Direct Plasma Analysis

Direct Plasma Analysis

Analysis of plasma and other biological fluids by HPLC usually requires several steps of sample preparation. Tasks such as solid-phase extraction, liquid-liquid extraction, centrifugation, and filtration are usually necessary steps prior to actual HPLC analysis in order to remove proteins and peptides from plasma to protect the HPLC column. When plasma is analyzed directly, the efficiency of the column degrades quickly due to irreversible adsorption of some components of the plasma. A multi-step process increases the cost and compromises the accuracy of determination.

SIELC Technologies now offers a simple solution which allows you to analyze a broad range of small molecules in plasma or other biofluids via a single-column system without any sample preparation or common mobile phase.
This approach is based on the unique stationary phase of Primesep® D column that is comprised of two types of functional groups — an anion exchange group and long alkyl chain — chemically bonded to silica support.

This combination of a very polar positively-charged group and a very hydrophobic alkyl group in a single ligand on a surface of the stationary support allows direct injection of plasma and other biofluids. At a pH around 3.0, which can be obtained by adding of formic acid to the mobile phase of MeCN-H2O, most proteins become positively charged and are excluded from interaction with the surface of the stationary phase. Small negatively charged molecules can be separated and retained on the Primesep D column by anion-exchange mode or reverse phase mode chromatography. Small hydrophobic molecules are retained and can be separated by regular RP mode on the Primesep D column.

The Primesep® D column demonstrates high analyte recovery and high selectivity. Proteins and peptides elute as a sharp peak in void or pre-void time and they don’t interfere with the analytes’ peaks. A simple bypass valve can be used to remove the protein peak from reaching the detector by diverting flow to waste in the first 40 seconds (for a 50 mm long column — time may need to be increased if longer columns are used).

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