Supercritical Fluid Chromatography and Gas Chromatography

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Food Safety and Toxicology

Supercritical fluid chromatography and gas chromatography coupled to tandem mass spectrometry for the analysis of pyrethroids in vegetable matrices. A comparative study. María Murcia-Morales, Víctor Cutillas, and Amadeo R. Fernández-Alba J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00732 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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Journal of Agricultural and Food Chemistry

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Supercritical fluid chromatography and gas chromatography coupled to tandem

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mass spectrometry for the analysis of pyrethroids in vegetable matrices. A

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comparative study

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María Murcia-Moralesa, Víctor Cutillasa, Amadeo R. Fernández-Albaa*

6 7

a

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University of Almeria, Agrifood Campus of International Excellence (ceiA3) Department

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of Hydrogeology and Analytical Chemistry, Ctra. Sacramento S/Nº, La Cañada de San

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European Union Reference Laboratory for Pesticide Residues in Fruit & Vegetables.

Urbano, 04120, Almería, Spain. [email protected] +34 950 015 034

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ACS Paragon Plus Environment


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Abstract

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This study describes a comprehensive comparison between supercritical fluid

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chromatography (SFC) and gas chromatography (GC) coupled to mass spectrometry for

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the analysis of pyrethroids in vegetable matrices. The ionization process used was

16

electrospray ionization in SFC and electron ionization in GC. In general, liquid

17

chromatography coupled to mass spectrometry with ESI sources provides poor results for

18

pyrethroids detection, as described in previous literature. A total of 14 pyrethroids were

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selected, together with 6 representative matrices. The differences in chromatographic

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separation and ionization process were assessed. Similar results were obtained in terms

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of sensitivity (limits of quantification close to 2 µg/kg injecting the same amount of

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sample), matrix effect and linearity. 17 real samples were analyzed by both systems

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obtaining similar results. These data suggest that supercritical fluid chromatography

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offers a suitable alternative to gas chromatography in the analysis of pyrethroids and

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allows their inclusion in a wider multiresidue method.

26 27 28 29

Keywords Supercritical fluid chromatography; gas chromatography; pyrethroids; vegetable matrices; electron ionization; electrospray ionization.

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1. Introduction

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Pyrethrins are natural compounds present in Chrysanthemum cinerariaefolium

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flowers. They are considered one of the most effective natural insecticides and have been

34

used to control pest insects since ancient times. Pyrethroids are a class of synthetic

35

insecticide consequence of a modification of pyrethrins’ chemical structures. These

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changes provide increased stability to the light and air exposition, also improving their

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biological performance though a more selective toxicity1. The mechanism of the

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insecticidal activity resides in an alteration of the sodium channels, thus altering the nerve

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action potential 2. Due to their efficiency and low toxicity compared to organophosphorus

40

pesticides, pyrethroids are pesticides that are applied regularly.

41

Most pyrethroids are chiral molecules, with only a few exceptions such as etofenprox.

42

Therefore, each one has a number of stereoisomers and their identification is in some

43

cases influenced by the number of chromatographic peaks and their resolution. Some

44

pyrethroids such as cypermethrin, cyhalothrin or cyfluthrin possess three chiral centers,

45

resulting in eight possible stereoisomers (even though only the four pairs of enantiomers

46

can be resolved by chromatography without the use of chiral columns). The existence of

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these isomers also affects their legislation, as in some cases only some of them are

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approved. For example, in the European Union, cyhalothrin (as the sum of 4 pairs of

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stereoisomers) is not approved 3, whereas lambda-cyhalothrin (only 2 of these isomers)

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and gamma-cyhalothrin (1 isomer) are approved for their use as plant protection products

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4-6.

52

is no ambiguity in its residue definition.

In other cases such as deltamethrin, only one enantiomer form is formulated and there

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Pyrethroids are considered less toxic and safer to use compared to organophosphate

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insecticides. Their toxicity by dermal exposure is low due to their limited capability to be

55

absorbed through the skin. However, the average acute oral LD50 for vegetable oils is in

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the range of 50-500 mg/kg, which is considered to be moderately toxic 7. Due to their

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high lipophilicity, they tend to remain in the organism, which leads to bioaccumulation.

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Corcellas et al. reported the bio-accumulation of 11 pyrethroids in human breast milk,

59

even though these pesticides were assumed to hydrolyze in mammals 8.

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Pyrethroids show very low polarity, with log Kow values higher than 4. As a

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consequence, most of the analytical methods described for the analysis of these

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compounds use gas chromatography (GC) instead of liquid chromatography (LC) 9-11. In



63

general terms, liquid chromatography usually provides lower sensitivity in the analysis

64

of pyrethroids when reverse-phase and ESI sources are applied. Few studies have used

65

liquid chromatography for the analysis of pyrethroids and, in these cases, a very specific

66

sample extraction method including several clean-up steps or preconcentration stages is

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often applied to increase the sensitivity of the analysis 12-15. Some derivatization processes

68

have also been tested and the general metabolite 3-phenoxybenzoic acid (3-PBA) is more

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sensitive than the original pyrethroids 16.The main inconvenience is that a high number

70

of pyrethroids can be converted to this acid. On the other hand, due to their low polarity,

71

gas chromatography is the most widely used technique for the analysis of pyrethroids.

