Identification of in vitro metabolites of a new anticoccidial drug nitromezuril using HepG2 cells, rat S9 and primary hepatocytes by liquid chromatography/tandem mass spectrometry

Article abstract of DOI:10.1002/rcm.6953

RATIONALE Nitromezuril is a novel triazine compound possessing remarkable anticoccidial activity that could have possible future use in the prevention of coccidiosis; however, its metabolic characteristics have still not been revealed. METHODS In the present study, the in vitro metabolism of nitromezuril in HepG2 cells, rat S9 and primary hepatocytes was investigated using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. The structures of metabolites and their product ions were easily and reliably characterized based on the accurate MS² spectra and known structure of nitromezuril. RESULTS As expected, three metabolites (M1-M3) were detected in a HepG2 cells system, one metabolite was respectively detected and identified as M1 in rat S9 and M2 in rat primary hepatocytes. M1 and M2 were confirmed respectively based on comparing their retention times, full scan, product ion scan with available authentic standards and M3 was tentatively identified as hydroxyl compound of M2. CONCLUSIONS Pathways of nitromezuril were reported for the first time and no obvious species difference was shown. The proposed metabolic pathways of nitromezuril can be expected to play a key role in pharmacodynamics and food safety evaluations. Copyright

Full text of DOI:10.1002/rcm.6953

Research Article
Received: 14 January 2014
Revised: 15 May 2014
Accepted: 21 May 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1723–1734
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6953
Identification of in vitro metabolites of a new anticoccidial drug
nitromezuril using HepG2 cells, rat S9 and primary hepatocytes
by liquid chromatography/tandem mass spectrometry
Keyu Zhang, Sumei Li, Wenli Zheng, Lifang Zhang, Chunmei Wang, Xiaoyang Wang,
*
Chenzhong Fei, Feiqun Xue and Mi Wang
Key Laboratory of Veterinary Drug Safety Evaluation and Residues Research, Shanghai Veterinary Research Institute, Chinese
Academy of Agricultural Sciences, Shanghai 200241, P.R. China
RATIONALE: Nitromezuril is a novel triazine compound possessing remarkable anticoccidial activity that could have
possible future use in the prevention of coccidiosis; however, its metabolic characteristics have still not been revealed.
METHODS: In the present study, the in vitro metabolism of nitromezuril in HepG2 cells, rat S9 and primary hepatocytes
was investigated using high-performance liquid chromatography with electrospray ionization tandem mass
spectrometry. The structures of metabolites and their product ions were easily and reliably characterized based on the
accurate MS2 spectra and known structure of nitromezuril.
RESULTS: As expected, three metabolites (M1–M3) were detected in a HepG2 cells system, one metabolite was
respectively detected and identified as M1 in rat S9 and M2 in rat primary hepatocytes. M1 and M2 were confirmed
respectively based on comparing their retention times, full scan, product ion scan with available authentic standards
and M3 was tentatively identified as hydroxyl compound of M2.
CONCLUSIONS: Pathways of nitromezuril were reported for the first time and no obvious species difference was shown.
The proposed metabolic pathways of nitromezuril can be expected to play a key role in pharmacodynamics and food
safety evaluations. Copyright © 2014 John Wiley & Sons, Ltd.
