Determination of Pesticides in Foodstuffs by LC/MS/MS Application Food Safety Authors Masahiko Takino Agilent Technologies, Inc. Hachioji, Tokyo Japan Toshitsugu Tanaka Kobe Institute of Health Department of Food Chemistry Chuo-ku, Kobe Japan Abstract A sensitive and selective analytical method for the determination of pesticide residues in several foodstuffs using the Agilent G0AA Triple Quadrupole Mass Spectrometer (QQQ) was developed. This method use two different sample preparation methods followed by LC/MS/MS (liquid chromatography/tandem mass spectrometry). The limits of detection for all pesticides were less than 0 ng/ml in foodstuff. The sensitivity of QQQ easily met the maximum residue limits (MRLs) of all investigated pesticides in Japan Food Hygiene Law. Introduction Pesticides are widely used in agricultural practices. The main application can be classified in production and post-harvest treatment of agricultural commodities for transport purposes. In this sense, production agriculture comprises the main category of use of pesticides subject to control requirements and, therefore, maximum residue levels (MRLs) have been fixed to assess food safety. In recent years, the established regulations regarding MRLs in commodities have been more and more stringent. In Japan, the positive list system was introduced this year, and MRLs have been set for over 00 pesticides in all foodstuffs. This new system sets different MRLs for each pesticide within each food group. Typically, the MRLs range from 0.0 to 3 µg/g depending on the commodities and pesticides. The low MRLs fostered the development of more sensitive analytical methods to meet the requirements of complex samples. In this sense, liquid chromatography/tandem mass spectrometry (LC/MS/MS) with QQQ in multiple reaction monitoring (MRM) mode has become so far the most widely used techniques for the quantitation of polar pesticides in food. MRM mode provides for more specific detection in a complex matrix such as food. In this work, pesticides (Tables and ) are analyzed in two separate runs with sample analytical conditions. The sensitivity requirements set by the positive lost system for these pesticides are easily met. Experimental Chemicals The acetonitrile was of LC/MS grade from Wako Pure Chemical Ind (Japan). Toluene, acetone, n- hexane, formic acid, sodium chloride, and anhydrous sodium sulfate were of analytical grade from Wako Pure Chemical. All SPE cartridges were purchased from Spelco Japan (Japan). Pesticide standards were obtained from Hayashi Pure Chemical (Japan).
Sample Preparation Extraction Vegetable and fruit samples were obtained from the local markets. A sample of 0 to 00 g was chopped in a food processor to obtain thoroughly mixed homogenates. A 0-g portion of sample homogenate was weighed in a 00-mL PTFE centrifuge tube. Then 0 ml of acetonitrile was added and blended in a Polytoron. The extract was then filtered by applying vacuum. The filtrate was collected and the residue was re-extracted with 0 ml of acetonitrile. The filtrates were combined in a 00-mL volumetric flask and made up to volume with acetonitrile. A 0-mL portion of the extract was transferred into a PTFE centrifuge tube, and 0 g of NaCl and 0 ml of 0. M phosphate buffer (ph 7.0) were added to the extract followed by shaking for min. Five grams of anhydrous Na SO were added to the acetonitrile layer obtained after salting out. After removing anhydrous Na SO, the extract was evaporated to dryness by rotary evaporator (water bath temperature did not exceed 0 C). The residue was dissolved in ml of acetonitrile-toluene (3:). Cleanup Group - The extract was loaded into a GCB/amino propyl SPE cartridge (00 ng/00 mg) preconditioned with 0 ml of acetonitrile-toluene (3:). The 0 ml of acetonitrile-toluene (3:) was further added to the SPE cartridge. All eluate was collected and evaporated by rotary evaporator. The residue was dissolved in ml of methanol. Group - The extract was loaded into a silica gel SPE cartridge (00 mg) preconditioned with 0 ml each of methanol, acetone, and n-hexane (0 ml of methanol, 0 ml of acetone, and 0 ml of n-hexane, total volume is 30 ml). The 0 ml of acetone-triethylamine-n-hexane (0:0.:80) was further added to the SPE cartridge. All eluate was discarded. The 0 ml of acetone-methanol (:) was applied and the eluate was collected and evaporated by rotary evaporator. The residue was dissolved in ml of methanol. LC/MS/MS Instrument The LC/MS/MS system used in this work consists of an Agilent 00-series vacuum degasser, binary pump, well-plate autosampler, thermostatted column compartment, and the Agilent G0 Triple Quadrupole Mass Spectrometer with an electrospray ionization source (ESI). The objective of the method development was to obtain a fast and sensitive analysis for quantifying pesticides in fruits and vegetables. For chromatographic resolution and sensitivity, different solvents and columns were optimized. It was found that a simple solvent system using water, acetonitrile, formic acid, formic acetate, and a.8-µm particle size C8 column would work very well. LC Conditions Instrument: Column: Agilent 00 HPLC ZORBAX Extend C8, 00 mm. mm,.8 µm (p/n 78700-0) Column temp: 0 C Mobile phase: A = 0.% formic acid + mm ammonium formate in water B= Acetonitrile Gradient: 0% B at 0 min, 80% B at 30 min Flow rate: 0. ml/min Injection vol: µl MS Conditions Instrument: Agilent 0 QQQ Source: Positive ESI Drying gas flow: 0 L/min Nebulizer: 0 psig Drying gas temp: 30 C V cap: 000 V Scan: m/z 00 to 0 Fragmentor: Variable 00 V MRM ions: Shown in Tables and Collision energy: Shown in Tables and LC/MS/MS Method Quantitative analysis was carried out using MRM mode with time program. The parameters of MRM transition are shown in Tables and. Standard Preparation Stock solutions of individual pesticides were prepared in methanol at µg/ml. Serial dilutions using methanol produced a range of standard mixture solutions at 0.00 µg/ml to µg/ml. The blank matrix residues were fortified with a mixture of pesticides studied at 0 ng/g.
