Multiresidue Method for Determination of 183 Pesticide Residues in

Subscriber access provided by Penn State | University Libraries

Article

A multi-residue method for determination of 183 pesticide residues in leeks by rapid multiplug filtration cleanup and gas chromatography-tandem mass spectrometry Nan Zou, Yongtao Han, Yanjie Li, Yuhong Qin, Kejia Gu, Jingru Zhang, Canping Pan, and Xuesheng Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05132 • Publication Date (Web): 11 Dec 2015 Downloaded from http://pubs.acs.org on December 12, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

Journal of Agricultural and Food Chemistry

1

A multi-residue method for determination of 183 pesticide residues in

2

leeks by rapid multiplug filtration cleanup and gas

3

chromatography-tandem mass spectrometry

4

Nan Zou1, Yongtao Han1, Yanjie Li1, Yuhong Qin1, Kejia Gu1, Jingru Zhang, Canping

5

Pan1, *, Xuesheng Li2

6

1

7

University, Beijing, 100193, People’s Republic of China

8 9

Department of Applied Chemistry, College of Science, China Agricultural

2

Institute of Pesticide & Environmental Toxicology, Guangxi University, Nanning,

10

530005, China

11

*(Author for correspondence: e-mail: [email protected]; Fax: +86 10 62733620;

12

Tel: +86 10 62731978)

13

1

ACS Paragon Plus Environment


14

Abstract:

15

This study reports the development of a novel multiplug filtration cleanup (m-PFC)

16

procedure for analysis of pesticide residues in leek samples followed by gas

17

chromatography-tandem mass spectrometry (GC-MS/MS) detection. The leek

18

samples were initially purified following the dispersive solid phase extraction (d-SPE)

19

with different sorbents to find out the most suitable sorbent materials proportioning,

20

and then m-PFC method was carried out by applying the streamlined procedure with

21

syringes. Average recoveries of most pesticides were in the range from 70.2 to 126.0%

22

with the RSD < 20% with m-PFC process. The limits of detection (LODs) were

23

0.03-3.3 µg kg-1. The limits of quantitation (LOQs) were 0.1-10 µg kg-1. The m-PFC

24

process is convenient, time-saving, taking just a few seconds per sample. Finally, the

25

developed method was successfully applied to the determination of pesticide residues

26

in market samples. 35 pesticides were detected in 29 samples, with values ranged

27

from 2.0 to 9353.1 µg kg-1.

28

Keywords: d-SPE, leek, MWCNTs, m-PFC, pesticide residue analysis

29

2


Page 2 of 36

Page 3 of 36


30

Introduction

31

Leek (Allium porrum L.) belongs to the Allium genus which belongs on its turn to

32

the Liliaceae family.1 Leek is predominantly a Eurocentric crop with significant

33

cultivation in Turkey (10000 ha), France (6200 ha), Belgium (4700 ha) and Poland

34

(4800 ha). In 2008, 33,800 ha were dedicated to leek cultivation in Europe. This

35

coverage equates to ~0.72 M tonnes.2 Alliums with an average annual production

36

value of US$13.87 billion ranked 5th in 25 human food crops or crop groups in China

37

over the period 2002–2011.3 Leeks are one of the important vegetables in China,

38

which are delicious and nutritious.4 They are rich in carbohydrate, protein, vitamin,

39

cellulose and mineral. Moreover, they are reported to have the similar medicinal

40

values as garlic, which show some anticancer activity.5 However, the residues of

41

pesticide in leeks are quite serious.4 Leeks are known as complex matrices in pesticide

42

analysis.6 They contain a large amount of pigments and sulfur-containing compounds

43

that exhibit both partition behaviour and chromatographic characteristics similar to

44

some pesticides, and which may bring serious matrix effects and significant

45

interferences in MS analysis.6, 7

46

Due to the interferences in these troublesome matrices, much effort has been put

47

into extraction and clean-up processes for pesticides analysis: solid phase extraction,8

48

d-SPE,

49

permeation chromatography,14 matrix solid-phase dispersion15 and so on. Some of

50

these pretreatment methods demand a large amount of organic solvent and several

51

clean-up procedures, which may be laborious, time-consuming and expensive.