Coccidiosis is one of the most important diseases affecting the
poultry industry. The estimated total annual cost derived
directly or indirectly from diseases caused by Eimeria species
in raising fowl reaches up to US $800 millions.[1] Prophylactic
anticoccidial drugs as feed additives are widely used to
control coccidiosis.[2] However, the development of resistance
to most drugs has prompted a constant search for products
that could replace drugs against which organisms have
acquired resistance.[3] Since the 1980s, triazine coccidiostats,
including diclazuril and toltrazuril, have been widely used
in chickens and turkeys for their remarkable clinical effects
on the members of the genus Eimeria but have generated
resistance problems in recent years. The structure of 2-(3-
methyl-4-(4-nitrophenoxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-
dione, also known as nitromezuril (CAS:1352755-63-5,
Scheme 1), is similar to that of diclazuril and toltrazuril. It is a
novel anticoccidial triazine compound developed using
systematic structure–activity relationship studies by the
Shanghai Veterinary Research Institute of the Chinese Academy
of Agricultural Sciences in recent years.[4] The chemical synthesis
of this compound has been published in China Patent No. CN
102285930. Relevant preclinical research indicated good safety
and high effect of anticoccidiosis, anticoccidia indices (ACI) of
which were 185–190.[4] Furthermore, nitromezuril can exert
effects on coccidia that resist diclazuril and toltrazuril, which
illustrates its future wide application prospective.[4]
Owing to its selectivity, sensitivity, and speed of analysis
with minimal sample processing, liquid chromatography/
tandem mass spectrometry (LC/MS/MS) has been proven to
be a powerful modern analytical tool for the identification
and structural characterization of drug metabolites in biological
matrices and has become the preferred method for metabolite
identification in the fast paced environment of drug discovery
and development.[5] Complex metabolite samples from
biological matrices can be separated on a high-performance
liquid chromatography (HPLC) column and full-scan MS and
product ion scan MS/MS data generated on-line. By producing
the MSn ions associated with these basic structural features as a
substructural template based on the parent drug, the structures
of the metabolites may be rapidly characterized by comparing
their product ions with those of the parent drug, even without
standards.[6] Moreover, multiple reaction monitoring (MRM) as
a highly sensitive acquisition method in triple quadrupole mass
spectrometry can provide good signal intensity by parallel
monitoring of several product ions from the same precursor
ion and has been used as a method of detecting drug residues
in animal-borne food.[7]
On the basis of in vitro metabolism research, we have been
able to develop new drugs from possible metabolites, in some
cases the metabolite exhibits the same pharmacological
potential as the parent drug and is less toxic than the parent;
* Correspondence to: F. Xue, Shanghai Veterinary Research
Institute, Chinese Academy of Agricultural Sciences, No.
518 Ziyue Road, Shanghai 200241, P.R. China.
E-mail: zcole@shvri.ac.cn
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K. Zhang et al.
collagen and formic acid were purchased from Sigma (St.
Louis, MO, USA). Ethyl acetate and other analytical reagents
were of analytical grade and purchased from Shanghai Expe-
rimental Reagent Co., Ltd (Shanghai, China). Ultra-pure
water was prepared using a Milli-Q water purification system
(Millipore, Milford, MA, USA). All other chemicals and
reagents were of the highest analytical grade available.
Preparation of cells
The frozen tube of HepG2 cells was taken out of the liquid
nitrogen tank, put into a water bath at 37°C and shaken until
totally thawed. The cell suspension was transferred to a 15 mL
centrifuge tube, 4 mL of complete medium was added and the
tube was centrifuged at 1000 rpm for 5 min. The sediment was
resuspended in pre-heated 37°C culture medium, the cell
suspension was implanted to 24-well plates at a density
of 5.0 × 105 cmÀ2 (area 1.9 cm2), and the cells were culti-
vated in a humidified incubator at 37°C and atmosphere
containing 5% CO2.
Preparation of rat primary hepatocytes was performed using
the two-step perfusion method.[11] Isolated hepatocytes were
resuspended in DMEM. Then 180 μL of cell suspension was
mixed with 20 μL 0.4% Trypan blue solution, and the the viable
(white) and dead (blue) cells were counted in a hemocytometer
under a light microscope; four fields were counted per
chamber. The viability and concentration of the cells were
calculated and reached 95% and 9 × 107cells/mL, respectively.
Primary hepatocytes began to adhere after 4 h stabilization.
Scheme 1. Chemical structure of toltrazuril (a), diclazuril (b),
and nitromezuril (c).
besides, we have also conducted in vivo metabolism, as possible
metabolic pathways could provide a hint for appropriate
radioactive labeled spots which guarantee the success of
radioactive label research of pharmacokinetics. In addition,
the liver constitutes a crucial step before the distribution of oral
drugs, so hepatocytes are widely used in in vitro studies of
toxicity and the metabolism of xenobiotics.[8–10]
The comprehensive metabolism of nitromezuril in vitro and
in vivo has not been investigated till now. The study firstly
assumed possible metabolites of nitromezuril according to
common metabolic paths and made use of LC/MS/MS to
obtain the fragmentation behavior of nitromezuril and
predicted the product ions of possible metabolites. Then we
obtained the molecular weights of possible metabolites by
recording chromatograms from their peaks in total ion
chromatograms (TICs) and detected low levels of possible
metabolites by extracted ion chromatograms (EICs) on the basis
of assumed metabolites. By comparing product ion scan
spectra of possible metabolites of blank and incubation samples
with available authentic standards we were able to identify
them. Finally, the MRM method was used to detect blank and
incubation samples to compensate for the lower sensitivity
of the results obtained from product ion scans. The objective
of this study was to offer scientific grounds for research of
pharmacodynamics and food safety evaluations.
