Analysis of Polar Compounds in Biological Matrix with LC/MS/MS via “Normal Phase” LLE for Sample Preparation
authors
Kristopher W. King, Chris Tran, Guangyu Zhao, Ling Morgan
Tandem Labs, New England
introduction
Three sample preparation techniques are commonly used to extract analytes out of biological
matrices: protein precipitation (PPT), supported liquid extraction (SLE) or liquid-liquid extraction
(LLE), and solid phase extraction (SPE). Among of three, PPT is the most used sample preparation
approach in early drug discovery due to less method development time required, therefore
fast data turn around time. However, choosing which extraction approach is best is compound
dependent in real practice. Analysis of highly hydrophilic molecules in biological matrices presents
challenges due to low extraction recovery from biological matrices. Commonly, various solid phase
extraction (SPE) procedures are used due to the abundance of different retention mechanisms
available to retain the desired analytes, and extract them out of bio-matrices. Often this involves
time consuming method development, not suitable for quick turn around. SLE or LLE performed
with common organic solvents such as methyl t-butyl ether (MTBE), hexanes/ethyl acetate, and
1-chlorobutane commonly cannot apply due to their low LogP index. Also, PPT contributes to the
sample extracts a fair amount of phospholipids and dose vehicle that concentrate during the drydown
and reconstitution step. The competition between aqueous and organic phases may result
in a lower compound recovery. Also, lower mass compounds can be lost to evaporation during the
dry-down period. Herein, we evaluate common approaches and problems in dealing with small
hydrophilic molecules in bio-matrices in early stage drug discovery.
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