Simultaneous Determination of Caffeine, Theobromine and Theophylline in Food, Drinks and Herbal Products by HPLC

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Journal of Chromatographic Science http://mc.manuscriptcentral.com/jcs

Simultaneous determination of caffeine, theobromine and theophylline in
food, drinks and herbal products by HPLC
Journal: Journal of Chromatographic Science
Manuscript ID: JCS-07-150
Manuscript Type: Special Issue
Date Submitted by the
Author:
22-Jun-2007
Complete List of Authors: Srdjenovic, Branislava; Medical Faculty, Department of Pharmacy
Djordjevic-Milic, Vukosava; Medical Faculty, Department of
Pharmacy
Grujic, Nevena; Medical Faculty, Department of Pharmacy
Injac, Rade; Faculty of Pharmacy, Department of Pharmaceutical
Biology
Lepojevic, Zika; University of Novi Sad, Faculty of Technology
Keyword:
Caffeine, theobromine, theophylline, natural products, food and
beverage
Confidential materials. Please destroy hard copies upon completion of review
Journal of Chromatographic Science manuscript for review
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Title page: 1
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Simultaneous determination of caffeine, theobromine and 4
theophylline in food, drinks and herbal products by HPLC 5
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Branislava Srdjenovic
*1
, Vukosava Djordjevic-Milic
1
,
Nevena Grujic
1
, Rade Injac
2
, 9
Zika Lepojevic
3
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11
1
Medical Faculty, Department of Pharmacy, University of Novi Sad, Hajduk 12
Veljkova 3, 21000 Novi Sad, Serbia 13
2
Faculty of Pharmacy, Department of Pharmaceutical Biology, University of 14
Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia 15
3
Faculty of Technology, University of Novi Sad, Cara Lazara 1, 21000 Novi Sad, 16
Serbia 17
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* Corresponding author: Srdjenovic Branislava, Medical Faculty, Department of 20
Pharmacy, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia 21
E-mail address: srdjbr@yahoo.com 22
Tel.: +381 63 7694399 Fax: + 381 21 422 760 23
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Abstract 1
2
A rapid and selective high performance liquid chromatographic (HPLC) method has 3
been developed for the separation and determination of caffeine, theobromine and 4
theophylline. The chromatography was performed on a Zorbax C8 column (4.6 mm x 5
150 mm, i.d., 5 m particle size) at 25C, with a mobile phase of water/THF (0.1 % 6
THF in water, pH 8) acetonitrile (90:10, v/v). The flow rate was 0.8 mL/min, and 7
detection by UV at 273 nm. The method permits the simultaneous determination of 8
caffeine, theobromine and theophylline in food, drinks and herbal products with 9
detection limits of 0.07 0.2 mg/L and recoveries of 100.20 100.42 %. Correlation 10
coefficients, for calibration curves in the linear range of 0.2 100 mg/L, were greater 11
than 0.9999 for all compounds. The within- and between-day precision was 12
determined for both retention times and peak area. Data suggested that the proposed 13
HPLC method could be used for routine quality control of food, drinks and herbal 14
products. 15
16
Keywords: 17
Caffeine, theobromine, theophylline, natural products, food and beverage 18
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1. Introduction 1
Xanthine derivates caffeine, 1,3,7-trimethylxantine, theobromine, 3,7- 2
dimethylxantine, theophylline, 1,3 -dimethylxantine are widely found in the human 3
diet. These compounds naturally occur in food products such as tea, coffee, and cocoa 4
beans, with theobromine and caffeine being two most abundant xanthines in 5
chocolate. In recent years, xanthine derivatives have received increased attention in 6
the food and nutrition industry because they can cause various physiological effects. 7
Caffeine is used as a central nervous system, cardiac and respiratory stimulant. 8
