Simultaneous quantification of acetaminophen and structurally related compounds in human serum and plasma

The method described in this study allows the simultaneous quantification of acetaminophen (APAP) and nine structurally related compounds, namely acetaminophen metabolites and structurally similar analogs (acetaminophen‐glucuronide [APG], ‐sulfate [APS], mercapturate [APM], ‐cysteine [APC], p‐phenetidine, phenacetin), antidote (N‐acetylcysteine, NAC), and two tricyclic antidepressants (imipramine and amitryptiline). Due to the relatively high serum concentration levels in the µg/ml range, matrix effects were simply minimized by dilution. The samples were diluted with water and disulfide bonds between serum proteins and analytes reduced using tris(2‐carboxyethyl)phosphine. Chromatographic separation of the analytes was achieved by gradient elution using a pentafluorphenyl (PFP) column with subsequent detection by electrospray ionization (ESI) triple quadrupole mass spectrometry in positive and negative ionization multiple reaction monitoring (MRM) modes. Quantification was performed by means of deuterated analogues of the analytes as internal standards. Total run time of the assay was 19 min. The method was fully validated and allowed quantification of the analytes with lower limits of quantification between 50 and 0.5 ng/ml. The calibration curves were linear over the range 0.1–100 µg/ml for APAP, APG, NAC, p‐phenetidine and phenacetin, 0.03‐50 µg/ml for APS, and 0.01‐10 µg/ml for APM, APC, imipramine and amitriptyline with correlation coefficients r2 > 0.99. The intra‐assay precision was ≤5% for all analytes except NAC (CV