4, 7, 9-12

microwave pretreatment,4 solid phase microextraction,13 gel

3



Page 4 of 36

52

Therefore, new samples preparation methods for leek samples should be studied and

53

developed.

54

Carbon nanotubes (CNTs) are novel and interesting carbonaceous materials with

55

an ultra-high specific surface area first reported by Iijiama16 in 1991, which were

56

classified as single-walled carbon nanotubes (SWCNTs) and multi-walled carbon

57

nanotubes (MWCNTs). MWCNTs have been used as sorbent materials in purification

58

of inorganic elements, pollutants, and veterinary drugs.17-19 The QuEChERS method

59

(quick, easy, cheap, effective, rugged, and safe) has been widely used as pesticide

60

multi-residue methods in vegetables, fruits and many other matrices since it was

61

introduced by Anastassiades and Lehotay et al. in 2003.11, 12 Its main advantage is

62

comprehensiveness, being useful for the analysis of various pesticides with a wide

63

range of polarity and volatility. In our previous work, MWCNTs were used as d-SPE

64

materials in pesticide multiresidue analysis with the QuEChERS.20 They also were

65

mixed with PSA and GCB for cleanup of tea samples.21

66

In method development, any saving measures that can be achieved, such as saving

67

labor, time and/or cost, often carry out significant benefits when the measures can be

68

implemented in routine operations.22 A more simple and effective way meaning

69

m-PFC method was initially introduced by our research group

70

and elution steps. In m-PFC, MWCNTs and other sorbent mixtures were used as

71

solid-phase sorbents, which were packed in syringe tubes. The m-PFC process was

72

carried out by applying the streamlined procedure with the packed syringe. The

73

m-PFC method is shown to be very rapid, taking just a few seconds to perform and 4


23-25

without leaching

Page 5 of 36


74

eliminating the vortex and centrifugation steps without any solvent evaporation in

75

purification process. This method saves several minutes of time and labor per sample,

76

and can eliminate the need for a (mini-) centrifuge.

77

To evaluate this approach in practice, we chose to apply it in a multiresidue

78

monitoring method that serves to analyze a wide range of pesticides in leek samples.

79

In our work presented here, the leek samples were initially purified following d-SPE

80

cleanup method with different sorbents, to find out the most suitable sorbent materials

81

proportioning. Based on optimization results of d-SPE method, leek extracts were

82

developed using m-PFC followed by GC-MS/MS analysis.

83

Experimental

84

Standards, Reagents, and Materials. Pesticide standards used in this work

85

(Table S1) were provided by the Institute of the Control of Agrochemicals, Ministry

86

of Agriculture, Peoples’ Republic of China. The purities of the standard pesticides

87

were from 95 to 99%. Working standard mixture containing 10 mg L-1 of each

88

pesticide was prepared in acetone and stored at −20 °C. PSA, C18 and GCB were

89

obtained from Tianjin Bonna-Agela Technologies Co., Ltd. (China). HPLC grade

90

acetonitrile and acetone were obtained from Fisher Chemicals (Fair Lawn, NJ, USA).

91

Analytical reagent grade sodium chloride (NaCl) and anhydrous magnesium sulfate

92

(anh. MgSO4) were obtained from Sinopharm Chemical Reagent (Beijing, China).

93

MWCNTs with average external diameters of 5-10 nm were provided by National

94

Center for Nanoscience and Technology, and with average external diameters of 5



Page 6 of 36

95

10-20 nm were provided by Tianjin Bonna-Agela Technologies Co., Ltd. (China).

96

m-PFC syringes were packed with 8 mg MWCNTs, 10 mg PSA, 10 mg C18 and 150

97

mg anh. MgSO4 with assistance from Tianjin Bonna-Agela Technologies.

98

GC-MS/MS Analytical Conditions. The analysis was carried out on Thermo

99

Quantum GC with Trace GC ultra and Quantum triple quadrupole mass spectrometer

100

(mass range from m/z 10–3000). Samples were injected with AS 3000 auto-sampler

101

into a split/splitless injector. The capillary column was 30 m

102

TR-5MS and with 0.25 µm film thickness (Thermo Fisher Scientific, USA). At 0.75

103

min, split mode was switched on with the split flow of 60 mL min-1. At 2 min, gas

104

saver was turned on with the flow of 20 mL min-1. The column temperature was

105

initially at 80 °C (hold for 1 min), increased to 150 °C at a rate of 30 °C min-1 and

106

then to 210 °C at a rate of 3 °C min-1, and finally increased to 290 °C at a rate of

107

10 °C min-1, holding for 12 min. The temperature of the injector port was 260 °C, and

108

a volume of 1 µL was injected in splitless mode. The total running time was 43 min.

