Identification and elucidation of the structure of in vivo metabolites of diaveridine in chicken

Journal of Chromatography B, 965 (2014) 91–99

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Identification and elucidation of the structure of in vivo metabolites of diaveridine in chicken Hui Wang a , Bo Yuan a , Zhenling Zeng a,b , Limin He a,b , Huanzhong Ding a,b , Chunna Guo a , Xiangkai Kong a , Wei Wang a , Xianhui Huang a,∗ a College of Veterinary Medicine, National Reference Laboratory of Veterinary Drug Residues (SCAU), South China Agricultural University, Guangzhou 510642, PR China b Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, PR China

a r t i c l e

i n f o

Article history: Received 3 May 2013 Accepted 6 June 2014 Available online 23 June 2014 Keywords: Diaveridine Metabolites LC-LTQ-Orbitrap Chicken

a b s t r a c t Diaveridine (DVD) is a popular antibacterial synergist that is widely used in combination with sulfonamide. It has been reported to be genotoxic to mammalian cells, but more studies are required to clarify this. Moreover, there is very little information on its pharmacokinetics, metabolic elimination and mechanism of toxicity. Therefore, in order to gain a better understanding of the metabolism of DVD, we performed high-performance liquid chromatography linear ion trapped orbitrap mass spectrometer (LC-LTQ-Orbitrap). With this approach, we identified 15 metabolites of DVD in chicken after a single oral administration of DVD; 10 of these metabolites have been identified in vivo for the first time. Nine phase I and five phase II metabolites were detected in the plasma, and eight phase I and six phase II metabolites were found in feces. The major phase I metabolites were formed via the O-demethylation and N-oxidation pathways, and the major phase II metabolites were glucuronide conjugates. These results are essential for understanding this compound more clearly and lay the basis for further studies about the metabolism of DVD. Therefore, using this approach, we were able to identify and characterize metabolites of DVD with high sensitivity and resolution. We were able to detect a broad range of metabolites, even some trace ones and some so far unknown metabolites. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Diaveridine (DVD), 5-[(3 ,4 -dimethoxyphenyl)methyl]-2,4diaminopyrimidine, is an antibacterial synergist [1] which can interfere with bacterial nucleic acid synthesis. DVD has broadspectrum antibacterial activity against most Gram-negative and Gram-positive bacterium [2], including Escherichia coli, Clostridium spp., Salmonella spp., Staphylococcus aureus, and Bacillus anthracis. DVD also has remarkable activity against coccidia. Therefore, it is widely used to prevent chicken coccocidiosis, fowl cholera and pullorum [3,4]. DVD is rarely used by itself in the clinic; it is usually used in combination with sulfaguanidine and sulfamonomethoxine. This drug combination can block the metabolism of folic acid in bacteria by two different mechanisms, and even appears to have

Abbreviations: DVD, Diaveridine; LC-LTQ-Orbitrap, high-performance liquid chromatography linear ion trap orbitrap mass spectrometry; TMP, Trimethoprim; RDBs, ring and double-bond equivalents; CID, collision-induced dissociation. ∗ Corresponding author. Tel.: +86 20 87344801; fax: +86 20 87344801. E-mail address: [email protected] (X. Huang). http://dx.doi.org/10.1016/j.jchromb.2014.06.010 1570-0232/© 2014 Elsevier B.V. All rights reserved.

bactericidal effects [5]; the resistant strains of sulfanilamide can also be suppressed. The DVD absorption rate in animals is low, with the highest plasma concentration being only one-fifth that of Trimethoprims; but it has a high concentration in the intestines [1]. Therefore, DVD is used as a synergist to treat intestinal infections. DVD is thought to be genotoxic towards mammalian cells in vitro and in vivo [6,7]. However, there is not enough information on the genotoxicity of DVD, and its pharmacokinetics, metabolism, residual elimination and toxicology need to be better understood. Recently, liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry was used to study the in vitro metabolites of DVD in pig liver microsomes: six DVDrelated metabolites were detected, and O-demethylation was found to be the major metabolic route [8]. However, in vivo comprehensive studies of the metabolites are still missing. Therefore, in the present study, we have conducted what we believe to be the first in vivo study of DVD metabolism in chicken. We used liquid chromatography coupled with electrospray ionization linear ion trap orbitrap mass spectrometer (LC-LTQOrbitrap) [9–12], because it has been proven to be a successful approach for the structural elucidation of drug metabolites [13–17].

