Negligible depletion solid-phase microextraction with radiolabeled analytes to study free concentrations and protein binding:: An example with [3H]Estradiol

A new method is presented that enables sensitive measurement of free concentrations of radiolabeled ligands. Additionally, protein binding of radiochemicals in complex matrixes can be determined with this new technique that combines negligible depletion solid-phase microextraction (nd-SPME) with liquid scintillation counting (LSC) as detection. [H-3]Estradiol was taken as an example compound. Possible matrix effects of protein on fiber uptake kinetics were studied. No matrix effect was found, either by fouling of the fiber, or by changed uptake kinetics. The validity of the method was shown in the determination of the affinity constant (K-a) of estradiol for human serum albumin (HSA). The K-a was estimated at 8.9 x 10(4) M-1, which corresponds well with literature values. This study shows that nd-SPME is suitable to study the free concentration and protein binding of [H-3]estradiol. The method described in this paper combines the advantages of nd-SPME with the advantages of radiolabeled analytes, creating a timesaving, simple, and sensitive analytical tool that will be particularly useful in complex matrixes containing many potential interferences for chromatographic methods.