ELUENT CONDITIONS FOR DIRECT INJECTIONS OF INTACT PLASMA SAMPLES ONTO A DUAL MODE COLUMN

Abstract

The use of transmittance at 650nm was evaluated to measure plasma protein precipitation. The transmittance reflected the protein precipitation quite well and was useful as a facile method to find out suitable eluent conditions allowing a direct injection of plasma samples onto a dual mode column. By applying the method, precipitation of human plasma and human serum albumin were examined with various conditions. They did not precipitate quickly in water, physiological saline, phosphate/citrate and phosphate buffers of extensive pH range (2.32-7.12) when Incubated at 30°C. Methanol, acetonitrile, ethanol, isopropanol and acetone did not show any considerable effects on the precipitation when mixed with either saline or the buffers at neutral pHs (≥5) up to 30%(V/V). At low pHs, however. especially below pH 4, 30% of the organic solvents render the plasma proteins to precipitate in short time of the incubation. On the basis of the above findings, a direct HPLC separation of human plasma spiked with 23 β-lactam antibiotics successfully achieved on a dual mode column, Develosil 60C8-OH-5.