Direct untreated biological samples

In recent years, special SPE supports possessing restricted access properties have been developed [61-65] to allow the direct injection of untreated biological samples into online SPE-LC systems. 

Some chromatographic systems have been designed in which untreated biological samples can be applied directly to the paper. The impure sample on the paper is chromatographed with hexane, which elutes the lipids with the solvent front. After drying, the paper is developed with pure water (if the analyte is water insoluble) to elute salts, sugars, etc. at the front. Finally, development with the proper mobile phase for the analytical separation is carried out, to a point below the purification developments. 

Because of the detrimental effect of the presence of water on the column substrate, and the risk of irreversible interactions of some matrix constituents with the column material, direct application of an untreated biological sample is not advisable. Consequently, a sample pretreatment step is always required. This step may involve the removal of most of the matrix constituents by ion-exchange chromatography, ultrafiltration and/or dialysis followed by solvent extraction and derivatization of the species under study. The complexity of the method chosen would depend on the stability of the species and the extent to which other matrix constituents interfere with its isolation. 

Today, there is strong interest in the development of online sample treatment techniques that allow the handling of untreated biological samples. Thus, in online SPE-LC, deproteination of plasma and serum is required before extraction, especially if the same cartridge is used for repeated analysis. For this purpose, restricted-access materials (RAMs) have been developed, which combine size-exclusion and reversed phase mechanisms, allowing extraction and cleanup of samples in the same step. RAMs have became quite popular for the direct injection of biological fluids, since they prevent the access of matrix components (e.g., proteins) while retaining the analytes in the interior of the sorbent. 

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