Food Chemistry 276 (2019) 140–146

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Evaluation of cleanup procedures in pesticide multi-residue analysis with T

QuEChERS in cinnamon bark
Zihao Zhanga, Maofeng Dongb, Xianghong Haoa, Lijun Hana, , Shuangyu Songa, Wei Yaoa
⁎

a
College of Science, China Agricultural University, Beijing 100193, China
b
Pesticide Safety Evaluation Research Center, Institute for Agro-food Standards and Testing Technology, Shanghai Academy of Agricultural Sciences, Shanghai 201106,
China

ARTICLE INFO ABSTRACT

Keywords: Cinnamon bark is a very complex spices matrix for pesticide residue analysis. In this study, two cleanup pro-
QuEChERS cedures, dispersive solid-phase extraction (d-SPE) and repetitive d-SPE, combined with Quick, Easy, Cheap,
d-SPE Effective, Rugged and Safe (QuEChERS) extraction were both investigated and applied for the determination of
Repetitive d-SPE 60 pesticide residues in cinnamon bark by liquid chromatography − tandem mass spectrometry (LC-MS/MS)
Pesticides
detection. The extraction solvents, phase partition salts, and cleanup sorbents were optimized. The results
Cinnamon
showed that 150 mg MgSO4 + 50 mg C18 showed the best performance in d-SPE. The repetitive d-SPE achieved
less matrix effects than d-SPE, but several high water-solubility analytes were lost during the cleanup, and also
the 10 fold dilution compromised the sensitivities of analytes. With the validated QuEChERS-d-SPE method, 44
of 60 pesticides obtained satisfactory recoveries (71%–118%) and RSDs (2%–27%) at three to five spiking levels.
The method was applied to monitor 15 market cinnamon samples.

1. Introduction investigated the organochlorine pesticide residues in Chinese herbal

Cinnamon bark is obtained from the inner bark of several tree
species from the genus Cinnamomum, and is an important species of
medicinal and edible spices. These species are widely cultivated in
southern China and some countries of southern and eastern Asia areas
(Li et al., 2013). Since cinnamon bark is grown in humid and hot areas,
the damage of bacteria, fungi and pests is inevitable. For example,
imidacloprid, chlorpyrfos and acetamiprid are frequently used in-
secticides for the treatment of plant-bug, roll moth and wood moth
which are common and major threats to the production of cinnamon
(Cross, 2010; Yamada, Takahashi, & Hatano, 1999; Yue, Wilde, &
Arthur, 2003); and difenoconazole, carbendazim have a good preven-
tion to diseases from bacteria and fungi, such as, anthrax and brown
blotch (Li, 2009) (APS crop protection and management collection,
Evaluation of captan/topsin program and the experimental fungicide
difenoconazole for the control of sooty blotch, flyspeck, and rots http://
plantmanagementnetwork.org/pub/trial/PDMR/volume1/abstracts/
PF010.asp). Besides, use of herbicides can effectively prevent weeds in
the cinnamon tree orchard.
However, use of pesticides may lead to potential pesticide residues
in edible parts of cinnamon trees. Only two market investigations of
herbal plants (involved cinnamon) were found so far. Hwang et al.
formulations with solid-phase microextraction (SPME) and gas chro-
matography-mass spectrometry (GC–MS) detection which indicated a
handful residues of γ-HCH (3.7 ng/g), aldrin (5.1 ng/g), p,p′-DDT
(3.9 ng/g), and endosulfan (23.1 ng/g) in cinnamon and mahuang
(Hwang & Lee, 2000). Another report was for 8 kinds of medicinal
plants (cinnamon, rosemary, Chinese tea, etc.) from different markets in
Jeddah, Saudi Arabia, and the results showed that cinnamon were free
from 30 of 31 pesticides except chloropyrifos which is less than limit of
quantification (LOQ) (0.01 mg/kg) (Mosleh, Nafea, Almagrabi, &
Alkaladi, 2016). Therefore, it is necessary to do more researches to
investigate the pesticide and other contaminant residues in cinnamon
species.
For the safe use of pesticides on cinnamon, the maximum residue
limit (MRL) regulations (listed in Table S1) was issued by European
Union (EU), which shows that MRLs of most pesticides are between
0.05 and 0.2 mg/kg (http://ec.europa.eu/food/plant/pesticides/eu-
pesticides-database/public, http://bcglobalportal.com/), and also EU
set a default MRL of 0.01 mg/kg for those pesticides not regulated for a
specific matrix. There are no registered pesticides and established MRLs
in China and Codex Alimentarius Commission (CAC) (http://www.
chinapesticide.gov.cn/), and only USA has the MRL of azoxystrobin of
38 mg/kg in cinnamon.

⁎
Corresponding author.
E-mail address: hlj2000@cau.edu.cn (L. Han).