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LTQ provides valuable information about fragment ions in the MSn mode, and Orbitrap can provide high mass accuracy and resolution for small-molecule drugs and their metabolites present at a very low level in the complex biological matrix [18]. The combination of these two mass analysis methods enables both scan types include full-scan and data-based scan to be acquired simultaneously and consecutively, and can allow for unambiguous determination of the elemental composition of unknown metabolites. In the present study, therefore, we adopted a highly sensitive and specific LC-LTQ-Orbitrap method to identify and elucidate the structures of DVD metabolites in chicken plasma and feces on the basis of the mass spectra after oral administration of DVD. Based on the results, we have proposed the pathways for DVD metabolism in chickens. 2. Experimental 2.1. Chemicals and reagents Standard DVD was purchased from the China Institute of Veterinary Drug Control (Beijing, China). HPLC-grade methanol was obtained from Fisher Chemicals Co. (New Jersey, USA). HPLCgrade formic acid was supplied by Technologies GmbH (Düsseldorf, Germany). Ultrapure water was purified using a Milli-Q system (Millipore, Milford, MA, USA). All other chemicals and reagents used were of the highest analytical grade, and were used without further purification. 2.2. Animals Eight Kebao-500 chicken (four male, four female) that weighed 1.5–1.9 kg were purchased from Chia Tai Kang (Dongguan Co. Ltd., Guangdong, China), and chosen randomly for the animal experiments. Poultry were adapted to normal temperature (25 ◦ C), humidity (65%) and sunlight. The birds were freely fed standard food and water in metabolic cages for 1 week before the experiment. The chickens were not given food for 12 h before the experiment started, but water was still given. Six chicken (three male, three female) were administered a single dose of DVD (24 mg/kg, dissolved in 0.9% physiological saline) by oral gavage. The control chickens were housed and fed in the same way, but they were administered the same volume of 0.9% physiological saline. All studies on animals were performed with the approval of the Institutional Authority for Laboratory Animal Care. 2.3. Samples Blood was collected from the armpit vein into heparinized tubes at 0, 2, 6, 12, and 24 h after the injection was given. All blood samples were isolated by centrifuging at 4 ◦ C for 10 min at 1726 × g. Feces were collected before 0 h and at 0–2, 2–6, 6–12, and 12–24 h after the single dose. To avert cross-contamination, the metabolic cages were cleaned after every sampling. The samples were assayed immediately or stored in a freezer at −20 ◦ C before preparation.

(60 mg/3 mL) was used to remove salts and enrich the metabolites. The cartridge was conditioned using 3 mL methanol and 3 mL water, and then the extracts were loaded onto it. The cartridge was then washed with 3 mL water and 3 mL methanol, and the fluid was wiped off using an air stream and eluted with 5 mL ammonia water–methanol (4:96, by volume). The eluate was evaporated to dryness under nitrogen at 40 ◦ C, and the residue was re-dissolved in 1 mL of 20% methanol.

2.4.2. Feces Trichloroacetic acid–acetonitrile (7:3 by volume, 5 mL) was mixed with 1.0 g of fecal matter. After ultrasonic extraction for 15 min, vortex for 2 min, shaking for 15 min, and centrifugation for 8 min at 3500 × g, the extract was transferred to another tube. The residue was again subject to the same process of ultrasonic extraction, vortex, shaking, and centrifugation. The supernatant was loaded onto an Anpelclean MCX cartridge (60 mg/3 mL). The cartridge was processed as described for the plasma sample. Then, the eluate was evaporated under a nitrogen stream in a water bath at 40 ◦ C. The residue was re-dissolved in 1 mL of 20% methanol for analysis.

