HPLC and mass spectrometric characterization of a candidate reference material for the alcohol biomarker carbohydrate-deficient transferrin (CDT)

Background: Measurement of the alcohol-induced change of the serum transferrin glycoform pattern, carbohydrate-deficient transferrin (CDT), is used as a biomarker for heavy drinking. This study characterized a candidate reference material for CDT measurement derived from isolated human transferrin glycoforms. Methods: Four transferrin glycoforms were separated from human plasma by standard methods. The identity and purity of the fractions was evaluated by HPLC, using specific absorbance measurement of the iron-transferrin complex at 470 non, and by mass spectrometry, Using ESI Q-Tof MS. A primary candidate reference material was prepared by mixing isolated fractions in transferrin-free plasma in a proportion similar to that in serum and with 0-12% disialotransferrin. A secondary candidate reference material was prepared by spiking a serum pool with 1-9% disialotransferrin. Results: Initial identification of the isolated transferrin fractions as disialo-, trisialo-, tetrasialo- and pentasialotransferrin was based on agreement with established HPLC retention times for authentic serum samples (RRT 0.998-1.004). The presence of single symmetric peaks suggested that the fractions were sufficiently pure. The identity and purity was further based on MS agreement of observed with theoretical molecular masses (Delta(m) <0.03%). The %disialotransferrin target values for the secondary candidate reference material showed good correlation with the measured results by an HPLC candidate reference method (r(2) = 0.999). Conclusions: The separated human transferrin fractions used to prepare the CDT candidate reference material were indicated to contain distinct glycoforms. Having access to a CDT reference material in serum matrix will facilitate comparison of results between different methods and aid in the standardization process. (C) 2008 Elsevier B.V. All rights reserved.