Stability of nitromezuril solution
Nitromezuril was dissolved in dimethyl sulfoxide (DMSO) at
a concentration of 2 mg/mL. The stock solution was mixed
with complete medium (DMEM with 10% FBS) at
a
proportion of 1% (v/v), then the mixed solution was placed
in 37°C humidified incubator containing 5% CO2 for 48 h.
The culture medium was extracted with ethyl acetate then
centrifuged at 12000 rpm for 20 min at 4°C. The organic
supernatant was evaporated under nitrogen atmosphere at
40°C and the residue was dissolved in 200 μL acetonitrile. The
aliquot were filtered by a 0.22 μm membrane then analyzed.
EXPERIMENTAL
Preparation of sample with HepG2 cells
HepG2 cells were allowed to adhere for 12 h. Then, the
culture medium was replaced with DMEM culture medium
with nitromezuril (20 μg/mL). After 48 h incubation, the
medium was collected and extracted with ethyl acetate then
centrifuged at 12000 rpm for 20 min at 4°C. The organic
supernatant was evaporated under nitrogen atmosphere at
40°C, the residue was dissolved in 200 μL acetonitrile. The
aliquot were filtered using a 0.22 μm membrane to identify
unchanged nitromezuril and its metabolites. The blank was
conducted in the absence of nitromezuril.
Reagents and chemicals
Nitromezuril (purity 99.8%), M1 (purity 99.0%) and M2
(purity 98.9%) were synthesized by the Shanghai Veterinary
Research Institute of the Chinese Academy of Agricultural
Science and characterized by LC-UV, LC-MS and NMR
methods (data not shown). HepG2 cells were obtained from
the Institute of Biochemistry and Cell Biology, Shanghai
Institute for Biological Sciences, Chinese Academy of Sciences
(Shanghai, China). Dulbecco’s modified Eagle’s medium
(DMEM) for culturing and fetal bovine serum (FBS) were
obtained from Life Technologies (Carlsbad, CA, USA).
Glucose 6-phosphate dehydrogenase, glucose-6-phosphate,
β-nicotinamide adenine dinucleotide phosphate sodium salt
(NADP) and arochlor-induced rat liver S9 homogenate (S9
mix) were purchased from Roche Chemical Co. (Beijing,
China). Acetonitrile (LC grade) was obtained from Fisher
Chemicals (Fair Lawn, NJ, USA). Collagenase IV, rat tail
Preparation of sample with rat liver S9
To investigate rat liver S9 mix metabolism of nitromezuril, 2 mg
protein/mL of S9 mix and an NADPH-generating system
consisting of 1 mmol/L NADP, 10 mmol/L glucose-6-
phosphate, 0.8 U glucose-6-phosphate dehydrogenase, and
7 mmol/L MgCl2 were mixed. The mixture was preincubated
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Identification of in vitro metabolites of nitromezuril
at 37°C for 5 min. To initiate the reaction, 50 μL nitromezuril
was added, and the mixture was maintained at 37°C for
another 2.5 h. Control incubations were performed in the
absence of nitromezuril and added 50 μL 0.1 mol/L
phosphate buffer (pH 7.4). The reaction was terminated by
the addition of twice the volume of ethyl acetate, and the
mixture was vortexed and centrifuged at 5000 rpm for 15 min
at 4°C.[12] The organic supernatant was evaporated under
nitrogen atmosphere at 40°C, the residue was dissolved in
200 μL acetonitrile. The aliquot were filtered using a 0.22 μm
membrane to identify metabolites by LC/MS/MS.
LC/MS/MS analysis of nitromezuril and its metabolites
All experiments were performed with a Waters Alliance 2695
LC system (Waters) and a quattro micro API quadrupole
mass spectrometer (Micromass, Manchester, UK) equipped
with an electrospray ionization (ESI) source.
LC analysis was carried out on an XTerra C18 HPLC column
(2.1 mm × 100 mm, 3.5 μm; Waters, Milford, MA, USA) equipped
with a guard column (XTerra C18 2.1 mm × 100 mm, 3.5 μm;
Waters, Milford, MA, USA). The mobile phase consisted
of A (water containing 0.1% formic acid, v/v) and B
(acetonitrile) with a gradient elution: 0–2 min, 95% A;
2–10 min, 95%–50% A; 10.01 min, 95% A. The flow rate
during the complete runs was 0.2 mL/min. Following the
gradient elution, the initial conditions were used and stayed
under the initial conditions for 2 min for re-equilibration
and to get ready for the next injection. The injection of
the pretreated sample was 10 μL and the column oven
temperature was set at 30°C.
