Development and validation of a LC-MS² method for the simultaneous quantification of caffeine and adenosine from DBS

A liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous quantification of caffeine and adenosine in dried blood spot (DBS) samples. Caffeine is used in the treatment of apnea of prematurity in newborn children and adenosine levels play a major role in apnea. 40μL of whole blood spiked with the analytes was spotted onto specimen collection cards to prepare calibration DBS samples. 11.2 mm disks were punched from the card and was extracted in methanol and acetonitrile containing the internal standard. The extract was separated and quantified using a Phenomenex C18, 150x4.6mm, 3µm column as the stationary phase and 90 parts of 0.1% acetonitrile and 10 parts of 0.1% formic acid in water as the mobile phase delivered isocratically at 0.2mL/min. The injection volume and auto sampler temperature were 10µL and 35⁰C, respectively. The electrospray positive ion mode was used as the ionization source. The MS was operated using the single reaction monitoring at m/z 195→137.91 for caffeine and m/z 268→136.17 for adenosine. The method was seen to be linear from 0.050-10µg/ml and demonstrated 85.12-88.98% recoveries for both the analytes. The method depicted an accuracy within 99.52-100.57% and the precision (coefficient of variation) was 0.32-1.66% for both the analytes.