https://doi.org/10.1016/j.foodchem.2018.10.019
Received 13 April 2018; Received in revised form 23 August 2018; Accepted 3 October 2018
Available online 04 October 2018
0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
Z. Zhang et al.
Fig. 1. Flowchart of repetitive d-SPE clean-up procedure.
In order to monitor or screen the potential risks of the pesticide
residues in cinnamon products, it is essential to use efficient and reli-
able methods for the multi-residue analysis in cinnamon samples.
However, the reports related to the analysis methods of cinnamon bark
are very few and limited. Xie (2007) described a method which de-
termined twelve organochlorine pesticides (hexachlorocyclohexane
(BHC) and four structural isomers, dichlorodiphenyltrichloroethane
(DDT) and four structural isomers, pentachloronitrobenzene (PCNB)
and hexachlorobenzene (HCB) by GC-ECD in cinnamon bark. The
samples were extracted by acetone and cleaned-up by sulfonation
method. Similarly, Liu, Qiang, Zhong, and Wen (2014) developed a
method to determine BHC (four structural isomers included) and DDT
(four structural isomers included) which were also extracted by acetone
but cleaned up by SPE and detected by GC-μECD. The residue analysis
of chlorpyrifos in cinnamon was described by Li and Cai (2007) which
was extracted by dichloromethane and cleaned up by neutral alumina
column chromatography. In the previously mentioned pesticide residue
survey of eight types of medicinal plants conducted in Saudi Arabia, 31
pesticides were screened, using QuEChERS combined with a citrate
buffer. However, the samples were extracted and concentrated by ro-
tary evaporation and were directly injected into HPLC-MS/MS without
additional cleanup. A fully validated method is still needed for quan-
tification of those analytes. Also, we discovered that most LC-amenable
and other types of GC-amenable pesticides, except organochlorine and
organophosphorus, were rarely reported and the sample preparation
was complex and time consuming. Therefore, it is necessary to develop
an easy, fast and broad method to monitor the pesticides in cinnamon
bark.
The “quick, easy, cheap, effective, rugged, and safe” (QuEChERS)
approach was introduced in 2003 (Anastassiades, Lehotay, Stajnbaher,
& Schenck, 2003) and has demonstrated wide application in analysis of
pesticide residues in a variety of matrices. Dispersive solid-phase ex-
traction (“d-SPE”) was the general cleanup procedure in QuEChERS
method to remove residual water and matrix co-extracts (including
pigments, fatty acids, and essential oils, etc.). However, spices are
known to be difficult matrices in residue analysis due to the complex
components and significant matrix effects (Lacina et al., 2012). For
example, Yogendrarajah et. al. found 17%–66% of the compounds in
seven kinds of spices (black pepper, paprika, curcuma, etc.) showed
strong matrix suppression effects (≤−50%) when determining afla-
toxins, dyes and 21 insecticides and fungicides by QuEChERS and LC-
MS/MS using electrospray ionization (ESI) (Yogendrarajah, Van, De, &
De, 2013). A simple and efficient cleanup method is therefore critically
needed for residue analysis of complex matrices such as cinnamon bark.
However, no related information was reported on cinnamon so far.
The chemical composition of cinnamon bark is very complex, and
researches indicated that it consists of aromatic essential oil (about
0.5%–1%), diterpenoids, flavonoids, glycosides, and some polysomal
compounds (Lou & Qin, 1995). These components could be co-extracted
by the solvent and appear in chromatograms which potentially interfere
with the quantification of target analytes. Therefore, further cleanup
was essential after the extraction step. As a rapid cleanup method, d-
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Food Chemistry 276 (2019) 140–146
SPE was originally adopted in QuEChERS procedure, for which, various
kinds of sorbents with different adsorption characteristics can help re-
move co-extracted components, individually or simultaneously
(Anastassiades et al., 2003). For example, PSA is reported to have sig-
nificant retention of organic acids, fatty acids, sugars, and pigments,
whereas C18 is often helpful to retain lipophilic materials, such as
sterols, fat, and volatile oil (Lehotay, Mastovská, & Lightfield, 2005). As
one of the frequently used carbon materials, GCB has a good adsorption
effect on color impurities such as chlorophyll, although it also retains
some targeted planar compounds (Nguyen et al., 2008). MWCNTs is a
novel nanomaterial that features varying porosity and big surface areas,
and it has been successfully applied as a novel carbon material to re-
move pigments and other interferences in fruits, vegetables (Fan, Zhao,
Yu, Pan, & Li, 2014; Zhao, et al., 2012) and other complex matrix such
as tea (Hou, et al., 2014), and MWCNT was also reported as an effective
solid-phase extraction adsorbent in enrichment of pesticides from water
samples (Wang, Zhao, Min, & Fang, 2007)
C18 is a very common adsorbent in reversed phase SPE procedure for
enrichment of pesticides from water or other liquid matrices
(Rodríguez-Delgado & Hernández-Borges, 2007). Repetitive d-SPE is a
concept which also based on the same principle as in the reverse phase
C18-SPE. Fig. 1 demonstrates the procedure of the repetitive d-SPE
approach, which uses the reversed-phase adsorption ability of C18 to
concentrate the compounds of interest and remove the co-extracts with
the adsorption and desorption process. Recently, Sun et al. (2017) used
this method (it was called dispersive solid–liquid extraction (d-SLE)) in
the residue analysis of E/Z-fluoxastrobins in vegetables and fruits by
UHPLC-DAD, and it demonstrated a better cleanup efficiency than d-
SPE with the matrix effects of < 5%.