Preparation of samples with rat primary hepatocytes
Rat primary liver cells were seeded on collagen-coated six-well
plates (area 9.4cm2) at a density of 5 × 105 cells cmÀ2 and
allowed to adhere in 37°C humidified incubator containing
5% CO2 for 4 h, then the culture medium was replaced with
fresh warm complete medium and cells allowed to recover
for 12 h before incubation with nitromezuril (20 μg/mL) for
48 h.[13] The medium was collected and treated as for the
preparation of sample with HepG2 cells.
The mass spectrometer was used for analysis operating in
negative ion mode. The main parameters were optimized by
infusing 1 μg/mL of nitromezuril standard dissolved in
Figure 1. Total ion chromatogram (TIC) (a), full scan mass spectrum (b), and
low-energy collision-induced dissociation product ion spectrum (c) behavior
of authentic standard nitromezuril. The deprotonated molecule of nitromezuril
was m/z 339 and it was eluted at 9.4 min (a, b). MS/MS analysis of m/z 339
revealed prominent product ions at m/z 268 formed by the loss of CONHCO
(71 Da). The fragment ion at m/z 268 lost C6H4NO2 (122 Da) to form m/z 146.
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K. Zhang et al.
acetonitrile at 10 μL/min using a syringe needle (Hamilton,
Switzerland). Nitrogen was used as desolvation gas (400 L/h)
and nebulizing gas (50 L/h). The source temperature was
held at 100°C. The capillary and cone voltages were 4.5 kV
and 30 V, respectively. Argon was used as collision gas at a
pressure of 3.3 × 10À4 mbar, desolvation temperature 300°C
and collision energy of 25 eV. The spectra were recorded in
the range m/z 50–600 for full scan MS analysis.
elimination of endogenous substances and good extraction
efficiency. Therefore, ethyl acetate was selected to prepare
incubation samples. All the extracts were dried under nitrogen
atmosphere at 40°C and then dissolved; pure acetonitrile and
50% acetonitrile both were tried as solvents. Pure acetonitrile
indicated better extraction specificity of metabolites. The
injection volume was 10 μL for LC/MS analysis.
Identification of metabolites was based on comparing their
retention times, full scan, product ion scan to available
authentic standards. Multiple reaction monitoring (MRM)
was also used to confirm the conclusion. System control and
data acquisition were completed by using the software
Masslynx 4.0 (Waters, Milford, MA, USA).
LC/MS and LC/MS/MS analyses of nitromezuril
In the present study, both positive and negative ion modes
were tried and it turned out that the negative ion mode
provided a dramatically stronger signal for nitromezuril.
The characterization of nitromezuril was conducted with the
conditions mentioned above. Full scan analysis indicated that
the deprotonated molecule of nitromezuril was m/z 339 and it
was eluted at 9.4 min (Figs. 1(a) and 1(b)). MS/MS analysis of
m/z 339 revealed prominent product ions at m/z 268 formed
by the loss of CONHCO (71 Da). The fragment ion at m/z
268 lost C6H4NO2 (122 Da) to form m/z 146. The fragmentation
of m/z 339 resulted in the cleavage of the triazine ring and the
ether bond (Fig. 1(c)).
RESULTS AND DISCUSSION
Stability of nitromezuril solution
Stock solutions of nitromezuril was stable for at least 2 months
when stored at 4°C (data not shown). On this basis the stability
of nitromezuril in DMEM was investigated, and results show
that the DMEM samples were stable for 48 h at 37°C in a 5%
CO2 incubator at 95% humidity (data not shown). We assumed
nitromezuril was stable under incubation conditions for 48 h
and that the metabolism of nitromezuril could be investigated
in vitro.
The characteristic product ions and cleavage patterns
reported above provided us with scientific grounds for the
identification of possible metabolites of incubation samples of
nitromezuril with S9, HepG2 cells and primary hepatocytes.
LC/MS and LC/MS/MS for the analysis of the HepG2 sample
In recent years, scientists have taken advantage of quan-
titative polymer chain reaction (PCR) to measure the
expression levels of CYPs in both HepG2 cells and human
primary hepatocytes. The result showed that main CYPs,
such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6,
2E1 and 3A4, were expressed in HepG2 cells.[14] Besides,
HepG2 cells have the ability to proliferate and are easy to
obtain, which indicates a good model for metabolism research.
