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Approach for Pesticide Residue Analysis for Metabolite Prothioconazole-desthio in Animal origin food Hui Liu, Guojun Yao, Xueke Liu, Chang Liu, Jing Zhan, Donghui Liu, Peng Wang, and Zhiqiang Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00062 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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Journal of Agricultural and Food Chemistry

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Approach for Pesticide Residue Analysis for Metabolite

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Prothioconazole-desthio in Animal origin food

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Hui Liu1, Guojun Yao1, Xueke Liu, Chang Liu, Jing Zhan, Donghui Liu, Peng Wang, Zhiqiang

4

Zhou *

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Department of Applied Chemistry, China Agricultural University, Beijing, 100193, P. R. China.

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Beijing Advanced Innovation Center for Food Nutrition and Human Health.

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*Corresponding author: Zhiqiang Zhou, Department of Applied Chemistry and Beijing Advanced

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Innovation Center for Food Nutrition and Human Health, China Agricultural University,

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Yuanmingyuan west road 2, Beijing 100193, P.R. China.

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E-mail: [email protected]

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ABSTRACT：：The food safety problems such as the damage to immune system, nervous and

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endocrine leading to cancer and malformations has received people's increasing attention. In order

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to achieve the MRLs, the most discussed method of HPLC-MS/MS is widely used with a advantage

15

of high precision and resolution. Prothioconazole is a broad-spectrum thiocarbamate fungicide. It

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can rapidly metabolize to prothioconazole-desthio in different matrix. Rapid and effective methods

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for the determination of prothioconazole-desthio in five kinds of different animal food were

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developed. Samples were extracted with acetonitrile or acetonitrile/water and determined by

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HPLC-MS/MS. The LOD and LOQ values of prothioconazole-desthio were 0.015 and 0.05 mg/kg

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for pig liver and kidney, 0.0015 and 0.005 mg/kg for pork, 0.003 and 0.01mg/kg for eggs, together

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with 0.0012 and 0.004 mg/kg for milk of the detected method. A good linear regression trend can be

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observed in a certain concentration range for all the animal food. At fortified levels, recoveries

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were between 83.6%-105%, with relative standard deviations (RSDs) of 1.5%-10.3%. A sample

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survey of 150 samples with 30 samples for each kind of animal food across the country was

ACS Paragon Plus Environment

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conducted and found that there was no prothioconazole-desthio detected in all samples.

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KEYWORDS: Prothioconazole-desthio, Residue analysis, Animal origin food

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INTRODUCTION

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Pesticides have been playing an important role in the control of pests and diseases in recent

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decades. However, the widespread use of pesticides can lead to pesticide residues in plants and

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accumulation in animal products along with cereals and animal feed. Pesticides tend to bio

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accumulate in the fat layer, resulting in residues in animal origin such as meat, fish, fat, offal, eggs

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and milk.1Thus it may produce secondary toxicities through the food chain which is threatening

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human health. 2 Current studies have shown that metabolites of pesticides often have a higher

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detection rate than the parent, and their entry into environment can lead to greater adverse effects.

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Currently, metabolites are expensive to identify, and the study interest of metabolites are limited, 3

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which is a challenge in the study of pesticide residue.

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Prothioconazole is the ISO common name for 2-[2-(1-chlorocyclopropyl)-3-(2-chloropheny

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-l)-2-hydroxypropyl]-2,4-dihydro-1,2,4-triazole-3-thione which belongs to the class of fungicides

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commonly referred to as the triazole. It is a systemic fungicide with protective, curative and

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eradicative activities. Its mode of action is steroid demethylation, which is a part of ergosterol

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biosynthesis4. Prothioconazole was developed by Bayer CropScience and appeared on the market

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in 2004. Prothiocanazole-desthio is a major breakdown product via chiral metabolism of

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prothioconazole (See Figure 1). 5, 6

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The enforcement residue definition established for commodities of animal origin in the

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Regulation (EC) No 396/20057 refers to the concept that “sum of prothioconazole-desthio and its

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glucuronide conjugate, expressed as prothioconazole-desthio”. During the peer review under

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Directive 91/414/EEC, an analytical method using HPLC-MS/MS and its ILV were evaluated and

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validated for the determination of prothioconazole-desthio in food of animal origin with an LOQ of

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0.004 mg/kg in milk and an LOQ of 0.01 mg/kg in muscle, fat, liver and kidney. 8,9 This method has


