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Simple and Fast Extraction Coupled UPLC-MS/MS Method for the Determination of Mequindox and Its Major Metabolites in Food Animal Tissues Yanli You, Liting Song, Yanshen Li, and Yongtao Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05903 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 3, 2016

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Journal of Agricultural and Food Chemistry

Simple and Fast Extraction Coupled UPLC-MS/MS Method for the Determination of Mequindox and Its Major Metabolites in Food Animal Tissues

Yanli You, Liting Song, Yanshen Li *, Yongtao Wu

College of Life Science, Yantai University, Yantai, Shandong, 264005, P. R. China

Corresponding Author *(Yanshen) Tel: +86-535-691-3938; Fax:+86-535-690-2638; E-mail address: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT:

2

This research described a sensitive and rapid UPLC-MS/MS method for

3

determination of mequindox and its 6 major metabolites in chicken muscle, chicken

4

liver, swine muscle, and swine liver. Among the metabolites, Carbonyl

5

reduction-1,4-bisdesoxy-mequindox is novel. Target analytes could be extracted by

6

ethyl acetate without any acidolysis, enzymolysis steps. After purification by a Bond

7

Elut C18 cartridge, analysis was carried out by UPLC-MS/MS using positive ion

8

multiple reaction monitoring (MRM) mode. Validation was performed in spiked

9

samples, mean recoveries ranged from 64.3% to 114.4%, with intra-day and inter-day

10

variation less than 14.7% and 19.2%. The limit of detection (LOD) was less than 1.0

11

µg kg-1, while limit of quantification (LOQ) was less than 4.0 µg kg-1. This procedure

12

will help control animal derived food from mequindox residues, and it will also

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facilitate further pharmacokinetic of mequindox.

14 15

Keywords: Mequindox, Metabolites, Residue analysis, UPLC-MS/MS, animal

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derived food

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2


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INTRODUCTION

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Mequindox (3-methyl-2-quinoaxlinacetyl-1,4-dioxide, MEQ, structure was

20

shown in Figure 1), one of the quinoxaline-1,4-dioxides (QdNOs), has been

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developed as a broad-spectrum antibacterial agent since 1980s.1 MEQ has been

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widely used in young swine feed because of its high antimicrobial activity. It can

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promote growth; improve feed efficiency; control swine dysentery and bacterial

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enteritis.2 However, in recent years, the European Commission (EC) banned the use of

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another two QdNOs, carbadox (CBX) and olaquindox (OLA) in animal husbandry3

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because of the potential carcinogenicity and mutagenicity of QdNOs.4-6 MEQ, which

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exhibits corresponding structure to CBX and OLA, draws an increasing attention with

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the risk assessment. In addition, in recent researches, it was also reported that DNA

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damage and genotoxicity could be induced by MEQ.7,8

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In addition, the metabolites of MEQ might also exhibit high toxicity to liver and

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spleen as well as the precursor.9,10 Therefore, it should be paid close attention to MEQ

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and its metabolites residues in animal derived food. Till now, in the previous

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metabolism studies, numerous metabolites of MEQ were identified.11,12 Specially,

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N→O group reduction and carbonyl reduction metabolites might cause even more

35

severe toxicity than MEQ.13,14 From these studies, there were 6 major metabolites

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were confirmed, and the 6 metabolites covered almost most quantity of all the

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metabolites of MEQ. They were identified as 1-desoxy-mequindox (M1, 1-DMEQ),

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4-desoxy-mequindox (M2, 4-DMEQ), 2-isoethanol-mequindox (M3, 2-iso-MEQ),

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2-isoethanol-1-desoxy-mequindox

(M4, 3


2-iso-1-DMEQ),


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2-isoethanol-4-desoxy-mequindox

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reduction-1,4-bisdesoxy-mequindox (M6, 2-iso-BDMEQ). To control these chemical

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compounds in animal derived food, many literatures were reported for the

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determination of residues of QdNOs and the metabolites employing LC (high

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performance liquid chromatography) coupled with UV (ultraviolet) detector1,15-17 and

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LC-MS/MS

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spectrometry).18,19,20-25 Mass spectrometry allows obtaining a better sensitivity and

47

covering a wider range of analytes. Methods for the determination of MEQ and some

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of its metabolites in swine liver and kidneys have been reported in the previous

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literatures.18,20,21 However, these methods can not cover all the metabolites of MEQ,

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and need complicate extraction and purification procedures, including acidolysis and

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enzymolysis. These acidolysis and enzymolysis pretreatment procedures are

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complicated and time consuming. Beyongs, these current reported methods could not

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cover the whole range of MEQ and its major metabolites. Besides, the separation

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efficiency of liquid phase for analysis is also very low.

(high-performance

(M5,

liquid

2-iso-4-DMEQ),

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chromatography

and

tandem

Carbonyl

mass

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This research presented a new, sensitive, and rapid ultra-performance liquid

56

chromatography coupled with triple-quadrupole mass spectrometry (UPLC-MS/MS)

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method for the simultaneously determination of MEQ and its 6 major corresponding

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metabolites (M6 is novel) in chicken muscle, chicken liver, swine muscle, and swine

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liver. The extraction step was simplified with ethyl acetate as extract solution without

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complicated acidolysis, enzymolysis steps. Till now, there are no marker residue

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metabolites of MEQ in animal tissues. Therefore, in order to monitor MEQ residue in 4


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animal tissues, it is better to cover all the major metabolites as much as possible. This

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developed procedure covered all the characterized major metabolites of MEQ,

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including M6. And it will contribute to the control of MEQ in animal derived food.

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Moreover, this research will be benefit for the further pharmacokinetics

66

investigations.

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MATERIALS AND METHODS

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Chemicals and Reagents

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Analytical standards MEQ (purity > 98%) was purchased from the China

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Institute of Veterinary Drug Control (Beijing, China). 1-DMEQ (M1) (purity > 98%),

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4-DMEQ (M2) (purity > 99%), 2-iso-MEQ (M3) (purity > 98%), 2-iso-1-DMEQ (M4)

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(purity > 98%), 2-iso-4-DMEQ (M5) (purity > 98%), and 2-iso-BDMEQ (M6)

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(purity > 98%) (Figure 1) were synthesized by science of college, China Agricultural

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University (Beijing, China).

