Environmental Fate and Safety Management of Agrochemicals

Chapter 3

Multiresidue Analysis of Pesticides in Foods Using Liquid Chromatography/Mass Spectrometry

Downloaded by PENNSYLVANIA STATE UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: March 8, 2005 | doi: 10.1021/bk-2005-0899.ch003

Futoshi Sato, Hitoshi Iwata, Takakazu Nomura, and Kazuhiro Komatsu Japan Food Research Laboratories, Tama Laboratory, Tama-si, Tokyo 206-0025, Japan

We developed a multiresidue method (MRM) using LC/MS which enabled to analyze 50 thermolabile or involatile pesticides in foods because of remaining unavailable for the GC mehod. The sample wasa extracted with acetonitrile. After a salting-out with sodium chloride, the acetonitrile extract was purified by gel permeation chromatography. The pesticide fraction was purifeied by ENVI-Carb/NH2 column and determined by LC/MS. Apples, grapefruits, Japanese radishes, brownrice,spinach, and green tea were used for recovery tests in triplicates. The recoveries were more than 70% in 81% (264/324) of the tested items. The complemental use of this method for the conventional GC/MS methods would enable analysis of a wide range of pesticides in a large number of crops.

28

© 2005 American Chemical Society In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

29


Introduction At present, the Ministry of Health, Labor and Welfare (MHLW) in Japan has set the MRL of 229 pesticides in foods and plans a further increase of the number by more than 400 in a few years. The ministry is going to introduce a "positive list system" in which pesticides having no MRLs should be treated as the illegal substance if detected in foods. According to increasing the pesticides listed for MRLs, their physicochemical properties should expand widely. Therefore multiresidue method (MRMs) are required to cover considerably wider range of polarity. During the recent decades, capillary GC with different sensitive and/or selective detectors has been the most common tool for the multiresidue determination of pesticides in foods. Among these GC systems, the multi-channel mass spectrometer has superseded the element-selective detectors such as ECD and FPD for some years. However, the thermolabile and/or involatile pesticides still remained unavailable for the GC/MS method. On the other hand, it was obvious that the LC/MS suites for such relatively high-polar pesticides. Therefore we developed a MRM using LC/MS which enabled to analyze a total of 50 pesticides consisting of 11 categories (benzoylurea, carbamate, dicarboximide, neonicotinoid, organophosphosphate, oxim carbamate, pyazole, strobilurin, triazole, urea and the others).

Experimental Standard preparation Fifty pesticides were selected and their reference calibrants of garanteed grade or equivalent were obtained from Kanto Kagaku (Tokyo, Japan), Hayashi Pure Chemical (Osaka, Japan), and Wako Pure Chemical (Osaka, Japan). Each stock solution (500mg/L) of the individual pesticide was prepared in acetone. Portions of all the stock solutions were combined in a volumetric flask and made up with acetone to prepare a mixed standard solution. Pesticides concentrations of working solutions for LC/MS analysis were prepared between 0.02 and 1 mg/L using acetnitrile. Sample preparation Basically 20g of a homogenized sample (apples, grapefruits, Japanese radishes, and spinach) was weighed, added with 60ml acetnitrile, and blended thoroughly. As for brown rice and green tea, the sample size was scaled down to 10g and 2g, respectively, and 30ml of water was added to these samples before added with acetonitrile. The filtrate of the mixture was combined with 10g of sodium chloride and 10ml of 0.05mol/L phosphate buffer (pH=7), shaken for five minutes. The acetonitrile layer was collected and evaporated near to dryness. Then the residue was

In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

Table I. Instruments conditions of M S


#

1 2 3 4 5 6 7 8 9 10 11 11 11 12 13 14 14 15 16 17 18 19 20 20 20 21 22 23 24 25 25 26 27 28 28 29 30 31 32 33 34 35 36 37

a b c a b

a b c

a b a b

Pesticide oxamyl thiamethoxam imidacloprid acetamiprid aldicarb propoxur bendiocarb carbofiiran isouron carbaryl ethiofencarb ethiofencarb sulfoxide ethiofencarb sulfone pirimicarb isoprocarb metominostrobin-E metominostrobin-Z methabenzthiazuron diuron fenobucarb azoxysrtobin siduron methiocarb methiocarb sulfoxide methiocarb sulfone dimethomoφh linuron daimuron cumyluron triflumizole triflumizole-M procymidone tebufenozide iprodione iprodione-M diflubenzuron cyprodinil famoxadone phoxim etobenzanid bitertanol clofentezine difenoconazole hexaflumuron

