A multi-residue method for fast determination of pesticides in tea by ultra performance liquid chromatography–electrospray tandem mass spectrometry combined with modified QuEChERS sample preparation procedure

Food Chemistry 125 (2011) 1406–1411

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Analytical Methods

A multi-residue method for fast determination of pesticides in tea by ultra performance liquid chromatography–electrospray tandem mass spectrometry combined with modified QuEChERS sample preparation procedure Guoqiang Chen ⇑, Pengying Cao, Renjiang Liu Advanced Measurement & Data Modeling, Unilever R&D Centre, 66 Linxin Road, Linkong Economic Development Zone, Shanghai, 200335, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 13 July 2009 Received in revised form 3 November 2009 Accepted 1 October 2010

Keywords: Pesticide Multi-residue Tea UPLC/MS/MS QuEChERS SPE

a b s t r a c t A multi-residue method was developed for rapid determination of pesticide residues in tea by ultra performance liquid chromatography–electrospray tandem mass spectrometry (UPLC/MS/MS). The QuEChERS method was used for sample preparation. In order to minimise the matrix effects from tea, a solid phase extraction (SPE) cartridge layered with graphite carbon/aminopropylsilanized silica gel was applied as complementary to QuEChERS method. For accurate quantification, representative matrixmatched calibration curves were applied to compensate matrix effects. Limits of quantification varied with different pesticides but all can be measured at 0.01 mg kg 1 level in a 5 g tea sample except dichlorvos (0.02 mg kg 1). Recoveries ranged from 70% to 120% and relative standard deviation (RSD) met the European United Quality Control guideline. Efficiency and reliability of this method were investigated by the analysis of both fermented and unfermented Chinese tea samples. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Tea farming is vulnerable to a great multitude of pests, especially mites, leaf-eating beetles and caterpillars. Weeds and diseases can also be a problem. To minimise these problems, the most common practice in tea crop production is to use pesticides. However, similar to other raw agriculture commodities (RACs), unsafe pesticide residues in tea have been associated with neurological dysfunction and disease (Kamel & Hoppin, 2004). Consequently, determination of pesticide residues is at the forefront among preventive measures in public health safety. Furthermore, there are potential international trade barriers due to maximum residue limits (MRLs) in tea established by most countries and several international organisations, e.g. United States Environmental Protection Agency, Food and Agriculture organisation of the United Nations, and European Union, etc. The existing MRLs for some pesticides in many RACs including tea are periodically revised and become more strict and comprehensive. Moreover, there is a trend for regulators to temporarily reduce the MRLs if new data unexpectedly indicates certain risks to human or animal health. For reliable identification and confirmation of the target pesticide residues at trace levels, food analytical laboratories are increasingly interested in finding new analytical methods with shorter analysis time

⇑ Corresponding author. Tel.: +86 21 22125781; fax: +86 21 22125051. E-mail address: [email protected] (G. Chen). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.10.017

and higher sample throughput. (Frenich, Gonzalez-Rodrıguez, Arrebola, & Vidal, 2005). Gas chromatography (GC) seems to be the technical choice for analysis of pesticides in food commodities. However, many pesticides which are thermally unstable or non-volatile such as carbamates and benzimidazoles are difficult to be analysed with GC. High performance liquid chromatography (HPLC) offers an alternative and powerful tool for the determination of such compounds, as complementary to GC method (Gervais, Brosillon, Laplanche, & Helen, 2008). UPLC with columns packed with small particles (1.7 lm) and high linear velocities (accompanied by maximum back pressures up to 15,000 w) has been shown to give superior chromatographic resolution, reduce analysis time, consume less solvent and increase sensitivity (Gervais et al., 2008; Leandro, Hancock, Fussell, & Keely, 2006, 2007; Nguyen, Guillarme, Rudaz, & Veuthey, 2006; Picó, Farré, Soler, & Barceló, 2007). For pesticide residue analysis in tea, there are published methods available in literature including official and unofficial methods (China CIQ SN/T 1747-2006; Germany DFG method S 19, 1999; Hu, Song, Xie, & Shao, 2008; Huang, Li, Chen, & Yao, 2007; Ji, Deng, Zhang, Wu, & Zhang, 2007; Schurek, Portolés, Hajslova, Riddellova, & Hernández, 2008). Traditional analytical methods like solvent extraction cannot provide effective solution to minimising matrix effects (Huang et al., 2007). SPE is a common choice for clean-up (Huang et al., 2007). Japanese official method for residual compositional substances of agricultural chemicals, feed additives and veterinary drugs in food applies SPE cartridge packed with graphite

