Simultaneous detection of sulfoxaflor and its metabolites, X11719474 and X11721061, in

lettuce using a modified QuEChERS extraction method and liquid chromatography-

tandem mass spectrometry

Sung-Woo Kim1, Md. Musfiqur Rahman1, A. M. Abd El-Aty2,3*, Md. Humayun Kabir1,

Tae Woong Na4, Jeong-Heui Choi1, Ho-Chul Shin2, Jae-Han Shim1*

1
Biotechnology Research Institute, College of Agriculture and Life Sciences, Chonnam

National University, Yongbong-ro 77, Buk-gu, Gwangju 500-757, Republic of Korea

2
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine,

Konkuk University, Seoul 143-701, Republic of Korea

3
Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211-

Giza, Egypt
4
Jeonnam Bioindustry Foundation, Biocontrol Research Center Immyeon, Gokseonggun.

Jellanamdo, Republic of Korea

*Corresponding authors. Tel.: +82-10-5934-0701; fax: +82-2-444-4396. E-mail address:

abdelaty44@hotmail.com; amabdelaty@konkuk.ac.kr (A. M. Abd El-Aty)

and Tel.: +82-62-530-2135; fax: +82-62-530-0219. E-mail address: jhshim@chonnam.ac.kr

(J. H. Shim)

Short running title: Residue analysis of sulfoxaflor and its metabolites in lettuce

Pages: 23

Tables: 3

Figures: 3
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/bmc.3885

This article is protected by copyright. All rights reserved.

ABSTRACT

An analytical method has been developed to quantify the residual levels of sulfoxaflor and its

metabolites (X11719474 and X11721061) in/on cultivated lettuce grown under greenhouse

conditions. Samples were extracted and purified using a quick, easy, cheap, effective, rugged,

and safe “QuEChERS” method (original version) following systematic method optimization

and were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Good

linearity with coefficient of determination (R2) ≥ 0.9930 was obtained and the limits of

detection (LOD) and quantification (LOQ) were in the ranges of 0.003–0.006 and 0.01–0.02

mg/kg, respectively. The recovery rates of both the parent compound and its metabolites

(fortified at 10 and 50× the LOQ) estimated from six replicates ranged between 81.9 and

115.5% with a relative standard deviation (RSD) < 12%. The validated method was applied to

field-incurred samples (collected over 7 days) sprayed once or twice with a water-dispersible

granule formulation. Notably, a substantial reduction in rate was observed after 3 days and

the half-life was short, only 1.5 days. The developed method is simple and versatile and can

be used for various leafy vegetables.

Keywords: Sulfoxaflor, Metabolites; Residues; lettuce, QuEChERS, half-life, Liquid

chromatography-tandem mass spectrometry

This article is protected by copyright. All rights reserved.

Introduction

Lettuce (Lactuca sativa L.) is a widely grown and popularly consumed leafy vegetable

because it contains vitamin C, polyphenols, and a dietary fiber, which contribute to weight

loss (due to its low caloric content), lower the risk of cardiovascular diseases (via reducing

low-density lipoprotein (LDL) cholesterol and blood pressure), and reduce the risk of

diabetes (by improving glucose metabolism) and colon cancer (due to protective role of

dietary fiber) (Institute of Medicine, 2002; Kim et al., 2016). In Korean cuisine, lettuce is

usually consumed with seasonings such as soybean sauce and to wrap meat or other fillings.

In the Republic of Korea, it is considered a minor crop cultivated on less than 1000 ha of land

grown under greenhouse conditions, and a representative crop for Swiss chard and chicory to

establish the pre-harvest interval (PHI) (Ko et al., 2014; Im et al., 2016).

Insecticides such as organophosphates, carbamates, and pyrethroids have been used to

protect significant agricultural commodities from insect pests. As a consequence of their

extensive use, many insects have developed resistance against insecticides over the years.

Neonicotinoids are highly effective insecticides because they exhibit no cross-resistance and

are active against a wide range of insects. They are classified into first-generation chloro-

pyridyls, second-generation chloro-thiazolyls, third-generation furanyls, and fourth-

generation sulfoximines (Tanner and Czerwenka, 2011; Culter et al., 2013; Jovanov et al.,

2013; Longhurst et al., 2013; Jiao et al., 2016). Sulfoxaflor [methyl (oxo) {1-[6-

(trifluoromethyl)-3-pyridine] ethyl}-λ6-sulfanylidene] cyanamide (Figure 1) is a systemic

fourth-generation neonicotinoid acting on the nicotinic acetylcholine receptors (nAChRs) in

the nervous system of pests (FAO, 2011; Cutler et al., 2013). Its mode of interaction with

insect nAChRs was different from that of other neonicotinoids and nAChR-acting

insecticides. It is highly efficacious against sap-feeding insects; including those displaying

cross-resistance to other neonicotinoids (first up to third generation) and other insecticides

This article is protected by copyright. All rights reserved.