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not been validated for the determination of prothioconazole-desthio in eggs. The residue was

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considered to be not fat-soluble for the purposes of residue definition. Australia informed that the

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MRLs of prothioconazole and prothioconazole-desthio in meat, milks, eggs and edible offal were

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0.02, 0.004, 0.01 and 0.2 mg/kg, respectively. 10 Japan and the CAC have declared that the MRLs of

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prothioconazole and prothioconazole-desthio in meat, milk and edible offal were 0.01, 0.004 and

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0.5 mg/kg. 11,12 Regulations for United Kingdom indicated that the MRLs of prothioconazole and

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prothioconazole-desthio in meat, milk, edible offal and eggs were 0.01, 0.01 and 0.5 and 0.01

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mg/kg.

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prothioconazole-desthio in China (See Table 1). 14

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More concern is that there are not registered MRLs of prothioconazole and

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At present, there are few studies on the residue analysis methods of prothioconazole and its

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metabolites in food. Shi et al established the residue analysis method of prothioconazole and

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prothioconazole-desthio and found that prothioconazole will be quickly converted to

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prothioconazole-desthio in soil.

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prothioconazole and prothioconazole-desthio in peanut using HPLC-MS/MS.

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method of HPLC-MS/MS for prothioconazole-desthio has not been reported in the animal food. As

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is known, the residue definition for plant and animal commodities for enforcement and dietary risk

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assessment was prothioconazole-desthio. So the detection of prothioconazole-desthio seems to be

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more meaningful.

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A sensitive method was developed for determination of 16

However, the

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The establishment of the residual method is an important research content to establish the

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MRL value. The residual analysis method introduced in JMPR is relatively old and

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time-consuming due to the complexity of the matrix of animal food samples. It is difficult to deal

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with a large number of samples. MRL values of prothioconazole abroad still remain the temporary

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residue limit for the animal source of food in the residue limit development. Now we need to

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improve the method and transform the MRL from CAC to China to develop China's residue limit

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standards. In this study, methods for the determination of metabolite prothioconazole-desthio in

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animal origin food by HPLC-MS/MS were developed by acetonitrile extraction, liquid-liquid


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distribution and C 18 dispersion solid-phase purification. This method can meet the requirements of

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existing international limits. In order to understand the residual status of prothioconazole in market

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samples, the samples in the local large supermarkets were monitored at the same time.

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EXPERIMENTAL SECTION

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Chemicals and reagents. Prothioconazole-desthio was from Dr. Ehrenstorfer, Germany with a

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purity of 99.5 %. NaCl and NaSO4 were provided by Beijing Chemical Reagent Co, Ltd. (Beijing,

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China). Acetonitrile was obtained by Fisher Scientific (Shanghai, China). n-Hexane was purchased

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from CNW company of Germany (95%, Shanghai, China). C

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was used for purifying of fat and ultra-pure water was used for HPLC-MS/MS analysis.

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Equipment. The balance, 50-mL Corning CentristarTM, XHF-D HI H-Speed Disperastor (Ningbo

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Scientz Biotechnology Co.,Ltd) multi-tube vortexer (TARGIN VX-III), 100-mL heart-shaped

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bottles, evaporation instrument (EYELA N-1100), low-speed Centrifuge (Shanghai anting

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instrument factory) and palm centrifuge (LX-100) were used for extraction and purification.

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Residue analysis methods were conducted on DionexTM UltiMateTM 3000 Open Sampler XRS

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UHPLC system and ThermoFisher TSQ Quantum Access MAX system from Tewksbury,

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Massachusetts, USA. A Waters Atlantis T3 C18 (3µm，2.1mm×150mm) column was employed and

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mobile phases A and B used for HPLC were acetonitrile and water, respectively. The gradient

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program started with 15 % A and 85 % B. From 1 to 7 min, the gradient changed to 85 % A and

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15 % B. Since 7 to 8 min, the gradient changed from 85 % A and 15 % B to 15 % A and 85 % B.

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Then 15 % A and 85 % B were kept to the endpoint of 10 min. ESI positive ion mode was

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performed. The mass spectrometry conditions were shown as follows：Molecular ion peak [M-Cl]

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was 312.0; Two pair of ions for 312.0/70.3 and 312.0/125.0, in which 312.0/70.3 ions was

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conducted for quantitative analysis; The tube lens voltage was set up to be 85 V ; CE collision

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voltage was 21V; Electrospray voltage was 3000V; Vaporization temperature was 300 ºC；

18 (Hebei


Jin Yang Filter Co., Ltd.)

