B American Society for Mass Spectrometry (outside the USA), 2014
J. Am. Soc. Mass Spectrom. (2014) DOI: 10.1007/s13361-014-0912-1
RESEARCH ARTICLE
Accurate Mass Fragment Library for Rapid Analysis of Pesticides on Produce Using Ambient Pressure Desorption Ionization with High-Resolution Mass Spectrometry Sara E. Kern, Lora A. Lin, Frederick L. Fricke U.S. Food and Drug Administration, Forensic Chemistry Center, 6751 Steger Drive, Cincinnati, OH 45237, USA
Abstract. U.S. food imports have been increasing steadily for decades, intensifying the need for a rapid and sensitive screening technique. A method has been developed that uses foam disks to sample the surface of incoming produce. This work provides complimentary information to the extensive amount of published pesticide fragmentation data collected using LCMS systems (Sack et al. Journal of Agricultural and Food Chemistry, 59, 6383–6411, 2011; Mol et al. Analytical and Bioanalytical Chemistry, 403, 2891–2908, 2012). The disks are directly analyzed using transmission-mode direct analysis in real time (DART) ambient pressure desorption ionization coupled to a high resolution accurate mass-mass spectrometer (HRAM-MS). In order to provide more certainty in the identification of the pesticides detected, a library of accurate mass fragments and + isotopes of the protonated parent molecular ion (the [M+H] ) has been developed. The HRAM-MS is equipped with a quadrupole mass filter, providing the capability of “data-dependent” fragmentation, as opposed to “all -ion” fragmentation (where all of the ions enter a collision chamber and are fragmented at once). A temperature gradient for the DART helium stream and multiple collision energies were employed to detect and fragment 164 pesticides of + varying chemical classes, sizes, and polarities. The accurate mass information of precursor ([M+H] ion) and fragment ions is essential in correctly identifying chemical contaminants on the surface of imported produce. Additionally, the + inclusion of isotopes of the [M+H] in the database adds another metric to the confirmation process. The fragmentation data were collected using a Q-Exactive mass spectrometer and were added to a database used to process data collected with an Exactive mass spectrometer, an instrument that is more readily available for this screening application. The commodities investigated range from smooth-skinned produce such as apples to rougher surfaces like broccoli. The minimal sample preparation and absence of chromatography has shortened the analysis time to about 15 min per sample, and the simplicity and robustness of the technique make it ideal for rapid screening. Keywords: DART ionization, Exactive Orbitrap, Q-Exactive Orbitrap, Pesticide analysis, Surface screening technique, Rapid analysis Received: 27 December 2013/Revised: 4 April 2014/Accepted: 5 April 2014
Introduction
O
ver the past three decades, fruit and vegetable consumption has increased in the U.S. with much of that produce being grown overseas. In 2010, 48.8% of fresh fruits and 25% of fresh vegetables consumed in the U.S. were imported, an increase of 8.4% for fresh fruits and 15.1% for fresh vegetables since 1990 [3]. The U.S. Food Electronic supplementary material The online version of this article (doi:10.1007/s13361-014-0912-1) contains supplementary material, which is available to authorized users. Correspondence to: Sara E. Kern; e-mail:
[email protected]
and Drug Administration (FDA) is charged with inspecting domestic and imported produce for food safety purposes, including analyzing the produce for pesticide residues and enforcing established pesticide tolerances. There are two ways that pesticide residue violations may occur. In one instance, a pesticide residue may be present at a level that exceeds the tolerance that is established for a particular pesticide on a specific food commodity. The more commonly observed violation occurs when a residue is detected on a food item for which a tolerance has not been established (i.e., the pesticide is not allowed to be used on that commodity at any level). Pesticides such as insecticides, herbicides, and fungicides have been used frequently in agriculture for decades. Pesticides typically kill pests and fungi or render them ineffective. This
S. E. Kern et al.: Accurate Mass Fragments of Pesticides