G. Chen et al. / Food Chemistry 125 (2011) 1406–1411

carbon/aminopropylsilanized silica gel to clean-up tea matrix (Department of Food Safety Ministry of Health, Labour and Welfare, Japan, 2006). Gel permeation chromatography (GPC) is also an effective clean-up method which has been widely applied (Kerkdijk, Mol, & Nagel, 2007). There is a trend to shift from labour intensive traditional methods to fast and simple approaches, such as QuEChERS (Anastassiades, Lehotay, Stajnbaher, & Schenck, 2003), which symbolizes a new milestone for pesticide residue analysis. Some laboratories (Lesueur, Knittl, Gartner, Mentler, & Fuerhacker, 2008; Payá et al., 2007) have applied this clean-up method for fruits and vegetables. Pesticide multi-residue methods applying gas chromatography–mass spectrometry (GC/MS/MS or GC/MS) and liquid chromatography–tandem mass spectrometry (LC/MS/MS) are increasingly popular, which enable us to analyse more pesticides in one injection and with higher sensitivity. For the confirmation of legal substances and banned substances, a minimum of three and four identification points, respectively, are required according to EU Decision (2002/657/EC, 2002). UPLC coupled with triple quadruple MS/MS can improve the speed of analysis and provide higher sensitivity and accuracy. To the best of our knowledge, until now, there has been no literature or publications reporting about multi-residue methods for the determination of pesticides in tea by UPLC/MS/MS. The purpose of this paper is to develop a multi-residue method based on the application of UPLC/MS/MS combined with modified QuEChERS sample preparation procedure for rapid determination of 65 selected pesticide residues in tea.

1407

and data acquisition. The capillary voltage was 3.50 kV and the source temperature was 100 °C. The desolvation gas temperature was set at 350 °C with nitrogen flow rate of 300 L h 1. The collision gas (argon) pressure was approximately set at 3  10 3 mbar. The multiple reaction monitoring (MRM) mode was operated for each pesticide. All the parameters for MRM transitions, cone voltage and collision energy were optimised in order to obtain highest sensitivity and resolution (Table 1). 2.3. Sample preparation For both unfermented and fermented tea, weigh about 50 g and comminute in a small dis-integrator for 1 min. Transfer 5 g comminuted sample to a 50 mL centrifugal tube. Add 10 mL H2O and 10 mL acetonitrile (containing 1% acetic acid), vortex for 3 min and then set for 1 h. Add 1.5 g anhydrous CH3COONa and 4 g anhydrous MgSO4, vortex for 1 min. Cool the tube in an ice-water bath immediately, for 5 min. Centrifuge for 5 min at 5000 r min 1. The samples were then subjected to SPE clean-up. SPE column was conditioned with 10 mL acetonitrile/toluene (3:1, 1% acetic acid). Transfer 1 mL extracted solution to the column. Elute the column with 20 mL Acetonitrile/toluene (3:1, 1% acetic acid). Concentrate the effluent to 1 mL or less by evaporating with weak nitrogen stream at 40 °C. The residue was reconstituted in 1 mL acetonitrile (1% acetic acid) and filtered with a 0.2 lm organic filter (Millipore), ready for injection into UPLC/MS/MS. 2.4. Method performance