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Transport capillary temperature was 350 ºC; Value of sheath gas and auxiliary gas was 30 and 10

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Arb, respectively.

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Sample Preparation. Test samples were purchased from supermarkets, wholesale markets supply

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stations from major cities in China. The samples of milk were collected from the milk stations and

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farmers of Ningxia, Inner Mongolia and Xinjiang provinces. Pork, porcine liver and kidney

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samples were provided from wholesale markets in 10 provinces of Hebei, Sichuan, Hunan, Jiangsu,

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Hubei, Shandong, Henan, Guangdong, Yunnan, and Anhui. In addition, the eggs were purchased

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from wholesale markets and large supermarkets in 4 provinces of Beijing, Zhejiang, Shandong,

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Hainan. The samples were divided into two masses with 200g each mass and homogenized in HI

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H-Speed Disperastor, after which the samples were stored in a freezer at -20°C before analysis.

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Extraction procedure

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Porcine liver and porcine kidney. An aliquot (2.00 ± 0.05 g) of the homogenized sample was

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weighed into a 50 mL plastic centrifuge tube. 10 mL of the extract solvent was added (acetonitrile /

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water, 85/15) and vortexed for 5 min, after which 1.5 g of sodium chloride was mixed and vortexed

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for 1 min. After centrifugation for 5 min at 4000 r / min, the supernatant was transferred to a 50 mL

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plastic centrifuge tube through 5 g of anhydrous sodium sulfate. The sample was extracted again

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with the same method, and the supernatant was combined eluted with 4 mL of acetonitrile.

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Pork. An aliquot (5.00 ± 0.05 g) of the homogenized pork was weighed into a 50 mL plastic

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centrifuge tube. 15 mL of the extraction solvent (acetonitrile / water, 85 / 15) was added. After

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vortexing for 5 min, 1.5 g of sodium chloride was added and vortexed for another 1 min. The

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sample was centrifuged for 5 min at 4000 r / min and the supernatant was transferred to a 50 mL

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plastic centrifuge tube before passing through 5 g of anhydrous sodium sulfate. Then another 15

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mL of the extract solvent was used to extract one more time. At last, 4 mL of acetonitrile was

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employed to rinse.

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Egg. An aliquot (2.00 ± 0.05 g) of the homogenized eggs were weighed into a 50 mL plastic


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centrifuge tube, add 10 mL acetonitrile. Then 1 g of sodium chloride was added after fully

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vortexing for 5 min. Before centrifugation at 4000r / min for 5 min, the mixture was vortexing for 1

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min. The supernatant was transferred to a 50 mL plastic centrifuge tube after passing through 5 g of

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anhydrous sodium sulfate, and then 10 mL of acetonitrile was added again and extraction was

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repeated. Ultimately Anhydrous sodium sulfate was rinsed with 4 mL of acetonitrile.

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Milk. An aliquot (5.00 ± 0.05 g) of milk and 15mL acetonitrile were mixed in a 50 mL plastic

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centrifuge tube, then 2 g of sodium chloride was added after fully vortexing for 5 min. 1 min

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vortexing was needed and the samples were centrifugation at 4000r / min for 5 min. The

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supernatant was transferred to a 50 mL plastic centrifuge tube after passing through 5 g of

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anhydrous sodium sulfate and the extraction was repeated with 4 mL of acetonitrile to rinse.

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Purification. The mixture was purified by n-hexane-acetonitrile liquid –liquid distribution and

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then purified by C

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amount of n-hexane (5 mL for porcine liver, porcine kidney and eggs; 8 mL for pork and milk) and

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fully vortexed for 3 min, after which the mixture was centrifuged at 4000 r / min for 3 min. The

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upper layer of n-hexane was removed, and another same amount of n-hexane was added for purify

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again. The lower layer of acetonitrile was transferred to a 100 mL heart-flask to be evaporated to

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nearby dry at temperature of 35 ºC and the sample was dissolved in 1 mL acetonitrile, after which it

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was transferred to a 2 mL plastic centrifuge tube containing 15 mg of C18. The sample was vortexed

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for 1 min and centrifuged on a palm centrifuge for 1 min. The supernatant was over 0.22 µm

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organic phase membrane before the HPLC-MS / MS analysis.

