unstable stereo-configuration [89] and could undergo enantiomer interconversion
during degradation but also during sample preparation and storage. This is a very
sensitive step. For MTP, pure enantiomers were commercially available and separately incubated in soil slurry experiments. Enantiomer interconversion was never
observed. Due to the lack of enantiopure standards of CBZ, potential CBZ
enantiomerization was investigated in the presence of D 2 O, where the formation of
deuterated CBZ enantiomers was observed. However, the rate constants for deuteration reaction (K deut ) were found to be slightly higher for CBZ (E1) than for CBZ
(E2) (K deut ¼ 0.91 and 1.12 Â 10
À5 min
À1 (T 1/2 ¼ 52.9 days and 43.0 days) for CBZ
(E1) and CBZ (E2), respectively). This result could be explained by the very weak
hydrogen acidity of the α-carbonyl carbon precluding the C–H bond cleavage, the
formation of a carbocation, and at the end the possibility of CBZ
enantiomerization [87].
Chiral analytical methods were then developed for the determination of the ER of
MTP and CBZ in soil slurries. In routine chiral analysis, several rules should be
respected. The most important one is the removal as much dissolved organic matter
as possible because humic and fulvic substances are good chiral selectors which
usually degrade the quality of the enantiomer separation after a few injections.
Consequently, a resolution >1 should be obtained for accurate ER calculation.
Finally, quantification by isotopic dilution is compulsory because matrix effect
causes different ion suppression of a pair of enantiomers in electrospray with
negative implications in ER calculation. In practice, 10-mL supernatant of soil
slurries were percolated through mixed-mode cation exchange cartridges after
water acidification at pH 3 to remove as much dissolved organic matter as possible.
The use of a sequential elution protocol allows for the removal of neutral and acidic
interferences before analyte elution with methanol containing ammonia. This led to a
significant reduction of matrix effects in LC-HRMS analysis [87]. Enantiomers of
MTP and those of its major transformation products were separated using an ASTEC
vancomycin-based analytical column (Chirobiotic V) using a reverse phase isocratic
mode of elution with a mobile phase consisting of water +30 mM ammonium
acetate/methanol, 10/90 (v/v) (see Fig. 3a). Enantiomers of CBZ were separated
using a Phenomenex Lux Amylose-2 analytical column only using water/acetonitrile
(35/65, v/v) as mobile phase in an isocratic mode of elution (see Fig. 3b). LODs
down to 10 ng L
À1 were obtained with both analytical methods, which made them
suitable for the analysis of MTP and CBZ in soil slurries.
The validated chiral analytical methods were then applied to investigate the
relationship between the evolution of the enantiomeric ratio of MTP and CLB and
the extent of their biodegradation rates in soil slurry experiments under laboratory
control conditions. Soil slurries were spiked with MTP or CBZ at concentrations
close to environmental concentrations, that is, 20 μg L
À1 . MTP incubations were
carried under aerobic conditions by bubbling the reactor with air, and CBZ incubations were conducted under anoxic conditions in 100 mL serum bottles, sealed with
butyl rubber stoppers and aluminum caps in which syringes were inserted for sample
collection. Serum bottles were charged with 10 g of an agricultural soil and filled
with secondary treated wastewater collected at a biological wastewater treatment
164
M. Brienza et al.
during degradation but also during sample preparation and storage. This is a very
sensitive step. For MTP, pure enantiomers were commercially available and separately incubated in soil slurry experiments. Enantiomer interconversion was never
observed. Due to the lack of enantiopure standards of CBZ, potential CBZ
enantiomerization was investigated in the presence of D 2 O, where the formation of
deuterated CBZ enantiomers was observed. However, the rate constants for deuteration reaction (K deut ) were found to be slightly higher for CBZ (E1) than for CBZ
(E2) (K deut ¼ 0.91 and 1.12 Â 10
À5 min
À1 (T 1/2 ¼ 52.9 days and 43.0 days) for CBZ
(E1) and CBZ (E2), respectively). This result could be explained by the very weak
hydrogen acidity of the α-carbonyl carbon precluding the C–H bond cleavage, the
formation of a carbocation, and at the end the possibility of CBZ
enantiomerization [87].
Chiral analytical methods were then developed for the determination of the ER of
MTP and CBZ in soil slurries. In routine chiral analysis, several rules should be
respected. The most important one is the removal as much dissolved organic matter
as possible because humic and fulvic substances are good chiral selectors which
usually degrade the quality of the enantiomer separation after a few injections.
Consequently, a resolution >1 should be obtained for accurate ER calculation.
Finally, quantification by isotopic dilution is compulsory because matrix effect
causes different ion suppression of a pair of enantiomers in electrospray with
negative implications in ER calculation. In practice, 10-mL supernatant of soil
slurries were percolated through mixed-mode cation exchange cartridges after
water acidification at pH 3 to remove as much dissolved organic matter as possible.
The use of a sequential elution protocol allows for the removal of neutral and acidic
interferences before analyte elution with methanol containing ammonia. This led to a
significant reduction of matrix effects in LC-HRMS analysis [87]. Enantiomers of
MTP and those of its major transformation products were separated using an ASTEC
vancomycin-based analytical column (Chirobiotic V) using a reverse phase isocratic
mode of elution with a mobile phase consisting of water +30 mM ammonium
acetate/methanol, 10/90 (v/v) (see Fig. 3a). Enantiomers of CBZ were separated
using a Phenomenex Lux Amylose-2 analytical column only using water/acetonitrile
(35/65, v/v) as mobile phase in an isocratic mode of elution (see Fig. 3b). LODs
down to 10 ng L
À1 were obtained with both analytical methods, which made them
suitable for the analysis of MTP and CBZ in soil slurries.
The validated chiral analytical methods were then applied to investigate the
relationship between the evolution of the enantiomeric ratio of MTP and CLB and
the extent of their biodegradation rates in soil slurry experiments under laboratory
control conditions. Soil slurries were spiked with MTP or CBZ at concentrations
close to environmental concentrations, that is, 20 μg L
À1 . MTP incubations were
carried under aerobic conditions by bubbling the reactor with air, and CBZ incubations were conducted under anoxic conditions in 100 mL serum bottles, sealed with
butyl rubber stoppers and aluminum caps in which syringes were inserted for sample
collection. Serum bottles were charged with 10 g of an agricultural soil and filled
with secondary treated wastewater collected at a biological wastewater treatment
164
M. Brienza et al.