In this article, we focused on the in vitro metabolism of
Sample preparation
In this study, protein precipitation with acetonitrile and
liquid-liquid extraction (LLE) methods were used in order
to extract metabolic substances from samples. It turned out
that direct protein precipitation was less effective because of
its inferior ability to remove endogenous substances.
Dichloromethane and ethyl acetate were tried to compare
the extraction effects: ethyl acetate demonstrated good
Figure 2. Total ion chromatograms (TICs) of the extracts of both blank (a) and
incubation (b) samples in the HepG2 cells system.
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Identification of in vitro metabolites of nitromezuril
Figure 3. Deprotonated molecular ions of metabolites by taking spectra at the top
of the peaks in the TIC of the incubation group. The deprotonated molecules of
these metabolites were m/z 309 (M1), m/z 351 (M2) and m/z 367 (M3), respectively.
The response of m/z 367 is far lower than that of the other metabolites.
Figure 4. Extracted ion chromatograms of both blank (a) and incubation (b) samples with HepG2 cells.
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K. Zhang et al.
nitromezuril in HepG2 cell and rat primary hepatocyte
systems and aimed to find out possible metabolites, which
could provide scientific grounds for further research.
signal intensities as those found in the TICs. The
retention times of M1, M2 and M3 were 5.9, 7.0 and 6.8
min, respectively (Fig. 4).
We firstly speculated possible metabolites according to the
most common biotransformation pathways, the structure of
the parent drug and similarly structured drugs, and eight
possible metabolites were assumed. Then, the full scan mass
spectra and extracted ion chromatograms (EICs) of the
extraction of incubation sample were compared with those
obtained from the extraction of blank culture medium to find
out possible metabolites.
According to comparing both TICs (Fig. 2) and EICs of
pretreated samples, taking the chromatogram at the top
of the peaks in the TICs produced the deprotonated
molecules of nitromezuril and its metabolites. The dep-
rotonated molecules of these metabolites were m/z 309
(M1), m/z 351 (M2) and m/z 367 (M3), respectively. The
response of m/z 367 is far lower than that of the other
metabolites (Fig. 3). Detecting low levels of possible
metabolites according to EICs, metabolites had the same
Combining the results reported above with common
metabolic pathways, we tentatively identified the two main
metabolites as 2-(4-(4-aminophenoxy)-3-methylphenyl)-1,2,4-
triazine-3,5-(2H,4H)-dione and N-(4-(4-(3,5-dioxo-4,5-dihydro-
1,2,4-triazin-2-(3H)-yl)-2-methylphenoxy)phenyl) acetamide (M1
and M2 standards) (Scheme 2). Then the two main possible
metabolites were synthesized.
The characterization of the two possible metabolites
standards was studied in both positive and negative ion mode.
The signal response intensity of the M2 standard was the same
as nitromezuril. The signal of the M1 standard was a little
stronger in the positive ion mode than that in the negative ion
mode because of the influence of its amino moiety. Finally, we
chose the negative ion mode to analyze the pretreated samples.
Their retention times were 5.9 and 7.0 min, respectively, which
were the same as those of the metabolites. MS/MS analysis of
M1 and M2 standards showed that prominent product ions of
m/z 309 (M1 standard) were m/z 238 formed by the loss of
CONHCO (71 Da) and m/z 146 formed by the consecutive loss
of C6H4NH2 (92 Da), which revealed the cleavage was the same
with nitromezuril as well as m/z 351 (M2 standard), fragment
ions of which were at m/z 280 and m/z 146. The MS/MS spectra
of M1 and M2 are shown in Fig. 5.
Studies on metabolism have shown that nearly 90%
unchanged diclazuril excretes through faeces, which was the
main metabolic pathway. Only four minor metabolites of
diclazuril were detected and each of them was less than 1%.[15]
Scheme 2. Chemical structures of M1 (a) and M2 (b) standards.
Figure 5. LC/MS/MS product ion spectra of the authentic standards of M1 (a)
and M2 (b) standards.
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Identification of in vitro metabolites of nitromezuril
Moreover, unchanged toltrazuril was the main component, four
metabolites were identified, one major metabolite, toltrazuril
sulfone, represented 4.6–16%.[16] Thus we can infer that the
metabolism of an anticoccidial drug of the triazine family is
relatively stable. Diclazuril possesses the same trizaine ring as
nitromezuril, the only fragment of which is formed by the loss
of CONHCO (71 Da).[16] So we can infer that the 1,2,4-triazine
ring is inclined to cleave by the loss of CONHCO (71 Da).