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HPLC grade methanol and acetonitrile were purchased from Dima Technology

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Inc. (Muskegon, MI, USA). Formic acid (HPLC grade) was obtained from Fisher

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Scientific Inc. (Pittsburgh, PA, USA). Water was purified using a Milli-Q Synthesis

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system from Millipore (Bedford, MA, USA). Bond Elut C18 cartridge (500 mg, 6 cc)

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were purchased from Agilent Technologies (CA, USA). ethyl acetate, metaphosphoric

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acid，dimethylsulfoxide (DMSO) and other reagents were purchased from Sinopharm

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Chemical Reagent Beijing Co., Ltd (Beijing, China).

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Standard stock solutions (1 mg mL-1) were prepared by dissolving 5 mg of each

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compound in 5 mL of methanol, while M6 were in 5 mL of DMSO due to its low 5



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solubility in methanol. These solutions were stored at -20°C in brown amber bottles

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and stable for at least 3 months.

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Mixed working standard solution (100 µg mL-1) was prepared by diluting stock

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solutions with methanol. These working solution were stored at -20°C in brown

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amber bottle and stable for at most 1 week.

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Sample

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Blank samples (chicken muscle, chicken liver, swine muscle and swine liver)

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which were characterized using UPLC-MS/MS were purchased from local

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supermarket. Samples were homogenized by a domestic food blender and then stored

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at -20°C till use.

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Preparation

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An amount of 2 g (± 0.02 g) of each sample was weight into a 50 mL

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polypropylene centrifuge tube. Samples were divided into three groups. Each group

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was fortified by adding mixed working standard solution to yield final concentrations

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of 10, 50, and 200 µg kg-1 for validation. One unfortified sample was set as the

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negative control. Each was vortexed for 30 s and incubated at 25°C for 20 min before

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the next proceeding. After that, 10 mL of ethyl acetate were added for extraction.

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Each sample was shaken at 300 rpm for 25 min, and then centrifuged at 9000 rpm for

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15 min at 4°C. The supernatant was transferred to another tube and then the extract

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procedure was repeated. Combine the extraction and evaporated to dryness under a

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gentle flow of nitrogen at 45°C. The residues were re-dissolved by adding 5 mL of 10%

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methanol in water and vortex for 2 min. For further purification, a C18 cartridge was 6


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previously conditioned with methanol (5 mL) and water (5 mL). Then the extract

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solution was loaded onto the C18 cartridge by gravity, and the cartridge was rinsed

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with 5 mL of water. The compounds adsorbed on each cartridge were eluted with

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methanol (5 mL) and the eluate was taken to dryness under a gentle flow of nitrogen

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at 45°C. The residues were re-dissolved and filtered through a 0.22 µm nylon filter

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into an autosampler vial for UPLC-MS/MS analysis.

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Instrumental conditions

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Chromatographic separation for all the target analytes was performed on a

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Waters AcquityTM UPLC system with column oven temperature maintained at 30°C

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via an Acquity BEH C18 column (50 mm × 2.1 mm i.d., 1.7 µm particle size) (Waters,

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Milford, MA, USA). The mobile phase was composed of solvent A (water containing

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0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) with a

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flow rate was 0.3 mL/min. Gradient elution program was performed as follows: 0-1.5

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min, 85% A; 1.5 to 2.2 min, 85-75% A; 2.2-4.0 min, 75-30% A; 4.0-5.0 min, 30-85%

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A; 5.0-6.0 min 85% A. The injection volume was 10 µL. Weak and strong wash

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solvents were 10% and 90% acetonitrile in water, respectively.

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The UPLC system was coupled to a Mass Quattro Premier XE triple quadrupole

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mass spectrometer (Waters, Manchester, UK) fitted with an electrospray ionization

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source (ESI). Typical source conditions for maximum intensity of precursor ions were

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as follows: desolvation gas was maintained at 650 L/h with the temperature of 280°C;

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capillary voltage was 3.0 kV; source temperature was set at 80°C, and cone gas flow

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rate was 30 L h-1. For all compounds, MS instrument was operated in ESI positive 7



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(ESI+) multiple reaction monitoring (MRM) mode. Optimized MS/MS parameters

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were summarized in table 1.

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Method validation

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In order to evaluate the performance of this method, the linearity, limit of

132

detection (LOD), limit of quantification (LOQ), and accuracy and precision were

133

validated.

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Linearity. To evaluate the linearity, matrix-matched calibration curves were prepared

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by external standard calibrations for each analyte with seven points of concentrations.

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A series of calibration curve was prepared for each matrix with seven different

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concentrations (Table 2). The matrix-standard solutions were prepared with the

138

prepared samples at the end of sample preparation and correlation coefficient (r2) was

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determined.

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LOD and LOQ. LOD was defined as the lowest concentration for the detection of

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each analyte, and it is determined by a signal-to-noise ratio (S/N) ≥ 3. LOQ was

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defined as the lowest measured concentration for each analyte, and it was determined

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by S/N ≥ 10. Each was obtained on the basis of the chromatographic conditions.

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Accuracy and precision. Accuracy and precision were evaluated by determining

145

recoveries of each analyte in spiked samples with six replicates on three separate days.

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The spiked levels were 10, 50, and 200 µg kg-1. Concentrations of the samples were

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calculated according to the calibration curves. The recovery was determined by means

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of the measured concentration compared with the spiked concentration.

8


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RESULTS AND DISCUSSION

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UPLC-MS/MS analysis

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The MRM associated parameters were optimized for the maximum abundance of

152

fragmented ions under ESI+ mode conditions by injecting matrix-standard solutions of

153

the target compounds to the tandem mass spectrometer, and the most intense ion was

154

used for quantitation (Table 1).

155

In order to improve the ionization efficiency in positive ESI mode, formic acid

156

was added to the mobile phases (acetonitrile and water) to a final percentage of 0.1%.

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Typical UPLC-MS/MS chromatograms of fortified samples were shown in Figure 2.

158

Optimized minimal run times were within 6 min for all the 7 compounds. Compared

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to the previously reported HPLC separation method15,16,18,19, it exhibited high

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separation efficiency for all compounds.

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Optimization of sample preparation

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Optimization of extraction procedure.

