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Caffeine content of Energy Drinks

Article · July 2011

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 Caffeine content of Energy Drinks

Investigators

Dr Sapna Johnson
Mr Ramakant Sahu
Dr Nimisha Jadon

Advisors
Prof. H. B. Mathur
Prof. H. C. Agarwal

DATE
July 2011

CENTRE FOR SCIENCE AND ENVIRONMENT

41, TUGHLAKABAD INSTITUTIONAL AREA, NEW DELHI –110062
PH: 91-11-2995 6110/5124/6394/6399
FAX: 91-11-2995 5879 EMAIL: cse@cseindia.org
WEBSITE: www.cseindia.org

POLLUTION MONITORING LABORATORY

INDIA HABITAT CENTER, CORE-6A, FOURTH FLOOR
LODHI ROAD, NEW DELHI – 110003
PH: 91-11-24645334/5

 
Caffeine content of Energy Drinks

Contents

1. Pollution Monitoring Laboratory of CSE 3

2. Introduction 3

3. Caffeine 4

4. Regulations for Caffeine in Energy Drinks 4

5. Health Impacts 5

6. Objective of the study 5

7. Materials and Methods 5

8. Results and Discussion 7

9. Conclusions 7

10. References 8

11. Annexures 1-2 9

12. Figures 1- 3 12

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Caffeine content of Energy Drinks

1. Pollution Monitoring Laboratory of CSE

The Centre for Science and Environment (CSE), a non-governmental organization based in New
Delhi, has set up the Pollution Monitoring Laboratory (PML) to monitor environmental pollution.
PML is an ISO 9001:2008 certified laboratory accredited by SWISO, CH-5610, Wohlen,
Switzerland, conducting Pollution Monitoring and Scientific Studies on Environmental Samples.
The Lab has highly qualified and experienced staff that exercise Analytical Quality Control (AQC)
and meticulously follow what is called Good Laboratory Practices (GLP). It is equipped with most
sophisticated state-of-the-art equipments for monitoring and analysis of air, water and food
contamination, including Gas Chromatograph with Mass Detector (GC-MS), Gas Chromatograph
(GC) with ECD, NPD, FID and other detectors, High Performance Liquid Chromatograph (HPLC),
Atomic Absorption Spectrometer (AAS), UV-VIS Spectrophotometer, Mercury Analyzer,
Respirable Dust Sampler etc. Its main aim is to undertake scientific studies to generate public
awareness about food, water and air contamination. It provides scientific services at nominal cost
to communities that cannot obtain scientific evidence against polluters in their area. This is an
effort to use science to achieve ecological security.

2. Introduction

Caffeine, a representative of the group of three methylxanthine compounds, acts as a stimulant

on the central nervous system. Energy drinks are non-alcoholic beverages containing caffeine,
guarana, glucuronolactone, taurine, ginseng, inositol, carnitine, B-vitamins etc. as main
ingredients that act as stimulants. In recent years, a number of different energy drinks have been
introduced in the Indian market to provide an energy boost or as dietary supplements. These
drinks contain high levels of caffeine which stimulates the nervous system. Energy drinks are
heavily marketed to young adults and others and manufacturers compare the effects of the drinks
to the use of drugs like cocaine. Many of these drinks are heavily promoted in bars or for use in
combination with alcohol, which could further increase the health risk to consumers. There are a
number of scientific reports on the adverse consequences of excessive consumption of caffeine.
The main sources of caffeine are tea, coffee and soft drinks. In energy drinks, caffeine is added at
levels of up to 80 mg per serve. The drinks usually have a number of added water soluble
vitamins such as, niacin, pantothenic acid, vitamin B6 and vitamin B12 and other substances,
such as amino acids. There are, at present, no standards for caffeine in energy drinks. The
present study was undertaken to determine the caffeine content of energy drinks.

