Integrated on-line sample clean-up using cation exchange restricted access sorbent for the LC determination of atropine in human plasma coupled to UV detection

Abstract

A new, simple and fully automated liquid chromatographic (LC) method with UV detection has been developed for the direct determination of atropine in plasma. Sample clean-up was based on the use of cation exchange restricted access material (RAM) in a pre-column, coupled to LC by means of a column switching system. After direct injection of a 200 μl-volume of plasma sample, the biological matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate adjusted to pH 3.0 and methanol (97:3; v/v). By rotation of the switching valve, atropine was then eluted in the back-flush mode for 2 min and transferred to the analytical column packed with octadecyl silica by the LC mobile phase constituted of a mixture of acetonitrile and potassium phosphate buffer (pH 3.0; 50 mM) containing 2 mM sodium heptanesulfonate (16:84; v/v). The UV detection was performed at 220 nm. The method was validated according to a new approach based on accuracy profile over a concentration range from 25 ng/ml, corresponding to the limit of quantitation, to 1000 ng/ml. The method was then applied for the determination of atropine in plasma after intravenous administration to hospitalised patients.