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Toxicokinetics, tissue distribution and excretion of dufulin racemate and its R (S)- enantiomers in rats Huaguo Chen, XIN ZHOU, and Baoan Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01101 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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Journal of Agricultural and Food Chemistry

Toxicokinetics, tissue distribution and excretion of dufulin racemate and its R (S)- enantiomers in rats Huaguo Chen #, §, Xin Zhou §, Baoan Song #, * #

State Key Laboratory Breeding Base of Green Pesticide and Agricultural

Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, China §

Guizhou Engineering Laboratory for Quality Control & Evaluation Technology of

Medicine, Guizhou Normal University, 116 Baoshan North Rd., Guiyang, 550001, China Corresponding author * Tel.: +86-851-8362-0521; Fax: +86-851-8362-2211 E-mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Dufulin is a plant antiviral agent with a novel molecular structure and

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has been used widely to prevent and control tobacco and rice viral diseases. In this

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study, an UHPLC-MS/MS method was developed for rapid determination of dufulin

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racemate (rac-DFL) and its R (S)-enantiomers in rat plasma, tissues, urine and feces.

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A MALDI-MSI method was further used to visual research on tissue distribution after

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intragastric administration of the three analytes. Toxicokinetic study showed that both

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(R)-enantiomers of dufulin ((R)-DFL) and (S)-enantiomers of dufulin ((S)-DFL) had a

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faster ability to reach Cmax than that of rac-DFL. (R)-DFL and (S)-DFL had a similar

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T1/2, though both were significantly lower than rac-DFL. Cmax of rac-DFL was

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obviously higher than (R)-DFL or (S)-DFL. Meanwhile Cmax of (S)-DFL was only

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about 60 % of (R)-DFL. Rac-DFL and its R (S)-enantiomer had a dose-dependent

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toxicokinetic profile. Tissue distribution results revealed rac-DFL, (R)-DFL and

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(S)-DFL mainly distributed in liver and kidney, but the maximum concentration was

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only ng/g grade and could significant degradation within 3 hours. This indicates that

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dufulin does not cause liver and kidney toxicity in animals. In addition, rac-DFL and

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its R (S)-enantiomers were not been detected in brain tissue. Cumulative excretion of

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rac-DFL and its R (S)-enantiomers within 24 h in urine and feces were less than 20 %,

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indicating that they mainly excreted as metabolites. These results could provide

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evidence for the in-depth toxicity evaluation of dufulin pesticide. In addition, its

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metabolic selectivity information in vivo was also been obtained.

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KEY WORDS: Dufulin; toxicokinetics; tissue distribution; excretions

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INTRODUCTION

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Dufulin is a new type of plant antiviral agent with a novel amino phosphate chemical

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structure invented by Guizhou university1, and granted registration as a new chemical

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entity by the Ministry of Agriculture of China. It has high activities on tobacco,

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cucumber and tomato virus and has been used widely to prevent and control tobacco2,

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agent to meet China's environmental friendly standards.

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As a kind of highly active antiretroviral pesticide, dufulin has gained the researchers'

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more and more interests, in the recent years. Its effectiveness, functional mechanism,

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environmental biological toxicity and safety evaluation have been widely studied. For

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example, Xiangyang Li et al.

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infection of southern rice black-streaked dwarf virus. Chen Zhuo et al.2 reported the

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protein target and mechanism of dufulin on prevent and control tobacco virus. Wang

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Hua Zi et al6 reported the biotic and abiotic degradation of dufulin in soils.

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Enantioselective degradation of dufulin in four types of soil was reported by Zhang

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Kankan et al7. Fan Huitao et al8 reported the acute toxicity of dufulin and safety

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evaluation to environmental biology. As a whole, the antiviral effect of dufulin has

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confirmed by various kinds of studies and its environmental biological toxicity or

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safety extensively surveyed. However, there are limited research publications

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reporting the metabolic processes and changes of dufulin in vivo. In fact, with the

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continuous progress and development of science and technology, the metabolic

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processes and changes of pesticide in vivo should become one of the important

and rice viral diseases4, et al. In addition, dufulin is also the first anti-plant viral

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reported the inhibitory effect of dufulin on the

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indexes for pesticide’s toxicity evaluation. To evaluate the safety of a pesticide, its

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metabolic processes and changes in vivo should not been ignored. As for dufulin, it is

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a kind of racemic mixture and has two chemical configurations of (R)-enantiomers or

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(S)-enantiomers. Except for metabolic processes and changes in vivo, metabolic

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selectivity of dufulin racemate and its R (S)-enantiomers should also been taken into

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the value.

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Toxicokinetics is mainly concerning the absorption, distribution, metabolism,

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excretion process and characteristics of toxic substances in animal9-11. The

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pharmacokinetic parameters can describe the systemic exposure of poisons and the

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relationship between dose and time, to evaluate the toxicity of a drug in different

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species, sex, age, and physical state, such as disease or pregnancy12-15. Thus, in order

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to clarify the metabolism process, changes and metabolic selectivity of dufulin in vivo,

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we examined and compared the pharmacokinetics, tissue distribution and excretion of

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dufulin racemate and its R (S)-enantiomers after oral administration in rats, by using a

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validated UHPLC-MS/MS assay and a matrix assisted laser desorption/ionization

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mass spectrometry imaging (MALDI-MSI) method.

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MATERIALS AND METHODS

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Materials Dufulin racemate (rac-DFL), (R)-enantiomers of dufulin ((R)-DFL) and

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(S)-enantiomers of dufulin ((S)-DFL) were obtained from Guizhou University, and

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their purity were more than 99 %. The internal standard substance (IS) of Bergenin

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(Batch No. 20150322, purity > 99 %) was purchased from the National Institute for

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Food and Drug Control of China. HPLC grade methanol and acetonitrile were 4


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obtained from TEDIA company (USA). Formic acid of MS grade purchased from Roe

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Scientific Inc (USA). Ultra-pure water prepared by a Millipore Milli-Q purification

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system (Bedford, USA). All other chemicals were analytical grade.

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Animal and experimental design

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(weighting 250 ± 20 g ) were obtained from Changsha Tianqin Bio-technology Co.,

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Ltd (Changsha, China, Certificate No. SCXK2016-0015). All rats need to adapt to the

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environment (25 ± 1 °C, 12/12 h circadian cycle, free feeding and water) for at least

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one week. The rats were required to fast over night before the experiments but could

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supply with water. All experimental programs were been conducted according to the

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Guide of the Care and Use of Laboratory Animal, Eighth Edition (2011) and approved

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by the Guizhou Normal University Animal Care and Use Committee.

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Different concentration of rac-DFL, (R)-DFL and (S)-DFL solutions for toxicokinetics,

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tissue distribution and excretion studies were prepared by dissolving the appropriate

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amount of control in 0.2 % DMSD aqueous solution, respectively.

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For the toxicokinetic studies16, 108 male rats were randomly divided into 9 groups (n

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= 12). Group 1 to 3 were intragastric administration of rac-DFL (2.5, 5.0 and 10.0

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mg·kg-1), group 4 to 6 were orally administration of (R)-DFL (2.5, 5.0 and 10.0

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mg·kg-1), group 7 to 9 were orally administration of (S)-DFL (2.5, 5.0 and 10.0

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mg·kg-1), respectively. The blood samples (500 µL) were collected from the

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suborbital vein at 0, 10, 20, 30, 40, 60, 80, 100, 120, 180, 300 and 480 min

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respectively, and were been placed into the EP tube containing heparin sodium. After

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blood collecting at each collection point, the equal volume of physiological saline

Adult male pathogen-free Sprague-Dawley rats

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supplied in time. The plasma was been immediately separated by high-speed

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centrifugation (2000 rpm, 10 min) for UHPLC-MS/MS analysis. Toxicokinetics

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parameters including the elimination half-life time (T½), area under the time

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concentration curve (AUC), apparent volume of distribution (Vd), the number of Vd

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removed from the body within a unit time (CL) and mean retention time (MRT) were

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calculated by non-compartmental analysis mode of Phoenix WinNonlin 6.4 (Pharsight,

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Missouri, USA). Drug peak time (Tmax) and drug peak concentration (Cmax) acquired

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directly from the concentration versus time curve.

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Tissue distribution studies17 were conducted in 120 rats randomly divided into 10

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groups (n = 12). Animal grouping and administration of Group 1 to Group 9 were the

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same as the toxicokinetic study section. The tenth group given a physiological saline

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orally. Tissues including liver, heart, lung, spleen, kidney and brain harvested at 60

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min and 3 h and thoroughly eliminate blood and other interfering substances by

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ice-cold saline rinsing, then dried with filter paper. Half of tissue samples in the

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middle dose group were placed at -80 ºC for MALDI-MSI analysis18-21, and the other

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tissue samples were accurately weighed and homogenized using 3 times 50 %

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acetonitrile (v/v) solution, then kept in the freezer (-80 °C) until UHPLC-MS/MS

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analysis.

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Excretion studies were performed in 54 rats which were divided into 9 groups (n = 6),

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with a same mode of administration as toxicokinetics study section

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rat was been reared in an independent metabolic cage after orally administration of

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rac-DFL, (R)-DFL and (S)-DFL, respectively. The sampling time of urine samples 6


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. Then each

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was set to 0-3, 3-6, 6-12 and 12-24 h after oral administration of the analyte, while

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feces sample collecting was set at intervals of 0-6, 6-12 and 12-24 h. During the

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experiment, all rats allowed to take both food and water freely. After the feces sample

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was accurately weighed, the homogenate was treated with 3 times 50 %

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acetonitrile(v/v) solution, then the supernatant liquor was immediately separated by

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high-speed centrifugation (5000 rpm, 10 min) and stored at -80 °C until analysis.

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Sample preparation for UHPLC-MS/MS analysis

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precipitation method

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biological samples including plasma, tissue homogenates, urine and feces supernatant

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liquor. An aliquot of 100 µL plasma samples were transferred into the 1.5 ml EP tubes,

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then 20 µL bergenin solution (IS, 12 µg·mL-1) was added into each tube except the

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blank. The mixtures were vortex-mixed for 50 s and then centrifuged (15000 rpm) 8

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min at 4 °C. After centrifugation, the supernatant was separated and a 5 µL aliquot

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was used for UHPLC-MS/MS analysis. Other biological samples (tissue homogenates,

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urine and feces supernatant liquor) followed with the same procedure.

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Sample preparation for MALDI-MSI analysis

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at -18 °C using a Cryostar NX70 freezing microtome (Thermo Fisher Scientific Ltd.,).

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The heart, brain, and kidney tissues were been sliced in sagittal directions, and the

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lung, spleen and liver tissues were sliced in lateral directions. Eight microns thick

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sections were been cut and mounted onto conductive Indium Tin Oxide (ITO) glasses.

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Matrix deposition for MALDI-MSI analysis was performed by spraying 2, 5-

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Dihydroxybenzoic acid (30 g/L in 50 % MeOH within 0.2 % trifluoroacetic acid)

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In this study, a simple protein

was used to remove impurities and extract the analytes from

All rat tissue sections were sliced

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using the ImagePrep automated spraying device (Bruker Daltonics, Bremen,

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Germany).

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Preparation of stock, standard and quality control samples

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rac-DFL (503 µg·mL-1), (R)-DFL (505 µg·mL-1) and (S)-DFL (508 µg·mL-1) were

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prepared in methanol, and then diluted into 0.0503-5.0300 µg·mL-1, 0.0505-5.0500

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µg·mL-1 and 0.0508-5.0800 µg·mL-1 for calibration curves, respectively. All the

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solutions were stored at 4 °C and reverted to room temperature before analysis. The

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final calibration standard solutions prepared by transferring appropriate amount of the

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stock solution into 100 µL of blank biological matrices to get a series of rac-DFL

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(concentration range from 5.03 to 5030.00 ng·mL-1), (R)-DFL (concentration range

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from 5.05 to 5050.00 ng·mL-1) and (S)-DFL solutions (concentration range from 5.08

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to 5080.00 ng·mL-1) in plasma, respectively. The same procedure was followed to

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gain rac-DFL (15.09-5030.00 ng·mL-1), (R)-DFL (15.15-5050.00 ng·mL-1) and

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(S)-DFL solutions (15.24-5080.00 ng·mL-1) in tissue homogenates, feces and urine,

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respectively. Quality control samples (QCs) were prepared by the similar procedure,

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to achieve three different concentrations of 15, 420, 4200 ng·mL-1 in plasma and 35,

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400, 4000 ng·mL-1 in tissue homogenates, feces and urine.

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UHPLC-MS/MS analysis

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were performed on an UHPLC-MS/MS system, consisting of an Accela1250 UHPLC

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system and a TSQ quantum ultra-triple-quadrupole mass spectrometer detector

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(Thermo Fisher Scientific Inc., USA). Chromatographic separation was performed on

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an Agilent XB-C18 column (2.1×150 mm, 1.7 µm) maintaining temperature at 40 °C

Stock solutions of

The quantification of rac-DFL, (R)-DFL and (S)-DFL

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and flow rate of 0.2 mL·min-1. The mobile phase system consisted of 0.1 % formic

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acid in acetonitrile (A) and 0.1 % formic acid in water (B). The gradient elution

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program was as follows: 0-3 min, linear change from A to B (3:97, v/v) to A-B (5:95,

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v/v); 3-10 min, linear change from A to B (5:95, v/v) to A-B (12:88, v/v); 10-17 min,

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linear change from A to B (12:88, v/v) to A-B (20:80, v/v). The injection volume was

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5 µL. Mass spectrometric analyses were performed on a TSQ quantum ultra-triple-

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quadrupole mass spectrometer (Thermo Fisher Scientific Inc., USA) equipped with an

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electrospray ionization interface in negative mode. The three analytes and the IS were

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detected by negative ion mode and quantified in multiple reactions monitoring (MRM)

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mode with transitions of m/z 407.432-125.268 for rac-DFL, 407.427-125.261 for

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(R)-DFL, 407.429-125.263 for (S)-DFL and m/z 326.978-192.152 for IS. Sheath gas

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flow rate, auxiliary gas flow rate, spray voltage, vaporizer temperature and capillary

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temperature were set to 40 arbitrary units, 10 arbitrary units, 2500 V, 340 °C and

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340 °C, respectively.

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Statistical analysis carried out by SPSS 18.0 software (SPSS, Inc., Chicago, USA).

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Data were presented as mean ± S.D. and P value < 0.05 suggested a statistically

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significance.

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UHPLC-MS/MS method validation

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biological samples (plasma, tissues, urine and feces ), according to the guide of

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European Medical Agency (EMA) Guideline for Bioanalytical Method Validation and

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FDA Guidance for Industry, Bioanalytical Method Validation (US-FDA, 2001). The

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validation content including selectivity, matrix effects, calibration curves, accuracy,

The analytical method was verified on all

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precision and stability of the method.

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MALDI-MSI analysis

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MALDI Tissuetyper (Bruker, Germany) in negative-ion mode, using 600 laser shots

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per spot and 50 × 50 µm pixel size. Data were acquired in the m/z range from 380-420.

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Peptide Calibration Standard II (Bruker product no. #8217498) was been used for

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external instrument calibration. The average mass spectra was been normalized to

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their total-ion-count (TIC). Data acquisition and visualizations performed using the

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flex software-package (flexControl 3.4; flexImaging 4.1, Bruker, Germany).

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RESULTS AND DISCUSSION

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Optimization of chromatography and mass spectrometry conditions

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(R)-DFL, (S)-DFL and IS were monitored under negative ion mode and quantified in

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multiple reactions monitoring (MRM) conditions. Under the UHPLC-MS/MS

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conditions, there were no interfering peaks at the elution times of rac-DFL, (R)-DFL,

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(S)-DFL and IS. Figure 1 shows the representative chromatograms of blank plasma,

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spiked plasma containing rac-DFL, (R)-DFL, (S)-DFL and IS at LLOQ level and

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plasma collected at 1 h after oral administration of the three analytes (5.0 mg·kg-1).

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Retention times of three compounds and internal standard were in vicinity of 7.11 min,

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9.66 min, 9.55 min and 12.17 min, respectively. The chromatographic condition was

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also optimized for separation of rac-DFL, (R)-DFL, (S)-DFL and IS with higher

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resolution. Different types of chromatographic columns were also screened, and

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Agilent XB-C18 column (2.1×150 mm, 1.7 µm) was been considered to be the most

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appropriate one. Different mobile phase systems, such as water-acetonitrile,

MALDI-MSI analyses were performed using a rapiflex

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Rac-DFL,

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water-methanol, water (0.1 % formic acid)-methanol, water (0.1 % formic

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acid)-methanol (0.1 % formic acid), water (0.1 % formic acid)-acetonitrile and water

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(0.1 % formic acid)-acetonitrile (0.1 % formic acid) were evaluated for

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chromatographic behaviors. As a result, acetonitrile (0.1 % formic acid)-water (0.1 %

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formic acid) system was found to has a satisfactory resolution value, sharp and

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symmetrical peaks.

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Figure 1

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Method validation

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Assay linearity and sensitivity To evaluate linearity, calibration solutions of

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rac-DFL, (R)-DFL and (S)-DFL were prepared and detected. The peak area ratios of

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rac-DFL, (R)-DFL and (S)-DFL to IS in rat plasma and other biological samples

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changed linearly over the concentration ranges. The calibration curves showed

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excellent linearity with correlation coefficients (γ) ≥ 0.99 in all matrices. The

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weighing factor 1/X2 was been selected for back calculation of nominal value because

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it produced best linear fit with minimum bias. The regression equations for rac-DFL,

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(R)-DFL and (S)-DFL were listed in Table 1, respectively, where Y refers to the peak

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area ratios (analyte/IS) and X is the concentration of rac-DFL or (R)-DFL or (S)-DFL.

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The developed method offered LLOQ of 10 ng·mL-1 in plasma, urine, feces and

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tissues and was quantified with acceptable accuracy and precision (≤ 15 %).

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Table 1

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Precision and accuracy

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rac-DFL, (R)-DFL and (S)-DFL in biological samples were assessed, according to the

The intra-day and inter-day precision and accuracy of

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guidance of the US Food and Drug Administration. The intra-day and inter-day

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precision (RSD, %) values were ≤ 10.56 % and ≤ 11.27 %, respectively. While the

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intra-day and inter-day accuracy (%) were in the range of 92.5-107.4 % and

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93.1-108.2 %, respectively. All the results are within the technical scope stipulated in

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the management guidelines, indicating that the analytical method established was

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suitable for quantitative determination of rac-DFL, (R)-DFL and (S)-DFL in rat

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biological samples.

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Recovery and matrix effects

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effects for rac-DFL, (R)-DFL and (S)-DFL in biological samples. The recoveries of

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rac-DFL, (R)-DFL and (S)-DFL in all biological samples were reproducible,

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concentration independent and consistent. The recovery for rac-DFL, (R)-DFL and

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(S)-DFL was between 77.65 % and 87.59 %. Meanwhile, the recovery for IS was

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ranged from 76.95 % to 88.55 %. They were both according with the guideline for

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validation of bioanalytical method, which issued by the U. S. Food and drug

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administration, drug evaluation and Research Center. The matrix effects for rac-DFL,

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(R)-DFL and (S)-DFL in all biological matrices were within ± 11.00 % with RSD %

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of ≤ 7.78 % which was considered as negligible or insignificant matrix effects.

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Table 2 shows the extraction recovery and matrix

Table 2

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Stability

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change under different storage conditions. As shown in Table 3, the stability of

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rac-DFL, (R)-DFL and (S)-DFL in different conditions (short-term, freeze/thaw and

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long-term stability) were within the acceptable levels (RSD % of precision values

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