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Assay Validation. A series of working standard solutions of prothioconazole-desthio were

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prepared in different animal tissues. Linear regression analysis was performed with the LOD and

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the LOQ calculated. LOQ of method was defined as the lowest concentration in the calibration

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curve with acceptable precision and accuracy for 20 % variability, also to be the concentration that

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produced a S/N ratio of 10. LOD of method was considered to be the concentration that produced a

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S/N ratio of 3. The concentration of prothioconazole-desthio in samples was calculated by the

18

before HPLC-MS/MS analysis. The supernatant was added with a certain


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linear regression. Absolute recovery of prothioconazole-desthio was determined in all animal

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origin food fortified with prothioconazole-desthio at three levels of concentrations.

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RESULTS AND DISCUSSION

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Method validation. LOD and LOQ. The LOD and LOQ values of prothioconazole-desthio were

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calculated in different substrates. The prothioconazole-desthio was detectable at 0.0015 mg/kg for

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pork, 0.015 mg/kg for porcine liver and kidney, 0.003 mg/kg for eggs and 0.0012mg/kg for milk,

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respectively. It was found that LOQs were 0.005 mg/kg for pork, 0.05 mg/kg for porcine liver and

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kidney, 0.01mg/kg for eggs, together with 0.004 mg/kg for milk of the method detected.

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Linearity. There was a good linear relationship between the peak area and the injection

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concentration among the studied concentration range (Table 3). In the porcine liver and kidney, the

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solution with concentration of 0.1 mg/L, 0.2 mg/L, 1 mg/L, 2 mg/L and 4 mg/L were prepared and

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the standard curve equation was y = 32358822 x + 2166764 with a determination coefficient of

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0.9988 in porcine liver, besides, the standard curve equation was y = 9957101x - 350340 with a

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determination coefficient of 0.9986 in porcine kidney. For the other three matrixes, the standard

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curve equations were also calculated. In the pork, the standard curve equation was y = 32529653 x

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+ 1222763 with a determination coefficient of 0.9992 with five concentrations of 0.025, 0.05, 0.25,

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0.5 and 2.5mg/L were set up. Then solutions of 0.02, 0.1, 0.2, 1 and 2mg/L were used to get the

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standard curve equation in the matrix. The results showed that the standard curve equation was y =

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38015502 x + 1125777 with a determination coefficient of 0.9986. At last, the method was

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established in milk and a good linear equation was gotten in 0.02, 0.05, 0.1, 0.2 and 0.5mg/L that is

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y = 35618552 x + 955353 with a determination coefficient of 0.9936.

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Accuracy and Precision. Based on the MRLs of prothioconazole in porcine liver, kidney, pork,

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egg and milk and the sensitivity of the method, three added concentrations of 0.05 mg/kg, 0.5

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mg/kg and 2 mg/kg for porcine liver and kidney, 0.005 mg/kg, 0.01 mg/kg and 0.1mg/kg for pork,


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0.01 mg/kg, 0.05 mg/kg and 0.5mg/kg for eggs, besides, 0.004 mg/kg, 0.04 mg/kg and 0.1mg/kg

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were set up. The recovery rate and the relative standard deviation for porcine liver, porcine kidney,

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pork, eggs and milk were shown in the following table. The average recoveries ranged from 83.6%

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to 105% and the relative standard deviations were from 1.5% to 10.3% (Table 4 and Figure 2).

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Market research and testing. In order to investigate the use of prothioconazole in China and the

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residue of prothioconazole-desthio, we conducted a sample survey of 150 samples with 30 samples

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for each kind of animal origin food across the country and found that there was no

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prothioconazole-desthio detected in all samples (Table 5). The daily maximum intake of

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prothioconazole in the general population was 0.223 mg, accounting for 35.5% of the daily

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allowable intake according to the registration of pesticides and the dietary structure of Chinese

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residents. In the JMPR of 2014,9 based on the dietary data of prothioconazole in STMR and WHO

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GEMS / Food project for 13 regions, the calculated IEDI accounted for 0% - 3% of the daily

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allowable intake. The proposed MRLs for pesticides generally do not create unacceptable risks to

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the health of the general population (Table 6).

