Simultaneous determination of caffeine, theobromine and theophylline in food, drinks and herbal...

8/11/2019 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 PharmacyDjordjevic-Milic, Vukosava; Medical Faculty, Department ofPharmacyGrujic, Nevena; Medical Faculty, Department of PharmacyInjac, Rade; Faculty of Pharmacy, Department of PharmaceuticalBiology

Lepojevic, Zika; University of Novi Sad, Faculty of Technology

Keyword: Caffeine, theobromine, theophylline, natural products, food andbeverage

Journal of Chromatographic Science manuscript for review


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Title page:1

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Simultaneous determination of caffeine, theobromine and4

theophylline in food, drinks and herbal products by HPLC5

6

7

8

Branislava Srdjenovic*1, Vukosava Djordjevic-Milic

1,Nevena Grujic

1, Rade Injac

2,9

Zika Lepojevic3

10

111

Medical Faculty, Department of Pharmacy, University of Novi Sad, Hajduk12

Veljkova 3, 21000 Novi Sad, Serbia13

2Faculty of Pharmacy, Department of Pharmaceutical Biology, University of14

Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia15

3Faculty of Technology, University of Novi Sad, Cara Lazara 1, 21000 Novi Sad,16

Serbia17

18

19

* Corresponding author: Srdjenovic Branislava, Medical Faculty, Department of20

Pharmacy, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia21

E-mail address: [email protected]

Tel.: +381 63 7694399 Fax: + 381 21 422 76023

24

Page 1 of 26 Journal of Chromatographic Science manuscript for review

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mailto:[email protected]:[email protected]


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Abstract1

2

A rapid and selective high performance liquid chromatographic (HPLC) method has3

been developed for the separation and determination of caffeine, theobromine and4

theophylline. The chromatography was performed on a Zorbax C8 column (4.6 mm x5

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, and7

detection by UV at 273 nm. The method permits the simultaneous determination of8

caffeine, theobromine and theophylline in food, drinks and herbal products with9

detection limits of 0.07 0.2 mg/L and recoveries of 100.20 100.42 %. Correlation10

coefficients, for calibration curves in the linear range of 0.2 100 mg/L, were greater11

than 0.9999 for all compounds. The within- and between-day precision was12

determined for both retention times and peak area. Data suggested that the proposed13

HPLC method could be used for routine quality control of food, drinks and herbal14

products.15

16

Keywords:17

Caffeine, theobromine, theophylline, natural products, food and beverage18

19

20

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1. Introduction1

Xanthine derivates caffeine, 1,3,7-trimethylxantine, theobromine, 3,7-2

dimethylxantine, theophylline, 1,3 -dimethylxantine are widely found in the human3

diet. These compounds naturally occur in food products such as tea, coffee, and cocoa4

beans, with theobromine and caffeine being two most abundant xanthines in5

chocolate. In recent years, xanthine derivatives have received increased attention in6

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 three9

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 dietary11

intake and the resulting biological effects of these compounds on humans [1].12

Recently appeared methods, reporting the determination of caffeine, theophylline and13

theobromine in various sample mixtures, cover a broad spectrum of instrumental14

analysis. The most popular techniques for the determination of caffeine in different15

mixtures, especially in recent reports, comprise of high performance liquid16

chromatography (HPLC) and its variants [1-9]. Other methods include batch UV-VIS17

spectrophotometry [10-12], thin-layer chromatography (TLC) and its variants [1, 13-18

17], ion chromatography [18], FT-Raman spectrometry [19], FT-IR19


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The main objective of this study is to produce a quick and reproducible method for1

the routine, simultaneous analyses of caffeine, theobromine and theophylline in food,2

drinks and herbal products.3

2. Materials and methods4

2.1 Materials5

All solvents and reagents were of analytical grade unless indicated otherwise.6

Solutions were prepared with deionised water (Milli-Q-quality). Standards of7

caffeine, theobromine and theophylline were obtained from Sigma (Deisenhofen,8

Germany). Acetonitrile and methanol (HPLC grade) were obtained from Sigma9

(Deisenhofen, Germany). Chloroform and tetrahydrofurane (THF) were HPLC grade,10

obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA)11

2.2 Instrumentation and chromatographic conditions12

A HPLC-DAD model Agilent HP 1100 system equipped with an autosampler13

(Waldbronn, Germany), was used. Analytical column was Zorbax C8 column (4.6 mm14

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 rate17

of 0.8 mL/min, and column temperature of 25C. Analytes were detected at 273 nm.18

