Human insulin was fluorescently labelled with fluorescein isothiocyanate (FITC) and the conjugate species produced were identified using high performance liquid chromatography and electrospray mass spectroscopy. Mono-labelled FITC-insulin conjugate (A1 or B1) was successfully produced using human insulin at short reaction times (up to 5 h) however the product always contained some unlabelled native human insulin. As the reaction time was increased over 45 h, no unlabelled native human insulin was present and more di-labelled FITC-insulin conjugate (A1B1) was produced than mono-labelled conjugate with the appearance of tri-labelled conjugate (A1B1B29) after 20 h reaction time. The quantities switch from mono-labelled to di-labelled FITC-insulin conjugate between reaction times 9 and 20 h. In the presence of phenol or m-cresol, there appears to be a 10 % decrease in the amount of mono-labelled conjugate and an increase in di-labelled conjugate produced at lower reaction times. Clinically used insulin analogues present in commercially available preparations were successfully fluorescently labelled for future biomedical applications.
Keywords: FITC-insulin; Fluorescent labelling; HPLC; Insulin; Mono-labelled.