Thus, we expected to investigate a rapid multi-residue pesticide
analysis method with less matrix effects on cinnamon bark samples by
evaluating these two alternative methods–QuEChERS coupled with d-
SPE and repetitive-d-SPE cleanup. In this study, sixty targeted analytes
were selected, including the 47 pesticides which have MRLs of EU as
shown in Table S1. The toxic metabolites of three pesticides, six her-
bicides and other few frequently detected LC-amenable pesticides are
also included in this study. The extraction and cleanup conditions or
parameters were optimized for both methods, and the two methods
were further validated and compared in the analysis of 60 target ana-
lytes. The applicability of the two methods was assessed by analyzing
the multi-residue of pesticides in real world cinnamon bark samples
obtained from local markets.
2. Materials and methods
2.1. Chemicals and materials
MeCN (HPLC Grade) was obtained from Fisher Scientific (Pittsburg,
PA, USA). Standards of the 60 pesticides (including 23 insecticides, 15
fungicides, and 22 herbicides), and triphenyl phosphate (TPP) which
was used as internal standard, were obtained from the Department of
Applied Chemistry, China Agricultural University and Beijing
Z. Zhang et al.
Qinchengyixin Technology Development Company (Beijing, China).
The stock standard solutions of the 60 individual pesticides were pre-
pared at 1000 µg/mL in MeCN and diluted into a mixed standard so-
lution of 20 µg/mL. All solutions were stored at −20 °C.
Analytical grade formic acid was purchased from Beijing Chemical
Reagent Company (Beijing, China). Anhydrous MgSO4 and ammonium
formate were obtained from Shanghai Reagent Company (Shanghai,
China) and NaCl was from Sinopharm Chemical Reagent Co., Ltd
(Beijing, China). Cleanert C18 (50 μm particle size, 60 Å pore size),
graphitized carbon black (GCB, 120–400 μm) and multi-walled carbon
nanotubes (MWCNTs, 10–20 nm) were purchased from Tianjin Bonna
Agela Technology (Tianjin, China) and primary secondary amine (PSA,
40–60 μm) was from Anpel Laboratory Technologies (Shanghai, China).
The blank samples of cinnamon bark were purchased from local
supermarkets in Beijing, China, and 15 cinnamon bark market samples
were collected from 4 different cities of Guangxi province
(Fangchenggang city, Wuzhou city, Yulin city and Hezhou city) and
Sichuan province (Chengdu city). The cinnamon bark samples were
grounded into fine powder before analysis.
2.2. Apparatus and conditions
The HPLC-MS/MS system of Agilent (Little Falls, DE; USA) 1200 LC
equipped with an Athena C18-WP column (2.1 mm × 50 mm i.d., 3 μm
particles) and 6410B triple-quadrupole tandem mass spectrometry was
used in the study. The mobile phase performed following the gradient
elution procedure listed in Table S2 with flow rate of 0.2 mL/min, and
injection volume was 5 μL. The MS/MS detection was performed using
ESI+ and multiple reaction monitoring (MRM) mode. The source
temperature was 350 °C with nitrogen gas flow rate of 8.0 L/min and
nebulizer pressure of 35 psi. Table S3 lists the retention times (tR) and
qualitative and quantitative MRM transitions for the 60 target pesti-
cides.
2.3. Sample preparation
QuEChERS method was employed and optimized for the complex
matrix of cinnamon bark sample, and with the aim of finding a simple
and efficient enough cleanup method, the two different cleanup ap-
proaches d-SPE and repetitive d-SPE were investigated for the cin-
namon bark extracts.
Extraction procedure: grounded cinnamon samples (2 g) were
weighed into 50 mL centrifuge tubes, and appropriate volume of stan-
dard solutions with internal standards were spiked (except for the blank
samples) and placed at room temperature for 15 min. Then 5 mL
deionized water and 10 mL MeCN (for d-SPE cleanup) or 5 mL water
and 4 mL MeCN (for repetitive d-SPE cleanup) were added for extrac-
tion. After the tubes were shaking (VX-III TRAGIN multi-tube vortex
generator) for 5 min, 2.5 g ammonium formate was added (or 2.5 g
NaCl was added for comparison). Then the content was immediately
vortexed for 5 min and centrifuged at 3800 rpm (2260 rcf) for 5 min.
Then the upper layer was subject to further cleanup.