(Watson et al., 2011; Sparks et al., 2013; Nugent et al., 2015; Loso et al., 2016). In the

Republic of Korea, sulfoxaflor has been used as an insecticide for various grains, vegetables,

and fruits against aphids, whiteflies, hoppers, lygus, and mulberrythrips, with safety

guidelines for three types of formulation products including suspension concentration (SC),

granule (GR), and water-dispersible granule (WG) (Korea Crop Protection Association,

2015). Sulfoxaflor, as a parent compound, can be degraded, forming two metabolites:

X11719474 and X11721061 (Figure 1), which show low acute and oral toxicity in rats and no

in-vitro genotoxic potential in mammalian or microbial test systems. The X11719474 is a

major soil and plant metabolite (FAO, 2011); its acceptable daily intake (ADI) and the acute

reference doses (ARfD) are 0–0.05 mg/kg body weightand 0.3 mg/kg body weight,

respectively (FAO, 2011).

The residues of the parent compounds and their degraded metabolites in/on agricultural

commodities may cause hazardous effects, if consumed either raw or processed. Although the

residual levels of both the parent compound and the metabolites are expected to be low, it is

crucial to evaluate their safety and to minimize risks by monitoring dissipation patterns in/on

crops, setting safety use guidelines (pre-harvest interval, and maximum residue limits (MRL).

The Ministry of Food and Drug Safety (MFDS) has established the MRL of sulfoxaflor (and

its dimers) between 5.0 (lettuce) and 7.0 mg/kg (perilla leaves) (MFDS, 2015). Therefore, for

safety purposes and dietary risk assessment, an effective analytical method must be

developed to quantitatively determine both the parent compound as well as its metabolites in

specific crops, even though the metabolites (approximately seven times less toxic than parent

sulfoxaflor) were not considered in residue definition for sulfoxaflor(FAO, 2011).

Various extraction and purification methods have been reported for determination of

parent sulfoxaflor in/on agricultural commodities and animal food products (Table 1). For

instance, a liquid–liquid extraction (using dichloromethane as extractant), amino cartridge

This article is protected by copyright. All rights reserved.

(NH2) purification, and HPLC-UV detection were used to determine the analyte in apple,

pear, pepper, hulled rice, potato, mandarin, and soybean ((Do et al., 2013). A solid-phase

extraction (Cleanert PestiCarb/PSA cartridge) and HPLC-diode-array detection (DAD)

method has also been reported for stereoselective estimation of sulfoxaflor in brown rice,

cucumber, and apple (Chen et al., 2014a). Furthermore, Xu et al. (2012) has developed a

method for detection of sulfoxaflor in vegetables, fruits, and soil, using primary-secondary

amine (PSA) and ultra-performance liquid chromatography-tandem mass spectrometry

(UPLC-MS/MS). Chen et al. (2014b; 2016) reported the stereoselective separation and

pharmacokinetic dissipation of parent sulfoxaflor in greenhouse vegetables and soil using

multi-walled carbon nanotubes (MWCNTs) and UPLC-MS/MS. Sulfoxaflor residue in

animal-based food has recently been determined using dispersive-solid phase extraction (d-

SPE), a multi-plug filtration cleanup method, and UPLC-MS/MS (Tian et al., 2016). The

parent compound and its metabolites (X11719474 and X11721061) were simultaneously

determined using LC-MS/MS, following extraction with acetonitrile/water, hydrolysis with

sodium hydroxide (to release base-labile conjugates), and purification with SPE cartridge

(FAO, 2011). . Notably, none of the above-mentioned methodologies used QuEChERS nor

were they used to determine the residues in leafy vegetables like lettuce. Therefore, the

present study was conducted to develop and validate a “QuEChERS”-based extraction

method (Anastassiades et al., 2003) with modification (in purification step) for simultaneous

determination of the parent compound, sulfoxaflor and its metabolites (X11719474 and

X11721061) using LC-MS/MS to estimate the dissipation pattern in/on lettuce grown under

greenhouse conditions.

This article is protected by copyright. All rights reserved.