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Using standard extraction methods such as QuEChERS, an adequate analysis of

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pyrethroids by GC can be achieved in multiple matrices 17-18.

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Supercritical fluid chromatography (SFC) literature focused on pyrethroid analysis is

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not very extensive. Most of the articles emphasize the use of this type of chromatography

76

for the separation of enantiomers 19 and, when it is used for the analysis of pyrethroids,

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usually it is not coupled to mass spectrometry. However, there are numerous studies of

78

the use of supercritical fluid extraction (SFE) coupled or not to SFC for the analysis of

79

pyrethroids

80

view resides in the absence of water in the system 22. Some authors achieve an increase

81

in the sensitivity of pyrethroids by liquid chromatography when analyzing them using an

82

isocratic flow with very low percentage of water in the mobile phase 23. These methods

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are useful if the scope is mainly focused on pyrethroids, but could be impractical when

84

considering a multiresidue method. Supercritical fluid chromatography provides high

85

sensitivity for both polar and non-polar compounds and therefore allows the introduction

86

of pyrethroids in a broad multiresidue method.

20-21.

One of the main advantages of SFC regarding the ionization point of

87

The main objective of the present work is to highlight the advantages that SFC with

88

ESI sources can provide for the analysis of pyrethroids, allowing to enlarge the common

89

electrospray scope compounds with this group of pesticides. The capabilities of this new

90

approach facilitate the inclusion of pyrethroids in a wide scope SFC multiresidue method.

91

For the comparative study, 6 different vegetable matrices were investigated (tomato, pear,

92

zucchini, orange, onion and tea). These samples represent a wide difficulty range

93

expected in an analysis laboratory, including complex matrices such as onion, orange and

94

tea. The nature of the samples includes high-water matrices (tomato, pear, zucchini,

95

onion), acid matrix (orange) and dry matrix (tea). The QuEChERS standard extraction


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method was used in all cases, together with common chromatographic conditions for each

97

separation technique.

98 99 100

2. Materials and methods 2.1 Reagents and materials

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Pesticide standards were acquired from two different manufacturers: LGC

102

(Teddington, United Kingdom) and Sigma-Aldrich (Steinheim, Germany). The purity of

103

these standards was higher than 96% in all cases except for permethrin (94.5%) and

104

flucythrinate (87.5%). The standards were stored at -30ºC. Prior to the preparation of the

105

mixed solution with all pyrethroids, individual stock solutions were prepared with a

106

concentration around 1000 mg L-1 for each pyrethroid.

107

Reagents employed in the citrate QuEChERS extraction method (anhydrous

108

magnesium sulphate, sodium hydrogenocitrate sesquihydrate, sodium chloride, and

109

sodium citrate tribasic dihydrate) as well as formic acid and ammonium formate (for the

110

mobile phase preparation) were obtained from Sigma-Aldrich (Steinheim, Germany). All

111

gases used by the chromatographic systems (CO2, N2, He) have been supplied by Air

112

Liquide (Madrid, Spain).

113 114

2.2 Sample preparation

115

Five representative fruits and vegetables with different matrix compositions were

116

purchased from a local market in Almeria (Spain). These matrices (tomato, zucchini, pear,

117

orange, onion and tea) were extracted and analyzed in a first step to ensure the absence

118

of pyrethroids. Citrate buffer QuEChERS method with PSA dSPE clean-up was applied.

119

The procedure followed for the extraction did not have any modification and its

120

parameters have been previously described22,

121

extraction method contained 1 g of matrix per mL.

24.

The final extract resulting from the

122 123

2.3 Vials preparation

124

In order to prepare the vials for the injection in the system, the extracts were

125

spiked with the 14 pyrethroids as follow: each blank extract (50 µL for GC-MS/MS, 100



126

µL for SFC-MS/MS) was evaporated under a gentle stream of nitrogen and reconstituted

127

with the same volume of an organic solvent (ethyl acetate for GC-MS/MS, acetonitrile

128

for SFC-MS/MS) containing the mixture of the analyzed pyrethroids at 2, 5, 10, 20 or

129

100 µg/L.

130 131

2.4 SFC-MS/MS analysis

132

The SFC analysis was performed using a Nexera UC (Shimadzu Corporation,

133

Kyoto, Japan). In addition to the common devices of a liquid chromatograph, this system

134

is equipped with a CO2 pump and a back-pressure regulator (BPR) splitless device just

135

before the MS source.

