Simultaneous Determination of Caffeine, Theobromine, and Theophylline by High-Performance Liquid Chromatography

Abstract

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 microg/mL and 3.21 microg/mL to 71.2 microg/mL for caffeine; < 0.1 pg/mL to 32 microg mL and < 0.1 pg/mL to 13.2 microg/mL for theobromine; < 0.1 pg/mL to 47 microg/mL and < 0.1 pg/mL to 66.3 microg/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.

Analytical methods applied for the characterization and the determination of bioactive compounds in coffee

“…Sample preparation before this analysis is usually very simple and contains only filtration and dilution steps [54,57,58]. Some methods include clarification with Carrez reagents [15,36,53] or with lead acetate [59].…”

“…The samples prepared according to one of these procedures were analyzed in a reverse phase elution mode with octadecylsilicapacked column. Caffeine was detected with the ultraviolet absorbance detector at 270-280 nm [11,15,27,36,53,54,[56][57][58]60]. Alternatively, HPLC with mass spectrometry Extraction with aqueous methanol and sonication for 10 min, followed by shaking at 300 rpm for 30 min 5-CQA and 3-CQA [53] was used for determination of caffeine [59] or theobromine and theophylline [36].…”

Ritual drinks in the pre-Hispanic US Southwest and Mexican Northwest

“…Groundwater tested in areas with native stands of I. vomitoria also has detectible traces of caffeine (14), which presumably may contaminate porous ceramics in those areas; because neither holly nor T. cacao was native or cultivated in the US Southwest and Mexican Northwest before contact, contamination from groundwater is not a source for the residues we identified. Direct contact with urine from individuals consuming caffeinated foods might also contaminate porous ceramics, because the methylxanthines are excreted in urine (15); however, this also is an unlikely source for the residues recovered from most archaeological ceramics. It is possible that southwestern populations used ceramics for many different varieties of drinks, possibly diluting or mixing the compounds found (13), so we cannot assume that the ratios determined represent a single episode of use.…”

Bioactive compounds content of chimarrão infusions related to the moisture of yerba maté (Ilex Paraguariensis) leaves

“…Infusions were analyzed with no other modification than the appropriate dilution to fit the standard curves, as described by several authors (Ewald et al, 1999;Astill et al, 2001;Bispo et al, 2002). A Shimadzu HPLC chromatograph, equipped with an LC-10ATvp quaternary pump, a Rheodyne manual injection valve with a 20 µL sample loop and a diode array detector SPD M10AVP, was used for the determinations.…”