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 approach to increasing sample throughput involves precipitated protein removal by filtration in the 96-well format, thereby eliminating the need for centrifugation and manual handling of individual tubes. The potential for such a new technique has been investigated for the determination of an iron chelator, a highly protein-bound compound (> or =99.5%) in plasma. An analog was used as internal standard. Acetonitrile and plasma were sequentially aspirated, separated by an air gap, using a manual electronic pipettor. They were then dispensed into the channel of an Empore filter PPT plate above the filter, and a slight vacuum was applied. The eluate was collected and diluted prior to injection. The compounds were then separated by reversed-phase chromatography and detected by UV at 295 nm. The chromatographic run time was 6 min. The mean recovery following protein precipitation was 78%, which shows that the technique can apply to a highly protein-bound compound. Replicate quality control samples were prepared in drug-free normal human plasma at four different concentrations. The mean accuracy ranged from 87 to 108% with the CV ranging from 3 to 8%. The described procedure is simple, fast and reproducible. It requires minimal equipment. The time required to prepare a plate manually is only about 20 min. The use of 12-channel repeater pipettors reduces the risk of error and improves productivity. Automation should be an aid to further increasing sample throughput when more than one plate a day is to be prepared.
Rouan
M C
MC
Drug Metabolism and Pharmacokinetics, Novartis Pharma SA, 2-4 rue Lionel Terray, 92500, Cedex, Rueil-Malmaison, France. marie-claude.rouan@galderma.com
Buffet
C
C
Marfil
F
F
Humbert
H
H
Maurer
G
G
D001565:Benzoates / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D000077588:Deferasirox; D005374:Filtration; D007502:Iron Chelating Agents / Q000737:chemistry; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet; D014230:Triazoles / Q000737:chemistry
D001565:Benzoates; D001798:Blood Proteins; D007502:Iron Chelating Agents; D014230:Triazoles; D000077588:Deferasirox