and RBF-ANN models

Several equilibrium studies to characterise the different Ce4+ The orthogonal array design was selected for the building of the
species in aqueous H2SO4 media have been carried out (Das, calibration models because this approach minimises the number of
2001). The predominant equilibria are: calibration samples required for the model without any loss of statis-
tical rigor. For the simultaneous prediction of three analytes sixteen
Ce4þ þ HSO4 ¼ CeðSO4 Þ2þ þ Hþ ð1Þ
calibration samples were required to build a four-level model with
concentrations of 0.4, 2.8, 5.2 and 6.8 lg mL1 for caffeine, theobro-
CeðSO4 Þ2þ þ HSO4 ¼ CeðSO4 Þ2 þ Hþ ð2Þ mine and theophylline, respectively, and these data were organised
into a concentration matrix, C (16  3). These levels were selected to
CeðSO4 Þ2 þ HSO4 ¼ HCeðSO4 Þ3 ð3Þ allow for a wide distribution of concentrations, which also covered
Given the variety of cerium species in aqueous H2SO4, there is the range of levels found in real samples. Then, the kinetics data were
obtained according to the experimental procedures (Section 2.3),
little tendency for Ce4+ to hydrolyse, and thus, Ce(SO4)2+ is the ma-
and arranged in a kinetics data matrix, A (16  150), where 150 rep-
jor species in this medium. Consequently, the postulated redox
resents the sampling time points from 0 to 600 s at intervals of 4 s.
reaction between the methylxanthine analytes and cerium (IV) is:
An equation between these two data matrices, A and C, was estab-
MethylxanthinesðRedÞ þ CeðSO4 Þ2þ þ Hþ lished: A = C  K (Ni, Deng, & Kokot, 2010).
Two different types of chemometrics methods, PLS and RBF-
! Ce3þ þ MethylxanthinesðOxÞ þ H2 O ð4Þ
ANN – linear and nonlinear modelling, respectively, were used to
Literature suggests that the C@N bonds in the methylxanthine investigate the analytical data from the three methylxanthine ana-
structure were converted to C–N, and the C–H bond was then oxi- lytes. Relative standard error of prediction for single or combined
dised to C@O (Sanghavi & Sricastava, 2010). analyte results, RPES and RPET, were calculated, and the mean
recoveries (%) were used to investigate the reliability of the meth-
3.4. Calibrations for the analysis of the three methylxanthine od (Table 1). Of the two types of model constructed, PLS and RBF-
ANN, the latter performed somewhat better (Table 1), and thus, it
Calibration graphs for single methylxanthines were constructed was applied for the analysis of the real samples.
from the measured kinetics data at different concentrations of the
three analytes obtained at 320 nm according to Beer’s law (condi- 3.6. Analysis of methylxanthines in real samples
tions – Section 3.1). Kinetics data for each analyte are summarised
in Fig. 3; the individual linear calibration models were established The novel kinetic spectrophotometric method described in this
at a selected time (600 s; parameters – insert Fig. 3). The obtained work was applied for the determination of the three analytes from
correlation coefficients (0.9998, 0.9992 and 0.9996) suggested cola drinks, tea samples, cocoa powder and caffeine powder. For
good linearity over the concentration range of 0.4–8.2 lg mL1 this analysis, the real samples were treated as previously described
for caffeine, theobromine and theophylline, respectively. The LODs (Section 2.4), and then a suitable volume of each extract was
4092 Z. Xia et al. / Food Chemistry 141 (2013) 4087–4093

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The authors are grateful for financial support from the National method for determination of three sulphanilamide artificial sweeteners with
Natural Science Foundation of China (NSFC-201065007), and the the aid of chemometrics. Food Chemistry, 113, 1339–1345.
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State Key Laboratory of Food Science and Technology of Nanchang
of theobromine and caffeine in saliva, plasma and urine via liquid
University (SKLF-ZZA-201302 and SKLF-ZZB-201303).
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