2.5. LC-LTQ-Orbitrap conditions The experiments were performed on a Thermo Electron LTQOrbitrap XL hybrid mass spectrometer (Thermo Electron, Bremen, Germany) equipped with an electrospray (ESI) ion source and coupled to a Surveyor solvent delivery pump and a Surveyor autosampler (Thermo Electron, Bremen, Germany). The liquid chromatography conditions were as follows: a Luna ODS C18 column (150 mm × 2.1 mm; i.d., 5 ␮m; Phenomenex, Torrance, CA, USA) and a SecurityGuard C18 guard column (4.0 mm × 3.0 mm; i.d., 5 ␮m; Phenomenex, Torrance, CA, USA), with the column temperature set at 30 ◦ C. The mobile phases were 0.1% formic acid in water (A) and methanol (B). Gradient elution was linearly programmed as follows: 0.00–9.00 min, 98% B to 55% B; 9.00–10.00 min, 55% to 10% B; 10.00–12.00 min, 10% B; 12.00–13 min, linear gradient back to 98% B; 13–22 min, 98% B at a constant flow rate of 0.25 mL/min. The injection volume was 10 ␮L. The LTQ-Orbitrap conditions were as follows: ESI, positive mode; ion spray voltage, 4.5 kV; capillary temperature, 300 ◦ C. Nitrogen was used as the sheath gas (25 arbitrary units) and auxiliary gas (11 arbitrary units), and helium served as the collision gas. The tube lens and capillary voltages were set to 120 and 46 V, respectively. Collision-induced dissociation (CID) was conducted with an isolation width of 2 Da, and the activation time was set at 30 ms. CID was conducted in the dynamic exclusion mode, with repeat counts of 2, repeat duration of 30 s, exclusion list size of 50, and exclusion duration of 180 s. All ion acquisition experiments were performed using the Orbitrap mass spectrometer, and scanning was performed at a resolution of 30,000 for both full-scan MS and data-dependent scan MS, MS2 and MS3 .

2.6. Metabolite analysis and data processing 2.4. Sample pretreatment 2.4.1. Plasma Aliquots of the blank plasma and plasma samples were allowed to completely thaw at room temperature and treated separately following the same process as that for sequential comparison. In brief, 1 mL of plasma from each chicken was taken in a 15-mL CORNING tube and mixed with 5 mL of trichloroacetic acid–acetonitrile (7:3, by volume), and then vortexed for 2 min. The mixture was centrifuged at 3500 × g for 8 min. Then, an Anpelclean MCX cartridge

First, the characteristic product ions and fragmentation routes of DVD were determined in the MSn mode, in order to identify possible metabolites. Then, full-scan MS/MS was conducted in the dynamic exclusion mode to obtain mass data which was analyzed with MetWorks 1.2 (Thermo Electron, Bremen, Germany) to identify metabolites. Following this, fragment ions were obtained via the LC-MS2 and LC-MS3 mode to identify the metabolites that had similar fragmentation routes to DVD. Finally, the elemental composition of the metabolites was determined based on mass accuracy

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Fig. 1. Accurate MS2 spectra of DVD showing product ions at m/z 246.11, m/z 245.10, m/z 217.11 and m/z 123.06.

and ring and double-bond equivalent (RDB) values, which were provided by the Orbitrap analyzer.

product ions were used as references to interpret the product ions of the metabolites, as well as to examine the high resolution and mass accuracy of the instrument. The elemental compositions, the observed and calculated masses, and the mass errors of protonated DVD and its fragments are listed in Table 1. The observed and calculated mass errors were <5 ppm.

3. Results and discussion 3.1. General observations

3.2.1. Metabolic profiles of DVD in plasma Fig. 3 shows the accurate mass extracted ion chromatogram (EIC) for plasma from female and male chickens at 6 h after oral administration of DVD: 14 DVD metabolites were detected. No more metabolites were detected at 2, 12 and 24 h. The predicted elemental composition, observed and calculated masses, mass errors, and characteristic fragment ions for the proposed metabolites are presented in Table 2. The agreement between accurate mass measurement in MS spectra and the predicted formula calculation was within 5 ppm, indicating a high level of confidence in the proposed elemental composition of the metabolites.

