Tobacco is a high-value production crop for the United States and ranks 6th in the amount of pesticides applied per acre in American agriculture. Even after the processing of tobacco, some pesticide residues remain on the final product. We used the Quick\u2013Easy\u2013Cheap\u2013Effective\u2013Rugged\u2013Safe (QuEChERS) sample preparation approach to isolate residues prior to analyzing pesticides in tobacco. We evaluated the cleanup efficacy and pesticide recoveries for different formulations of QuEChERS dispersive solid phase extraction (dSPE) cleanup and the more traditional cartridge solid phase extraction (cSPE) cleanup. Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) was used to determine pesticide residues in the resulting extracts. The results of the cleanup evaluation indicated that the dSPE cleanup formulation with 7.5 mg of carbon (verses 50 mg) provided the best recovery of targeted pesticides. The average recoveries for the 500 ppb spike level and 50 ppb spike level were 92% (13% RSD) and 91% (22% RSD) respectively.<\/p>\n\n\n\n
Tobacco has a rich history in the United States and around the world. Christopher Columbus made notes in his journal about the custom of indigenous Americans smoking a \u201cstrange leaf\u201d and within a century tobacco was in global use. Tobacco is now grown widely, with China, India, Brazil, the United States, and Turkey producing two-thirds of the world\u2019s supply [1].<\/p>\n\n\n\n
Pesticides are used heavily on tobacco in order to increase crop production value. In fact, tobacco ranks 6th out of all crops in the U.S. in terms of pesticide application, falling only behind potatoes, tomatoes, citrus, grapes, and apples [2]. Although these fruits and vegetables are grown using more pesticides per acre, the final residue levels on these foods is regulated. No such controls exist for tobacco. Although the U.S. Environmental Protection Agency (EPA) regulates and approves the pesticides that can be applied to tobacco based on worker safety, environmental quality, and crop protection, it does not set allowable levels for pesticides in finished tobacco products. For some time, the U.S. Department of Agriculture (USDA) has analyzed pesticides in tobacco for non-EPA approved pesticides on both imported and domestic products, and recently it has also began including pesticides that are EPA-approved for application to the tobacco plant. In spite of this additional monitoring, the lack of set regulatory limits creates the potential for high levels of pesticide residues to remain on final tobacco products. A few countries that import U.S. tobacco products do have regulations on maximum residue levels of pesticides in either cigarettes or the tobacco leaf itself [2].<\/p>\n\n\n\n
The QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology was developed for the determination of multiresidue pesticides in fruits and vegetables [3]. This methodology uses a simple shake extraction where the pesticides are extracted and partitioned using acetonitrile and a salt\/buffer solution. The resultant extract is then cleaned using a very quick dispersive solid phase extraction (dSPE) step that requires no additional solvent usage. While this method was originally developed for high water content produce, we have successfully adapted the method for dry commodities, such as dietary supplements [4]. This approach utilized a modified QuEChERS extraction and a cartridge solid phase extraction cleanup (cSPE). The cSPE cleanup provides the potential for enhanced cleanup capacity for complex matrices like dietary supplements and tobacco, but it requires additional solvent and extra time for sample elution and concentration. For the more complex dietary supplement finished products we employed comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) as the determinative technique [5]. Analyzing tobacco using GCxGC proved to be a powerful technique in separating matrix interferences from the pesticides of interest.<\/p>\n\n\n\n
Here we used the QuEChERS extraction approach and GCxGC-TOFMS and evaluated several cleanup methods for finished tobacco product. The tobacco extract can be very complex, so we explored both dSPE and cSPE cleanup approaches and monitored their performance for pesticide recovery and matrix reduction. The wide range of pesticides chosen for this study covered many, but not all, of the 37 pesticides that have been approved by the EPA for use on tobacco.<\/p>\n\n\n\n
Two types of bulk loose cigarette tobacco, a light and dark, were provided by Global Laboratory Services. A custom stock standard that included organochlorine, organonitrogen, and organophosphorus pesticides was prepared at Restek and diluted to 10 ng\/\u00b5L and 1 ng\/\u00b5L concentrations in acetonitrile. Recovery experiments were performed at two fortification levels, 500 ppb and 50 ppb. Fortified samples were prepared by adding 100 \u00b5L of the appropriate diluted standard (10 ng\/\u00b5L or 1 ng\/\u00b5L) to tobacco samples. Unfortified samples were also prepared in order to find potential incurred pesticides in tobacco and make matrix-matched standards for quantification.<\/p>\n\n\n\n