For d-SPE cleanup, aliquot of 1 mL upper layer of the extract was
transferred into a 2 mL disposable centrifuge tube containing sorbents
of 150 mg anh. MgSO4 and 50 mg C18. The other different sorbent
combinations were also tested in this step for comparison. After the
tubes were vortexed for 1 min and centrifuged for 2 min at 10,000 rpm
(4472 rcf), the extract was filtered through a 0.22 μm nylon organic
filtration for analysis by HPLC-MS/MS.
For repetitive d-SPE cleanup, aliquot 0.1 mL of upper MeCN layer
and 0.9 mL deionized water were transferred into a 2 mL disposable
centrifuge tube containing 100 mg C18 (or 20–30 mg MWCNTs for the
optimization experiments), and then immediately vortexed for 1 min
and centrifuged for 2 min at 10,000 rpm (4472 rcf). Remove all the li-
quid layer using an 1 mL disposal syringe, then add 50 mg Anh. MgSO4,
and accurately add 1.0 mL MeCN into the tube. The mixture was
142
Food Chemistry 276 (2019) 140–146
vortexed for 1 min and then centrifuged for 2 min at 10,000 rpm
(4472 rcf). Then the supernatant were filtered through a 0.22 μm nylon
organic filtration for analysis by LC-MS/MS.
2.4. Method validation
Trueness and precision of both d-SPE and repetitive-d-SPE methods
were evaluated with regards to the recoveries and RSDs of the 60
analytes at five spiking levels: 0.01, 0.02, 0.05, 0.1, and 0.5 mg/kg (5
replicates for each level). At these spiking levels most individual ana-
lytes were fortified at least between 0.5X, 1X, and 2X of their MRLs
(according to MRLs of EU as in Table S1). Since TPP showed high re-
coveries of 96–142% and RSDs of 11–19% in cinnamon bark samples,
all the recoveries were calculated by external standard method (without
using the internal standard to avoid the misleading results). LOQ was
defined as the lowest spiking level which met the requirement of re-
sidue analysis per the Codex Alimentarius Commission (CAC) guideline
(CAC/GL 90-2017) that recoveries within 70–120% and RSDs ≤ 20%
can be demonstrated as acceptable, whereas broader requirements with
recoveries of 60%–120% and RSD ≤ 30% were employed for lower
spiking levels (> 0.001–≤0.01 mg/kg). Linearity was studied using
matrix-matched calibration curves at 5–6 equivalent concentrations in
the final extracts. Slopes of solvent-based and matrix-matched calibra-
tion curves were used to evaluate the matrix effects (MEs) of the two
methods by the equation of ME (%) = (slope of matrix-matched cali-
bration curve − slope of solvent-based calibration curve)/(slope of
solvent-based calibration curve) × 100 (Lehotay, Mastovska, Lightfield,
& Gates, 2010).
3. Results and discussion
3.1. Method optimization for QuEChERS-d-SPE
3.1.1. Extraction and partitioning
In QuEChERS extraction procedure with MeCN, for the low water
content(≤25%) matrices like spices, cereals and dried fruits, sample
amount was reduced to 2 g in 10 mL MeCN and usually 5 mL water had
to be added to make sample pores more accessible to the extraction
solvent (Abbas, Soliman, Elgammal, Amer, & Attallah, 2017; Kolberg,
Prestes, Adaime, & Zanella, 2011; Lozano et al., 2012). Cinnamon
powder is the kind of dry matrix with low density and large volume.
Therefore, we also used 2 g cinnamon bark powder and 5 mL water with
10 mL MeCN as the initial conditions for extraction. Also, it was re-
ported that using of buffer (salt and acid) have a great influence on the
extraction efficiency and stability of some pesticides in the analysis of
multi-residues (Lehotay et al., 2005). So in this study, 5 mL water with
or without 0.1% formic acid (FA) was explored in the extraction pro-
cedure. At the same time, a volatile salt ammonium formate (AF) was
used in comparison with traditional NaCl as the phase separation salts,
which meant that MeCN along with four extraction + phase-out con-
ditions, namely water + NaCl, water + AF, FA + water + NaCl and
FA + water + AF, were compared for the 60 analytes at spiking level of
0.1 mg/kg with 3 replicates. To avoid the effect of the sorbents, further
cleanup was omitted in this case.
The results indicated that 49, 50, 50, 50 pesticides gave acceptable
recoveries of 70%–116% with RSDs of 0.3%–19% under the condition
of water + NaCl, water + AF, FA + water + NaCl and
FA + water + AF, respectively, which showed that different combina-
tions of solvent and salt had no significant effect for most pesticides.
Although most of the recoveries did not show much difference
under the four extraction conditions, the responses (the peak area of
matrix-matched standard solution) of most pesticides using water + AF
showed some relative increases (0.2%–29%) compared to
water + NaCl. Fig. S1 presents the 10 pesticides that peak areas in-
creased significantly (increased up to 31%–360%) vs. using
water + NaCl. It is obvious that great improvement in response was
Z. Zhang et al.
Table 1
Different sorbent combinations in d-SPE cleanup for cinnamon bark sample.