Clinical examination of all the chickens after each administration did not reveal any abnormalities or local or systemic adverse reactions. 3.2. Identification of the metabolites of DVD The theoretical elemental composition of DVD is found to be C13 H17 N4 O2 + ([M + H]+ ), m/z 261.13, with a retention time of 8.80 min. As shown in Fig. 1, the MS2 product ions of protonated DVD are mainly the displayed product ions at m/z 246.11, m/z 245.10, m/z 217.11, and m/z 123.06. The product ions at m/z 246.11 and m/z 245.10 were the result of loss of a CH3 and CH4 radical from m/z 261.13, respectively. The fragment ion at m/z 217.11 was formed by loss of CO from m/z 245.10. Subsequently, the fragment ion at m/z 123.06 was generated by the separation of methylene carbon from the protonated DVD at m/z 246.11, m/z 245.10. The proposed fragmentation pattern of DVD is shown in Fig. 2. These

3.2.2. Metabolic profiles of DVD in feces Fig. 4 shows the accurate EIC for feces from female and male chickens at 6 h after oral administration. Fourteen DVD metabolites were found in the fecal samples too, but DM5 in the plasma samples was not observed; a new metabolite, annotated as DM15, was

NH2

NH2

OH H

N NH2

N

MS3:217.11 C6H6O

NH2 H2C

N

NH2

NH2

NH2

H CH2

N

NH2

O

MS2:245.10

C7 H 6 O 2

CH4

NH2

H

O

N

N

O

N

C8H10 O2

H2C

O

N

N

C7H7O2

NH2 O

N NH2

N

MS2:246.11

NH2

MS2:123.07

CH3

M/Z 261.13

N

OH

Fig. 2. The proposed fragmentation pathways for DVD.

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Table 1 Elemental compositions observed and calculated masses, ring and double-bond equivalents (RDBs), and mass errors of protonated DVD and its fragmentations. Elemental compositions ([M + H])+

Observed m/z (Da)

Calculated mass (Da)

RDB

Error (mDa)

[C13 H16 N4 O2 + H]+ [C12 H13 N4 O2 + H]+ [C12 H12 N4 O2 + H]+ [C11 H12 N4 O + H]+ [C5 H6 N4 + H]+

261.13431 246.11081 245.10303 217.10811 123.06641

261.13460 246.11112 245.10330 217.10838 123.06652

7.5 8.0 8.5 7.5 4.5

−0.052 −0.317 −0.272 −0.278 −0.113

observed. There were no qualitative differences in the metabolite profiles of DVD in the feces of female and male chickens. 3.3. Identification of phase I metabolites of DVD The accurate MS2 spectra for phase I metabolites are shown in Fig. 5 (DM1–DM9).

3.3.1. DM1 and DM2 DM1 and DM2 were eluted at 8.21 and 8.49 min, respectively. Both showed a protonated molecule at m/z 247.12, which indicated a loss of 14 Da when compared to the protonated molecule of DVD at m/z 261.13; this most likely corresponded to demethylation, indicating that O-demethylation had occurred. In addition, the MS2 fragment ion at m/z 232.09, which was 14 Da lower than

Fig. 3. The accurate extracted ion chromatogram (EIC) of plasma from male (A) and female (B) chicken at 6 h after oral administration. As can be seen, 14 metabolites were identified.

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Table 2 Predicted formulas, retention time, observed m/z, predicated mass, mass errors and observed m/z values of DVD and its metabolites. observed m/z (Da)

Calculated mass (Da)

Error (ppm)

8.85

261.13464

261.13460

0.144

[C12 H14 O2 N4 + H]+

8.21

247.11903

247.11895

0.315

DM2

+

[C12 H14 O2 N4 + H]

8.49

247.11908

247.11895

0.517

DM3

[C14 H18 O2 N4 + H]+

9.42

275.15033

275.15025

0.282

DM4

[C13 H16 O3 N4 + H]+

8.31

277.12970

277.12951

0.661

DM5

[C13 H16 O3 N4 + H]+

1.08

277.12747

277.12951

1.743

DM6

[C13 H16 O3 N4 + H]+

8.78

277.12976

277.12951

0.877

DM7

[C13 H16 O3 N4 + H]+

9.86

277.12967

277.12951

0.552

DM8

[C13 H16 O3 N4 + H]+

9.08

277.12955

277.12951

0.119

DM9

[C14 H18 O3 N4 + H]+

9.21

291.14514

291.14516

−0.093

DM10

[C19 H24 O8 N4 + H]+

8.33

437.16684

437.16669

0.343

DM11

[C19 H24 O8 N4 + H]+

8.57

437.16687

437.16669

0.411

DM12

[C18 H22 O8 N4 + H]+

7.68

423.15112

423.15104

0.189

DM13

[C18 H22 O8 N4 + H]+

7.88

423.15118

423.15104

0.331

DM14

[C19 H24 O9 N4 + H]+

15.06

453.16745

453.16160

−3.541

DM15

[C19 H24 O9 N4 + H]+

8.46

453.16153

453.16160

−0.165

Metabolite

Elemental compositions (M + H+ )

