International Journal of Environmental Analytical

Chemistry

ISSN: 0306-7319 (Print) 1029-0397 (Online) Journal homepage: https://www.tandfonline.com/loi/geac20

A comparative study of biochar, multiwalled

carbon nanotubes and graphitized carbon black as
QuEChERS absorbents for the rapid determination
of six triazole fungicides by UPLC-MS/MS

Junli Cao, Yunxi Zheng, Abdul Kaium, Xingang Liu, Jun Xu, Fengshou Dong,
Xiaohu Wu & Yongquan Zheng

To cite this article: Junli Cao, Yunxi Zheng, Abdul Kaium, Xingang Liu, Jun Xu, Fengshou Dong,
Xiaohu Wu & Yongquan Zheng (2019) A comparative study of biochar, multiwalled carbon
nanotubes and graphitized carbon black as QuEChERS absorbents for the rapid determination
of six triazole fungicides by UPLC-MS/MS, International Journal of Environmental Analytical
Chemistry, 99:3, 209-223, DOI: 10.1080/03067319.2019.1586892

To link to this article: https://doi.org/10.1080/03067319.2019.1586892

Published online: 15 Mar 2019. Submit your article to this journal

Article views: 311 View related articles

View Crossmark data Citing articles: 2 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=geac20
INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY
2019, VOL. 99, NO. 3, 209–223
https://doi.org/10.1080/03067319.2019.1586892

ARTICLE

A comparative study of biochar, multiwalled carbon

nanotubes and graphitized carbon black as QuEChERS
absorbents for the rapid determination of six triazole
fungicides by UPLC-MS/MS
Junli Caoa,b, Yunxi Zhengc, Abdul Kaium a
, Xingang Liua, Jun Xua, Fengshou Donga,
Xiaohu Wua and Yongquan Zhenga
a
State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese
Academy of Agricultural Sciences, beijing, China; bCollege of Chemistry, Central China Normal University,
Wuhan, China; cBeijing Bayi School, Beijing, China

ABSTRACT ARTICLE HISTORY

A rapid, effective and sensitive method to quantitatively determine six Received 24 November 2018
fungicide residue was developed using ultra-performance liquid chro- Accepted 9 February 2019
matography coupled with tandem mass spectrometry (UPLC–MS/MS). KEYWORDS
The target compounds were extracted by using acetonitrile and the Vegetable; multiwalled
sorbent used for clean-up in this modified QuEChERS analytical carbon nanotubes; biochar;
method were biochar, multiwalled carbon nanotubes (MWCNT) and graphitized carbon black
graphitized carbon black (GCB). Results indicated that the MWCNT
(10 mg) was the most effective sorbent in removing pigment. This
method was validated on spinach, tomato, cucumber, celery, lettuce,
rape, pakchoi, romaine lettuce and eggplant matrices spiked at three
concentration levels of 0.01, 0.1 and 1 mg kg−1. It exhibited recoveries
between 73.1% and 118.2% with RSD values below 20%. Matrix-
matched calibrations were performed with the coefficients of deter-
mination >0.9901 between concentration levels of 0.01–1 mg kg−1.
The limit of quantity (LOQ) for six pesticides ranged from 0.0036 to
0.011 mg kg−1. The developed method was satisfactorily applied to
determine pesticide residues in market vegetable samples.

1. Introduction
Fungal plant pathogens cause extensive loss of crops in all parts world. Fungicides are
crucial agronomic measures to protect the crops from their disease-causing organisms.
However, most of the fungicides have ecotoxicity and bioaccumulating properties which
make it harmful to humans, animals and the environment. Triazole fungicides belong to
heterocyclic compounds, which have the characteristics of high efficiency, broad spec-
trum, long persistence and strong internal absorbability [1-3]. Its main varieties include
myclobutanil, simeconazole, triadimenol, tebuconazole, flusilazole and propiconazole,
which are widely used in fungus control of cereals, vegetables and fruits all over the
world [4–6]. They are a group of fungicides belongs to ergosterol biosynthesis inhibitor
(EBI) groups [7], it inhibits the mycelia growth and germination of spores in fungus by

CONTACT Xingang Liu liuxingang@caas.cn

This article has been republished with minor changes. These changes do not impact the academic content of the article.
© 2019 Informa UK Limited, trading as Taylor & Francis Group
210 J. CAO ET AL.

