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Published on: June 10, 2018
HPLC and mass spectrometric characterization of a candidate reference material for the alcohol biomarker
Walter Oberrauch1, Ann-Charlotte Bergman, Anders Helander
1Bio-Rad Laboratories GmbH, Munich, Germany.
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 (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.