109

Helium at a constant flow rate of 1.2 mL min-1 was used as carrier gas; argon at a

110

pressure in the range of 1.0 mTorr was used as collision gas. The QqQ mass

111

spectrometer was operated in EI at 70 eV in the selected reaction monitoring (SRM)

112

mode. The specific MS/MS conditions were shown in Table S1.

╳

0.25 mm i.d.,

113

Sample Preparation. The leek samples obtained from a local organic farm. An

114

approximately 1000 g portion of sample was weighed and homogenized at 10000 rpm

115

for 1 min. For recovery determination, the homogenized samples (10.0 ± 0.1 g) were

116

spiked by the addition of the working standard solution at two concentration levels of 6


Page 7 of 36


117

10 and 100 µg kg-1 and vortex mixed into the samples. The spiked samples were set

118

aside for at least 20 min before their extraction.

119

An amount (10.0 ± 0.1 g) of leek samples was weighed into a 50 mL centrifuge

120

tube, followed by the addition of 10 mL of acetonitrile. Then the centrifuge tube was

121

shaken by the vortex for 1 min. 3 g of NaCl was added, and the tube was shaken

122

vigorously for 1 min before centrifugation at 3800 rpm for 5 min. The supernatant

123

was used for further d-SPE and m-PFC process.

124

d-SPE Procedures.1 mL of the supernatant was introduced into a 2.0 mL

125

micro-centrifuge tube containing sorbent mixtures. Then the tube was shaken

126

vigorously for 1 min and centrifuged for 1 min at 10000 rpm with a microcentrifuge.

127

Finally the acetonitrile layer was filtered through a 0.22 µm filter membrane and

128

introduced into an autosampler vial for GC–MS/MS analysis.

129 130

Different combination and proportion sets of sorbents were evaluated for d-SPE cleanup in experiments:

131

Combination (A): 8 mg MWCNTs + 150 mg anh. MgSO4;

132

Combination (B): 5 mg MWCNTs + 30 mg PSA + 30 mg C18 + 150 mg anh.

133 134 135 136 137 138

MgSO4; Combination (C): 5 mg MWCNTs + 20 mg PSA + 20 mg C18 + 5 mg GCB +150 mg anh. MgSO4; Combination (D): 8 mg MWCNTs + 10 mg PSA + 10 mg C18 + 150 mg anh. MgSO4; Combination (G): 15 mg MWCNTs with external diameters 10-20 nm + 150 mg 7



139

anh. MgSO4;

140

Combination (H): 80 mg PSA + 150 mg anh. MgSO4.

141

The fortified recoveries and GC-MS/MS total ion current (TIC) chromatograms of

142

the blank leek samples were compared. The results showed that considerably

143

satisfactory recoveries and fewer interferences were obtained when combination D

144

was applied in samples purification, and combination D was used in the final sorbents

145

combination and proportion for m-PFC procedures.

146

m-PFC Procedures. Schematic diagram of m-PFC syringe and cleanup procedure

147

diagram were shown in Figure 1. m-PFC syringe structure contains two PE sieve plate

148

and sorbent materials. The sorbent materials used in this research contain 8 mg

149

MWCNTs, 10 mg PSA, 10 mg C18 and 150 mg anh. MgSO4. A 1 mL aliquot of the

150

initial extract was used for purification, which was introduced into a m-PFC syringe

151

tube. Then push the piston, all of the extract filtered through the sorbent at the speed

152

of 1 drop s-1. At the same time, the acetonitrile layer was filtered through a 0.22 µm

153

filter membrane and introduced into an autosampler vial for GC–MS/MS analysis.

154

Method Performances. The following parameters were determined during

155

validation of the analytical method: linearity, LOQs, LODs, accuracy and precision.