Experimental

Chemicals and reagents

Analytical sulfoxaflor standard (purity 99.7%) and its metabolites, X11719474 (purity

99.9%) and X11721061 (purity 99.0%), were provided by Dow Agro Sciences (Seoul,

Republic of Korea). HPLC-grade acetonitrile (MeCN) was supplied by Burdick and Jackson

(SK Chemical, Ulsan, Republic of Korea). Analytical-grade anhydrous magnesium sulfate

(MgSO4, purity 99.5%) and sodium chloride (NaCl, purity 99.5%) were purchased from

Junsei Chemical Co. Ltd. (Kyoto, Japan). Formic acid (purity > 95%) was supplied by Sigma

Aldrich (St. Louis, MO. USA). Primary secondary amine (PSA), C18, and graphitized carbon

black (GCB) were provided by Agilent Technologies (Palo Alto, CA. USA).

Field trial

The field trial was conducted at an experimental plot of Naju, Jeonnam Province, Republic of

Korea. The trial was carried out between April 30th and May 7th, 2014. A commercial product

of sulfoxaflor, in the form of a water-dispersible granule (Straight®, 7% active ingredient,

Dongbang Agro, Seoul, Republic of Korea) was sprayed once or twice in/on lettuce at

recommended dose rates (10 mL/20 L). The humidity and temperature were recorded

throughout the experimental period. Samples were randomly collected at 0 (2 h after

application), 1, 3, 5, and 7 days after application. The collected samples were kept on ice,

transferred to the laboratory, chopped, homogenized with dry ice (Hanil Mixer, HMF-3000S,

Hanil, Seoul, Republic of Korea), and stored at −20 °C pending analysis.

This article is protected by copyright. All rights reserved.

Sample preparation

Homogenized samples (10 g) were weighted in a 50-mL polytetrafluoroethylene (PTFE,

Gyeongi-do, Republic of Korea) centrifuge tube to which 20 mL MeCN was added. The

tubes were vigorously shaken for 1 min using a vortex-mixer (Thermolyne MaxiMix® II,

Thermo Scientific, USA). For salting out, 6 g MgSO4 and 1 g NaCl were added, vortex-

mixed for 1 min and centrifuged (Combi-514R, Hanil Science Industrial, Daejeon, Republic

of Korea) at 4,500 rpm for 5 min. 1.5 mL of the upper supernatant layer was transferred to a

2-mL d-SPE tube (Simport, Bernard-pilon Beloeil, QC, Canada) containing 75 mg PSA, 75

mg C18, and 25 mg GCB. The tubes were then vortex-mixed for 1 min and then centrifuged

at 4,500 rpm for 5 min. The final supernatant was filtrated through a 0.45-µm PDVF filter

(Advantec, Saijo, Japan) for LC-MS/MS analysis.

LC/ESI-MS/MS analysis

A Waters Alliance 2695 Separation Module HPLC system, equipped with Waters TQ detector

API tandem quadrupole mass spectrometer (Waters Corp., Massachusetts, USA) was used for

analysis. A Phenomenex-Kinetex 2.6-µm C18 100-Å column (100 × 2.1 mm, Torrance, CA,

USA) was used for the separation. The binary solvent system comprising 0.1% formic acid in

distilled water (A) and 0.1% formic acid in MeCN (B) was used in a gradient mode. The

starting composition of the mobile phase (A:B) was 95:5 (v/v) (0–1 min), was ramped to

25:75 (1–20 min), was held at 25: 75 (20–24 min), ramped to 95:5 (24–25 min), and

maintained at 95:5 (25–30 min). The flow rate and injection volume were 0.35 mL/min and 5

µL, respectively.

Triple-quadrupole MS/MS (QQQ) was conducted in positive electrospray ionization (ESI+)

mode with multiple reaction monitoring (MRM) mode with two mass transitions, of which

the higher-intensity product ion was selected for quantitation and the other was used for

This article is protected by copyright. All rights reserved.

confirmation (Figure 1). The MS source conditions were as follows: capillary voltage, 3.6

kV; source temperature, 150 °C; desolvation temperature, 350 °C; desolvation gas (N2) flow,

600 L/h; and cone gas (N2) flow, 50 L/h. Masslynx software version 4.1 program (Waters

Corp., Massachusetts, USA) was used for data analysis.

