Separating and quantifying unbound target analytes from biological samples

A method of determining an amount of free target analyte in an aqueous sample, comprising:

• obtaining an aqueous sample that contains a target analyte in a free form (free target analyte) and the target analyte in a protein-bound form (protein-bound target analyte) in which the target analyte is bound to a target analyte binding protein;
• passing the aqueous sample through a size exclusion chromatography matrix with a molecular weight cut off sufficient to allow the free target analyte to permeate into pores of the size exclusion chromatography matrix and exclude the protein-bound target analyte,
• whereupon the free target analyte adheres to and is immobilized by the size exclusion chromatography matrix and the protein-bound target analyte does not adhere to by the size exclusion chromatography matrix;
• separating the free target analyte from the protein-bound analyte by removing the protein-bound target analyte from the size exclusion chromatography matrix;
• eluting the free target analyte (eluted free target analyte) from the size exclusion chromatography matrix with an organic solvent;
• and
• quantifying the amount of the free target analyte in the aqueous sample from the amount of the eluted free target analyte in the eluent.