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The prothioconazole is not widely used in China. Although many countries abroad have

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developed the residual limit of prothioconazole, but there is no residual limit of prothioconazole in

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national standard in China.

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prothioconazole-desthio in different animal tissues, but also the residue of prothioconazole-desthio

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in animal products was explored to provide data for pesticide risk assessment.

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Development and comparison of analytical methods. In the development of pesticide residue

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analysis in animal food, MSPD technology was firstly used, however, which eliminated the

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formation of latex with a high recovery rate and low consumption of organic solvent. For

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technology of MSPD, the extraction and purification is simple. But it is not suitable for solid

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sample with high fat content. During the extraction, the consumption of adsorbents is very high,

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and biological samples require additional purification steps, which are complex and

14

Through this study, we not only established the residue method of


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time-consuming. 17-19 Animal-derived foods are very complex matrices that contain proteins, lipids,

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saturated and unsaturated fatty acids. The matrix effect comes from co-eluting sample components,

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which affect the degree of ionization of the analyst and may lead to erroneous results. Even if the

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purification step is cumbersome, a good purification is also needed so as not to cause damage to the

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instrument in order to get more accurate test results. The traditional SPE operation is troublesome

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until the QuEChERS method appears and makes purification more convenient. PSA is commonly

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used in the QuEChERS process to absorb fatty acids, sugars and other acidic compounds while C 18

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removes lipid and non-polar interference. GCB can remove pigments such as chlorophyll,

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hemoglobin, sterols and other matrix components. 20,21 In order to meet the requirement of residue

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analysis, it is always a problem that people need to overcome to reduce the detection line and the

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limit of quantification. The analysis method of liquid quality has short analysis time and low

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detection line, so the use for analysis of the animal origin food has great advantages. 22, 23

213

This

present

study

gave

us

a

quick

and

straightforward

residue

analysis

of

214

prothioconazole-desthio for representative food of animal origin, using high precision detection

215

instruments like HPLC-MS/ MS. This method was well validated in a broad range of possiblity to

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be applied to other analogous animal tissues and food. Although the metabolite

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prothioconazole-desthio was not detected in domestic animal food, there is a need to arouse

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people's attention to the pollution in agricultural products.

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220

221

222

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English abbreviation

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HPLC-MS/MS: High Performance Liquid Chromatography - Mass Spectrometry / Mass Spectrometry;

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LOD: Limit of Detection;

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LOQ: Limit of Quantification;

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MRL(s): Maximum Residue Limit(s);

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CAC: Codex Alimentarius Commission;

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JMPR: (the WHO/FAO) Joint Meeting on Pesticide Residues;

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S/N: Signal-to-Noise;

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STMR: Standard Residue Test Median;

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WHO: World Health Organization;

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GEMS / Food: Global Environmental Monitoring System / Food Contamination Monitoring and Assessment Project;

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IEDI: the Daily Estimated Intakes;

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MSPD: Matrix Solid-Phase Dispersion;

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QuEChERS: Quick, Easy, Cheap, Effective, Rugged, Safe;

241

PSA: Primary Secondary Amine;

242

GCB: Graphatized Carbon Black.

243

244

245

246

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AUTHOR INFORMATION

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*Corresponding Author

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Zhiqiang Zhou. Phone: 86-10-62733547. Fax: 86-10-62733547. E-mail: [email protected]

253

Funding

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This work was supported by National Natural Science Foundation of China (Contract Grants

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21337005, 21307155) and Chinese Universities Scientific Fund 2016LX001.

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Notes

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The authors declare no competing financial interest.

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259

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REFERENCES

269 270

(1) Castillo, M.; Carbonell, W.; González, C.; Miralles, M., A.; In: Soundararajan RP (ed) Pesticides- Recent Trends in Pesticide Residue Assay, InTech. 2012.

271 272 273

(2) Karolina, L.; Roman, F.; Franz, X. S.; Hermann, U.; Sonja, M.; Alternative QuEChERS-based modular approach for pesticide residue analysis in food of animal origin. Anal Bioanal Chem. 2015, 407,3727–3742.

274 275 276

(3) Srinivas ,S.; Marley, W.; Vijay, T.; John, R. L.; Allan J. C.; and Nancy, G.; Effects of Glyphosate and Two Herbicide Mixtures on Microbial Communities in Prairie Wetland Ecosystems: A Mesocosm Approach. J. Environ. Qual. 2012,41,732–743.