2. Experimental 2.1. Reagents, chemicals and materials Pesticide reference standards, all 95% or higher purity, were obtained from Dr. Ehrenstorfer (Augsburg, Germany), Chemservice (West Chester, PA, USA) and Accustandard (New Haven, CT, USA). Stock solutions of mixture pesticides were prepared in acetonitrile or acetone stored in freezer ( 18 °C). The working solutions should be prepared daily. All chemicals used in the experiment were analytical grade or above: HPLC grade acetonitrile, HPLC grade formic acid and acetic acid. A.R. anhydrous CH3COONa and anhydrous MgSO4 were baked at 650 °C for 3 h to remove phthalates and any residual water. Graphite carbon/aminopropylsilanized silica gel layered SPE cartridge (Sep-Pak Carbon NH2, 6 cc) was purchased from Waters Corporation (P.N. 186003369). Analytically confirmed pesticides-free green tea from Shiru Tea Company in Guangxi province and black tea from Unilever UK were used as blank samples for matrix-matched calibration and recovery evaluations. 2.2. Apparatus Waters ACQUITY UPLC System (Waters, UK) was employed. An AQUITY UPLC BEH Shield RP18, 2.1 mm (I.D.)  150 mm, 1.7 lm column (Waters, Ireland) was applied in this method. The mobile phase consisted of 0.02% formic acid in acetonitrile (A) and 0.02% formic acid in water (B). Elution was performed in the gradient mode [time 0 min, 10% A; 12 min, 98% A; 12.5 min, 10% A] and total analysis time of 18 min. The flow rate was 0.3 mL min 1 and injection volume was 2 lL. The temperatures of column and sample room were set at 30 and 8 °C, respectively. Waters Quattro Micro API mass spectrometer (Waters, UK) equipped with electrospray source was used for all experiments. MassLynx software (version 4.1) was used for instrument control

The sensitivity and precision of the method were evaluated by spiked blank tea samples. Recoveries and RSD were determined for five replicates at two concentration levels (0.050 and 0.010 mg kg 1). The accuracy of this method was evaluated externally by participating in a 31-laboratories-proficiency-test for pesticide residue analysis in tea which was organised by FAPAS (http://www.fapas.com) in 2008. All the target compounds (Acetamiprid, Bifenthrin, Cypermethrin, DDT [four homologues], Ethion, Fenpropathrin, Fenvalerate, lamda-Cyhalothrin, Propargite, S-421) covered by the proficiency test were properly identified and the respective z-score values obtained were satisfactory (|z| < 2).

3. Results and discussion 3.1. Optimisation of UPLC/MS/MS conditions Application of UPLC in this method provides superior chromatographic resolution, shorter analysis time and higher sensitivity. The total analytical time for instrumentation was only 18 min including 6 min for column equilibrium. At the initial developmental stage, 5 mM ammonium acetate was used in LC mobile phase. However, the UPLC column was very easy to be jammed when ammonium acetate buffer was used. It was proven that 0.02% formic acid provided the same sensitivity and resolution as 5 mM ammonium acetate. Formic acid was then selected as the replacement for acetic ammonium. Some pesticides including fenxoycarb, indoxacarb, clethodim or flufenoxuron showed poor peak shapes when they were dissolved into the initial gradient of mobile phase (acetonitrile/H2O = 1:9). It could be explained by their poor solubility in water. For other pesticides, there were no differences in sensitivity when acetonitrile/ H2O (1:9 and 5:5) or pure acetonitrile was used as solvent. Consequently, the residue was reconstituted in 1 mL acetonitrile (1% acetic acid) for better peak formation and sensitivity.

