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A modified QuEChERS method for simultaneous determination of flonicamid and its metabolites in paprika using tandem mass spectrometry
Food Chemistry 157 (2014) 413–420
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
A modified QuEChERS method for simultaneous determination of flonicamid and its metabolites in paprika using tandem mass spectrometry Ah-Young Ko a, A.M. Abd El-Aty a,b,⇑, Md. Musfiqur Rahman a, Jin Jang a, Sung-Woo Kim a, Jeong-Heui Choi a, Jae-Han Shim a,⇑ a b
Biotechnology Research Institute, College of Agriculture and Life Sciences, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Republic of Korea Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt
a r t i c l e
i n f o
Article history: Received 9 November 2013 Received in revised form 4 February 2014 Accepted 11 February 2014 Available online 22 February 2014 Keywords: Method development Validation Improved sample preparation Tandem mass spectrometry Paprika
a b s t r a c t A modified quick, easy, cheap, effective, rugged and safe (QuEChERS) acetate-buffered sample preparation method was developed to improve extraction recovery of flonicamid and its two metabolites (4-trifluoromethylnicotinic acid and N-(4-trifluoromethylnicotinoyl)glycine) in paprika followed by analysis using tandem mass spectrometry. Acidified acetonitrile (containing 5% acetic acid) was used as an extraction solvent and partitioning was carried out using sodium chloride. The extract was then cleaned up using C18. The linearity over a concentration range of 0.005–1 lg/mL was good with a determination coefficient (R2) > 0.9997. Recovery at three different fortification levels was 82.2–101.7% with a relative standard deviation <10 for all analytes. The limit of quantitation of 0.01 mg/kg was quite lower than the maximum residue level set by the Korea Food and Drug Administration (2 mg/kg). The method was successfully applied to determine flonicamid and its metabolites from field incurred samples. The undulating residue pattern observed for the parent analyte together with its metabolites could explain the movement behavior of systemic pesticides into plants over time. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Various kinds of pesticides have been used regularly to increase production, quality and storage longevity of agricultural commodities throughout the globe. Pesticide residues in food naturally pose a potential hazardous risk to consumers. Thus, pesticide and/or metabolite residues should be analyzed in all agricultural commodities to improve public health and food safety (Park et al., 2012). Among these pesticides; flonicamid, (N-cyanomethyl-4-trifluoromethylnicotinamide), a selective systemic pesticide, is highly effective against aphids and other sucking insects (Morita et al., 2000). Flonicamid blocks type-A potassium channels, which leads to loss of direct movement and suppression of aphid feeding (Joost et al., 2006). This analyte can be used in integrated pest management programmes as it has a minimal cross-resistance
⇑ Corresponding authors. Address: Biotechnology Research Institute, College of Agriculture and Life Sciences, Chonnam National University, Buk-gu, Gwangju 500-757, Republic of Korea. Tel.: +20 2 27548926; fax: +20 2 35725240 (A.M. Abd El-Aty). Tel.: +82 62 530 2135; fax: +82 62 530 0219 (J.-H. Shim). E-mail addresses: [email protected] (A.M. Abd El-Aty), jhshim@chonnam. ac.kr (J.-H. Shim). http://dx.doi.org/10.1016/j.foodchem.2014.02.038 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
characteristics and lack of toxicity to beneficial arthropods (Dixon, 2002). Analyte metabolism is comparable in wheat, potato and peach, and the residues are composed of the parent compound (flonicamid) and its two major metabolites TFNA (4-trifluoromethylnicotinic acid) and TFNG (N-(4-trifluoromethylnicotinoyl)glycine. The ratios between both metabolites are substantially different depending on the crop (European Food Safety Authority, 2010). Additional metabolites, including TFNA-AM (4-trifluoromethylnicotinamide) and TFNG-AM are not expected to exist in plants at significant levels and, consequently, the residue definition for risk assessment is limited to the sum of flonicamid, TFNA and TFNG (European Food Safety Authority, 2010). In the Republic of Korea, the Korea Food and Drug Administration (KFDA refers to the residue for monitoring flonicamid to the parent analyte only and set its maximum residue level (MRL) to 2 mg/kg in paprika (KFDA, 2011). However, the Japanese define the residue to the parent compound (flonicamid) and its metabolites; TFNA and TFNG and set the MRL to 2 mg/kg in paprika (The Japan Food Chemical Research Foundation, 2012). Such differences between countries causes obstacles to trade, and interferes with international trade of agricultural commodities (Lee et al., 2011). The Rural Development Administration (RDA, 2012), the
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organisation responsible for pesticide registration and safety guideline in Republic of Korea, changed their concept and used the same definition set by the Japan Food Chemical Research Foundation (2012); however, the KFDA, the organisation responsible for setting and implementing the MRL, still refer to the flonicamid value only. We expect that this change will happen in the upcoming upgrade of the KFDA databases. Paprika (Capsicum annuum) is widely used as a vegetable and food additive, as this fruit is a good source of carotenoid pigments. These fruits are often grown under glasshouse conditions in highvalue production systems. Carotenoids are effective as free-radical scavengers (Matsufuji, Nakamura, Chino, & Takeda, 1998). Therefore, consumers consume paprika for its nutritive value. Paprika contains more than 2500 natural compounds (Ferrer, Fernandez-Alba, Zweigenbaum, & Thurman, 2006) that may mask the detection of some pesticide residues and make it difficult or impossible to identify the target compounds. Efficient sample preparation is absolutely necessary for an accurate determination of pesticide residues in foods consisting of complicated matrices (Watanabe, Kobara, & Yogo, 2012). Pesticide residues are distributed heterogeneously in/on crops; thus, the sample must be homogenized thoroughly. A sufficiently homogeneous sample is extracted, with the result that matrix components are often co-extracted together with the target pesticides. Consequently, cleanup processes are necessary (Watanabe et al., 2012). High-pressure liquid chromatography (HPLC) coupled with mass spectrometry (MS) is available to detect flonicamid and its metabolites in agricultural matrices (Chen, 2002; Zywitz, Anastassiades, & Scherbaum, 2003). Hengel and Miller (2007) conducted liquid–liquid extraction (LLE) with a mixture of acetonitrile (ACN) and water (50/50, v/v) and a two-step solid-phase extraction (SPE) cartridge cleanup followed by liquid–liquid partitioning for liquid chromatography–tandem mass spectrometry (LC/MS/MS) analysis to extract flonicamid, TFNA and TFNG. Chen, Sun, Yang, and Wu (2012) detected flonicamid and its metabolite (TFNG, TFNA and TFNAAM) in cucumbers and apples by LC/MS/MS with LLE. However, these methods are tedious, time-consuming and not environmentally friendly. Xu, Shou, and Wu (2011) detected flonicamid and its metabolites TFNG, TFNA and TFNA-AM in spinach and cucumber using QuEChERS and primary secondary amine (PSA) and graphite carbon black (GCB) as a cleanup procedure followed by LC/MS/MS. In the current study, we used the same amount of PSA (0.1 g) for paprika; however, the recoveries of metabolites were not satisfactory. The aim of this study was to detect the residues of flonicamid and its major metabolites (TFNG and TFNA) in paprika grown under greenhouse conditions using a modified acetate-buffered QuEChERS method for extraction and LC/MS/MS for analysis.
