Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: dose-response, mechanisms, and clinical implications.

At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.

Quantification of a biomarker of acetaminophen protein adducts in human serum by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: clinical and animal model applications.

The aims of this study were to develop, validate, and apply a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantification of protein-derived 3-(cystein-S-yl)-acetaminophen (APAP-Cys) in human serum. Formation of acetaminophen (APAP) protein adducts is thought to be a critical, early event in the development of APAP-induced hepatotoxicity, and quantification of these protein adducts in human serum represents a valuable tool for assessment of APAP exposure, metabolism, and toxicity. In the reported procedure, serum samples were first dialyzed or passed through gel filtration columns to remove APAP-Cys not covalently bound to proteins. Serum eluates were then subjected to enzymatic protease digestion to liberate protein-bound APAP-Cys. Norbuprenorphine-D3 was utilized as an internal standard (IS). APAP-Cys and IS were recovered from digested serum by protein precipitation with acetonitrile, and sample extracts were analyzed by HPLC-ESI-MS/MS. The method was validated by assessment of intra- and inter-assay accuracy and imprecision on two different analytical instrument platforms. APAP-Cys could be accurately quantified from 0.010 to 10µM, and intra- and inter-assay imprecision were <15% on both analytical instruments. APAP-Cys was stable in human serum for three freeze-thaw cycles and for 24h at ambient temperature. Extracted samples were stable when stored in refrigerated autosamplers for the typical duration of analysis or when stored at -20°C for six days. Results from process efficiency and matrix effect experiments indicated adequate recovery from human serum and insignificant ion suppression or enhancement. The utility and sensitivity of the reported procedure were illustrated by analysis of clinical samples collected from subjects taking chronic, therapeutic doses of APAP. Applicability to other biological matrices was also demonstrated by measurement of protein-derived APAP-Cys in plasma collected from APAP-treated mice, a common animal model of APAP-induced hepatotoxicity.

A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry.

A novel validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-beta-D-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1'-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-beta-D-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC-MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R(2)) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52-88% in plasma and 51-118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-beta-D-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were < or = 14% and < or = 17%, respectively. Matrix effect (%) was sufficiently minimized to < or = 19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze-thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 degrees C) and 1 week in the freezer (-20 degrees C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 degrees C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1'-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.

Identification and quantification of nicotine biomarkers in human oral fluid from individuals receiving low-dose transdermal nicotine: a preliminary study.

The objective of this preliminary study was to identify and quantify potential nicotine (NIC) biomarkers in post-exposure oral fluid samples collected from 10 NIC-abstinent human participants administered 7 mg transdermal NIC using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Oral fluid samples were collected prior to NIC patch application and at 0.5 and 0.75 h after patch removal using the Quantisal() oral fluid collection device. The validated LC-MS-MS analyte panel included nicotine-Nbeta-D-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1'-N-oxide, cotinine (COT), nornicotine, NIC, anatabine, anabasine, and cotinine-N-beta-D-glucuronide. Analytes and corresponding deuterated internal standards were extracted by solid-phase extraction. NIC and COT concentrations were quantifiable in oral fluid samples collected from 6 of the 10 participants 0.5 h after patch removal and in oral fluid samples collected from 7 of the 10 participants 0.75 h after patch removal. Based on the mean NIC and COT concentrations in oral fluid and plasma for the participants with both quantifiable NIC and COT at the 0.5 and 0.75 h collection times, the oral fluid-plasma ratio was 6.4 for NIC and 3.3 for COT. An ELISA procedure was also validated and successfully applied as a screening tool for these oral fluid samples in conjunction with LC-MS-MS confirmation. An ELISA cut-off concentration of 5.0 ng/mL provided excellent sensitivity for discrimination of COT-positive post-exposure oral fluid samples collected after low-level transdermal NIC exposure and oral fluid samples collected prior to patch application.