Plasma deproteinization by precipitation and filtration in the 96-well format

The need for fast bioanalytical methods within the pharmaceutical sector is rapidly growing. Sample preparation is often the bottleneck step. A new ap proach to increasing sample throughput involves precipitated protein remova l by filtration in the 96-well format, thereby eliminating the need For cen trifugation and manual handling of individual tubes. The potential for such a new technique has been investigated for the determination of an iron che lator, a highly protein-bound compound (greater than or equal to 99.5%,) in plasma. An analog was used as internal standard. Acetonitrile and plasma w ere sequentially aspirated, separated by an air gap, using a manual electro nic pipettor. They were then dispensed into the channel of an Empore (TM) f ilter PPT plate above the filter, and a slight vacuum was applied. The elua te was collected and diluted prior to injection. The compounds were then se parated by reversed-phase chromatography and detected by UV at 295 nm. The chromatographic run time was 6 min. The mean recovery following protein pre cipitation was 78%,. which shows that the technique can apply to a highly p rotein-bound compound. Replicate quality control samples were prepared in d rug-free normal human plasma at four different concentrations. The mean acc uracy ranged from 87 to 108% with the CV ranging from 3 to 8%. The describe d procedure is simple, fast and reproducible. It requires minimal equipment . The time required to prepare a plate manually is only about 20 min. The u se of 12-channel repeater pipettors reduces the risk of error and improves productivity. Automation should be an aid to further increasing sample thro ughput when more than one plate a day is to be prepared. (C) 2001 Elsevier Science B.V. All rights reserved.