① MgSO4 + PSA + C18 + GCB ② MgSO4 + PSA + C18 + MWCNTs
150 + 50 + 50 + 7.5 mg 150 + 50 + 50 + 5 (7.5, 10) mg*
*Note: For combination ②, different amount (5, 7.5, and 10 mg) of MWCNTs were optimized first, and 7.5 mg was determined afterwards.
achieved by addition of ammonium formate (AF) for many pesticides.
In the research of Gonzalez-Curbelo et al, it was found that as a volatile
salt, ammonium formate could reduce the accumulation of ammonium
formate crystals on spray shield or inlet liner in conventional LC- or
GC–MS. Ammonium formate might also aid in the ionization of analytes
in ion source which may contribute to higher responses of analytes in
this study. The overall results indicate that water + AF gave the best
results and 22 pesticides of 60 had the highest responses with
0.2%–304% increase compared to water + NaCl, followed by
FA + water + AF with responses of 16 pesticides were highest (in-
creased 4%–360%). Therefore, water + AF was chosen as the optimized
combination for extraction and salt-out condition of the 60 analytes in
cinnamon bark samples.
3.1.2. Dispersive-SPE clean-up
With the aim to achieve efficient removal of the diverse matrix in-
terferences in cinnamon bark, six types of sorbents combinations listed
in Table 1 were investigated in the d-SPE procedure regarding re-
coveries and RSDs of the 60 analytes in cinnamon bark spiked at
0.1 mg/kg. Matrix-matched standard solutions at 0.1 mg/kg were used
for calculation of recoveries, and the responses (peak area) were also
compared for determination of optimal sorbents combination.
MWCNTs are relatively novel in pesticide multi-residue analysis,
and due to its large surface area and strong absorbing capability, the
amount of MWCNTs required for cleanup is only 5.0 mg for fruits and
vegetables (Han, et al., 2016; Song et al., 2017). For use as cleanup
sorbent for cinnamon sample, the optimal amount of MWCNTs should
be first determined. In this study, the amount of 5.0, 7.5, and 10 mg
MWCNTs were tested along with MgSO4 + PSA + C18 combination,
and our results demonstrated that no obvious differences or trends were
observed in both recoveries and peak areas (responses) for most pesti-
cides. The number of pesticides obtained acceptable recoveries and
RSDs were 41, 42 and 40, respectively out of 60. Nevertheless, as shown
in Fig. S2, only five pesticides showed a relative decrease in recoveries
with increasing amounts of MWCNTs (i.e. 7.5 or 10 mg). According to
Zhao’s research (Zhao, Fan, Yu, Zhang, & Pan, 2013), the loss of re-
covery occurred during the multi-plug filtration clean-up (m-PFC) using
MWCNTs (10 mg) on prochloraz and four benzo-heterocyclic com-
pounds including thiabendazole, carbendazim, fenoxaprop-P-ethyl, and
quizalofop-P-ethyl. In our research, the similar situation occurred with
thiabendazole and carbendazim, and in addition, the recoveries of di-
notefuran, tebuconazole, and anilofos also decreased with increasing
amounts of MWCNTs (5–10 mg). The above indicated these analytes
could be retained by MWCNTs from the extracts. In this study, we chose
7.5 mg as the optimal amount of MWCNTs due to its better performance
in recoveries. Its performance was further compared with 7.5 mg GCB
and no carbon sorbent (i. e. the sorbents combination of ①②③ in
Table 1).
The comparison of sorbents combination of ①②③ showed that for
most analytes, 7.5 mg MWCNTs, 7.5 mg GCB, and no carbon sorbent
didn’t show big differences in recoveries and RSDs with the number of
qualified pesticides 42, 43 and 44, respectively. However, it is ob-
viously that recoveries of approximately 18 pesticides (Fig. 2) were
decreased to some extent (13%–44%) with the addition of MWCNTs or
GCB, although the recoveries of 13 pesticides were still within
70–120%. Four pesticides (dinotefuran, thiabendazole, tricyclazole,
carbendazim) were dramatically retained by both MWCNTs and GCB
which caused unsatisfactory low recoveries ranging from 20 to 71%,
Food Chemistry 276 (2019) 140–146
③ MgSO4 + PSA + C18 ④MgSO4 + C18 ⑤MgSO4 + PSA ⑥MgSO4
150 + 50 + 50 mg 150 + 50 mg 150 + 50 mg 150 mg
and pymetrozine had low recoveries throughout (Fig. 3). Besides that,
we observed that the pigments contents were light in cinnamon extracts
and no carbon sorbents was therefore used in this study to avoid the
potential loss of pesticides.