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in the sample preparation process for a high recovery results. Based on the polarity

164

and solubility of all the target compounds, they were insoluble in water. In the

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previous research, ultrasound assistant extractions of MEQ using a mixture solution

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have been reported.20 However, most extraction procedures of MEQ in tissues need

167

acidolysis or enzymolysis previously.18 In this research, the extraction procedure was

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performed without any acidolysis or enzymolysis steps, saving at least one hour of

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hydrolyzing process. Different conditions for the extraction were evaluated, including

Extraction is one of the most critical steps

9



170

ultrasound assistant, temperature, and different organic solvent. The resulted showed

171

that the recovery exhibited no difference with or without ultrasound assistant

172

extraction. In this research, different temperatures of 25°C, 35°C, 45°C, 55°C, and

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65°C were tested. It was observed that low recovery was obtained when the

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temperature was over 45°C. It might because that MEQ and its metabolites were not

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stable under high-temperature condition.26 Therefore, all the pretreatment procedures

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were process at the temperature below 45°C. Different organic solvents were

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optimized for extraction, and methanol resulted in a low recovery (≤60%). As to

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acetonitrile, it could lead to tissue samples conglomeration, which might lead to a low

179

recovery of target analytes. Ethyl acetate exhibited the most satisfactory recovery

180

without any acidolysis or enzymolysis steps. And it was adopted as the extract

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solution for its high efficiency, simplicity.

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Optimization of clean-up procedure. For trace amount analysis, it is important to

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eliminate possible interferences from crude sample extract. Based on the previous

184

literatures, Oasis HLB, Bond Elut C18, and MAX cartridges were the most common

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SPE cartridges for the purification of MEQ and its metabolites.23 As ion-exchange

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cartridge, MAX cartridge was associated with pKa value of each compound and the

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pH value of loading solution. MAX was suitable for quinoxaline-2-carboxylic acid

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(QCA) and 3-methylquinoxaline-2-carboxylic acid (MQCA) with the corresponding

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structure of carboxyl group.22,26 During preliminary research, it was observed that the

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Bond Elut C18 cartridge led to a more satisfactory recovery result (92.7%) than the

191

HLB cartridges (90.1%). Thus, Bond Elut C18 cartridge was adopted in this research. 10


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192

Considering that the percentage of organic solvent in sample solution could influence

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the binding affinity between C18 cartridge and the target analytes. For optimization,

194

different percentages (0-100%) of methanol were evaluated, and it was observed that

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when the percentage of methanol is less than 10% all the 7 compounds could bind to

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the C18 cartridge, and when it is over 85% the binding efficiency could be neglected

197

(Figure 3). Taking the extraction step together, the residues should be re-dissolved

198

after the extract was taken to dryness. And the solubility of all the 7 compounds is

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very low in water. Therefore, in order to obtain both a suitable solution for SPE

200

purification and maximum re-dissolve efficiency, 10% methanol was adopted as the

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re-dissolved solvent. As shown in the figure 3, when the percentage of methanol was

202

over 85%, it could be applied to elute all the 7 compounds. Considering the following

203

concentration step, 85% methanol was hardly to concentrate to a constant volume

204

because of the 15% water. Therefore, pure methanol was adopted as the elute solvent

205

in this research.

206

Method validation

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20 blank samples of different origin were processed using this proposed

208

procedure to evaluate the specificity. MRM chromatograms of blank and fortified

209

chicken muscle, chicken liver, swine muscle, and swine liver were shown in Figure 2.

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From the figure, it was shown that no interference was obtained at the retention time

211

of each analyte. Matrix-matched liner regression calibration curves were summarized

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in Table 2 with correlation coefficients (r2) for each analyte over 0.99.

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LOD was determined from fortified samples based an S/N ratio over 3, while 11



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LOQ based on an S/N ratio over 10. With the UPLC-MS/MS method applied in this

215

work, LOD and LOQ obtained for each samples were at a very low level (Table 2).

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From the table, it was found that LOD and LOQ obtained in this research ranged from

217

0.1 to 1.0 µg kg-1 and 0.4 to 4.0 µg kg-1 in all the samples.

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Table 3 exhibited the accuracy and precision for this developed procedure. It was

219

evaluated by determining recoveries as well as intra-day and inter-day RSD (Relative

220

Standard Deviation) of each analyte in fortified samples. Each was processed at three

221

different concentrations with six replicates on three separate days. Mean recoveries

222

ranged from 64.3%~114.4% for all the analytes with the intra-day RSD less than 14.7%

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and inter-day RSD less than 19.2%, respectively.

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As to the four matrixes, mean recoveries of all the analytes were 77.3%, 88.1%,

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79.3%, and 86.1% for chicken muscle, chicken liver, swine muscle, and swine liver,

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respectively. It can be concluded that both in chicken and swine samples, the recovery

227

in liver samples are higher than that in muscle samples. It might due to the component

228

percentage of different matrix. In chicken muscle, the percentage of protein and fat

229

are 21.5% and 2.5%, while they are 18.2% and 3.4% in chicken liver. All the analytes

230

were lipophilic, and higher percentage of fat will lead to a higher recovery of these

231

analytes. On the other hand, protein could bind the analytes and it will bring down the

232

recoveries. Protein in chicken muscle takes up more percentage than that in chicken

233

liver, which keeps consistent with the lower recovery in chicken muscle than chicken

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liver. In swine samples, the percentage of fat is higher in swine muscle (6%) than

235

swine liver (4.5%), and the percentage of protein is higher in swine muscle (29%) 12


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236

than swine liver (21.3%) as well. The recovery of the analytes in swine muscle with

237

higher percentage of fat and protein is lower than that in swine liver. It seems that the

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protein plays the dominant role in the extract efficiency of the target analytes.

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Application to real samples

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The developed analytical method was successfully applied for the determination

241

of all the 7 compounds in 40 samples obtained from the local supermarket (10 chicken

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muscle samples, 10 chicken liver samples, 10 swine muscle samples, and 10 swine

243

liver samples). 12 positive samples were detected of M1 and M6 with the

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concentrations ranged from 5.88 to 53.39 µg kg-1 and from 2.08 to 3.62 µg kg-1. As to

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MEQ, M2, M3, M4, and M5, these compounds were not detected in all the samples

246

(Table 4). To verify this development experiment, the 12 positive samples were

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process according to the previous reported protocol with enzymolysis extraction

248

procedure.18 The results were in agreement with our research, with M1 and M6 at the

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concentrations ranging from 6.77 to 56.72 µg kg-1 and from 4.32 to 5.73 µg kg-1,

250

respectively.