mode of action means that they may also cause ill effects in unintended organisms such as humans [4]. The potential for human harm from pesticide usage has necessitated the development of analytical techniques capable of detecting many different types of compounds at the residue level [5]. The variety of insecticides, herbicides, and fungicides currently available presents a real analytical challenge, as they span a range of physico-chemical characteristics that are not amenable to just one technique. Gas chromatography (GC) with mass spectral detection (GCMS) was the standard technique for many years, although liquid chromatography (LC) with MS detection (LCMS) has become a much valued complementary technique that is preferred for pesticides that are thermally unstable or highly polar [6]. Owing to the variety of pesticide classes and functionality present on the market, it is not uncommon to have many different pesticides present on a particular crop. More recently, an ambient pressure desorption ionization technique has proven to be useful for detecting pesticides [7–9] as well as many other chemical compounds. Direct analysis in real time (DART) is a highly versatile ionization technique that saves time and solvent because it requires little or no sample preparation and eliminates the need for chromatography [10]. This technique offers many advantages in terms of ease of sample handling and increased throughput. To date, the DART ionization source has been successfully utilized in a variety of applications, including metabolic fingerprinting [11, 12], screening for toxic glycols in glycerin-containing products [13], detection and characterization of synthetic cathinone designer drugs or “bath salts” [14], analysis of mycotoxins in cereal [15], and detection of counterfeit drugs [16]. A surface sampling technique in which polyurethane foam swabs were used to collect pesticides and other chemical contaminants from the skins of imported produce has been previously reported [17, 18]. Transmission mode analysis of the foam swabs using a DART ionization source coupled to a high resolution Exactive mass spectrometer was conducted, and hundreds of pesticides from different classes were detected rapidly and with excellent mass accuracy, offering a quick screening procedure to process large numbers of samples [17, 18]. However, the original technique suffered from potential false positives because of a lack of verifying data such as chromatographic retention time data and fragmentation information. In order to gain more confidence in the identification of potential contaminants, an accurate mass database containing precursor ions, fragments, and isotopes of the [M+H] + ion has been developed using a Q-Exactive mass spectrometer. This instrument has the ability to perform data-dependent fragmentation through the use of a quadrupole mass filter. The fragmentation data collected using the QExactive were added to a database used to process data collected with an Exactive mass spectrometer, which is an instrument that is more readily available in the discipline.
Herein, we report this database and describe the method developed, including a temperature gradient for the DART ionization source (to facilitate separation attributable to the varying thermal desorption profiles of the compounds of interest) and alternating normalized collision energies utilized by the high energy collision dissociation (HCD) cell of the Exactive. The method was developed to be versatile enough to ionize, fragment, and detect a wide range of chemical compounds rapidly and with confidence.
Experimental Materials and Reagents Polyurethane foam (ITW Texwipe, USA) was obtained from Fisher Scientific (Pittsburgh, PA, USA). The following HPLCgrade solvents were purchased from Fisher Scientific and used without further purification: methanol, isopropyl alcohol, acetone, 95% hexane, ethyl acetate, and acetonitrile. Formic acid (88%) was purchased from Aldrich (St. Louis, MO, USA). Anhydrous caffeine was acquired from Sigma (St. Louis, MO, USA). Agilent (Santa Clara, CA, USA) ESI-L G1969-8500 Low Concentration Tuning Mix was used to calibrate the Q-Exactive and Exactive mass spectrometers using the DART source.
Pesticide Standard Mixtures Pesticide standards were obtained from the USEPA Pesticide Repository (Ft. Meade, MD, USA), Fluka/Sigma Aldrich (St. Louis, MO, USA), Wako Chemicals USA (Richmond, VA, USA), Honeywell Riedel-de Haëm (Seelze, Germany), and EQ Laboratories (Atlanta, GA, USA). Individual stock standards were prepared at a concentration of 1 mg ml−1 in acetonitrile. From the stock standards, 17 pesticide mixtures were prepared, each containing up to 10 pesticides at concentrations of 10 μg ml−1 in acetonitrile.
