Biological Sample preparation is a technique used to clean up a sample before analysis and /or to concentrate a sample to improve its detection. When samples are biological fluids such as plasma, serum or urine, this technique is described as bioanalytical sample preparation. Removal of unwanted matrix components primarily protein that would interfere with analyte determination. Concentration of analyte to meet the detection limits of the analytical instrument.  Exchange of the solvent or solution in which the analyte resides so that it is compatible with mobile phase for injection into a chromatographic system. Dilution to reduce solvent strength or avoid solvent incompatibility. Stabilization of analyte to avoid hydrolytic or enzymatic degradation. After selection of biological fluid the required analyte should be extracted from it. This step in bioanalytical method is more important because sample preparation can be done by different methods of extraction .The sample preparation is a time taking process and it should be done carefully because of its importance. If biological matrix is in liquid state like blood, plasma and urine then liquid-liquid extraction is used or it is solid then liquid-solid extraction can be done.

Protein precipitation is based on the interaction between the precipitation reagent and protein groups. Soluble proteins generally have a hydrophobic core surrounded by a hydrophilic surface including ionic groups that are not involved in intra-molecular binding. Organic solvents interfere with the intra-molecular hydrophobic interactions of proteins. The addition of a volume of solvent (frequently acetonitrile) to the serum causes the proteins of the serum to precipitate and leaves the analyte of interest in the solvent, which can either be injected directly or dried down and reconstituted in a smaller volume to concentration before injection. While this is the fastest and simplest method for sample preparation, it is the most likely to cause ion suppression issues, especially in ESI, where the coelution of endogenous compounds such as lipids, phospholipids and fatty acids affect the ESI droplet desolvation process.

To the biological fluid two immiscible solvents are added and centrifuged, then the organic solvent is evaporated. To ensure rapid equilibrium more surface area is required that is achieved by thoroughly mixing or manually shaking. The residue thus obtained is reconstituted with the suitable solvent of small volume which is compatible with HPLC separation while analytes extracted in aqueous phase can be directly injected into HPLC. More than one sample can be extracted in this method. To do this method effectively some steps are present, they are,  facilitate removal of the extraction solvent at the end with low boiling point is used.  Solvent with low viscosity is used to facilitate mixing with the sample. Analyte must be soluble in extracting solvent. The organic solvent selected should have low solubility in water, high purity and should be compatible for the HPLC detection techniques.Solid-phase extraction (SPE) is a sample preparation process by which compounds that are dissolved or suspended in a liquid mixture are separated from other compounds in the mixture according to their physical and chemical properties.

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 Amelia Johnson
 Managing Editor
Single Cell Biology.