136

Methanol with 1 mM of ammonium formate was used as modifier and mixed with

137

the CO2 to build the method gradient. The composition of the make-up was methanol with

138

0.1% formic acid and 5mM of ammonium formate. The make-up solvent was introduced

139

in the system isocratically at 0.080 mL min-1. The SFC separation was performed on a

140

C18 stationary phase column Shimpack UC-X RP (3 µm, 250 x 2.1 mm). The oven

141

temperature was set at 40ºC. The BPR pressure and temperature were established at 150

142

bar and 50ºC respectively. A total mobile phase flow of 1.3 mL/min was used. The

143

gradient started with an isocratic flow of 1% of modifier that was kept for 2 minutes. The

144

modifier percentage increased linearly to 5% at minute 5 and to 40% at minute 8. This

145

condition was kept for 2 minutes. The modifier percentage was then reduced from 40%

146

to 1% to recover initial conditions and maintained over 3 minutes. Autosampler

147

temperature was set at 10ºC and 2µL were established as the injection volume.

148

The SFC system is coupled to a triple quadrupole mass spectrometer LCMS 8060

149

(Shimadzu Corporation, Kyoto, Japan). The study was carried out employing an

150

electrospray ionization source (ESI) operating with 5 msec of switching polarity time.

151

The interface temperature was set at 300ºC, 250ºC for desolvation line and 400ºC in the

152

case of heat block. The interface voltage used was 4 kV. Regarding nebulizer, heating

153

and drying gas flows: 3 L min-1, 10 L min-1 and 10 L min-1 were used respectively.

154 155

2.5 Optimization of the SFC-MS/MS parameters


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Shimadzu Extended MRM Library was used for the creation of the multiresidue

157

method. This feature shows many transitions for each pesticide; three of them were

158

selected following the sensitivity rank. Individual standard solutions of the pesticides

159

were injected to confirm the transition with higher signal (quantifier) and the second most

160

sensitive (qualifier). Some compounds such as internal standards (dimethoate-d6,

161

carbendazim-d3, malathion-d10, and dichlorvos-d6) were not present in the library and

162

must be manually optimized using precursor ion search. For a proper identification, two

163

transitions must be detected with an ion ratio difference less than 30% and a retention

164

time shift under 0.1 min. Acquisition windows of ± 0.35 min were established for each

165

pesticide in the multiresidue method.

166 167

2.6 GC-MS/MS analysis

168

The analyses of pyrethroids by gas chromatography were performed with an

169

Agilent Intuvo 9000 GC coupled to an Agilent 7010 GC-MS/MS triple quadrupole and

170

equipped with an Agilent 7693 autosampler. The samples were injected in splitless mode

171

using a multimode injector through ultra inert inlet liners with glass wool (Agilent). A

172

temperature ramp (80 for 0.1 min, then increased to 300 ºC at 600 ºC min-1) was used in

173

the injector and the total injection volume was 1 µL.

174

The run time was 12.4 min, followed by 2.1 min for backflush (310 °C). The

175

temperature program of the oven started at 60 ºC (0.5 min), then it was increased to 170

176

ºC (80 °C min-1) and finally to 310 °C (20 °C min-1). The flows were kept constant during

177

the analyses (1.28 mL/min for the first column and 1.48 mL/min for the second column).

178

Helium was employed as the carrier gas.

179

1.5 mL/min, and the quenching gas (helium) flow was 2.25 mL/min. The high efficiency

180

electron ionization source and the transfer line were kept at 280 ºC during the analyses,

181

and the quadrupoles were maintained at 150 ºC.

The collision gas (nitrogen) flow was

182 183

2.7 Optimization of the GC-MS/MS parameters

184

The optimization of the GC-MS/MS parameters was performed according to a

185

previously published article25. The individual pyrethroids were first analysed in full-scan

186

mode to select the precursor ion/s and the retention time. Then, each pyrethroid was re-

187

analysed with a product ion method and the fragmentation patterns were obtained at



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188

different collision energies (5, 10, 15, 20, 25 and 30 eV). The quantifier and qualifier

189

transitions, together with their corresponding collision energies, were selected.

190 191 192

3. Results Acrinathrin,

bifenthrin,

cyfluthrin,

cypermethrin,

deltamethrin,

etofenprox,

193

fenpropathrin, fenvalerate, flucythrinate, lambda-cyhalothrin, permethrin, phenothrin,

194

tau-fluvalinate and tetramethrin were the 14 pyrethroids selected to carry out this study.

195

These pesticides were analysed using supercritical fluid chromatography and gas

196

chromatography, both coupled to tandem mass spectrometry. The comparative study of

197

these techniques was carried out in terms of limits of quantification (LOQs), linearity and

198

matrix effect. The differences in chromatographic separation and ionization process were

199

also assessed.

200 201

3.1 Ionization

202

The ionization processes applied differ considerably between the studied

203

techniques due to the type of ion source used in both equipments. In SFC-MS/MS, an ESI

204

source was used producing soft ionization. Consequently, the most intense ion of each

205

compound corresponded to the protonated molecular ion or ammonium adduct. Except

206

for phenothrin, tau-fluvalinate and tetramethrin, all pyrethorids formed an ammonium

207

adduct instead for the usual protonation of the molecular ion. This could be related to

208

their proton affinity and the functional groups present in these compounds, which share

209

similar structures in most cases 26-27.

