compound or different modes of action regarding their biological activity. Last, but
not least, metabolites of PPCPs formed eventually during the passage through
WWTPs may get transformed back to the parent substance when taken up by plants.
In general, the metabolization of xenobiotics in plants follows the pathways
known from mammalian organisms. During phase I, xenobiotics are transformed
into more polar compounds (that also exhibit a higher water solubility) by reactions
like hydroxylation, which are catalyzed by enzymes such as cytochrome P450
oxidases. Phase II involves the conjugation of phase I metabolites with small
molecules like hexoses (whereas in mammalian organisms glucuronic acid is commonly attached), amino acids, sulfate, or glutathione. Various transferases are
involved in this step, such as glutathione S-transferases (GSTs) with structures
close to that in mammalian organisms except for a catalytic serine in place of a
tyrosine residue [27]. During phase III the metabolites may be further transformed
(an example is the reaction of glutathione conjugates to acetylcysteine conjugates),
and in case of mammalian organisms, the metabolites are excreted. In case of plants,
metabolites undergo compartmentalization and binding to cell walls. This fraction is
not readily available for extraction and analysis, but its extent can be estimated by
using
14 C-labeled parent drugs and measuring the activity in the plant cells, as
recently shown for investigations of the metabolism of naproxen and ibuprofen in
the Arabidopsis plant [28].
The investigation of the uptake and metabolization of a certain PPCP by a plant
may be done in the form of two different approaches, namely, either by a targeted
analysis or by an untargeted analysis. In case of targeted analysis, a limited number
of metabolites are defined for quantitation prior to the analysis itself. These potentially present metabolites may be selected according to existing knowledge about
metabolization pathways in plants or on the basis of studies done on mammalian or
microbial organisms. One can also use in silico prediction approaches and tools
available as commercial software packages or freeware for selecting the target
metabolites (for a review dealing with such tools, see, e.g., [29]). Such a targeted
analysis makes an effective optimization of the sample preparation and analyte
preconcentration methods for the selected analytes possible, so that quantitation
limits can be achieved that may be low enough for investigating plants irrigated with
real reclaimed water.
Contrary to targeted analysis, the untargeted approach aims at a comprehensive
detection of known and unknown metabolites formed from PPCPs taken up by the
plant. For that purpose techniques are available that are nowadays commonly used in
metabolomics of mammalian systems and rely on high-performance liquid chromatography (HPLC) hyphenated with high-resolution mass spectrometry (HPLC-HRMS) [30–32]. When unknown metabolites are the focus, the sample pretreatment
including preconcentration steps can hardly be fully optimized which may compromise quantitation limits. Therefore, untargeted analysis of metabolites in plants is
often done by treating model plants (or plant cell cultures) with relatively high
concentrations of PPCPs. In this way, the detection of new metabolites and their
subsequent identification may become possible, and in a second step, a targeted
approach can be added for real samples.
Analytical Approaches for the Determination and Identification of Drug. . .
497
not least, metabolites of PPCPs formed eventually during the passage through
WWTPs may get transformed back to the parent substance when taken up by plants.
In general, the metabolization of xenobiotics in plants follows the pathways
known from mammalian organisms. During phase I, xenobiotics are transformed
into more polar compounds (that also exhibit a higher water solubility) by reactions
like hydroxylation, which are catalyzed by enzymes such as cytochrome P450
oxidases. Phase II involves the conjugation of phase I metabolites with small
molecules like hexoses (whereas in mammalian organisms glucuronic acid is commonly attached), amino acids, sulfate, or glutathione. Various transferases are
involved in this step, such as glutathione S-transferases (GSTs) with structures
close to that in mammalian organisms except for a catalytic serine in place of a
tyrosine residue [27]. During phase III the metabolites may be further transformed
(an example is the reaction of glutathione conjugates to acetylcysteine conjugates),
and in case of mammalian organisms, the metabolites are excreted. In case of plants,
metabolites undergo compartmentalization and binding to cell walls. This fraction is
not readily available for extraction and analysis, but its extent can be estimated by
using
14 C-labeled parent drugs and measuring the activity in the plant cells, as
recently shown for investigations of the metabolism of naproxen and ibuprofen in
the Arabidopsis plant [28].
The investigation of the uptake and metabolization of a certain PPCP by a plant
may be done in the form of two different approaches, namely, either by a targeted
analysis or by an untargeted analysis. In case of targeted analysis, a limited number
of metabolites are defined for quantitation prior to the analysis itself. These potentially present metabolites may be selected according to existing knowledge about
metabolization pathways in plants or on the basis of studies done on mammalian or
microbial organisms. One can also use in silico prediction approaches and tools
available as commercial software packages or freeware for selecting the target
metabolites (for a review dealing with such tools, see, e.g., [29]). Such a targeted
analysis makes an effective optimization of the sample preparation and analyte
preconcentration methods for the selected analytes possible, so that quantitation
limits can be achieved that may be low enough for investigating plants irrigated with
real reclaimed water.
Contrary to targeted analysis, the untargeted approach aims at a comprehensive
detection of known and unknown metabolites formed from PPCPs taken up by the
plant. For that purpose techniques are available that are nowadays commonly used in
metabolomics of mammalian systems and rely on high-performance liquid chromatography (HPLC) hyphenated with high-resolution mass spectrometry (HPLC-HRMS) [30–32]. When unknown metabolites are the focus, the sample pretreatment
including preconcentration steps can hardly be fully optimized which may compromise quantitation limits. Therefore, untargeted analysis of metabolites in plants is
often done by treating model plants (or plant cell cultures) with relatively high
concentrations of PPCPs. In this way, the detection of new metabolites and their
subsequent identification may become possible, and in a second step, a targeted
approach can be added for real samples.
Analytical Approaches for the Determination and Identification of Drug. . .
497