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Chromatography methods for the determination of stable isotope traced metabolites and their apllication in the clinical research
ŽABOKRTSKÁ, Jana
In medical diagnostics substances labelled with radioactive or stable isotope tracers are used. At present stable isotope-traced substrates 13C or 2H are most commonly used for metabolism evaluation in pathologic situations. A modern approach is a combination of microdialysis method and an application of stable isotopes. In this study analytic methods for the determination of glucose and lactate in microdialysate are described. Due to used substances, labelled with stable isotope-tracers, these substances were determined by the method of gass chromatography with detection by mass spectrometry (GC-MS). The aim of the study was the development, determination of parameters, validation and optimalization of analytic methods oriented on measuring of isotope enrichment of molecules of chosen analytes. The isotope enrichment of glucose was determined after its derivatization by hydroxylamine hydrochloride and acetanhydride. A derivate aldonitril pentacetyl-D-glucose was determined. The method described in the study is the result of optimalization of time and temperature of derivatization of samples of microdialysates and chromatographic conditions. The obtained data were evaluated by the software Chemstation and statistics software SigmaStat. The precision was verified by the method of standard addition and was stated as 1,48%, the precision was determined by repeated measuring of a real sample {--} variation coefficient was 2,51%. Parameters of linear regression for concentrations 0,5 {--} 15 mmol/l were under given conditions with regression coefficient (R2) 0,9997 and the regression equation y = -0,0185 + 0,142x. Glucose for determination by gass chromatography is usually derivatized only by acetanhydride that however using capillary column CP-Sil 8 CB-MS (60 m x 0,32 mm) provides for interference in chromatographic recording {--} a double peak of likely formed anomers of pentacetyl-glucopyranose. For this reason it was necessary to make derivatization of aldehydic group of glucose first by hydroxylamin hydrochloride. The result method shows suitable parameters for using in analysis of microdialysate samples. Lactate was determined by derivatization by dimethoxypropane, propylamine and further by heptafluorobutyranhydride by formation of derivate of L-lactin-n-propylamidheptafluorobutyrate. Following parameters were determined {--} accuracy 3,25%, precision 3,15%, parameters of linear regression for concentrations wihtin 0,5 {--} 10 mmol/l were under given conditions R2 = 0,999 and the regression equation y = 0,215 + 1,039x. This relatively complicated derivatization method was taken over from an associated workshop at the University of Lausanne and its parameters were compared with a simpler method of derivatization by N-(butyl-dimethyl-silyl)-2,2,2-trifluoro-N-methyl-acetamide. The formed derivate di(tert-butyldimethylsilyl) lactate was determinated by the method GC-MS. The precision was 3,02%, accuracy 3,8%, parameters of linear regression R2 = 0,999, the regression equation y = -0,0809 + 0,792x. The presented methods were used in the pilot study of microdialyse of muscle and hepar of rats in various metabolic situations. At present the methods are used in clinical research on patients suffering from diabetes mellitus type I and have extended a spectrum of methods applied by the laboratory of the Clinic of Gerontology and Metabolism of the University Hospital of Hradec Králové.
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