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Caffeine content of Energy Drinks

3. Caffeine

Caffeine (C8H10N4O2) is the common name for trimethylxanthine (systematic name is 1,3,7-
trimethylxanthine or 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione) and is an alkaloid that
occurs naturally in the leaves, seeds and fruits of tea coffee, cacao, kola trees an many other
plants(Reid, 2005). The chemical is also known as coffeine, theine, mateine, guaranine, or
methyltheobromine. The molecule was first isolated by the German chemist Friedrich Ferdinand
Runge in 1819. When purified, caffeine is an intensely bitter white powder. It is added to colas
and other soft drinks to impart a pleasing bitter note. However, caffeine is also an addictive
stimulant. When ingested, it is a stimulant of the central nervous system and can temporarily
increase blood pressure and heart rate(Hartley et al, 2004). A normal dose of caffeine is generally
considered to be 100 mg, which is roughly the amount found in a cup of coffee. Caffeine is
quickly and completely removed from the brain. Its effects are short-lived and it tends not to
negatively affect concentration or higher brain functions. However, continued exposure to caffeine
leads to developing a tolerance to it. Too much caffeine can result in caffeine intoxication, which
is characterized by nervousness, irritability, anxiety, tremulousness, muscle twitching
(hyperreflexia), insomnia, headaches, respiratory alkalosis, and heart palpitations(Leson, 1988)

4. Regulations for Caffeine in Energy drinks

The regulation of beverages to which caffeine is added has been challenging, partly because of
the widespread and long-term use of beverages such as coffee and tea in which caffeine is a
natural constituent. Nonetheless, several countries have enacted measures to regulate the
labeling, distribution, and sale of energy drinks that contain significant quantities of caffeine. US
FDA regulates caffeine content in soft drinks but does not regulate caffeine contained in energy
drinks. Caffeine is considered ‘generally regarded as safe’ under US Code of Federal
Regulations.The European Food Safety Authority mandates that energy drinks with over 150
parts per million (ppm) or 150 mg per litre caffeine content should be labeled as ones with “high
caffeine content” and the exact amount should be indicated; Australia has banned energy drinks
with over 320 ppm caffeine level and proposes to classify them as pharma products. Countries
like Denmark, Uruguay and Turkey have banned energy drinks altogether while Sweden has
banned its sale among children The health implications of caffeine have also been enquired into
by several countries.
In India, there are no standards for energy drinks under PFA Act, 1954. The standards of
carbonated water under PFA Rules, 1955 specify the maximum limits of caffeine of 200ppm
which subsequently on recommendations by Central Committee on Food Standards were

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Caffeine content of Energy Drinks

reduced to maximum level of 145ppm and notified vide notification GSR 431(E) dated
19.06.2009.

5. Health impacts

Caffeine is a stimulant, which has different health effects concerning stimulation of the central
nervous system and can produce restlessness, headaches, and irritability. Large amount of
caffeine consumption can cause physiological and psychiatrical dependence. Research has
shown links between heavy caffeine consumption and osteoporosis, high blood pressure, heart
disease, heart burn, ulcers, severe insomnia and infertility. Caffeine has diuretic properties when
administered in sufficient doses to subjects who do not have a tolerance for it.The diuretic effect
of coffee causes excretion of fluid through the kidneys, which may lead to dehydration (Maughan
& Griffin, 2003). Pregnant women who consume high amounts of caffeine have increased risk of
miscarriage, difficult birth and delivery of low-weight babies (Weng et al, 2008). The major
behavioral problems associated with heavy caffeine use are anxiety and depression. Over the
long-term as the body gets used to caffeine, it requires higher amounts to get the same
effects.Common features of caffeine intoxication include nervousness, anxiety, restlessness,
insomnia, gastrointestinal upset, tremors, tachycardia, psychomotor agitation (American
Psychiatric Association, 1994) and in rare cases, death (Kerrigan and Lindsey, 2005).

6. Objective of the Study

In view of the importance of Caffeine in energy drinks, the Caffeine content of Energy Drinks by
HPLC-DAD

7. Materials and Methods

Sampling
16 Energy drink samples - 2 samples each of 8 brands –XXX( Rejuve , Nicofix, Minus), Burn,
Redbull, Cloud 9 Monster, Tzinga were randomly purchased from a local market in June
2011(Annexure 1). All samples were analysed for caffeine and kept sealed in the refrigerator
until analysed.

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Caffeine content of Energy Drinks

Chemicals
Caffeine (1,3,7-trimethylmethylxanthine, anhydrous) – purity (>99%) was obtained from Fluka
Chemie GmbH (Buchs, Switzerland). Acetonitrile (HPLC grade) was purchased from Waligens
(Mumbai, India) and water (HPLC grade) was obtained from Merck Limited (Mumbai, India).

Equipment
High Performance Liquid Chromatograph of Agilent 1100 series equiped with Diode Array
Detector and HP Chemstation software, Zorbax ODS, 4.6mmID x 250mm (5µm) column and
50µL Hamilton syringe.

