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  • 1
    Electronic Resource
    Electronic Resource
    Analysis of reduced glutathione using a reaction with 2,4′-dichloro-1-(naphthyl-4-ethoxy)-s-triazine (EDTN) (1999)
    Springer
    Microchimica acta 130 (1999), S. 225-231 
    Details
    ISSN: 1436-5073
    Keywords: 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine ; EDTN ; reduced glutathione ; EDTN-glutathione analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A fluorescent adduct was formed between 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine (EDTN) and reduced glutathione in a reaction at 37 °C and pH 9.2. This reaction was used as the basis of an assay for reduced glutathione. The fluorescence was examined at an excitation wavelength of 319 nm and an emission wavelength of 425 nm after extraction of residual unreacted EDTN with methylene dichloride and subsequent dilution of the aqueous phase with ethanol containing 0.01 percent Triton X-100. The reaction rate was low at pH 7 but was accelerated by addition of preparations containing the enzyme glutathione-S-transferase. The adduct gave a discrete peak using isocratic elution with HPLC on a Nova-pak C18 3 μm reverse phase column and a solvent system of methanol: 0.1 M phosphate buffer pH 6.3 (40∶60). An analytical concentration range of 24 to 240 μM reduced glutathione was obtained with an ultraviolet detection system but the concentration range was 7.5 to 75 μM when a fluorescence detection system was used. Adducts of other mercapturic acid pathway thiol compounds were not formed at 37 °C under the conditions used and hence did not interfere in the assay. They were formed by heating EDTN and the respective thiol compound at 60 °C for 30 min and they clearly separated from the reduced glutathione compound on HPLC analysis. A strong reaction was observed with digitonin while solutions of tyrosine, at 10 mM concentration, also reacted but these reactants are unlikely to interfere with reduced glutathione analysis in biological systems. When adduct formation was used to estimate reduced glutathione concentrations in some mammalian and plant tissues the reaction using 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine and HPLC separation gave the same results as ano-phthaldialdehyde assay for liver and muscle but the HPLC method gave slightly lower values for other mammalian and plant tissues. The differences were attributed to other material in the tissue extracts which was fluorescing at the same wavelengths as the reduced glutathione adduct.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01242910
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  • 2
    Electronic Resource
    Electronic Resource
    Mixed Mode Retention and the Use of Competing Acid for the Ag+-HPLC Analysis of Underivatized Conjugated Linoleic Acids (2000)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 23 (2000), S. 317-323 
    Details
    ISSN: 0935-6304
    Keywords: Conjugated linoleic acids ; underivatized ; Ag+-HPLC ; resolution ; mixed mode retention ; hydrogen bonding ; competing acid ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Fatty acids (FAs) and fatty acid residues are generally methylated and analyzed by GC. The reasons for this are partly historic and partly because of the sensitivity advantage of flame ionization detection over UV absorption by the carboxylic acid functionality in saturated FAs. However, for strongly absorbing unsaturated acids such as the conjugated linoleic acids (CLAs), the sensitivity advantage is greatly reduced. Hence there seems little reason to waste time and introduce errors associated with methylation. Remarkably, this appears not to have been recognized. In this paper we describe our method development for the analysis of underivatized CLAs by silver ion HPLC separation on the ChromSpher Lipids column. Using mobile phases previously optimized for the analysis of the methylated CLAs, retention is excessive and a competing acid is required. Various combinations of small concentrations of acetic acid (3.0-2.5%) with acetonitrile (0.0-0.025%), respectively, yield similar resolution and run times. As well as its role as a competing acid, acetic acid acts as a general strong solvent and thus can be used alone as a modifier (without acetonitrile). However, for slightly shorter run times a mobile phase of 2.5% acetic acid and 0.025% acetonitrile was chosen as the optimum mobile phase for analysis. The separation of the free CLAs is clearly superior to those previously published and obtained in this study for the methylated CLAs. The additional specific strong interactions of the underivatized CLAs seem certain to be due to hydrogen bonding between the CLA carboxylic acid functionality and the large number of residual silanols on the surface of the silica support of the stationary phase.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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