DM0

[C13 H16 O2 N4 + H]+

DM1

retention time (min)

the DVD fragment ion at m/z 246.11, and the fragment ion at m/z 123.06 were the characteristic fragment ions of DVD. We found that the intensity of DM1 was higher than that of DM2: this can be explained by an electronic effect caused by reduction of the CH2 group at the 4 site, as the 2,4-pyrimidinediamine group of DVD is an electron-donating group. Moreover, DM1 had an earlier retention time than DM2 because of its higher polarity; therefore, DM1 was identified as 4 -OH-DVD and DM2 was identified as 3 -OH-DVD.

3.3.2. DM3 DM3 was eluted at 9.42 min and had a protonated ion at m/z 275.15, which was 14 Da higher than that of the parent compound. The elemental composition of the product ion was C14 H19 O2 N4 (predicted molecular weight, 275.15025 Da), which indicates that the CH2 radical was present in the molecule. Therefore, it was proposed to be a methylated metabolite of DVD. In the MS2 spectra of DM3, fragment ions at m/z 123.06 and 245.10 are the same as the characteristic fragment ions of DVD. From the product ion m/z 245.10, it is clear that the methylated position was not on the

Fragment ions’ observed m/z values

Metabolite description

123.06652; 217.10829; 245.10336; 246.11105 123.06638; 232.09523 123.06646; 232.09532 123.06633; 245.10284; 256.98216 230.92860; 248.93815; 259.11862 217.10565; 245.10091; 260.10068 139.06114; 230.92845; 248.93848; 260.12646 139.06139; 230.92862; 248.93881 123.06654; 230.92894; 248.93866; 259.11526; 262.10590 228.19553; 244.09525; 259.11865 123.06662; 232.09552; 247.11884 123.06660; 232.09550; 247.11865 123.06662; 232.09554; 247.11882 123.06660; 232.09549; 247.11879 139.06145; 277.12909 139.06157; 277.12915

DVD

Hydroxylation Hydroxylation Ethoxy

C␣ -hydroxylation

Hydroxylation

N-oxidation

N-oxidation

Hydroxylation

C␣ -OH-methylation

N-glucuronidation

N-glucuronidation

O-glucuronide

O-glucuronide

N-glucuronidation N-glucuronidation

aromatic ring, but at the 4-methoxy joint. Thus, we assumed that DM3 was a 4 -ethoxy metabolite of DVD.

3.3.3. DM4, DM5, DM6, DM7, DM8 and DM9 DM4, DM5, DM6, DM7 and DM8 were eluted at 8.29, 1.08, 8.79, 9.89 and 9.08 min, respectively. All of these metabolites showed a protonated molecule at m/z 277.13, which was 16 Da higher than that of the protonated molecule of DVD; this suggests that they were oxidized metabolites of DVD. As shown in Fig. 5, in the case of DM4, the product ion at m/z 277.13 showed loss of H2 O and subsequent formation of the fragment ion at m/z 259.12, accompanied with high intensity; this suggests that H2 O loss from the metabolite was easy and resulted in formation of the oxygen atom and OH radical. The product ion at m/z 248.94 was formed as a result of loss of CO (predicted molecular weight, 27.99436 Da) from m/z 277.13, and the latter fragment ion at m/z 230.93 was formed by H2 O loss from m/z 248.94. However, in the MSn spectrum, the fragment ions at m/z 123.06, 245.10 and 246.11 were not found, so DM4 was identified as C␣-OH-DVD.

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Fig. 4. The accurate extracted ion chromatogram (EIC) of feces from male (A) and female (B) chicken at 6 h after oral administration. As can be seen, 14 metabolites were identified.