Standard preparation:
1000 µg/mL (ppm) stock solution of caffeine standard was prepared by dissolving solid caffeine in
HPLC grade water. Working solutions ranging from 1-500ppm were prepared from the stock
solution of caffeine and filtered through a 0.45µm syringe filter.

Sample preparation and analysis

The determination of caffeine was performed by an Agilent 1100 series high performance liquid
chromatograph equipped with DAD detector and HP chemstation software following AOAC
official method 979.08 for the simultaneous determination of benzoate, caffeine and saccharin in
soda beverage.The method after validation was applied to quantify samples (16) of 8 different
popular energy drinks XXX( Rejuve , Nicofix, Minus), Burn, Redbull, Cloud 9 ,Monster and
Tzinga. Mobile phase used was acetonitrile/water mixure, 20/80 (v/v) with a flow rate of 1ml/min.
Retention time of Caffeine was 3.6 minutes (Figure 1)
Approximately 100mL of sample of carbonated beverage were degassed in ultrasonic bath and
filtered through a 0.45µm syringe filter. There was no further clean up procedure.20µL of filtered
sample were injected directly into HPLC. Every sample was injected in triplicate (n=3). The
retention times of the peaks for the sample were compared with the retention time of the
reference standard and confirmed when the retention time of the sample was identical to those of
the pure standards.

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Caffeine content of Energy Drinks

8. Result and Discussion

The standard curve was linear between 1- 500 µg/ml (ppm) and the detection limit was 0.05
µg/ml. The mean correlation coefficient was 0.999±0.0004 (n=5). Results for caffeine analyzed in
triplicate for two different samples of 8 brands of energy drinks have been summarized in ppm
(Annexure 2). Caffeine was detected in the range from 112.23- 314.45 ppm.Chromatograms from
two typical products (one with a lower caffeine level and one with a higher caffeine level) are
shown in Figure 2 and 3. The mean amount of caffeine in energy drinks was XXX (Rejuve) -
117.14 ppm, XXX (Nicofix) -121.71 ppm, Redbull - 310.08 ppm, Burn -291.73ppm, XXX (Minus)-
153.30ppm, Cloud 9 - 142.25 ppm, Monster - Ripper - 314.09 ppm and Tzinga was 258.37ppm.
Caffeine was also detected in samples which did not mention on the label that it had caffeine.
38% of the samples breached the limit mentioned on the label by up to 24%. 38% of the samples
were within the limit mentioned on the label. 25% of the samples i. e 4 out of 12 samples did not
mention the caffeine content on the label. 44% of the samples breached the limit set by Central
Committee on Food Standards limit of 145 ppm.

9. Conclusions

The results showed that the caffeine levels in most brands exceeded 145 ppm. Only two
brands—XXX Rejuve and XXX Nicofix— stuck to the 145 ppm limit XXX Minus and Cloud 9
flouted labeling rules. The former did not mention the caffeine levels and the latter did not
mention that it had caffeine. The tests showed they had 153.30 ppm and 142.25 ppm caffeine,
respectively. The warning that lactating mothers should not consume energy drinks is missing
from Cloud 9, Burn and XXX Minus labels. The label on XXX Minus was not legible. Though
caffeine levels in Tzinga and Red Bull vary, both claim to have caffeine content equivalent to that
in an average cup of coffee—80 mg (320 ppm) in a 250 ml can.

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Caffeine content of Energy Drinks

10. References

American Psychiatric Association(1994). Diagnostic and Statistical Manual of Mental Disorders

(4th ed.). American Psychiatric Association. ISBN 0-89042-062-9.

Hartley TR, Lovallo WR, Whitsett TL (2004) Cardiovascular effects of caffeine in men and women
Am J Cardiol 93: 1022-1026

Kerrigan S and Lindsey T (2005) Fatal caffeine overdose: Two case report. Forensic Science
International 153 (1): 67–69

Leson CL, McGuiga MA,Bryson SM (1988) Caffeine overdose in an adolescent male Journal of
toxicology Clinical toxicology 26 (5–6): 407–15

Maughan RJ and Griffin J (2003) Caffeine ingestion and fluid balance: a Review Journal of
Human Nutrition and Dietetics 16 (6): 411–20.

Reid TR (2005) Nat Geogra 1/19/2005:15-32

Verkhratsky A (2005) Physiology and Pathophysiology of the Calcium Store in the Endoplasmic
Reticulum of Neurons Physiological Reviews 85: 381–3.