SupelcleanTM

LC-18 SPE cartridges 6 mL (0.5 g) used for solid phase extraction were19


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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 (Novi2

Knezevac, Serbia), respectively. Different energy drinks and beverages as Nestea3

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 HBC6

(Belgrade, Serbia), Coca cola HBC (Belgrade, Serbia), Red bull Gmbh (Vienna,7

Austria), Shark Gmbh (Vienna, Austria), Pepsi-Cola Co. (Ljubljana, Slovenia), Max8

Co (Novi Sad, Serbia), Nectar (Backa Palanka, Serbia) and Ljubljanske mlekarne9

(Ljubljana, Slovenia), respectively. Baking chocolate Ideal and Milka chocolate10

were obtained from Pionir (Subotica, Serbia) and Kraft Foods (Germany)11

respectively.12

2.4 Preparation of standard stock solution13

Standard stock solution of caffeine, theobromine and theophylline was prepared by14

weighing 30 mg, 10 mg and 10 mg of the standard substances respectively and15

dissolving in 10 mL water, pH 8 adjusted with 0.1 M NaOH. Solution was stable16

approximately 3 days under refrigeration (4C).17

2.5 Preparation of working standard solution18

A working solution was prepared by diluting 100 L of stock solution to 1.00 mL19


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caffeine, theobromine and theophylline using the method described is shown in Figure1

1. Linear standard curves for caffeine, theobromine and theophylline were obtained2

separately by plotting concentration versus area.3

2.7 Sample preparation and extraction4

Water extracts of Indian tea (5.00 g), green tea (5.00 g) and mate tea (5.00 g) were5

made by mixing for 30 min in hot water (200 mL, first boiled) in a thermal flask, on6

magnetic stirrer. The extracts were then filtered though filter paper to remove7

particulate matter. 10 mL of filtrate, adjusted to pH 8 with 0.1 M NaOH was8

subjected to the cleanup procedure as described below.9

Coffee powder samples were weighed (5 g) and extracted with boiling hot water (20010

mL) by mixing in thermal flask for 5 min, magnetic stirrer. The extracts were then11

filtered though filter paper to remove particulate matter. 10 ml of filtrate, adjusted to12

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 (2514

mL) and Milka chocolate (8.22 g) were filled up to 200 mL with water in plastic15

container and extracted for 30 min at 60C in ultrasonic bath. The extracts were then16

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 as18

described below.19


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SPE tubes, washed with 6 mL HPLC grade water, air dried under vacuum for 101

minutes and elutes were rejected. Caffeine, theobromine and theophylline were eluted2

from SPE tubes with 10 mL of chloroform into an evaporating flask. Solution was3

evaporated to dryness under nitrogen. The residue of all samples was reconstituted in4

1 mL of water pH 8 except for cocoa powder, chocolate and chocolate milk which5

were reconstituted in 2 mL. Prior analysis samples were filtered through a 0.22 m6

nylon filter and injected on the HPLC.7

2.9 Recovery study for the cleanup procedure8

A solution for this study was prepared by diluting 100 L of stock solution to 10.009

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, theobromine11

and theophylline after SPE extraction procedure was calculated.12

3. Results and discussion13

3.1 Study of chromatographic variables14

The development of the method was based on the experience obtained with the15

methods previously developed for the analysis of CF and some other compounds of16

interest [1-9]. Of the columns tested (Hypersil ODS C18100 x 4.6 mm, Cosmosil

C1817

150 x 4.6 mm, CosmosilC18250 x 4.6 mm, Phenomenex

Luna 5 m C8150 x 4.618

mm, PhenomenexLuna 5 m C8250 x 4.6 mm and Zorbax 5 m C8 column 150 x19


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three injections) are shown in Table 1. According to results from the Table 1 the1

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 analyte3

retention time (sampling rate), separation efficiency (number of theoretical plates) and4

consumption of solvents.5

Three factors were considered when the pH of the mobile phase was chosen. Firstly6

xanthines have to be in stable form, secondly the lifetime of the column stationary7

phase is reduced at the low pH and finally because of similar chemical properties of8

all three compounds, pH is very important for good separation. In view of these9

considerations a pH value of 8, was chosen.10

3.2 Validation of the HPLC method11

The characteristics and the procedures used for validation were those described in12

