Isocratic high-performance liquid chromatography-photodiode-array detection method for determination of lysine- and arginine-vasopressins and oxytocin in biological samples.

Abstract

A simple, isocratic, sensitive (1 ng), and specific high-performance liquid chromatographic (HPLC) method based on photodiode-array detection (PAD) is described for simultaneous quantitation of the bioactive peptides, lysine vasopressin (LVP), arginine vasopressin (AVP) and oxytocin (OXY). Acidified pig plasma and left ventricular (LV) tissue samples were first extracted with Sep-Pak C18 columns, and the bioactive peptides were eluted with methanol, then dried at 37 degrees C and reconstituted with HPLC mobile phase. The bioactive peptides were separated by HPLC on a Dynamax 3009-A C8 column with a mobile phase of 0.1% trichloroacetic acid-50 mM heptanesulfonic acid-30mM triethylamine-20% acetonitrile in water, pH 2.5 and identified with a Waters 990-PAD system (spectrum index plots in the range 200-400 nm). Standards of LVP, AVP and OXY and their mixtures showed a linear increase in the range 5 to 100 ng and were eluted at 6.1, 6.9 and 4.6 min, respectively. Spectrum analysis showed a distinct absorption peak at 280 nm, corresponding to peptide bonds. The reproducibility of the method coefficient of variation for standards is 6.9, 5.8 and 4.7% for LVP, AVP and OXY, respectively. In plasma and tissue it is much higher: 12.9% (LV tissue) and 18.6% (plasma) for LVP. Pig plasma contains negligible amounts of AVP and OXY; LVP is much higher (0.28 +/- 0.19 ng/ml). In pig tissue, LVP predominates (6.95 ng/g wet weight) compared to AVP (1.45) and OXY (1.50). Spectral analysis is necessary to identify the bioactive peptide peaks among interfering substances and to increase the sensitivity four-fold. The method described here is useful for the simultaneous determination of LVP, AVP and OXY in the nanogram range and can be extended to picogram levels by employing PAD spectral analysis techniques.