A semi-automated 96-well protein precipitation method for the determination of montelukast in human plasma using high performance liquid chromatography/fluorescence detection

Abstract

A simple, semi-automated, protein precipitation assay for the determination of montelukast (SINGULAIR™, MK-0476) in human plasma has been developed. Montelukast is a potent and selective antagonist of the cysteinyl leukotriene receptor used for the treatment of asthma. A Packard MultiPROBE®II EX is used to transfer 300 μl of plasma from sample, standard, and QC sample tubes to a microtiter plate (96-well). After addition of the internal standard by a repeating pipettor, a Tomtec QUADRA 96® adds 400 μl of acetonitrile to all plasma sample wells, simultaneously, in the microtiter plate. The Tomtec is also used to transfer the acetonitrile supernatant from the plasma protein precipitation step, batchwise, to another microtiter plate for analysis by HPLC with fluorescence detection. This assay has been validated and implemented for a clinical study of over 1300 plasma samples and is comparable to manual assays in the LLOQ (lower limit of quantitation, 3 ng/ml) and in stability. This is the first semi-automated protein precipitation assay published for the analysis of montelukast in human plasma and it results in significant time savings over the manual methods, both in sample preparation and in HPLC run time.