2. Experimental 2.1. Chemicals and reagents Reference standards for flonicamid (CAS Registry No. 15806267-0; purity 98.5%), TFNA (CAS Registry No. 158063-66-2; purity 94.9%), and TFNG (CAS Registry No. 207502-65-6; purity 99.4%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). HPLC-grade acetonitrile (ACN) was purchased from Burdick and Jackson (Ulsan, Republic of Korea). Sodium chloride (NaCl, purity 99.5%) was obtained from Merck (Darmstadt, Germany), and sodium acetate (NaOAc, purity 98.0%), anhydrous magnesium sulfate (MgSO4, purity 99.5%), and triethylamine (TEA, purity 99.0%) were provided by Junsei Chemical Co., Ltd. (Kyoto, Japan). Primary secondary amine (PSA) and C18 were supplied by Agilent Technologies (Palo Alto, CA, USA). Acetic acid (purity 99.5%) and formic acid (purity 85.0%) were obtained from Daejung Chemicals & Mate-
rials (Siheung, Republic of Korea), and Duksan Pure Chemicals Co., Ltd. (Ansan, Republic of Korea), respectively. 2.2. Standard solution preparation Individual standard solutions of flonicamid and its metabolites (TFNA and TFNG) were prepared in ACN at a concentration of 1000 lg/mL. A working solution (10 lg/mL) was prepared by diluting the stock solution with blank sample extracts, which were confirmed previously to contain none of the tested analytes. Matrix-matched calibration standard solutions were prepared by mixing the matrix-matched working standard solutions with additional blank sample extracts to reach the multi-compound concentrations of 0.005–1 lg/mL. All standards solutions were stored at 20 °C in a bottle and freshly prepared matrix standards were used before each analysis. 2.3. Field trails Experimental trials were carried out in a greenhouse located at Chonnam National University, Gwangju, Republic of Korea. Paprika was cultivated either in soil or using hydroponics. The commercial formulation was a water dispersible granule (WG), which contained 10% of the active ingredient of flonicamid (SetisÒ, Dongbu HiTek, Seoul, Republic of Korea). The formulation was applied as a direct spray treatment for soil cultivation, whereas direct spray and drench treatments were used for paprika cultivated in hydroponic soilless media. The recommended dose was sprayed either once or twice with a 7 day interval in case of the direct spray or 30 days in case of the drench treatment. Samples were randomly collected at 0 (2 h later) 1, 3, 5, 7 and 10 days after application; however, the drench treated paprika samples were collected at 1, 4, 7, 14 and 21 days following the final application. The samples were transferred to the laboratory in sealed bags, chopped, mixed with other samples from the same plot, packed in plastic bags, and stored at 20 °C pending analysis. 2.4. Sample preparation Extraction and cleanup were carried out using acetate-buffered QuEChERS after major modification (Lehotay, 2007). Approximately 10 g of homogenized paprika samples were placed into a 50-mL Teflon centrifuge tube. Twenty mL of 5% acetic acid in ACN was added to 10 mL of water, and the tubes were vortexmixed for 2 min. Then, 10 g NaCl was added to each sample and vortex-mixed vigorously for an additional 2 min. The extract was centrifuged for 5 min at 5000 rpm and room temperature, and the supernatant (6 mL) was transferred to a 15-mL Teflon centrifuge tube containing 0.3 g of C18. The tubes were shaken for 30 s for cleanup and centrifuged again for 5 min at 3000 rpm. One mL of the upper layer was filtered through a polytetrafluoroethylene (PTFE) membrane filter (0.2 lm, ADVANTECÒ, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) and subsequently analyzed via LC/MS/MS. 2.5. LC/MS/MS The LC/MS/MS system consisted of a Waters Alliance 2695 LC Separations Module and a Micromass Quattro Micro triple quadrupole tandem mass (Waters Crop., Milford, MA, USA). The analytes were separated on a X-bridge C18 (2.1 mm i.d 150 mm, 3.5 lm, Waters Crop.) that had been kept in an oven at 35 °C. A binary solvent system containing ACN (mobile phase A) and 0.1% formic acid in water (mobile phase B) was run in a gradient mode to detect flonicamid and its metabolites; TFNA, and TFNG, simultaneously. A linear mobile phase gradient started at 5% A (0–1.5 min), increased to 50% A (1.5–3 min), increased to 90% A (3–6 min),
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maintained at 90% A (6–8 min), and decreased to 5% A (8–8.1 min), after which the column was equilibrated at 5% A (8.1–12 min). Flow rate, and injection volume were set at 0.25 mL/min, and 5 lL, respectively. Tandem mass spectrometric analysis was carried out via electrospray ionization in positive mode and operated in multiple reactions monitoring (MRM) mode. The MS source condition were as follows: capillary voltage, 3.5 kV; source temperature, 150 °C; desolvation temperature, 350 °C; desolvation gas (N2) flow, 650 L/h; cone gas (N2) flow, 50 L/h; and collision gas (argon) pressure of 4.0 10 3 mbar. Optimization of the precursor ion, product ions, and collision energy (CE) was performed via direct injection of the analyte and metabolite standard solution (1 lg/mL) into the MS/MS system. The most intense transition was used for quantitation, while the other was employed for confirmation. The optimization parameters are presented in Supplementary Table. Mass Lynx V4.1 software (Waters Corp.) was used for instrument control, data acquisition and processing.
2.6. Validation of the analytical method The method was validated using the single laboratory validation approach such as linearity, specificity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. Linearity was assessed by the determination coefficient (R2) of a matrixmatched calibration curve that was created based on eight points of different flonicamid concentrations (0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 lg/mL) and its metabolites TFNA and TFNA. The LOD and LOQ were determined using signal-to-noise ratios of 3 and 10, respectively. Recovery experiments were conducted by fortifying blank paprika samples with mixed standard solutions at three different fortification levels (0.05, 0.1, and 0.5 mg/kg) with three replicates per level. Precision (repeatability and intermediate precision) of the analytical method was estimated from the relative standard deviations (RSD%) analyzed on the same day (intra-day) and across 3 days (inter-day precision), respectively. Matrix effects in terms of signal suppression or enhancement due to co-elution of matrix components were evaluated by post-extraction spiking and compared with the solvent standards. Higher and lower slopes of the matrix calibration equation with reference to the solvent-based calibration equation represent matrix-induced enhancement and suppression, respectively (Cho et al., 2013).
3. Results and discussion
3.1.1. Sample preparation Paprika was hydrated before being extracted through salting out with ACN followed by cleanup procedures. Cajka et al. (2012) clearly documented that adding water to the sample is a key to achieve maximum extraction yield (and therefore accurate results). 3.1.2. Effect of salting out agents The effect of salting out agents during the partitioning step to extract flonicamid and its metabolites was tested using NaCl, NaOAc, or MgSO4 in paprika samples. No substantial differences were observed when the salts were used alone or in combination regarding the recovery of flonicamid; however, recovery of the metabolites was significantly altered (Table 1). The TFNA and TFNG metabolites were completely unrecovered when 1% acetic acid in ACN was used with the QuEChERs salts (6 g of MgSO4 and 1.5 g of NaOAc) (Lehotay, 2007). When the same experiment was conducted with MgSO4 without NaOAc, recovery of TFNA and TFNG increased to 34.24% and 47.85%, respectively. In contrast, the highest recovery for TFNA (53.99%) and TFNG (47.85) was observed when NaCl was used. Therefore, we used NaCl as a salting out agent to separate the compounds from the aqueous fraction into the organic layer during the extraction partitioning step. 3.1.3. Effect of acidic and basic agents The effect of acetic acid as an acidic agent or triethylamine (TEA) as a basic agent was evaluated to successfully recover flonicamid and its metabolites. Kamel (2010) reported that 2% TEA in ACN is an appropriate solvent to extract nitro-substitute compounds, but it failed to completely recover the analytes and the metabolites used here. However, the recovery of TFNA and TFNG improved substantially when acetic acid in ACN was used. Different volumes of acetic acid from 100–1200 lL were tested to optimize the amount. Fig. 1 shows that 1000 lL of acetic acid produced a satisfactory recovery. 3.1.4. Optimization of cleanup We tried QuEChERS dispersive-SPE cleanup using PSA. Lehotay (2007) reported that PSA strongly binds pesticides containing a carboxylic acid group, which reduces their recovery rates. TFNA and TFNG yielded recovery of <70% when PSA was used (Fig. 1). The C18 absorbent does not have a substantial effect on metabolite recovery, even though the amount was increased. Thus, PSA was replaced with C18 absorbent. Fig. 1 shows the effect of PSA and C18 on the recovery of flonicamid, TFNA and TFNG. C18 results in decreasing chromatographic interference and increases sensitivity to quantitatively identify the tested analytes (Grimalt et al., 2011).
3.1. Sample extraction and cleanup
3.2. Chromatographic optimization
Given the matrix-dependent issues associated with co-extractive interference, rapid and effective cleanup procedures are required for complex matrices. The extraction step is followed by a cleanup step involving either SPE or dispersive-solid phase extraction (d-SPE). QuEChERS is used to reduce sample handling and processing time for analysis of pesticides in fruit and vegetable samples (Grimalt et al., 2011). QuEChERS is a method composed of an extraction step with ACN and partitioning using MgSO4, followed by d-SPE using PSA that was developed to detect pesticide residues in crops and other foods by Anastassiades, Lehotay, Stajnbaher, and Schenck (2003). When the above mentioned conditions were applied to paprika, good recovery was observed for the parent compound; however, that of the metabolites was <50%. The poor recovery of metabolites encouraged us to find a way to improve it. Thus, the effects of various extraction factors, including sample preparation, salting out, acidic and basic agents were investigated.
