Protein Precipitation Fundamentals

Most biological matrices contain protein to varying extents. Protein-binding phenomena are known to influence drug-drug interactions in the clinical setting. Among the various plasma proteins, serum albumin is the most widely studied and is regarded as the most important carrier for drugs. The presence of these materials for bioanalysis is problematic, and they must be removed before chromatographic separation and detection. When injected into a chro-matographic system, proteins will precipitate upon contact with the organic solvents used in LC mobile phases. The precipitated mass of protein builds up within the column inlet, reduces the column lifetime and increases the system back pressure. When protein is carried through the analytical system, it may reach the mass spectrometer to foul the interface and require frequent cleaning; the result is system downtime.

A common approach to remove proteins from an injected sample is precipitation with an organic solvent (methanol, ethanol, or acetonitrile). Typically, a volume of sample matrix (1 part) is diluted with a volume of precipitating agent (3 or 4 parts), followed by vortex mixing, and then centrifugation or filtration to isolate or remove the mass of precipitated protein (Figure 17.1). The supernatant or filtrate is then analyzed directly. Note that ionic salts (saturated

Sample Spiked with IS

Precipitating Agent (150 |XL Acetonitrile)

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