Determination of Fungicides in Fruits and Vegetables by Time-of Flight and Ion Trap LC/MS

Abstract

This application examines the feasibility of the new instrumentation of electrospray and liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) in conjunction with liquid chromatography/ ion trap mass spectrometry (LC/ITMS) to analyze five major fungicides in fruit extracts (apple, lemon, melon, and orange) and in salad vegetables (tomato, broccoli, and pepper). Included are the LC/TOFMS and LC/ITMS MS/MS spectra of five important fungicides (carbendazim, thiabendazole, azoxystrobin, dimethomorph, and triflumizole), the TOF empirical formula and MS/MS fragmentation and diagnostic ion(s) for these fungicides in the matrices of variousimportant fruits and vegetables. A detailed rapid procedure for sample preparation and extraction of these fungicides from the fruit and vegetables is given. Spectral quality at the limit of detection (LOD), linearity, and quantitation of the fungicides in pure solvent and in the fruit and vegetable extracts using TOF and ion trap are provided. Last are the results of the analysis of real samples from the marketplace for these ungicides in fruits and vegetables: apples, oranges, lemons, and melons.

Introduction

The importance of the identification and quantitation of fungicides in fruits and vegetables was described [1, 2]. Increasingly, LC/MS is being used for the analysis of many polar fungicides of the European Union (EU) Directives and is rapidly becoming an accepted technique in pesticide residue analysis for regulatory monitoring. In particular LC/MS works well on the various families of fungicides that are polar and labile. Thus, often LC/MS methods are preferred over the older gas chromatography/mass spectrometry (GC/MS) methods. For example, the use of MS and MS/MS for pesticides (including fungicides) in food was reviewed by Pico et al. [3], and there are currently about 100 published papers dealing with the analysis of pesticides in food. Of these, there are approximately 25 papers that deal with fungicides by LC/MS, the majority published in the early 2000s. Thus, LC/MS is an emerging technology for the analysis of food products. Interestingly though, none of the reported papers in this most recent review deal with the use of LC/TOFMS and accurate mass analysis for fungicides in food.

Thus, there is an important need for research studies and methods development on the analysis of pesticides in food by accurate mass using LC/TOFMS [1, 2]. Our study in this report is one of the first of its kind to examine the new Agilent LC/MSD TOF time-of-flight mass spectrometer for the analysis of fungicides in food. This topic was chosen because of the relevance of these fungicides and their significant use on apples, oranges, lemons, melons, tomatoes, broccoli, and peppers.

Agilent Technolgy

Furthermore, LC/ITMS will be demonstrated as a companion technique to LC/TOFMS for the analysis of fungicides in fruits and vegetables. The companion use of LC/ITMS was described in more detail [2].

The molecular structures of the common fungicides of this study (carbendazim, thiabendazole, azoxystrobin, dimethomorph, and triflumizole) are

shown in Figure 1.

Experimental Methods

Fruit and Vegetable Extraction (QuEChERS)

QuEChERS is the acronym for the method of extraction, which stands for quick, easy, cheap, effective, rugged, and safe [4]. It is a method that is receiving wide acceptance for rapid extraction of pesticides in food.

1. Weigh 15 g of a previously homogenized sample into a 40-mL Teflon centrifuge tube. Add 15 mL of acetonitrile (containing 1% acetic acid), 6 g of anhydrous MgSO4 and 2.5 g of NaAc•3H2O (sodium acetate trihydrate) and shake the sample vigorously for 1 min by using a Vortex mixer at maximum speed or by hand shaking. Afterwards, centrifuge for 3 min at 3700 rpm.

2. Take 5 mL of the supernatant into a 15-mL tube, add 250 mg of PSA adsorbent and 750 mg of MgSO4, and vortex for 20 s. Then microcentrifuge again for 3 min at 3700 rpm. Transfer 1.0 mL into an autosampler vial. Then evaporate the fruit and vegetable residues to dryness and redissolve in 8/92% methanol/ water.

Standards were prepared by fortifying with a mixture of carbendazim, thiabendazole, azoxystrobin, dimethomorph, and triflumizole at concentrations ranging from 0.01 to 0.5 mg/kg for analysis using the Agilent LC/MSD TOF and the Agilent 1100 Series LC/MSD Trap.

Nonfortified samples were analyzed directly at this same point by either the LC/MSD TOF or the LC/MSD Trap by injecting 50 μL.

The extraction method is summarized in Figure 2.

Agilent LC/MSD TOF Methods

• LC Pumps were Agilent 1100 binary pumps, injection volume 50 μL with standard Agilent 1100 ALS.