Different gradients of water/ACN as a mobile phase are good ionization media to separate flonicamid and its metabolites. A gradient elution programme provided baseline resolution for the analyte and its metabolites with a high detection signal, improved peak shapes, and shortened the detection time of all analytes (within 10 min). Additionally, the selected programme facilitated efficient removal of matrix constituents, and reduced noise level Table 1 Effect of salting out agents on recovery of flonicamid and its metabolites. Compound
Flonicamid TFNA TFNG
Salting out agents (mean ± SD) MgSO4
NaCl
MgSO4 + NaOAc
NaCl + NaOAc
96.74 ± 2.66 34.24 ± 6.42 47.85 ± 3.45
99.06 ± 1.18 53.99 ± 6.28 67.76 ± 0.99
95.9 ± 1.20 1.97 ± 0.68 N.D
116.88 ± 3.45 16.73 ± 2.44 11.23 ± 6.54
N. D. Not detected.
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Fig. 1. Effect of acetic acid, primary secondary amine (PSA), and C18 amounts on the extraction efficiency of flonicamid and its metabolites in paprika.
and risk for carry-over effects and column deterioration (Cho et al., 2013). As shown in Fig. 2, no interfering peaks were observed around the retention time of the analytes. The incorporation of formic acid into the mobile phase at a low concentration of 0.1% facilitated ionization of the analyte and its metabolites. Filtering the extracts prior to LC/-MS/MS analysis is necessary to prevent instrument and column damage (Cho et al., 2013). We used a PTFE membrane filter prior to analysis. Optimal ionization occurred at a 350 °C desolvation temperature. Among the two mass transitions, flonicamid and it metabolites (TFNA and TFNG) showed their greatest intensity at m/z 203.22, 148.03, and 203.17, respectively, and were considered a quantitation ion. However, the lower intensities at m/z 148.05, 98.12, and 148.24 were considered a confirmation ion.
metabolites in paprika. The matrix-matched calibration curve was obtained by plotting peak area vs. the concentration of the analytes. Good linearity (over a concentration range of 0.005– 1 lg/mL) with determination correlation coefficients (R2) of 0.9997 were achieved for the three analytes as given in Table 2. 3.3.3. LOD and LOQ The LOD and LOQ of all analytes were 0.003 and 0.01 mg/kg, respectively, (Table 2). The LOQ value was below flonicamid (total sum) residue MRLs set by the EU for pepper in general (0.15 mg/kg, EU pesticides database, 2005), the Japan MRL (2 mg/kg) for paprika (The Japan Food Chemical Research Foundation, 2012) and the Republic of Korea MRL (2 mg/kg) (RDA, 2012).
3.3.1. Specificity Specificity was validated by analyzing representative blank paprika samples (n = 3) to confirm the absence of potential interfering compounds at various retention times. No interfering peaks appeared at the retention times of flonicamid or its two metabolites (Fig. 2).
3.3.4. Recovery Recovery tests were carried out at three different concentrations (0.05, 0.1, and 0.5 mg/kg) in three replicates. Fig. 2 shows the LC/MS/MS chromatograms obtained from the blank samples fortified with 0.1 mg/kg flonicamid, TFNA and TFNG. The recoveries of the flonicamid and its metabolites were good (82.2–101.7% with RSD <10%) and were independent of sample matrix and the fortified level (Table 2). All analytes showed results consistent with the acceptable range specified by SANCO, 2009 (70–120%).
3.3.2. Linearity and the matrix effect A comparison between calibration curves obtained from standards prepared in pure solvent and calibration curves constructed using matrix spiked with standards was performed to evaluate the matrix effect (Cho et al., 2013). The responses obtained were comparable, and no significant effect was observed (Table 2). Matrixmatched calibration was used to quantify flonicamid and its
3.3.5. Accuracy and precision Accuracy and precision were evaluated via intra- and inter-day analysis, both of which were conducted in a single laboratory. Precision was calculated in terms of inter-day repeatability and inter-day reproducibility. Accuracy was described as the mean of recovery and precision was expressed as the RSD. Recovery percent was calculated by comparing the results of the intra- and inter-day
3.3. Validation performance
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Fig. 2. Representative MRM (quantitation ion) chromatograms of (a) Blank sample, (b) standard (flonicamid or its metabolites) at 0.1 mg/kg, (c) fortified paprika sample at 0.1 mg/kg, and (d) field incurred paprika sample. Flonicamid (A), TFNA (B), and TFNG (C).
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Fig. 2 (continued)
Table 2 Linearity, LOD, LOQ, and recovery of flonicamid and its metabolites in paprika. Linearity (R2)
Compounds
Flonicamid TFNA TFNG
Recovery (RSD%)
Solvent
Matrix-matched
0.05 mg/kg
0.1 mg/kg
0.5 mg/kg
0.9996 0.9995 0.9985
0.9998 0.9997 0.9997
94.0 (3.84) 86.8 (3.84) 101.1 (9.04)
101.1 (3.52) 82.9 (6.76) 88.7 (2.47)
101.7 (3.14) 82.2 (4.80) 85.4 (2.79)
repeatability with the matrix-matched standard solutions prepared at the same concentration in a blank extract. The mean intra- and inter day accuracy ranged from 85.4% to 103.2% and from 78.0% to 101.1% for all analytes, whereas intra- and interday precision was 3.14–7.22 and 2.22–9.04%, respectively. As shown in Table 3, these values were consistent with the ranges listed in the Codex guidelines (Codex, 1993) suggesting that the present method is reliable, reproducible and accurate.
LOQ (mg/kg)
0.003
0.01
Table 3 Intra-day and inter-day accuracy and precision of flonicamid and its metabolites in paprika. Compounds
Flonicamid
TFNA
3.4. Method application TFNG
The method was applied to two different types (direct spray and drench treatment) of treated paprika samples obtained from soil cultivation and hydroponic conditions. The total residue was estimated by the sum of flonicamid and its metabolite residues (SANCO, 2009). Total flonicamid residual concentrations (containing metabolites residues) in field samples are reported in Table 4. The residue level of the parent compound in the direct spray under soil cultivation treatment decreased slightly from day 0 to day 2 post-application. Then, the residues increased gradually from days 2 to 5 and decreased thereafter, both for the one and two time applications, respectively. The highest residual concentration of parent compound was detected on day 7 post-application. TFNA
LOD (mg/kg)
Fortified concentration (mg/kg)
0.05 0.1 0.5 0.05 0.1 0.5 0.05 0.1 0.5
Accuracy (%) (RSD) Intra-daya
Inter-dayb
94.0 (3.84) 101.1 (3.52) 101.7 (3.14) 86.8 (8.84) 82.9 (6.76) 82.2 (4.40) 101.1 (9.04) 88.7 (2.47) 85.4 (2.79)
85.4 (7.22) 101.0 (6.59) 103.2 (5.81) 79.7 (7.22) 78.4 (7.58) 78.0 (6.88) 88.8 (8.58) 85.5 (4.85) 85.6 (2.22)
a Intra-day repeatability was estimated by analyzing three replicate samples at three concentration levels on the same day. b Inter-day repeatability was estimated by analyzing three replicate samples at three concentration levels on 3 consecutive days.