blocking the synthesis of 1,4-α-demethylase enzyme which is essential for ergosterol

biosynthesis in fungus [8,9]. Triazole fungicides that accumulate in water, soil and crops
due to poor degradation and migration will affect soil microbes, animal and human
health [10–12]. Due to the wide-ranging use of triazole fungicides in agricultural com-
modities and the risk aspects, there is an increasing need to monitor the triazole
fungicide residue in the agricultural and environmental commodities at trace level
concentration [13,14].
The triazole fungicides residue analysis in vegetables is a challenging task due to the
complexity of matrices and trace amounts content. Vegetables contain a large amount
of pigments. Several methods were previously developed for determining triazole
fungicide residue in various samples such as liquid-liquid extraction (LLE) [15–17], solid-
phase extraction (SPE) [18], dispersive liquid-liquid microextraction (DLLME) [19,20], stir-
bar sorptive extraction (SBSE) [21,22] and accelerated solvent extraction (ASE) [23]. In
previous LLE and SPE methods were used frequently to extract the triazole fungicides
from crops and environmental samples, but these methods are time-consuming, expen-
sive and required a large volume of solvents [24]. On the other hand, the SBSE and ASE
methods required specific equipment and lower performance for multiresidue analysis
of large-scale samples. In recent years, QuEChERS (quick, easy, cheap, effective, rugged,
and safe) has become a universal method for sample processing due to its easy
operation and low solvent consumption [25]. Since 2003, it has been mostly used
sample preparation method for the determination of the broad range of pesticides in
high water content matrix [26–28]. In the QuEChERS extraction methods, the clean-up is
the most important step to reduce the matrices and co-extractives from the extract, but
there have alternative options to choose the sorbents which are less expensive, envir-
onment-friendly and more efficient regarding the removal of interferences [29]. Some
previous modified QuEChERS methods were used graphitized carbon black (GCB)
[30,31]、N-propyl ethylenediamine (PSA)、multiwalled carbon nanotubes (MWCNT)
[32] as a sorbent for better clean-up complex matrix [33]. PSA,
N-propylethylenediamine, contains a large amount of primary/secondary amino groups,
which can effectively remove fatty acids, organic acids and sugars [34,35]. However, it
does not have a strong ability to absorb pigments. GCB can remove sterols and
pigments [36] and MWCNT gives higher efficiency to remove matrices and co-extracts
from the samples with better pesticides recoveries [37,38]. Biochar is a carbonaceous
residue derived from the pyrolysis of waste biomass under little or absence of oxygen
[39]. In some recent studies showed that biochar has a profound sorptive ability to
remove organic and inorganic contaminants from different liquid and solid samples like
as wastewater or soil samples [40–42]. What is more, biochar is less expensive and
environment-friendly than other dSPE sorbent used before. It is interesting, but no study
has been reported that biochar as clean-up material combined with QuEChERS analytical
method for analysis pesticide residues.
The purpose of this study was to validate a novel and efficient analytical method to
simultaneous determine six commonly used fungicides residue in different vegetables
and compare the purification effects of GCB, MWCNT and biochar. All analyses were
successfully separated with good specificity. Satisfactory recoveries were obtained, as
well as precision and accuracy. And the developed method was validated to the analysis
of authentic samples.
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 211

2. Experimental
2.1. Chemicals and reagents
The standard myclobutanil (99.2% pure) was purchased from the national quality
inspection centre of pesticide products (Shenyang, China). Simeconazole (98% pure)
was purchased from Suzhou fumeishi. Triadimenol (99.5% pure) standard purchased
from the national pesticide quality supervision and inspection centre (Beijing, China).
Standard tebuconazole (95.6% pure) was purchased from Bayer Crop Science Ltd
(Germany). One hundred percent flusilazole standard was the purchase from DuPont
Co in the United States of America. Propiconazole (93.8% pure) was recruited from the
Shanghai Pesticide Research Institute (Shanghai, China). HPLC grade acetonitrile (ACN)
was gained from Sigma–Aldrich (Steinheim, Germany). Ultrapure water was prepared
using a Milli-Q reagent water system (Bedford, MA, USA). Analytical standard sodium
chloride (NaCl) and anhydrous magnesium sulfate (anhydrous MgSO4) were bought
from Beijing Chemical Company (Beijing, China). Graphitized carbon black (GCB,
120–400 meshes) and multiwalled carbon nanotubes (MWCNT, 20-30 nm) were pur-
chased from Bonna-Agela Technologies (Tianjin, China) and biochar (200–400 mesh) was
getting from Panzhihua Xiyu Biological Technology Co. Ltd.
The standard stock solution of six triazole fungicides was prepared separately at the
concentration of 100 mg L−1 in acetonitrile. Standard working solutions of six triazole
fungicides at 0.01, 0.05, 0.1, 0.5 and 1 mg L−1 were prepared by the dilution of the appropriate
amount of the standard stock solution with acetonitrile and stored at 4°C in the dark.

