Food Research 5 (2) : 325 - 330 (April 2021)

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Spectrophotometric determining of caffeine content in the selection of teas, soft
and energy drinks available on the Croatian market
1,*
Vuletić, N., 2Bardić, L. and 1Odžak, R.
1
Department of Chemistry, Faculty of Science, University of Split, R. Boškovića 33, 21 000 Split, Croatia
2
University Department of Forensic Sciences, University of Split, R. Boškovića 33, 21000 Split, Croatia
Article history:
Abstract
Received: 4 September 2020
Received in revised form: 19
October 2020
This study aimed to determine concentrations of caffeine in the samples of the selected
Accepted: 17 December 2020 brands of teas, soft carbonated and energy drinks that are commercially available on the
Available Online: 3 April Croatian market. Quantitative analysis of caffeine content in the chosen beverage samples
2021 was done with UV/Vis spectrophotometric method. Chloroform was used as the solvent
Keywords:
and concentrations of caffeine were measured at the wavelength of 274 nm for three
Caffeine, samples of each of the selected beverages. Among the analyzed tea samples the highest
Tea, caffeine concentration was found in Franck's Black Tea (1471.021 ppm) and the lowest in
Soft drinks, Naturavita's Green Tea with Ginger (588.138 ppm). Between the analyzed carbonated soft
Energy drinks,
Spectrophotometry drinks, the highest caffeine concentration was measured in Fresh Cola (136.036 ppm) and
the lowest in Sky Cola (48.198 ppm). The highest caffeine concentration in the analyzed
DOI: energy drink samples was measured in Hell energy drink (394.670 ppm) while the lowest
https://doi.org/10.26656/fr.2017.5(2).482
was in Coca Cola Energy drink (173.574 ppm). The results of this study gave preliminary
information about caffeine levels in often consumed teas, carbonated soft drinks and
energy drinks in Croatia. The caffeine content in the analyzed teas and drink samples in
this study was under the allowed one except for Red Bull and Hell.

1. Introduction predator insects (Caballero et al., 2015). Usually, it is

isolated from coffee beans, tea leaves and cola seeds
Caffeine is a bitter white crystalline alkaloid from (Andrews et al., 2007).
the methylxanthine group. The systematic name of
caffeine is 1,3,7-trimethylxanthine. The structure of Tea is a big source of caffeine. Some types of tea
caffeine is given in Figure 1. The molar mass of caffeine such as black or oolong tea contain higher concentrations
is 194,19 g/mol and its density is 1,2 g/mL. It has low of caffeine compared with other types of tea such as
solubility in cold water. Its solubility is somewhat better green tea (Wanyika et al., 2010). Tea originates from
in hot water, ethyl-acetate, pyrimidine, pyrrole and China where it was/is used for medical purposes. It is
acetone. It is very well dissolved in ether, petroleum produced from Camellia sinensis, a plant that is native to
ether, benzene and chloroform (Schlager et al., 2006). China and India and is cultivated in tropical and
subtropical areas (Atomssa and Gholap, 2011). Tea
contains flavonoids and has anti-inflammatory and
neuroprotective properties (Maidon et al., 2012).
Differences in tea types are consequences of different
geographical origin, cultivation conditions and ways of
processing. Green tea is the most popular tea in Asia. It
is made of unfermented leaves, tastes mild and contains a
Figure 1. Chemical structure of caffeine large amount of caffeine. Black tea has a rich bitter taste
and is prepared with fermented leaves. It contains the
Caffeine can be found in the leaves and seeds of highest amounts of caffeine and polyphenols such as
different plants. Most commonly it is derived from flavonoids. Flavonoids are important because of their
coffee, cocoa or cola seeds and tea leaves. About 60 effect against harmful agents (Sharangi, 2009).
species of plants native to Africa, East Asia and South
America contain caffeine which helps them against Caffeine is the most common component of soft