was first detected on days 5 and 7 and thereafter increased substantially. In contrast, the TFNG residues increased steadily after day 2 to day 10. Although the parent compound residue decreased over time, flonicamid (as total residues) had a tendency to increase over time. This was because the metabolites were produced from
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A.-Y. Ko et al. / Food Chemistry 157 (2014) 413–420 Table 4 Field-incurred flonicamid residues and its metabolites in paprika grown under soil cultivation and hydroponic conditions using direct spraying and drench treatment. Cultivation
Direct spray Soil cultivation
Treatment
Once
Twice
Hydroponic
Once
Twice
Drench treatment Hydroponic
Once
Twice
Days after application
Residual concentration (Mean ± SD) (mg/kg) Flonicamid
TFNA
TFNG
Total residuea
0 1 2 3 5 7 10 0 1 2 3 5 7 10
0.159 ± 0.011 0.144 ± 0.008 0.160 ± 0.002 0.229 ± 0.004 0.229 ± 0.004 0.230 ± 0.013 0.210 ± 0.026 0.263 ± 0.004 0.198 ± 0.016 0.193 ± 0.008 0.328 ± 0.027 0.451 ± 0.034 0.225 ± 0.017 0.211 ± 0.021
N.D N.D N.D N.D N.D 0.028 ± 0.003 0.029 ± 0.002 N.D N.D N.D N.D 0.028 ± 0.002 0.030 ± 0.004 0.035 ± 0.005
0.049 ± 0.007 0.046 ± 0.006 0.036 ± 0.003 0.017 ± 0.001 0.025 ± 0.004 0.033 ± 0.003 0.063 ± 0.007 0.035 ± 0.008 0.031 ± 0.005 0.023 ± 0.003 0.054 ± 0.011 0.058 ± 0.007 0.069 ± 0.009 0.094 ± 0.003
0.205 ± 0.014 0.187 ± 0.013 0.194 ± 0.003 0.245 ± 0.005 0.253 ± 0.007 0.317 ± 0.019 0.313 ± 0.036 0.295 ± 0.010 0.226 ± 0.021 0.215 ± 0.006 0.394 ± 0.027 0.547 ± 0.040 0.347 ± 0.019 0.367 ± 0.029
0 1 2 3 5 7 10 0 1 2 3 5 7 10
0.169 ± 0.002 0.125 ± 0.003 0.131 ± 0.007 0.158 ± 0.006 0.184 ± 0.005 0.113 ± 0.007 0.123 ± 0.003 0.261 ± 0.024 0.280 ± 0.015 0.385 ± 0.014 0.280 ± 0.024 0.290 ± 0.008 0.229 ± 0.012 0.201 ± 0.005
N.D N.D N.D N.D 0.027 ± 0.003 0.024 ± 0.002 0.021 ± 0.002 N.D N.D N.D 0.022 ± 0.001 0.030 ± 0.002 0.043 ± 0.004 0.064 ± 0.005
0.014 ± 0.001 0.018 ± 0.002 0.010 ± 0.001 0.010 ± 0.000 0.036 ± 0.005 0.021 ± 0.002 0.023 ± 0.002 0.027 ± 0.005 0.021 ± 0.004 0.027 ± 0.005 0.030 ± 0.002 0.039 ± 0.009 0.060 ± 0.002 0.135 ± 0.004
0.183 ± 0.002 0.142 ± 0.005 0.140 ± 0.008 0.167 ± 0.006 0.272 ± 0.003 0.180 ± 0.013 0.186 ± 0.006 0.287 ± 0.029 0.299 ± 0.017 0.410 ± 0.012 0.351 ± 0.028 0.387 ± 0.016 0.370 ± 0.017 0.452 ± 0.019
1 4 7 14 21 1 4 7 14 21
N.D N.D N.D N.D 0.011 ± 0.002 0.019 ± 0.003 0.033 ± 0.003 0.068 ± 0.007 0.069 ± 0.005 0.043 ± 0.004
N.D N.D N.D N.D 0.024 ± 0.002 0.066 ± 0.004 0.089 ± 0.009 0.085 ± 0.011 0.114 ± 0.14 0.077 ± 0.003
N.D N.D N.D N.D 0.015 ± 0.004 0.235 ± 0.022 0.333 ± 0.009 0.461 ± 0.021 0.696 ± 0.032 0.798 ± 0.028
N.D N.D N.D N.D 0.073 ± 0.002 0.366 ± 0.022 0.518 ± 0.019 0.662 ± 0.039 0.938 ± 0.037 0.933 ± 0.031
Correction factor for TFNA (1.20) = Flonicamid MW (229.2)/TFNA MW (191.2). Correction factor for TFNG (0.92) = Flonicamid MW (229.2)/TFNG MW (248.2). N.D.: not detected. a Total residue = Residue of P + (MW of P/MW of M) Residue of M (SANCO, 2009).
degradation of the parent compound. Such a residue pattern was similarly found in samples treated twice and cultivated under hydroponic conditions. Park (2010) found that metabolite residue concentrations increase consistently until day 42 post-application. We suggest that the undulating residue pattern observed could explain the movement behavior of systemic pesticides into plants over time. Bound pesticide residues on plant surfaces move from the hydrophobic epicuticular wax layer to inside the plant tissue and are then distributed to hydrophilic plant cells (Yukimoto & Hamada, 1985). Variations in the distribution speed of pesticides across plants are related to differences in their water solubilities as described by Yukimoto and Hamada (1985). The drench-treated paprika showed no residues (until 14 days) or decreasing flonicamid and TFNA residue levels compared to those of TFNG. As drench is one way of direct injection, the analytes take some time to be transported from the root to different parts of the plant. Flonicamid is metabolized in plants via hydrolysis of –CN and – CONH2 groups (Health Canada, 2010). The overall TFNG residue was higher than that of TFNA, suggesting that flonicamid was primarily metabolized to TFNG followed by TFNA. However, various crops should be investigated to demonstrate this flonicamid metabolic pathway(s).
4. Conclusion The improved QuEChERS and validated LC/MS/MS methods allowed for accurate and precise quantitation of flonicamid and its metabolites in paprika with an LOQ of 0.01 mg/kg. The QuEChERS method was improved with acidified ACN and 1 mL acetic acid and NaCl as a salting-out agent. Such modifications have not previously reported in other studies. Our method could be applied to analyze flonicamid and its metabolites in other food commodities. A proportional relationship between pesticide application rate and the resulting residues in paprika were existed. The up and down residue pattern of the parent analyte together with its metabolites could explain the movement behavior of systemic pesticides into plants over time. Acknowledgement This study was supported by the MSIP (Ministry of Science, Ict & future Planning and the Rural Development Administration) (Grant No. PJ008979).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 02.038.
References Anastassiades, M., Lehotay, S. J., Štajnbaher, D., & Schenck, F. J. (2003). 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, 86, 412–431. Cajka, T., Sandy, C., Bachanova, V., Drabova, L., Kalachova, K., Pulkrabova, J., et al. (2012). Streamlining sample preparation and gas chromatography–tandem mass spectrometry analysis of multiple pesticide residues in tea. Analytica Chimica Acta, 743, 51–60. Chen, A. W. (2002). Analytical methodology for IKI-220 (F1785) and its major metabolites in/on peach, potato tuber, and wheat straw. Princeton, NJ, USA: FMC Corporation. Analytical report (P-3561M). Chen, G., Sun, Y., Yang, T., & Wu, Y. (2012). Simultaneous determination of flonicamid and its metabolites in cucumbers and apples by liquid chromatography–tandem mass spectrometry. Se Pu, 30(6), 555–559. Cho, S. K., Abd El-Aty, A. M., Park, K. H., Park, J. H., Assayed, M. E., et al. (2013). Simple multiresidue extraction method for the determination of fungicides and plant growth regulator in bean sprouts using low temperature partitioning and tandem mass spectrometry. Food Chemistry, 136(3–4), 1414–1420. Codex, 1993. Codex Guidelines for the establishment of a regulatory programme for control of veterinary drug residues in foods, CAC/GL 16. Available from:. Dixon, A. (2002). OP alternative status granted to new insecticide, flonicamid (F 1785 GH) for use on ornamentals grown in indoor greenhouses. Alternative to OP’s and other chemistries. Washington, DC, USA: U.S. Environmental Protection Agency. European Food Safety Authority (2010). Conclusion on the peer review of the pesticide risk assessment of the active substance flonicamid. EFSA Journal, 8(5), 1445. EU pesticides database. Pesticide EU-MRL. Regulation (EC) No 396/2005 . Ferrer, I., Fernandez-Alba, A., Zweigenbaum, J. A., & Thurman, E. M. (2006). Exactmass library for pesticides using a molecular-feature database. Rapid Communications in Mass Spectrometry, 20, 3659–3668. Grimalt, S., Thompson, D. G., Coppens, M., Chartrand, D. T., Shorney, T., Meating, J., et al. (2011). Analytical study of azadirachtin and 3-tigloylazadirachtol residues in foliage and phloem of hardwood tree species by liquid chromatography– electrospray mass spectrometry. Journal of Agricultural and Food Chemistry, 59(15), 8070–8077. Health Canada Pest Management Regulatory Agency (2010). Flonicamid. Ontario, Canada: Health Canada.
Hengel, M. J., & Miller, M. (2007). Analysis of flonicamid and its metabolites in dried hops by liquid chromatography–tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 55, 8033–8039. Joost, H., Staetz, C., Black, B., Hayashi, J., Kinne, L., Kelly, G., & Treacy, K. (2006). FMC Corporation. Flonicamid – a novel mode of action for piercing sucking insects. Proceedings – Beltwide Cotton Conferences. Kamel, A. (2010). Refined methodology for the determination of neonicotinoid pesticides and their metabolites in honey bees and bee products by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Journal of Agricultural and Food Chemistry, 58, 5926–5931. Korea Food and Drug Administration (KFDA). (2011). Maximum residue limits of pesticide.. Lee, S. W., Choi, J. H., Cho, S. K., Yu, H. A., Abd El-Aty, A. M., & Shim, J. H. (2011). Development of a new QuEChERS method based on dry ice for the determination of 168 pesticides in paprika using tandem mass spectrometry. Journal of Chromatography A, 1218(28), 4366–4377. Lehotay, S. J. (2007). Determination of pesticide residues in foods by acetonitrile extraction and partitioning with magnesium sulfate: Collaborative study. Journal of AOAC International, 90, 485–520. Matsufuji, H., Nakamura, H., Chino, M., & Takeda, M. (1998). Antioxidant activity of capsanthin and the fatty acid ester in paprika (Capsicum annuum). Journal of Agricultural and Food Chemistry, 46, 3468–3472. Morita, M., Ueda, T., Yoneda, T., Koyanagi, T., Murai, S., Matsuo, N., Stratmann, B., & Ruelens, R. (2000). Ishihara Sangyo Kaisha, Ltd. IKI-220 – a novel systemic aphicide. BCPC Conference – Pests & disease, 59–65. Park, J. H. (2010). Determination of Flonicamid, TFNG, and TFNA in sweet pepper by LC/ MS/MS. (Master thesis). Republic of Korea (Korean): Chonbuk National University. Park, K. H., Choi, J. H., Abd El-Aty, A. M., Cho, S. K., Park, J. H., Kim, B. M., et al. (2012). Determination of spinetoram and its metabolites in amaranth and parsley using QuEChERS-based extraction and liquid chromatography–tandem mass spectrometry. Food Chemistry, 134(4), 2552–2559. RDA (2012). Rural Development Administration, National Academy of Agricultural Science. Pesticide residue definitions for agricultural products. SANCO, (2009). Document No. 10684. Method validation and quality control procedures for pesticide residues analysis in food and feed. The Japan Food Chemical Research Foundation, (2012). Table of MRLs for Agricultural Chemicals. (Available at . Watanabe, E., Kobara, Y., & Yogo, Y. (2012). Rapid and simple analysis of pesticides persisting on green pepper surfaces swabbing with solvent-moistened cotton. Journal of Agricultural and Food Chemistry, 60(36), 9000–9005. Xu, Y., Shou, L. F., & Wu, Y. L. (2011). Simultaneous determination of flonicamid and its metabolites in vegetables using QuEChERS and reverse-phase liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 1218(38), 6663–6666. Yukimoto, M., & Hamada, K. (1985). Eds In Genshoku Sakumotsu No Yakugai, Zenkoku Noson Kyoiku Kyokai: Tokyo, Japan, (in Japanese). Zywitz, D., Anastassiades, M., & Scherbaum, E. (2003). Simultaneous determination of neonicotinoid insecticides in fruits and vegetables by LC–MS and LC–MS/MS. Deutsche Lebensmittel-Rundschau, 99(5), 188–219.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
A modified QuEChERS method for simultaneous determination of flonicamid and its metabolites in paprika using tandem mass spectrometry Ah-Young Ko a, A.M. Abd El-Aty a,b,⇑, Md. Musfiqur Rahman a, Jin Jang a, Sung-Woo Kim a, Jeong-Heui Choi a, Jae-Han Shim a,⇑ a b
Biotechnology Research Institute, College of Agriculture and Life Sciences, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Republic of Korea Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt
a r t i c l e
i n f o
Article history: Received 9 November 2013 Received in revised form 4 February 2014 Accepted 11 February 2014 Available online 22 February 2014 Keywords: Method development Validation Improved sample preparation Tandem mass spectrometry Paprika
a b s t r a c t A modified quick, easy, cheap, effective, rugged and safe (QuEChERS) acetate-buffered sample preparation method was developed to improve extraction recovery of flonicamid and its two metabolites (4-trifluoromethylnicotinic acid and N-(4-trifluoromethylnicotinoyl)glycine) in paprika followed by analysis using tandem mass spectrometry. Acidified acetonitrile (containing 5% acetic acid) was used as an extraction solvent and partitioning was carried out using sodium chloride. The extract was then cleaned up using C18. The linearity over a concentration range of 0.005–1 lg/mL was good with a determination coefficient (R2) > 0.9997. Recovery at three different fortification levels was 82.2–101.7% with a relative standard deviation <10 for all analytes. The limit of quantitation of 0.01 mg/kg was quite lower than the maximum residue level set by the Korea Food and Drug Administration (2 mg/kg). The method was successfully applied to determine flonicamid and its metabolites from field incurred samples. The undulating residue pattern observed for the parent analyte together with its metabolites could explain the movement behavior of systemic pesticides into plants over time. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Various kinds of pesticides have been used regularly to increase production, quality and storage longevity of agricultural commodities throughout the globe. Pesticide residues in food naturally pose a potential hazardous risk to consumers. Thus, pesticide and/or metabolite residues should be analyzed in all agricultural commodities to improve public health and food safety (Park et al., 2012). Among these pesticides; flonicamid, (N-cyanomethyl-4-trifluoromethylnicotinamide), a selective systemic pesticide, is highly effective against aphids and other sucking insects (Morita et al., 2000). Flonicamid blocks type-A potassium channels, which leads to loss of direct movement and suppression of aphid feeding (Joost et al., 2006). This analyte can be used in integrated pest management programmes as it has a minimal cross-resistance
⇑ Corresponding authors. Address: Biotechnology Research Institute, College of Agriculture and Life Sciences, Chonnam National University, Buk-gu, Gwangju 500-757, Republic of Korea. Tel.: +20 2 27548926; fax: +20 2 35725240 (A.M. Abd El-Aty). Tel.: +82 62 530 2135; fax: +82 62 530 0219 (J.-H. Shim). E-mail addresses: [email protected] (A.M. Abd El-Aty), jhshim@chonnam. ac.kr (J.-H. Shim). http://dx.doi.org/10.1016/j.foodchem.2014.02.038 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
characteristics and lack of toxicity to beneficial arthropods (Dixon, 2002). Analyte metabolism is comparable in wheat, potato and peach, and the residues are composed of the parent compound (flonicamid) and its two major metabolites TFNA (4-trifluoromethylnicotinic acid) and TFNG (N-(4-trifluoromethylnicotinoyl)glycine. The ratios between both metabolites are substantially different depending on the crop (European Food Safety Authority, 2010). Additional metabolites, including TFNA-AM (4-trifluoromethylnicotinamide) and TFNG-AM are not expected to exist in plants at significant levels and, consequently, the residue definition for risk assessment is limited to the sum of flonicamid, TFNA and TFNG (European Food Safety Authority, 2010). In the Republic of Korea, the Korea Food and Drug Administration (KFDA refers to the residue for monitoring flonicamid to the parent analyte only and set its maximum residue level (MRL) to 2 mg/kg in paprika (KFDA, 2011). However, the Japanese define the residue to the parent compound (flonicamid) and its metabolites; TFNA and TFNG and set the MRL to 2 mg/kg in paprika (The Japan Food Chemical Research Foundation, 2012). Such differences between countries causes obstacles to trade, and interferes with international trade of agricultural commodities (Lee et al., 2011). The Rural Development Administration (RDA, 2012), the
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organisation responsible for pesticide registration and safety guideline in Republic of Korea, changed their concept and used the same definition set by the Japan Food Chemical Research Foundation (2012); however, the KFDA, the organisation responsible for setting and implementing the MRL, still refer to the flonicamid value only. We expect that this change will happen in the upcoming upgrade of the KFDA databases. Paprika (Capsicum annuum) is widely used as a vegetable and food additive, as this fruit is a good source of carotenoid pigments. These fruits are often grown under glasshouse conditions in highvalue production systems. Carotenoids are effective as free-radical scavengers (Matsufuji, Nakamura, Chino, & Takeda, 1998). Therefore, consumers consume paprika for its nutritive value. Paprika contains more than 2500 natural compounds (Ferrer, Fernandez-Alba, Zweigenbaum, & Thurman, 2006) that may mask the detection of some pesticide residues and make it difficult or impossible to identify the target compounds. Efficient sample preparation is absolutely necessary for an accurate determination of pesticide residues in foods consisting of complicated matrices (Watanabe, Kobara, & Yogo, 2012). Pesticide residues are distributed heterogeneously in/on crops; thus, the sample must be homogenized thoroughly. A sufficiently homogeneous sample is extracted, with the result that matrix components are often co-extracted together with the target pesticides. Consequently, cleanup processes are necessary (Watanabe et al., 2012). High-pressure liquid chromatography (HPLC) coupled with mass spectrometry (MS) is available to detect flonicamid and its metabolites in agricultural matrices (Chen, 2002; Zywitz, Anastassiades, & Scherbaum, 2003). Hengel and Miller (2007) conducted liquid–liquid extraction (LLE) with a mixture of acetonitrile (ACN) and water (50/50, v/v) and a two-step solid-phase extraction (SPE) cartridge cleanup followed by liquid–liquid partitioning for liquid chromatography–tandem mass spectrometry (LC/MS/MS) analysis to extract flonicamid, TFNA and TFNG. Chen, Sun, Yang, and Wu (2012) detected flonicamid and its metabolite (TFNG, TFNA and TFNAAM) in cucumbers and apples by LC/MS/MS with LLE. However, these methods are tedious, time-consuming and not environmentally friendly. Xu, Shou, and Wu (2011) detected flonicamid and its metabolites TFNG, TFNA and TFNA-AM in spinach and cucumber using QuEChERS and primary secondary amine (PSA) and graphite carbon black (GCB) as a cleanup procedure followed by LC/MS/MS. In the current study, we used the same amount of PSA (0.1 g) for paprika; however, the recoveries of metabolites were not satisfactory. The aim of this study was to detect the residues of flonicamid and its major metabolites (TFNG and TFNA) in paprika grown under greenhouse conditions using a modified acetate-buffered QuEChERS method for extraction and LC/MS/MS for analysis.
