Quantitative determination of caffeine in tea, chocolate products and in carbonated beverages by HPLC and fragmentation pattern of Caffeine by MS

1. Quantitative determination of caffeine in tea, chocolate
products and carbonated beverages by HPLC and
fragmentation pattern of Caffeine by MS
Hajira Mahmood
Roll No
629

2. INTRODUCTION
• Caffeine, an alkaloid structurally 1,3,7- trimethylxanthine having molecular
formula C8H10N4O2
• Molecular weight: 194.19 g/mol
• most widely consumed psychoactive substance in the world from coffee,
tea, chocolate products and carbonated beverages.
• acts as a central nervous system (CNS) stimulant in humans .
• Recognized that an excessive consumption of caffeine by people from
dietary sources may reach levels that induce pharmacological effects.

3. Necessary Conditions
Analysis was carried out using an HPLC apparatus equipped with a
Waters 6000A pump, a Waters Model 440 fixed wavelength detector
Detector UV-vis detector at 254nm.
Column reverse phase C18
particle size 5mm
sample injector system (Rheodyne) with a 5ml sample loop.
Mobile phase
Flow rate of 1.0ml min-1
• methanol±water
• (30:70, v/v)
For tea
• acetonitrile±water
• (10:90, v/v)
for chocolate products
• methanol±water
• (25:75, v/v).
for soft drinks

4. Materials And Method
Sample preparation
Soft drinks
• Samples are decarbonated in an ultrasonic bath for 10min
and injected into a liquid chromatograph.

5. The extraction procedure for mate and black tea
(bags and leave)
• 1.80g Bag mate and black tea extracted with 150ml of
boiling water
• mate leaf tea extracted using 8.52 g spoon to 1L of water.
• On reaching room temperature, an aliquot of 20ml mixed
with 0.1M hydrochloric acid and four times more saturated
basic acetate solution (8ml) used.
• centrifuged at 1057.5g for 5min.
• Added NaHCO3, centrifuged again at 1057.5g for 5min.
• Solution diluted with water & injected into a liquid
chromatograph.

6. Chocolate Products
• A clean-up step was added before injection into the chromatograph.
Samples of chocolate bars (0.60g) and chocolate powders (1.0g)
weighed into test tubes equipped with Teflon-lined screw caps.
• Fats are extracted by shaking twice with 30ml portions of petroleum
ether, centrifuging at 470.0g for 10min.
• Evaporation of residual solvent occurs by placing the test tubes in a
warm water bath.
• 30ml water and saturated basic acetate solution (5ml) added, centrifuge
again at 1057.5g for 5min.
• NaHCO3 added, repeated the centrifugation.
• Added 0.1M HCl , diluted, mixed and injected into a liquid
chromatograph.

7. Quantitation
• External standard plot method.
• used to make a calibration line for quantification with different
concentrations
• also determine the recovery rate to know how much of your analyte is
lost during sample preparation.
• Preparation of stock standard solution.
• Triplicate injections of 5ml caffeine standard solution used to
construct linear regression lines (peak area ratios versus caffeine
concentrations).
• The concentration corresponding to a peak height of three times the
baseline noise level is 0.10mgm-1 (detection limit ).

9. Recovery study
• To verify the accuracy and precision of the analytical procedure,
recovery studies were carried out by spiking selected samples of
mate tea and chocolate bars with caffeine.
• Samples are spiked, just before the addition of the saturated basic
acetate solution, by adding 1ml of a caffeine standard water solution
at different concentrations (0.05±0.25mgml-1).
• Recoveries are calculated from the differences in total amount of
caffeine between the spiked and unspiked samples.

10. HPLC Chromatogram
Dark sweet chocolate
Black Tea
Diet Cola

11. Results and discussion
• Clean-up set up to avoid loss of caffeine.
• A better resolution obtained using acetonitrile±water (10:90, v/v). Fig
1(A).
• Among the mobile phases tested, methanol± water (30:70, v/v) was the
one that gave the best resolution for the three types of tea in a short
retention time Fig 1(B).
• black tea, irrespective of the brand, showed the highest amounts of
caffeine (32.21±36.23mg per cup) (cup=150ml), while the lowest
concentrations of this alkaloid (1.05± 6.75mg per cup) found in mate
leaf tea.
• the caffeine content in diet cola A (45.89mg per can) (can=350ml) is
approximately 30% higher than in regular cola A (33.08mg per can).

12. Results and discussion
Caffeine content range per cup per can
mate tea 1.05 to 15.83mg per cup
black tea 32.21 to 36.23mg per cup
chocolate products 32.21 to 36.23mg
soft drinks 2.73 to 7.49mg per can
cola soft drinks 19.81 to 45.89mg per can

13. Fragmentation pattern of Caffeine

14. EI Mass spectra for Caffeine
Molecular ion peak
M+1
Base peak
Isotopic peaks
Fragment ion peaks
137 165

15. •