*Corresponding author. eISSN: 2550-2166 / © 2021 The Authors. Published by Rynnye Lyan Resources
Email: [email protected]
 326 Vuletić et al. / Food Research 5 (2) (2021) 325 - 330

drinks like colas. It is deliberately added to these drinks coordination (Stranton and Gray, 1995). Acute caffeine
because of their taste and to cause the addiction to a overdose, usually more than 250 mg (more than 2-3 cups
drink (Khalid et al., 2016). The caffeine content in soft of cooked coffee) can lead to the state of overstimulated
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drinks varies depending on drink type. It can be from 10 CNS and caffeine intoxication as well (Khalid et al.,
to 50 mg of caffeine per serving portion (Violeta et al., 2016).
2010). US Food and Drug Administration restricted the
maximum amount of caffeine in carbonated drinks to 6 There are many advantages of caffeine consumption.
mg/oz. The allowed amount of caffeine in soft drinks can It is used to reduce physical fatigue and for its medical
be in the range of 30 to 72 mg/335 mL (12 oz) or 8,45 to characteristics also (Amos-Tautua et al., 2014). It can be
20,28 mg/100 mL (Amos-Tautua et al., 2014). used in combination with certain pain relievers for
treating migraines (Khalid et al., 2016). In the big
Energy drinks such as Red Bull, Monster etc. are non research that included 217 883 people were shown that
-alcoholic drinks that contain caffeine and herbal extracts there was lower kidney stones formation if caffeine was
of guarana, ginseng or Ginkgo biloba, B vitamins, amino consumed (Ferraro et al., 2013). Caffeine can induce hair
acid derivatives like carnitine and sugar derivatives growth, protects from eyelid spasm, cataract and
(ribose, glucuronolactone) (Boyle and Castillo, 2006). It prevents skin cancer. It can reduce the risk of some types
has been noted that there is 80 to 350 mg of caffeine and of cancer like liver cancer or colon cancer. It reduces the
35 g of processed sugar in 8-oz per serving portion of risk of Type 2 diabetes and Parkinson's disease (Graham,
energy drink (Clauson et al., 2008). One can of 1978).
commercially available energy drink contains 80 to 280
mg of caffeine depending on the size of a can (Weinberg There are many disadvantages to caffeine. It can
and Bealer, 2004). cause anxiety if taken in the amount of 300 mg or more
(Smith, 2002). Caffeine increases the concentration of
Drinks which contain caffeine like coffee, tea, soft dopamine in the brain and that eases depression. It
and energy drinks are very popular. Caffeine is the most accelerates bone tissue loss in postmenopausal older
frequently used psychoactive matter worldwide. Adults women. It can cause auditory hallucinations. If in
take 3/4 of a daily amount of caffeine through coffee. overdose, caffeine causes high blood pressure,
Usually, a cup of coffee contains 100 mg of caffeine. restlessness, insomnia, accelerated pulse, nausea,
Even decaf coffee is not without caffeine. It can contain increased urination, cardiac palpitations, gastrointestinal
12 mg of caffeine (Frary et al., 2005). A cup of tea disorders (diarrhea) and dizziness (Graham, 1978).
contains 85 mg of caffeine. The effect of caffeine on the
human body can appear 15 minutes after ingestion and The main goal of this study was to determine the
can last for hours. Children get caffeine from soft and concentrations of caffeine in different samples of
energy drinks in the fast-growing beverage market commercially available types of teas, carbonated soft
(Kamijo et al., 1999). drinks and energy drinks in the supermarkets in Croatia.

Most people do not go through mood changes with

2. Materials and methods
less than 300 mg of caffeine taken. Sleep is more
sensitive and can be disturbed with 200 mg of caffeine 2.1 Chemicals
(Frary et al., 2005). Dehydration is a big drawback in The chemicals used in this study include caffeine
caffeine consummation because of its diuretic action standard (C8H10N4O2) from Sigma-Aldrich (Sigma-
(Lau et al., 1992). Caffeine creates strong addiction, Aldrich Chemic GmbH, Munich, Germany), chloroform
increases the amount of stress, accelerates ageing and the (CHCl3) and sodium carbonate (Na2CO3) obtained from
appearance of wrinkles (Mrvos et al., 1992). Caffeine is Merck (Merck, Darmstadt, Germany). All reagents used
metabolized in the liver through the P450 oxidase in this study were of analytical grade and all solutions
enzyme system and each of these metabolites has its were prepared by using distilled water.
effect on the body. Paraxantine (84%) increases the
2.2 Sample collection
amount of free fat acids in blood plasma. Theobromine
(12%) increases urine volume. Theophylline (4%) Different samples of black and green tea (Franck
releases smooth muscles of bronchi and is used to treat Green Tea, Franck Black Tea, Naturavita Green Tea,
asthma (Newton et al., 1981). Naturavita Green Tea with Ginger, Agristar Green Tea,
Agristar Black Tea and Orange, Dar Vitalis Eko Green
Ingestion of 100-200 mg of caffeine causes increased
Tea), soft drinks (Coca-Cola, Coca-Cola Zero, Pepsi
alertness and vigilance, faster and more clear thought
Cola, Cockta, Fresh Cola, Fresh Cola Light, Sky Cola)
flow, increased focus and better general body
and energy drinks (Red Bull, Monster Assault, Burn