251

In conclusion, a quantitative and confirmatory UPLC-MS/MS procedure for the

252

determination of MEQ and all the 6 characterized major metabolites (metabolite M6

253

is novel) in chicken muscle, chicken liver, swine muscle, and swine liver was

254

developed in this study. Specially, target analytes were extracted with ethyl acetate

255

without any complicated acidolysis, enzymolysis steps and then further purified by

256

C18 cartridge to minimizing the matrix effect in order to obtain high sensitivity and

257

low LOD and LOQ. The validation of this developed procedure proved the suitability 13



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258

of the method for the confirmatory analysis of MEQ and its metabolites with mean

259

recoveries from 64.3%~114.4%, intra-day RSD < 14.7%，inter-day RSD < 19.2%,

260

LOD < 1.0 µg kg-1, and LOQ < 4.0 µg kg-1. Applying to real samples, 30% were

261

detected as positive. This proposed analytical procedure could thus be employed for

262

the control of MEQ in animal derived food, and moreover it will also be benefit for

263

the further pharmacokinetics investigations.

264

ABBREVIATIONS USED

265

1-desoxy-mequindox, M1, 1-DMEQ; 2-isoethanol-1-desoxy-mequindox, M4,

266

2-iso-1-DMEQ;

2-isoethanol-4-desoxy-mequindox,

267

2-isoethanol-mequindox, M3, 2-iso-MEQ; 3-methylquinoxaline-2-carboxylic acid,

268

MQCA;

269

reduction-1,4-bisdesoxy-mequindox, M6, 2-iso-BDMEQ; Correlation coefficient, r2;

270

Dimethylsulfoxide,

271

Commission, EC; High performance liquid chromatography, LC; High-performance

272

liquid chromatography tandem mass spectrometry, LC-MS/MS; Limit of detection,

273

LOD; Limit of quantification, LOQ; Mequindox, MEQ; Multiple reaction monitoring,

274

MRM;

275

Quinoxaline-2-carboxylic acid, QCA; Signal-to-noise ratio, S/N; Ultra-performance

276

Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry,

277

UPLC-MS/MS; Ultraviolet, UV.

278

ACKNOWLEDGMENTS

4-desoxy-mequindox,

DMSO;

Olaquindox,

M2,

4-DMEQ;

Electrospray

OLA;

M5,

Carbadox,

ionization

source,

2-iso-4-DMEQ;

CBX;

ESI;

Quinoxaline-1,4-dioxides,

14


Carbonyl

European

QdNOs;

Page 15 of 33


279

The authors would like to thank Sasha Stone (Department of Microbiology,

280

Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104,

281

USA) for critical reading of this manuscript and writing assistance.

282

15



283

REFERENCES

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Life Sci. 2012, 881-882, 96-106.

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13. Huang, L.; Xiao, A.; Fan, S.; Yin, J.; Chen, P.; Liu, D.; Qiu, Y.; Wang, Y.; Yuan, Z.

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Development of liquid chromatographic methods for determination of

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quinocetone and its main metabolites in edible tissues of swine and chicken. J.

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Aoac. Int. 2005, 88, 472-478.

325

14. Zhang, H.; Huang, C. H. Reactivity and transformation of antibacterial N-oxides

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in the presence of manganese oxide. Environ. Sci. Technol. 2005, 39, 593-601. 17



Page 18 of 33

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15. Ding, H.; Liu, Y.; Zeng, Z.; Si, H.; Liu, K.; Liu, Y.; Yang, F.; Li, Y.; Zeng, D.

328

Pharmacokinetics of mequindox and one of its major metabolites in chickens after

329

intravenous, intramuscular and oral administration. Res. Vet. Sci. 2012, 93,

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374-377.

331

16. Zhang, J.; Gao, H.; Peng, B.; Li, Y.; Li, S.; Zhou, Z. Simultaneous determination

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of four synthesized metabolites of mequindox in urine samples using

333

ultrasound-assisted dispersive liquid-liquid microextraction combined with

334

high-performance liquid chromatography. Talanta 2012, 88, 330-337.

335

17. He, Q.; Fang, B.; Su, Y.; Zeng, Z.; Yang, J.; He, L.; Zeng, D., Simultaneous

336

determination of quinoxaline-1,4-dioxides in feeds using molecularly imprinted

337

solid-phase extraction coupled with HPLC. J Sep Sci 2013, 36, (2), 301-10.

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18. Zeng, D.; Shen, X.; He, L.; Ding, H.; Tang, Y.; Sun, Y.; Fang, B.; Zeng, Z. Liquid

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chromatography tandem mass spectrometry for the simultaneous determination of

340

mequindox and its metabolites in porcine tissues. J. Sep. Sci. 2012, 35,

341

1327-1335.

342

19. Liu, K.; Cao, X.; Wang, Z.; Li, L.; Shen, J.; Cheng, L.; Zhang, S. Analysis of

343

mequindox and its two metabolites in swine liver by UPLC-MS/MS. Anal.

344

Methods 2012, 4, 859-863.

345

20. Wu, C.; Li, Y.; Shen, J.; Cheng, L.; Li, Y.; Yang, C.; Feng, P.; Zhang, S. LC–MS–

346

MS

347

Chromatographia 2009, 70, 1605-1611.

348

Quantification

of

Four

Quinoxaline-1,4-Dioxides

in

Swine

Feed.

21. Li, Y.; Liu, K.; Ross, C. B.; Cao, X.; Shen, J.; Zhang, S. Simultaneous 18


Page 19 of 33


349

determination of mequindox, quinocetone, and their major metabolites in chicken

350

and pork by UPLC–MS/MS. Food chem. 2014, 160, 171-179.

351

22. Boison, J. O.; Lee, S. C.; Gedir, R. G., A determinative and confirmatory method

352

for residues of the metabolites of carbadox and olaquindox in porcine tissues.

353

Anal. Chim. Acta 2009, 637, (1-2), 128-134.

354

23. Merou, A.; Kaklamanos, G.; Theodoridis, G., Determination of Carbadox and

355

metabolites of Carbadox and Olaquindox in muscle tissue using high performance

356

liquid chromatography-tandem mass spectrometry. J. Chromatogr. B Analyt.

357

Technol. Biomed. Life Sci. 2012, 881-882, 90-95.