• Column: ZORBAX® Eclipse XDB-C8, 4.6 mm × 150 mm, 5 μm, part number 993967-906

• Mobile Phase A=acetonitrile and B=0.1% formic acid in water, gradient was 10% A for 5 min, then to 100% A in 25 min at a flow rate of 0.6 mL/min

• Agilent LC/MSD TOF with electrospray source

• Positive ESI, Capillary 4000 V

• Nebulizer 40 psig, drying gas 9 L/min, gas temp 300 °C

• Fragmentor 190 V, Skimmer 60 V, Oct DC1 37.5 V, OCT RF V 250 V

• Reference Masses: m/z 121.0509 and 922.0098, resolution: 9500±500 @ m/z 922.0098, Reference A Sprayer 2 is constant flow rate during the run

Agilent 1100 Series LC/MSD Trap Methods

• Chromatographic methods were identical to Agilent LC/MSD TOF for direct comparison of peaks

• LC Pumps were Agilent 1100, injection volume 50 μL

• Column: ZORBAX Eclipse XDB-C8, 4.6 mm × 150 mm, 5 μm, part number 993967-906

• Mobile Phase A=acetonitrile and B=0.1% formic acid in water, gradient was 10% A for 5 min, then to 100% A in 25 min at a flow rate of 0.6 mL/min.

• Agilent 1100 Series LC/MSD Trap

• Positive electrospray ionization (ESI), Capillary 3200 V

• Nebulizer 40 psig, drying gas 9 L/min, gas temp 300 °C

• Capillary exit 70 V

• Trap accumulation, 50,000 counts

Results and Discussion

Fragmentation and Mass Accuracy

Figure 3 shows the LC separation of five fungicides: carbendazim, thiabendazole, azoxystrobin, dimethomorph, and triflumizole. The sample contained the ungicides at 0.125 mg/kg (ppm) in a fortified lemon extract. The extracted ions for each of the compounds are shown in Figure 3b. The window of extraction was clean, which is commonly a feature of accurate mass extraction of ions, where the width of the window of extraction may be narrowed to (0.02 amu) or ∼100 ppm. In Figure 3, the window of extraction was 0.1 amu.

At concentrations as low as 0.05 mg/kg, the extracted ions (m/z 192, 202, 346, 388, and 404) still yielded clean chromatograms testifying to the importance of accurate mass and its ability to give clean extracted ion chromatograms (EICs) with a narrow mass window. This accurate mass window is reflected in the determinations of the accurate mass of the protonated molecule of each of the fungicides. Table 1 shows the elemental composition, the exact mass, and errors, in mDa and ppm, for each of the fungicides at the 0.50 mg/kg concentration. Mass accuracy was always better than 2 ppm in all the fruit and vegetable matrices, except for dimethomorph.

These results show that the use of continuous calibration is effective for accurate mass across an order of magnitude concentration range in complex fruit and vegetable matrices.

Figures 4a-4e show the fragmentation pathway of each of the five fungicides, based on accurate mass spectra with pure standards in a methanol solution. The fragmentation ions of the LC/MSD TOF were verified with the LC/MSD Trap spectra at MS2 with identical chromatographic conditions. Beginning with carbendazim, the MS2 shows that the protonated molecule loses methanol (32 Da) to give the m/z 160 fragment ion (Figure 4a). This fragment ion of m/z 160 is also seen in the LC/MSD TOF.

The fragmentation pathway for thiabendazole is shown in Figure 4b with the protonated molecule, m/z 202, fragmenting to give the m/z 175, with the neutral loss of 27 Da or HCN.

The fragmentation pathway for azoxystrobin is shown in Figure 4c with the protonated molecule, m/z 404, fragmenting to m/z 372 (loss of methanol of 32 Da) at MS2, and to m/z 342 at MS^3.

The fragmentation pathway for dimethomorph is shown in Figure 4d with the protonated molecule, m/z 388, fragmenting to give the m/z 301 at MS2, then m/z 165 at MS3.

The fragmentation pathway for triflumizole, m/z 346, is to m/z 278 at MS2, then to m/z 250 at MS3 (Figure 4e). The combination of the LC/MSD Trap and LC/MSD TOF is extremely valuable for interpretation of spectra in that both instruments work well for these compounds and give complementary information on the structure and identity.

