Salting-out assisted liquid-liquid extraction for the determination of biogenic amines in fruit juices and alcoholic beverages after derivatization with 1-naphthylisothiocyanate and high performance liquid chromatography.

A new method for determining biogenic amines in fruit juices and alcoholic beverages is described involving reaction of biogenic amines with 1-naphthylisothiocyanate followed by extraction of 1-naphthylthiourea derivatives with water-miscible organic solvent acetonitrile when solvents phase separation occurred using ammonium sulphate, a process called salting-out assisted liquid-liquid extraction. The extract was analyzed by high-performance liquid chromatography with UV detection at 254nm. The new reagent avoided many of the inconveniences as observed with existing derivatizing agents, such as dansyl chloride and benzoyl chloride, in regard to their inselectivity, instability, adverse effect of excess reagent, and necessity to remove excess reagent. The procedure has been optimized with respect to reaction time and temperature, water-miscible extraction solvent, and salt for solvents phase separation. Use of reagent as dispersed phase in aqueous medium produced derivatives in high yield. A linear calibration was obtained between the amount of biogenic amines in range 1-1000µgL(-1) and peak areas of corresponding thioureas formed; the correlation coefficient was 0.9965, and the limit of detection and limit of quantification found were 1.1µgL(-1) and 3.2µgL(-1), respectively. The pre-concentration method gave an average enrichment factor of 94. The application of the method has been demonstrated in the determination of biogenic amines in commercial samples of fruit juices and alcoholic beverages. In spiking experiments to real samples, the average recovery found by the present method was 94.5% that agreed well with 95.8% obtained by established comparison methods.

Fluorometric sensing of Hg2+ ions in aqueous medium by nano-aggregates of a tripodal receptor.

Two new tripodal receptors (1­2) have been synthesized and characterized by various spectroscopic techniques. The nano-aggregates of 1 and 2 (N1 and N2) have been prepared by a re-precipitation method in aqueous medium and have shown different photo-physical properties. Nano-aggregates of 1 (N1) can selectively recognize Hg(2+) in aqueous medium in the presence of other metal ions with enhancement in fluorescent intensity. The response was linearly proportional to the concentration of Hg(2+) in the range 0­10 µM with a detection limit of 2.4 nM. The mechanism of selective binding of Hg(2+) by N1 has also been supported by theoretical studies. To the best of our knowledge, this work represents the first report on substituted thiourea based nano-aggregates for nano-molar detection of mercury in aqueous medium.

Metabonomics: evaluation of nuclear magnetic resonance (NMR) and pattern recognition technology for rapid in vivo screening of liver and kidney toxicants.

The purpose of this study was to evaluate the feasibility of metabonomics technology for developing a rapid-throughput toxicity screen using 2 known hepatotoxicants: carbon tetrachloride (CCl(4)) and alpha-naphthylisothiocyanate (ANIT) and 2 known nephrotoxicants: 2-bromoethylamine (BEA) and 4-aminophenol (PAP). In addition, the diuretic furosemide (FURO) was also studied. Single doses of CCl(4) (0.1 and 0.5 ml/kg), ANIT (10 and 100 mg/kg), BEA (15 and 150 mg/kg), PAP (15 and 150 mg/kg) and FURO (1 and 5 mg) were administered as single IP or oral doses to groups of 4 male Wistar rats/dose. Twenty-four-h urine samples were collected pretest, daily through Day 4, and on Day 10 (high dose CCl(4) and BEA only). Blood samples were taken on Days 1, 2, and 4 or 1, 4, and 10 for clinical chemistry assessment, and the appropriate target organ was examined microscopically. NMR spectra of urine were acquired and the data processed and subjected to principal component analyses (PCA). The results demonstrated that the metabonomic approach could readily distinguish the onset and reversal of toxicity with good agreement between clinical chemistry and PCA data. In at least 2 instances (ANIT and BEA), PCA analysis suggested effects at low doses, which were not as evident by clinical chemistry or microscopic analysis. Furosemide, which had no effect at the doses employed, did not produce any changes in PCA patterns. These data support the contention that the metabonomic approach represents a promising new technology for the development of a rapid throughput in vivo toxicity screen.