Method validation

To validate the analytical method, linearity, specificity, recovery, accuracy, selectivity, LOD,

and LOQ were evaluated. A standard stock solution of sulfoxaflor and its metabolites (100

mg L-1) were prepared in MeCN. For testing recovery in the samples and for generating a

calibration curve, working solutions were prepared by serially diluting the stock solution

using the same solvent. Matrix-matched standard solutions (sulfoxaflor, 0.02, 0.04, 0.1, 0.2,

0.4, 1.0, 2.0, and 6.0 mg/kg; metabolites, 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 1.0, and 2.0 mg/kg)

were prepared in blank lettuce extracts. The LOD and LOQ were calculated at signal-to-noise

(S/N) ratios of 3 and 10, respectively. Specificity was tested via comparing the blank and

standard chromatograms with the absence of any interference in blank samples at or around

the standard retention time. Recovery was evaluated to assure the accuracy of the method at

two different concentration levels (10 × LOQ and 50 × LOQ) in six replicates. The

repeatability was estimated in terms of RSD.

Results and discussion

In the present study, the original QuEChERS method (neutral pH) was modified for the

analysis of the parent compound (stable under hydrolysis conditions) (EFSA, 2014) and its

metabolites in lettuce, a model for leafy vegetables.

This article is protected by copyright. All rights reserved.

Method optimization

Although, the extraction efficiency of the original QuEChERS method was satisfactory for

sulfoxaflor, 89.0 ± 7.5%; and its metabolites (X117194, 88.3 ± 7.6%; X11721061, 92.2 ±

7.0%), we didn’t use such results. The absence of chlorophyll removal (matrix interference)

is behind the reason, as it may affect the column and MS/MS instrument. Therefore, a

cleanup procedure was added as a step of modification to the original methodology. In this

context, d-SPE sorbent efficiency was optimized with four different types of sorbents: PSA,

C18, graphitized carbon black (GCB, and a combination of these three. The recovery range of

sulfoxaflor, X11719474, and X11721061 ranged between 94.2 and 108.2% (Table 2). As the

effect of the amounts of different sorbents (individually or conjointly) on the recovery rate

was not substantial, we selected the mixture of all the individual sorbents to remove matrix

interferences during LC-MS/MS analysis.

Method validation

The potential interference peaks in the blank lettuce sample were monitored by comparing

the LC-MS/MS chromatograms of the blank sample with those of the standard and fortified

samples. Figure 2 demonstrated that there was no interference peak at the retention time of

the expected standard peak. Additionally, the fortified peak matched the retention time for the

standard, which confirms the method’s specificity and selectivity. Interestingly, Figures 2a

(sulfoxaflor) and 2b (X11719474) have shown double peaks, however, this is not the feature

of Figure 2c. Although, chromatographic conditions that would promote chiral separations

were not used throughout the experimental protocol, this is most probably related to the

presence of sulfur atom in both analytes. Thanks to the Editor in Chief who gives this

suggestion.

This article is protected by copyright. All rights reserved.

 A matrix-matched calibration curve was constructed to avoid signal suppression and

enhancement due to matrix effect. The calculated matrix effect of sulfoxaflor, X11719474,

and X11721061 were 4.84, −6.62, and 7.06%, respectively; the values are not substantial

enough to affect the analytical results. Linearity was evaluated using the matrix-matched

calibration curve at 8 concentration levels. Satisfactory linearity was obtained with

coefficient of determinationR2 ≥ 0.9930. The LOQs of sulfoxaflor and its metabolites were

0.02 and 0.01 mg/kg, good enough to determine the residual levels of the parent compound

with its metabolites below the MRL set by MFDS (5 mg/kg) (MFDS, 2015), EU (4 mg/kg)

(EU), and Codex (6 mg/kg in leafy vegetables). As shown in Table 2, the average recoveries

of sulfoxaflor and its metabolites were in the range of 81.88 and 115.52% with RSD <12%.

These values are satisfactory and in line with the SANTE guideline (60-140%)

(SANTE/11945/2015). Therefore, the developed method is suitable for the determination of

total sulfoxaflor residues in lettuce samples.

Method application

The developed analytical method was successfully applied to field-incurred lettuce samples.

The residual levels of sulfoxaflor and its metabolites in lettuce during the experimental period

are shown in Table 3. The total initial residue levels of sulfoxaflor and its metabolites in

lettuce were 3.52–4.74 mg/kg, barely below the MRL (5.0 mg/kg) in lettuce set by MFDS

(2015). After treatment, the residual level steadily decreased with time. The reduction in

residual levels after 7 days following one and two treatments were 96.59 and 96.04%,

respectively, and the half-life was 1.5 days. A similar biological half-life (1.6–2.9 days) was

found in cucumber; however, the half-life was longer in soil (1.5–7.2 days) (Xu et al., 2012;

Chen et al., 2014b; Chen et al., 2016). Chen et al. (2016) reported that the uptake pattern of

the sulfoxaflor stereoisomers was prolonged under the foliage (t1/2, 3.4–14.1 days) compared

This article is protected by copyright. All rights reserved.

to the roots (t1/2, 2.7–5.1 days). The dynamic dissipation patterns could be determined using

the following equation C = 4.3646e-0.474x (R2 = 0.9639) for one-time treatment, and C =

5.4696e-0.453x (R2 = 0.9233) for two-time treatment (Figure 3). The dynamic behavior of the

sum of the parent compound and its metabolites was best fit to a first-order kinetic model.