2. Experimental 2.1. Chemicals and reagents Reference standards for flonicamid (CAS Registry No. 15806267-0; purity 98.5%), TFNA (CAS Registry No. 158063-66-2; purity 94.9%), and TFNG (CAS Registry No. 207502-65-6; purity 99.4%) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). HPLC-grade acetonitrile (ACN) was purchased from Burdick and Jackson (Ulsan, Republic of Korea). Sodium chloride (NaCl, purity 99.5%) was obtained from Merck (Darmstadt, Germany), and sodium acetate (NaOAc, purity 98.0%), anhydrous magnesium sulfate (MgSO4, purity 99.5%), and triethylamine (TEA, purity 99.0%) were provided by Junsei Chemical Co., Ltd. (Kyoto, Japan). Primary secondary amine (PSA) and C18 were supplied by Agilent Technologies (Palo Alto, CA, USA). Acetic acid (purity 99.5%) and formic acid (purity 85.0%) were obtained from Daejung Chemicals & Mate-
rials (Siheung, Republic of Korea), and Duksan Pure Chemicals Co., Ltd. (Ansan, Republic of Korea), respectively. 2.2. Standard solution preparation Individual standard solutions of flonicamid and its metabolites (TFNA and TFNG) were prepared in ACN at a concentration of 1000 lg/mL. A working solution (10 lg/mL) was prepared by diluting the stock solution with blank sample extracts, which were confirmed previously to contain none of the tested analytes. Matrix-matched calibration standard solutions were prepared by mixing the matrix-matched working standard solutions with additional blank sample extracts to reach the multi-compound concentrations of 0.005–1 lg/mL. All standards solutions were stored at 20 °C in a bottle and freshly prepared matrix standards were used before each analysis. 2.3. Field trails Experimental trials were carried out in a greenhouse located at Chonnam National University, Gwangju, Republic of Korea. Paprika was cultivated either in soil or using hydroponics. The commercial formulation was a water dispersible granule (WG), which contained 10% of the active ingredient of flonicamid (SetisÒ, Dongbu HiTek, Seoul, Republic of Korea). The formulation was applied as a direct spray treatment for soil cultivation, whereas direct spray and drench treatments were used for paprika cultivated in hydroponic soilless media. The recommended dose was sprayed either once or twice with a 7 day interval in case of the direct spray or 30 days in case of the drench treatment. Samples were randomly collected at 0 (2 h later) 1, 3, 5, 7 and 10 days after application; however, the drench treated paprika samples were collected at 1, 4, 7, 14 and 21 days following the final application. The samples were transferred to the laboratory in sealed bags, chopped, mixed with other samples from the same plot, packed in plastic bags, and stored at 20 °C pending analysis. 2.4. Sample preparation Extraction and cleanup were carried out using acetate-buffered QuEChERS after major modification (Lehotay, 2007). Approximately 10 g of homogenized paprika samples were placed into a 50-mL Teflon centrifuge tube. Twenty mL of 5% acetic acid in ACN was added to 10 mL of water, and the tubes were vortexmixed for 2 min. Then, 10 g NaCl was added to each sample and vortex-mixed vigorously for an additional 2 min. The extract was centrifuged for 5 min at 5000 rpm and room temperature, and the supernatant (6 mL) was transferred to a 15-mL Teflon centrifuge tube containing 0.3 g of C18. The tubes were shaken for 30 s for cleanup and centrifuged again for 5 min at 3000 rpm. One mL of the upper layer was filtered through a polytetrafluoroethylene (PTFE) membrane filter (0.2 lm, ADVANTECÒ, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) and subsequently analyzed via LC/MS/MS. 2.5. LC/MS/MS The LC/MS/MS system consisted of a Waters Alliance 2695 LC Separations Module and a Micromass Quattro Micro triple quadrupole tandem mass (Waters Crop., Milford, MA, USA). The analytes were separated on a X-bridge C18 (2.1 mm i.d 150 mm, 3.5 lm, Waters Crop.) that had been kept in an oven at 35 °C. A binary solvent system containing ACN (mobile phase A) and 0.1% formic acid in water (mobile phase B) was run in a gradient mode to detect flonicamid and its metabolites; TFNA, and TFNG, simultaneously. A linear mobile phase gradient started at 5% A (0–1.5 min), increased to 50% A (1.5–3 min), increased to 90% A (3–6 min),
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maintained at 90% A (6–8 min), and decreased to 5% A (8–8.1 min), after which the column was equilibrated at 5% A (8.1–12 min). Flow rate, and injection volume were set at 0.25 mL/min, and 5 lL, respectively. Tandem mass spectrometric analysis was carried out via electrospray ionization in positive mode and operated in multiple reactions monitoring (MRM) mode. The MS source condition were as follows: capillary voltage, 3.5 kV; source temperature, 150 °C; desolvation temperature, 350 °C; desolvation gas (N2) flow, 650 L/h; cone gas (N2) flow, 50 L/h; and collision gas (argon) pressure of 4.0 10 3 mbar. Optimization of the precursor ion, product ions, and collision energy (CE) was performed via direct injection of the analyte and metabolite standard solution (1 lg/mL) into the MS/MS system. The most intense transition was used for quantitation, while the other was employed for confirmation. The optimization parameters are presented in Supplementary Table. Mass Lynx V4.1 software (Waters Corp.) was used for instrument control, data acquisition and processing.
2.6. Validation of the analytical method The method was validated using the single laboratory validation approach such as linearity, specificity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. Linearity was assessed by the determination coefficient (R2) of a matrixmatched calibration curve that was created based on eight points of different flonicamid concentrations (0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 lg/mL) and its metabolites TFNA and TFNA. The LOD and LOQ were determined using signal-to-noise ratios of 3 and 10, respectively. Recovery experiments were conducted by fortifying blank paprika samples with mixed standard solutions at three different fortification levels (0.05, 0.1, and 0.5 mg/kg) with three replicates per level. Precision (repeatability and intermediate precision) of the analytical method was estimated from the relative standard deviations (RSD%) analyzed on the same day (intra-day) and across 3 days (inter-day precision), respectively. Matrix effects in terms of signal suppression or enhancement due to co-elution of matrix components were evaluated by post-extraction spiking and compared with the solvent standards. Higher and lower slopes of the matrix calibration equation with reference to the solvent-based calibration equation represent matrix-induced enhancement and suppression, respectively (Cho et al., 2013).