Scheme 3. Possible chemical structures of M3.
Figure 6. LC/MS/MS product ion scan spectra of metabolites in incubation
sample with HepG2 cells: (a) (M1 were m/z 238 formed by the loss of
CONHCO (71 Da) and m/z 146 formed by the loss of C6H4NH2 (92 Da));
(b) (M2 were m/z 280 formed by the loss of CONHCO (71 Da) and m/z 146
formed by the loss of C8H8NO (134 Da);and (c) (M3 was the product of
phenyl hydroxylation of M2).
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K. Zhang et al.
The molecular weight of M3 was 16 Da more than that of
M2, so we inferred that M3 was the product of phenyl
hydroxylation of M2. However, we were unsure about the
position of the hydroxyl moiety. According to the cleavage
pattern of the parent drug and other metabolites, we
speculated possible ion pairs of M3, which were m/z
367→296, m/z 367→162 and (or) m/z 367→146 (Scheme 3).
Product ion scans were conducted in both the blank and
incubation groups to obtain the product ions of the possible
metabolites and compare them with the authentic standards.
The product ions of the possible metabolites were the same as
those of the M1 and M2 standards (Figs. 6(a) and 6(b)), and the
main product ions of M3 were m/z 296 and 146 (Fig. 6(c)), so
we tentatively identified M3 as structure a shown in Scheme 3.
Figure 7. Qualitative detection of M1, M2 and M3 in both blank (a) and incubation (b)
samples by the multiple reaction monitoring (MRM) method in the HepG2 cell sytem.
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Identification of in vitro metabolites of nitromezuril
Neutral loss scan analysis was also performed and this
facilitated the detection of the glucuronide and sulfate
conjugates by monitoring losses of 176 Da and 80 Da,
respectively. However, no sulfate or glucuronide conjugates
of nitromezuril, M1, M2 or M3 were found.
Six transitions were used as follows: m/z 367→296, m/z
367→146, m/z 351→280, m/z 351→ 146, m/z 309→238, and
m/z 309→146. No signal was shown in the blank sample
whereas ion pairs could be detected in the incubation sample
(Fig. 7).
Multiple reaction monitoring (MRM) was applied to
compensate for for the lower sensitivity of the product ion
scan and enhance the reliability of the metabolism results.
Drug metabolism often converts lipophilic chemical compounds
into more readily excreted hydrophilic products. If the
metabolites are sufficiently polar, they may be readily
Figure 8. Proposed metabolic pathways of nitromezuril incubated
with HepG2 cells in vitro.
Figure 9. Total ion chromatograms (TICs) of the extracts of both blank (a) and
incubation (b) samples in the S9 mix.
Figure 10. Extracted ion chromatograms (EICs) of both blank and incubation samples with S9.
No more metabolites were detected in the EICs of the potential metabolites of nitromezuril.
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K. Zhang et al.
excreted. Based on these results, the major metabolic
pathways of nitromezuril metabolism in HepG2 cells are
completed and summarized in Fig. 8. Nitromezuril was
metabolized by reduction of nitro to amino followed by
acetylation of amino to amide and followed by hydroxylation
of the benzene ring of the amide.
LC/MS and LC/MS/MS for the analysis of the S9 sample
S9 is a crude cell preparation and contains both cytosolic
enzymes (e.g. sulfotranferase, aldehyde oxidase) and
membrane-bound enzymes (e.g. CYP450, FMO). The S9 mix is
useful for studying both phase I and phase II metabolism. Thus,
incubation of potential drug candidates with the S9 fraction
can help understand the metabolic fate of compounds.[17]
We firstly speculated possible metabolites according to the
most common biotransformation pathways, the structure of
the parent drug and similarly structured drugs, and eight
possible metabolites were assumed. Then, the full scan mass
spectra and extracted ion chromatograms (EICs) of the
extraction of incubation sample were compared with the
extraction of a blank culture medium to discover possible
metabolites. Detecting possible metabolites in rat S9, only
one metabolite was found and identified as M1.
The total ion chromatograms (TICs) are shown in Fig. 9; by
taking the chromatogram at the top of the peaks, a possible
metabolite eluting at 5.9 min was detected, which could be M1.
No more metabolites were detected according to the EICs (Fig. 10).
Figure 11. LC/MS/MS product ion scan spectra of
metabolite in incubation sample with S9.