M.W. 219.3 291.7 255.7 222.7 190.3 209.2 223.2 221.3 211.3 201.2 225.3 241.3 257.3 238.3 193.2 284.3 284.3 221.3 233.1 207.3 403.4 232.3 225.3 241.3 257.3 387.9 249.1 268.4 302.8 345.7 294.6 284.1 352.5 330.2 330.2 310.7 225.3 374.4 298.3 340.2 337.4 303.1 406.6 461.1

m/z mode pos 237 292 pos 256 pos 223 pos 213 pos 210 pos 224 pos 222 pos 212 pos 202 pos 226 pos 242 pos 258 pos 239 pos 194 pos 285 pos 285 pos 222 pos 233 pos 208 pos 404 pos 255 pos 226 pos 242 pos 258 pos 388 pos 249 pos 269 pos 303 pos 346 pos 295 pos 284 pos 297 pos 330 pos 330 pos 311 pos 226 pos 373 neg 299 pos 340 pos 338 pos pos 303 pos 406 pos 461

F.V. 60 80 100 100 120 120 120 120 120 80 80 100 100 120 120 100 80 120 120 120 120 120 120 100 100 120 120 120 120 80 120 120 80 80 80 80 80 140 80 80 80 80 120 120


31


Table I. Instruments conditions of MS (continue) # Pesticide M.W. m/z Mode F.V. pos 120 furathiocarb 38 382.5 383 39 tebufenpyrad 333.9 334 pos 120 40 tolfenpyrad 383.9 384 pos 80 41 a fenpyroximate-E 421.5 422 pos 120 41 b fenpyroximate-Z 421.5 422 pos 120 42 piperonyl butoxide 338.4 356 pos 120 43 teflubenzuron 381.1 379 neg 100 44 hexythiazox 352.9 353 pos 120 45 flufenoxuron 488.8 489 pos 120 46 lufenuron 511.2 511 pos 80 47 chlorfluazuron 540.7 540 pos 120 48 methamidophos 141.1 142 pos 120 49 acephate 183.2 184 pos 120 50 monocrotophos 223.2 224 pos 120 #: Peak number assigned by the order of elutionfromLC/MS column, M.W.: molecular weight, m/z: mass-to-charge ratio, Mode: mode of API-ES, F.V.: fragmenter voltage, -M: metabolite

dissolved with 30mL of ethyl acetate and the solution was dehydrated with anhydrous sodium sulfate. After evaporating the solvent, the residue was dissolved with ethyl acetate/cyclohexane (1:1), and a 2.5g portion of the solution was loaded on a gel permeation chromatography (GPC) column. As for green tea, the volume loaded to GPC was changed to a lg portion to avoid interfering effects from the matrix. The corresponding fraction from the GPC was collected and evaporated to dryness. The residue was dissolved in toluene/acetnitrile (1:3) and loaded on an ENVICarb/NH ™ column, and then eluted with 30ml toluene/acetnitrile (1:3). The eluate was evaporated to dryness and the residue was dissolved in 1ml acetonitrile. To verify the accuracy and precision of the method we conducted recovery tests in triplicates at 0.2mg/kg as the spiking level. 2

Instruments and conditions The GPC was performed using a LC-10 Series GPC system (Shimadzu, Kyoto, Japan). A glass column (φ25 χ 600mm) packed with 60g of Envirobeads S-X3 Select (O.I.Analytical, Houston, USA) was operated at room temperature and at a flow-rate of 5 ml/mi η. The mobile phase was ethyl acetate/cyclohexane (1:1), and the sample injection volume was 5ml. LC/MS determination was achieved using "1100 series" LC system equipped with "G1946D" MS instrument (Agilent Technologies, CA, USA). The chromatographic separations were carried out in gradient modes using methanol and 2mmol/L ammonium acetate solution as the solvent. The gradient was started from 20% methanol, increased linearly to 95% methanol in 45 min, and held


32 Table II. Elution profiles on GPC and ENVI-Carb/NlI ™ 2


Pesticide benzovlurea chiorfluazuron diflubenzuron flufenoxuron hexaflumuron teflubenzuron carbamate bendiocarb carbaryl carbofiiran ethiofencarb ethiofencarb sulfone ethiofencarb fenobucarb furathiocarb isoprocarb methiocarb methiocarb sulfone methiocarb sulfoxide pirimicarb propoxur dicarboximide iprodione iprodione-M procymidone neonicotinoid acetamiprid imidacloprid thiamethoxam oreanonhosnhosnhate acephate methamidophos monocrotophos phoxim oxime carbamate aldicarb oxamyl nvrazole fenpyroximate-E fenpyroximate-Z tebufenpyrad tolfenpyrad

GPC Elution volume (ml) Rec(%)

ENVI-Carb/NH* Elution volume (ml) Rec(%)

105-135 130-160 105-135 110-130 120-150

76 97 83 57 67

10-70 0-15 0-15 0-15 5-30

108 95 100 90 106

140-180 150-190 140-180 150-170 140-180 145-190 135-170 145-175 135-170 145-180 150-180 150-200 160-200 140-170