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G. Chen et al. / Food Chemistry 125 (2011) 1406–1411

Table 1 Parameters for 65 pesticide residue analysis by UPLC/MS/MS. Name

Rt (min)

MRM transitiona

Cone (v)

CE (eV)

192:1>160:2 192:1>132:1 189:3>102:1 189:3>144:2 142:1>94:1 142:1>112:1 184:1>143:1 184:1>125:1 214:0>155:1 214:0>183:2 223:1>148:1 223:1>166:2 224:1>127:0 224:1>193:1 239:2>72 239:2>182:3 162:9>105:9 162:9>87:8 225:1>127:1 225:1>67:0 203>175:1 203>145:1 220:1>163:1 220:1>107:0 256:1>175:1 256:1>209:1 268:1>76:0 268:1>161:2 230:0>199:1 230:0>125 223:1>126:1 223:1>55:7 297:0>159:1 297:0>69:0 191:2>75:0 191:2>116:0 300:0>127:1 300:0>174:2 411:0>192:2 411:0>213:2 191:1>116:0 191:1>88:8 253:0>126:1 253:0>98:9 298:3>144:3 298:3>100:2 221:1>109:0 221:1>127:1 200:2>107:2 200:2>168:3 388:0>167:3 388:0>141:3 355:2>88:1 355:2>108:1 210:1>111:0 210:1>168:2 224:1>167:1 224:1>109:0 222:1>165:2 222:1>123:1 402:0>167:2 402:0>141:2 202:2>145:2 202:2>127:2 207:2>72:0 207:2>134:1 226:1>107:1 226:1>164:2 376:0>308:1 376:0>266:1 216:2>174:2 216:2>146:2 215:1>99:0 215:1>126:1 226:2>93:1 226:2>108:2 411:0>149:2 411:0>182:2 233:1>72:0 233:1>160:1 296:1>70:0 296:1>99:1 273:9>194:0 273:9>78:9 226:1>169:2 226:1>121:1 321:2>119:2 321:2>144:2 404:1>372:3 404:1>329:2 318:0>132:1 318:0>160:1 208:2>109:1 208:2>107:1 306:1>201:3 306:1>106:1 493:1>264:2 493:1>238:3 294:1>197:3 294:1>155:2 308:2>70:1 308:2>151:1 369:8>127:0 369:8>214:9 284:1>252:1 284:1>176:3 302:1>97:1 302:1>55:0 314:1>162:2 314:1>119:1 302:2>116:1 302:2>256:2 346:0>132:0 346:0>160:1

25

18 30 16 12 11 11 7 17 15 10 8 6 16 8 18 16 10 8 18 20 16 14 10 23 20 16 10 16 9 20 20 16 19 17 20 3 24 10 18 26 3 12 23 39 18 31 9 9 23 27 25 20 12 11 15 14 9 18 12 20 14 19 13 25 15 24 15 8 10 16 16 19 27 15 30 24 18 19 14 24 10 11 30 26 10 17 19 14 12 28 13 7 20 26 12 30 22 26 13 20 18 21 29 34 15 26 21 33 15 33 11 14 15 6

311.0 > 158.1

30 13

LOQ (lg kg

1

)