USP 24 [23] and in the International Conference of Harmonization (ICH) Guidelines13

(Q2A, Q2B) [24, 25]. Also some other literature data were used [26].14

We studied selectivity (different samples with different matrices) and linearity in15

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 ratio17

3:1) and limit of quantification (LOQ S/N ratio 10:1) of each compound are18

determined and shown in Table 2. There was no interference in HPLC results by the19


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P 9 f 26 J l f Ch hi S i i f i


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= 6 for each of presented concentration), proving a good accuracy of the method1

(Table 3).2

A repeatability test was performed to determine intra-day variation in peaks areas3

and migration times. RSD 0.84 % (n= 6) indicate that repeatability of the method is4

acceptable (Table 4).5

Intermediate precision was evaluated over three days (inter-day repeatability) using6

working solution. This solution (0.2-10.0 L) was injected daily under the same7

conditions and the results were used for the repeatability study. The solution was8

stored at room temperature (25 2C) in sunlight, decreasing recovery values9

approximately from 100.42 to 96.6 % for all compounds. When stored in refrigerator10

in the dark, the recovery ranged from 100.42 to 98.7 % over three days for all11

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 to14

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. No17

significant variations in specificity, accuracy and precision were found over the tested18

ranges, which indicated good robustness of the method (RSDs were lower then 2.2819


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P 10 f 26J l f Ch t hi S i i t f i


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which implies high efficacy and selectivity of used method. Use of C8 column1

packing results in better resolution, intensity, shape and symmetry of the obtained2

peaks comparing to C18 [1,2,7,8]. Recoveries for, caffeine, theobromine and3

theophylline were 96.8 0.92 %, 93.7 0.87 %, 92.00 0.67% respectively. High4

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 of6

impurities of matrix, no matter of type of sample. Run time for analysis is less than 87

min. The lowest concentration that can be quantified (LOQ) with acceptable accuracy8

and precision was 0.5, 0.4 and 0.2 mg/L for theobromine, theophylline and caffeine9

respectively. Furthermore, the limit of detection (LOD) defined as signal to noise ratio10

(S/N)>3 was 0.2 mg of theobromine /L, 0.1 mg theophylline /L and 0.07 mg of11

caffeine /L. These findings are in good correlation with literature [8].12

4. Conclusion13

The present method was tested to simultaneously measure the caffeine, theobromine14

and theophylline in food, beverages and natural products. In this work a fast, accurate15

and sensitive method was developed for determination of caffeine, theobromine and16

theophylline in food, beverages and natural products. Use of SPE pretreatment for17

samples and results for recoveries for this procedure confirmed that there is no matrix18

effect, so the extracts can be assessed with a calibration curve set from the analytes19


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1

Acknowledgment2

This work was supported by the Ministry of Science and Environment protection,3

Belgrade, Serbia, Grant No 145060.4

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References1

[1] J.B. Thomas, J.H. Yen, M.M. Schantz, B.J. Porter and K.E. Sharpless.2

Determination of caffeine, theobromine and theophylline in standard reference3

material 2384, baking chocolate, using reversed-phase liquid chromatography. J.4

Agric. Food Chem. 52: 3259-63 (2004).5

[2] P.D. Tzanavaras and D.G. Themelis. Development and validation of a high-6

throughput high-performance liquid chromatography assay for the determination of7

caffeine in food samples unsing a monolithic column. Anal. Chim. Acta 581: 94-88

(2007).9

[3] D. Satinsky, I. Neto, P. Solich, H. Sklenrova, M. Conceio, B.S. Montenegro10

and A.N. Arajo. Sequential injection chromatographic determination of paracetamol,11

caffeine, and acetylsalicylic acid in pharmaceutical tablets. J. Sep. Sci. 27: 529-3612

(2004).13

[4] M. Kartal. LC method for the analysis of paracetamol, caffeine and codeine14

phosphate in pharmaceutical preparations. J. Pharm. Biomed. Anal. 26: 857-6415

(2001).16

[5] F.L. Coco, F. Lanuzza,G. Micali and G. Cappellano. Determination of17

theobromine, theophylline, and caffeine in by-products of cupuacu and cacao seeds by18

high-performance liquid chromatography. J. Chromatogr. Sci. 45: 273-5 (2007).19


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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Cappellano%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Neto%20I%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Solich%20P%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Sklen%C3%A1kova%20H%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Concei%C3%A7%C3%A3o%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Montenegro%20BS%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Ara%C3%BAjo%20AN%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Lo%20Coco%20F%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Lanuzza%20F%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Micali%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Cappellano%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Neto%20I%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Solich%20P%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Sklen%C3%A1kova%20H%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Concei%C3%A7%C3%A3o%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Montenegro%20BS%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Ara%C3%BAjo%20AN%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Lo%20Coco%20F%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Lanuzza%20F%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Micali%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Cappellano%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus


