Quantification of 25OHD in serum by ID-LC-MS/MS based on oriented immobilization of antibody on magnetic materials

Magnetic nanomaterials are widely used, but co-adsorption of impurities will lead to saturation. In this study, the aim was to prepare a magnetic nano-immunosorbent material based on orienting immobilization that can purify and separate 25-hydroxyvitamin D (25OHD) from serum and provides a new concept of sample pretreatment technology. Streptococcus protein G (SPG) was modified on the surface of the chitosan magnetic material, and the antibody was oriented immobilized using the ability of SPG to specifically bind to the Fc region of the monoclonal antibody. The antigen-binding domain was fully exposed and made up for the deficiency of the antibody random immobilization. Compared with the antibody in the random binding format, this oriented immobilization strategy can increase the effective activity of the antibody, and the amount of antibody consumed is saved to a quarter of the former. The new method is simple, rapid, and sensitive, without consuming a lot of organic reagents, and can enrich 25OHD after simple protein precipitation. Combining with liquid chromatography-tandem mass spectrometry (LC-MS/MS), the analysis can be completed in less than 30 min. For 25OHD₂ and 25OHD₃, the LOD was 0.021 and 0.017 ng mL^-1, respectively, and the LOQ was 0.070 and 0.058 ng mL^-1, respectively. The results indicated that the magnetic nanomaterials based on oriented immobilization can be applied as an effective, sensitive, and attractive adsorbent to the enrichment of serum 25OHD.