Linearity and Detection Limits

Calibration curves were established for the five fungicides using both the LC/MSD TOF and LC/MSD Trap over the analyte concentration range of interest, which was from 0.01 mg/kg to 0.5 mg/kg (ppm) in solvent and in each of the fruit and vegetable matrices (orange, lemon, melon, broccoli, and pepper). Results showed the similarity among matrices with the LC/MSD TOF and LC/MSD Trap, given the variability in fruit and vegetable matrices. For example, Figures 5a-5b show the standard curves for two representative fungicides, carbendazim and azoxystrobin. These figures demonstrate that there was little or no matrix suppression in the LC/MSD TOF system for the fruit extracts up to 0.5 ppm of parent compound for carbendazim, thiabendazole, and azoxystrobin. However, triflumizole and dimethomorph showed both enhanced signal and suppressed signal (data not shown here). The enhancement occurred for the fruit extracts of melon, orange, and lemon. The vegetable extracts of pepper showed no enhancement and some suppression for broccoli was observed. One possible explanation

for these results for triflumizole has to do with the late retention time of the analyte (∼26 min), which means that the mobile phase is approximately 80% acetonitrile. The ESI signal is susceptible to matrix effects at these high concentrations of organic solvent [5], which indicates the importance of using

matrix matched standards for unknown analysis of food extracts. Thus, best accuracy for reporting concentrations was with the standard curve made up in matrix for each fruit or vegetable. This was the procedure that was used for unknown analysis in real fruit and vegetable samples.

Furthermore, Figures 5a-b also show that the standard curves were linear across the range of concentration tested with correlation coefficients of 0.990 to 0.999 for all matrices tested and for all four of the fungicides (Table 2). Similar results for standard curves were seen with the LC/MSD Trap, with correlation coefficients typically of 0.990±0.001.

Table 3 shows the limits of detection (LODs) of the fungicides for various matrices. Typically, the values of Table 3 vary from 0.001 ppm to 0.010 ppm. The European standard for pesticides with no regulatory standard is 0.010 ppm. Thus, the LC/MSD TOF is sufficiently sensitive to detect these compounds in all matrices. A similar result was achieved for the LC/MSD Trap for LODs (Table 4) using single MS. The accurate mass capabilities of the LC/MSD TOF appear capable of seeing through the complexity of the fruit and vegetable matrices without interferences to the LODs. These LOD results of the various fungicides in fruits and vegetables are capable of meeting the regulation limits of Spain and at those of the EU.

For example, carbendazim is regulated at 0.10 mg/kg in tomatoes, thiabendazole is 0.50 mg/kg for tomatoes, while azoxystrobin is not regulated in tomatoes in Spain; therefore, the maximum residue limit is automatically 0.01 mg/kg. The LC/MSD TOF is capable of these LODs for each of the fungicides in the various fruit and vegetable matrices.

The quantitation for real fruit extracts from actual grocery store samples are shown in Table 5 for LC/MSD TOF analysis of fungicides with LC/MSD Trap confirmation. Thus, these data show that the LC/MSD TOF is capable of accurate mass measurements of the fungicides in fruit and vegetable extracts with LODs needed for their monitoring in the EU.

Conclusions

• LC/MSD TOF analysis is a powerful tool for identification of fungicides in fruits and vegetables and is a new tool for environmental food chemistry.

• Quantitation is easily possible over 2 orders of magnitude with accuracy better than 3 ppm, typically less than 2 ppm, and in this work was an amazing 1 ppm in ESI+ ion for most compounds!

• Elemental composition of fungicides and fragment ions are possible with LC/MSD TOF, and further confirmed with LC/MSD Trap.

• LODs of the five fungicides in fruit and vegetables were from 0.001 to 0.010 μg/g. These concentrations are equal to or better than the EU directives for controlled fungicides in fruits and vegetables.

References

1. Imma Ferrer and E. Michael Thurman, (2004) "Determination of chloronicotinyl insecticides in salad vegetables by LC/MSD TOF and LC/MSD ion trap", Agilent Technologies, publication 5989-1842EN www.agilent.com/chem

2. E. Michael Thurman and Imma Ferrer, (2005) "Identification of unknown pesticides in food using both LC/MSD TOF and Ion Trap MSn", Agilent Technologies, publication 5989-1924EN www.agilent.com/chem

3. Y. Pico, C. Blasco, and G. Font, (2004) Mass Spectrometry Reviews, 23, 45-85. 4. M. Anastassiades, S.J. Lehotay, D. Stajnbaher, and F.J. Schenck, (2003) Journal of AOAC International, 86: 412-431.

5. E.M. Thurman, I. Ferrer, and A.R. Fernández-Alba, Chapter 8: "LC/MS I. Basic Principles and Technical Aspects of LC/MS for Pesticide Analysis",

in Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Ed. A.R. Fernández-Alba, Elsevier, Amsterdam, 2005.

Acknowledgements

We acknowledge Amadeo Fernández-Alba, Department of Hydrogeology and Analytical Chemistry, University of Almería for analytical support.

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