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

Abstract

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 nm, 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 (Δ 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.