277 278

(4) EFSA (European Food Safety Authority), Modification of the existing maximum residue level (MRL) for prothioconazole in shallots. J. EFSA. 2015,13(5), 4105

279 280

(5) Australian Pesticides and Veterinary Medicines Authority.; Evaluation of the new active prothioconazole in the product redigo fungicidal seed treatment. 2007, ISSN 1443-1335.

281 282

(6) United States Environmental Protection Agency.;Pesticide factsheet: prothioconazole. 2007, 7501 P.

283 284 285

(7) EFSA (European Food Safety Authority), Conclusion on the peer review of the pesticide risk assessment of the active substance prothioconazole 2007b.. EFSA Scientific Report , 2007,106, 1-98.

286 287 288

(8) United Kingdom., Draft assessment report on the active substance prothioconazole prepared by the rapporteur Member State United Kingdom in the framework of Council Directive 91/414/EEC, 2004.

289 290

(9) Evaluations Part I – Residues. Joint meeting of the FAO Panel of Experts on Pesticide Residues in food and the Environment and the WHO Core Assessment Group Rome., 2014.

291 292

(10) Australian Pesticides and Veterinary Medicines Authority., Agricultural and Veterinary Chemicals Code Instrument No. 4 (MRL Standard)., 2012.

293 294 295

(11) The Japan Food Chemical Research Foundation, Maximum Residue Limits (MRLs) List of Agricultural Chemicals in Foods, http: / / www. m5. ws001. squarestart. ne. jp / foundation / search. html. 2015.

296

(12) CAC, Pesticide Residues in Food and Feed, http://www.fao.org/fao-who-codexalimentarius

297 298 299 300 301

/standards/pestres/pesticide-detail/en/?p_id=232. (13) Health and Safety Executive of UK. https://secure.pesticides.gov.uk/MRLs/mrls.asp?page =14.2016. (14) National food safety standard-- Maximum residue limits for pesticides in food. GB 2763-2016, 31-32.


12

Page 13 of 23


302 303 304

(15) Wei, K. S.; Li, L.; Mao, F. L. Determination and dissipation of prothioconazole and its metabolite prothioconazole-desthio in soil. Chinese Journal of Pesticide Science. 2016, 18(5): 659-663.

305 306 307 308

(16) Mei, X. S., Jin, F.; Wei, X., D.; Ting., Y. H.; Miao W.; Jing W.; Jun, M., J.; Hua, S.; Determination of Prothioconazole and Prothioconazole-desthio in Peanut Samples by Liquid Chromatography-Tandem Mass Spectrometry. Chinese Journal of Analytical Chemistry.2012, 40 (8):1284-1288.

309 310

(17) Steven, A. B.; Applications of matrix solid-phase dispersion in food analysis. J. Chromatogr. A, 2000, 880: 63–68.

311

(18) Steven A. B. Matrix solid-phase dispersion. J. Chromatogr. A, 2000, 885:115–127.

312 313

(19) Jozef, T.; Tatiana H.; Katerˇina P.; Isolation and Purification Techniques for Pesticide Residue Analyses in Samples of Plant or Animal Origin. Eur Food Res Technol, 2001, 213:250–258

314 315 316

(20) Li, H.Z.; Hua, J. W.; Li, L.; Ying, W.; Determination of 103 Pesticides and Their Main Metabolites in Animal Origin Food by QuEChERS and Liquid Chromatography–Tandem Ma ss Spectrometry. Food Anal. Methods, 2016. doi:10.1007/s12161-016-0736-7

317 318 319 320 321

(21) Yan, C. T.; Jun, X.; Shou, F. D.; Gang, X. L.; Hu X. W.; Huan, H. Z.; Chao, J.; Mei, D. W.; and Quan, Y. Z.; Determination of Sulfoxaflor in Animal Origin Foods Using Dispersive Solid-Phase Extraction and Multiplug Filtration Cleanup Method Based on Multiwalled Carbon Nanotubes by Ultraperformance Liquid Chromatography/Tandem Mass Spectrometry. J. Agric. Food Chem. 2016, 64, 2641−2646.

322 323 324 325 326

(22) Min, M. L.; Gang, X. L.; Shou, F. D.; Jun X.; Qiang, Z. K.; Bo, Y. L.; Quan Y. Z.; Simultaneous determination of cyflumetofen and its main metabolite residues in samples of plant and animal origin using multi-walled carbon nanotubes in dispersive solid-phase extraction and ultrahigh performance liquid chromatography–tandem mass spectrometry. J. Chromatogr. A, 2013, 1300: 95–103.

327 328 329

(23) Eva M.; Gloria M.;, laura P.; Eulalia S.; and Francesc C.; Multiresidue Method for Pesticide Residue Analysis in Food of Animal and Plant Origin Based on GC or LC and MS or MS/MS. J AOAC INT, 2012 95,6:1777-1796.

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335

336

Figures and tables Captions

337

Table 1 Development of MRLs for prothioconazole in different countries in animal origin food.

338

Table 2 Summary of extraction methods for prothioconazole-desthio in different animal origin

339

food.

340

Table 3 Validation analysis of prothioconazole-desthio.

341

Table 4 Recoveries (%) of prothioconazole-desthio (N=5) in different animal food.

342

Table 5 Market research and testing of prothioconazole-desthio in different animal food from

343

different places in China.

344

Table 6 Dietary risk assessments.

345

Figure 1 Chemical structure and information of prothioconazole and prothioconazole-desthio.

346

Figure 2 Typical chromatograms for prothioconazole-desthio in animal origin food. Figure A stood

347

for the standard solution of prothioconazole-desthio; Figure B to figure F were the samples of liver,

348

kidney, pork, eggs and milk. The mark 1 and 2 were taken as the chromatograms for substrate blank

349

and matrix samples respectively, whose concentrations were 0.05 mg/kg, 0.05 mg/kg, 0.005 mg/kg,

350

0.01 mg/kg, 0.004 mg/kg for the five kinds of food.

351

352


14

Page 15 of 23


Table 1 Development of MRLs for prothioconazole in different countries in animal origin food

Food name

Meat (mammals, except marine mammals)

Food classification

Livestock and poultry

Edible viscera (mammal)


Egg

Eggs and their products

Milk

Milk and its products

CAC

China

The United States

0.01

Beef 0.02; beef fat 0.1; mutton 0.02; sheep fat 0.1

0.5

Bovine liver and bovine kidney 0.2; liver and pig kidney 0.05; sheep liver and sheep kidney 0.2

0.004

0.02

The European Union

Japan

0.01

Meat and adipose tissue of cattle, pigs, sheep, goats, horses and other farms land animals 0.05

Cattle, pigs and other land mammals 0.01, Fat 0.05

0.2

0.5

The liver, kidney and edible viscera of cattle, pigs, sheep, goats, horses and other farm animals 0.5

The liver, kidney and edible viscera of cattle, pigs and other terrestrial mammals 0.5

0.01

0.01

0.05

0.004

0.01

0.01

Australia

0.02

UK

Korea

0.004

Note: Standard of China (GB 2763); China registration situation (Pesticide Information Network); All listed food should check the corresponding MRL from CAC, China, the United States, Australia, Korea, the European Union, Japan and UK) TMDI was calculated with underlined limit values.


15


Page 16 of 23

Table 2 Summary of extraction methods for prothioconazole-desthio in different animal origin food

Our method Matrix

JMPR (2014)

Liver Kidney Pork Egg Milk

The residue was extracted into acetonitrile / water (4/1 v / v) followed by refluxing through 5 M HCl for two hours. The residue was separated from the conjugate of the glycoside by hydrolysis. HPLC-MS / MS was used to for quantitative analysis.

Sample amount （g）） 2.00 ± 0.05

Solvent type acetonitrile /water (85/15)

The solvent amount (mL) 10

Extraction times

NaCl (g)

n-hexane (mL)

C18 （mg））

2

1.5

5

15

2.00 ± 0.05

acetonitrile /water (85/15)

10

2

1.5

5

15

5.00 ± 0.05

acetonitrile /water (85/15)

15

2

1.5

8

15

2.00 ± 0.05

acetonitrile

10

2

1.0

5

15

5.00 ± 0.05

acetonitrile

15

2

2.0

8

15


16

Page 17 of 23


Table 3 Validation analysis of prothioconazole-desthio

Matrix

Liner equation

R2

Liner range (mg / L)

LOD (mg / kg)

LOQ (mg / kg)

Liver

y = 32358822 x + 2166764

0.9988

0.1-4

0.015

0.05

Kidney

y = 9957101x - 350340

0.9986

0.1-4

0.015

0.05

Pork

y = 32529653 x + 1222763

0.9992

0.025-2.5

0.0015

0.005

Egg

y = 38015502 x + 1125777

0.9986

0.02-2

0.003

0.01

Milk

y = 35618552 x + 955353

0.9936

0.02-0.5

0.0012

0.004

Note: In the liner equation, “x” stood for the concentration of prothioconazole-desthio, of which the measurement unity was “mg/L”; “y” stood for the peak area.


17


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Table 4 Recoveries (%) of prothioconazole-desthio (N=5) in different animal food

Fortification

Recovery

Average recovery

RSD

(mg/kg)

(%)

(%)

(%)

0.05

103, 105, 106, 106, 107

105

1.7

0.5

98.6, 99.1, 99.4, 101, 103

100

1.5

2

102, 102, 106, 106, 107

104

2.3

0.05

90.9, 93.8, 108, 110, 111

103

9.4

0.5

96.5, 100, 101, 101, 101

100

1.9

2

86.5, 89.5, 95.5, 96.7, 98.6

93.4

5.5

0.005

89.0, 98.8, 102, 110, 111

102

8.7

0.01

97.5, 97.5, 100, 111, 112

104

7.1

0.1

101, 102, 103, 105, 105

103

1.5

0.01

86.7, 89.6, 103, 107, 109

99.1

10.3

0.05

91.7, 93.7, 103, 109, 111

102

8.6

0.5

77.7, 86.4, 86.5, 88.6, 89.5

85.7

5.4

0.004

76.1, 83.7, 84.5, 86.6, 87.4

83.6

5.4

0.04

72.2, 86.1, 87.1, 89.9, 90.1

85.1

8.7

0.1

83.7, 88.5, 93.9, 95.9, 99.6

92.3

6.8

Matrix

Liver

Kidney

Pork

Egg

Milk


18

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Table 5 Market research and testing of prothioconazole-desthio in different animal food from different places in China Agricultural Products / Food Liver

MRLs (mg/kg)

Sampling location

0.5a

Kidney

0.5a

Pork

0.01a

Egg

0.01b

Milk

0.004a

Hebei, Sichuan, Hunan, Jiangsu, Hubei, Shandong, Henan, Guangdong, Yunnan, Anhui Hebei, Sichuan, Hunan, Jiangsu, Hubei, Shandong, Henan, Guangdong, Yunnan, Anhui Hebei,Sichuan,Hunan, Jiangsu, Hubei, Shandong,Henan, Guangdong, Yunnan, Anhui Beijing, Zhejiang, Shandong, Hainan Ningxia, Inner Mongolia, Xinjiang

Number of samples 30

The rate of detection (%) 0

Excessive rate (%) 0

30

0

0

30

0

0

30

0

0

30

0

0

Note: a: The MRLs from the CAC Codex；b: The MRLs from Regulations for UK and Australia.


19


Page 20 of 23

Table 6 Dietary risk assessment Food type

Dietary intake (kg)

MRL (mg/kg)

TMDI (mg)

Rice and its products

0.2399

Flour and its procucts

0.1385

1

0.1385

Other cereals

0.0233

1

0.0233

Potatoes

0.0495

Dried beans and their products

0.016

1

0.016

Dark vegetables

0.0915

Light vegetables

0.1837

Pickles

0.0103

Fruit

0.0457

Nut

0.0039


0.0795

0.5

0.03975

Milk and its products

0.0263

0.004

0.000105

Eggs and their products

0.0236

0.05

0.00118

Fish and shrimp

0.0301

Vegetable oil

0.0327

0.1

0.00327

Animal oil

0.0087

Sugar, starch

0.0044

0.3

0.00132

Salt

0.012

Soy sauce

0.009

Sum

1.0286

-

0.223425

Day allowable intake (mg)

Risk probability (%)

ADI×63


0.63

35.46%

20

Page 21 of 23


Prothioconazole-desthio

Prothioconazole Compound information Mol. wt. 344.26 M.f. C14H15Cl2N3OS CAS 178928-70-6 Form Pure white or light gray powder crystal. M.p. 139.1～144.5℃ -7 V.p.

Approach for Pesticide Residue Analysis for ... - ACS Publications

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