Figure 12. Qualitative detection of M1 in both blank (a) and incubation (b) samples by the multiple reaction
monitoring (MRM) method in the S9 mix.
Figure 13. Total ion chromatograms (TICs) of the extracts of both blank (a) and
incubation (b) samples in the rat primary hepatocytes system.
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Identification of in vitro metabolites of nitromezuril
Figure 14. Extracted ion chromatograms (EICs) of both blank and incubation samples with
primary hepatocytes. No more metabolites were detected in the EICs of the potential metabolites
of nitromezuril.
Product ion scans were conducted in both the blank and
incubation samples to obtain the product ions of possible
metabolites and compare them with the authentic standards.
MS/MS analysis of metabolite M1 and authentic standard M1
showed that prominent product ions of m/z 309 both were m/z
238 formed by the loss of CONHCO (71 Da) and m/z 146
formed by the consecutive loss of C6H4NH2 (92 Da), which
revealed the product ions of metabolite were the same as
those of the M1 standard (Figs. 5 and 11). The MRM
comparison between blank and incubation samples is shown
in Fig. 12: m/z 309→238, m/z 309→146, m/z 339→268, m/z
339→146 were set as the detecting ion pairs.
LC/MS and LC/MS/MS for the analysis of primary
hepatocytes sample
The same procedure of identification was used to detect
possible metabolites in rat primary hepatocytes. Only one
metabolite was found and identified as M2.
The TICs are shown in Fig. 13; by taking the chromatogram
at the top of the peaks, a possible metabolite that eluted at 7.0
min was detected, which could be M2. No more metabolites
were detected according to the EICs (Fig. 14).
Figure 15. LC/MS/MS product ion scan spectra of
metabolite in incubation sample with primary hepatocytes.
Figure 16. Qualitative detection of M2 in both blank (a) and incubation (b) samples by
the multiple reaction monitoring (MRM) method in the primary hepatocytes system.
Rapid Commun. Mass Spectrom. 2014, 28, 1723–1734
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

K. Zhang et al.
[4] C. Fei, C. Fan, Q. Zhao, Y. Lin, X. Wang, W. Zheng,
M. Wang, K. Zhang, L. Zhang, T. Li, F. Xue. Anticoccidial
effects of a novel triazine nitromezuril in broiler chickens.
Vet. Parasitol. 2013, 198, 39.
[5] S. Ma, S. K. Chowdhury, K. B. Alton. Application of mass
spectrometry for metabolite identification. Curr. Drug
Metab. 2006, 7, 503.
[6] Z. Liu, L. Huang, M. Dai, D. Chen, Y. Tao, Y. Wang,
Z. Yuan. Metabolism of cyadox in rat, chicken and pig liver
microsomes and identification of metabolites by accurate
mass measurements using electrospray ionization hybrid
ion trap/time-of-flight mass spectrometry. Rapid Commun.
Mass Spectrom. 2009, 23, 2026.
[7] S. Dresen, N. Ferreirós, H. Gnann, R. Zimmermann,
W. Weinmann. Detection and identification of 700 drugs by
multi-target screening with a 3200 Q TRAP LC-MS/MS system
and library searching. Anal. Bioanal. Chem. 2010, 396, 2425.
[8] P. Kosina, J. Vacek, B. Papoušková, M. Stiborová, J. Stýskala,
P. Cankař, E. Vrublová, J. Vostálová, V. Simánek, J. Ulrichová.
Identification of benzo[c]phenanthridine metabolites in
human hepatocytes by liquid chromatography with
electrospray ion-trap and quadrupole time-of-flight mass
spectrometry. J. Chromatgr. B. 2011, 879(15–16), 1077.
[9] M. Patrick. The use of adult human hepatocytes in primary
culture and other in vitro systems to investigate drug
metabolism in man. Adv. Drug Deliv. Rev. 1996, 22, 105.
[10] Z. Dvorák, P. Kosina, D. Walterová, V. Simánek, P. Bachleda,
J. Ulrichová. Primary cultures of human hepatocytes as a
tool in cytotoxicity studies: cell protection against model
toxins by flavonolignans obtained from Silybum marianum.
Toxicol. Lett. 2003, 137, 201.
Product ion scans were conducted in both the blank and
incubation samples to obtain the product ions of possible
metabolites and compare them with the authentic standards.
The product ions of the metabolite were the same as those of
the M2 standard (Fig. 15). An MRM comparison between the
blank and incubation samples is shown in Fig. 16: m/z
351→280 and m/z 351→146 were set as detecting ion pairs.
