Determination of a potent urokinase-type plasminogen activator, UK-356,202, in plasma at pg/mL levels using column-switching HPLC and fluorescence detection

A rapid, sensitive and selective method using column-switching HPLC with fluorescence detection has been developed for the determination of UK-356,202, a potent urokinase-type plasminogen activator, in human plasma. A structural isomer of UK-356,202 is used as an internal standard. The lower limit of quantification is 20 pg/mL and the method is linear over a 100-fold concentration range. UK-356,202 is extracted from plasma simply through the removal of proteins by precipitation with acetonitrile. The HPLC system comprises three columns and the cycle time is 9.5 min per sample. The eluate from the extraction column is heart-cut onto a trace enrichment cartridge which is then back-flushed onto a narrow-bore Supelco ABZ+ Plus™ analytical column. The method has been used to analyze many thousands of samples from clinical and toxicological studies support. Its ruggedness is demonstrated by the use of a single extraction column for the analysis of over 1200 clinical samples.