100 101 96 14 75 146 83 103 91 110 17 82 84 91

0-10 0-15 0-15 0-10 0-15 0-15 0-10 0-10 0-15 0-15 0-10 0-10 0-15 0-10

91 99 100 93 103 100 92 102 92 100 94 100 97 95

135-170 140-170 145-175

90 64 98

0-20 0-10 0-10

96 102 105

150-200 150-200 165-195

105 103 79

0-10 0-15 0-10

94 99 97

155-185 155-185 145-185 140-180

89 83 98 89

10-70 0-10 0-10 0-10

106 100 115 99

150-180 160-190

76 87

0-10 0-15

89 99

130-180 130-180 135-165 145-175

97 118 131 116

0-25 0-15 0-10 0-10

99 109 128 119


33 Table II. Elution profiles on GPC and ENVI-Carb/NH ™ (continue) 2


Pesticide strobilurin azoxysrtobin famoxadone metominostrobin triazole bitertanol difenoconazole triflumizole triflumizole-M urea diuron isouron linuron lufenuron methabenzthiazuron siduron others clofentezine cumyluron cyprodinil daimuron dimethomoφh etobenzanid hexythiazox piperonyl butoxide tebufenozide

GPC Elution volume (ml) Rec(%)

ENVI-Carb/NH,™ Elution volume (ml) Rec(%)

150-200 145-165 165-185

102 90 87

0-10 0-10 0-10

103 103 102

125-170 155-185 125-155 125-155

90 109 98 88

0-15 0-10 0-10 0-10

99 104 102 96

135-180 145-175 140-170 105-140 180-220 135-170

102 100 135 119 92 117

0-15 0-10 0-10 0-15 0-25 0-15

97 100 108 118 91 107

170-210 135-170 150-190 130-160 170-210 145-190 145-200 130-170 120-160

98 117 89 117 97 97 110 108 110

0-20 0-10 0-20 0-10 0-15 10-70 0-10 0-15 0-10

96 106 94 106 101 114 114 104 101

Rec: Recovery (%) of elution test, -M: metabolite at 90% for 10min except for acephate, methamidophos and monocrotophos. A Develosil C30™ (φ2 X 250mm, Nomura Chemical, Aichi, Japan) was used as the column. For acephate, methamidophos and monocrotophos, the gradient was from methanol 5% to 50% linearly in 15 min and held at 50% for 10 min, and the column was a Hydrosphere C18 (φ2 x 150mm, YMC, Kyoto, Japan). The temperature, flow rate, and injection volume were at 40°C, 0.2ml/min, and 2μί, respectively, in all cases. The API-ES interface was operated in both positive and negative modes. The drying gas was operated at 350°C under 10 L/min of flow rate. The nebulizer gas pressure was 35 psi. The capillary voltages were 4000V and 3500V for the positive and negative modes, respectively. SIM mode was used for the determination. The details of the fragmenter voltage and m/z were shown in Table-1.


34

oxamyl

acetamiprid


JL 5

_j

10

30

methabenzthiazuro

tebufenozide

32

min

chlorfluazuron

i

_ b c d — e ^

34

36

50

52

min

a: standard (O.lng), b: apples, c: grapefruits, d: J. radishes, e: brown rice, f: spinach, g:standard (0.04ng), h: green tea Figure J. Chromatograms for blank extracts.

Results and discussion Shibata et. al. (1) have reported that ENVI-Carb/NH was efficient for purification and enough recoveries of pesticides in foods by MRMs. Table-2 shows typical elution profiles of the pesticides on the GPC and ENVI-Carb/NH . We have chosen to collect the fraction of the GPC from 105ml to 215ml based on the result shown in Table II. As for ENVI-Carb/NH ™, all pesticides were eluted with 30ml, except for chlorfluazuron, acephate, and etobenzanid. To separate interfering materials which elutes from 30ml to 70ml in the eluate, as well as to save the analytical time, we chose to collect first 30ml for the elution volume despite incomplete elution of 3 pesticides. For most of the pesticides, the highest response was obtained in the positive mode, where [M+H] was the highest abundant ion (Table I). In contrast, the highest abundant ion for famoxadone and teflubenzuron was [M-H]" in negative mode. 2

TM

2

2

+


35

benzoylurea chlorfluazuron diflubenzuron flufenoxuron hexaflumuron teflubenzuron carbamate bendiocarb 7 carbaryl 10 8 carbofiiran ethiofencarb 11 a fenobucarb 17 furathiocarb 38 isoprocarb 13 20 a methiocarb pirimicarb 12 6 propoxur dicarboximide iprodione 28 a 28 b iprodione-M procymidone 26 neonicotinoid 4 acetamiprid 3 imidacloprid 2 thiamethoxam organophosphoras 49 acephate 48 methamidophos 50 monocrotophos 32 phoxim oxime carbamate 5 aldicarb oxamyl 1 pyrazole 41 a fenpyroxim-E fenpyroxim-Z 41 b 39 tebufenpyrad 40 tolfenpyrad 47 29 45 37 43

