Determination of caffeine, paraxanthine, theophylline and theobromine in premature infants by HILIC-MS/MS.

Aim: Caffeine is a central nervous system stimulant, used to treat apnea of prematurity. A hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) approach was developed to detect caffeine, paraxanthine, theophylline and theobromine in premature infants. Method: Protein precipitation of plasma samples (10 μl) was carried out by treating with acetonitrile containing caffeine- 13 C 3 . The separation was carried out on an ACQUITY HPLC ® BEH HILIC column. Caffeine and its metabolites were quantified by multiple reaction monitoring modes with positive electrospray ionization. Results: The established method had a good linear relationship in the range of 0.0600-60.0 μg/ml for caffeine, 0.0250-7.50 μg/ml for theobromine and 0.0150-4.50 μg/ml for paraxanthine and theophylline. Conclusion: A HILIC-MS/MS method was developed and validated to determine caffeine and its major metabolites in plasma of premature infants.