Analysis of phenols by high-performance liquid chromatography with pre-column derivatization by 2-(9-carbazole)-ethyl-chloroformate

2-(9-Carbazole)-ethyl-chloroformate (CEOC), a novel pre-column fluorescence labeling reagent, has been synthesized and applied for the derivatization of phenols. Taken phenol, p-chlorophenol, 2,5-dimethylphenol, 2,4-dichlorophenol and 1,4-dihydroxybenzene as testing standards, the effects of derivatization conditions, such as pH of borate buffer, reaction time and fluorescent tagging reagent concentration, have been systematically studied. Under the optimized conditions, CEOC reacts readily with the phenols to form stable derivatives with excitation and emission wavelengths, respectively, at 293 and 360 nm. The single step derivatization reaction could be finished within 20 min even at room temperature. Such a method has been successfully applied to the analysis of phenols in printing ink by high-performance liquid chromatography. (c) 2005 Elsevier B.V. All rights reserved.

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 turn x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges. (c) 2005 Elsevier B.V. All rights reserved.

High-performance liquid chromatography-tandem mass spectrometry for identification of isoflavones and description of the biotransformation of kudzu root

High-performance liquid chromatography-tandem mass spectrometry has been used to identify isoflavone aglycones and glycosides in kudzu root. Fourteen isoflavones were detected. Among these, six were identified by comparison with authentic standards. Tentative identifications of the other isoflavones are based on UV spectra, mass spectra of protonated and deprotonated molecules, and MS-MS data. Several are reported for the first time in kudzu root. The bioactivity and bioavailability of isoflavone aglycones are usually greater than those of their glycosides. To improve the bioavailability of kudzu root isoflavones, crude beta-glycosidases prepared from microbes were used to hydrolyze the isoflavone glycosides. Several MS modes are combined not only to identify the isoflavones in kudzu root, but also to describe the biotransformation of kudzu root isoflavone glycosides. It is also proved that crude beta-glycosidases have high selectivity toward the O-glycosides of isoflavones.

Studies on the persistence of estradiol benzoate and nortestosterone decanoate in hair of cattle following treatment with growth promoters, determined by ultra-high-performance liquid chromatography-tandem mass spectrometry

Measurement of steroid esters in bovine hair samples, using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides a powerful tool for identifying animals treated illicitly with growth promoters. The successful application of such testing requires appropriate sampling of hair from treated animals. This paper describes the results of hair analysis by LC-MS/MS for two animal studies in which animals were treated with estradiol-3-benzoate and nortestosterone decanoate. The results from the first animal study indicate that animals treated with these anabolic steroids may not always be identified from analysis of hair samples; positive test results occur sporadically and only for some of the treated animals. The results from the second animal study identify conditions attaching to positive hair samples, such as, that concentrations of steroid esters in hair are related to distance of sampling from point of injection and to time post-treatment, that concentrations of steroid esters in hair are related to dose given to the animal but that this relationship may vary over time post-treatment, and that steroid esters may be measured in regrowth hair taken some weeks after treatment. Steroid esters are determined along the length of the hair, confirming that accumulation of steroid esters into hair occurs from various sources, including blood, sweat and sebum. The reported research provides some useful insights into the mechanisms governing the persistence of steroid esters in bovine hair following illicit treatment with growth promoters. (C) 2009 Elsevier B.V. All rights reserved.

Denaturing high performance liquid chromatography for the detection of microsatellite instability using Bethesda and pentaplex marker panels

Microsatellite instability (MSI) is a characteristic molecular phenotype of tumors from the hereditary nonpolyposis colorectal cancer (Lynch) syndrome. Routine MSI screening of tumors in patients is an efficient prescreening tool for the population-based detection of Lynch syndrome in the absence of family cancer history. We describe here the optimization of a denaturing high performance liquid chromatography (DHPLC) assay for MSI analysis with the