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 Vuletić et al. / Food Research 5 (2) (2021) 325 - 330 327

Passion Punch, Hell Strong Apple, Coca-Cola Energy) content was heated and then boiled for 10 mins. A total
were purchased from the local markets in Croatia. of 2 g of sodium carbonate was added to each sample for
precipitating tannins. Samples were filtered and filtrates

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2.3 Instrument were concentrated to 5 mL by heating. From the given
The UV/Vis spectrophotometer (Secomam, UVi volume caffeine was extracted by adding 5 mL of
Light-XTD5, Ales, France) was used for the analysis of chloroform in the separatory funnel. Caffeine was
caffeine in different samples of teas, soft and energy extracted by stirring in the separatory funnel for a few
drinks. minutes. The lower caffeine-containing layer was
separated and analyzed for caffeine content with UV/Vis
2.4 Wavelength selection spectrophotometer. 0.1 mL of each tea extract was mixed
with 10 mL of chloroform and placed in a quartz cuvette.
The wavelength at which caffeine absorbs maximum
Absorbance was measured at 274 nm. Three samples of
was determined by scanning the range of 190-400 nm.
each brand of tea were analyzed for caffeine content.
The wavelength at which caffeine absorbs maximum was
Average values are given in Table 2.
found to be 274 nm which was selected for further
analyses. The same wavelength was reported by Rehman Table 2. Caffeine contents of tea and beverage samples
and Ashraf, 2017 (Rehman et al., 2017). Caffeine
Sample
Sample name Mark concentration
type
2.5 Calibration solutions preparation (ppm)
Franck Green Tea T1 1209.76
Caffeine stock solution (100 ppm) was prepared by Franck Black Tea T2 1471.021
dissolving 0.01 g of recrystallized caffeine in 100 mL of Naturavita Green Tea T3 904.204
chloroform in the volumetric flask. From the caffeine Naturavita Green Tea T4 588.138
stock solution following dilutions were prepared: 1 ppm, Teas
with Ginger
5 ppm, 10 ppm, 15 ppm, 20 ppm and 25 ppm. Their Agristar Green Tea T5 1423.724
absorbances were measured at the wavelength of 274 nm Agristar Black Tea T6 1413.964
in quartz cuvettes three times for each dilution. Average and Orange
values of measured absorbances are given in Table 1. Dar Vitalis Eko Green T7 625.676
The absorbance values were used to make the calibration Tea
line for caffeine content analysis as shown in Figure 2. Coca-Cola SD1 102.252
Coca-Cola Zero SD2 95.12
Table 1. Absorbance of calibration solutions of caffeine
Pepsi Cola SD3 86.486
No Concentration (ppm) Absorbance Carbonated
Fresh Cola SD4 136.036
1 1 0.143 soft drinks
2 5 0.437 Fresh Cola Light SD5 49.7
3 10 0.644 Sky Cola SD6 48.198
4 15 0.776 Cockta SD7 0
5 20 1.079 Red Bull ED1 350.751
6 25 1.319 Monster ED2 317.342
Energy
Hell ED3 394.67
drinks
Burn ED4 252.778
Coca-Cola Energy ED5 173.574

2.7 Extraction of caffeine from soft and energy drinks

and preparation of the sample solutions

Soft and energy drink samples were heated and then

boiled for 10 minutes to remove CO2. The samples were
left to cool to room temperature. A total of 10 mL of
each sample was taken and placed in the separatory
Figure 2. Standard calibration curve of caffeine funnel. 1 mL of 20% (w/v) sodium carbonate solution
and 5 mL of chloroform was added to each sample.
2.6 Extraction of caffeine from black and green tea Caffeine was extracted by stirring the funnel for a few
samples and preparation of the sample solutions minutes and then the lower layer was separated. An
aliquot (0.1 mL) of each extract was mixed with 5 mL of
Exactly 2 g of each tea sample was weighed. 20 mL chloroform and placed in a quartz cuvette. The
of distilled water was added to the sample and the absorbance was measured at 274 nm. Three samples of

eISSN: 2550-2166 © 2021 The Authors. Published by Rynnye Lyan Resources

 328 Vuletić et al. / Food Research 5 (2) (2021) 325 - 330

each brand of drink were analyzed for caffeine content. It is visible in the present study that the caffeine content
Average values are given in Table 2. in analyzed carbonated soft drinks is under the allowed
one. It is observed that the caffeine concentrations in the
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energy drinks are higher than the caffeine concentrations