Instrumentation Thermo Scientific Q-Exactive and Exactive Orbitrap Mass Spectrometers (Thermo Fisher Scientific, West Palm Beach, FL, USA).
Mass Spectrometer Parameters The data for this study were obtained through two sets of experiments. The first set of experiments was to identify high resolution fragments of the pesticide standards using the QExactive instrument. Data acquisition was performed with Thermo Scientific Q-Exactive 2.0 SP1 software. Data analysis was performed with Thermo Scientific ToxID 2.1.2 SP2. The mass spectrometer was mass calibrated daily by infusing calibration solution into the DART source using a flow rate of 5 μL min−1 as reported previously [17]. Table 1 lists the experimental parameters for the Q-Exactive.
S. E. Kern et al.: Accurate Mass Fragments of Pesticides
Table 1. Q-Exactive Parameters for Fragment Collection
Table 3. DART Transmission Gradient Parameters
Q-Exactive (full scan)
Q-Exactive (MS/MS)
Polarity
Positive
Positive
Resolution Automatic gain control (AGC) target Maximum injection time Acquisition time Capillary temperature Capillary voltage Normalized collision Energy (NCE)
140,000 1 × 106 1000 ms 2.2 mina 150°C 3.0 kV None
35,000 1 × 105 120 ms 2.2 mina 150°C 3.0 kV 15 eV or 45 eV
Ion mode
Positive
Start temperature First stabilization time First increment Second start temperature Second stabilization time Second increment Scan speed Sample time Standby temperature DART position
100°C 10 s 100°C 200°C 10 s 100°C 0.3 mm/s 5s 100°C 6–8 mm from the inlet of the MS
a
Final data file is 0.8 min long
The second set of experiments consisted of spiking the produce with the pesticide standard mixtures and then swabbing the produce with foam disks to determine which pesticides could be recovered and confirmed using the Exactive mass spectrometer, which does not have datadependent fragmentation capabilities. Data acquisition was performed with Thermo Scientific Exactive 1.1 SP5 software. Data analysis was performed with Thermo Scientific ToxID 2.1.2 SP2. The mass spectrometer was mass calibrated daily as noted above. Table 2 lists the experimental parameters for the Exactive. DART Ionization Source, Model Number SVP100 (IonSense, Inc., Saugus, MA, USA)
DART Parameters The DART parameters were set such that a heated gradient was utilized. The DART paused at each foam disk to allow maximum ionization of the analytes and entrance into the mass spectrometer. Following are the DART parameters that were employed for these experiments. Table 3 lists the experimental parameters for the DART ionization source. Table 2. Exactive Parameters for Produce Spiking Experiments Exactive (full scan)
Exactive (all ion fragmentation)
Polarity
Positive
Positive
Resolution Automatic gain control (AGC) target Maximum injection time Acquisition time Spray voltage Capillary temperature Capillary voltage Tube lens voltage Skimmer voltage Collision energy (CE)
100,000 1 × 106
100,000 1 × 106
1000 ms 2.2 mina 1.50 kV 150°C 45 V 120 V 24 V None
1000 ms 2.2 mina 1.50 kV 150°C 45 V 120 V 24 V 15 eV or 45 eV
a
Final data file is 0.8 min long
Produce Sample Preparation Seventeen multi-pesticide standard mixtures were prepared by combining and diluting stock standards for a final concentration of 10 ug mL−1 of each pesticide in acetonitrile. Each mixture was spiked onto apples, oranges, and broccoli using a pipette for a total amount of 10 ng of each pesticide in the mix per gram of produce (10 ng g−1). The spiking solution was allowed to dry. The produce was then lightly spritzed with a solvent mixture and the moistened surface was immediately swabbed clean for pesticides using polyurethane foam disks. Ten foam disks were used per sample, and each sample typically consisted of 10 pieces of produce (1:1 foam disk:produce ratio). This procedure has been described previously, although the broccoli was treated differently [17, 18]. The broccoli was not washed prior to spiking as it was very difficult to dry and the remaining water resulted in saturation of the foam. If the foam was overly wet (from water or solvent), the signal intensity was greatly reduced. Additionally, the broccoli was not swabbed in the same manner as the smoother-skinned produce. The foam was pressed gently onto the florets to remove the pesticide standards. After the produce had been was swabbed, the foam was analyzed using the method described below.
