High-performance size-exclusion chromatographic procedure for the determination of fluoresceinyl isothiocyanate dextrans of various molecular masses in biological media.
A high-performance size-exclusion chromatographic procedure, using Nucleosil Diol, for the quantitative analysis of fluoresceinyl isothiocyanate dextrans of various molecular masses (10,000-150,000) in biological media was developed. The influence of the molecular mass and the degree of substitution of the conjugates on the chromatographic behaviour are discussed. In addition to quantitation, the molecular mass of the conjugates with degree of substitution below 1.6 could be estimated from the chromatograms. Linear standard calibration curves were obtained at concentrations down to 0.050 micrograms ml-1 in rabbit plasma and urine and homogenates of rabbit liver, lymph node and muscle when the derivative (degree of substitution 0.85) was monitored by fluorescence detection (lambda ex = 493 nm, lambda em = 520 mm). The fluoresceinyl isothiocyanate dextrans were found to be stable for more than three days at 37 degrees in all biological media under investigation. A pH-rate profile for the alkaline hydrolysis of fluoresceinyl isothiocyanate dextrans was constructed. The applicability of the method to pharmacokinetic studies was demonstrated by recording the plasma concentration-time profile of a fluoresceinyl isothiocyanate dextran T-70 conjugate following intravenous injection to a rabbit. In relation to future pharmacokinetic investigations on dextran conjugates, the results reported indicate that labelling of the parent dextran with fluoresceinyl isothiocyanate and monitoring of the fluoresceinyl isothiocyanate dextran conjugate throughout the organism using the described method is a promising development.
Kurtzhals
P
P
Royal Danish School of Pharmacy, Department of Pharmaceutics, Copenhagen.
Larsen
C
C
Johansen
M
M
D000818:Animals; D000941:Antigens / Q000032:analysis*; D002850:Chromatography, Gel; D003911:Dextrans / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000941:Antigens; D003911:Dextrans; D005452:Fluoresceins; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate