658 Meijing Lan Shuying Li Zhenzhen Xu Chunxia Tian Mei Yang Wenjun Gui Guonian Zhu Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou, P.R. China Received October 12, 2013 Revised December 8, 2013 Accepted December 18, 2013

J. Sep. Sci. 2014, 37, 658–664

Research Article

Simultaneous determination of saflufenacil and its two metabolites in soil samples by ultra-performance liquid chromatography with tandem mass spectrometry Saflufenacil is a new protoporphyrinogen-IX-oxidase inhibitor herbicide. When used, it can enter the soil and has a high risk to reach and contaminate groundwater and aquatic systems. A rapid and sensitive method of ultra-performance LC with MS/MS was developed for the simultaneous determination of saflufenacil and its two metabolites in soil samples. A modified quick, easy, cheap, effective, rugged, and safe method was applied as the pretreatment procedure. The method was validated by five types of soil samples collected from several regions of China, which all showed good linearity (R2 ≥ 0.9914) and precision (RSD ≤ 26.2%). The average recoveries of the three analytes ranged between 74.1 and 118.9% at spiking levels of 3–300 ␮g/kg. The method limits of detection (S/N 3:1) and method limits of quantification (S/N 10:1) achieved are in the ranges of 0.25–2.75 and 0.83–9.16 ␮g/kg, respectively. This indicated that the developed ultra-performance LC with MS/MS method is a promising analytical tool for monitoring the environmental risks posed by saflufenacil. Keywords: Metabolites / MS/MS / Saflufenacil / Soil / Ultra-performance liquid chromatography DOI 10.1002/jssc.201301121



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Introduction Saflufenacil (BAS 800H) (N -{2-chloro-4-fluoro-5-[1,2,3,6-tetrahydro-3-methyl-2,6-dioxo-4-(trifluoromethyl)pyrimidin-1-yl] benzoyl}-N-isopropyl-N-methylsulfamide) is a new protoporphyrinogen-IX-oxidase inhibitor herbicide developed by BASF [1]. It can control more than 70 kinds of broadleaf weeds including troublesome and herbicide-resistant broadleaf weeds, such as horseweed (Conyza canadensis [L.] Cronq.) [2, 3], giant ragweed (Ambrosia trifida L.) [4] in corn, soybean, and other crops when used alone or with other partner herbicides [1, 5, 6]. Differently from glyphosate, saflufenacil is a selective herbicide, and the selectivity is based on physical placement and rapid metabolism in tolerant crop species [7]. Based on the above characteristics, saflufenacil is mainly used as a pre-emergence herbicide, a crop desiccating harvest aid or used in burndown treatments [8]. Correspondence: Dr. Wenjun Gui, Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, P.R. China E-mail: [email protected] Fax: +86-571-88982517

Abbreviations: ME, matrix effect; MLOD, method LOD; MLOQ, method LOQ; QuEChERS, quick, easy, cheap, effective, rugged, and safe; SLOQ, sample LOQ; UPLC, ultraperformance LC

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Saflufenacil was first registered in the United States in 2009, and then expanded to Argentina, Chile, Nicaragua, and Canada under the trade names: Kixor, Sharpen, OpTill, Verdict, Integrity, Treevix, and Heat. As a herbicide, saflufenacil enters the soil when used for various field and row crops, and can leach to soil depths >60 cm. Due to a combination of properties that favor leaching (persistence, high solubility, low-binding potential, and low volatility), saflufenacil and its transformation products have high risk to reach and contaminate the groundwater and aquatic systems [9]. Consequently, a methodology with high sensitivity for monitoring and analyzing saflufenacil in environmental samples is urgently needed. M800H11 (N -{2-chloro-5-[2,6-dioxo-4-(trifluoromethyl)3,6-dihydro-1(2H)-pyrimidinyl]-4-fluorobenzoyl}-N-isopro-pylsulfamide) and M800H35 (N-[4-chloro-2-fluoro-5-({[(isopropylamino)sulfonyl]amino}carbonyl)phenyl]urea) are two major metabolites of saflufenacil. Furthermore, the European Food Safety Authority (EFSA) established the risk assessment and enforced the residue definition as “the sum of saflufenacil, metabolites M800H11, and M800H35, expressed as saflufenacil” and set new maximum residue limits in a wide range of food commodities all ≥0.03 mg/kg, which were calculated by summing up the individual LOQs