Conclusions

A simultaneous analytical method was developed to determine sulfoxaflor and its metabolites

(X11719474 and X11721061) in lettuce following the original QuEChERS method (with

modification) coupled with LC-MS/MS detection. The developed method was reliable,

simple, and sensitive for the analysis of sulfoxaflor and its metabolites in field-incurred

lettuce samples and should be capable of being extended to other leafy vegetables. The

residual analyte concentration rapidly declined with a very short half-life of 1.5 days. The

residual levels following both treatments were lower than the MRL values set by some

regulatory authorities.

Acknowledgements

This study was supported by the Rural Development Administration (PJ01035908), Republic

of Korea.

Conflict of Interest

The authors have declared no conflict of interest

This article is protected by copyright. All rights reserved.

References

Anastassiades M, Lehotay SJ, Stajnbaher D and Schenck FJ. Fast and easy multiresidue

method employing acetonitrile extraction/partitioning and "dispersive solid-phase

extraction" for the determination of pesticide residues in produce. Journal of AOAC

International 2003; 86(2): 412-431.

Chen Z, Dong F, Xu J, Liu X, Cheng Y, Liu N, Tao Y and Zheng Y. Stereoselective

determination of a novel chiral insecticide, sulfoxaflor in brown rice, cucumber and

apple by normal-phase high-performance liquid chromatography. Chirality 2014a; 26

(2): 114-120.

Chen Z, Dong F, Xu J, Liu X, Cheng Y, Liu N, Tao Y, Pan X and Zheng Y. Stereoselective

separation and pharmacokinetic dissipation of the chiral neonicotinoid sulfoxaflor in soil

by ultraperformance convergence chromatography/tandem mass spectrometry. Analytical

and Bioanalytical Chemistry 2014b; 406 (26): 6677-6690.

Chen Z, Dong F, Pan X, Xu J, Liu X, Wu X and Zheng Y. Influence of uptake pathways on

the stereoselective dissipation of chiral neonicotinoid sulfoxaflor in greenhouse

vegetables. Journal of Agricultural and Food Chemistry 2016; 64 (13), 2655-2660.

Codex Alimentarius Commission., Available from:

http://www.fao.org/fao-who-codexalimentarius/standards/pestres/pesticide-detail/en/?p_i

d=252

Cutler P, Slater R, Edmunds AJF, Maienfisch P, Hall RG, Earley FG, Pitterna T, Pal S, Paul

VL, Goodchild J, Blacker M, Hagmann L and Crossthwaite AJ. Investigating the mode

of action of sulfoxaflor: a fourth-generation neonicotinoid, Pest Management Science

2013; 69 (5), 607-619.

This article is protected by copyright. All rights reserved.

Do JA, Lee MY, Park HJ, Kwon JE, Jang HJ, Cho YJ, Kang IH, Lee SM, Oh JH Hwang IG.

Development and validation of an analytical method for the insecticide sulfoxaflor in

agricultural commodities using HPLC-UVD. Korean Journal of Food Science and

Technology 2013; 45(2): 148-155.

EU., European commission. Available from: http://ec.europa.eu/food/plant/pesticides/eu-

pesticides-database/public/?event=pesticide. residue.CurrentMRL&language=EN

European Food Safety Authority. Conclusion on the peer review of the pesticide risk

assessment of the active substance sulfoxaflor. EFSA Journal 2014;12 (5): 3692.

Food and Agricultural Organization/World Health Organization., Available from:

http://apps.who.int/pesticide-residues-jmpr-database/pesticide?name=SULFOXAFLOR

2011.

Institute of Medicine. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty

Acids, Cholesterol, Protein, and Amino Acids, Institute of Medicine, Washington D.C,

2002.

Jiao W, Xiao Y, Qian X, Tong M, Hu Y, Hou R and Hua R. Optimized combination of

dilution and refined QuEChERS to overcome matrix effects of six types of tea for

determination eight neonicotinoid insecticides by ultra performance liquid

chromatography-electrospray tandem mass spectrometry. Food Chemistry 2016; 2010,

26-34.