Theophylline and theobromine are widely used as smooth muscle relaxants. All three 9
of these compounds can cause diuresis. The lack of reference procedures and well- 10
characterized food-based products makes it difficult to accurately evaluate the dietary 11
intake and the resulting biological effects of these compounds on humans [1]. 12
Recently appeared methods, reporting the determination of caffeine, theophylline and 13
theobromine in various sample mixtures, cover a broad spectrum of instrumental 14
analysis. The most popular techniques for the determination of caffeine in different 15
mixtures, especially in recent reports, comprise of high performance liquid 16
chromatography (HPLC) and its variants [1-9]. Other methods include batch UV-VIS 17
spectrophotometry [10-12], thin-layer chromatography (TLC) and its variants [1, 13- 18
17], ion chromatography [18], FT-Raman spectrometry [19], FT-IR 19
spectrophotometry [20, 21] etc. 20
In most cases, however, these methods involve tedious and laborious pre-treatment 21
steps before the chromatographic determination, long analyses time or tedious or 22
limited applications (only caffeine or only theobromine). All of HPLC methods 23
mentioned earlier were using C18 columns [1-9, 22] to determinate xantine derivates. 24
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The main objective of this study is to produce a quick and reproducible method for 1
the routine, simultaneous analyses of caffeine, theobromine and theophylline in food, 2
drinks and herbal products. 3
2. Materials and methods 4
2.1 Materials 5
All solvents and reagents were of analytical grade unless indicated otherwise. 6
Solutions were prepared with deionised water (Milli-Q-quality). Standards of 7
caffeine, theobromine and theophylline were obtained from Sigma (Deisenhofen, 8
Germany). Acetonitrile and methanol (HPLC grade) were obtained from Sigma 9
(Deisenhofen, Germany). Chloroform and tetrahydrofurane (THF) were HPLC grade, 10
obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA) 11
2.2 Instrumentation and chromatographic conditions 12
A HPLC-DAD model Agilent HP 1100 system equipped with an autosampler 13
(Waldbronn, Germany), was used. Analytical column was Zorbax C8 column (4.6 mm 14
x 150 mm, i.d., 5 m particle size). The mobile phase used was water/THF (0.1 % 15
THF in water, pH 8) acetonitrile (90:10, v/v). pH was adjusted with 0.1 M NaOH. 16
The mobile phase was filtered (0.45 m nylon filter), run time 8 min, with a flow rate 17
of 0.8 mL/min, and column temperature of 25C. Analytes were detected at 273 nm. 18
Supelclean
TM
LC-18 SPE cartridges 6 mL (0.5 g) used for solid phase extraction were 19
obtained from Supelco, USA. SPE was performed in a 12-position Vacuum Manifold, 20
Supelco, USA. 21
2.3 Real samples 22
The natural products Indian tea, Green tea, Mate tea, Barcaffe classic
, Barcaffe 23
dekote
, Barcaffe light
, Nescafe classic
and Cocoa powder were obtained from 24

Macval tea D.O.O. (Novi Sad, Serbia), Macval tea D.O.O. (Novi Sad, Serbia), Macval 25
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tea D.O.O. (Novi Sad, Serbia), Droga (Portoroz, Slovenia), Droga (Portoroz, 1
Slovenia), Droga (Portoroz, Slovenia), Nestle (Belgrade, Serbia) and Aleva (Novi 2
Knezevac, Serbia), respectively. Different energy drinks and beverages as Nestea 3
lemon
, Cockta
, Coca Cola
, Coca Cola light
, Red bull
, Shark
, Pepsi
, Fast, 4
Energis, Booster and chocolate milk were obtained from Coca cola Co. (Ljubljana, 5
Slovenia), Palanacki kiseljak (Smederevska Palanka, Serbia), Coca cola HBC 6
(Belgrade, Serbia), Coca cola HBC (Belgrade, Serbia), Red bull Gmbh (Vienna, 7
Austria), Shark Gmbh (Vienna, Austria), Pepsi-Cola Co. (Ljubljana, Slovenia), Max 8
Co (Novi Sad, Serbia), Nectar (Backa Palanka, Serbia) and Ljubljanske mlekarne 9
(Ljubljana, Slovenia), respectively. Baking chocolate Ideal
and Milka chocolate 10

were obtained from Pionir (Subotica, Serbia) and Kraft Foods (Germany) 11
