Specific assay for the quantitation of indocyanine green in rat plasma using high‐performance liquid chromatography with fluorescence detection

A rapid and sensitive reversed‐phase HPLC assay employing fluorescence detection was developed for quantitating indocyanine green (ICG) in rat plasma. Sample preparation entailed precipitation of plasma proteins with acetonitrile prior to injection on the column. The assay was linear from 0.4 to 200 μg/mL, with a detection limit of 3 ng on column. The plasma concentration–time profile of ICG was characterized by this HPLC method and compared with the traditional spectrophotometric assay following iv bolus and iv infusion administration of 5 mg/kg of ICG to rats. Concentrations of ICG obtained using the spectrophotometric assay were consistently higher than those determined by HPLC. In animals receiving ICG by infusion, the maximum difference between the two assays was observed 1 min post‐infusion and became negligible by 5 min post‐infusion. The calculated pharmacokinetic parameters for ICG, systemic clearance and apparent volume of distribution, were higher using the HPLC assay as compared with the spectrophotometric procedure. The data suggest that a biotransformation or degradation product of ICG is formed in the rat and interferes with the determination of ICG by the spectrophotometric assay. Since the HPLC assay is specific for the parent dye, it is suggested that this assay method be used when determining pharmacokinetic parameters of ICG in rats.