High-performance liquid chromatographic determination of hormonal peptides and their fluorenylmethoxycarbonyl...

Reference methods for the quantitation of peptide and protein hormones in blood are urgently needed and high-performance liquid chromatography (HPLC) is potentially applicable. Owing to the low concentrations of these substances in body fluids, very low detection limits have to be achieved. This can be done on the one hand by reducing the diameter of the separation column and increasing the number of theoretical plates, and on the other by derivatization. Angiotensin II was chosen as a model compound. Reduction of the inner diameter from 4 to 2 mm increased the peak height by a factor of 3.4 (theoretical value 4.0). The peptide was derivatized with 9-fluorenyl methylchloroformate in lithium carbonate-sodium hydrogencarbonate or sodium borate buffer at different pH values. The precision (coefficient of variation) was 10.4%, the linear range 1:40 (r = 0.998) and the detection limit 500 fmol of derivative on-column. The volume injected was 2 microliter. However, this is not sufficiently sensitive for the quantitation of most peptide hormones using an acceptable maximum specimen volume of 5 ml of serum or plasma.