3. Results and discussion
3.1.1. Sample preparation Paprika was hydrated before being extracted through salting out with ACN followed by cleanup procedures. Cajka et al. (2012) clearly documented that adding water to the sample is a key to achieve maximum extraction yield (and therefore accurate results). 3.1.2. Effect of salting out agents The effect of salting out agents during the partitioning step to extract flonicamid and its metabolites was tested using NaCl, NaOAc, or MgSO4 in paprika samples. No substantial differences were observed when the salts were used alone or in combination regarding the recovery of flonicamid; however, recovery of the metabolites was significantly altered (Table 1). The TFNA and TFNG metabolites were completely unrecovered when 1% acetic acid in ACN was used with the QuEChERs salts (6 g of MgSO4 and 1.5 g of NaOAc) (Lehotay, 2007). When the same experiment was conducted with MgSO4 without NaOAc, recovery of TFNA and TFNG increased to 34.24% and 47.85%, respectively. In contrast, the highest recovery for TFNA (53.99%) and TFNG (47.85) was observed when NaCl was used. Therefore, we used NaCl as a salting out agent to separate the compounds from the aqueous fraction into the organic layer during the extraction partitioning step. 3.1.3. Effect of acidic and basic agents The effect of acetic acid as an acidic agent or triethylamine (TEA) as a basic agent was evaluated to successfully recover flonicamid and its metabolites. Kamel (2010) reported that 2% TEA in ACN is an appropriate solvent to extract nitro-substitute compounds, but it failed to completely recover the analytes and the metabolites used here. However, the recovery of TFNA and TFNG improved substantially when acetic acid in ACN was used. Different volumes of acetic acid from 100–1200 lL were tested to optimize the amount. Fig. 1 shows that 1000 lL of acetic acid produced a satisfactory recovery. 3.1.4. Optimization of cleanup We tried QuEChERS dispersive-SPE cleanup using PSA. Lehotay (2007) reported that PSA strongly binds pesticides containing a carboxylic acid group, which reduces their recovery rates. TFNA and TFNG yielded recovery of <70% when PSA was used (Fig. 1). The C18 absorbent does not have a substantial effect on metabolite recovery, even though the amount was increased. Thus, PSA was replaced with C18 absorbent. Fig. 1 shows the effect of PSA and C18 on the recovery of flonicamid, TFNA and TFNG. C18 results in decreasing chromatographic interference and increases sensitivity to quantitatively identify the tested analytes (Grimalt et al., 2011).
3.1. Sample extraction and cleanup
3.2. Chromatographic optimization
Given the matrix-dependent issues associated with co-extractive interference, rapid and effective cleanup procedures are required for complex matrices. The extraction step is followed by a cleanup step involving either SPE or dispersive-solid phase extraction (d-SPE). QuEChERS is used to reduce sample handling and processing time for analysis of pesticides in fruit and vegetable samples (Grimalt et al., 2011). QuEChERS is a method composed of an extraction step with ACN and partitioning using MgSO4, followed by d-SPE using PSA that was developed to detect pesticide residues in crops and other foods by Anastassiades, Lehotay, Stajnbaher, and Schenck (2003). When the above mentioned conditions were applied to paprika, good recovery was observed for the parent compound; however, that of the metabolites was <50%. The poor recovery of metabolites encouraged us to find a way to improve it. Thus, the effects of various extraction factors, including sample preparation, salting out, acidic and basic agents were investigated.
Different gradients of water/ACN as a mobile phase are good ionization media to separate flonicamid and its metabolites. A gradient elution programme provided baseline resolution for the analyte and its metabolites with a high detection signal, improved peak shapes, and shortened the detection time of all analytes (within 10 min). Additionally, the selected programme facilitated efficient removal of matrix constituents, and reduced noise level Table 1 Effect of salting out agents on recovery of flonicamid and its metabolites. Compound
Flonicamid TFNA TFNG
Salting out agents (mean ± SD) MgSO4
NaCl
MgSO4 + NaOAc
NaCl + NaOAc
96.74 ± 2.66 34.24 ± 6.42 47.85 ± 3.45
99.06 ± 1.18 53.99 ± 6.28 67.76 ± 0.99
95.9 ± 1.20 1.97 ± 0.68 N.D
116.88 ± 3.45 16.73 ± 2.44 11.23 ± 6.54
N. D. Not detected.
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Fig. 1. Effect of acetic acid, primary secondary amine (PSA), and C18 amounts on the extraction efficiency of flonicamid and its metabolites in paprika.
and risk for carry-over effects and column deterioration (Cho et al., 2013). As shown in Fig. 2, no interfering peaks were observed around the retention time of the analytes. The incorporation of formic acid into the mobile phase at a low concentration of 0.1% facilitated ionization of the analyte and its metabolites. Filtering the extracts prior to LC/-MS/MS analysis is necessary to prevent instrument and column damage (Cho et al., 2013). We used a PTFE membrane filter prior to analysis. Optimal ionization occurred at a 350 °C desolvation temperature. Among the two mass transitions, flonicamid and it metabolites (TFNA and TFNG) showed their greatest intensity at m/z 203.22, 148.03, and 203.17, respectively, and were considered a quantitation ion. However, the lower intensities at m/z 148.05, 98.12, and 148.24 were considered a confirmation ion.
metabolites in paprika. The matrix-matched calibration curve was obtained by plotting peak area vs. the concentration of the analytes. Good linearity (over a concentration range of 0.005– 1 lg/mL) with determination correlation coefficients (R2) of 0.9997 were achieved for the three analytes as given in Table 2. 3.3.3. LOD and LOQ The LOD and LOQ of all analytes were 0.003 and 0.01 mg/kg, respectively, (Table 2). The LOQ value was below flonicamid (total sum) residue MRLs set by the EU for pepper in general (0.15 mg/kg, EU pesticides database, 2005), the Japan MRL (2 mg/kg) for paprika (The Japan Food Chemical Research Foundation, 2012) and the Republic of Korea MRL (2 mg/kg) (RDA, 2012).
3.3.1. Specificity Specificity was validated by analyzing representative blank paprika samples (n = 3) to confirm the absence of potential interfering compounds at various retention times. No interfering peaks appeared at the retention times of flonicamid or its two metabolites (Fig. 2).
3.3.4. Recovery Recovery tests were carried out at three different concentrations (0.05, 0.1, and 0.5 mg/kg) in three replicates. Fig. 2 shows the LC/MS/MS chromatograms obtained from the blank samples fortified with 0.1 mg/kg flonicamid, TFNA and TFNG. The recoveries of the flonicamid and its metabolites were good (82.2–101.7% with RSD <10%) and were independent of sample matrix and the fortified level (Table 2). All analytes showed results consistent with the acceptable range specified by SANCO, 2009 (70–120%).
3.3.2. Linearity and the matrix effect A comparison between calibration curves obtained from standards prepared in pure solvent and calibration curves constructed using matrix spiked with standards was performed to evaluate the matrix effect (Cho et al., 2013). The responses obtained were comparable, and no significant effect was observed (Table 2). Matrixmatched calibration was used to quantify flonicamid and its
3.3.5. Accuracy and precision Accuracy and precision were evaluated via intra- and inter-day analysis, both of which were conducted in a single laboratory. Precision was calculated in terms of inter-day repeatability and inter-day reproducibility. Accuracy was described as the mean of recovery and precision was expressed as the RSD. Recovery percent was calculated by comparing the results of the intra- and inter-day
3.3. Validation performance
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417
Fig. 2. Representative MRM (quantitation ion) chromatograms of (a) Blank sample, (b) standard (flonicamid or its metabolites) at 0.1 mg/kg, (c) fortified paprika sample at 0.1 mg/kg, and (d) field incurred paprika sample. Flonicamid (A), TFNA (B), and TFNG (C).
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Fig. 2 (continued)
Table 2 Linearity, LOD, LOQ, and recovery of flonicamid and its metabolites in paprika. Linearity (R2)
Compounds
Flonicamid TFNA TFNG
Recovery (RSD%)
Solvent
Matrix-matched
0.05 mg/kg
0.1 mg/kg
0.5 mg/kg
0.9996 0.9995 0.9985
0.9998 0.9997 0.9997
94.0 (3.84) 86.8 (3.84) 101.1 (9.04)
101.1 (3.52) 82.9 (6.76) 88.7 (2.47)
101.7 (3.14) 82.2 (4.80) 85.4 (2.79)
repeatability with the matrix-matched standard solutions prepared at the same concentration in a blank extract. The mean intra- and inter day accuracy ranged from 85.4% to 103.2% and from 78.0% to 101.1% for all analytes, whereas intra- and interday precision was 3.14–7.22 and 2.22–9.04%, respectively. As shown in Table 3, these values were consistent with the ranges listed in the Codex guidelines (Codex, 1993) suggesting that the present method is reliable, reproducible and accurate.
