The invention discloses a dissociation method of 25-hydroxyvitamin D
binding protein. The dissociation method comprises the following steps: preparing a mother solution, taking a serum sample, addingthe mother solution, carrying out vortex mixing for 1min, uniformly mixing the obtained solution, allowing the obtained
mixed solution to stand for 5min, and centrifuging the
mixed solution in a
centrifuge; and taking the obtained supernatant of the solution, adding
ethanol, carrying out vortex mixing, allowing the obtained
mixed solution to stand, placing the obtained solution in the
centrifuge,centrifuging the solution, taking the obtained supernatant, placing the supernatant in a new
centrifuge tube, blow-
drying the supernatant by using a
nitrogen blow-
drying instrument, adding a separation mobile phase, re-dissolving the dried supernatant, uniformly mixing the re-dissolved supernatant and the separation mobile phase, and carrying out LC-MS / MS detection. The solution uses
dithiothreitol with a certain concentration, so the 25-hydroxyvitamin D to be analyzed can be well separated, the detection result of the 25-hydroxyvitamin D can be improved, and the overall content of the vitaminD is further determined; and the
dithiothreitol has strong reducibility, so the stability of the 25-hydroxyvitamin D in the purification and extraction process is improved.