Linearity/r

Recovery/%b 50 lg kg

Carbendazim

1.76

Propamocarb

1.78

Methamidophos

2.07

Acephate

2.31

Omethoate

2.56

Aldoxycarb

3.56

Monocrotophos

3.63

Pirimicarb

3.86

Methomyl

3.88

Mevinphos

4.80

Metamitron

4.84

Carbofuran-3-hydroxy

5.06

Imidacloprid

5.16

Thiofanox-sulfon

5.31

Dimethoate

5.33

Acetamiprid

5.49

Imazalil

5.70

Butocarboxim

5.88

Phosphamidon

5.97

Nicosulfuron

6.12

Aldicarb

6.21

Thiacloprid

6.23

Spiroxamine

6.36

Dichlorvos

6.67

Pyrimethanil

6.79

Thifensulfuron-methyl

6.83

Thiodicarb

7.01

Propoxur

7.13

Bendiocarb

7.21

Carbofuran

7.25

Triasulfuron

7.30

Carbaryl

7.73

Isoproturon

7.73

Ethiofencarb

7.84

Prochloraz

7.90

Atrazine

7.93

Monolinuron

7.98

Cyprodinil

8.03

Bensulfuron-methyl

8.16

Diuron

8.26

Triadimenol

8.37

Bromoxynil

8.59

Methiocarb

8.84

Iprovalicarb

8.94

Azoxystrobin

8.97

Azinphos-methyl

8.98

Promecarb

9.06

Buprofezin

9.09

Triflusulfuron-methyl

9.14

Triadimefon

9.15

Tebuconazole

9.31

Ioxynil

9.38

Metolachlor

9.47

Fenhexamid

9.57

Triazophos

9.60

Fenoxycarb

9.73

Azinphos-ethyl

9.80

Diflubenzuron Tebufenozid

9.82 10.00

Indoxacarb

10.76

Quizalofop-ethyl

10.94

Clethodim

11.13

353:2>133:2 353:2>297:3 528:1>150:2 528:1>218:2 373:0>299:2 373:0>271:2 360:2>164:1 360:2>206:3

25 21 15 20 23 17 25 15 17 28 25 22 10 11 23 30 10 24 22 14 25 28 25 35 20 15 14 18 20 25 15 20 15 17 30 19 38 18 21 14 38 17 15 19 14 17 17 25 21 28 35 20 31 21 20 16