210

On the other hand, stronger ionizations are obtained when using an EI source in

211

GC-MS/MS. The parent ion of each transition corresponds in most cases to a fragment of

212

the original molecule and the molecular ion is not present. In the case of pyrethroids,

213

whose chemical structures are similar, this may affect the method selectivity, as less

214

selective transitions are formed with smaller ions. Therefore, it may be necessary to

215

consider coelutions of other pyrethroids that could result in isobaric interferences and

216

interfere with their quantification. This happened, for instance, in the case of permethrin

217

(retention time 9.58 min), cyfluthrin (10.609 min) and cypermethrin (10.862 min), which

218

share the transition 163 > 127 (Figure 1a). These three compounds are very close to each


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219

other in the chromatographic window and each one shows two (permethrin) or three

220

(cyfluthrin, cypermethrin) separated peaks due to the presence of stereoisomers (Figure

221

1b). Therefore, the qualifier transition plays an essential role in their proper identification.

222

Softer ionizations can be also achieved in gas chromatography with the use of CI

223

(chemical ionization) instead of EI. a) Cypermethrin

Permethrin

Cyfluthrin F

O O

O Cl

O

O

Cl

O

O O

N

Cl

Cl

Cl

O Cl

N

Most abundant transition 163 > 127 Cl Cl Cl Chemical Formula: C7H9Cl2•

Chemical Formula: C7H8Cl•

b)

224

Figure 1. a) Molecular formulas of permethrin, cyfluthrin and cypermethrin and one common

225

transition (163 > 127); b) Chromatogram of cyfluthrin obtained by gas chromatography showing

226

an isobaric interference of permethrin and cypermethrin in the quantifier transition (163 > 127)

227 228

3.2 Chromatographic considerations



229

Very short run times were achieved with both techniques, being 13 minutes for

230

SFC and 12.4 minutes for GC. In SFC, the compressibility properties of CO2 make it

231

possible to apply high flow rates (1.3 mL min-1), which are necessary to improve the

232

chromatographic performance of some compounds included in the multiresidue method.

233

Therefore, a very short chromatogram was obtained with all the compounds eluting

234

during the first 7 minutes. This strong eluent flow hinders a proper separation of the

235

pyrethroid isomers which, in some cases, are not fully resolved. Figure 2 shows the

236

chromatograms of permethrin in zucchini matrix at the concentration of 2 ppb obtained

237

by both SFC and GC. It can be observed that the peaks are only resolved in GC. This

238

could have been an inconvenience if the pyrethroids residue definition required individual

239

quantification of a certain isomer. However, for the analyzed pyrethroids, the maximum

240

residue levels (MRLs) are defined for the sum of isomers, so a low chromatographic

241

resolution is not a problem for their quantitative analysis. Counts

163.0 -> 127.0 , 183.0 -> 153.0 x10 5 Ratio = 75.1 (100.0 %) 1.05 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 -0.05 -0.1 9.4 9.45

242

9.5

9.55

9.6

9.65

9.7 9.75 Acquisition Time (min)

243

Figure 2. Chromatograms of permethrin (2 transitions) obtained by gas chromatography (left)

244

and supercritical fluid chromatography (right).

245 246

The main inconvenience of the short chromatograms obtained in SFC-MS/MS

247

consists generally of the presence of isobaric interferences with the matrix. This effect is

248

more intense when working with complex matrices that possess a large number of

249

coeluting compounds, and it is especially troublesome at low concentration levels. It is

250

sometimes necessary to add more selective transitions to the affected compounds in order

251

to identify them 22. However, pyrethroids are a pesticide group with molecular masses

252

above the average, ranging from 331.4 g mol-1 (tetramethrin) to 514.4 g mol-1

253

(acrinathrin) for the 14 pyrethroids included in this study. These high molecular weights

254

result in more selective transitions and, therefore, the probability of finding isobaric


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interferences decreases dramatically. In the present study, only cyfluthrin showed a

256

transition affected by interferences that modified the ion ratio in complex matrices

257

(orange, onion and tea). The use of different transitions avoided the effect of this

258

interference, but their lower sensitivity did not allow the identification of this compound

259

at the lowest concentration levels.

260

This problem is less common in GC-MS/MS, since this technique employs longer

261

chromatographic columns that allow for a better separation efficiency and, therefore, a

262

lower number of compounds co-elute with the analytes.