Product M1 which, according to chemical transformation
formula, was the premise for producing M2 was not detected
in the system. We considered the reason responsible for this
result: the acetylase catalyzing this reaction was more active
in rat primary hepatocytes than that in HepG2 cells, M2
was the main metabolic component in the rat primary
hepatocytes system.[18]
A neutral loss scan was also conducted by monitoring the
loss of 176 and 80 to detect common conjugates, no sulfate
or glucuronide conjugates of nitromezuril, M1, M2 or M3
were found.
CONCLUSIONS
The metabolism of the new anticoccidial drug nitromezuril
was studied for the first time. HepG2 cells, rat liver S9 and
primary hepatocytes incubation systems were used to study
the metabolism. Apart from its convenience and easy
availability, HepG2 cells contained the main CYPs that
anticipated drug metabolism, S9 and primary hepatocytes
were regarded as gold standards for metabolism research.
Finally, a total of three metabolites (M1–M3) were confirmed
or tentatively identified in HepG2 cells and one metabolite
was confirmed respectively as M1 in rat S9 and M2 in the
rat hepatocytes system. Overall, nitromezuril demonstrated
no obvious species difference in metabolism. M1 and M2
could be an important objective in pharmacodynamics
and residue research. This study supports further research
with scientific data and hints.
[11] P. O. Seglen. Preparation of isolated rat liver cells. Methods
Cell Biol. 1976, 13, 29.
[12] M. Rajanikanth, K. P. Madhusudanan, R. C. Gupta.
Simultaneous quantitative analysis of three drugs by high-
performance liquid chromatography/electrospray ionization
mass spectrometry and its application to cassette in vitro
metabolic stability studies. Rapid Commun. Mass Spectrom.
2003, 17, 2063.
[13] F. Bourdon, M. Lecoeur, V. Verones, C. Vaccher, N. Lebegue,
T. Dine, N. Kambia, J. F. Goossens. In vitro pharmacokinetic
profile of a benzopyridooxathiazepine derivative using rat
microsomes and hepatocytes: identification of phases I and
II metabolites. J. Pharm. Biomed. Anal. 2013, 80, 69.
[14] W. M. Westerink, W. G. Schoonen. Cytochrome P450
enzyme levels in HepG2 cells and cryopreserved primary
human hepatocytes and their induction in HepG2 cells.
Toxicol. In Vitro 2007, 21, 1581.
[15] Committee for Veterinary Medicinal Products Diclazuril
summary report (1) EMEA/MRL/086/ 96-FINAL.
[16] Committee for Veterinary Medicinal Products Toltrazuril
summary report (1) EMEA/MRL/314/ 97-FINAL.
[17] N. R. Varkhede, S. Jhajra, D. S. Ahire, S. Singh. Metabolite
identification studies on amiodarone in in vitro (rat liver
microsomes, rat and human liver S9 fractions) and in vivo
(rat feces, urine, plasma) matrices by using liquid
chromatography with high-resolution mass spectrometry
and multiple-stage mass spectrometry: Characterization of
the diquinone metabolite supposedly responsible for the
drug’s hepatotoxicity. Rapid Commun. Mass Spectrom. 2014,
28, 311.
Acknowledgements
This work was financially supported by the Special Fund for
Agro-scientific Research in the Public Interest (201303038–1)
and the National High Technology Research and Develop-
ment Program of China (863 program) (2011AA10A214).
The authors wish to thank the Key Laboratory of Animal
Parasitology of Ministry of Agriculture for assistance in
animal arrangement.
REFERENCES
[1] R. B. Williams. Epidemiological aspects of the use of live
anticoccidial vaccines for chickens. Int. J. Paristiol. 1998, 28, 1089.
[2] H. W. Peek, W. J. Landman. Coccidiosis in poultry:
anticoccidial products, vaccines and other prevention
strategies. Vet Q. 2011, 31, 143.
[3] J. J. Zhang, L. X. Wang, W. K. Ruan, J. An. Investigation into
the prevalence of coccidiosis and maduramycin drug
resistance in chickens in China. Vet. Parasitol. 2013, 191, 29.
[18] S. Wilkening, A. Bader. Influence of culture time on the
expression of drug-metabolizing enzymes in primary
human hepatocytes and hepatoma cell line HepG2.
J. Biochem. Mol. Toxicol. 2003, 17, 207.
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 1723–1734