A modified QuEChERS method for simultaneous

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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A.-Y. Ko et al. / Food Chemistry 157 (2014) 413–420
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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A.-Y. Ko et al. / Food Chemistry 157 (2014) 413–420
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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A.-Y. Ko et al. / Food Chemistry 157 (2014) 413–420
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
A.-Y. Ko et al. / Food Chemistry 157 (2014) 413–420
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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A.-Y. Ko et al. / Food Chemistry 157 (2014) 413–420
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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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).
420
A.-Y. Ko et al. / Food Chemistry 157 (2014) 413–420
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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