Colour Online: See the article online to view Fig. 2 in colour. www.jss-journal.com

J. Sep. Sci. 2014, 37, 658–664

(0.01 mg/kg) for saflufenacil, M800H11, and M800H35 [10]. As a result, M800H11 and M800H35 were selected as target analytes as well. While there are few peer-reviewed publications reporting analytical methods of the parent of saflufenacil in soil matrices [11], methodological studies remain rare for the simultaneous determination of saflufenacil, M800H11, and M800H35. The only reference was BASF Analytical Method D0603/02, which established an analytical method for the three analytes in various plant matrices with the LOQs of 10 ␮g/kg for each analyte. In this method, the three analytes were extracted with methanol/water (70:30, v/v), partitioned in a mixture of ethyl acetate/cyclohexane (70:30, v/v) and 0.1% TFA, and detected by HPLC–MS/MS. Still, so far there is no published methodology to quantify saflufenacil, M800H11, and M800H35 in environmental samples. Several multiresidue extraction methods have been developed already, such as the Mills method [12], which is focused on the analysis of nonpolar organochlorine pesticides, and the Luke method [13] and its modifications, which recover plenty of polar pesticides. Anastassiades et al. reported a novel method named QuEChERS (quick, easy, cheap, effective, rugged, and safe) that presented good results with few steps [14]. QuEChERS has been successfully used for the extraction and purification of a variety of chemicals in different matrices, such as fruit, vegetable, soil, and meat [14–16]. One of the most prominent characteristics of this method is the salting-out effect that makes a perfect phase separation and leads to increased recoveries of polar compounds [17,18]. The QuEChERS method has been applied to the pesticides from soils with or without clean-up steps [19, 20] achieving recoveries from 53 to 128% [21]. The aim of the present study was to develop and validate a QuEChERS with ultra-performance LC (UPLC) coupled to MS/MS for the simultaneous determination of BAS 800 H, M800H11, and M800H35 in soil samples The main advantage of the methodology herein was related to the elimination of the SPE clean-up step after the extraction, which made the whole procedure simpler, faster, cheaper, and minimized the errors associated with this step [22, 23]. As far as we know, this is the first report to extract BAS 800 H, M800H11, and M800H35 from soil samples, which would be a step toward the establishment of a standard analytical method used for monitoring the residues of saflufenacil in the environment and help in the assessment of environmental risks when improperly used.

2 Materials and methods

Liquid Chromatography

659

Figure 1. Chemical structures of the three target analytes.

was obtained from a water purification system (Pall Corporation, USA). Magnesium sulfate and sodium chloride (99.5%) of analytical grade were purchased from Shisihewei Chemical (Shanghai, China). Formic acid was provided by Wenzhou Chemical (China). The 0.22 ␮m one-off membrane (polytetrafluoroethylene) microfilters (MITEX, Millipore, USA) were used to filter the extracts. Other chemicals and solvents were of analytical grade or better. Five types of soil samples were used to evaluate the proposed methodology: black soil (S1, form Harbin, Heilongjiang Province), paddy soil (S2, from Wuxi, Jiangsu Province), moist soil (S3, from Jiaxing, Zhejiang Province), powder soil (S4, from Hangzhou, Zhejiang Province), and red soil (S5, from Quzhou, Zhejiang Province). The physical and chemical properties of these five kinds of soil are listed in Table 1. Samples were air dried and then passed through a 2 mm sieve for the removal of particles and nondecomposed plant residues. The prepared soil samples were stored at room temperature. 2.2 Preparation of standard solutions Individual standard stock solutions of 1000 mg/L were prepared in methanol. A 10 mg/L mixed-standard solution for the fortification of BAS 800 H, M800H11, and M800H35 was prepared by combining 1 mL of each stock solution into a 100 mL volumetric flask, and then diluting to the mark with methanol. A set of standard mixed-working solutions (1–500 ␮g/L) were prepared from the stock solutions through serial dilution with methanol. All of the solutions were stored at 4⬚C in the dark and the mixed-working solution was made fresh every week, whereas the stock solutions of 1000 mg/L could be used for three months.