Kim MJ, Moon YY, Tou JC, Mou B and Waterland NL. Nutritional value, bioactive

compounds and health benefits of lettuce (Lactuca sativa L.). Journal of Food

Composition and Analysis 2016; 49: 19–34.

Ko AY, Rahman MM, Abd El-Aty AM, Jang J, Park JH, Cho SK and Shim JH.

Development of a simple extraction and oxidation procedure for the residue analysis of

imidacloprid and its metabolites in lettuce using gas chromatography. Food Chemistry

This article is protected by copyright. All rights reserved.

 2014; 148: 402–409.

Korea Crop Protection Association (2015). p. 526. www.koreacpa.org.

Longhurst C, Babcock JM, Denholm L, Gorman K, Thomas JD and Sparks TC. Cross-

resistance relationships of the sulfoximine insecticide sulfoxaflor with neonicotinoids

and other insecticides in the whiteflies Bemisia tabaci and Trialeurodes vaporariorum.

Pest Management Science 2013; 69 (7), 809-813.

Loso MR, Benko Z, Buysse A, Johnson TC, Nugent BM, Rogers RB, Sparks TC, Wang NX,

Watson GB and Zhu Y. SAR studies directed toward the pyridine moiety of the sap-

feeding insecticide sulfoxaflor (IsoclastTM active), Bioorganic and Medicinal Chemistry

2016; 24(3): 378-382.

MFDS (2015). Ministry of Food and Drug Safety, Pesticide and Veterinary Drugs

information. Available from:

http://www.foodnara.go.kr/residue/prd/info/list.do?currentPageNo=1&searchCode=P016

18&menuKey=1&subMenuKey=9&subChildMenuKey=&searchConsonantFlag=&searc

hFlag=prd&searchValue2=&searchConsonantFlag2=&excelSave=&excelSaveInput=&et

cFlag=&searchValue= 2015.

Nugent BM, Buysse AM, Loso MR, Babcock JM, Johnson TC, Oliver MP, Martin TP, Ober

MS, Breaux N, Robinson A and Adelfinskaya Y. Expanding the structure-activity

relationship of sulfoxaflor: the synthesis and biological activity of N-heterocyclic

sulfoximines. Pest Management Science 2015; 71 (7): 928-936.

SANTE/11945/2015.

Guidance document on analytical quality control and method validation procedures for

pesticides residues analysis in food and feed. Available from:

http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_guidelines_wrkdoc_11945_en.p

df

This article is protected by copyright. All rights reserved.

Sparks TC, Watson GB, Loso MR, Geng C, Babcock JM and Thomas JD. Sulfoxaflor and the

sulfoximine insecticides: Chemistry, mode of action and basis for efficacy on resistant

insects. Pesticide Biochemistry and Physiology 2013; 107: 1-7.

Tanner G and Czerwenka C. LC-MS/MS analysis of neonicotinoid insecticides in honey:

Methodology and residue findings in Austrian Honeys. Journal of Agricultural and Food

Chemistry 2011; 59 (23): 12271-12277.

Tian C, Xu J, Dong F, Liu X, Wu X, Zhao H, Ju C, Wei D and Zheng Y. Determination of

sulfoxaflor in animal origin foods using dispersive solid-phase extraction and multiplug

filtration filtration cleanup method based on multiwalled carbon nanotubes by

ultraperformance liquid chromatography/tandem mass spectrometry. Journal of

Agricultural and Food Chemistry 2016; 64 (12): 2641-2646.

Watson GB, Loso MR, Babcock JM, Hasler JM, Letherer TJ, Young CD, Zhu Y, Casida JE

and Sparks TC. Novel nicotinic action of the sulfoximine insecticides sulfoxaflor. Insect

Biochemistry and Molecular Biology 2011; 41 (7): 432-439.

Xu J, Dong F, Liu X, Li J, Li Y, Shan W and Zheng Y. Determination of sulfoxaflor residues

in vegetables, fruits and soil using ultra-performance liquid chromatography/tandem

mass spectrometry. Analytical Methods 2012; 4: 4019.

This article is protected by copyright. All rights reserved.

