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

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

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