2.1 Chemicals and materials

2.3 Instrumentation and chromatographic conditions

Saflufenacil (99.6% purity), M800H11 (98.0% purity), and M800H35 (94.8% purity) standards (Fig. 1), and the formulations (69.7% saflufenacil water-dispersible granule) were supplied from BASF. Acetonitrile and methanol of HPLC grade were purchased from Sigma–Aldrich (USA). Ultrapure water

Centrifugation was performed in an Anke DL-5-B centrifugation (Shanghai Flying Pigeon, China). Mechanical shaking extraction was performed by using a ZHWY-2012C constant temperature incubator shaker (Shanghai Zhicheng Analysis Instrument Manufacturing, China). The rotary evaporator

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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J. Sep. Sci. 2014, 37, 658–664

M. Lan et al.

Table 1. Physical and chemical properties of five types of soil samples

Sample

Resource

Texture class

pH

Sand (%)

Silt (%)

Clay (%)

OMCa) (%)

CECb) (cmol/kg)

S1 S2 S3 S4 S5

Harbin Wuxi Jiaxing Hangzhou Quzhou

Black soil Paddy soil Moisture soil Powder soil Red soil

6.8 6.18 6.52 6.8 4.2

39.7 10.1 7.6 21.5 44.8

43.9 80.7 69.3 71.1 44

16.4 9.2 23.1 7.4 11.2

2.94 2.95 5.02 3.1 0.93

22.2 14.8 21.3 10.6 21.4

a) Organic matter content. b) Cation exchange capacity. Table 2. Molecular weight, retention time, and optimized MS/MS parameters for BAS 800H, M800H11, and M800H35

Analyte

MW

RT (min)

Q1 mass (Da)

Q3 mass (Da)

DP (V)

EP (V)

CE (V)

CXP (V)

BAS 800H

500.9

2.83

499

M800H11

472.8

2.6

470.9

M800H35

352.8

2.26

351

347.8 328.0 a) 332 350.0 a) 308 187 138.0 a)

−10 −10 −20 −20 −18.7 −18.7 −18.7

−3 −3 −5.6 −5.6 −3.1 −3.1 −3.1

−40 −50 −26.68 −26.68 −23.8 −34.69 −24

−10 −18.59 −18.79 −18.79 −16.36 −10 −22

MW, molecular weight; RT, retention time; DP, declustering potential; EP, entrance potential, CE, collision energy; CXP, cell exit potential. a) Q3 used for quantification.

RE-2000 was purchased from Yarong Biochemical Instrument (Shanghai, China). Chromatographic analyses were conducted by an Acquity Ultra Performance LC (Waters, Milford, MA, USA). Three R HSS T3 analytes were separated on an ACQUITY UPLC column (2.1 × 100 mm, with a 1.8 ␮m particle size; Waters). The mobile phases consisted of a mobile phase A (0.1% formic acid in ultrapure water) and B (methanol) with a flow rate of 0.2 mL/min, and the gradient elution program was as follows: gradient starting from 40% methanol at 0 min; increased to 85% methanol at 0–1 min, held for 3 min; then reversed to 40% methanol, held for 1 min before the next injection. The injection volume was 10 ␮L, and the column temperature was maintained at 30⬚C. Mass spectrometric detection was carried out by using an Applied Biosystems Triple Quad 5500 (ESI–MS/MS; Foster, CA, USA) in electrospray negative-ion multiple reaction modes. Compound-dependent MS parameters (including declustering potential, entrance potential, collision energy (CE), and collision cell exit potential as well as compound multiple reaction modes transitions were optimized by the direct infusion of individual standard solution of each analyte at 50 ␮g/L. A summary of these parameters is listed in Table 2. Source-dependent parameters were determined by flow injection analysis and were as follows: ion spray voltage, −4000 V; atomization air pressure (GS1), 20 psi; auxiliary gas (GS2), 20 psi; curtain gas (CUR), 20 psi; ion source temperature (TEM), 450⬚C; collision-activated dissociation (CAD), 4 V. Instrument control and data acquisition and evaluation were performed with the AB Sciex Analyst 1.6 software (Applied Biosystems).

