of 4′-OH-DCF.7 With respect to microbial transformation pathways
of DCF in the aquatic environment, one investigation carried out
in a fixed-bed column bioreactor filled with riverine sediment
under aerobic conditions revealed the transient formation of the
p-benzoquinone imine of 5-hydroxy-DCF, which was characterized
by LC-electrospray (ESI)-MS and NMR spectroscopy.8 Neither
the presumed intermediate 5-hydroxy-DCF nor the formation of
4′-OH-DCF, constituting the main human metabolite, was ob-
served. In fact, studies on DCF conducted in a pilot WWTP,9 gas
chromatographic separation of the silylated sample extracts
indicated the formation of three degradation products, which were
tentatively identified through their electron impact-mass spectra
as the intramolecular lactam, an alcohol corresponding to the
reduction of the carboxylic acid, and a methoxy derivative thereof.
In full-scale WWTP relying on continuous activated sludge
(CAS) treatment for the degradation of organic compounds, the
biotransformation of the acidic DCF has generally been estimated
to be low based on comparisons of influent and effluent concentra-
tions. Despite the consensus viewing DCF as fairly recalcitrant
to microbial attack, for an unbiased interpretation of differences
in influent and effluent levels of the WWTP, it needs to be taken
into consideration that conjugated DCF can be liberated during
the biological wastewater treatment process. As mentioned above,
a fraction of DCF is excreted from the human body as acyl-
glucuronide, which is potentially cleavable by ꢀ-glucuronidase
enzymes, thus releasing the parent drug in its native form. In
addition to this, a second possible source for DCF formation in
the activated sludge tank is the ester hydrolysis of aceclofenac
(ACF: 2-(2-(2-(2,6-dichlorophenylamino)phenyl)acetoxy)acetic acid;
Figure 3C), which is a potent anti-inflammatory and analgesic drug
with efficacy similar to DCF but with improved gastrointestinal
tolerance.10 In analogy to DCF, the major human metabolite of
ACF derives from hydroxylation at the 4′-position of the halo-
genated aromatic ring, which is then partly excreted as the
glucuronide conjugate.11,12 Minor amounts of DCF and 4′-OH-
DCF, predominantly conjugated, have also been identified in urine.
Given the lack of field data on 4′-OH-DCF and the fact that
ACF and 4′-OH-ACF are potential precursors of DCF and its major
hydroxylated metabolite, respectively, the present study aimed
at investigating still uncovered aspects in the environmental life
cycle of DCF. The two principal objectives were (a) to determine
for the first time concentrations of the four target analytes in
influent and effluent samples from a WWTP operating in parallel
a CAS process and a pilot-scale membrane bioreactor (MBR), and
(b) to conduct biodegradation experiments under controlled
laboratory settings in order to gain further insight into the
biodegradability and metabolic pathways of ACF, DCF, and their
hydroxylated metabolites. Due to the difficulty of obtaining
commercially available reference standards of 4′-OH-DCF and 4′-
OH-ACF, these metabolites were synthesized from DCF and ACF,
respectively, by selective biocatalysis using recombinant human
CYP2C9 enzyme. For the trace determination of DCF and the
related compounds in wastewater, a quantitative analytical meth-
odology was developed based on solid-phase extraction (SPE)
followed by LC/ESI-MS/MS analysis on a hydrid quadrupole-
linear ion trap (QqLIT) instrument. Moreover, samples from the
biodegradation studies were screened for the presence of stable
intermediates and these were characterized by hybrid quadrupole-
time-of-flight (QqTOF)-MS in combination with H/D-exchange
experiments leading to the discovery of unusual microbial
transformation products.
EXPERIMENTAL SECTION
ChemicalandBiologicalReagents.DCF(CASNo.15307-79-6)
was purchased from Jescuder (Rub´ı, Spain) and ACF (CAS No.
89796-99-6) was provided by Laboratorios Almirall S.A. (Bar-
celona, Spain). All HPLC organic solvents were Chromasol LC
grade. Deuterium oxide (g99.9%) was obtained from Euriso-top
(Gif-Sur-Yvette, France). Ultrapure water, acetic acid-d4, glucose-
6-phosphate dehydrogenase from baker’s yeast 1 KU (Saccharo-
mycer cerevisiae), D-glucose 6-phosphate disodium salt hydrate
(98-100%), and ꢀ-glucuronidase (Escherichia coli) were purchased
from Sigma Aldrich (Munich, Germany). Acetonitrile and metha-
nol were from Riedel de Ha¨en (Steinheim, Germany). Dichloro-
methane, hydrochloric acid (25%), ammonium acetate, sodium
bicarbonate, disodium hydrogen phosphate, magnesium chloride,
and disodium EDTA were obtained from Merck (Darmstadt,
Germany). All reagents were of ACS grade. Microsomes contain-
ing recombinant human CYP2C9, expressed in baculovirus-
infected insect cells, and recombinant rabbit NADPH-P450 re-
ductase were obtained from Sigma Aldrich.
CYP2C9-Mediated Synthesis of 4′-OH-DCF and 4′-OH-
ACF. Given the difficulty in finding commercially available
reference compounds of the hydroxy metabolites of DCF and ACF,
they were produced by CYP2C9-mediated oxidation of the parent
compounds at the C-4′ position of the dichlorophenyl ring. The
regioselective biotransformation13 of DCF and ACF was ac-
complished by using recombinant human CYP2C9, affording the
two desired metabolites. To this end, a 25 µM solution of each
substrate was prepared in 50 mM phosphate buffer (pH 7.4) at a
final volume of 900 µL containing 1% acetonitrile. After addition
of a 50-µL aliquot of recombinant hCYP2C9 (80 pmol/mg of
protein) and a preincubation period of 3 min, the reaction was
initiated by adding 50 µL of a 10 mM NADPH-generating system.
The addition of four volumes of ice-cold acetonitrile terminated
the reaction after incubating 3 h at 37 °C, the precipitated proteins
were then separated by centrifugation for 10 min at 12000g, and
the recovered supernatants were evaporated to dryness under a
gentle stream of nitrogen. After reconstitution of the samples in
1 mL of water/acetonitrile (1:1), purity and concentration of the
synthesized standard were verified by LC-diode array detector
(DAD)-(+)ESI-MS/MS (Agilent 1100 Series coupled to Applied
Biosystems API QTRAP 4000, see below). Inspection of the UV
trace at the absorption maximum of 270 nm confirmed the
complete conversion of the substrates into their 4′-hydroxy
derivatives with a purity of >95%. The compound identities were
corroborated by recording the (+)ESI product ion mass spectra
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(9) Kosjek, T.; Heath, E.; Kompare, B. Anal. Bioanal. Chem. 2007, 387, 1379–
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(10) Pasero, G.; Marcolongo, R.; Serni, U.; Parnham, M. J.; Ferrer, F. Curr. Med.
Res. Opin. 1995, 13, 305–315
(11) Bort, R.; Ponsoda, X.; Carrasco, E.; Go´mez-Lecho´n, M. J.; Castell, J. V. Drug
Metab. Dispos. 1996, 24, 969–975
(12) Bort, R.; Ponsoda, X.; Carrasco, E.; Go´mez-Lecho´n, M. J.; Castell, J. V. Drug
Metab. Dispos. 1996, 24, 834–841
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8136 Analytical Chemistry, Vol. 80, No. 21, November 1, 2008