Determination of vitamin D metabolite and displacement from plasma or serum binding proteins

A method for measuring vitamin D metabolite present in a plasma or serum sample, the method comprising:

• (a) adding to a serum or plasma sample a non-competitive displacement agent comprising 8-anilino-1-napthalenesulfonic acid ammonium salt, 3-(acetonylbenzyl)-4-hydroxycoumarin and a water miscible solvent, which separates any vitamin D metabolite in the sample from protein to which the vitamin D metabolite is bound, such that any separated vitamin D metabolite can be captured;
• (b) contacting the sample with a support having immobilised thereon a binding factor, the binding factor capturing any vitamin D metabolite separated in step (a);
• and
• (c) measuring the amount of vitamin D metabolite captured in step (b);
• wherein capture of separated vitamin D metabolite occurs in step (b) without prior extraction from the sample of the protein separated from the vitamin D metabolite in step (a).