2.2. Apparatus for sample preparation

For sample preparation, an HR 7735 Cucin food processor (Philips, Holland) was used to
comminute samples. A Vortex-Genie 2 mixer (Sigma–Aldrich, Steinheim, Germany) and
Thmorgan CK 2000 (Beijing Tuomo root Biotechnology Co., Ltd; China) shaker were used
for mixing the samples. One TG16-WS Centrifuge (Xiangyi Centrifuge machines Ltd.,
Changsha, China) and one Sigma compact microcentrifuge 1–6 (Sigma Laborzentrifugen
GmbH, Germany) were used for separation and clean-up step in sample preparation.
Polytetrafluoroethylene syringe filter with a pore size of 0.22 μm was purchased in
Agilent Technology Co., Ltd (China).

2.3. Chromatographic and mass spectrometry condition

A Waters Acquity UPLC instrument with Waters Acquity UPLC binary solvent manager
and an Acquity column heater equipped with a Waters Acquity UPLC BEH C18 column
(100 mm × 2.1 mm, 1.7 µm particle sizes) (Milford, MA, USA) was used. The column
temperature was 30 °C.
A Waters triple-quadrupole mass spectrometer (TQD, Waters Crop., Milford, USA)
equipped with an electrospray ionization (ESI) source and MassLynx software (version 4.1)
were applied to analyze the six triazole fungicides. Argon (99.999%) and nitrogen (99.95%)
were used as the collision and nebulizer gas, respectively, with a pressure of 2 × 10−3 mbar in
the T-wave cell. Multiple reaction monitoring (MRM) mode and electrospray ionization (ESI)
in the positive ion mode was fitted for MS detection. The following MS condition was
212 J. CAO ET AL.

optimized for triazole fungicides detection: the capillary voltage: 3.0 kV; ion source tem-
perature: 120 °C; desolvation gas temperature: 350°C; cone gas flow: 50 L h−1 desolvation
gas flow: 5000 L h−1.

2.4. Sample preparation

Rape, tomato, eggplant, lettuce, spinach, pakchoi, celery, cucumber and romaine lettuce
samples were chopped and blended by an HR 7735 Cucin food processor (Philips,
Holland). Thoroughly homogenized 10 g samples into a 50 mL teflon centrifuge tube,
then added 0.1 mL 100 mg kg−1,10 mg kg−1 and 1 mg kg−1 standard working solution of
mixed six triazole fungicides in the blank samples for recoveries test. The centrifuge
tubes with spiked samples were vortexed 30 s and allowed to stand for 2 h at room
temperature to distribute and interact the fungicides evenly with the sample matrix.
Then, 5 mL ultrapure water and 10 mL acetonitrile was added to the centrifuge tube.
After the tube was shaken vigorously for 10 min, 1 g NaCl and 4 g anhydrous MgSO4
were added to the centrifuge tubes and shaking step was done again for 5 min. After
centrifugation (4000 RPM, 5 min) of the sample containing centrifuge tubes, 1.5 mL
upper layers (acetonitrile) supernatant transferred to a 2 mL centrifuge tube containing
150 mg MgSO4 and different clean-up sorbents were evaluated as follows: 5 mg
biochar (sorbent 1), 10 mg biochar (sorbent 2), 20 mg biochar (sorbent 3), 5 mg
MWCNT (sorbent 4), 10 mg MWCNT (sorbent 5), 20 mg MWCNT (sorbent 6), 5 mg GCB
(sorbent 7), 10 mg GCB (sorbent 8), 20 mg GCB (sorbent 9). Afterward, the centrifuge
tubes were vortexed for 1 min and centrifuged for 5 min at 5000 RPM. Finally, the upper
acetonitrile layer was made available through a 0.22 µm hydrophilic PTFE filter and
transferred to an auto-sampler vial for UPLC-MS/MS analysis.
Standard check samples were produced in the same way as the sample treatment
procedure mentioned above. The matrix-match working standard solution was worked
out by blowing 1 mL of the standard solution with a nitrogen blower and then adding
1 mL standard check solution. The matrix-match working standard and the standard
check solution were compared to evaluate the matrix effect in samples.