156

External standard method was used for quantitative. Linearity was studied using

157

matrix-matched calibration by analyzing leek samples. The recovery and

158

reproducibility experiments were carried out for each sample in five replicates at two

159

fortification levels (10 and 100 µgkg−1). LODs and LOQs of the proposed method

160

were calculated as the concentration giving signal-to-noise ratios of 3 (S/N = 3) and 8


Page 8 of 36

Page 9 of 36


161

10 (S/N = 10), respectively.

162

Results and Discussion

163

Optimization of Triple-quadruple MS/MS Conditions. Optimization of

164

triple-quadruple MS/MS was a rather demanding task because specific experimental

165

conditions were required for each target compound to conduct analyses. 26

166

The MS/MS detection method was first optimized with individual injections in

167

full scan mode of each target compound in order to obtain their retention times and to

168

select the appropriate precursor ions, generally selecting the ion with higher m/z ratio

169

(increase in selectivity) and abundance (increase in sensitivity).

170

precursor ion was carried out to try to choose the product ions, and optimization of

171

collision energies (from 5 to 40 eV) for best response. The aforementioned criterion

172

was also applied to choose the suitable product ions (higher m/z ratio and abundance).

173

The collision energy was optimized for two selective ion transitions for every

174

compound. The characteristic ion transition and collision energy for each compound

175

were listed in Table S1.

27

Selection of the

176

Optimization of d-SPE Condition. The goal of sample preparation was to test

177

whether the mixtures of MWCNTs, PSA, C18 and GCB had superior ability to remove

178

selectively interfering substances from acetonitrile extracts of the troublesome leek

179

matrices. Traditionally, PSA is a weak anion exchanger which can remove various

180

organic acids, fatty acids and some sugars, C18 can absorb weak polarity substances

181

like lipids, and GCB is used for pigment and sterols removal.28, 29 MWCNTs have 9



182

been applied as effective clean-up sorbents for removing fatty acids, pigments and

183

other matrix compounds in pesticide multiresidue analysis in our previous work.20, 21,

184

23

185

and GCB were investigated in the d-SPE procedure. The experiments were performed

186

using 1 mL of the acetonitrile extracts that was placed into 2.0 mL micro-centrifuge

187

tubes containing 150 mg MgSO4 and different amounts of sorbent mixtures

188

(combination A, B, C, D, G and H) at the spiked level of 100 µg kg-1.

To obtain the best cleanup performance, different amounts of MWCNTs, PSA, C18

189

As shown in Figure 2, the leek samples purified by MWCNTs with external

190

diameters 5-10 nm (Figure 2A, 2B, 2C and 2D) appeared light green in color, and the

191

other samples purified by 15 mg MWCNTs with external diameters 10-20 nm ( Figure

192

1G) and 80 mg PSA only ( Figure 2H) had a deeper color. Therefore, MWCNTs with

193

external diameters 5-10 nm were selected for further optimization combination with

194

other adsorbents.

195

For four methods of Figure 2(A-D), it is difficult to distinguish from the color.

196

Figure 3 (A-D) shows GC-MS/MS total ion current (TIC) chromatograms of the blank

197

leek samples after d-SPE process (corresponding combination A-D and Figure

198

2(A-D)). The blank samples chromatographic response intensity (Y-axis) of

199

combinations (A-C) method could reach 106, however, the response of combination

200

(D) method was only 105. Combination D had superior clean-up performance, and we

201

observed fewer interferences.

202

Relevant histograms about recoveries (I) and RSD (II) by four different cleanup

203

combinations with GC−MS/MS are shown in Figure 4. Considerably satisfactory 10


Page 10 of 36

Page 11 of 36


204

recoveries were obtained when combination D was applied in samples purification.

205

Recoveries of 172 of 183 pesticides were in the range of 70-110% at a spiked level of

206

100 µg kg-1 in leek samples (Table S2). When combination A or combination B was

207

applied in samples purification, recoveries of 140 or 170 of 183 pesticides were in the

208

range of 70-110% (Table S2). However, recoveries of combination C were lower than

209

those obtained in combination A, B and D, and 28 pesticides recoveries were less than

210

70%. The results were consistent in some of the literature, that using plenty of GCB

211

can sufficiently remove the chlorophyll, but it can also resulted in loss of target

212

compounds.21,

213

essentially identical. Precision of the recovery results showed that optimization results

214

of four different cleanup combinations were reliable.