LOQ (mg/kg)
0.003
0.01
Table 3 Intra-day and inter-day accuracy and precision of flonicamid and its metabolites in paprika. Compounds
Flonicamid
TFNA
3.4. Method application TFNG
The method was applied to two different types (direct spray and drench treatment) of treated paprika samples obtained from soil cultivation and hydroponic conditions. The total residue was estimated by the sum of flonicamid and its metabolite residues (SANCO, 2009). Total flonicamid residual concentrations (containing metabolites residues) in field samples are reported in Table 4. The residue level of the parent compound in the direct spray under soil cultivation treatment decreased slightly from day 0 to day 2 post-application. Then, the residues increased gradually from days 2 to 5 and decreased thereafter, both for the one and two time applications, respectively. The highest residual concentration of parent compound was detected on day 7 post-application. TFNA
LOD (mg/kg)
Fortified concentration (mg/kg)
0.05 0.1 0.5 0.05 0.1 0.5 0.05 0.1 0.5
Accuracy (%) (RSD) Intra-daya
Inter-dayb
94.0 (3.84) 101.1 (3.52) 101.7 (3.14) 86.8 (8.84) 82.9 (6.76) 82.2 (4.40) 101.1 (9.04) 88.7 (2.47) 85.4 (2.79)
85.4 (7.22) 101.0 (6.59) 103.2 (5.81) 79.7 (7.22) 78.4 (7.58) 78.0 (6.88) 88.8 (8.58) 85.5 (4.85) 85.6 (2.22)
a Intra-day repeatability was estimated by analyzing three replicate samples at three concentration levels on the same day. b Inter-day repeatability was estimated by analyzing three replicate samples at three concentration levels on 3 consecutive days.
was first detected on days 5 and 7 and thereafter increased substantially. In contrast, the TFNG residues increased steadily after day 2 to day 10. Although the parent compound residue decreased over time, flonicamid (as total residues) had a tendency to increase over time. This was because the metabolites were produced from
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A.-Y. Ko et al. / Food Chemistry 157 (2014) 413–420 Table 4 Field-incurred flonicamid residues and its metabolites in paprika grown under soil cultivation and hydroponic conditions using direct spraying and drench treatment. Cultivation
Direct spray Soil cultivation
Treatment
Once
Twice
Hydroponic
Once
Twice
Drench treatment Hydroponic
Once
Twice
Days after application
Residual concentration (Mean ± SD) (mg/kg) Flonicamid
TFNA
TFNG
Total residuea
0 1 2 3 5 7 10 0 1 2 3 5 7 10
0.159 ± 0.011 0.144 ± 0.008 0.160 ± 0.002 0.229 ± 0.004 0.229 ± 0.004 0.230 ± 0.013 0.210 ± 0.026 0.263 ± 0.004 0.198 ± 0.016 0.193 ± 0.008 0.328 ± 0.027 0.451 ± 0.034 0.225 ± 0.017 0.211 ± 0.021
N.D N.D N.D N.D N.D 0.028 ± 0.003 0.029 ± 0.002 N.D N.D N.D N.D 0.028 ± 0.002 0.030 ± 0.004 0.035 ± 0.005
0.049 ± 0.007 0.046 ± 0.006 0.036 ± 0.003 0.017 ± 0.001 0.025 ± 0.004 0.033 ± 0.003 0.063 ± 0.007 0.035 ± 0.008 0.031 ± 0.005 0.023 ± 0.003 0.054 ± 0.011 0.058 ± 0.007 0.069 ± 0.009 0.094 ± 0.003
0.205 ± 0.014 0.187 ± 0.013 0.194 ± 0.003 0.245 ± 0.005 0.253 ± 0.007 0.317 ± 0.019 0.313 ± 0.036 0.295 ± 0.010 0.226 ± 0.021 0.215 ± 0.006 0.394 ± 0.027 0.547 ± 0.040 0.347 ± 0.019 0.367 ± 0.029
0 1 2 3 5 7 10 0 1 2 3 5 7 10
0.169 ± 0.002 0.125 ± 0.003 0.131 ± 0.007 0.158 ± 0.006 0.184 ± 0.005 0.113 ± 0.007 0.123 ± 0.003 0.261 ± 0.024 0.280 ± 0.015 0.385 ± 0.014 0.280 ± 0.024 0.290 ± 0.008 0.229 ± 0.012 0.201 ± 0.005
N.D N.D N.D N.D 0.027 ± 0.003 0.024 ± 0.002 0.021 ± 0.002 N.D N.D N.D 0.022 ± 0.001 0.030 ± 0.002 0.043 ± 0.004 0.064 ± 0.005
0.014 ± 0.001 0.018 ± 0.002 0.010 ± 0.001 0.010 ± 0.000 0.036 ± 0.005 0.021 ± 0.002 0.023 ± 0.002 0.027 ± 0.005 0.021 ± 0.004 0.027 ± 0.005 0.030 ± 0.002 0.039 ± 0.009 0.060 ± 0.002 0.135 ± 0.004
0.183 ± 0.002 0.142 ± 0.005 0.140 ± 0.008 0.167 ± 0.006 0.272 ± 0.003 0.180 ± 0.013 0.186 ± 0.006 0.287 ± 0.029 0.299 ± 0.017 0.410 ± 0.012 0.351 ± 0.028 0.387 ± 0.016 0.370 ± 0.017 0.452 ± 0.019
1 4 7 14 21 1 4 7 14 21
N.D N.D N.D N.D 0.011 ± 0.002 0.019 ± 0.003 0.033 ± 0.003 0.068 ± 0.007 0.069 ± 0.005 0.043 ± 0.004
N.D N.D N.D N.D 0.024 ± 0.002 0.066 ± 0.004 0.089 ± 0.009 0.085 ± 0.011 0.114 ± 0.14 0.077 ± 0.003
N.D N.D N.D N.D 0.015 ± 0.004 0.235 ± 0.022 0.333 ± 0.009 0.461 ± 0.021 0.696 ± 0.032 0.798 ± 0.028
N.D N.D N.D N.D 0.073 ± 0.002 0.366 ± 0.022 0.518 ± 0.019 0.662 ± 0.039 0.938 ± 0.037 0.933 ± 0.031
Correction factor for TFNA (1.20) = Flonicamid MW (229.2)/TFNA MW (191.2). Correction factor for TFNG (0.92) = Flonicamid MW (229.2)/TFNG MW (248.2). N.D.: not detected. a Total residue = Residue of P + (MW of P/MW of M) Residue of M (SANCO, 2009).
degradation of the parent compound. Such a residue pattern was similarly found in samples treated twice and cultivated under hydroponic conditions. Park (2010) found that metabolite residue concentrations increase consistently until day 42 post-application. We suggest that the undulating residue pattern observed could explain the movement behavior of systemic pesticides into plants over time. Bound pesticide residues on plant surfaces move from the hydrophobic epicuticular wax layer to inside the plant tissue and are then distributed to hydrophilic plant cells (Yukimoto & Hamada, 1985). Variations in the distribution speed of pesticides across plants are related to differences in their water solubilities as described by Yukimoto and Hamada (1985). The drench-treated paprika showed no residues (until 14 days) or decreasing flonicamid and TFNA residue levels compared to those of TFNG. As drench is one way of direct injection, the analytes take some time to be transported from the root to different parts of the plant. Flonicamid is metabolized in plants via hydrolysis of –CN and – CONH2 groups (Health Canada, 2010). The overall TFNG residue was higher than that of TFNA, suggesting that flonicamid was primarily metabolized to TFNG followed by TFNA. However, various crops should be investigated to demonstrate this flonicamid metabolic pathway(s).
4. Conclusion The improved QuEChERS and validated LC/MS/MS methods allowed for accurate and precise quantitation of flonicamid and its metabolites in paprika with an LOQ of 0.01 mg/kg. The QuEChERS method was improved with acidified ACN and 1 mL acetic acid and NaCl as a salting-out agent. Such modifications have not previously reported in other studies. Our method could be applied to analyze flonicamid and its metabolites in other food commodities. A proportional relationship between pesticide application rate and the resulting residues in paprika were existed. The up and down residue pattern of the parent analyte together with its metabolites could explain the movement behavior of systemic pesticides into plants over time. Acknowledgement This study was supported by the MSIP (Ministry of Science, Ict & future Planning and the Rural Development Administration) (Grant No. PJ008979).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 02.038.
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