 Table 1. Summary of reported methodology for analysis of sulfoxaflor

Targeted Analysis Recovery LOD LOQ

Author Matrix Extraction Clean-up
analyte tool range (%) (㎍/kg) (㎍/kg)
Sulfoxaflor
FAO MeCN, Water
X11719472 Crop SPE cartridge LC-MS/MS - - -
Sodium hydroxide
X11721061
Cucumber 86.0-91.2 0.3 0.9
Tomato 89.4-98.7 0.5 1.6
MeCN (10 mL)
Xu et al., 2012 Cabbage PSA (75 mg) 78.4-96.9 0.6 2.0
Sulfoxaflor NaCl (1 g) LC-MS/MS
Apple MgSO4 (150 mg) 80.8-93.9 0.3 1.1
MgSO4 (4 g)
Grape 83.8-91.5 0.2 0.8
Soil 88.0-90.0 0.4 1.3
Apple 93.2-101.1
Pear 91.8-94.8
Pepper LLE; Dichloromethane 88.2-101.1
Do et al., 2013 MeOH (45 mL)
Sulfoxaflor Hulled rice (100 mL, 2 times) LC-UVD 87.9-94.0 10 50
1N HCl (5 mL)
Potato Purification; SPE-NH2 cartridge 97.2-99.4
Mandarin 90.0-108.2
Soybean 82.8-93.1
Brown rice MeCN (20 mL) 83.7-97.7 50-60 170-210
Chen et al., 2014a
Sulfoxaflor Cucumber NaCl (1 g) Cleanert PestiCarb/PSA cartridge LC-DAD 82.9-95.2 50-60 170-220
Apple MgSO4 (4 g) 77.1-99.3 50-70 180-230
Cucumber MeCN (10 mL) 72.9-99.2 0.38-0.50 1.28-1.68
Chen et al., 2014b MgSO4 (150 mg) 2
Sulfoxaflor Tomato NaCl (1 g) UPC -MS/MS 73.3-103.7 0.40-0.55 1.34-1.83
MWCNTs (5 mg, <8 nm in size)
Soil MgSO4 (4 g) 79.5-102.4 0.30-0.47 1.21-1.58
Chen et al., 2016 Sulfoxaflor Cucumber MeCN (5 mL) MgSO4 (150 mg) UHPSFC- 72.9-103.7 _ 1.3-1.7

This article is protected by copyright. All rights reserved.

 NaCl (0.75 g) MWCNTs (5 mg, <8 nm in size) MS/MS
Tomato 1.3-1.8
MgSO4 (3 g)
Milk 91.0-109.7
Eggs 81.4-114.9
Chicken MeCN (10 mL) C18 (50 mg) + m-PFC cleanup 76.2-104.7
Tian et al., 2016 UPLC-
Sulfoxaflor Pork NaCl (1 g, milk; 4 g) 89.9-106.6 _ 1
MS/MS
Porcine fat MgSO4 (4 g) 82.1-102.0
Porcine Liver C18 (30 mg) + PSA (20 mg) 75.5-104.5
Porcine kidney + GCB (10 mg) + m-PFC cleanup 81.1-103.4
Sulfoxaflor MeCN (20 mL) 92.3115.5 6 20
C18 (75 mg) + PSA (75 mg)
Current study X11719474 Lettuce NaCl (1 g) LC-MS/MS 81.9-91.2 3 10
+ GCB (25 mg)
X11721061 MgSO4 (6 g) 91.1-97.4 3 10

This article is protected by copyright. All rights reserved.

Table 2. Optimization of d-SPE with different sorbents and the recovery results of sulfoxaflor and its metabolite in lettuce

Optimization and
Optimization result
validated method

Sorbent PSA (P) C18 (C) GCB (G) P+C+G

QuEChERS Sulfoxaflor 99.7 99.5 98.9 104.3

sorbent filtration Recovery (%) X11719474 104.2 108.2 115 103.4

X11721061 97.9 94.2 101.8 99.8

Recovery
Linearity LOD LOQ
Pesticide (Mean ± RSD), %, n = 6)
(R2) (mg/kg) (mg/kg)
10 LOQ 50 LOQ
Final recovery
Sulfoxaflor 0.9979 0.006 0.02 115.5 ± 4.21 92.3 ± 9.89

X11719474 0.9930 0.003 0.01 81.9 ± 5.47 91.2 ± 11.61

X11721061 0.9997 0.003 0.01 91.1 ± 6.98 97.4 ± 7.48

This article is protected by copyright. All rights reserved.

 Table 3. Residue levels of sulfoxaflor and its metabolite in lettuce grown under greenhouse conditions

Application Residue level (mg/kg) Reduction Half-life

DATa
time Sulfoxaflor X11719474 X11721061 Total residueb ratio (%) (day)

0 3.50 0.02 <0.01 3.52 -

1 3.03 0.06 0.01 3.10 11.97

1 times 3 1.04 0.01 <0.01 1.05 70.18 1.5

5 0.58 0.01 <0.01 0.59 83.16
7 0.12 <0.01 <0.01 0.12 96.59

0 4.66 0.07 0.01 4.74 -

1 4.62 0.07 0.01 4.70 0.91
2 times 3 0.89 0.03 <0.01 0.91 80.78 1.5
5 0.88 0.02 <0.01 0.91 80.92
7 0.15 0.04 <0.01 0.19 96.04
a
DAT = Days after treatment

Correction factor for X11719474 (0.94) = Sulfoxaflor M.W (277.27)/X11719474 M.W (295.29) (SANTE/11945/2015).

