Biomedical Engineering Reference
In-Depth Information
solid phase extraction (SPE). The optimum extraction technique depends on the
analytical requirements, biological matrix, chromatographic separation, or the need
for high throughput.
Several authors described a minimum sample pretreatment by plasmatic pro-
tein precipitation using acetonitrile (ACN), MeOH, acid solutions, or a mixture
of them, for the analysis of one antidepressant and its metabolite(s) [
46-
52
] .
Single analyte procedures are useful for TDM or to perform pharmacokinetic,
bioequivalence and pharmacogenomic studies, where the targeted antidepressant
is known; however, this is an unrealistic situation in clinical and forensic toxicol-
ogy, and for those applications, multianalyte procedures are preferable. Only
Kirchherr et al. [
53
] used protein precipitation with ACN/MeOH as the only
sample treatment procedure for the determination of 48 psychopharmaceuticals
in human serum, including the main antidepressant drugs. No matrix effect was
reported for any of the analytes, except for olanzapine, for which a 185% signal
enhancement was observed.
However, protein precipitation usually produces severe matrix effect [
54-
56
] .
Therefore, in most LC-MS published methods, more extensive sample extraction
procedures were used, being liquid-liquid (LLE) and SPE the most frequent
techniques.
2.1
Liquid-Liquid Extraction (LLE)
In LLE, analytes are isolated from the biological matrix by means of an organic
solvent immiscible with aqueous solution. Due to the basic character of antidepres-
sants, samples are initially alkalinized with NaOH, NH
4
OH or a basic buffer to
achieve a pH >8.5. The polarity range of the analytes included in the analytical
method, as well as the biological matrix, determine the organic solvent of choice.
Hexane, dichloromethane, butyl chloride, butyl acetate, isoamylic acid, or a mixture
of them, have been used for the extraction of several antidepressants from the bio-
logical samples [
39,
57-
61
]. Apart from those previously mentioned, other solvents
employed for the extraction of only one antidepressant and/or metabolite(s) were
diethylether, ethyl acetate or methyl
tert
-butylether [
62-
64
] . After centrifugation,
the organic layer is evaporated to dryness and reconstituted in a small volume for
LC-MS analysis. In some cases, the organic layer was reextracted before evapora-
tion by addition of aqueous acid solution to obtain cleaner extracts. De Santana
et al. [
42
] and Halvorsen et al. [
65
] determined one or several antidepressants,
respectively, in whole blood and/or plasma, using liquid-phase microextraction
(LPME). LPME is a minituarized LLE procedure, where analytes are extracted
from the biological matrix through an organic solvent impregnated in the pores of a
hollow fiber and into a micro-liquid phase (acceptor solution) inside the fiber, which
is subsequently injected into the LC-MS system. The main advantage of this tech-
nique is that it enables simultaneous analyte preconcentration and sample cleanup,
using small sample and solvent volumes.
