Development of a method to determine caffeine in human serum by HPLC

Background: Caffeine-a methylxanthine- is used as semi luxury food and stimulant but also has a medical use in bronchopulmonary dysplasia in premature infants and is added to painkillers. Theophylline, a related methylxanthine and metabolite of caffeine, was used as bronchodilator for years and is known as convulsant poison. As a CYP1A2 inhibitor caffeine becomes important in psychiatry since some psychotropic drugs are metabolized by CYP1A2. So caffeine intake might increase serum concentrations of psychotropic drugs and cause side effects. Method: Besides caffeine, its metabolites theophylline, theobromine and paraxanthine are determined. Separation is performed on a 5 µm particle size RP 18 column using an eluent of acetonitrile and potassiumdihydrogenphosphate-buffer (10:90) and pH2. The UV detector is set to a wavelength of 280 nm. Results: I. Development of the method in buffer The four substances are detected in potassiumdihydrogenphosphate-buffer pH2. Retention times are below 20 minutes. There are four separate peaks. II. Method used in human serum Retention times are below 20 minutes. All substances are identified but theophylline and paraxanthine are not completely separated. Conclusion: To show that caffeine intake can increase serum levels of psychotropic drugs a method to determine caffeine in human serum is required. However, caffeine metabolites also show pharmacological effects and so the next step is to separate theophylline and paraxanthine completely.