Determination of insulin in a single islet of Langerhans by high-performance liquid chromatography with fluorescence detection

Rodents (rat and mouse) have two types of insulin (insulin I and II; each contains a universal chain A and a different composition of each type BI chain or type BII chain). The physiological role for each isomer is not yet clarified because of the lack of an appropriate separative determination method for these isomers. Thus, in this paper, a sensitive and selective HPLC-fluorescence determination method for the isomers was developed, which includes derivatization with a fluorogenic reagent for thiols, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, in the presence of a reducing agent, TCEP, a nonionic surfactant, n-dodecyl beta-D-maltopyranoside, and EDTA. The resultant chain A, BI, and BII derivatives were separated on a reversed-phase column (TSK gel ODS-120T, 250 x 4.6 mm i.d.) with a mobile phase containing 5 mM phosphate buffer (pH 7.0) and were detected at 505 mu with excitation at 380 nm. The detection limits for chain A, BI, and BII derivatives were 2.2, 3.4, and 3.7 fmol on column, respectively. The method was applicable to the determination of rodent insulin in a single islet of Langerhans, and the results indicated its feasibility for the investigation of the pathophysiological roles of the isomers in diabetes in the rodent.