Simultaneous Determination of Theobromine, (+)-Catechin, Caffeine, and (-)-Epicatechin in Standard Reference Material Baking Chocolate 2384, Cocoa, Cocoa Beans, and Cocoa Butter

Abstract

A reverse-phase liquid chromatography analysis is used to access the quantity of theobromine, (+)-catechin, caffeine, and (-)-epicatechin in Standard Reference Material 2384 Baking Chocolate, cocoa, cocoa beans, and cocoa butter using water or a portion of the mobile phase as the extract. The procedure requires minimal sample preparation. Theobromine, (+)-catechin, caffeine, and (-)-epicatechin are detected by UV absorption at 273 nm after separation using a 0.3% acetic acid-methanol gradient (volume fractions) and quantified using external standards. The limit of detection for theobromine, (+)-catechin, caffeine, and (-)-epicatechin averages 0.08, 0.06, 0.06, and 0.06 microg/mL, respectively. The method when applied to Standard Reference Material 2384 Baking Chocolate; baking chocolate reference material yields results that compare to two different, separate procedures. Theobromine ranges from 26000 mg/kg in cocoa to 140 mg/kg in cocoa butter; (+)-catechin from 1800 mg/kg in cocoa to below detection limits of < 32 mg/kg in cocoa butter; caffeine from 2400 mg/kg in cocoa to 400 mg/kg in cocoa butter, and (-)-epicatechin from 3200 mg/kg in cocoa to BDL, < 27 mg/kg, in cocoa butter. The mean recoveries from cocoa are 102.4 +/- 0.6% for theobromine, 100.0 +/- 0.6 for (+)-catechin, 96.2 +/- 2.1 for caffeine, and 106.2 +/- 1.7 for (-)-epicatechin.

Health Benefits of Methylxanthines in Cacao and Chocolate

“…[15], a Srdjenovic et al . [16]; and/or Risner [17]; b Bruinsma and Taren [18]; The Hershey Company 2012; UK Joint Food Safety and Standards Group 1998.5. Safety of caffeine and theobromine.…”

“…IC 50 values were 45 and 98 µM for, respectively, theophylline and caffeine, and 2500 µM for theobromine. To our knowledge theobromine has not been tested on A 3 receptors although the partner compounds (caffeine and theophylline) display very poor affinity for them [17]. Actual affinities of caffeine and theophylline for the four human adenosine receptor subtypes expressed in heterologous cells are in the range: 4–39 µM with the affinity of theophylline higher by a factor of two to four [22].…”

Determination of Caffeine, Myosmine, and Nicotine in Chocolate by Headspace Solid‑Phase Microextraction Coupled with Gas Chromatography‑Tandem Mass Spectrometry

“…Caffeine contents are in the range of published data: Graham () determined caffeine in cocoa beans from 700 to 17000 mg/kg, Risner () detected in cocoa from 1500 to 2400 mg/kg, and Zoumas and others () detected in milk chocolate 50 to 540 mg/kg caffeine and in sweet chocolate 170 to 1250 mg/kg. Only Rudolph () reported approximately 10‐fold lower concentrations of caffeine in chocolate (milk chocolate 17 mg/kg and dark chocolate 114 mg/kg).…”

“…The analysis of pharmaceuticals in water, caffeine, and nicotine included (Benotti and Brownawell ; Huerta‐Fontela and others ), and the determination of caffeine from cocoa and chocolate extracts (Graham ; Zoumas and others ; Risner ; Rudolph and others ) are well described with different liquid chromatography approaches. The determination of volatile xanthine and pyridine alkaloids using different SPME approaches were mostly described in fluids (Hawthorne and others ; Yang and others ; Brčić Karačonji and others ).…”

Roadmap of cocoa quality and authenticity control in the industry: A review of conventional and alternative methods

“…Risner () determined both methylxanthines (theobromine and caffeine) and flavan‐3‐ols (catechin and epicatechin) by HPLC in different cocoa products, including standard reference material baking chocolate 2384, cocoa powder, cocoa beans, and cocoa butter. Miller et al.…”