24 28 19

14 19 7 25 23 18 18 19 18

1

RSD

10 lg kg

1

RSD

5

0.9918

85.06

19.2

109.57

5

0.9992

71.23

9.2

81.49

3.7

5

0.9959

80.86

7.5

61.86

23.9

10

0.9932

84.96

17.8

77.57

28.9

5

0.9979

76.73

9.8

78.49

11.4

5

0.9992

95.12

5.9

78.49

17.7

5

0.9998

78.79

6.1

70.94

8.3

5

0.9999

91.64

8.6

76.60

5.7

5

0.9997

70.64

10.3

81.91

20.1

5

0.9996

100.17

8.4

82.60

14.4

5

0.9977

78.55

18.2

78.49

30.2

5

0.9988

98.55

16.6

60.81

17.5

5

0.9956

84.77

15.4

73.70

10.2

5

0.9985

84.32

14.3

64.98

18.2

5

0.9997

88.42

11.5

62.17

15.1

5

0.9992

90.27

17.9

70.98

12.1

5

0.9993

80.41

5

0.9993

91.45

5

0.9994

87.49

5

0.9999

67.73

5

0.9968

5 5

6.4

9.1

64.34

45

66.18

12.4

9.9

82.01

15.5

6.7

72.25

15.1

85.95

11.3

122.67

17.6

0.9985

106.09

8.3

90.40

10.7

0.9999

83.12

7.7

74.73

20

0.9966

96.77

17.5

5

0.9988

89.64

3.5

106.61

8.3

5

0.9992

76.54

12.6

77.13

12.1

5

0.9989

97.17

8.9

70.08

11.1

5

0.9991

84.31

11.2

81.52

8.3

5

0.9990

85.95

18.8

62.23

21.9

5

0.9980

73.35

16.2

69.40

5.0

5

0.9986

69.97

15.7

89.62

12.0

5

0.9975

103.78

8.5

103.90

12.0

5

0.9992

90.59

12.6

95.82

8.2

5

0.9952

80.55

12.1

75.11

19.9

5

0.9997

82.39

10.9

81.55

31.6

5

0.9993

99.06

9.6

86.00

9.2

5

0.9976

111.21

16.2

104.17

23.1

5

0.9998

69.28

9.5

74.15

12.8

5

0.999

89.71

8.1

85.26

12.0

5

0.9987

91.06

13.1

103.92

11.0

5

0.9997

90.28

6.0

80.83

33.7

5

0.9975

83.12

17.7

72.39

27.3

5

0.9993

75.81

12.2

87.45

30.4

5

0.9997

87.77

6.9

94.58

3.3

5

0.9988

93.28

10.5

109.45

11.8

5

0.9995

83.81

1.7

84.26

9.7

5

0.9999

80.83

9.6

97.01

7.8

5

0.9995

80.11

4.7

65.85

16.8

5

0.9999

91.37

8.0

71.65

14.3

5

0.9998

96.14

11.3

79.45

10.7

5

0.9993

96.97

11.9

85.49

6.2

5

0.9994

77.24

8.9

90.91

9.3

5

0.9998

87.27

17.8

66.53

5.0

5

0.9997

88.66

7.7

87.13

10.2

5

0.9997

88.21

8.3

124.53

9.5

5

0.9996

89.07

9.8

89.36

7.6

5

0.9996

82.64

11.3

114.55

22.7

5 5

0.9888 0.9999

67.33 92.77

9.0 7.9

64.17 103.64

8.1 12.9

5

0.9976

107.33

14.1

88.07

7.6

5

0.9999

88.35

8.9

93.31

10.5

5

0.9998

72.23

13.6

60.08

16.1

23


3.4 –

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G. Chen et al. / Food Chemistry 125 (2011) 1406–1411 Table 1 (continued) Name

Rt (min)

MRM transitiona

Cone (v)

CE (eV)

Furathiocarb

11.19

20

Fluazifop-p-butyl

11.31

Flufenoxuron

11.73

383:1>195:2 383:1>52:2 384:1>282:2 384:1>328:2 488:9>158:2 488:9>141:2

17 11 20 17 22 32

LOQ (lg kg

1

)

Linearity/r

Recovery/%b 50 lg kg

a b

24 25

1

RSD

10 lg kg

1

RSD

5

0.9992

101.66

5.5

115.80

1.5

5

0.9999

86.22

8.6

76.11

11.8

5

0.9998

81.84

11.2

78.18

6.2

The underlining MRM transitions are used for qualitative analysis. n = 5.

Tea matrix is very ‘‘dirty”, which contain pigments, caffeine, sugars, organic acids and other interferences. A tandem mass detector, which has high selectivity and sensitivity, provides an effective solution. MRM parameters including ion transition, collision energy and cone voltage of UPLC/MS/MS are listed in Table 1. Each pesticide was tuned using a single standard solution at 1 lg mL 1 which was infused into the MS detector at a flow rate of 0.3 mL min 1. Product ion mass spectra for the pesticides were obtained in electrospray ionisation using collision induced dissociation (CID). Variations in collision energy influence both sensitivity and fragmentation. The collision energy was optimised for two selective ion transitions for every pesticide. Both pairs of the MRM transitions were used for confirmation analysis, which can meet the EU Decision (2002/657/EC, 2002), and the most sensitive transitions were selected for quantification analysis. Dwell times for different transitions were optimised to achieve higher sensitivities, as well. Some compounds like bromoxynil and ioxynil were analysed in negative ESI mode ([M H] ) while other pesticides were on positive ESI mode ([M + H]+). In this investigation, a total of 65 pesticides were determined in tea. A combined MRM chromatogram of fortified tea sample by 15 representative LC amenable pesticides at 0.1 mg kg 1 is shown in Fig. 1.

trile was chosen as the extraction solvent in the QuEChERS method. Application of MgSO4 for partitioning could yield a significant volume of the upper layer and give high recoveries. Acetic acid with CH3COONa makes up of a buffer (pH 4–5), which could give adequately high recoveries of acephate and imazalil etc. Moreover, the usage of buffer could improve stability of the base-sensitive pesticides for their analysis (Hiemstra & de Kok, 2007). Several sorbents can be used in the clean-up method for pesticide residue analysis, e.g. PSA (primary secondary amine), NH2, graphitized carbon black (GCB) and ODS SPE cartridges. The use of PSA + GCB SPE (Department of Food Safety Ministry of Health, Labour and Welfare, Japan, 2006) could remove more matrix materials. The mechanism of PSA (or NH2) sorbent is based on the weak ion exchange. It removes fatty acids, sugars and other components that form hydrogen bonds. The use of GCB is easy to remove pigment especially chlorophyll. However, GCB strongly retains planar pesticides. In this research, QuEChERS method (dispersive-SPE) was proven to be unable to remove the pigment from tea effectively. SPE cartridge (NH2 + GCB) was used for further clean-up as complementary to the QuEChERS method. 1% acetic acid in the elution solvent was proven to be able to reduce the absorption of planar pesticides in GCB and thus improve the recoveries of pesticides residues, with the exception of pymetrozin, diflubenzuron and thiabendazole (Fig. 2).