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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 caffeine2

in cocoa samples by a high-performance liquid cromatografphic method with on-line3

sample cleanup in a switching-column system. Food Chem. 100: 459-67 (2007).4

[9] J.P. Naik. Imporoved high-performance liquid cromatographymethod to determine5

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 and8

F. Bani. Simultaneous spectrophotometric determination of caffeine and theobromine9

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 and12

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 spectrophotometric15

determination of caffeine in some beverages. Turk. J. Chem. 26: 295-302 (2002).16

[13] G.J. Van Berkel, M.J. Ford and M.A. Deibel. Thin-layer cromatography and17

mass spectrometry coupled using desorption electrospray ionization. Anal. Chem. 77:18

1207-15 (2005).19


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sampling probe electrospray ionization tandem mass spectrometry system. Anal.1

Chem. 77: 4385-9 (2005).2

[16] M. Aranda and G. Morlock. Simultaneous determination of riboflavin,3

pyridoxine, nicotinamide, caffeine and taurine in energy drinks by planar4

chromatography-multiple detection with confirmation by electrospray ionization mass5

spectrometry. J. Chromatogr. A.1131: 253-60 (2006.)6

[17] M. Aranda, G. Morlock. Simultaneouos determination of caffeine, ergotamine7

and metamizol in solid pharmaceutical formulation by HPTLC CUV-FLD with mass8

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

[19] S. Armenta, S. Garrigues and M.Guardia. Solid-phase FT-Raman determination13

of caffeine in energy drinks. Anal. Chim. Acta 547: 197-203 (2005).14

[20] N.M. Najafi, A.S. Hamid and R.K. Afshin. Determination of caffeine in black tea15

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 soft18

drinks using FTIRATR spectroscopy. Food Chem. 78: 261-266 (2002).19

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[24] International Conference on Harmonization, guideline Q2A, Text on validation1

of analytical procedures. Federal Register 1995, 60, 11260.2

[25] International Conference on Harmonization, guideline Q2B, Validation of3

analytical procedures: methodology. Federal Register 1997, 62, 27463.4

[26] Y.V. Heyden, A. Nijhuis, J. Smeyers-Verbeke, B.G.M. Vandeginste and D.L.5

Massart. Guidance for robustness/ruggedness tests in method validation. J. Pharm.6

Biomed. Anal. 24: 723-53 (2001).7

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g g p p

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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 the3

optimized conditions4

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(caffeine8

free), c) Red bull, d) Mate tea.9

10

Table 1.11

Study of chromatographic variables12

Table 2.13

Statistical parameters of the calibration curve for each compound (linear regression),14

with LODs and LOQs15

Table 3.16

Determination of accuracy in samples of known concentration17

Table 4.18

Determination of repeatability19

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Figure 1. Srdjenovic B. et al.

min

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mA

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4.

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theobromine3.245

theophylline 4.532

caffeine 7.577

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Figure 2a. Srdjenovic B. et al.

min0 1 2 3 4 5 6 7 8

mAU

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caffeine 7.605

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Figure 2b. Srdjenovic B. et al.

min0 1 2 3 4 5 6 7 8

mAU

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Figure 2c. Srdjenovic B. et al.

min0 1 2 3 4 5 6 7 8

mAU

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500caffeine

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Figure 2d. Srdjenovic B. et al.

min0 1 2 3 4 5 6 7 8

mAU

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theobromine3.198

caffeine6.952

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Table 1. Srdjenovic B. et al.

Chromatographic

conditions

tR(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 11399.10Effects 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

Confidential materials. Please destroy hard copies upon completion of review

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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 (R2) 0.9999 0.9999 0.9999

LOD (mg/L) 0.20 0.10 0.07

LOQ (mg/L) 0.50 0.40 0.20


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Drug Caffeine Theobromine Theophylline

Recovery SD (%) 100,20 0.96 100,27 0.91 100,42 0.82


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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


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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


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Simultaneous determination of caffeine, theobromine and theophylline in food, drinks and herbal...

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