Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
40, Number 1, January 2002, pp. 45-48
Simultaneous Determination of Caffeine, Theobromine, and Theophylline
by High-Performance Liquid Chromatography
Marcia S. Bispo[2], Márcia
Cristina C. Veloso[1], Heloísa Lúcia C. Pinheiro[1], Rodolfo F.S.
De Oliveira[2], José Oscar N. Reis[2], and Jailson B. De Andrade[2]
[1]Centro Federal de Educação Tecnologica da Bahia- CEFET and
[2]Instituto de Química, Universidade Federal da Bahia – UFBA, 40.170-290
– Salvador, Bahia, Brazil
This work relates the development of an analytical methodology
to simultaneously determine three methylxanthines (caffeine, theobromine, and
theophylline) in beverages and urine samples based on reversed-phase high-performance
liquid chromatography. Separation is made with a Bondesil C18 column using methanol–water–acetic
acid or ethanol–water–acetic acid (20:75:5, v/v/v) as the mobile phase
at 0.7 mL/min. Identification is made by absorbance detection at 273 nm. Under
optimized conditions, the detection limit of the HPLC method is 0.1 pg/mL for
all three methylxanthines. This method is applied to urine and to 25 different
beverage samples, which included coffee, tea, chocolate, and coconut water.
The concentration ranges determined in the beverages and urine are: < 0.1
pg/mL to 350 µg/mL and 3.21 µg/mL to 71.2 µg/mL for caffeine;
< 0.1pg/mL to 32 µg mL and < 0.1 pg/mL to 13.2 µg/mL for theobromine;
< 0.1 pg/mL to 47 µg/mL and < 0.1 pg/mL to 66.3 µg/mL for
theophylline. The method proposed in this study is rapid and suitable for the
simultaneous quantitation of methylxanthines in beverages and human urine samples
and requires no extraction step or derivatization.
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