Greener solvents for microelution solid phase extraction of proteins from biological fluids followed by their top-down CE-MS analysis

In recent years, there has been a growing demand for making the analytical processes sustainable and environmentally friendly. In proteomic analysis, sample preparation plays an important role, employing different extraction techniques to isolate proteins of interest from biological fluids to be able to analyze them. These techniques usually use large volumes of organic solvents, such as acetonitrile, which has been recognized as hazardous and difficult to dispense. According to green analytical chemistry principles, the environmental impact of the methods can be reduced either by miniaturizing the processes or by replacing organic solvents with less toxic alternatives. This work compares the effect of different greener solvents on the solid-phase extraction process of low molecular weight proteins spiked in biological fluids (pooled human serum, plasma, urine, and saliva) and suggests that ethanol (60 %, v/v) offers, when used as an elution solvent, comparable results to acetonitrile (75 % v/v) in terms of extraction recovery. The addition of acidic modifiers to the elution solvent did not improve the extraction recovery, and the nitrogen evaporation of the eluate resulted in a 17–53 % loss of the proteins compared to the simple dilution of the eluate.