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SPE ( EluChrom™) Chocolate Extraction
for the Analysis of Methylxanthines

Caffeine and Theobromine belong to a group of substances known as methylxanthines. Caffeine occurs naturally in coffee, tea, cola and to some degree cocoa beans. It may also be added to cola drinks and is a component of certain over-the-counter and prescription medications. Theobromine is found in cocoa beans; tea contains trace amounts. Although theobromine is chemically related to caffeine, it lacks caffeine’s stimulant effect on the central nervous system. In fact, theobromine is virtually inert as a CNS stimulant. Theobromine does however pose a toxicity issue in dogs. Dogs are unable to excrete these chemicals as efficiently as humans, and thus as the compounds travel via the liver and bile duct, they are converted back into the original methylxanthines for another circuit through the dog’s system. Instead of excreting the substance, dogs keep re-poisoning themselves. Listed below is a very simple yet robust method for extraction of methylxanthines from chocolate.


Extraction Procedure
3cc EluChrom™washed with two column volumes of MeOH followed by two column volumes of HOH. 

Add 10mg of pulverized chocolate, add 1mL of HOH, cap cartridge and shake for 5 minutes.  Place cartridge on SPE manifold and drain off HOH extract. 

Add 1mL MeOH and drain to waste.  Add iml CH3CN and collect in 2mL sample vial. 

Chromatograph via HPLC
Chromatographic Conditions: HP1090 HPLC, 80:20 HOH:MeOH, 0.2mL/min. Detection: UV 272nm. HPLC Column Advantage ARMOR C18 10cm x 2.1mm I.D. (ADV7009). Sample size 10mg, 2.0µL Injection.

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