Matrix-matched standards were prepared at 100 pg\/\u00b5L and 10 pg\/\u00b5L by adding 5 \u00b5L of a standard solution (1 ng\/\u00b5L and 0.1 ng\/\u00b5L) to 45 \u00b5L of the final cleaned extract of the unfortified tobacco samples for each type of cleanup.<\/p>\n\n\n\n
A 2 g sample of tobacco was weighed into a 50 mL polypropylene centrifuge tube (Restek, cat. # 26239). After the addition of 10 mL of organic-free water to the sample, 100 \u00b5L of QuEChERS internal standard mix for GC-MS analysis (Restek,\u00a0cat. # 33267) was added to each sample. For samples that were fortified, 100 \u00b5L of the fortification standard was then added. Next, 10 mL of acetonitrile was added and the samples were vortexed for 30 min using a digital Vortex-Genie 2 (Scientific Industries, cat. # SI-A236). Immediately after vortexing, pre-packaged QuEChERS European EN 15662 method formulation extraction salts containing 4 g MgSO4<\/sub>, 1 g NaCl, 1 g trisodium citrate dihydrate, and 0.5 g disodium hydrogen citrate sesquihydrate (Restek, cat. # 25849) were added to each centrifuge tube. The tubes were immediately shaken for 1 min and then centrifuged in the Q-sep 3000 centrifuge (Restek,\u00a0cat. # 28295) for 5 min at 3000 g. The top acetonitrile layer was collected and aliquots were taken for subsequent cleanup.<\/p>\n\n\n\n Two formulations of pre-packaged dispersive solid phase extraction tubes were evaluated, the AOAC 2007.01 formulation containing 150 mg MgSO4<\/sub>, 50 mg primary secondary amine (PSA), 50 mg graphitized carbon black (GCB), and 50 mg C18 (Restek,\u00a0cat. # 26219); and the mini-multiresidue, European EN 15662 formulation containing 150 mg MgSO4<\/sub>, 25 mg PSA and 7.5 mg GCB (Restek,\u00a0cat.# 26218). For cleanup, a 1 mL aliquot of each extract was fortified with 5 \u00b5L of an anthracene standard (Restek,\u00a0cat. # 33264) and added to the dSPE tubes. The tubes were gently shaken for 2 min and then centrifuged for 5 min using a Q-sep 3000 centrifuge (Restek,\u00a0cat. # 28295). A 0.5 mL portion of the supernatant extract was removed and placed into an autosampler vial and 5 \u00b5L of a 5% formic acid solution in acetonitrile was added to each sample.<\/p>\n\n\n\n For the cartridge solid phase extraction cleanup, a 6 mL pesticide residue cleanup SPE cartridge packed with 500 mg CarboPrep 90 material and 500 mg PSA (Restek,\u00a0cat. # 26194) was used. Approximately 0.5 cm of anhydrous MgSO4<\/sub>\u00a0was added to the top of the cartridge bed. The cartridge was rinsed with 20 mL of acetone prior to the sample being loaded. After the cartridge rinsing, 1 mL of tobacco extract was loaded onto the cartridge and eluted with 15 mL of a 3:1 acetone:toluene mixture. The eluent was collected and evaporated to 1 mL under a stream of nitrogen using a TurboVap II concentration workstation (Biotage, cat. # 103187).<\/p>\n\n\n\n A LECO Pegasus 4D GCxGC-TOFMS equipped with an Agilent 6890 GC and 7683 autoinjector was used to determine pesticide recoveries and levels of incurred pesticides in tobacco. A 30 m x 0.25 mm x 0.25 \u00b5m Rxi-5Sil MS column (Restek,\u00a0cat. #13623) was installed in the primary oven and connected via a press-fit (BGB Analytik AG, cat. # 2525LD) to a 1.3 m x 0.25 mm x 0.25 \u00b5m Rtx-200 column (Restek,\u00a0cat. # 15124) installed in the secondary oven. The primary oven temperature conditions were 90 \u00b0C (hold 1 min) to 310 \u00b0C (hold 2 min) at 5 \u00b0C\/min. The secondary oven temperature program tracked the primary oven with a +5 \u00b0C offset. The second dimension separation time was 3 sec with a +20 \u00b0C modulator temperature offset. The carrier gas was helium operated under corrected constant flow conditions at 2 mL\/min. 1 \u00b5L fast autosampler splitless injections were made with a 1 min purge valve time and an inlet temperature of 250 \u00b0C. A 4 mm Restek Premium single taper inlet liner with wool (Restek, cat. # 23303) was used for all analyses. Data were acquired from 45 to 550 u with an acquisition rate of 100 spectra\/sec. The transfer line was 300 \u00b0C and electron ionization at 70 eV was used with a source temperature of 225 \u00b0C.