358

24. Sniegocki, T.; Gbylik-Sikorska, M.; Posyniak, A.; Zmudzki, J., Determination of

359

carbadox

and

olaquindox

metabolites

in

swine

muscle

by

liquid

360

chromatography/mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed.

361

Life Sci. 2014, 944, 25-29.

362

25. Souza, D. W.; de Alkimin, F. J.; Da, S. O. F.; Sampaio, D. A. D.; Camargos, L. L.;

363

de Figueiredo, T. C.; de Vasconcelos, C. S., HPLC-MS/MS method validation for

364

the detection of carbadox and olaquindox in poultry and swine feedingstuffs.

365

Talanta 2015, 144, 740-744.

366

26. Wu, Y.; Yu, H.; Wang, Y.; Huang, L.; Tao, Y.; Chen, D.; Peng, D.; Liu, Z.; Yuan, Z.

367

Development of a high-performance liquid chromatography method for the

368

simultaneous

369

methyl-3-quinoxaline-2-carboxylic acid in animal tissues. J. Chromatogr. A 2007,

370

1146, 1-7.

quantification

of

quinoxaline-2-carboxylic

19


acid

and


371 372 373

NOTE. This work was financially supported by National Natural Science Foundation of China (Grant no. 31402246).

20


Page 20 of 33

Page 21 of 33


Figure captions Figure 1.

Chemical structure of MEQ and 6 major metabolites

Figure 2.

MRM chromatograms for MEQ and major metabolites in blank chicken

muscle samples Figure 2-1 (A), chicken muscle sample fortified at 10 µg kg-1 Figure 2-1 (B), blank chicken liver samples Figure 2-2 (A), chicken liver sample fortified at 10 µg kg-1 Figure 2-2 (B), blank swine muscle samples Figure 2-3 (A), swine muscle sample fortified at 10 µg kg-1 Figure 2-3 (B), blank swine liver samples Figure 2-4 (A), swine liver sample fortified at 10 µg kg-1 Figure 2-4 (B). Figure 3.

Optimization of SPE C18 loading and elute condition with different

percentages of methanol.

21



Page 22 of 33

Table 1 Mass Spectrum Parameters of MEQ and 6 Major Metabolites Analyte MEQ

M1 (1-DMEQ)

Retention time

Precursor ion

Product ion

Cone voltage

Collision energy

(min)

(m/z)

(m/z)

(V)

(eV)

1.24

219.0

142.9a

18

15

159.9

18

15

18

20

18

20

a

18

20

2.88

203.0

144.0

a

161.0 M2 (4-DMEQ)

3.27

203.0

158.0 186.0

18

20

M3 (2-iso-MEQ)

0.92

221.0

168.9 a

18

21

187.9

18

21

20

22

18

18

20

16

M4 (2-iso-1-DMEQ)

1.33

205.0

168.9

a

169.7 M5 (2-iso-4-DMEQ)

1.92

205.0

160.8 187.9

M6 (2-iso-BDMEQ) a

2.47

189.0

a

10

18

142.8

22

24

170.8 a

22

20

Ion for Quantification

22


Page 23 of 33


Table 2 Parameters (Including Standard Curve, LOD, and LOQ) of MEQ and Metabolites in Different Tissues Matrix

Chicken muscle

Chicken liver

Swine muscle

Swine liver

Analyte

Liner range (µg kg-1)

Regression equitation

r2

LOD

LOQ

(µg kg-1)

(µg kg-1)

MEQ

1.0~500

y=111.16x+243.66

0.9999

0.2

0.9

M1 (1-DMEQ)

0.5~500

y=155.55x+166.53

0.9996

0.1

0.5

M2 (4-DMEQ)

1.0~500

y=159.28x+382.95

0.9986

0.2

1.0

M3 (2-iso-MEQ)

1.0~500

y=36.587x+142.03

0.9986

0.2

1.0

M4 (2-iso-1-DMEQ)

0.5~500

y=213.11x+598.04

0.9986

0.2

0.4

M5 (2-iso-4-DMEQ)

0.5~500

y=268.91x+2434.3

0.9958

0.1

0.4

M6 (2-iso-BDMEQ)

0.5~500

y=198.65x+173.06

0.9999

0.1

0.5

MEQ

2.0~1000

y=69.507x+2453.2

0.9932

0.6

2.0

M1 (1-DMEQ)

0.5~1000

y=167.26x+2269.5

0.9946

0.2

0.5

M2 (4-DMEQ)

1.0~1000

y=101.36x+642.06

0.9976

0.2

0.7

M3 (2-iso-MEQ)

1.0~1000

y=15.971x+341.14

0.9904

0.3

0.7

M4 (2-iso-1-DMEQ)

1.0~1000

y=132.82x+1389.3

0.9990

0.3

0.8

M5 (2-iso-4-DMEQ)

0.5~1000

y=122.39x+1859.8

0.9950

0.1

0.4

M6 (2-iso-BDMEQ)

1.0~1000

y=262.93x+2082.1

0.9984

0.2

0.7

MEQ

5.0~1000

y=90.236x+2323.7

0.9972

1.0

4.0

M1 (1-DMEQ)

1.0~1000

y=192.09x+2720.1

0.9954

0.3

1.0

M2 (4-DMEQ)

1.0~1000

y=150.53x+849.73

0.9994

0.2

0.7

M3 (2-iso-MEQ)

1.5~1000

y=20.282x+468.61

0.9908

0.4

1.5

M4 (2-iso-1-DMEQ)

2.0~1000

y=147.80x+1631.5

0.9982

0.5

1.7

M5 (2-iso-4-DMEQ)

1.0~1000

y=155.53x+2727.4

0.9918

0.2

0.7

M6 (2-iso-BDMEQ)

5.0~1000

y=307.43x+4211.0

0.9910

1.0

3.5

MEQ

1.0~1000

y=57.417x+1108.5

0.9950

0.3

1.0

M1 (1-DMEQ)

0.5~1000

y=126.77x+1323.1

0.9954

0.1

0.3

M2 (4-DMEQ)

0.5~1000

y=81.831x+465.92

0.9986

0.1

0.2

M3 (2-iso-MEQ)

0.5~1000

y=12.421x+182.13

0.9968

0.1

0.3

M4 (2-iso-1-DMEQ)

0.5~1000

y=101.43x+1501.8

0.9948

0.2

0.6

M5 (2-iso-4-DMEQ)

