Liquid chromatography-mass spectrometric analysis of urinary metabolites and their pattern recognition for the prediction of drug-induced hepatotoxicity.

Multivariate pattern recognition (PR) analysis combined with LC/MS was utilized to evaluate the feasibility of predicting chemical-induced toxicity in rats. Urine samples were collected from rats treated with vehicles or four hepatotoxins, alpha-naphthyl isothiocyanate (ANIT), carbontetrachloride (CCl4), acetaminophen, and diclofenac, and analyzed by HPLC coupled with electrospray mass spectrometry. Chromatographic data were normalized using modified Z score transformation with those of the control group, to remove the vehicle effects for a further enhanced multivariate analysis. The LC/MS-based profiles of the urine samples showed different levels of endogenous metabolites, which were characteristic of each hepatotoxin. In the principal component (PC) map of the urinary spectra from rats treated with ANIT, the metabolic trajectory moved away from the predose position, reaching a maximum separation at the 32-48 h time period. The metabolic profiles partially recovered to the basal conditions on 7 days postdose. A principal component analysis was performed on the urinary spectra of rats treated with the vehicles or four hepatotoxins. Each group formed a distinct and isolated cluster in the PC map, indicating drug-induced perturbation in the urine profiles. To construct mathematical models for predicting drug-induced hepatotoxicity, supervised analyses, such as linear discriminant analysis and soft independent modeling of class analogy with residual distance (SIMCA-RD), were performed. The SIMCA-RD showed high predictability, over 95%, in the results of cross-validation using the leaving-one-out method. The developed LC/MS-PR approach might be a useful tool for the prediction of drug-induced hepatotoxicity and for the understanding of hepatotoxic mechanisms.

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.

NMR and pattern recognition studies on the time-related metabolic effects of alpha-naphthylisothiocyanate on liver, urine, and plasma in the rat: an integrative metabonomic approach.

We present here a novel integrative metabonomic approach to probe toxic effects of drugs in experimental animals using alpha-naphthylisothiocyanate (ANIT) as a model hepatotoxicant. Male Han-Wistar rats were dosed with ANIT (150 mg/kg, n = 25), and plasma and liver samples were collected for NMR and magic-angle spinning (MAS) NMR spectroscopy at 3, 7, 24, 31, and 168 h postdosing. Urine was collected continuously for 3 days prior to dosing and up to 168 h postdose. Histopathology and plasma clinical chemistry was also performed at all time points. Liver samples were analyzed either intact by 600 MHz 1H MAS NMR techniques or using high resolution (liquid state) 1H NMR of water-acetonitrile extracts. These data were related to sequential 1H NMR measurements in urine and plasma using pattern recognition methods. 1D 1H NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by ANIT toxicity. From the eigenvector loadings of the PCA, those regions of the 1H NMR spectra and hence the combinations of endogenous metabolites marking the main phase of the toxic episode were identified. The ANIT-induced biochemical manifestations included a hepatic lipidosis associated with hyperlipidaemia; hyperglycaemia and glycosuria; increased urinary excretion of taurine and creatine; a shift in energy metabolism characterized by increased plasma ketone bodies with reduced urinary excretion of tricarboxylic acid cycle intermediates and raised hepatic bile acids leading to bile aciduria. The integration of metabolic data derived from several sources gives a holistic approach to the study of time-related toxic effects in the intact system and enables the characterization of key metabolic effects during the development and recovery from a toxic lesion.