respectively. 12
2.4 Preparation of standard stock solution 13
Standard stock solution of caffeine, theobromine and theophylline was prepared by 14
weighing 30 mg, 10 mg and 10 mg of the standard substances respectively and 15
dissolving in 10 mL water, pH 8 adjusted with 0.1 M NaOH. Solution was stable 16
approximately 3 days under refrigeration (4C). 17
2.5 Preparation of working standard solution 18
A working solution was prepared by diluting 100 L of stock solution to 1.00 mL 19
with water, pH 8 adjusted with 0.1 M NaOH to give concentrations of caffeine, 20
theobromine and theophylline of 0.3 g/ L, 0.1 g/ L and 0.1 g/ L respectively. 21
2.6 Calibration 22
Working standard solutions (0.2-10.0 L) of caffeine, theobromine and theophylline 23
were injected into the HPLC, and peak area responses were obtained. Method of 24
external standard calibration was used. The separation of the standard mixture of 25
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caffeine, theobromine and theophylline using the method described is shown in Figure 1
1. Linear standard curves for caffeine, theobromine and theophylline were obtained 2
separately by plotting concentration versus area. 3
2.7 Sample preparation and extraction 4
Water extracts of Indian tea (5.00 g), green tea (5.00 g) and mate tea (5.00 g) were 5
made by mixing for 30 min in hot water (200 mL, first boiled) in a thermal flask, on 6
magnetic stirrer. The extracts were then filtered though filter paper to remove 7
particulate matter. 10 mL of filtrate, adjusted to pH 8 with 0.1 M NaOH was 8
subjected to the cleanup procedure as described below. 9
Coffee powder samples were weighed (5 g) and extracted with boiling hot water (200 10
mL) by mixing in thermal flask for 5 min, magnetic stirrer. The extracts were then 11
filtered though filter paper to remove particulate matter. 10 ml of filtrate, adjusted to 12
pH 8 with 0.1 M NaOH was subjected to the clean up procedure as described below. 13
Cocoa powder (5 g), baking chocolate (8.85 g of crude powder), chocolate milk (25 14
mL) and Milka chocolate (8.22 g) were filled up to 200 mL with water in plastic 15
container and extracted for 30 min at 60C in ultrasonic bath. The extracts were then 16
firstly filtered though filter paper to remove particulate matter. 10 ml of filtrate, 17
adjusted to pH 8 with 0.1 M NaOH was subjected to the cleanup procedure as 18
described below. 19
The samples of fizzy drinks were degassed for 15 min in an ultrasonic bath for 20
releasing of CO
2
. Prior to analysis samples were adjusted to pH 8 with 0.1 M NaOH, 21
filtered through a 0.22 m nylon filter and injected directly into the HPLC. 22
2.8 Cleanup procedure 23
Supelclean
TM
LC-18 SPE cartridges were conditioned with 2 x 6 mL methanol, 24
followed by 2 x 6 mL HPLC grade water. Sample extracts were than passed trough 25
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SPE tubes, washed with 6 mL HPLC grade water, air dried under vacuum for 10 1
minutes and elutes were rejected. Caffeine, theobromine and theophylline were eluted 2
from SPE tubes with 10 mL of chloroform into an evaporating flask. Solution was 3
evaporated to dryness under nitrogen. The residue of all samples was reconstituted in 4
1 mL of water pH 8 except for cocoa powder, chocolate and chocolate milk which 5
were reconstituted in 2 mL. Prior analysis samples were filtered through a 0.22 m 6
nylon filter and injected on the HPLC. 7
2.9 Recovery study for the cleanup procedure 8
A solution for this study was prepared by diluting 100 L of stock solution to 10.00 9
mL with water, pH 8 and subjected to the cleanup procedure as described above. 10
Recovery study was carried out in five replicates. Recovery for caffeine, theobromine 11
and theophylline after SPE extraction procedure was calculated. 12
3. Results and discussion 13
3.1 Study of chromatographic variables 14
The development of the method was based on the experience obtained with the 15