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.4 Sample pretreatment Soil sample was prepared with a modified QuEChERS treatment as follows. A previously fully homogenized sample (20 g) was weighed in a plastic centrifuge bottle (250 mL). Then deionized water (12 mL) was added in order to make pores in the sample more accessible to the extraction solvent and to homogenize the water content in different soil samples. After 20 min, an accurate volume of 50 mL acetonitrile was added and the mixture was shaken on a shaker at 180 rpm for 60 min (25⬚C). After centrifugation (3000 rpm, 5 min), the supernatant was transferred to the mixing cylinder with stopper preloaded with 2 g NaCl and 8 g MgSO4 . Then the mixtures were shaken tempestuously for 3 min and kept still for stratification. Subsequently, an accurate volume of 25 mL clarified supernatant (acetonitrile) was evaporated to dryness at 45⬚C. The residue was redissolved with 10 mL methanol, then filtered through a 0.22 ␮m one-off membrane and transferred into autosampler vial for UPLC–MS/MS analysis.

3 Results and discussion 3.1 Optimization of UPLC–MS/MS Different from the BASF Method D0603/02, in this study, BAS 800H, M800H11, and M800H35 were all detected in the negative-ion mode, which had higher intensity than the positive-ion mode. The identification of the precursor ions

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J. Sep. Sci. 2014, 37, 658–664

and optimum ionization conditions was performed in the full scan mode. As a result, m/z 499.0, 470.9, and 351.0 were defined as the precursor ions of BAS 800H, M800H11, and M800H35, respectively. After that, the identification of fragment ions was performed in the product ion scan mode. Observing the full scan MS/MS spectra under different values of collision energies at a concentration of 50 ␮g/L (Supporting Information Fig. S1), m/z 347.8 and 328.0 of m/z 499.0; m/z 332.0 and 350.0 of m/z 470.9; m/z 308.0, 187.0 and 138.0 of m/z 351.0 were selected as the product ions in the present work. Subsequently, further optimization of the CE and cell exit potential for each product ion was carried out to achieve the highest response, and the optimal results are summarized in Table 2. It is known that the additives of mobile phases can affect the LC resolution and MS response of chemicals. In the current work, we study the role by adding ammonia and formic acid at various concentrations in the mobile phases. As shown in Supporting Information Fig. S2), the additives of ammonia and formic acid all increased the MS response of M800H35. Particularly, the addition of 0.1% formic acid not only enhanced the sensitivities of the M800H11 and BAS 800H but also achieved the complete separation with perfect peak shape. As a result, the addition of 0.1% formic acid in the elution was used in the study.

3.2 Optimization of sample pretreatment For soil sample preparation, our goal was to adapt the liquid– liquid extraction method of our previous study, which was developed for the analysis of BAS 800H in soil samples. The liquid–liquid extraction was performed with paddy soil: 20 g homogenized soil, spiked with mixed-standard working solutions to obtain the final fortified concentration 100 ␮g/kg, were placed in a 250 mL centrifuge tube. The tube was hermetically sealed and left overnight to let the analytes penetrate into the samples matrix. After soaking with 20 mL deionized water, 50 mL acetone, acetonitrile, and methanol were added individually to extract the analytes and mixture was agitated on a shaker for 60 min. After centrifugation (3000 rpm, 5 min), the supernatant mixtures were concentrated on the RE-2000 rotary evaporator. Residues were then partitioned with dichloromethane twice (50 + 40 mL). The organic phase was evaporated to dryness and redissolved with methanol for UPLC–MS/MS. From the data of fortified recoveries (Table 3), we learned that good recoveries were gained for BAS 800H with the three extraction solvents (82.3–108.1%) and for M800H11 with acetonitrile (87.8%). However, the recoveries of M800H35 obtained were all 100% indicated enhancement, whereas those

Simultaneous determination of saflufenacil and its two metabolites in soil samples by ultra-performance liquid chromatography with tandem mass spectrometry.

Saflufenacil is a new protoporphyrinogen-IX-oxidase inhibitor herbicide. When used, it can enter the soil and has a high risk to reach and contaminate...
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