0.5~1000

y=96.168x+1237.2

0.9964

0.1

0.3

M6 (2-iso-BDMEQ)

1.0~1000

y=207.06x+440.69

0.9999

0.5

1.6

23



Page 24 of 33

Table 3-1 Accuracy and Precision of MEQ and Metabolites in Chicken Muscle and Chicken Liver Matrix

Analyte MEQ

M1 (1-DMEQ)

M2 (4-DMEQ)

Chicken

M3 (2-iso-MEQ)

muscle M4 (2-iso-1-DMEQ)

M5 (2-iso-4-DMEQ)

M6 (2-iso-BDMEQ)

MEQ

M1 (1-DMEQ)

M2 (4-DMEQ)

chicken

M3 (2-iso-MEQ)

liver M4 (2-iso-1-DMEQ)

M5 (2-iso-4-DMEQ)

M6 (2-iso-BDMEQ)

Fortified level

Mean recovery

Intra-day RSD%

Inter-day RSD%

(µg kg-1)

（%）

(n=6)

(n=18)

10

64.4

5.6

6.0

50

64.3

3.2

3.2

200

79.0

2.8

17.8

10

76.4

5.2

15.3

50

72.5

6.3

9.8

200

87.4

7.0

19.1

10

74.7

7.8

11.7

50

74.6

5.7

14.2

200

84.8

7.0

18.7

10

77.6

4.5

9.5

50

73.5

4.5

7.4

200

87.1

3.3

10.3

10

71.5

6.8

8.8

50

73.7

6.1

6.4

200

81.6

7.3

19.2

10

77.0

4.8

15.2

50

75.6

4.1

4.0

200

82.3

4.0

16.7

10

77.4

7.6

8.5

50

81.1

5.1

16.4

200

87.0

10.7

11.8

10

82.0

5.7

8.7

50

76.4

7.5

10.6

200

88.4

8.0

11.0

10

92.2

4.6

5.9 6.7

50

82.5

6.6

200

85.9

6.2

9.8

10

90.0

5.4

8.0 8.6

50

84.4

7.1

200

84.4

7.5

8.8

10

91.0

5.0

6.2

50

78.8

6.5

9.2

200

79.6

9.4

10.9

10

92.0

6.0

6.2 6.2

50

87.6

5.9

200

92.0

3.5

5.8

10

114.4

10.2

17.2 5.8

50

96.0

5.5

200

94.6

3.0

3.5

10

89.1

6.6

6.2

50

81.6

9.2

11.5

200

88.2

4.3

6.0

24


Page 25 of 33


Table 3-2 Accuracy and Precision of MEQ and Metabolites in Swine muscle and Swine Liver Matrix

Analyte MEQ

M1 (1-DMEQ)

M2 (4-DMEQ)

Swine

M3

muscle M4

M5

M6

MEQ

M1 (1-DMEQ)

M2 (4-DMEQ)

Swine

M3

liver M4

M5

M6

Fortified level

Mean recovery

Intra-day RSD%

Inter-day RSD%

(µg kg-1)

（%）

(n=6)

(n=18)

10

80.1

5.7

10.9 6.5

50

69.5

6.9

200

75.5

5.8

8.1

10

76.6

5.9

15.2

50

73.6

14.7

14.8

200

71.4

7.6

11.1

10

88.7

10.2

9.6

50

70.7

12.8

12.4

200

75.5

6.5

13.5

10

89.1

5.8

5.4 6.5

50

80.7

6.4

200

87.0

4.6

4.6

10

80.1

8.0

13.5 9.9

50

78.0

9.6

200

84.8

7.7

8.6

10

88.9

5.2

4.9 8.4

50

80.4

6.3

200

85.2

5.1

6.3

10

84.4

7.7

11.1

50

75.6

9.7

15.7

200

69.8

10.8

13.9

10

78.0

5.3

5.2 9.1

50

74.5

8.2

200

85.7

4.1

3.6

10

86.9

7.0

8.9 10.7

50

82.3

5.4

200

87.2

3.0

5.6

10

88.2

5.6

6.0 6.2

50

87.2

4.5

200

86.0

1.6

5.0

10

83.5

3.1

4.7 2.5

50

84.6

2.7

200

92.2

5.5

6.8

10

90.0

9.6

11.5 6.2

50

86.0

7.0

200

91.3

2.9

3.2

10

88.7

7.7

9.8 4.0

50

85.3

3.8

200

92.9

2.6

2.3

10

88.6

4.6

4.8

50

82.9

5.6

9.8

200

85.9

6.0

6.5

25



Page 26 of 33

Table 4 Concentrations (µg kg-1) of MEQ and metabolites contamination in commercial real samples (chicken muscle, chicken liver, swine muscle, and swine liver). Commercial sample

chicken muscle

chicken liver

Sample code

Concentration of M1 (µg kg-1)


1

ND

2

Commercial sample

Sample code



ND

21

ND

ND

5.88

2.83

22

ND

ND

3

ND

ND

23

ND

ND

4

16.01

ND

24

ND

ND

5

ND

ND

25

ND

ND

6

ND

ND

26

ND

ND

7

12.22

ND

27

ND

ND

8

ND

ND

28

ND

ND

9

ND

ND

29

ND

ND

10

8.9

2.08

30

ND

ND

11 12 13 14 15 16 17 18 19 20

ND ND 33.86 ND 42.18 29,06 ND 50.02 53.39 30.09

ND ND ND ND ND ND ND 3.18 3.62 ND

31 32 33 34 35 36 37 38 39 40

ND ND ND 20.11 ND ND 15.53 ND ND ND

ND ND ND ND ND ND ND ND ND ND

swine muscle

swine liver

ND: not detected MEQ, M2, M3, M4, M5 were not detected in all commercial samples, which were not listed in the table. 26


Page 27 of 33


Figure 1

27



M EQ 111201000 100 %

3 .2 2 3 .3 2

Page 28 of 33

7 : M R M o f 2 C h a n n e ls E S + 3 .7 3 203 > 158 108 3 .8 1

0 1 .0 0 M EQ 111201000

2 .0 0

3 .0 0 2 .9 9 2 .5 6 2 .8 8 3 .0 6

4 .0 0 5 .0 0 6 : M R M o f 2 C h a n n e ls E S + 203 > 144 88

%

100

0 1 .0 0 M EQ 111201000

2 .0 0

3 .0 0 2 .5 5

100

2 .6 9

3 .2 5

4 .0 0 5 .0 0 5 : M R M o f 2 C h a n n e ls E S + 3 .8 0 3 .9 6 1 8 9 > 1 7 0 .8 91