methods previously developed for the analysis of CF and some other compounds of 16
interest [1-9]. Of the columns tested (Hypersil ODS C
18
100 x 4.6 mm, Cosmosil
C
18
17
150 x 4.6 mm, Cosmosil
C
18
250 x 4.6 mm, Phenomenex
Luna 5 m C
8
150 x 4.6 18
mm, Phenomenex
Luna 5 m C
8
250 x 4.6 mm and Zorbax 5 m C8 column 150 x 19
4.6 mm) it was only by using Zorbax 5 m C8 column 150 x 4.6 mm that good 20
separation of TB and TF was achieved, from each other as well as from CF. 21
The effect of the flow rate and composition of the mobile phase on the retention time 22
(t
R
), the peak width () and number of theoretical plates (N) for TB, TF and CF was 23
studied using working standard solution. The injection volume of working standard 24
solution was 5 L and the column temperature at 25C. The results (mean values of 25
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three injections) are shown in Table 1. According to results from the Table 1 the 1
combination of 0.8 mL/min flow rate and water/THF (0.1 % THF in water, pH 8) 2
acetonitrile (90:10, v/v) as mobile phase is selected as a compromise between analyte 3
retention time (sampling rate), separation efficiency (number of theoretical plates) and 4
consumption of solvents. 5
Three factors were considered when the pH of the mobile phase was chosen. Firstly 6
xanthines have to be in stable form, secondly the lifetime of the column stationary 7
phase is reduced at the low pH and finally because of similar chemical properties of 8
all three compounds, pH is very important for good separation. In view of these 9
considerations a pH value of 8, was chosen. 10
3.2 Validation of the HPLC method 11
The characteristics and the procedures used for validation were those described in 12
USP 24 [23] and in the International Conference of Harmonization (ICH) Guidelines 13
(Q2A, Q2B) [24, 25]. Also some other literature data were used [26]. 14
We studied selectivity (different samples with different matrices) and linearity in 15
range of 0.5 to 300 mg/L (0.5, 1.0, 3.0, 5.0, 10.0, 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, 16
250.0 and 300.0 mg/L). Also the results for assay, limit of detection (LOD S/N ratio 17
3:1) and limit of quantification (LOQ S/N ratio 10:1) of each compound are 18
determined and shown in Table 2. There was no interference in HPLC results by the 19
matrices ingredients in any of the tested sample, which indicates that the methods are 20
selective (Figure 2). 21
Accuracy of the method was determined by analyzing solutions of known 22
concentrations (working standard solutions) and comparing the measured and known 23
values. The mean recoveries for all compounds were in range of 100.20 100.42 % (n 24
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= 6 for each of presented concentration), proving a good accuracy of the method 1
(Table 3). 2
A repeatability test was performed to determine intra-day variation in peaks areas 3
and migration times. RSD 0.84 % (n = 6) indicate that repeatability of the method is 4
acceptable (Table 4). 5
Intermediate precision was evaluated over three days (inter-day repeatability) using 6
working solution. This solution (0.2-10.0 L) was injected daily under the same 7
conditions and the results were used for the repeatability study. The solution was 8
stored at room temperature (25 2C) in sunlight, decreasing recovery values 9
approximately from 100.42 to 96.6 % for all compounds. When stored in refrigerator 10
in the dark, the recovery ranged from 100.42 to 98.7 % over three days for all 11
compounds. The RSD values (0.11 0.78 % for migration time and 0.80 2.06 % 12
peak area) indicate that the intermediate precision is acceptable. 13
The parameters of the optimum HPLC conditions were slightly modified in order to 14
evaluate the robustness [24]. The effect of different concentration of THF ( 0.05 %), 15
as well as the effect of pH of the mobile phase ( 0.06), columntemperature ( 1 C), 16