Weng X, Odouli R and Li DK (2008) Maternal caffeine consumption during pregnancy and the
risk of miscarriage: A prospective cohort study American Journal of Obstetrics and Gynecology
198 (3) e1-8.

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 Caffeine content of Energy Drinks

Annexure 1 Details of the energy drink samples purchased from Delhi and analysed for Caffeine
S.No. Na me Manufacturer Date of Expiry date Batch Cost Other Information
manufacture Number

1 XXX-2(Rejuve) Union Beverages factory, P. O.Box 05.12.2009 Best before 24 months from 75 Made in Dubai, Marketed by GT& T India,
804, Jebel Ali Industrial Area 2, manufacture when stored in cool and JMJ House Orchard Avenue Hirandani
Dubai, UAE dry place away from direct sunlight. Gardens, Powai, Mumbai-400076
2 XXX-1(Rejuve) Union Beverages factory, P. O.Box 27.04.2010 Best before 24 months from 3 75 Made in Dubai, Marketed by GT& T India,
804, Jebel Ali Industrial Area 2, manufacture when stored in cool and JMJ House Orchard Avenue Hirandani
Dubai, UAE dry place away from direct sunlight. Gardens, Powai, Mumbai-400077
3 XXX-1(Nicofix) Union Beverages factory, P. O.Box 12.12.2009 Best before 24 months from Not given 75 Made in Dubai , Marketed by Goa
804, Jebel Ali Industrial Area 2, manufacture when stored in cool and technology and trade Pvt Limited India,
Dubai, UAE dry place away from direct sunlight. JMU House Orchard Avenue Hirandani
Gardens, Powai, Mumbai-400076
4 XXX-2(Nicofix) Union Beverages factory, P. O.Box 08.09.2010 Best before 24 months from 108101 75 Made in Dubai , Marketed by Goa
804, Jebel Ali Industrial Area 2, manufacture when stored in cool and technology and trade Pvt Limited India,
Dubai, UAE dry place away from direct sunlight. JMU House Orchard Avenue Hirandani
Gardens, Powai, Mumbai-400077
5 Red Bull 1 Rauche Fruchtsafte GmbH &Co 6.10.2010 Best before 24 months from 1168989C3 75/- Product of Austria , Imported for India by
OG, Kuhbruckweg 2, 6714 19:02 manufacture when stored in cool and Red Bull India Pvt Ltd, Kakad Chambers
Nuziders by Red Bull Asia FZE dry place away from direct sunlight. Annex, 132 Annie Besant Rd, Worli ,
Dubai Airport free Zone, UAE Mumbai, 40018
6 Red Bull 2 Rauche Fruchtsafte GmbH &Co 6.10.2010 Best before 24 months from 1168989C3 75/- Product of Austria , Imported for India by
OG, Kuhbruckweg 2, 6714 10:02 manufacture when stored in cool and Red Bull India Pvt Ltd, Kakad Chambers
Nuziders by Red Bull Asia FZE dry place away from direct sunlight. Annex, 132 Annie Besant Rd, Worli ,
Dubai Airport free Zone, UAE Mumbai, 40019
7 Burn Kian Joo Canpack DDN BHD, 107, 26.10.2010 Best before9 months from manufacture BN 01 75/- Made in Malaysia Imported by Hindustan
Jalan Permata 1, Kawasan Industri 14:37 when stored in cool and dry place away Coca Cola Beverages Pvt Ltd, 13, Abul
Arab Malaysian , 71800 Nilai , from direct sunlight. Fazal Road Bengali Market, New Delhi
Negeri Sembilan , Malaysia 110001
8 Burn Kian Joo Canpack DDN BHD, 107, 28.02.2011 Best before 9months from manufacture BN 01 75/- Made in Malaysia Imported by Hindustan
Jalan Permata 1, Kawasan Industri 10:02 when stored in cool and dry place away Coca Cola Beverages Pvt Ltd, 13, Abul
Arab Malaysian , 71800 Nilai , from direct sunlight. Fazal Road Bengali Market, New Delhi
Negeri Sembilan , Malaysia 110001

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 Caffeine content of Energy Drinks

S.No. Name Manufacturer Date of Expiry date Batch Cost Other Information
manufacture Number

