Rapid microtiter plate assay for determination of inulin in human plasma and dialysate

A rapid, sensitive, and reproducible microtiter plate assay for the determination of inulin in human plasma, dialysate, and phosphate-buffered saline (PBS) was developed. Plasma or PBS samples (100 μl aliquots) were prepared by the addition of indole-3-acetic acid (150 μl) and HCl (3 ml) and then briefly vortex-mixed. Samples were then incubated in a 60 °C water bath for 20 min, cooled in a room temperature water bath for 40 min, then diluted with deionized, distilled water (DDW; 3 ml) and again vortex-mixed. Finally, an aliquot (200 μl) of each sample was transferred to a 96-well microtiter plate and read spectrophotometrically at 490 nm. Dialysate samples were processed in a similar manner, but required an initial enzymatic step in order to remove dextrose and minimize assay interference. Samples (100 μl aliquots) were prepared by the addition of glucose oxidase/catalase solution (100 μl), briefly vortex mixed, and then incubated in a 37 °C water bath for 60 min, samples were then reacted with indole-3-acetic acid as before. Calibration curves were linear over the concentration range of 0.5–4 mg/ml or 0.025–0.4 mg/ml for plasma or PBS and dialysate, respectively; correlation coefficients ( r 2) were >0.99. The intra- and inter-day coefficients of variation in plasma, PBS, and dialysate were