%

2 .2 8

3 .3 9

0 1 .0 0 M EQ 111201000

2 .0 0

3 .0 0 2 .6 4

100 %

1 .4 3 1 .0 2 1 .2 5

2 .5 3

2 .0 0 1 .7 0

1 .1 4

%

2 .7 1

1 .8 1

0 1 .0 0 M EQ 111201000 100

4 .0 0 5 .0 0 3 : M R M o f 2 C h a n n e ls E S + 2 0 5 > 1 8 7 .9 89

3 .0 0


2 .5 8 2 .6 8 2 .5 1

1 .4 0 1 .9 3

1 .0 3

0 1 .0 0 M EQ 111201000

%

100

2 .0 0

0 .5 1 0 .9 1 0 .7 1 1 .0 9

1 .6 1

0 1 .0 0 M EQ 111201000

3 .0 0


3 .0 0


1 .8 3

2 .3 0 2 .0 8

2 .0 0 1 .6 8

%

100 1 .3 4

0 .6 8

1 .4 9

2 .0 7

2 .2 0

0 1 .0 0

Figure 2-1(A)

2 .0 0

3 .0 0

4 .0 0

T im e 5 .0 0

MRM chromatograms for MEQ and major metabolites in blank chicken muscle samples

M EQ 111207005 3 .3 2

%

100

4-DMEQ

3 .0 2

0 1 .0 0 M EQ 111207005

2 .0 0

3 .0 0

4 .0 0 5 .0 0 6 : M R M o f 2 C h a n n e ls E S + 203 > 144 1 .8 7 e 4

2 .9 2

%

100

7 : M R M o f 2 C h a n n e ls E S + 203 > 158 1 .8 8 e 4

3 .3 1

1-DMEQ

0 1 .0 0 M EQ 111207005

2 .0 0

3 .0 0 2 .5 1

%

100

4 .0 0 5 .0 0 5 : M R M o f 2 C h a n n e ls E S + 1 8 9 > 1 7 0 .8 1 .4 6 e 4

2-iso-BDMEQ

0 1 .0 0 M EQ 111207005

2 .0 0

3 .0 0

1 .9 9

%

100


2-iso-1-DMEQ

1 .1 0

0 1 .0 0 M EQ 111207005

2 .0 0

3 .0 0

1 .3 3

%

100


2-iso-4-DMEQ

1 .9 6

0 1 .0 0 M EQ 111207005

2 .0 0

3 .0 0

2 .0 0

3 .0 0

1 .2 8

%

100


MEQ

0 .5 1

0 1 .0 0 M EQ 111207005 0 .9 2

%

100 0 .5 0

1 .0 0

Figure 2-1(B)

2-iso-MEQ

2 .0 2

0


2 .0 0

3 .0 0

4 .0 0

T im e 5 .0 0

MRM chromatograms for MEQ and major metabolites in chicken muscle sample fortified at 10 µg kg-1

28


Page 29 of 33


M EQ 111207000 3 .3 2

3 .1 2

7 : M R M o f 2 C h a n n e ls E S + 3 .7 3 3 .8 5 203 > 158 66

%

100

0 1 .0 0 M EQ 111207000

2 .0 0

3 .0 0

2 .5 6

3 .1 6

%


3 .4 5

2 .9 2

100

0 1 .0 0 M EQ 111207000

2 .0 0

3 .0 0

%

100 2 .8 4

2 .0 6

4 .0 0 5 .0 0 5 : M R M o f 2 C h a n n e ls E S + 3 .8 0 1 8 9 > 1 7 0 .8 3 . 9 2 141 2 .9 1 3 .3 4 3 .4 9 3 .1 5

0 1 .0 0 M EQ 111207000

2 .0 0

1 .2 8 1 .3 6 1 .2 1

%

100

3 .0 0

2 .7 8 2 .8 9 1 .9 6 2 .4 1

0 1 .0 0 M EQ 111207000

2 .0 0

3 .0 0

2 .2 3 1 .4 0 1 .7 0 1 .8 5 1 .2 9 1 .0 7

%

100


1 .8 1

0 1 .0 0 M EQ 111207000

2 .5 8


2 .7 9

2 .0 0

3 .0 0


3 .0 0


0 .5 1

%

100

0 .9 3 1 .3 9 1 .5 0 1 .9 1

0 .7 3

0 1 .0 0 M EQ 111207000

2 .4 4

2 .0 0

1 .0 0

100

1 .5 6

0 .6 0

%

1 .1 2 1 .8 2

2 .2 9 2 .3 9

0 1 .0 0

2 .0 0

3 .0 0

4 .0 0

T im e 5 .0 0

MRM chromatograms for MEQ and major metabolites in blank chicken liver samples

Figure 2-2(A)

M E Q 111207006 3 .3 2

100


%

4-DMEQ

0 1 .0 0 M E Q 111207006

2 .0 0

3 .0 0


2 .9 2

%

100

3 .3 1

1-DMEQ

0 1 .0 0 M E Q 111207006

2 .0 0

3 .0 0


3 .0 0


3 .0 0


2 .0 0

3 .0 0


2 .0 0

3 .0 0


2 .5 1

%

100

2-iso-BDMEQ

0 1 .0 0 M E Q 111207006

2 .0 0 1 .9 9

%

100 1 .1 7

2-iso-1-DMEQ

1 .3 2

0 1 .0 0 M E Q 111207006

2 .0 0 1 .3 3

%

100

2-iso-4-DMEQ

1 .9 6

0 1 .0 0 M E Q 111207006 1 .3 1

%

100

MEQ

0 .6 1

0 1 .0 0 M E Q 111207006 0 .9 4

%

100

1 .1 5

0 .5 4

0 1 .0 0

Figure 2-2(B)

2-iso-MEQ

1 .7 1 2 .0 2

2 .0 0

3 .0 0

4 .0 0

T im e 5 .0 0

MRM chromatograms for MEQ and major metabolites in chicken liver sample fortified at 10 µg kg-1