263 264

3.3 Limits of quantification (LOQs)

265

The limits of quantification for the 14 pyrethroids were evaluated by the use of

266

matrix-matched standards with a concentration range of 2-100 µg L-1. The results are

267

detailed in table 1 as the lower limits of the instrumental concentration range. Similar

268

results were obtained with both techniques, being slightly better in SFC (figure 3). The

269

high sensitivity achieved in SFC is related to the absence of water in the mobile phase

270

and the low flows that reach the ESI source, resulting in low LOQs 22. In GC, for its part,

271

the good results are associated to the high sensitivity of the instrument used. The LOQ

272

values achieved are in most cases lower than the European MRLs for pyrethroids. Table 1. LOQs (in µg L-1) of the 14 pyrethroids in the matrices included in the comparative studies. Tomato Pear Zuccini Orange Onion Tea SFC GC SFC GC SFC GC SFC GC SFC GC SFC GC Achrinathrin 2 2 2 2 2 2 2 2 2 2 2 2 Bifenthrin 2 2 2 2 2 2 2 2 2 2 2 2 Cyfluthrin 2 2 2 2 2 2 20 5 20 2 10 2 Cypermethrin 2 5 2 5 2 5 2 10 2 5 5 10 Deltamethrin 2 2 2 2 2 2 2 2 2 2 2 100 Etofenprox 2 2 2 2 2 2 2 2 2 2 2 2 Fenprotathrin 2 2 2 2 2 2 2 5 2 2 2 5 Fenvalerate 2 2 2 2 2 2 5 5 2 2 5 5 Flucythrinate 2 2 2 2 2 2 5 2 5 2 10 2 Lambda2 2 2 2 2 2 2 2 2 2 5 2 cyhalothrin Permethrin 2 2 2 2 2 2 2 2 2 2 2 5 Phenothrin 2 10 2 10 2 10 5 20 2 5 10 10 Tau-Fluvalinate 2 2 2 2 2 2 2 2 2 2 2 2 Tetramethrin 2 2 2 2 2 2 2 2 2 2 2 2

273



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80 70 60 50 SFC/MS-MS

40

GC/MS-MS 30 20 10 0 2

274 275

5

≥10

Figure 3. Number of results with an LOQ of 2, 5 and 10 or higher in the different matrices.

276 277

The following pesticides: acrinathrin, bifenthrin, etofenprox, tau-fluvalinate and

278

tetramethrin could be identified at the lowest concentration level of 2 µg L-1 in all matrices

279

in both SFC and GC. Deltamethrin also showed LOQs of 2 µg L-1 in all matrices, with

280

the exception of tea in gas chromatography. In this case, an interference modified the ion

281

ratios and made it impossible to identify the pesticide at concentration levels lower than

282

100 µg L-1, even with the use of different qualifier transitions (figure 4). Cyfluthrin

283

showed the highest LOQs in SFC (10-20 µg L-1) in tea, orange and onion due to the use

284

of less sensitive transitions in order to avoid the effects of an isobaric interference (see

285

section 3.2). This pesticide showed LOQs of 2 µg L-1 in most matrices with the use of

286

GC. On the other hand, phenothrin and cypermethrin showed higher LOQs in all cases

287

with GC than with SFC. Counts

253.0 -> 172.0 , 253.0 -> 93.0 x10 5 Ratio = 589.4 (337.7 %) 4 3.8 3.6 3.4 3.2 3 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -0.2

288

11.25

11.3

Counts

253.0 -> 172.0 , 253.0 -> 93.0 x10 6 Ratio = 186.2 (106.7 %) 1.05 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 -0.05

11.35

11.4

11.45

11.5

11.55

11.6

11.65 11.1511.7 11.2 11.75 11.25 Acquisition Time (min)


11.3

11.35

11.4

11.45

11.5

11.55

11.6

11.65 11.7 Acquisition Time (min)

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289 290


Figure 4. Chromatogram of deltamethrin in tea by gas chromatography at 10 µg/kg (left, ion ratio 337.7%) and 100 µg/kg (right, 106.7%).

291 292

With regard to the matrices, LOQs in tea were in general higher than the rest, with

293

several pesticides reaching values of 5-10 µg L-1. In tomato, pear and zucchini, all

294

pesticides showed LOQs of 2 µg L-1 by SFC and GC, with the exception of cypermethrin

295

and phenothrin in the latter technique.

296

The responses were linear across the whole concentration range in all samples,

297

with the lowest limit corresponding to the LOQ of each pesticide in the matrix (Table 1).

298

Coefficients of determination (r2) were higher than 0.99 for all the studied cases.

299 300

3.4 Matrix effect

301

Complex matrices such as orange or tea tend to have a high number of interfering

302

matrix compounds. These compounds can coelute with the analytes in the source

303

producing signal suppression or signal enhancement. Coeluting matrix compounds

304

produce signal suppression in most cases when ESI source are employed

305

competition for the available charges between the analytes and the co-eluting matrix

306

compounds produces a decrease of the ionization efficiency in the interface. However,

307

the signal suppression using an ESI source performs differently in supercritical fluid

308

chromatography compared to liquid chromatography. In SFC, the analytes reach the

309

ionization source together with the modifier and the make-up flow. Methanol is

310

commonly used in SFC as a co-solvent. This solvent provides lower density and surface

311

tension compared to water, increasing the solvent evaporation rate and improving the

312

ionization efficiency. This fact, combined with the low flow reaching the source and the

313

small amount of sample injected (2 mg per injection), produces lower matrix effect

314

compared to liquid chromatography.