Experimental Design–Exactive MS Analysis This method was created to screen for a large number of pesticides from a variety of different classes. The wide range of compounds investigated required a temperature gradient to thermally desorb all of the pesticides. Additionally, multiple collision energies were used to successfully fragment the compounds. ToxID was selected as the data processing software because of its simplicity, speed, and easy-to-interpret reports. However, its simplicity does result in some limitations of its capabilities. For example, it cannot process data files with multiple segments within the data acquisition method. The data collection takes 7.4 min as the module containing 10 pieces of foam travels between the DART source and the mass spectrometer inlet. The module travels
S. E. Kern et al.: Accurate Mass Fragments of Pesticides
at a speed of 0.3 mm/s and once the center of each foam disk is lined up with the mass spectrometer inlet, it is analyzed for 5 s before the module resumes moving. A total of four data files were collected during that time, with the first three data files each consisting of three pieces of foam (lasting 2.2 min each) and the last data file consisting of one piece of foam (lasting 0.8 min). The normalized collision energy (NCE) was alternated from low (15 eV for the first and third data files) to high (45 eV for the second and fourth data files) during the 7.4 min. Additionally, the DART helium temperature was increased during the acquisition, exposing the foam to increasing temperatures as well as varying collision energies. Figure 1 shows an image of the module and how the data were collected. The four data files from the 10 pieces of foam were treated as one sample. The temperature was held constant at 100 oC for the first two foam disks, increased to 200 oC across the next three disks, held constant at 200 oC for the next two disks, and then increased to 300 oC across the last three disks.
Pesticide Fragment Ion Collection and the ToxID Database–Q-Exactive MS Analysis The pesticide standard fragment ions were collected using the Q-Exactive Orbitrap mass spectrometer. Each pesticide standard was analyzed individually to definitively identify its fragments. Ten mL aliquots from each stock standard were added to each of the 10 pieces of foam for fragmentation studies. The exact mass fragment ions were tabulated for each data file in the sequence. The fragment ions were not only evaluated for their intensities but also by how many of the four data files contained them. The ToxID software allows for the detection of the molecular ion and up to three fragment ions. The three most intense fragment ions detected were entered into the
database. In addition to the molecular ion and three fragment ions, two isotope ions from the [M+H]+ were added to the database. For a given compound, line one of the database includes the molecular formula of the protonated ion ([M+H]+ ion) and the exact masses of the fragment ions. Line two of the database is the molecular formula of a +1 isotope ion ([M+H+1]+ ion) of the pesticide. This was generated by using a carbon 13 isotope of the pesticide. Line three of the database is the molecular formula of a +2 isotope ion ([M+H+2]+ ion) of the pesticide. If possible, this was generated using a chlorine 37 isotope, a bromine 81 isotope, or a sulfur 34 isotope. If none of these atoms was present in the compound, a second carbon 13 isotope was used.
