A sensitive method for confirming the clearance of caffeine and its three primary metabolites in sheep tissues destined for human consumption

Caffeine is a naturally occurring stimulant that is consumed in coffee, tea, and energy and soft drinks, but may also be used therapeutically in humans and animals. Caffeine initially degrades rapidly via demethylation to theobromine, paraxanthine and theophylline, prior to further degradation to other metabolites. A method was developed to separate and quantify the four chemicals, which is generally complicated by co-elution of paraxanthine and theophylline, which are the two bioactive metabolites. Processes to extract and purify the four analytes from liver, kidney, muscle, brain, perirenal and abdominal fats, plasma and milk were also developed and provided limits of quantitation as low as 1 ng/g in solid tissues and 1 ng/mL in milk and plasma using a combination of solvent and solid phase extractions. Sample homogenisation considerations and recovery at different stages was investigated to identify the areas of greatest analyte loss. A study was conducted involving the daily oral caffeine dosing of pregnant merino ewes at a rate of 25 mg/kg for 52 days until the birth of lambs. Tissue samples and milk were collected from ewes and lambs up to 28 days after birth and analysed using the new methodologies. Caffeine and the three primary metabolites appeared in all tissues analysed at high concentrations at day 0 (birthing), but declined by 99.9 % in all tissues in ewes by day 7, and all tissues except plasma (98.5 %) in lambs by day 7. All analytes were only detected in milk from ewes at day 0, but provided a source of caffeine and its metabolites to suckling newborn lambs, which may be responsible for the slower clearance from lamb plasma. Theophylline was the dominant metabolite in both ewes and lambs, followed by paraxanthine, while theobromine was only detected at much lower concentrations. The results derived from this animal study using this new method indicate that accumulation of caffeine and its three primary metabolites does not occur in the milk or tissues of ewes or lambs.