3.2. Sample preparation 3.3. Matrix effects In the process of pesticide residue analysis, sample pretreatment and preparation are the most time-consuming, labour intensive and complicated procedures. According to the characteristics of pesticides, several solvents can be selected as the extraction solvent, e.g. acetone (Germany DFG method S 19, 1999; Payá et al., 2007), ethyl acetate (Frenich et al., 2005), and acetonitrile (Hajou, Afifi, & Battah, 2004). In comparison to other solvents, acetonitrile shows more advantages such as higher recoveries, less interference from lipids and proteins, better compatibility with LC and GC, and less co-extracted matrix components. For these reasons, acetoni-

The matrix effects may differ for different teas, e.g. green, black, Oolong and Puer. Consequently, it is required to compensate the matrix effects by a matrix-matched calibration (European Council N° SANCO/2007/3131, 2007) which could be more efficient than by use of an isotope labeled standard, or ECHO technique (Aldera, Lüderitz, Lindtner, & Stan, 2004) in LC/MS system. In this study, analytically confirmed pesticides-free organic green tea from Shiru Tea Company and black tea from Unilever UK were used as blank matrix. Organic green tea was selected as

Fig. 1. Combined UPLC/MS/MS chromatogram of fortified green tea at 0.1 mg kg 1 1. Propamocarb. 2. Pirimicarb. 3. Carbofuran-3-hydroxy. 4. Mevinphos. 5. Acetamiprid. 6. Thiofanox-sulfon. 7. Spiroxamine. 8. Triasulfuron. 9. Bromoxynil. 10. Promecarb. 11. Triadimefon. 12. Fenhexamid. 13. Fenoxycarb. 14. Clethodim. 15. Flufenoxuron.

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G. Chen et al. / Food Chemistry 125 (2011) 1406–1411

no acid in SPE elution

120

1%acetic acid in SPE elution

100

Recovery %

80 60 40 20 0 Bensulfuronmethyl

Nicosulfuron

ThifensulfuronMethyl Pesticides

Triasulfuron

TriflusulfuronMethyl

Fig. 2. Comparison for recovery of sulphonyl and ureas pesticides with and without 1% acetic acid in SPE elution (n = 5, spiked concentration = 0.05 mg kg expressed as mean ± standard deviation.

1

). Each value is

the representative matrix for green and Puer tea samples while organic black tea was selected as the representative matrix for black and Oolong tea samples. For the determination of matrix effects, the responses of the standard solutions prepared in solvent were compared with the responses of the standard solutions prepared in pesticides-free blank tea sample. In addition, the complex matrix from tea could play a negative impact on the separation performance of the UPLC column. Moreover, the UPLC column was prone to being blocked if the clean-up of tea samples was not complete.

cept for dichlorvos (0.02 mg kg 1). The mixed standard solution was added into the pesticides-free black tea samples to make the concentration of 10 and 50 lg kg 1, and then the method was carried as described in part 2.3. The majority of recoveries for these pesticides were in the range from 70% to 120%. But some recoveries (60–70% or 120–130%) could also be accepted (European Council N° SANCO/2007/3131, 2007). Reproducibility of this method is shown in Table 1 as RSD.