Results and Discussion Criteria for Confirmation of a Pesticide Although the fragment and isotope ions were easily detected with standards placed directly onto the foam disks, sometimes not all of the ions were detected when swabbed from a piece of produce. A set of criteria were developed to confirm a piece of produce as positive for pesticide contamination. First, the molecular ion must be detected. In addition to the molecular ion, at least two fragment ions, two isotope ions, or a fragment ion and an isotope ion must be detected. ToxID will only report a fragment ion if the molecular ion is also present. Additionally, the mass error of the molecular ion and isotope ion must G3 ppm and the intensity of the molecular ion must be 91000. All four data files in the sequence were considered to be one sample. If one fragment ion was detected in one data file and another fragment ion was detected in a second data file, the produce was considered positive for the pesticide since both data files were from a single sample. Since the isotope ions are listed in the database on separate lines from the molecular ion, it was possible to detect an isotope ion without detecting the molecular ion. When this occurred, the produce was not considered positive for the pesticide since the molecular ion must be detected. In summary, the four data files comprising the sample must satisfy the following criteria to be considered a positive identification of a pesticide:
Mass error of [M+H]+, isotopes, and fragments must be ≤3 ppm The intensity of [M+H]+ must be ≥1000 In addition to the [M+H]+ ion, the following must also be detected: Two fragments or [M+H+1]+ and [M+H+2]+ isotopes or One fragment and one isotope Figure 1. Temperature gradient and varying normalized collision energies across the DART module containing 10 foam disks (one module contains 10 pieces of foam and is analyzed in four separate data files, which is considered one sample)
Table 4 lists the pesticide standards spiked onto apples, broccoli, and oranges, and demonstrates that 100% of the
S. E. Kern et al.: Accurate Mass Fragments of Pesticides
pesticide standards spiked onto apples and oranges were positively identified and 80% of the pesticide standards spiked onto the broccoli (a more difficult surface to swab) were positively identified. Table 5 lists pesticides where the [M+H]+ ion was detected although they were not contained in the spiking solution. The most striking example of improving the ability to confirm detection of pesticides occurred in the apple experiment. Fourteen pesticides that were not present in the spiking mixture were initially detected by the [M+H]+ ion. Of those 14, five satisfied the abovementioned criteria for a positive identification (36%). Four of these five pesticides were positively identified on the unwashed apples (carbaryl was not detected in the unwashed apples, indicating that the batch of apples studied was not homogeneously contaminated). These five pesticides have tolerances (i.e., acceptable limits
Table 4. Positive Identification (ID) of Spiked Pesticides onto Three Different Matrices Spiking mixture
[M+H]+
Apples Thiamethoxam
292.02656
Table 5. Pesticides Detected that were not Present in the Spiking Solution Pesticides detected that were not spiked Apples Cyprodinil Diphenylamine Ethoxyquin Fenpyroximate Metalaxyl Methabenzthiazuron Metoxuron Napthalene acetamide Prometon Pyraclostrobin Pyrimethanil Tebuthiruon Broccoli Myclobutanil Oranges Dimethatryna Imazalil Phoxim Trietazinea
[M+H]+
ID criteria met (Y/N)
Tolerance
226.13387 170.09643 218.15394 422.20743 280.1546 222.06956 229.07383 186.09131 226.16624 388.10586 200.11822 229.11176
N Y N N N N N Y N N Y N
1.7 ppm 10 ppm N/T 0.3 ppm 0.2 ppm N/T N/T 0.15 ppm N/T 0.6 ppm 14 ppm N/T
289.12177
N
N/T
256.15904 297.05551 299.06171 230.11652
Y Y N Y
N/T 10 ppm N/T N/T
ID = identification; N/T = no tolerance has been established by the EPA. a Indicates isomers of pesticides present in the standard spiking mixture
ID criteria met