For the sorbent combinations without carbon sorbents, we com-
pared the recoveries and response of four combinations
(③MgSO4 + PSA + C18, ④MgSO4 + C18, ⑤MgSO4 + PSA, and
⑥MgSO4). The recovery data indicate that the number of qualified
pesticides for the above sorbent combinations were 44, 49, 42 and 47,
respectively, and it was observed (Fig. 3) that with the addition of PSA,
five pesticides (florasulam, penoxsulam, pyrazosulfuron-ethyl, fenox-
aprop-p and trifloxystrobin acid) had far low recoveries and knowing
that PSA retains acidic compounds (Niell, Pareja, Geis, Cesio, &
Heinzen, 2010; Pareja, Cesio, Heinzen, & Fernándezalba, 2011). When
considering the pKa values of the above pesticides, we found that
florasulam and penoxsulam are sulfonylurea herbicides with pKas be-
tween 4.54 and 5.1, and pyrazosulfuron-ethyl is sulfonamide herbicide
with a pKa of 3.7. Fenoxaprop-p and trifloxystrobin acid are the acid
metabolites of fenoxaprop-p-ethyl and trifloxystrobin, respectively. We
concluded that the loss of recovery of these five pesticides was due to
the retention properties of PSA.
As a summary of the optimization of d-SPE clean-up sorbents, both
MWCNTs and GCB failed to obtain satisfactory results on cinnamon
bark due to their retention of several planar or aromatic compounds,
and PSA retained five acidic analytes and caused low recoveries for
them. Overall, C18 along with MgSO4 showed the best performance and
49 pesticides had the acceptable recoveries and RSDs. Thus, 150 mg
MgSO4 + 50 mg C18 were finally chosen as the sorbents of the d-SPE
clean-up method.
Until now, the sample preparation of QuEChERS-d-SPE method for
determination of the 60 pesticides was optimized and MeCN with water
and ammonium formate were chosen together with 150 mg
MgSO4 + 50 mg C18 as the d-SPE clean-up sorbents. The method vali-
dation results will be discussed and compared together with the results
of repetitive d-SPE method.
3.2. Method optimization for QuEChERS with repetitive d-SPE
While exploring a more efficient cleanup method for cinnamon
samples, the QuEChERS with repetitive d-SPE method was also in-
vestigated in this study. Since little research on the application of this
method was performed, optimization of the basic parameters was ne-
cessary before applying it to actual cinnamon bark matrix. We initially
used solvent standard solutions of the 60 analytes instead of matrix
extracts to determine the varieties and amount of sorbents, as well as
the ratio of organic phase (MeCN) and water in the repetitive d-SPE
procedure.
3.2.1. The optimization of sorbent
Actually, repetitive d-SPE was developed on the basis of C18-SPE in
reverse-phase system, which means in high water/organic solvent ratio
conditions, pesticides in water can be absorbed and concentrated in C18
cartridge, and then eluted by organic solvents. While taking advantages
of this principle, we tried using C18 (100 and 150 mg) to conduct the
repetitive d-SPE cleanup for 60 pesticides under the condition of
MeCN:water (1:9, v/v). Due to the unique structural characteristics and
large surface area, MWCNTs have excellent adsorption ability and have
been applied to the pre-concentration of nicosulfuron, thifensulfuron-
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Z. Zhang et al. Food Chemistry 276 (2019) 140–146

Fig. 2. Typical pesticides with recoveries decreased more than 15% using MWCNTs or GCB vs. no carbon sorbent in cleanup of cinnamon bark (0.1 mg/kg, n = 3).

Fig. 3. Recoveries of pesticides which were obviously retained by PSA (0.1 mg/kg, n = 3).

methyl (Zhou, Wang, & Xiao, 2006; Zhou et al., 2006), phthalate esters,
bisphenol A, and 4-n-nonylphenol (Cai, Cai, Mou, & Lu, 2005; Cai,
Jiang, Liu, & Zhou, 2003) from environmental water samples. Therefore
the adsorption properties of MWCNTs (20, 30 mg) were also in-
vestigated in combination with the reverse-phase d-SPE system in this
research.
As shown in Fig. 4, the recoveries using C18 were higher than
MWCNTs which suggested the adsorption capability of C18 to target
pesticides is much better using the reverse phase system. When 100 mg
C18 was used, the number of pesticides with satisfactory recoveries
(70–120%) was slightly more than using 150 mg C18 (39 pesticides vs.
35 pesticides), and far more than using 20 or 30 mg MWCNTs (14, 12
pesticides, respectively). This shows that although MWCNTs had ad-
sorption ability for some analytes, but it is less suitable to be used in
reverse system for multi-residue pesticide analysis than C18. As we ex-
pected, C18 performed very well for 43 analytes, although for some
analytes with high water solubility (as shown of log (water solubi-
lity) > 3.6 in Fig. 4), the recoveries were much low (10–40%) for at
least 6 pesticides including nitenpyram, propamocarb, etc. There were
some other analytes, such as fenoxaprop-p and trifloxystrobin acid,
showed high recoveries (> 120%) maybe due to matrix interferences or
possibly signal enhancement using ESI.