flow rate ( 0.05 mL/min), and detection wavelength ( 3 nm), were determined. No 17
significant variations in specificity, accuracy and precision were found over the tested 18
ranges, which indicated good robustness of the method (RSDs were lower then 2.28 19
% for migration time and peak area). 20
3.3 Results of sample measurement 21
Contents of caffeine, theobromine and theophylline which are obtained from the 22
measurement of numerous different samples of food, beverages and natural products 23
are shown in Table 5. These results show strong correlation of declared and 24
determined values of caffeine, theobromine and theophylline for all analyzed samples, 25
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which implies high efficacy and selectivity of used method. Use of C8 column 1
packing results in better resolution, intensity, shape and symmetry of the obtained 2
peaks comparing to C18 [1,2,7,8]. Recoveries for, caffeine, theobromine and 3
theophylline were 96.8 0.92 %, 93.7 0.87 %, 92.00 0.67% respectively. High 4
values for recovery implies efficient extraction method as well as clean up procedure. 5
This is confirmed by chromatograms which are clean and without presence of 6
impurities of matrix, no matter of type of sample. Run time for analysis is less than 8 7
min. The lowest concentration that can be quantified (LOQ) with acceptable accuracy 8
and precision was 0.5, 0.4 and 0.2 mg/L for theobromine, theophylline and caffeine 9
respectively. Furthermore, the limit of detection (LOD) defined as signal to noise ratio 10
(S/N)>3 was 0.2 mg of theobromine /L, 0.1 mg theophylline /L and 0.07 mg of 11
caffeine /L. These findings are in good correlation with literature [8]. 12
4. Conclusion 13
The present method was tested to simultaneously measure the caffeine, theobromine 14
and theophylline in food, beverages and natural products. In this work a fast, accurate 15
and sensitive method was developed for determination of caffeine, theobromine and 16
theophylline in food, beverages and natural products. Use of SPE pretreatment for 17
samples and results for recoveries for this procedure confirmed that there is no matrix 18
effect, so the extracts can be assessed with a calibration curve set from the analytes 19
aqueous standard. The use of Zorbax C
8
column allowed to simultaneously 20
determinate xantine derivates in short time [5]. During the development of the 21
method, approximately 400 samples injections were made, showing no signs of 22
deterioration. Finally, data for sensitivity, accuracy, reproductively and high analyses 23
frequency suggest that the proposed HPLC method could be used for routine quality 24
control of food, drinks and herbal products. 25
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1
Acknowledgment 2
This work was supported by the Ministry of Science and Environment protection, 3
Belgrade, Serbia, Grant No 145060. 4
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theobromine, theophylline, and caffeine in by-products of cupuacu and cacao seeds by 18
high-performance liquid chromatography. J. Chromatogr. Sci. 45: 273-5 (2007). 19
[6] D.D. Rio, A.J. Stewart, W. Mullen, J. Burns, M.E.J. Lean, F. Brighenti and A. 20
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n
analysis of phenolic compounds and purine alkaloids in green 21
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Application of solid-phase extraction to brewed coffe caffeine and organic acid 24
determination by UV/HPLC. J. Food Compost. Anal. 20: 440-8 (2007). 25
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[8] M.R. Brunetto, L. Gutierrez, Y. Delgado, M. Gallignani, A. Zambrano, A. Gomez, 1
G. Ramos and C. Romero. Determination of theobromine, theophylline and caffeine 2
in cocoa samples by a high-performance liquid cromatografphic method with on-line 3
sample cleanup in a switching-column system. Food Chem. 100: 459-67 (2007). 4