28,

as the

315

On the other hand, using gas chromatography, matrix interferents usually cause

316

an enhancement of the analyte signal. This fact is due to the occupation of some available

317

sites in the liner by the co-extractive matrix components producing the transfer of a larger

318

amount of analyte to the chromatographic column 29.



319

The matrix effect obtained for the pyrethroids analyzed in the 6 matrices studied

320

is detailed in table 2. Three different ranges of matrix effect can be defined depending on

321

the percentage of signal suppression/enhancement. Between 0-20% is considered low or

322

non-existent matrix effect, however, an alteration of the signal between 20-50% and

323

>50% are considered medium and strong matrix effect respectively. In all cases, in SFC

324

the medium and strong matrix effect correspond to signal suppression. Considering gas

325

chromatography, the situation was the opposite: all pyrethroids with matrix effect higher

326

than 20% showed signal enhancement with one exception, tetramethrin, which presented

327

a signal suppression of -38% in tea. Table 2. Matrix effect (%) of the 14 pyrethroids in the matrices studied. Pear Zuccini Orange Onion Tea SFC GC SFC GC SFC GC SFC GC SFC GC Achrinathrin 5 10 5 7 0 94 0 44 -22 52 Bifenthrin 10 4 8 8 -7 33 -24 28 -3 5 Cyfluthrin 10 0 15 -1 -4 40 -2 23 -57 -5 Cypermethrin -2 1 4 -2 -8 43 -17 26 -32 3 Deltamethrin 1 11 3 -3 -14 23 -63 15 -18 Etofenprox 0 1 -1 0 -8 21 -65 15 -10 -12 Fenprotathrin 18 5 20 4 -7 41 -25 30 -1 13 Fenvalerate 0 -1 4 10 -6 24 -40 12 -32 1 Flucythrinate -5 6 -5 1 -8 52 -37 34 -86 16 Lambda-cyhalothrin -7 2 -6 2 -9 43 -18 25 -19 18 Permethrin 2 3 8 5 2 48 -11 28 -10 2 Phenothrin 6 9 11 12 -8 53 -17 41 -75 18 Tau-Fluvalinate 6 9 10 -4 -6 62 -12 22 -50 35 Tetramethrin -3 4 3 6 -4 64 -2 35 -11 -38

328 329

Low matrix effect was observed in pear and zucchini matrices by both techniques.

330

These are high water content matrices and present lower co-extracted matrix compounds.

331

Regarding onion, 4 pesticides presented medium matrix effect (bifenthrin, fenpropathrin,

332

fenvalerate, flucythrinate) and 2 pesticides showed strong matrix effect (etofenprox and

333

deltamethrin) in SFC. In GC, there was no matrix effect higher than 50% but most of the

334

pesticides presented medium matrix effect except for deltamethrin, etofenprox and

335

fenvalerate. Green tea was the most complex matrix in SFC: 4 compounds showed matrix

336

effect between 20-50% (acrinathrin, cypermethrin, fenvalerate, fluvalinate-tau) and 3

337

pesticides presented matrix effect higher than 50% (cyfluthrin, flucythrinate and

338

phenothrin). Better results were obtained in GC when analyzing green tea. Using this

339

technique, only two pesticides were affected: fluvalinate-tau and acrinathrin, which

340

showed medium and strong matrix effect respectively. No matrix effect was obtained


Page 14 of 19

Page 15 of 19


341

analyzing orange by SFC, however, this matrix produced a remarkable enhancement of

342

the signal for all the compounds in GC except for fenvalerate.

343

Focusing on each pesticide, the matrix effect performance was different

344

depending on the type of chromatography used. For example, tetramethrin did not present

345

any matrix effect in SFC for the matrices studied; however, in GC, medium and strong

346

matrix effect were observed in orange, onion and green tea. A similar situation takes place

347

with lambda-cyhalothrin and permethrin.

348 349

3.5 Real samples

350

Seventeen real samples of fruits and vegetables were analyzed by both systems to

351

prove the similarity and reliability of these two types of chromatography regarding

352

pyrethroids quantitation. A variety of matrices were acquired from local markets in

353

Almeria (apple, aubergine, banana, broccoli, carrot, green beans, kiwi, leek, lemon,

354

mandarina, pear, pepper, potato, pumpkin, spinach and zucchini). Seven of the 14

355

pyrethroids validated in the method were detected in 6 different samples (table 3). The

356

pepper sample showed 3 pyrethroids (acrinathrin, cypermethrin and lambda-cyhalothrin).

357

The 5 remaining samples contained only 1 pesticide each. Cypermethrin was the only

358

pyrethroid present in more than one sample (pepper and potato). Regarding quantitation,

359

the concentrations obtained using both instruments were similar. Taking the

360

concentrations obtained by supercritical fluid chromatography as reference, the difference

361

with those of gas chromatography is lower than 25% in all cases.

