Protein precipitation from biological fluids

Biological matrices are not directly compatible with LC-MS analysis, since these samples tend to block LC columns and contaminate the ion source. Extraction of compounds of interest from biological fluids is required prior to LC-MS/MS analysis [20]. Sample extraction can be achieved off-line with protein precipitation (PP), liquid-liquid extraction (LLE), or solid-phase extraction (SPE) [21]. With the ease of use and sophistication of automated liquid-handling systems, sample extraction procedures in a 96-well format can handle microliter volumes with multiple sorbents per plate and can simplify and expedite SPE method development [22,23]. The technique can be used to routinely develop methods for multiple analytes and examine a set of eluent compositions for each analyte [16]. 

A potential problem with the analysis of biological fluids, especially plasma samples from in vivo studies, is the risk of clogging of SPE cartridges and/or analytical columns. Therefore, filtration of such samples prior to LC or SPE is recommended. Combined filtering and protein precipitation in 96-well plate format was described [97-98]. The samples are collected and stored frozen in sealed 96-well polypropylene filter plates. Prior to SPE and LC-MS analysis, the seals are removed and the plate is placed on top of a 96-well SPE manifold As the plasma thaws, it passes through the filter and into the SPE device. 

Specimens may be subjected to distillation, protein precipitation, or solvent extraction for the separation of ethanol from the biological fluid. Most recently, the determination of ethanol and other volatiles has been accompanied by introducing the sample into the gas chromatograph either directly as a liquid or as a... 

An immunochemical technique for the determination of specific proteins in biological fluids. It consists of placing the samples in wells cut out of a sheet of agar gel into which specific antiserum has been incorporated. Diffusion of the proteins from the well into the gel results in the formation of antibody-antigen complexes which precipitate in the form of a ring around the well. The concentration of the protein antigen is proportional to the area (and therefore the diameter squared) of the precipitin ring. 

For biological fluids, SPE has been widely employed for the extraction of doping agents and veterinary drugs from urine and plasma. However, a number of authors have reported the use of simple sample treatments such as plasma protein precipitation or urine dilute-and-shoot (64,65). These procedures were only used during the initial screening step and not the confirmation step. In addition, TOF/MS devices were generally employed to compensate for the lack of selectivity and sensitivity afforded by these sample preparation procedures. 

Initially fermentation broth has to be characterised on the viscosity of the fluid. If the presence of the biomass or cells causes trouble, they have to be removed. Tire product is stored inside the cells, the cells must be ruptured and the product must be freed. Intracellular protein can easily be precipitated, settled or filtered. In fact the product in diluted broth may not be economical enough for efficient recovery. Enrichment of the product from the bioreactor effluents for increasing product concentration may reduce the cost of product recovery. There are several economical methods for pure product recovery, such as crystallisation of the product from the concentrated broth or liquid phase. Even small amounts of cellular proteins can be lyophilised or dried from crude solution of biological products such as hormone or enzymes.2,3... 

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