30, 31

RSDs of four different cleanup combinations recoveries were

215

Consequently, 8 mg MWCNTs + 10 mg PSA + 10 mg C18 + 150 mg anh. MgSO4

216

were used as the optimum amounts for the d-SPE cleanup and for further m-PFC

217

process studies since acceptable recoveries and good cleanup performances were

218

obtained at this combination.

219

Optimization of m-PFC Process. The m-PFC process was performed to remove

220

the sample matrix in the extract prior to chromatographic analysis. The m-PFC

221

method was performed using a 5.0 mL syringe where the sample matrix compounds

222

in the acetonitrile extraction interact with the mixture sorbents. In our previous work,

223

the cycles of pulling and pushing during the m-PFC procedure were optimized to

224

obtain the satisfactory recovery and cleanup performance. However, we found that

225

some impurities could be desorbed when the extract was through adsorbents by 11



226

pulling and pushing the piston. From the Figure S1, the final extract looked much

227

transparent in color (Figure S1 II) with the process of the extract through adsorbents

228

from top to bottom one time, and the cleanup performance was better than the process

229

of pulling and pushing two times(Figure S1 III), and the recoveries of fortified

230

samples could meet the requirements. The cycle times of extract through adsorbents

231

were also tested, but there was no significant difference for the cleanup performance

232

between 1 time and 2 times or more times. In addition, the cleanup performances were

233

far from satisfactory if the speed of extract through adsorbents was too fast, because

234

extract couldn’t interact with sorbents well. So, in this study, 1 mL of the extracts

235

filtered through the sorbents from top to bottom one time at the speed of 1 drop s-1 by

236

pushing the piston.

237

Figure 3E showed GC-MS/MS chromatogram of the blank leek sample with

238

cleanup procedures using m-PFC method. The blank sample chromatographic

239

response intensity (Y-axis) of m-PFC method was 6.46×105. Compared with d-SPE

240

process, the least interference appeared in the chromatogram of m-PFC cleaning-up

241

samples. The result was consistent in our research group, that m-PFC method had

242

better cleanup performance with less interference than d-SPE process.25 Finally, 8 mg

243

MWCNTs, 10 mg PSA, 10 mg C18 mixed with 150 mg MgSO4 (1 mL extract) were

244

used for the m-PFC process in the further method validation because acceptable

245

recoveries and good cleanup performances were both obtained for most compounds.

246

Method Validation. One of the main problems in trace analysis of complex

247

matrices is the matrix effect enhancement. In our work, matrix-matched standard 12


Page 12 of 36

Page 13 of 36


248

calibration was used for the more accurate results in order to avoid matrix effect.

249

The calibration curves of all pesticides were from 10 to 500 µg L-1 by the

250

calculation of a five-point plot (10, 50, 100, 200, 500 µg L-1). As shown in Table 1,

251

good linearity was found for all pesticides, and regression coefficient (R2) ranged

252

from 0.9920 to 1.0000.

253

In this study, LODs and LOQs of the proposed method were calculated as the

254

concentration giving signal-to-noise ratios of 3 (S/N = 3) and 10 (S/N = 10),

255

respectively, which was analyzed by the Xcalibur Data System and Microsoft Excel.

256

As shown in Table 1, the LODs for these pesticides studied ranged from 0.03 to 3.3

257

µg kg-1 and the LOQs ranged from 0.1 to 10 µg kg-1 in the leek matrices.

258

To study the accuracy and precision of the proposed m-PFC method using

259

GC-MS/MS analysis, leek matrices were spiked with standard pesticides at a low

260

level (10 µg kg−1) and a high level (100 µg kg−1). The results of the average recoveries

261

and relative standard deviations (RSDs) of pesticides spiked at the two levels and

262

replicated five times were shown in Table 1. Average recoveries of most pesticides

263

were in the range from 70.2 to 126.0% with the RSDs from 1.1 to 19.5%. However,

264

low recoveries (

Multiresidue Method for Determination of 183 Pesticide Residues in

Recommend Documents