3.2.2. The ratio of MeCN/H2O
From the discussion above, it was shown that the water solubility
had some influence on the recoveries of pesticides when cleaned-up
using repetitive d-SPE procedure. Therefore, five different ratios of
MeCN to water (1/9, 2/8, 3/7, 4/6, 5/5) were compared to explore the
optimum solvent ratio of MeCN to water. The results in Fig. S3 clearly
demonstrate that the average recoveries of most pesticides decrease
dramatically by increasing the ratio of MeCN/H2O, although the de-
creasing trend is more gradual for the late eluting pesticides, especially

Fig. 4. Average recoveries of 58 pesticides at 0.1 mg/kg using different amount of C18 and MWCNTs in repetitive d-SPE clean-up (Note: no water solubility data for
fenoxaprop-p and trifloxystrobin acid).

144
Z. Zhang et al.
Table 2
The number of pesticides with different matrix effects behavior using d-SPE and repetitive d-SPE method in cinnamon bark.
Method |ME| ≦ 20% 20% < |ME| ≦ 50%
No effect Medium matrix effect
d-SPE 35 19
Repetitive d-SPE 47 8 5
for those retention times later than difenoconazole (tR = 14.8 min).
Among them, chlorpyrifos is an exception for which recoveries rising
with the increasing ratio of MeCN/H2O. Based on the results, we can
conclude the adsorption efficiency of C18 was better under the condi-
tion of a high percentage of water. The ratio of 1/9 (MeCN: water) was
finally chosen since 37 pesticides had satisfactory recoveries and RSDs
when compared to higher ratios of MeCN/H2O such as 2/8, 3/7, 4/6,
and 5/5). These showed the qualified pesticides numbers of 32, 21, 7,
and 8, respectively.
3.2.3. Solvent volume for sample extraction
As a summary of the above optimization of repetitive d-SPE clean-
up, the optimal ratio of MeCN/water was determined as 1/9 and
100 mg C18 sorbent was chosen for the multi-residue analysis of 60
analytes. When the method was applied to the QuEChERS extracts of
cinnamon bark, the 1/9 meant only 0.1 mL extracts was transferred and
mixed with 0.9 mL water for the repetitive d-SPE clean-up. This in-
troduced a 10 fold dilution factor before injection to HPLC-MS/MS
which caused much lower sensitivity for the whole method. To solve
this problem, we tried to reduce the volume of extraction solvent
(MeCN) from 10 mL to 4 mL or even 2 mL per 2 g sample. The results in
Fig. S4 demonstrate that, with the reduction of the extraction volume of
MeCN, the recoveries were getting greater. When 2 mL MeCN was
employed, the recoveries of most pesticides were more than 120%
which may be caused by strong matrix interference and the fluctuation
of the small volume of supernatants. For 4 mL and 10 mL MeCN, the
recoveries were mostly satisfactory with recoveries between 60 and
130%. In order to meet the needed sensitivity, 4 mL MeCN was finally
selected as the volume of MeCN solvent for repetitive d-SPE method for
60 pesticides in cinnamon bark sample.
3.3. Method validation for both QuEChERS-d-SPE and repetitive d-SPE
Using the optimized sample preparation conditions, the linearity,
accuracy (trueness and precision), sensitivity, and matrix effects of both
QuEChERS-d-SPE and repetitive d-SPE methods were evaluated for
analysis of 60 pesticides in cinnamon bark. Per the MRLs of 60 pesti-
cides in cinnamon bark from EU, five spiking levels were used at the
concentration of 0.01, 0.02, 0.05, 0.1 and 0.5 mg/kg.
Tables S4 and S5 list the recoveries and RSDs of 60 pesticides cal-
culated by matrix-matched calibration method with QuEChERS-d-SPE
and repetitive d-SPE, respectively. For QuEChERS-d-SPE, 44 out of 60
pesticides achieved satisfactory recoveries (71%–118%) and RSDs
(2%–27%) at three to five levels, however, for the repetitive d-SPE, 37
of 60 pesticides met the method validation criteria with recoveries of
71%–120% and RSDs of 3%–30% at 3–5 levels. All these 37 analytes
were also qualified in d-SPE method. Both of the methods were quick
and easy and reliable for determination of 44 pesticides (QuEChERS-d-
SPE) or 37 pesticides (repetitive d-SPE) in cinnamon bark samples.
As for the 16 problematic pesticides in both methods, quinclorac,
cyhalofop-butyl and chlorpyrifos failed to obtain satisfactory recoveries
and RSDs because of the inaccurate quantification due to the low re-
sponse (not detectable at more than 3 spiking levels). Pymetrozine,
propamocarb, nitenpyram and nicosulfuron showed consistent low re-
coveries (< 50%) due to the extraction problem and possible stability
issues, and for the remaining 9 pesticides, the high RSDs (> 20%) were
145
Food Chemistry 276 (2019) 140–146
|ME| > 50% ME > 0% ME < 0%
Strong matrix effect matrix enhancement matrix suppression
6 21 39
25 35
the main reason of failure to get acceptable results which was probably
caused by the complex matrix interferences.