2.5. Method validation

Laboratory blank samples and spiked sample analyses were used for quality assurance
and quality control of this method according to the European Commission. The follow-
ing parameters were committed to validate the method: specificity, linearity, matrix
effect, limit of quantification (LOQ), precision and accuracy. Blank samples (spinach,
tomato, cucumber, eggplant, celery, lettuce, pakchoi, rape, romaine lettuce) were ana-
lysed to identify the absence of interfering peaks around the retention of the com-
pound. The linearity of the method was studied by calculating a standard curve,
correlation coefficient (R2), intercept and slope in the solvent blank solution and matrix-
matched standard solution in triplicate at six concentrations ranging from 0.01 to 1 mg
kg−1. Matrix effect (ME) of the sample was determined as follows:
slope of calibration curves in matri  slope of calibration curves in solvent
ME ¼  100%
slope of calibration curves in solvent
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 213

The accuracy and precision of the method were determined by conduction the
recoveries studies of blank spiked samples in five replicate each at three fortification
levels (0.01, 0.1 and 1 mg kg−1). The limit of quantifications (LOQ) was determined from
10-time ratio of signal to noise ratio (S/N) of the target analyses.

3. Results and discussion

3.1. Optimization of chromatography condition
Chromatographic separation was performed with the mobile phase A (acetonitrile) and
B (0.2% formic acid in water) at a flow rate of 0.4 mL/min and the gradient elution
procedure is shown in Table 1. The injection volume was 5 μL. Figure 1 shows the
chromatograms of six fungicides in nine samples. The compounds were eluted in the
following order: myclobutanil (4.52 ~ 4.66 min), simeconazole (4.16 ~ 4.27 min), triadi-
menol (3.39 ~ 3.46 min), tebuconazole (5.15 ~ 5.29 min), flusilazole (5.54 ~ 5.68 min) and
propiconazole (6.57 ~ 6.71 min).

3.2. Optimization of MS/MS

To obtain high specificity, MS/MS spectra were obtained from the infusion of 1 mg L−1
standard solution of myclobutanil, simeconazole, triadimenol, tebuconazole, flusilazole and
propiconazole in methanol at a flow rate of 0.4 mL min−1. Both negative and positive ion
modes were evaluated, and the positive mode can get a better fragmentation pattern for all
of the six compounds. For the analyses, positive ion mode was used to identify the precursor
ions. Different cone voltages were tested for the precursor ion to find the most suitable
settings. Then, the dissociation was induced and different collision energies were tested in
order to find the most abundant product ion. The most sensitive transition in the mode was
selected for quantification. Identification was conducted based on the retention time, two
selected ion transitions and their relative abundance. Table 2 displays the parameters of the
MRM transitions used for quantification and identification of targeted analyses.

3.3. Optimization of clean-up procedure

This paper emphasizes on the choice of different clean-up sorbents for dSPE step in
QuEChERS analytic technique to obtain satisfactory extraction. It is generally accepted
that GCB was mainly used to remove hydrophobic interaction-based compounds, con-
cluding chlorophyll and carotenoids [43–45]. MWCNT exhibit good adsorption capacity

Table 1. Flow phase gradient elution program.

Time/min A/% B/% Curve
Initial 10.0 90.0 Initial
1.5 90.0 10.0 6
3.5 90.0 10.0 6
3.6 55.0 45.0 6
9 55.0 45.0 6
9.1 10.0 90.0 6
10 10.0 90.0 6
214 J. CAO ET AL.

Figure 1. UPLC-MS/MS MRM chromatograms of six fungicides in (A) a blank spinach sample, (B)
a standard sample (0.01 mg L−1), (C) an eggplant sample spiked at 0.01 mg kg−1, (D) a rape sample
spiked at 0.01 mg kg−1.

in pesticide residue analysis due to surface extreme and hydrophobicity [46,47]. Biochar,
known as ‘black gold’, has a wide range of applications in contaminant remediation and
water purification [48–50]. Considering the many types of chlorophyll derivatives in
vegetables, we tested the abilities of the biochar, MWCNT and GCB in three dosages
(5 mg, 10 mg, 20 mg) to recover the pesticides from the nine samples. The recoveries of
the six fungicides (myclobutanil, simeconazole, triadimenol, tebuconazole, flusilazole
and propiconazole) spiked at 0.1 mg kg−1 in spinach, tomato, cucumber, eggplant,
celery, lettuce, pakchoi, rape and romaine lettuce are varies. As shown in Figure 2,
propiconazole had the lowest recoveries (between 41.63% and 71.27%) in nine matrix
when sorbents 1, 2 and 3 were used. However, much higher recoveries (between 92.8%
and 147.9%) of all six fungicide from rape were observed with the use of sorbent 7, 8
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 215