Table. Data Acquisition Parameters of MRM Transitions of Each Pesticide in Group RT Molecular Precursor Product Collision No Pesticides (min) weight ion (m/z) ion (m/z) energy(v) Thiabendazole.08 0 0 7 0 Thiamethoxam. 3 Clothianidin 7.83 0 0 Chloridazon 8. 0 0 Imidacloprid 8.3 0 0 Dimethirimol 8.8 0 0 7 0 7 Oxycarboxine.0 7 8 7 0 8 Thiacloprid.03 3 0 Azamethiophos.87 3 3 83 0 0 Ferimzone(E) 3. 0 Ferimzone(Z) 3.7 3 0 Phenmedipham 7.77 37 38 3 0 3 Azinphos-methyl 7. 38 3 77 Simeconazole 8. 3 70 Isoxaflutol 8.7 3 30 Pyriftalid 8.7 38 3 3 0 7 Tridemorph. 7 8 30 8 Methoxyfenozide 0.0 3 33 0 Chromafenozide 0.7 3 7 0 0 Fenoxycarb 0.3 30 30 88 Naproanilide.7 7 0 Butafenacil. 33 0 3 Cyazofamide.7 3 3 08 0 Anilofos. 37 38 0 Pyrazolate 3. 38 3 73 Benzofenap 30 3 0 0 7 Cyflufenamid.3 3 0 8 Indoxacarb.37 7 8 0 Clomeprop.78 37 373 30 Cloquincet-mexyl.8 33 33 38 3 Furathiocarb.7 3 383 3 Lactofen.3 78 7 3 33 Tralkoxydim.7 3 330 8 0 Table. Data Acquisition Parameter of MRM Transitions of Each Pesticide in Group RT Molecular Precursor Product Collision No Pesticides (min) weight ion (m/z) ion (m/z) energy (V) Flumetsulam. 3 3 0 Thidiazuron. 0 0 0 3 Imazaquin. 3 3 7 0 Thifensulfuron-methyl.8 387 388 7 0 Florasulam 3.7 3 30 0 Forchlorfenuron-methyl.3 7 8 0 7 Clorasulam-methyl. 30 38 0 8 Diclosulam.83 0 0 0 Fomesafen 8.7 38 3 0 0 Triflusulfuron-methyl. 3 Haloxyfop.7 3 3 3 3
Results and Discussion Optimization of MRM Transitions Determination of the optimal MRM transitions for each pesticide was carried out using full scan mode followed by product ion scan mode using two pesticide standard mixtures at µg/ml. TICs of these standard mixtures in full scan mode and product ion scan mode are shown in Figures and. The mass spectrum of each pesticide by full scan mode exhibited protonated molecular ions; [M+H] + as the base peak ion except azinphos-methyl, furathiocarb, and fomesafen, which exhibited fragment ion and ammonium adduct ion [M+NH ] +. These ions were selected as precursor ions for MRM mode. It was possible to generate individual product ion MS/MS spectrum of each pesticide by using multiple acquisition and time programming mode. As shown in Tables and, 0 time segments for 33 pesticides in group and 7 time segments for pesticides in group were used for MRM mode. Total ion chromatograms of pesticide standard mixture corresponding to the minimum MRL value for pesticides (0 ng/ml) are shown in Figure 3. These show excellent signal-to-noise (S/N) ratios for all pesticides. The limit of detection (LOD) for each pesticide was determined using an S/N ratio of 3 with an MRM chromatogram of each pesticide at ng/ml (see Table 3). To evaluate the linearity of the calibration curves, various concentrations of pesticide standard solutions ranging from 0.00 ng/ml to ng/ml were analyzed. As shown in Table 3, the linearity was very good for all pesticides with correlation coefficients (r ) greater than 0.8 The