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

Abstract

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 r(2) > 0.99. The intra-assay precision was ≤5% for all analytes except NAC (CV < 10%). The inter-day precision was ≤10% for all analytes except NAC (inter-assay precision <11%). This method was used to analyze 77 patient and spiked samples and results were consistent with expected values from a round robin test.

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

“…By applying a high declustering voltage (40 V) in the ionization source of the mass spectrometer, GPChos readily undergo in‐source collision‐induced dissociation (CID) and form the product ion trimethylammonium‐ethylphosphate ( m/z 184) that can further dissociate to trimethylammoniumethylate ( m/z 104) . Stability of the samples had already been assessed in our previous work; these experiments were therefore not repeated.…”

“…To evaluate performance and accuracy of the new extraction method, 68 patient samples were analyzed and values compared to results obtained for the same samples using a previously established LC‐MS method, which also lists information on ethics approval of the study. This experiment was conducted for the main target APAP as well as for the metabolites APG, APS, APC, and APM.…”

“…In this study, magnetic beads were applied to the quantitative analysis of acetaminophen (APAP), which is a widely used over‐the‐counter drug that can have severe toxic side effects in case of an overdose or long‐term abuse . We have recently presented a LC‐MS/MS assay for simultaneous quantification of APAP and other pharmaceutically relevant substances in human serum and plasma . That assay was developed for a group of analytes with broad range of polarities, namely APAP, its metabolites acetaminophen‐glucuronide (APG), ‐sulfate (APS), ‐cysteine (APC) and ‐mercapturate (APM) as well as phenacetin, p ‐phenetidine, imipramine and amitriptyline.…”

NSAIDs Determination in Human Serum by GC-MS

“…These drugs exhibit toxicity in the upper concentration levels or after a long time of intake [2,3]. Some of the toxic side effects are related with gastrointestinal disorders, intestinal ulceration, aplastic anemia, myocardial infarction, cerebrovascular events, inhibition of platelet aggregation, and renal dysfunction [4].…”

“…Some of the toxic side effects are related with gastrointestinal disorders, intestinal ulceration, aplastic anemia, myocardial infarction, cerebrovascular events, inhibition of platelet aggregation, and renal dysfunction [4]. Especially, acetaminophen and nimesulide exhibit significant hepatotoxicity [2,[5][6][7]. In addition, there are several cases of suicide attempts or crime commissions which are related with NSAIDs [8].…”