9 XXX-1(Minus) Pushpam Food and beverages Pvt 27.11.2010 Best before 24 months from MO910 100/- Made in India , Market ed by XXX Energy
Ltd Gat No 104/2A, Barote Vasti 5:18 manufacture when stored in cool and drinks Pvt Ltd JMJ House , Orchard
(Nandur) Tal Daund, District Pune dry place away from direct sunlight. Avenue, Hirandani Gardens, Powai,
412202 Mumbai-400076by
10 XXX-2(Minus) Pushpam Food and beverages Pvt 27.11.2010 Best before 24 months from MO810 100/- Made in India , Market ed by XXX Energy
Ltd Gat No 104/2A, Barote Vasti 5:53 manufacture when stored in cool and drinks Pvt Ltd JMJ House , Orchard
(Nandur) Tal Daund, District Pune dry place away from direct sunlight. Avenue, Hirandani Gardens, Powai,
412202 Mumbai-400076by
11 Cloud 9(Wild Pushpam Food and beverages Pvt 21.02.2010 Best before 18 months from W0710 75/- Made in indiaGoldwin Health Care Pvt
Berry) Ltd Gat No 104/2A, Barote Vasti manufacture when stored in cool and Ltd.ISO 9001 &140001:2004 Certified
(Nandur) Tal Daund, District Pune dry place away from direct sunlight. company 7, Zaver arcade , Zaver road
412202 Mulund (W) Mumbai-400080
12 Cloud 9(Premium) Pushpam Food and beverages Pvt 23.02.2010 Best before 18 months from A1610 75/- Made in indiaGoldwin Health Care Pvt
Ltd Gat No 104/2A, Barote Vasti manufacture when stored in cool and Ltd.ISO 9001 &140001:2004 Certified
(Nandur) Tal Daund, District Pune dry place away from direct sunlight. company 7, Zaver arcade , Zaver road
412202 Mulund (W) Mumbai-400081
13 Monster(Ripper) Monster Energy Limited South 10:00 Best before end 04.2012 when stored in D10060 Manufactured in EU
Bank House Barrow street, Dublin cool and dry place away from direct
4, Ireland sunlight.
14 Monster(Ripper) Monster Energy Limited South 12:47 Best before end 04.2012 when stored in D10060 Manufactured in EU
Bank House Barrow street, Dublin cool and dry place away from direct
4, Ireland sunlight.
15 Tzinga Hector Beverages Pvt Ltd 132, 13.04.2011 Best before 3 months from manufacture B2823 20/-
sector 3 Manesar , Gurgaon when stored in cool and dry place away
122050 Haryana from direct sunlight.
16 Tzinga Hector Beverages Pvt Ltd 132, 30.04.2011 Best before 3 months from manufacture BN-2816 20/-
sector 3 Manesar , Gurgaon when stored in cool and dry place away
122050 Haryana from direct sunlight.

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Caffeine content of Energy Drinks

Annexure 2 Caffeine Content of Energy drink samples

Caffeine Concentration No of times above PFA

Brand Name detected * Concentration on Percent Deviation from standard of 145ppm
S. No. (ppm) the label (ppm) the claim on the label
1 XXX-1(Rejuve) 112.23 100 12.23 within limit
2 XXX-2(Rejuve) 122.05 100 22.05 within limit
3 XXX-1(Nicofix) 119.48 100 19.48 within limit
4 XXX-2(Nicofix) 123.94 100 23.94 within limit
5 Red Bull 1 308.80 320 within limit 2.1
6 Red Bull 2 311.35 320 within limit 2.1
7 Burn-1 294.34 300 within limit 2.0
8 Burn-2 289.12 300 within limit within limit
9 XXX-1(Minus) 153.69 not given -- within limit
10 XXX-2(Minus) 152.90 not given -- within limit
11 Cloud 9(Wild Berry) 148.05 not given -- within limit
12 Cloud 9(Premium) 136.44 not given -- within limit
13 Monster-1(Ripper) 314.45 300 14.45 2.2
14 Monster-2(Ripper) 313.72 300 13.72 2.2
15 Tzinga-1 259.68 300 within limit 1.8
16 Tzinga-2 257.05 300 within limit 1.8

*Average of triplicate run

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Caffeine content of Energy Drinks

Figure 1: HPLC Chromatogram of Caffeine Standard(100ppm)

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Caffeine content of Energy Drinks

Figure 2: HPLC Chromatogram of Sample 03 with low level of Caffeine, retention time 3.62 min)

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 Caffeine content of Energy Drinks

Figure 3: HPLC Chromatogram of Sample 05 with higher level of Caffeine, retention time 3.62
min)

14

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