<\/p>\n\n\n\n An Agilent 6890 GC with a flame ionization detector (FID) was used to quickly evaluate the removal of the tobacco matrix for each type of cleanup. We used a 15 m x 0.25 mm x 0.25 \u00b5m Rxi-5Sil MS column (Restek,\u00a0cat. #13620) with helium carrier gas operated in constant flow at 2 mL\/min. The oven temperature program was 80 \u00b0C (hold 1 min) to 350 \u00b0C (hold 5.5 min) at 20 \u00b0C\/min and yielded a 20 min analysis time. A 4 mm Restek Premium single taper liner with wool was installed in the inlet, which was set to 250 \u00b0C. 1 \u00b5L fast splitless injections (0.75 min purge valve time) were performed with a 7683 autoinjector. The FID temperature was 350 \u00b0C and the makeup flow plus column flow was held constant at 50 mL\/min. Data were collected at 5 Hz.<\/p>\n\n\n\n Gravimetric analyses of the nonvolatile residue remaining in the final extracts were performed for both the dSPE and cSPE cleanup procedures. Cleanups for the two formulations of dSPE tubes and the cSPE procedure were each performed in triplicate. The resultant replicate extracts were combined and added to tared conical vials that were placed on a 60 \u00b0C hotplate and evaporated under a stream of dry nitrogen gas (Thermo Scientific, Reacti-Therm I [cat.# TS-18821] and Reacti-Vap [cat. # TS-18825]). The vials were reweighed after all solvent was evaporated to determine the amount of nonvolatile material present after the extract cleanup.<\/p>\n\n\n\n All data were processed with the LECO ChromaTOF software. Recoveries of pesticides in the fortified tobacco samples were quantified using the GCxGC-TOFMS data, matrix-matched standards, and the internal standard PCB 52 for each cleanup type. The matrix-matched standards represented 100% recovery and were used for single-point calibration and quantification. Evaluation of the tobacco extract prior to cleanup and following each cleanup type were performed by overlaying the FID traces to visually inspect gross differences in cleanup efficacy.<\/p>\n\n\n\n The goal of this work was to use the quicker dSPE cleanup methodology for the extracts as long as it was effective when analyzing tobacco using GCxGC-TOFMS. From previous work with complex matrices, we have found that dSPE does not have the capacity to clean the extract enough for GC-MS analysis, including GC-TOFMS or even GCxGC-TOFMS analysis [5]. In order to quickly determine if the time- and solvent-intensive cSPE was necessary, we evaluated the extracts using GC-FID. Pigment reduction from extracts is important for GC work, since many pigments are nonvolatile and quickly degrade the performance of the GC inlet and column. With a first visual inspection of the resultant extracts, it was clear that as the amount of carbon was increased, the pigment in the extract decreased (Figure 1). When overlaying the GC-FID traces for each cleanup type, it was apparent that some matrix components that were not removed by either dSPE cleanup were significantly reduced by the cSPE cleanup (Figure 2). The most notable were the fatty acids that are eluting in the middle of the chromatogram. The PSA sorbent is a weak anion exchange material and will remove fatty acids. Therefore, the 500 mg of PSA in the cSPE cartridge provided a more effective cleanup of fatty acids in the tobacco matrix than either of the dSPE cleanups, which contained just 50 mg or 25 mg of PSA. However, not all matrix interferences were further removed by the cSPE, which limits the benefits of this technique over the much quicker dSPE cleanup.<\/p>\n\n\n\n The next step in evaluating the efficacy of the different types of cleanups in removing matrix interferences was to determine the amount of nonvolatile residue that was removed by each cleanup. While nonvolatile residue will not necessarily cause interference in the actual chromatogram, it is an important aspect to evaluate when developing a cleanup method. Large amounts of residue from injected samples will quickly collect in the inlet liner and at the front of the column, degrading method performance and requiring more frequent injection port and column maintenance. In this respect, the use of wool in the splitless liner is also important in further protecting the analytical column from the residue of the tobacco matrix. The gravimetric analysis determined that the 25 mg PSA and 7.5 mg GCB dSPE formulation provided a 50% reduction in nonvolatile material from the raw tobbacco extract. The other dSPE formulation that had 50 mg PSA, 50 mg C18, 50 mg GCB removed 70% of the nonvolatile residue. The cSPE, which contained 500 mg PSA and 500 mg carbon, also removed 70% of the matrix material. The cSPE procedure took approximately 3 hours compared to just 20 minutes for the dSPE process, so the lack of additional removal of nonvolatile matrix components and only minimal improvement in cleanup of matrix intereferences did not outweigh the extra time and solvent usage of cSPE. Only the dSPE extracts were further evaluated for recovery of pesticides in tobacco on the GCxGC-TOFMS system.<\/p>\n\n\nExtract Cleanup<\/h3>\n\n\n\n
GCxGC-TOFMS Analysis<\/h3>\n\n\n\n
QuEChERS Extract Cleanup Evaluation<\/h3>\n\n\n\n
Results and Discussion<\/h2>\n\n\n\n
Removal of Matrix Interferences<\/h3>\n\n\n\n