362 Table 3. Concentrations (µg/kg) of the pyrethroids detected by both systems. Pesticide Matrix SFC-ESI-MS/MS GC-EI/MS/MS Acrinathrin Pepper 397 321 Pepper 639 633 Cypermethrin Potato 135 119 Deltamethrin Tangerine 158 137 Etofenprox Green Beans 502 516 Lambda-Cyhalothrin Pepper 75 80 Permethrin Broccoli 228 184 Tau-Fluvalinate Tangerine 142 137 363 364



365

Abbreviations

366

BPR, back pressure regulator; CI, chemical ionization; DL, desolvation line; EI, electron

367

ionization; ESI, electrospray ionization; GC, gas chromatography; LC, liquid

368

chromatography; LOQ, limit of quantification; MRL, maximum residue limit; MRM,

369

multireaction monitoring; MS, mass spectrometry; PSA (primary secondary amine);

370

PTFE, polytetrafluoroethylene; SFC, supercritical fluid chromatography; SFE,

371

supercritical fluid extraction.

372 373

Acknowledgments

374

The authors acknowledge support from The European Commission, DG SANTE

375

(Document Nº SANTE/11813/2017) and the European Union Reference Laboratory for

376

Fruits and Vegetables (EURL-FV).

377 378

References

379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403

1. Kaneko, H., Chapter 76 - Pyrethroid Chemistry and Metabolism. In Hayes' Handbook of Pesticide Toxicology (Third Edition), Krieger, R., Ed. Academic Press: New York, 2010; pp 16351663. 2. Soderlund, D. M., Chapter 77 - Toxicology and Mode of Action of Pyrethroid Insecticides. In Hayes' Handbook of Pesticide Toxicology (Third Edition), Krieger, R., Ed. Academic Press: New York, 2010; pp 1665-1686. 3. Regulation 94/643/EC: Commission Decision of 12 September 1994 concerning the withdrawal of authorizations for plant protection products containing cyhalothrin as active substance. 4. Commission Implementing Regulation (EU) No 1334/2014 of 16 December 2014 approving the active substance gamma-cyhalotrin, in accordance with Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market, and amending the Annex to Commission Implementing Regulation (EU) No 540/2011 and allowing Member States to extend provisional authorisations granted for that active substance Text with EEA relevance. 5. Commission implementing Regulation (EU) 2015/1885 of 20 October 2015 amending Implementing Regulation (EU) No 540/2011 as regards the extension of the approval periods of the active substances 2,4-D, acibenzolar-s-methyl, amitrole, bentazone, cyhalofop butyl, diquat, esfenvalerate, famoxadone, flumioxazine, DPX KE 459 (flupyrsulfuron-methyl), glyphosate, iprovalicarb, isoproturon, lambda-cyhalothrin, metalaxyl-M, metsulfuron methyl, picolinafen, prosulfuron, pymetrozine, pyraflufen-ethyl, thiabendazole, thifensulfuron-methyl and triasulfuron. 6. Regulation (EC) No 1107/2009 of the Europena Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC.