3.4. Validation of the method

In order to achieve the accurate results, a 2-step analytical strategy was applied. The first step was a screening method, which monitored only one MRM transition for each compound. In this way, negative and positive samples were separated. The second step is a confirmation method, in which at least two MRM transitions for each compound were monitored. The most sensitive MRM transition was selected for quantification. This strategy was applied to pesticide residue analysis of 18 tea samples from different regions. Among the pesticide residues detected in these 18 tea samples, acetamiprid had the highest detected frequency (61.1%), and this was followed by imidacloprid

Linearity, sensitivity, accuracy and precision of the multi-residue method were validated. The mixed matrix-matched pesticides standard solutions of 5, 10, 20, 50, 100 lg L 1 were injected into the UPLC/MS/MS system. Relative coefficients (r) are listed in Table 1. Limits of quantification (LOQ) of mixed pesticides standards were determined by injecting a series of different matrix-matched pesticides standard solutions. Parameters are listed in Table 1. Although the LOQ of the method varies with different pesticides, all can be measured at 0.01 mg kg 1 level in a 5 g tea sample ex-

Detected frequency

3.5. Pesticide residues in tea samples

Detected level

-1

0.2

80

0.4 60 0.6 40

0.8

20

1.2 Triazophos

Triadimenol

Methomyl

Imidacloprid

Dimethoate

Carbendazim

Buprofezin

Acetamiprid

0

1

Detected level (mg kg

Detected frequency /%

0

100

)

-0.2

120

Pesticides Fig. 3. Detected frequencies and levels of the pesticide residues in 18 tea samples. The error bars indicated the ranges of pesticide residue levels detected in tea samples.

G. Chen et al. / Food Chemistry 125 (2011) 1406–1411

(56.8%), carbendazim (56.6%), triazophos (44.4%), dimethoate & methomyl & uprofezin (33.3%), and triadimenol (22.2%). For some pesticide residues, the detected levels varied greatly. The minimum value of acetamiprid, for example, was 0.02 mg kg 1 in a green tea from Anhui province while the maximum value of 1.03 mg kg 1 was found in an Oolong tea from Fujian province. However, all the residue levels of these pesticides in these 18 tea samples were below the MRL required by Chinese government, EU and Japanese government, except for dimethoate and methomyl in five tea samples resulted higher than the Chinese and EU MRL. The patterns of pesticide usages are different from one tea plantation to another because they have different pest problems. The fact that we found multiple residues in a number of samples may be due to the fact that some teas constitute are a high degree of custom blending to produce distinct finished products. For instance, there were five tea samples contaminated by eight pesticides simultaneously and there were six tea samples in which dimethoate, methomyl and uprofezin were detected simultaneously. The detected frequencies and detected levels of the pesticide residues in these 18 tea samples are showed in Fig. 3. In literature, some GC amenable pesticides like DDT, HCH and some pyrethroid could be detected easily in tea. According to the pesticide residue levels of the tea samples investigated in this paper, easily-detectable LC amenable pesticides in tea were found to be acetamiprid, imidacloprid, carbendazim, triazophos, dimethoate, methomyl, uprofezin, and triadimenol. 4. Conclusions A very quick, easy, effective, rugged, reliable and accurate multi-residue method based on modified QuEChERS method was developed for determination of pesticides in tea by ultra performance liquid chromatography with tandem mass spectrometry. The performance of the method was very satisfactory with results meeting validation criteria. The method has been successfully applied for the determination of tea samples and ostensibly has further application opportunities, e.g. dry vegetable and herb extracts. Acknowledgements The authors are very grateful for the support of Unilever Discover Shanghai, especially the Advanced Measurement and Data Modelling group. Yumo Zhang and Domenic Caravetta are acknowledged for their English language help in preparing this manuscript. References Aldera, L., Lüderitz, S., Lindtner, K., & Stan, H. (2004). The ECHO technique-the more effective way of data evaluation in liquid chromatography–tandem mass spectrometry analysis. Journal of Chromatography A, 1058, 67–79. Anastassiades, M., Lehotay, S. J., Stajnbaher, D., & Schenck, F. J. (2003). 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, 86, 412–431.

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