Biomedical Engineering Reference
In-Depth Information
different requirements on qualitative and quantitative analysis of TA. Therefore, the
following main part of this chapter is addressed to corresponding sample preparation
procedures and specific characteristics depending on the aim of TA measurement.
3
Analysis of Biological Samples
Selective and sensitive analysis of TA requires adequate sample preparation to
reduce sample matrix complexity and concentrate the analyte. Subsequent chroma-
tography and MS detection should also account for potential matrix interferences
allowing rapid measurement and a sufficient limit of quantification. The next sec-
tion describes most important and frequent techniques for sample preparation.
3.1
Sample Preparation
In principle, sample preparation is performed (a) to clean the analyte by separating
from matrix compounds that might deteriorate selectivity and sensitivity in subse-
quent LC-MS analysis and (b) to concentrate the target molecules to achieve opti-
mum limits of quantification. Several conventional, less selective techniques are
well established including simple protein precipitation, LLE and SPE. The appro-
priate choice of the procedure depends on the purpose and requirements of analy-
sis. Non-selective protein precipitation appears useful when a larger number of
analytes with differing polarity (e.g. the drug and its polar and conjugated biotrans-
formation products) has to be measured simultaneously. For this purpose LLE
might be less adequate as charged and highly polar derivatives exhibit minor ten-
dency to be transferred into a non-polar environment. In contrast, modern SPE
columns provide solid materials that allow interaction with analytes exhibiting a
broad range of polarity.
The following paragraphs present these techniques for sample preparation
applied to TTA and QTA LC-MS analysis.
3.1.1
Protein Precipitation
Protein precipitation represents one of the most simple and quite frequent prepa-
ration methods for protein-containing fluids allowing rapid performance to reduce
costs. The addition of organic solvents, e.g. acetonitrile (ACN), methanol (MeOH)
or ZnSO
4
, causes deproteinization allowing separation of particulate matter from
clear analyte containing supernatant with good recovery. Elimination of high
molecular weight proteins is an important step for subsequent chromatographic