29



M EQ 111215000

Page 30 of 33

7 : M R M o f 2 C h a n n e ls E S + 203 > 158 102

3 .6 2 3 .9 3 3 .4 1 3 .0 2 3 .1 7

%

100

0 1 .0 0 M EQ 111215000

2 .0 0

3 .0 0

100

2 .8 8


2 .9 5 3 .3 5

%

2 .5 6

0 1 .0 0 M EQ 111215000

2 .0 0

100

3 .0 0

2 .0 6 3 .3 0

%

2 .1 9 2 .6 9

4 .0 0 5 .0 0 5 : M R M o f 2 C h a n n e ls E S + 3 .9 6 1 8 9 > 1 7 0 .8 3 .3 9 3 .7 2 162

0 1 .0 0 M EQ 111215000

2 .0 0

1 .2 5

100

1 .5 1 1 .6 2

2 .2 2

2 .5 7

0 1 .0 0 M EQ 111215000

2 .0 0

3 .0 0

1 .2 6 1 .7 0 1 .8 9 2 .4 1 1 .9 6 1 .0 3

%

100


2 .9 3

%

1 .1 4

3 .0 0


2 .9 0

0 1 .0 0 M EQ 111215000

2 .0 0

1 .5 7

%

3 .0 0


3 .0 0


2 .4 0

0 .5 3

100

0 .9 5

1 .8 0

0 1 .0 0 M EQ 111215000

2 .1 2

2 .0 0 1 .4 9

%

100

1 .6 4

0 .8 0 1 .1 2

1 .8 6 2 .3 4 2 .4 3

0 1 .0 0

2 .0 0

3 .0 0

4 .0 0

T im e 5 .0 0

MRM chromatograms for MEQ and major metabolites in blank swine muscle samples

Figure 2-3(A)

M E Q 111215006 3 .2 7


%

100

4-DMEQ

0 1 .0 0 M E Q 111215006

2 .0 0

3 .0 0


2 .8 8

%

100

1-DMEQ

3 .2 6

0 1 .0 0 M E Q 111215006

2 .0 0

3 .0 0 2 .4 2

%

100


3 .0 5

2 .0 6

3 .4 4

2-iso-BDMEQ

3 .8 4

0 1 .0 0 M E Q 111215006

2 .0 0

3 .0 0

1 .8 8

%

100


2-iso-1-DMEQ

2 .2 2 2 .7 5

0 1 .0 0 M E Q 111215006

2 .0 0

3 .0 0


3 .0 0


2 .0 0

3 .0 0


2 .0 0

3 .0 0

1 .2 9

2-iso-4-DMEQ

%

100

1 .8 9

0 1 .0 0 M E Q 111215006

2 .5 4

2 .0 0

1 .2 4

MEQ

%

100

1 .6 1

0 1 .0 0 M E Q 111215006

1 .8 3

0 .9 0

%

100

0 1 .0 0

Figure 2-3(B)

4 .0 0

T im e 5 .0 0

MRM chromatograms for MEQ and major metabolites in swine muscle sample fortified at 10 µg kg-1

30


Page 31 of 33


M EQ 111219000 100 %

3 .2 2

7 : M R M o f 2 C h a n n e ls E S + 3 .7 0 203 > 158 125 3 .8 5

3 .1 2

0 1 .0 0 M EQ 111219000

2 .0 0

100

3 .0 0 2 .6 0

2 .6 7

%

2 .5 2

4 .0 0 5 .0 0 6 : M R M o f 2 C h a n n e ls E S + 203 > 144 3 .2 6 3 .3 6 90

0 1 .0 0 M EQ 111219000

2 .0 0

3 .0 0 3 .1 0 3 .2 0

100 %

2 .3 3

4 .0 0 5 .0 0 5 : M R M o f 2 C h a n n e ls E S + 1 8 9 > 1 7 0 .8 3 .8 4 99

3 .3 9

2 .4 7

2 .0 6

0 1 .0 0 M EQ 111219000

2 .0 0

3 .0 0 2 .3 1

%

100 1 .2 5 1 .4 0

%

2 .9 6

2 .1 3

1 .0 2

0 1 .0 0 M EQ 111219000 100

2 .6 0

1 .6 2


2 .0 0

3 .0 0

2 .4 6 1 .4 8 1 .6 3 2 .2 8 1 .0 3 1 .3 7 1 .9 6

2 .8 3 2 .9 3


0 1 .0 0 M EQ 111219000 0 .9 3

100

2 .0 0

3 .0 0


3 .0 0


1 .2 0

0 .6 5

1 .7 2

2 .4 4

%

1 .8 0

0 1 .0 0 M EQ 111219000 0 .6 0

2 .0 0 2 .0 7

0 .9 0 1 .4 5 1 .9 7

%

100

0 1 .0 0

2 .0 0

3 .0 0

T im e 5 .0 0

4 .0 0

MRM chromatograms for MEQ and major metabolites in blank swine liver samples

Figure 2-4(A)

M EQ 12050751 3 .1 7

100


%

4-DMEQ

3 .7 7 3 .8 9

0 1 .0 0 M EQ 12050751

2 .0 0

3 .0 0 2 .7 7

%

100


1-DMEQ

3 .2 1

0 1 .0 0 M EQ 12050751

2 .0 0

3 .0 0 2 .3 3

2 .9 8

%

100

4 .0 0 5 .0 0 5 : M R M o f 2 C h a n n e ls E S + 1 8 9 > 1 7 0 .8 7 .9 5 e 3 3 .5 7

2-iso-BDMEQ

3 .8 4

0 1 .0 0 M EQ 12050751

2 .0 0

3 .0 0


3 .0 0


3 .0 0


2 .0 0

3 .0 0


2 .0 0

3 .0 0

1 .8 4

%

100

2-iso-1-DMEQ

2 .5 7

1 .1 0

0 1 .0 0 M EQ 12050751

2 .0 0

1 .2 6

%

100

2-iso-4-DMEQ

1 .8 2 1 .8 9

0 1 .0 0 M EQ 12050751

2 .0 0

1 .2 0

%

100

1 .4 6

MEQ

1 .8 4

0 1 .0 0 M EQ 12050751 0 .8 8

%

100

2-iso-MEQ

1 .5 6 1 .7 1

0 .7 8

0 1 .0 0

Figure 2-4(B)

4 .0 0

T im e 5 .0 0

MRM chromatograms for MEQ and major metabolites in swine liver sample fortified at 10 µg kg-1

31



Figure 3

32


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Page 33 of 33


Table of Content

33


MS Method for the

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