AN INTEGRATED COUPLED-COLUMN LIQUID-CHROMATOGRAPHY SYSTEM FOR THE DETERMINATION OF LEUKOTRIENE METABOLITES IN BIOLOGICAL SAMPLES

A fully integrated chromatographic system was developed for the determ ination of leukotrienes in biological samples using photodiode-array d etection (PDAD), which eliminates time consuming manual sample handlin g steps. A special solid phase extraction (SPE) methodology for leucot riene metabolite stability was developed which increased the recoverie s and eliminates the contamination risk of biological samples. The inh erent instability (autooxidation) of many of the leukotriene mediators , and the adsorption effects onto exposed surfaces in vials and in the chromatographic system were found to be very important parameters to control in order to circumvent high loss of sample analytes. By bindin g the cell supernatants to the functionalities of the SPE support stab ilised these mediators. Cell culture samples were eluted through a dis posable C-18 SPE column. The SPE columns were allowed to thaw and depo sited in an automated sample handling unit (ASPEC XL). Desorption of t he analytes was followed by a second on-line SPE step, to eliminate re maining interfering matrix compounds. Typical recoveries when stored a t -70 degrees C were in-between 55-97 % except for (LTE4) which was fo und to be around 40 % after 72 days of storage. Seven reversed-phase p ackings were studied. Selectivity factors, as well as the separation e fficiencies, were found to differ for the various C-18 bonded silica s tationary phases. This integrated on-line column liquid chromatographi c system was applied to the determination of leukotriene B-4, leukotri ene C-4, leukotriene D-4, leukotriene and E-4 in human cell extracts u sing prostaglandin B-2 as the internal standard. More than 1500 biolog ical samples were analysed. Some validation data are presented for una ttended operations.