3. Results and discussion
in the carbonated soft drinks (Gerald et al., 2014).
The standard linear calibration curve obtained from
the standard solutions of caffeine is presented in Figure According to the Ordinance on food additives of the
2. It showed a good linear relation between the Ministry of Health and Welfare in Croatia, the maximum
absorbance and concentrations of standard solutions. allowed amount of caffeine in soft carbonated drinks is
150 mg/L while the maximum allowed amount of
Caffeine content levels in tea, carbonated soft drink caffeine in energy drinks is 320 mg/L (Narodne Novine
and energy drink samples are presented and illustrated in 173, 2004).
Table 2 and Figure 3.
None of the analyzed tea samples except Franck's
The concentration of caffeine in tea samples was in had a caffeine content mark on the packaging.
the range of 588.138 to 1471.021 ppm with an average
value of 1090.927 ppm. The highest caffeine The results of this study show that the concentrations
concentration was measured in Franck's Black Tea and of caffeine in the analyzed soft carbonated drinks from
the lowest was measured in Natura Vita's Green Tea with the Croatian local markets were lower than the maximum
Ginger. allowed one. Since caffeine is an addictive substance and
when consumed can cause different health concerns, it
The concentration of caffeine in carbonated soft would be appropriate that the amount of caffeine was
drinks was in the range of 48.148 to 136.036 ppm with specified on the labels of all carbonated caffeine-
an average value of 86.299 ppm. The highest containing drinks. None of the carbonated drinks
concentration of caffeine was measured in Fresh Cola analyzed in this study was labelled so. Labels only
and the lowest in Sky Cola. There was not any caffeine contained information that drinks contain caffeine.
present in the samples of soft drink Cockta that were
analyzed in this study. All energy drinks were labeled with a maximum
allowed concentration of caffeine which was 320 mg/L,
Caffeine concentrations that were measured in except the energy drink Hell which was labeled with 384
energy drink samples were in the range of 173.574 to mg/L of caffeine. Concentrations of caffeine in energy
394.670 ppm with an average value of 297.823 ppm. The drinks measured in this study were lower than the one
highest caffeine concentration was measured in Hell specified on their labels for all of the analyzed drinks
energy drink while the lowest was measured in samples except Red Bull and Hell which shown slightly higher
of Coca-Cola Energy. concentrations of caffeine than those labels specified.
The caffeine content in tea depends on the According to the European Food Safety Authority
environmental conditions during the growth of the plant (EFSA) (2015), single doses of caffeine that do not raise
just as of the type of the species (Heckman et al., 2010). safety concerns recommended for adults are up to 200
The caffeine content in carbonated soft drinks mg. About 3 mg per kilogram of body weight (mg/kg
around the world varies depending on the type of brand. bw) are generally appropriate for a healthy adult
US Food and Drug Administration (FDA) has limited the population. Single doses of 100 mg of caffeine (about
maximum amount of caffeine in soft drinks to 200 ppm. 1.4 mg/kg bw) especially if consumed before bedtime in

Figure 3. Chart showing caffeine content in the tea and beverage samples
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 Vuletić et al. / Food Research 5 (2) (2021) 325 - 330 329

some adults may affect sleep. When healthy adults Roseland, J.M., Brandt, M., Dwyer, J.T., Picciano,
consume caffeine at a dose of 400 mg throughout the day M.F., Saldanha, L.G., Fisher, K.D., Yetley, E., Betz,
(about 5.7 mg/kg bw per day) they do not need to worry J.M. and Douglass, L. (2007). The caffeine content

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Boyle, M. and Castillo, V.D. (2006). Monster on the
loose. Fortune, 154, 116-122.
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concentrations in tea and drink samples is sensitive, Clauson, K.A., Shields, K.M., McQueen, C.E. and
precise and correct. Additional advantages of this Persad, N. (2008). Safety issues associated with
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European Food Safety Authority (EFSA) Panel on
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Dietetic Products, Nutrition and Allergies. (2015).
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The authors declare no conflict of interest. doi.org/10.1016/j.jada.2004.10.027
Gerald, I., Arthur, D.E. and Adedayo, A. (2014).
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