f: 211.06485, 131.96688, 181.05425, I2 Fenamidone 312.11651 f: 236.11825, 264.11312, I1, I2 Febuconazole 337.12145 I1, I2 Clothianidin 250.01600 f: 131.96730, I1, I2 Dichlorobenzamide 189.98210 f: 172.95539, 171.97169, I1, I2 Ethirimol 210.16009 f: 140.10694, I1 Dinotefuran 203.11387 f: 129.08948, 157.12086, I1 Trifloxystrobin 409.13697 f: 186.05230, 206.08098, 145.02588, I1,I2 Fenhexamid 302.07091 I1, I2 Mepanipyrim 224.11822 f: 209.09512, 131.06053, I1, I2 100% of compounds spiked were detected Broccoli Metobromuron 259.00754 f: 169.95999, 148.06317, I1, I2 Metoxuron 229.07384 I1, I2 Pymetrozine 218.10350 N (only [M+H]+ was detected) Siduron 233.16475 f: 137.07076, I1 Sulfentrazone 386.98915 N (nothing was detected) Methabenzthiazuron 222.06947 f: 150.02449, I1, I2 Tebufenpyrad 334.16769 f: 145.05273, I1, I2 Tebuthiuron 229.11172 I1, I2 Hexaconazole 314.08215 I1, I2 Ipconazole 334.16769 I1, I2 80% of compounds spiked were detected Oranges Carfentrazone ethyl 412.04346 f: 384.01232, 366.00175, I1, I2 Diphenamid 240.13809 f: 167.08514, I1, I2 Dipropetryn 256.15904 f: 144.03371, 214.11207, 172.06498, I1, I2 Fluometuron 233.08940 f: 160.03682, 168.02554, I1 Chlorbromuron 292.96841 f: 203.92116, I1, I2 Myclobutanil 289.12131 f: 125.01510, 151.03086, I1, I2 Neburon 275.07108 I1, I2 Pinoxaden 401.24365 I1, I2 Prometon 226.16614 f: 184.11962, I1, I2 Propazine 230.11652 f: 146.02267, 188.06959, I1, I2 100% of compounds spiked were detected The [M+H]+ ion was detected in all cases, unless otherwise noted. f = fragments, I1=[M+H+1]+, I2=[M+H+1]+
established by the EPA for apples) and four of the five are 10 ppm or above. Of the nine pesticides that were detected but did not meet the positive identification criteria, four had tolerances (G2 ppm) established by the EPA. It should be noted that while this method is not quantitative, limits of detection of 10 ng pesticide/g produce have been observed for most pesticides. They were not positively identified in the unwashed apples. The broccoli experiment only detected the [M+H]+ ion for two pesticides that were not in the spiking solution and neither of them satisfied the criteria for positive identification. The orange experiment detected the [M+H]+ ion for four additional pesticides, two of which were isomers of pesticides present in the mixture (denoted by a in Table 5). One of the other pesticides detected did not meet the identification criteria and the other pesticide had a tolerance of 10 ppm and was present on the unwashed oranges. The table in the Electronic Supplementary Material lists the theoretical masses for the 164 pesticides analyzed, along with the accurate mass information for their fragments and the formula of the isotopes used for confirmation. ToxID does not consider the isotope ratios during data analysis, but the spectra roughly reflect the expected ratios for 12C/13C, 35Cl/37Cl, 79Br/81Br, and 32S/34S. The ratios may be somewhat skewed due to the amount of ions entering the trap at once, not only from the pesticide standards but also from the foam and the various matrices. Figure 2 shows mass spectral data collected after swabbing plantains. Both imazalil and thiabendazole were positively identified.
S. E. Kern et al.: Accurate Mass Fragments of Pesticides
Figure 2. Mass spectral data for (a) full scan data of foam disks after swabbing unwashed plantains ([M+H]+ precursors shown), (b) fragmentation data for two of the detected pesticides
Conclusions A previously reported rapid screening technique for the determination of pesticides on the surface of imported produce has been updated to include fragment accurate
mass data. The addition of fragment information as well as the incorporation of isotopes in the target database has improved confirmation of identification of pesticides using this method, while still maintaining its speed and simplicity.
S. E. Kern et al.: Accurate Mass Fragments of Pesticides
Acknowledgments The authors thank to Travis Falconer, Greg Mercer, and Detlef Schumann for helpful discussions and assistance with preparing figures.
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