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Magnetic beads as an extraction medium for simultaneous quantification of acetaminophen and structurally related compounds in human serum.

This paper describes a sample preparation method that complements a previously published liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for acetaminophen and eight structurally-related compounds in human serum (C. Bylda, R. Thiele, U. Kobold, D.A. Volmer. Drug Test. Anal. 2014, 6, 451). The analytes (acetaminophen [APAP] + metabolites acetaminophen-glucuronide [APG], -cysteine [APC], -mercapturate [APM] and -cysteine [APC], structurally similar analogues phenacetin and p-phenetidine, as well as tricyclic antidepressants imipramine and amitryptiline) were extracted from serum using magnetized hyper-crosslinked polystyrene particles. The sample preparation protocol was developed by means of a design of experiments (DoE) statistical approach. Using three representative compounds from the analyte panel with different polarities (high, medium, and low), two screening designs were used to identify factors that exhibited significant impact on recovery of the analytes. These parameters were then optimized to permit extraction of the complete target panel exhibiting a broad range of chemical polarities. Liquid chromatographic separations were achieved by gradient elution using a pentafluorphenyl column with subsequent detection by electrospray ionization-triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode. The method was linear over the range 0.1-100 µg/mL for APAP, APG, p-phenetidine and phenacetin, 0.03-50 µg/mL for APS, and 0.01-10 µg/mL for APM, APC, imipramine and amitriptyline, with R(2)  > 0.99. The assay exhibited good precision with CVs ranging from 2 to 9% for all analytes; the accuracy was assessed by comparing two LC-MS/MS methods using a set of 68 patient samples.