Simultaneous LC-MS/MS quantitation of acetaminophen and its glucuronide and sulfate metabolites in human dried blood spot samples collected by subjects in a pilot clinical study

Background: In support of a pilot clinical trial using acetaminophen as the model compound to assess dried blood spot (DBS) sampling as the method for clinical pharmacokinetic sample collection, a novel sensitive LC-MS/MS method was developed and validated for the simultaneous determination of acetaminophen and its major metabolites, acetaminophen glucuronide and sulfate, in human DBS samples collected by subjects via fingerprick. Results: The validated assay dynamic range was from 50.0 to 5000 ng/ml for each compound using a 1/8 '' (3-mm) disc punched from a DBS sample. Baseline separation of the three analytes was achieved to eliminate the possible impact of insource fragmentation of the conjugated metabolites on the analysis of the parent. The overall extraction efficiency was from 61.3 to 78.8% for the three analytes by direct extraction using methanol. Conclusion: The validated method was successfully implemented in the pilot clinical study with the obtained pharmacokinetic parameters in agreement with the values reported in literature.