Correction factor for X11721061 (1.45) = Sulfoxaflor M.W (277.27)/X11721061 M.W (191.15) (SANTE/11945/2015).
b
Total residue = Residue of sulfoxaflor + X11719474ⅹ0.94 + X11721061 ⅹ1.45 (SANTE/11945/2015).

This article is protected by copyright. All rights reserved.

 H3C H3C H3C
CH3 CH3

S S OH
N CN N NH2
O O
F3C N F3C N O F3C N

Sulfoxaflor X11719474 X11721061

Precursor
Product ion Cone CE RT
Compound M.W. ion
(m/z) (V) (V) (min)
(m/z)

154 28 15.6
Sulfoxaflor 277 278 15
174a) 7 15.8

154 28
X1179474 295 296 15 13.0
174 a) 7

130 a) 19
X11721061 191 192 18 14.8
172 16

a) Quantitation ion

Figure 1. Chemical structures and mass spectrometry results of sulfoxaflor and its

metabolites

This article is protected by copyright. All rights reserved.

N-M-sul-05ppm 1: MRM of 2 Channels ES+
100 278 > 174 (Sulfoxaflor)
3.50e5

15.46 (A)
15.70
%

0 Time
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

SNL-con1 1: MRM of 2 Channels ES+

100 278 > 174 (Sulfoxaflor)
3.50e5

(B)
%

0 Time
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

SNL-S-High2 1: MRM of 2 Channels ES+

100 278 > 174 (Sulfoxaflor)
3.50e5

(C)

15.46

15.70
%

0 Time
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

ANL-S1-3d-2 1: MRM of 2 Channels ES+

100 278 > 174 (Sulfoxaflor)
15.52 3.50e5

15.78
(D)
%

0 Time
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Figure 2a. LC-MS/MS chromatograms of sulfoxaflor in lettuce: (A) matrix-matched standard

(sulfoxaflor 1.0 mg/kg); (B) a blank lettuce; (C) fortified lettuce; and (D) field-incurred samples
collected 3 days post application.

This article is protected by copyright. All rights reserved.

N-M-sul-02ppm 3: MRM of 2 Channels ES+
100 296 > 174 (X11719474)
1.00e5

12.86
(A)
%

12.95

0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

SNL-con1 3: MRM of 2 Channels ES+

100 296 > 174 (X11719474)
1.00e5

(B)
%

0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

SNL-S-High2 3: MRM of 2 Channels ES+

100 296 > 174 (X11719474)
1.00e5
12.84

(C)
%

12.93

0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

ANL-S1-3d-1 3: MRM of 2 Channels ES+

100 296 > 174 (X11719474)
1.00e5

(D)
%

13.05
0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Figure 2b. LC-MS/MS chromatograms of X11719474 in lettuce: (A) matrix-matched standard

(X11719474, 0.4 mg/kg); (B) a blank lettuce; (C) fortified lettuce; and (D) field-incurred samples
collected 3 days post application.

This article is protected by copyright. All rights reserved.

N-M-sul-02ppm 2: MRM of 2 Channels ES+
100 192 > 130 (X11721061)
2.00e5

(A)
14.72
%

0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

SNL-con1 2: MRM of 2 Channels ES+

100 192 > 130 (X11721061)
2.00e5

(B)
%

0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

SNL-S-High2 2: MRM of 2 Channels ES+

14.70 192 > 130 (X11721061)
100
1.98e5

(C)
%

0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

ANL-S1-3d-1 2: MRM of 2 Channels ES+

100 192 > 130 (X11721061)
2.00e5

(D)
%

0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Figure 2c. LC-MS/MS chromatograms of X11721061 in lettuce: (A) matrix-matched

standard (X11721061, 0.4 mg/kg); (B) a blank lettuce; (C) fortified lettuce; and (D) field-
incurred samples collected 3 days post application.

This article is protected by copyright. All rights reserved.

Figure 3. Dissipation pattern of the total sum of sulfoxaflor and its metabolites in lettuce

grown under greenhouse conditions.

This article is protected by copyright. All rights reserved.