.S 'EL

1

85 82 84 76 76

73 94 83 93 74

85 82 55 81 69

88 88 88 84 79 65 72 94 83 84

91 91 91 91 89 97 86 92 89 90

96 90 90 87 85 90 80 92 87 92

87 83 85 37 87 90 87 65 43 89

82 62 84

100 60 92

81 62 102

98 52 92

68 70 90

71 68 70

60 59 53

82 79 65

87 84 84

87 89 79

32 28 17

67 58 90 90

58 49 76 60

84 76 114 72

98 85 126 89

98 80 125 58

40 36 20 82

27 66

73 59

72 87

84 90

72 88

36 30

100 88 99 98

81 81 89 85

107 79 101 96

98 85 100 93

95 85 91 90

98 95 96 80

pies

Pesticide

8 s

le

&

grape fruits


#

own rice

Table III. Recoveries of pesticides for spiked test . Recovery (.%)

78 94 60 95 95

58 61 78 82 74

88 96 87 105 103

92 93 91 76 87 100 82 87 56 91

84 26 79 70 73 92 74 74 46 77

89 89 94

l a

H

&

Continued on next page


36 Table HI. Recoveries of pesticides for spiked test (continue) Recovery (%)

8 #

Pesticide

8 ο s i

J)


&1

•G

I 1

X3

ω

i

8

&

strobilurin azoxystrobin 93 84 88 90 89 85 famoxadone 92 86 91 97 95 86 metominostrobin-E 97 79 89 96 86 81 metominostrobin-Z 95 68 85 95 88 82 triazole 34 bitertanol 94 75 94 88 94 98 36 difenoconazole 94 74 97 98 97 87 25 a triflumizole 37 76 91 87 64 88 25 b triflumizole-M 78 59 77 90 84 76 urea 16 diuron 91 75 88 90 91 84 9 isouron 92 77 85 94 90 96 22 linuron 96 85 92 93 93 88 46 lufenuron 64 81 85 82 87 60 15 methabenzthiazuron 83 61 87 90 92 83 19 siduron 82 89 92 90 91 93 others 35 clofentezine 73 73 74 81 86 92 24 cumyluron 90 44 86 88 86 90 30 cyprodinil 82 50 89 90 89 83 23 daimuron 89 76 86 89 87 89 21 dimethomoφh 89 60 90 88 88 61 33 etobenzanid 40 26 46 46 48 79 44 hexythiazox 94 91 86 68 84 97 42 piperonyl butoxide 94 87 88 94 92 98 27 tebufenozide 94 70 89 91 90 107 #: Peak number assigned by the order of elutionfromLC/MS column, -M: metabolite, 18 31 14 a 14 b

Figure I shows ion chromatograms for blank extracts of some crops compared with six representative pesticides at two different doses. Using this method, the substantial responses of the standards were obtained at around 0.04ng with over 3 signal-noise ratio, except for iprodione and teflubenzuron. Matrix-derived interfering peaks observed in some samples such as grapefruits and green tea, make it difficult to determine at the level of 0.02mg/kg for carbaryl, triflumizole, methabenzthiazuron and tebufenozide. Except for these samples, no significant interfering peaks were observed for target pesticides. Table III shows the results of recovery tests. For Japanese radishes, brownrice,and spinach, the recoveries were above 70% for most pesticides. For green tea, high-polar pesticides such as neonicotinoid, oxime



37

Figure 2. Distribution map ofpesticides determined by this method and major pesticide classes with MRLs in Japan carbamate and a part of organophosphosphate showed less than 50% recoveries. The relatively lower recoveries for grapefruit and green tea can be accounted for the suppressed ionization due to the abundant interfering matrices in these samples. The recoveries of etobenzanid were less than 50% in all crops, because of their poor recoveries from ENVI-Carb/NH . However, chlorfluazuron and acephate showed enough recoveries. Figure 2 shows the distribution map of pesticides on the bases of their polarities and molecular weights. As the results, most pesticide classes were covered by this method with the exception of nonpolar pesticides like pyrethroid. In conclusion, our proposed method enabled to determine about 50 pesticides at LOD of 0.02mg/kg or below. The recoveries were more than 70% in 81% (264/324) of the tested items. The complemental use of this method for the conventional GC/MS methods would enable analysis of a wide range of pesticides in a large number of crops. 2

References 1.

Shibata, Y.; Oyama, M.; Sato, H.; Nakao, K,; Tsuda, M.; Sonoda, M.; Tanaka, F.: J.Food Hyg. Soc. Jpn. 1998, 39, 241-250.


Environmental Fate and Safety Management of Agrochemicals

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