matrix effect of this method was investigated by using orange, apple, potato, and cabbage extracts spiked with pesticide standards at 0 ng/ml. Typical MRM chromatograms of orange extract are shown in Figures and. The other chromatograms of apple, potato, and cabbage extract are shown in Figure. There was not additional peak from sample matrix in all food when compared with the pesticide standard mixture. These results indicate that MRM mode has very high selectivity. x0 7. (A),30 7,8 0 8, 0 0.8 0. 3, 3 7 3 7, 8 3 3 33 3 7 3 7 3 7 3 Abundance versus acquisition time (min) x0. (B). 7, 8 0, 0, 30 3. 0. Figure. 3 3 7 3 7 3 7 3 3, 7 8 Abundance versus acquisition time (min) 3 7, 8 TIC of 33 pesticides standard in full scan mode (A) and product ion scan mode (B) at µg/ml. 3 33
x0 7 (A) 3.0 3.0 0.0.0 7 8 x0 3.0 3 7 8 0 3 7 8 0 3 Abundance versus acquisition time (min) 3 (B).0 0.0 7 8 3 7 8 0 3 7 8 0 3 Abundance versus acquisition time (min) Figure. TIC of pesticides standard in full scan mode (A) and product ion scan mode (B) at µg/ml. Furthermore, the change on the peak intensity of each pesticide by sample matrix was calculated by comparing with the peak intensity of pesticide standards. As these results show in Table, the relative intensity of each pesticide ranged from to %. Thus, matrix effect such as ion suppression may be insignificant and it was possible to use external standards instead of matrix matched standards. The repeatability of each pesticide in orange extract is also shown in Table, and the RSD of each pesticide was in the range from.7 to.%.
7,8 000 (A),30 000 0 8,0, 000 000 3 0 3, 7 3 8 7 3 33 3 3 7 3 7 3 7 Abundance versus acquisition time (min) 000 (B) 0 000 8000 8 000 3 7 3 7 3 7 3 7 Abundance versus acquisition time (min) Figure 3. TIC of 33 pesticide standards (A) and pesticides standard (B) at 0 ng/ml in MRM mode. Table 3. Linearity and LOD of Pesticide Standard Solutions No Pesticides r LOD (ng/ml) No Pesticides r LOD (ng/ml) Group Thiabendazole 0. <0. 8 Methoxyfenozide 0.3 0. Thiamethoxam 0. <0. Chromafenozide 0. 0. 3 Clothianidin 0. <0. 0 Fenoxycarb 0.88 <0. Chloridazon 0.3 <0. Naproanilide 0.3 <0. Imidacloprid 0. <0. Butafenacil 0. <0. Dimethirimol 0.8 <0. 3 Cyazofamide 0.87 0.3 7 Oxycarboxine 0.3 <0. Anilofos 0. <0. 8 Thiacloprid 0. <0. Pyrazolate 0.0 0. Azamethiophos 0.88 <0. Benzofenap 0.8 0. 0 Ferimzone(E) 0.3 0.3 7 Cyflufenamid 0.3 0.3 Ferimzone(Z) 0. 0.3 8 Clomeprop 0.3 0. Phenmedipham 0.3 <0. Indoxacarb 0..0 3 Azinphos-methyl 0.7 <0. 30 Quinclorac-methyl 0.88 0.3 Simeconazole 0. <0. 3 Furathiocarb 0.87 <0. Isoxaflutol 0. <0. 3 Lactofen 0.87.0 Pyriftalid 0.88 <0. 33 Tralkoxydim 0. 0. 7 Tridemorph 0.. Group Flumetsulam 0. <0. 7 Clorasulam-methyl 0.87 <0. Thidiazuron 0. <0. 8 Diclosulam 0.8 <0. 3 Imazaquin 0. <0. Fomesafen 0.8 0.3 Thifensulfuron-methyl 0.8 <0. 0 Triflusulfuron-methyl 0. <0. Florasulam 0. <0. Haloxyfop 0. 0. Forchlorfenuron-methtyl 0.77 <0.