[9] J.P. Naik. Imporoved high-performance liquid cromatographymethod to determine 5
theobromine and caffeine in cocoa and cocoa products. J. Agric. Food Chem. 49: 6
6579-83 (2001). 7
[10] A.R. Khanchi, M.K. Mahani, M. Hajihosseini, M.G. Maragheh, M. Chaloosi and 8
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in Iranian tea by artificial neural networks and its comparison with PLS. Food Chem. 10
103: 1062-8 (2007). 11
[11] D.K. Sinhg and A.Sahu. Spectrophotometric determination of caffeine and 12
theophylline in pure alkaloids and its application in pharmaceutical formulations. 13
Anal. Biochem. 349: 176-80 (2006). 14
[12] G. Alpdogan, K. Karabina and S. Sungur. Derivative spectrophotometric 15
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[14] M. Arenda and G. Morlock. Simultaneous determination of caffeine, ergotamine 20
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sampling probe electrospray ionization tandem mass spectrometry system. Anal. 1
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[16] M. Aranda and G. Morlock. Simultaneous determination of riboflavin, 3
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chromatography-multiple detection with confirmation by electrospray ionization mass 5
spectrometry. J. Chromatogr. A.1131: 253-60 (2006.) 6
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and metamizol in solid pharmaceutical formulation by HPTLC CUV-FLD with mass 8
confirmation by online HPTLC CESI-MS. Journal of Chromatographic Science 45: 9
251-5 (2007). 10
[18] V.V. Khasanov, K.A. Dychko, T.T. Kuryaeva, G.L. Ryzhova and E.V. Maltseva. 11
A new procedure for caffeine determination. Russ. J. Appl. Chem. 78: 1427-9 (2005). 12
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of caffeine in energy drinks. Anal. Chim. Acta 547: 197-203 (2005). 14
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leaves by Fourier transform infrared spectrometry using multiple linear regression. 16
Microchem. J. 75: 151-8 (2003). 17
[21] M.M. Paradkar and J. Irudayaraj. Rapid determination of caffeine content in soft 18
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theophylline by High performance liquid chromatography. Journal of 22
Chromatographic Science 40: 45-8 (2002). 23
[23] United States Pharmacopoeia, 24
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Convention: Rockville, MD 2002. 25
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[24] International Conference on Harmonization, guideline Q2A, Text on validation 1
of analytical procedures. Federal Register 1995, 60, 11260. 2
[25] International Conference on Harmonization, guideline Q2B, Validation of 3
analytical procedures: methodology. Federal Register 1997, 62, 27463. 4
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Massart. Guidance for robustness/ruggedness tests in method validation. J. Pharm. 6
Biomed. Anal. 24: 723-53 (2001). 7
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Figure legends (Srdjenovic B. et al.) 1
Figure 1. 2
Chromatograms of working stock solution (0.2 - 10.0 L inject volume) under the 3
optimized conditions 4
Figure 2. 5
Chromatograms of some real samples under the optimized conditions, at 273 nm. 6
Mobile phase was water/THF (0.1% THF in water, pH 8) acetonitrile (90:10, v/v), 7
flow rate 0.8 mL/min, column temperature 25C: a) Coca cola
, b) Cockta
(caffeine 8
free), c) Red bull
, d) Mate tea. 9
10
Table 1. 11
Study of chromatographic variables 12
Table 2. 13
Statistical parameters of the calibration curve for each compound (linear regression), 14
with LODs and LOQs 15
Table 3. 16
Determination of accuracy in samples of known concentration 17
Table 4. 18
Determination of repeatability 19
Table 5. 20
Application results for natural products, beverages and food samples 21
22
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Figure 1. Srdjenovic B. et al.
min
3 4 5 6 7
8
mA
0
200
400
600
800
4.
7.
72
7.
68
theobromine
3.245
theophylline
4.532
caffeine
7.577
7.
66
7.
64
7.
62
4
7.