The MS2 spectrum of the protonated molecule obtained from DM5 showed the major fragment ion at m/z 260.10, which was formed by loss of OH from the precursor ion at m/z 277.13. The product ion at m/z 260.10 lost a CH3 radical to form m/z 245.10, which was followed by the loss of a CO radical to form m/z 217.10. Moreover, the retention time of DM5 was 1.07 min, which suggests that DM5 had strong polarity and that the position of OH was on the 4 -methoxy group. DM6 and DM7 showed the protonated molecule ion at m/z 277.13 and contained a product ion at m/z 139.06, which was 16 Da higher than that of the protonated molecule of DVD at m/z 261.13; its fragment ion was at m/z 123.06. Based on this, DM6 could be an N-oxide derivative of DVD. The MS2 spectrum of DM6 showed product ions at m/z 260.12 and m/z 248.94, resulting from the loss of OH and CO radicals from DM6, respectively. The resulting fragment

ion at m/z 230.93 was consistent with loss of H2 O from m/z 248.94, which indicates that the oxygen atom was on the N group. The MS2 spectrum of the protonated molecule obtained from DM7 also showed the same product ions as the product ions of DM6 at m/z 139.06 and m/z 248.94, but not the product ions at m/z 260.12; this indicated that the oxygen atom was steady. Therefore, we assumed that DM7 and DM6 were most likely 1-NO-DVD and 3-NO-DVD, respectively. In the MS2 spectra of DM8, the fragment ions at m/z 123.06 were the same as the characteristic fragment ions of DVD, which suggests that the oxygen atom was not on the methylene or the pyrimidine ring. Moreover, the product ion at m/z 262.10 and m/z 259.11 resulted from loss of the CH3 radical and H2 O, and the fragment ion at m/z 248.94 was consistent with loss of CO. The latter ion at m/z 230.93 was produced by loss of H2 O from the product ion at

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97

Fig. 5. Accurate MS2 spectra of phase I DVD metabolites (DM1–DM9).

m/z 248.94. DM8 was identified as the mono-hydroxyl group on the benzene ring, but the precise position of the hydroxyl group on the benzene ring could not be characterized. DM9 was eluted at 9.21 min and showed a protonated molecule at m/z 291.15, 30 Da higher than that at m/z 261.13. The MS2 spectra of DM9 showed the fragment ion at m/z 259.12, which was the same as that for DM4; this suggests that DM9 is an oxidized product formed by the addition of one oxygen atom to methylene and the resulting methylation of the hydroxy group. DM9 contained fragment ions at m/z 244.09 and 228.19 which correspond to loss of the CH3 and OCH3 radical from the fragment ion at m/z 259.12, respectively; however, it did not have a fragment ion at m/z 123.06. Thus, DM9 was identified as a methylated metabolite of DM4. 3.4. Identification of phase II metabolites of DVD The accurate MS2 spectra for phase II metabolites are shown in Fig. 6 (DM10–DM15). 3.4.1. DM10 and DM11 DM10 and DM11 were observed at retention times of 8.33 and 8.57 min, respectively, with the same protonated molecular ion [DM + H]+ at m/z 437.17, which was 176 Da more than the protonated DVD and had molecular weight equal to that of glucuronic acid. Therefore, we think that these metabolites are glucuronide conjugates of DVD. The MS2 spectrum of the protonated molecule obtained from DM10 had major product ions at m/z 261.13, m/z 247.12, and further MS3 analysis of m/z 247.12 revealed characteristic fragment ions at m/z 123.06 and the fragment ion at m/z