000 000 m/z = 0>>>7 700 3 m/z = 3>>>77 0 30 m/z = >>> 00 00 m/z = >>>70 00 3 m/z = 0>>> 00 00 m/z = 30>>> 0 3 7 8 0 700 m/z = >>>0 0 0 0 m/z = 3>>>3 00 m/z = >>>0 7 m/z = 8>>>30 800 00 m/z = 0>>>7 00 00 8 m/z = 33>>> 3 7 8 0 3 00 00 7 m/z = 8>>>7 00 00 m/z = 7>>> 000 000 8 m/z = 3>>> 00 0 00 m/z = 30>>>88 0 00 000 m/z = 3>>>83 00 m/z = >>>7 7 8 0 3 00 00 0 m/z = >>> 00 m/z = >>>33 00 3 00 m/z = >>>3 00 m/z = 3>>>08 0 000 m/z = 38>>>3 0 3 7 8 0 00 000 m/z = 38>> 7 8 0 3 Figure. MRM of 33 pesticides in orange extract spiked at 0 ng/ml. (Continued) 7
00 000 3 00 m/z = 3>>>73 00 m/z = 383>>> 00 0 3 80 m/z = 3>>>0 0 m/z = 7>>>3 80 7 33 00 m/z = 3>>> 00 m/z = 330>>>8 800 7 8 0 3 8 3 7 8 00 m/z = 373>>> m/z = 8>>>0 30 00 m/z = 33>>>38 3 7 8 Figure. MRM of 33 pesticides in orange extract spiked at 0 ng/ml. 000 000 m/z = 3>>> 000 000 7 m/z = 30>>>38 700 00 m/z = >>>0 8000 000 8 m/z = 0>>> 700 00 3 m/z = 3>>>7 800 00 m/z = >>>3 0000 0000 700 00 m/z = 388>>>7 m/z = 30>>> 0000 0000 000 000 0 m/z = 3>>> m/z = 3>>>3 0000 000 m/z = 8>>> 7 8 0 3 7 8 0 3 Figure. MRM of pesticides in orange extract spiked at 0 ng/ml. 8
7, 8 000 (A) 8, 0,, 30 0 000 3 0 3, 7 3 7 8 3 3 33 000 (B) 0 000 000 (C) 0 000 3 7 3 7 3 7 000 (D) 0 0000 000 3 7 8 000 (E) 0000 000 000 (F) 0000 000 3 7 3 7 Figure. TIC of spiked at 0 ng/ml.
Table. Relative Intensity of Each Pesticide in Sample Extracts No Pesticides Relative intensity(%) Group Orange* Cabbage Apple Potato Thiabendazole 0 (3.) 0 07 Thiamethoxam 03 (.) 8 0 0 3 Clothianidin 0 (.) 0 0 Chloridazon 0 (3.3) 0 0 0 Imidacloprid 0 (.7) 7 0 0 Dimethirimol 03 (.) 07 03 08 7 Oxycarboxine 0 (3.7) 0 0 0 8 Thiacloprid 0 (3.) 0 0 08 Azamethiophos 3 (.) 0 8 0, Ferimzone(E,Z) (.) 0 0 Phenmedipham (.3) 00 0 3 Azinphos-methyl 0 (.) 03 0 0 Simeconazole 0 (.) 0 0 0 Isoxaflutol 0 (.7) 0 08 03 Pyriftalid 7 (.) 03 0 3 7 Methoxyfenozide (3.) 0 7 8 Chromafenozide (.8) 0 03 0 Tridemorph 7 (3.) 00 0 Fenoxycarb (.) 0 0 0 Naproanilide (.3) 7 8 03 Butafenacil 0 (.) 0 3 Cyazofamide 3 (3.) 87 Anilofos 0 (.7) 0 03 07 Pyrazolate 03 (.7) 0 03 7 Benzofenap 08 (.) 8 08 7 Cyflufenamid 08 (3.) 0 0 0 Indoxacarb 0 (.) 0 00 8 Clomeprop 0 (.) 07 0 0 30 Cuinclorac-methyl 0 (.) 0 0 0 3 Furathiocarb 0 (.8) 0 0 0 3 Lactofen 00 (3.7) 0 0 33 Tralkoxydim 0 (3.3) 0 7 Group Flumetsulam 7 (.) 0 0 Thidiazuron 0 (.8) 0 0 3 3 Imazaquin 0 (3.) 00 00 0 Thifensulfuron-methyl 0 (.) 3 Florasulam (3.) 0 03 0 Forchlorfenuron-methyl 0 (.) 03 00 08 7 Clorasulam-methyl (3.) 0 7 8 Diclosulam (3.3) 0 07 Fomesafen (.) 0 0 0 Triflusulfuron-methyl 7 (.) 0 08 Haloxyfop 08 (.8) 0 *( ): RSD,% calculated based on five replicates within one day 0
Conclusions The multiresidue method by LC/MS/MS described here was suitable for the determination of pesticides in a variety of food samples due to its high sensitivity and high selectivity. Another advantage of this method is that ion suppression was not observed for all food samples studied. Thus, it may eliminate the need for matrix-matched standards, which make analysis more tedious for samples from different origins. For more details concerning this application, please contact masahiko_takino@agilent.com For More Information For more information on our products and services, visit our Web site at www.agilent.com/chem.
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