Semi-automatic liquid chromatographic analysis of pamidronate in urine after derivatization with 1-naphthylisothiocyanate.

An existing sensitive chromatographic assay for pamidronate in urine has considerably been automated. Using the same sample processor, the solid-phase extraction (SPE) was automated separately from the derivatization with 1-naphthylisothiocyanate, the two-fold ion-pair liquid-liquid-extraction and the treatment with hydrogen peroxide for the 2-20 ng/ml concentration range. The automatic procedure was preceded by a triple calcium precipitation and interrupted by evaporation of the SPE eluate under nitrogen. For the 0.5-5 microg/ml concentration range one automatic sequence was used by avoiding evaporation during the sample treatment. In addition to the labour-saving of the semi-automatic procedure, the daily sample-throughput was improved compared to the existing manual assay. Further, the validation showed marginal improvements in the precision, accuracy and lower limit of quantification.

Nuclear magnetic resonance spectroscopic and principal components analysis investigations into biochemical effects of three model hepatotoxins.

1H NMR spectroscopy of urine combined with pattern recognition (PR) methods of data analysis has been used to investigate the time-related biochemical changes induced in Sprague-Dawley rats by three model hepatotoxins: alpha-naphthyl isothiocyanate (ANIT), d-(+)-galactosamine (GalN), and butylated hydroxytoluene (BHT). The development of hepatic lesions was monitored by conventional plasma analysis and liver histopathology. Urine was collected continuously postdosing up to 144 h and analyzed by 600-MHz 1H NMR spectroscopy. NMR spectra of the urine samples showed a number of time-dependent perturbations of endogenous metabolite levels that were characteristic for each hepatotoxin. Biochemical changes common to all three hepatotoxins included a reduction in the urinary excretion of citrate and 2-oxoglutarate and an increased excretion of taurine and creatine. Increased urinary excretion of betaine, urocanic acid, tyrosine, threonine, and glutamate was characteristic of GalN toxicity. Both GalN and ANIT caused increased urinary excretion of bile acids, while glycosuria was evident in BHT- and ANIT-treated rats. Data reduction of the NMR spectra into 256 integrated regions was used to further analyze the data. Mean values of each integrated region were analyzed by principal components analysis (PCA). Each toxin gave a unique time-related metabolic trajectory that could be visualized in two-dimensional PCA maps and in which the maximum distance from the control point corresponded to the time of greatest cellular injury (confirmed by conventional toxicological tests). Thereafter, the metabolic trajectories changed direction and moved back toward the control region of the PR map during the postdose recovery phase. The combination of urinary metabolites which were significantly altered at various time points allowed for differentiation between biliary and parenchymal injury. This NMR-PR approach to the noninvasive detection of liver lesions will be of value in furthering the understanding of hepatotoxic mechanisms and assisting in the discovery of novel biomarkers of hepatotoxicity.

Determination of pamidronate in urine by ion-pair liquid chromatography after derivatization with 1-naphthylisothiocyanate.

A sensitive method for the determination of pamidronate disodium [(3-amino-1-hydroxypropylidene)bisphosphonate, APD] in urine has been developed and validated. The procedure involves a triple co-precipitation with calcium phosphate, solid-phase extraction on a quaternary ammonium column, derivatization with 1-naphthylisothiocyanate and ion-pair liquid-liquid extraction. From the two reaction products, naphthylthiocarbamyl-APD is converted into the other, naphthylcarbamyl-APD, by an oxidative desulphuration with hydrogen-peroxide prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The method has a coefficient of variation of 7% for the intra-assay precision of 99 ng ml-1 APD and 11% for the inter-assay precision. The lower limit of quantification is 3 ng ml-1 APD in 2.5 ml of human urine.