59
Page 17 of 26
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Figure 2a. Srdjenovic B. et al.
min
0 1 2 3 4 5 6 7 8
mAU
0
20
40
60
80
100
120
140
160
caffeine
7.605
Page 18 of 26
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Figure 2b. Srdjenovic B. et al.
min 0 1 2 3 4 5 6 7 8
mAU
0
25
50
75
100
125
150
175
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Figure 2c. Srdjenovic B. et al.
min 0 1 2 3 4 5 6 7 8
mAU
0
100
200
300
400
500
caffeine
7.598
Page 20 of 26
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Figure 2d. Srdjenovic B. et al.
min 0 1 2 3 4 5 6 7 8
mAU
0
50
100
150
200
250
300
350
400
theobromine
3.198
caffeine
6.952
Page 21 of 26
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Table 1. Srdjenovic B. et al.
Chromatographic
conditions
t
R
(min) (min) N
TB TF CF TB TF CF TB TF CF
Effect of flow rate (mL/min) of the mobile phase water/THF (0.1 % THF in water, pH 8) acetonitrile (90:10, v/v)
0.5 4.050 5.358 8.469 0.1264 0.0689 0.6974 5689.55 33502.46 816.97
0.8 3.249 4.552 7.626 0.0759 0.0151 0.1671 10151.39 503454.67 11538.53
1.2 2.458 3.458 6.854 0.0608 0.0103 0.1511 9054.54 624432.20 11399.10
Effects of mobile phase water/THF (0.1 % THF in water, pH 8) acetonitrile (0.8 mL/min)
85:15 3.025 4.235 7.349 0.0719 0.0139 0.1652 9806.25 514265.03 10963.42
90:10 3.249 4.552 7.626 0.0759 0.0151 0.1671 10151.39 503454.67 11538.53
5:95 3.455 4.901 8.021 0.0854 0.0181 0.1987 9067.55 406183.26 9027.57
Page 22 of 26
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Theobromine Theophylline Caffeine
Linear range (mg/L) 0.50 100.00 0.40 100.00 0.2 100.00
Slope 3658.8 3806.0 3499.5
Intercept 15.988 13.271 48.733
Correlation coefficients (R
2
) 0.9999 0.9999 0.9999
LOD (mg/L) 0.20 0.10 0.07
LOQ (mg/L) 0.50 0.40 0.20
Page 23 of 26
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Drug Caffeine Theobromine Theophylline
Recovery SD (%) 100,20 0.96 100,27 0.91 100,42 0.82
Page 24 of 26
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Drug Caffeine Theobromine Theophylline
RSD (%) migration time 0.19 0.11 0.20
RSD (%) peak area 0.80 0.84 0.82
Page 25 of 26
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Beverage and food samples Theobromine Caffeine Theophylline
Nestea lemon mg/L / 38.8 /
Cockta (caffeine free) mg/L / 0.0 /
Coca cola mg/L / 106.7 /
Coca cola light mg/L / 118.0 /
Red bull mg/L / 307.9 /
Guarana mg/L / 237.0 /
Booster mg/L / 313.2 /
Energis mg/L / 238.4 /
Pepsi mg/L / 119.1 /
Shark mg/L / 348.7 /
Choco milk mg/L 225.8 / 14.8
Indian tea mg/ 100 g of sample 39.5 1013.0 /
Green tea mg / 100 g of sample 32.0 1263.1 /
Mate tea mg / 100 g of sample 98.3 1116.7 /
Barcaffe light (0.1%) mg/100g of sample / 719.8 /
Barcaffe dekofe (1%) mg/100g of sample / 71.2 /
Barcaffe classic (2%) mg/100g of sample / 1328.5 /
Nescaffe mg/100g of sample / 3594.7 /
Cocoa mg/100g of sample 462.1 / 48.9
Baking chocolate mg/100 g of sample 1004.1 / 158.0
Milka chocolate mg/100 g of sample 100.4 5.6
Page 26 of 26
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Simultaneous Determination of Caffeine, Theobromine and Theophylline in Food, Drinks and Herbal Products by HPLC

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Simultaneous Determination of Caffeine, Theobromine and Theophylline in Food, Drinks and Herbal Products by HPLC

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F

and Cocoa powder were obtained from 24

, Coca Cola light

and Milka chocolate 10

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