Plasma protein binding of highly bound drugs determined with equilibrium gel filtration of non-radiolabeled compounds and LC-MS/MS detection.

Accurate determination of the free fraction of a drug in plasma can be challenging when it falls below 1% and even more so when below 0.1%. Equilibrium dialysis with diluted plasma has been used to determine unbound fraction below 1%, but some analytes are not amenable to this method. One robust alternative for accurately measuring very highly bound compounds is equilibrium gel filtration; however, radiolabeled compounds have been used with this technique to quantify the low analyte concentrations. This report examined results obtained using radiolabeled compounds with liquid scintillation detection and those obtained using their nonradiolabeled analogs with liquid chromatography–tandem mass spectrometry detection. The 2 methods provided comparable results over the range of 0.005%-4% free, with a slope of 1.0 and a R2 = 0.93. These results demonstrate that equilibrium gel filtration with liquid chromatography–tandem mass spectrometry detection can be used earlier in the drug discovery process to determine the unbound fraction of highly bound drugs and may help obviate the need for radiolabeled compound.