232.09. Thus, the fragment ions were the same as those of DM1. Based on these observations, we assumed that DM10 and DM11 were N-glucuronidation metabolites, but the precise position of the glucuronic acid could not be characterized. 3.4.2. DM12 and DM13 DM12 and DM13 were eluted at 7.68 and 7.88 min, respectively. These metabolites showed the protonated molecular ion at m/z 423.15, which was 176 Da more than that of DM1. They were identified as demethyl-O-glucuronides, which are glucuronidation metabolites of DM1. DM12 and DM13 had a neutral loss of 176 Da, which induced the same fragment ions at m/z 247.12 in datadependent scan MS2 spectra. Subsequent isolation and CID of m/z 247.12 in data-dependent scan MS3 spectra produced the characteristic fragment ions DM1 and DM2. Therefore, we assumed that DM12 and DM13 had undergone demethylation first, subsequently forming a hydroxyl ion conjugated with glucuronic acid. Considering the electron-absorbing effect of DM1 and DM2, DM12 and DM13 most likely correspond to 4 -demethyl-O-glucuronide and 3 -demethyl-O-glucuronide respectively. 3.4.3. DM14 and DM15 DM14 and DM15 were eluted at 15.06 and 8.46 min, respectively, and showed the same protonated ion at m/z 453.17, which was 176 Da higher than that at m/z 277.13 and 16 Da higher than that of m/z 437.17; these metabolites may therefore be the glucuronic acid derivatives of oxidized metabolites. The MS2 spectra of DM14 showed fragment ions at m/z 277.13, and the datadependent scan MS3 spectra showed the product ion at m/z 139.06,

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Fig. 6. Accurate MS2 spectra of phase II metabolites of DVD (DM10–DM15).

H. Wang et al. / J. Chromatogr. B 965 (2014) 91–99 NH2

N

H2 N

NH2

O

N

O

DM5

OH

H2N

NH2

O

N N

DM6

O

H2N

N NH2

N

N

O

H2N

N

O

O N

HO DM8

O

NH2 O

Glu

H2N

O N

OGlu

O

N

N

N

DM7

NH2

H2N

OGlu

DM13

N

N

NH NH2

O

OH

DM1

O

NH2

O

DVD

O

N

NH2

N

DM15

H2N

NH2

O

DM2

O

N

OH

H2N

H2N

Glu N

H2N

O

H2N

N

N

O

DM14

O

N

O

N

O

DM3

N

NH2

O

NH2

O

NH2

H2N

N

O

O

DM4

N NH2 N

Glu

O

N H2 N

O

N

O NH2 OH

99

O

NH2

Glu N HN

DM11

O

H2 N

N

DM9

O

O

N DM10

DM12

Fig. 7. The proposed metabolic pathways for DVD in chicken.

identical to M6; thus, DM14 may be an N-oxide derivative of DVD which then formed a hydroxyl and became conjugated with glucuronic acid. Meanwhile, we found DM15 showed the same fragment ions at m/z 277.13 and m/z 139.06. It may be also formed by N-oxide derivative of DVD. Considering the retention time and stability, we assumed that DM14 and DM15 were the N-oxide glucuronidation metabolites, but the precise position of the glucuronic acid could not be characterized.

We found that not only O-demethylation but also N-oxidation was the main metabolic pathway. This work therefore shows that LC-LTQ-Orbitrap is rapid, selective, and sensitive enough for the characterization of unknown metabolites in complicated matrices, and that the data procured here are an important basis for further estimations of the toxicity, food safety and marker residues of DVD. Acknowledgements

3.5. Proposed metabolic pathways Based on the above analyses, the proposed phase I and II metabolic pathways for DVD in chicken are shown in Fig. 7. DVD was first metabolized to phase I metabolites, the metabolic pathways were demethylation, methylation, oxidation, hydroxylation; then it took place the phase II metabolism, they are mainly glucuronide metabolites occurring at N-oxide position, NH2 position and O-demethylation position. 4. Conclusions The present work used LC-LTQ-Orbitrap and its analytical software tools as a rapid, reliable and sensitive method for identifying the expected and unexpected metabolites of DVD in chickens for the first time. In the previous in vitro study on DVD, six DVDrelated metabolites were characterized in pig liver microsomes, and O-demethylation was identified as the major metabolic route. Five (DM1, DM2, DM4, DM6 and DM7) of the metabolites found in the in vitro study were also detected in the present study. This means that the in vitro and in vivo DVD metabolisms are interrelated. We also found 10 other metabolites which included a 4 -ethoxy metabolite, a benzene ring-hydroxylation metabolite, a C␣-OH-methylation metabolite, two N-glucuronide metabolites, two O-demethylation-glucuronide metabolites, and two N-oxide glucuronide metabolites. The latter six were phase II metabolites.

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