Biomedical Engineering Reference
In-Depth Information
separation to minimize the risk of clotting, column obstruction and shifting reten-
tion properties caused by irreversible protein adsorption to the stationary phase.
Nevertheless, protein precipitation lacks selectivity and produces a still quite
complex matrix of salts and other ionic, hydrophilic or slight hydrophobic com-
ponents that might cause interferences in MS detection. Therefore, analyte con-
centrations have to be effectively high or additional preparation steps will be
necessary. Furthermore, co-precipitation of analytes bound to precipitated pro-
teins might reduce recovery. However, such effects have not been discussed in the
literature for TA analysis.
The use of organic solvents may constitute a matrix compatible to subsequent
liquid chromatography, thus not requiring any concentration or evaporation step.
However, protein precipitation seems to be inappropriate for automation and thus
requires a manual workflow.
Table 2 summarizes reports describing sample precipitation for TA analysis.
John et al. added twice the volume of ACN to plasma samples thus generating a
supernatant containing 66 % (v/v) ACN. Merely dilution with aqueous HPLC sol-
vent A [0.1 % formic acid (FA)] was necessary prior to injection allowing quantita-
tive analysis of R - and S -hyoscyamine [ 49 ] as well as atropine, cocaine, homatropine,
ipratropium, littorine, N -butyl-scopolamine and scopolamine, simultaneously [ 50 ] .
Recoveries were greater than 85 % (Table 2 ). Despite this crude preparation proce-
dure effects on ionization were negligible.
Chen et al. used MeOH for precipitation of rat plasma in a 3:1 volume ratio to
investigate a broad spectrum of biotransformation products generated from ani-
sodine [ 5 ] , anisodamine [ 6 ] , atropine [ 52 ] and scopolamine [ 87 ] . Biotransformation
products covered a broad spectrum of polarity including sulfo- and glucuronide
conjugates, oxidized, hydroxylated, methoxylated and demethylated metabolites of
the parent drug as well as its hydrolysis products. Unfortunately, recoveries were
not reported (Table 2 ).
Kajbaf et al. obtained 100 % recovery for cimetropium when adding aqueous
1 % (w/v) ZnSO 4 solution to buffered liver microsomes incubation mixtures.
Subsequently a SPE step was carried out to clean and concentrate the cimetropium
containing fraction [ 23, 62 ] .
In comparison to LLE and SPE, protein precipitation was applied less frequently
especially for quantitative measurement of TTA or QTA.
3.1.2
Liquid-Liquid Extraction
For LLE the liquid sample is mixed with a larger volume of a non-polar organic
solvent to induce a partition equilibrium of the analyte between the aqueous and
organic layer. Typically, the latter one contains the major fraction of analytes and is
further processed for LC-MS analysis. In contrast to protein precipitation, LLE thus
requires a subsequent drying step by evaporation or with a gentle stream of nitrogen
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