From Tables S5 and S6, there are seven problematic pesticides using
repetitive d-SPE (thiabendazole, methomyl, tricyclazole, clodinafop-
propargyl, metolachlor, imidacloprid and bentazone) which instead
had adequate recoveries and RSDs using QuEChERS-d-SPE method. In
repetitive d-SPE, the recovery loss of thiabendazole and tricyclazole
may be due to the difficulty of extraction from cinnamon matrix with
low volume of solvents, while low recoveries of methomyl were mainly
due to its high water solubility (55,000 mg/L). Clodinafop-propargyl,
metolachlor, imidacloprid and bentazone performed poorly due to low
responses and high RSDs at lower spiking levels which were caused by
the high dilution factors in the method.
The results show that most pesticides achieved satisfactory linearity
(r2 ranging from 0.90 to 1.00) by either d-SPE or repetitive d-SPE
method in the range of 1–100 μg/kg and 0.5–100 μg/kg, respectively,
except cyhalofop-butyl had a poor linearity (r2 = 0.6203) due to the
interferences when using d-SPE clean-up. The LOQ of most analytes was
10 μg/kg for both methods, except some analytes (such as methomyl
and metolachlor) were 20 μg/kg due to interference or high variability
at low spiking levels in d-SPE method, and in repetitive d-SPE, the LOQ
of some analytes (such as penoxsulam, pyraclostrobin, and bentazone)
increased to 50 μg/kg due to high dilution factor.
The MEs were calculated if the co-efficiency of linearity r2 > 0.90
independent of the recoveries or RSDs. Fig. S5 illustrates the MEs of 60
pesticides in cinnamon bark samples for both the established QuE-
ChERS-d-SPE and repetitive d-SPE methods. It is shown in Table 2 that
more analytes belonged to “No effects” and less analytes were
“medium” or “strong” MEs in repetitive d-SPE than in d-SPE, which
indicated significant improvements in reducing MEs in cinnamon
samples. Besides, the total ion chromatograms (TIC) of blank cinnamon
matrix after d-SPE and repetitive d-SPE cleanup (shown in Fig. S6)
indicated that the background peaks using repetitive d-SPE are much
lower than using d-SPE, especially in the retention time of 12–16 min
where many analytes eluted. This result may due to the selective ad-
sorption of interferences by C18 in reverse phase system, and moreover,
the higher dilution factor of repetitive d-SPE method may also attribute
to the lower MEs.
3.4. Application to market samples
To confirm the applicability of the two validated methods, 15 cin-
namon bark samples were collected from 5 cities of 2 provinces in
China and were analyzed by both the QuEChERS-d-SPE and repetitive
d-SPE methods. The results showed that most of the pesticides were
undetected from the 15 actual samples by both the two methods, except
epoxiconazole was detected in two samples with concentrations at
0.012 and 0.017 mg/kg using repetitive d-SPE, while the results were
0.009 and 0.013 mg/kg using d-SPE. The results were similar for the
two methods, and the concentrations are all lower than 0.1 mg/kg
which is the EU MRL for epoxiconazole in cinnamon bark. The other
pesticide pyraclostrobin was also detected at the concentration of
0.037 mg/kg in d-SPE, while it was not detectable (< 0.05 mg/kg) in
repetitive d-SPE due to its lower LOQ of 0.05 mg/kg. Nevertheless, the
residue is also lower than 0.1 mg/kg (the EU MRL of pyraclostrobin for
cinnamon bark).
Z. Zhang et al.
4. Conclusion
In this research, QuEChERS method coupled with d-SPE and re-
petitive d-SPE was optimized and evaluated systematically for the
multi-residue analysis of 60 frequently used pesticides in cinnamon
bark with LC-MS/MS detection. As expected, cinnamon is a complex
matrix, and the co-extracted interference was a critical influence factor
which caused high RSDs for about 9 analytes in both two methods. The
results illustrated that 150 mg MgSO4 + 50 mg C18 showed the best
performance among all the sorbent combinations in d-SPE cleanup. In
QuEChERS with repetitive d-SPE, less analytes showed satisfactory re-
coveries or RSDs mainly because of the large dilution ratio, which
caused low sensitivity and high RSDs at low levels, and also some
analytes had low recoveries due to their relative high water solubility.
The comparison results showed that the validated QuEChERS-d-SPE
method is simple, reliable and more efficient for the multi-residue
analysis of pesticides in cinnamon samples, with which 44 out of 60
pesticides achieved satisfactory recoveries. The method was applied to
15 market samples and only epoxiconazole and pyraclostrobin were
detected at the concentration of 0.012–0.037 mg/kg, which were all
below the MRL in EU of 0.1 mg/kg.
Acknowledgements
This work was supported by the Ministry of Science and Technology
of China, and China National Keynote Research & Development Plan,
Grant Number 2017YFD0800700.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2018.10.019.
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