Comparison of two HPLC columns: An attempt to improve analysis of carbohydrate-deficient transferrin

University essay from Uppsala universitet/Institutionen för kvinnors och barns hälsa

Abstract: 1ABSTRACTCarbohydrate-deficient transferrin (CDT) is a biomarker for excessive and long-running intake of alcohol. It is a form of transferrin called disialotransferrin that under normal circumstances is <2 % of total transferrin in human blood. An increase is seen when alcohol consumption exceeds450 g per week. CDT is analyzed in serum usinghigh performance liquid chromatography (HPLC) and UV/VIS detection. The purpose of this study was to investigate if an “in routine” method could be improved by switching columns.With ion exchange chromatography transferrin glycoforms are separated and quantified. The carbohydrate-deficient transferrin glycoforms have an isoelectric point between 5,7-5,9 that depends on the number of sialic acids on the molecule. With the use of a salt gradient and pH above the isoelectric point the glycoforms can beseparated depending on their affinity to the stationary phase. Batches with patient and control serum was first analyzed on the routine column Source 15Q PE and then on the alternative column Reprospher 200 SAX 5μm.Student’s t-test showed that the two methods’results correlated but were significantly different. A Bland-Altman plot illustrated differences between the two columns. High and low control serum values from Reprospher were lower than the accepted interval. In this study Reprospher’s stationary phase seemed to be affected to such an extent that stabile retention time, better resolution, and stabile values could not be achieved and because the information about the column was lacking an attempt to regeneratethe columnwas not conducted.

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