Normalización de un método de referencia para medir transferrina deficiente en carbohidratos CDT, por HPLC en pacientes alcohólicos
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This research has been performed within of the global initiative interested in validating the method of High Performance Liquid Chromatography (HPLC) for identification and quantification of CDT isoforms (Helander, Husa, & Jeppsson, 2003) and their application to assess this biomarker in the consumer alcoholic population in the Bogota city. We were used certified standards of blood serum obtained from Control Serum RECIPE Clinchek®. The HPLC system was A LACHROME ELITE -HITACHI chromatograph equipped with quaternary pump, UV-VIS detector, manual injector, EZChrom Elite software. The separation of CDT glycoforms was performed on a Source 15Q ® PE 4.6 / 100 anion exchange chromatography column at ambient temperature. We have used gradient elution with solvents as: (A) 10 mM Bis-Tris pH 7; (B) Bis-Tris 10 mm + Sodium Chloride (NaCl) 0.5 M, pH 6.2; (C) Bis-Tris 10 mm pH 6.2 and 0.5 M NaCl Buffer D. The HPLC system provided a good selectivity, the glicoforms of serum transferrin had a resolution factor, Rs > 1,5. The calibration curve at 470 nm was found to be linear over the range of concentrations from 0.25 % w / w and 5.09% w / w, with correlation coefficients for all major isoforms to 0.9943. The recovery percent was 99.4% and this curve showed a linear behavior with a coefficient 0.9975. The variation coefficient percent of (% CV) were below 10% (valid values for biological samples) which showed the repeatability of the method. There were not significant differences when the measurements were performed by different analysts and in different days, finding % CV <10%, which proved that the method has good reproducibility. The limits of detection and quantification were 0,24% w / w and 0,25% w / w. The serum blood samples was obtained from six hospitals and one clinic. The method was applied to the analysis of 315 blood serum samples of people between 18 and 65 years old, associated with consumer alcohol population and 50 control samples. The population with 18-24 years old were the heavy drinkers with addiction problems and the population with 35-65 years old were hazardous drinking.