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7. Soderlund, D. M.; Clark, J. M.; Sheets, L. P.; Mullin, L. S.; Piccirillo, V. J.; Sargent, D.; Stevens, J. T.; Weiner, M. L., Mechanisms of pyrethroid neurotoxicity: implications for cumulative risk assessment. Toxicology 2002, 171 (1), 3-59. 8. Corcellas, C.; Feo, M. L.; Torres, J. P.; Malm, O.; Ocampo-Duque, W.; Eljarrat, E.; Barcelo, D., Pyrethroids in human breast milk: occurrence and nursing daily intake estimation. Environ Int 2012, 47, 17-22. 9. Gopal, M.; Niwas, R.; Devakumar, C., Analysis of Synthetic Pyrethroids by Gas Chromatography–Mass Spectrometry. Agricultural Research 2015, 4 (2), 208-214. 10. Raeppel, C.; Appenzeller, B. M.; Millet, M., Determination of seven pyrethroids biocides and their synergist in indoor air by thermal-desorption gas chromatography/mass spectrometry after sampling on Tenax TA® passive tubes. Talanta 2015, 131, 309-314. 11. Gao, X.; Guo, H.; Wang, J.; Zhao, Q., Sensitive and rapid determination of pyrethroids in human blood by gas chromatography–tandem mass spectrometry with ultrasound-assisted dispersive liquid-liquid microextraction. Drug Testing and Analysis 2018, 10 (7), 1131-1138. 12. Feo, M. L.; Eljarrat, E.; Barceló, D.; Barceló, D., Determination of pyrethroid insecticides in environmental samples. TrAC Trends in Analytical Chemistry 2010, 29 (7), 692-705. 13. Petrarca, M. H.; Ccanccapa-Cartagena, A.; Masiá, A.; Godoy, H. T.; Picó, Y., Comparison of green sample preparation techniques in the analysis of pyrethrins and pyrethroids in baby food by liquid chromatography–tandem mass spectrometry. Journal of Chromatography A 2017, 1497, 28-37. 14. Moloney, M.; Tuck, S.; Ramkumar, A.; Furey, A.; Danaher, M., Determination of pyrethrin and pyrethroid residues in animal fat using liquid chromatography coupled to tandem mass spectrometry. Journal of Chromatography B 2018, 1077-1078, 60-70. 15. Ccanccapa-Cartagena, A.; Masiá, A.; Picó, Y., Simultaneous determination of pyrethroids and pyrethrins by dispersive liquid-liquid microextraction and liquid chromatography triple quadrupole mass spectrometry in environmental samples. Analytical and Bioanalytical Chemistry 2017, 409 (20), 4787-4799. 16. McCoy, M. R.; Yang, Z.; Fu, X.; Ahn, K. C.; Gee, S. J.; Bom, D. C.; Zhong, P.; Chang, D.; Hammock, B. D., Monitoring of Total Type II Pyrethroid Pesticides in Citrus Oils and Water by Converting to a Common Product 3-Phenoxybenzoic Acid. Journal of Agricultural and Food Chemistry 2012, 60 (20), 5065-5070. 17. Rawn, D. F. K.; Judge, J.; Roscoe, V., Application of the QuEChERS method for the analysis of pyrethrins and pyrethroids in fish tissues. Analytical and Bioanalytical Chemistry 2010, 397 (6), 2525-2531. 18. Singh, S.; Srivastava, A.; Singh, S. P., Inexpensive, effective novel activated carbon fibers for sample cleanup: application to multipesticide residue analysis in food commodities using a QuEChERS method. Analytical and Bioanalytical Chemistry 2018, 410 (8), 2241-2251. 19. Pérez-Fernández, V.; García, M. Á.; Marina, M. L., Characteristics and enantiomeric analysis of chiral pyrethroids. Journal of Chromatography A 2010, 1217 (7), 968-989. 20. El-Saeid, M. H.; Khan, H. A., Determination of Pyrethroid Insecticides in Crude and Canned Vegetable Samples by Supercritical Fluid Chromatography. International Journal of Food Properties 2015, 18 (5), 1119-1127. 21. Bagheri, H.; Yamini, Y.; Safari, M.; Asiabi, H.; Karimi, M.; Heydari, A., Simultaneous determination of pyrethroids residues in fruit and vegetable samples via supercritical fluid extraction coupled with magnetic solid phase extraction followed by HPLC-UV. The Journal of Supercritical Fluids 2016, 107, 571-580. 22. Cutillas, V.; Galera, M. M.; Rajski, Ł.; Fernández-Alba, A. R., Evaluation of supercritical fluid chromatography coupled to tandem mass spectrometry for pesticide residues in food. Journal of Chromatography A 2018, 1545, 67-74. 23. Li, W.; Morgan, M. K.; Graham, S. E.; Starr, J. M., Measurement of pyrethroids and their environmental degradation products in fresh fruits and vegetables using a modification of the quick easy cheap effective rugged safe (QuEChERS) method. Talanta 2016, 151, 42-50.



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24. Anastassiades, M.; Lehotay, S. J.; Stajnbaher, D.; Schenck, F. J., 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), 412-31. 25. Lozano, A.; Hernando, M. D.; Ucles, S.; Hakme, E.; Fernandez-Alba, A. R., Identification and measurement of veterinary drug residues in beehive products. Food Chem 2019, 274, 6170. 26. Gas phase acidity and proton affinity studies of organic species using mass spectrometry. 2011. 27. Swart, M.; Bickelhaupt, F. M., Proton Affinities of Anionic Bases: Trends Across the Periodic Table, Structural Effects, and DFT Validation. 2006; Vol. 2, p 281-287. 28. Kostiainen, R.; Kauppila, T. J., Effect of eluent on the ionization process in liquid chromatography–mass spectrometry. Journal of Chromatography A 2009, 1216 (4), 685-699. 29. de Sousa, F. A.; Guido Costa, A. I.; de Queiroz, M. E. L. R.; Teófilo, R. F.; Neves, A. A.; de Pinho, G. P., Evaluation of matrix effect on the GC response of eleven pesticides by PCA. Food Chemistry 2012, 135 (1), 179-185.

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Table of contents

474

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Page 18 of 19

LOQ

different matrices) PageJournal 19 of 19of Agricultural and Food (6 Chemistry 70

SFC MS/MS

CO2 MeOH

ESI source

Number of results

Achrinathrin Bifenthrin Cyfluthrin Cypermethrin Deltamethrin Etofenprox Fenprotathrin Fenvalerate Flucythrinate Lambdacyhalothrin Permethrin Phenothrin Tau-Fluvalinate Tetramethrin

60 50 40 30 20 10

EI GC 0 source ACS Paragon Plus Environment2 MS/MS Helium

5

≥10

Limit of quantification (µg/kg)

Supercritical Fluid Chromatography and Gas Chromatography

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