CPSA Digest 2002

Proceedings -Tuesday, October 8, 2002

TuOC2

Protein Precipitation Demonstrated in Microwell Filter Format for LC-MS/MS Bioanalytical Sample Preparation

The drug discovery process is increasingly focused upon LC-MS/MS as an analytical technique for the determination of drug concentrations in biological fluids. A reliable analytical method is achieved with the successful combination of efficient sample preparation, adequate chromatographic separation and a sensitive detector. An important component in the overall analysis is the choice of sample preparation technique, which influences the cleanliness of the sample introduced to the LC/MS/MS system. The success of fast LC has demanded faster methods for bioanalytical sample preparation, i.e., robust techniques that are easily multiplexed with high reproducibility.

Today there are at least three distinct methods that are used for sample preparation during the process of drug discovery bioanalysis. These techniques include protein precipitation and filtration, liquid-liquid extraction, and solid phase extraction. The choice of which procedure to use should be made following extensive analysis of criteria such as the number of samples in a batch, complexity of the sample matrix (such as urine, plasma or serum), the volumes of sample available, the need for reduction of ionization suppression from matrix interferences, and the level of automation that can be utilized. Automation in the 96-well format and integration of such procedures into either a task-oriented workstation or an application-customized liquid handling station are key features to attaining the reproducibility and speed required for sample preparation.

The extensive diversity in chemical structures seen in a drug discovery laboratory requires a generic, rapid concentration technique that can be performed with only a basic understanding of laboratory techniques. Removal of protein in the sample matrix through denaturation with acetonitrile affords such a simple, generic approach to sample preparation. A 96-well filter plate is now available from Orochem Technologies that provides a reactor-like environment by preventing leakage of aqueous and organic phase mixture for several hours until pressure is applied. This protein-crash plate offers the unique ability to conduct, in an automated fashion, the protein crash procedure, the mix step and the filtration of protein precipitants in the same 96-well flow-through plate. The extensive use of protein precipitation is indicative that the analyst is cognizant of the rapid speed requirements and general selectivity offered by this technique; co-eluting analytes are removed during the process of chromatographic separation.

Orochem 96-well Filter Plates are compatible with all vacuum accessories and liquid handling workstations. These plates are available in 1mL (left, OMNPPT20) and 2mL (right, OC21PPT20) formats.

Product information: www.orochem.com/products_top1_page3.htm
Application Note: www.orochem.com/products_top1_page3_info.htm

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