Table 2. LC–MS/MS conditions (retention time, ES mode used, transitions selected, cone voltage and
collision energy).
MW Rt CV CE
Analyte CS (g/mol) (min) ESI mode Transition (m/z) (V) (eV)
Myclobutanil 288.8 4.52 ~ 4.66 Positive 290.16→70.14 32 20
290.16→125.54 40

Simeconazole 293.4 4.16 ~ 4.27 Positive 294.37→69.79 32 28

294.37→134.88 25

Triadimenol 295.8 3.39 ~ 3.46 Positive 296.2→69.8 16 17

296.2→99.1 25

Tebuconazole 307.8 5.15 ~ 5.29 Positive 309.22→69.79 33 35

09.22→125.31 28

Flusilazole 315.4 5.54 ~ 5.68 Positive 316.48→246.94 30 25

316.48→164.94 40

Propiconazole 342.2 6.57 ~ 6.71 Positive 343.34→160.04 38 44

343.34→69.25 32

CS→chemical structure; MW→molecular weight; Rt = Retention time; CV→ cone voltage; CE→ collision energy

and 9 (Figure 2). The recoveries of myclobutanil, flusilazole and propiconazole were also
>120% when sorbents 4 and 6 were used for purification of those compounds from
eggplant and cucumber (Figure 2). Nevertheless, all analytes had a satisfactory recovery
(between 83.93% and 118.4%) when Sorbent 5 was used for purification of those
compounds from all nine matrices (Figure 2). MWCNT are new type of material based
on carbon nanomaterials. Compared with GCB and biochar, MWCNT has a larger surface
area and unique structure, which is the possible reason for its better adsorption effect
[47,51]. What is more, Figure 3 indicates that the MWCNT showed the best pigment
clean-up, followed by GCB and biochar for lettuce and eggplant.
Results show that MWCNT(10 mg) is the best choice as a clean-up sorbent in modified
QuEChERS analytic procedure for those vegetable samples containing high pigment.
216 J. CAO ET AL.

Figure 2. Effects of nine sorbents on recoveries for six fungicides (A, myclobutanil; B, simeconazole; C,
triadimenol; D, tebuconazole; E, flusilazole; F, propiconazole) in nine vegetables at 0.1 mg kg−1 (n = 5).

Hence, sorbent 5 (10 mg MWCNT) was selected to clean up the spinach, tomato,
cucumber, eggplant, celery, lettuce, pakchoi, rape and romaine lettuce matrices.
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 217

Figure 3. Lettuce (A) and eggplant (B) are processed: 1. without purification, 2. with biochar
(10 mg), 3. MWCNT (10 mg), 4. GCB (10 mg).

3.4. Linearity, LOQ and matrix effect

The linearity, LOD, LOQ and matrix effect for six fungicides (myclobutanil, simeconazole,
triadimenol, tebuconazole, flusilazole and propiconazole) were determined and the results
are presented in Table 3. The linearity of the method was determined by the linear regression
results of the standard solution and the matrix-matched calibration curves. The results
showed satisfactory linearity with a good linear correlation coefficient (R2 > 0.9901) for all
analyses in the extracts of rape, tomato, eggplant, spinach, lettuce, spinach, celery, cucumber
and romaine lettuce samples. In this method, LODs were calculated by analyzing matrix
matched standards at low concentrations and checking for peak signals with an S/N greater
than 3. LOQs were established as the concentration giving S/N ratios of 10. In this experiment,
LODs and LOQs were determined by the MassLynx software version B.4.01 and Microsoft
Excel. The LODs for the six fungicides were estimated to be 0.0005–0.0033 mg kg−1 and
analyses of spiked samples at the lowest spiked levels (0.01 mg kg−1). The LOQs in the nine
sample matrices were 0.0017−0.011 mg kg−1 for all compounds based on five replicates at
0.01 mg kg−1. The presence of a specific matrix component can affect the ionization of target
analyte by reducing or enhancing the detector response compared with that produced by
only the analyte in the solvent. Matrix effects are a major challenge in the analysis of pesticide
residues in complex samples [52,53]. Enhanced signal suppression between −20% and 20% is
considered to be a mild effect, whereas between −50% and 50% is considered to be a middle
effect, more than 50% or less than −50% is considered to be strong effect [54].
As shown in Table 3, for simeconazole, the spinach, tomato, cucumber, celery and
romaine lettuce showed a strong effect of signal enhancement (53%< ME<105%). But,
for flusilazole spinach, eggplant, celery, lettuce, rape and romaine lettuce presented
great signal suppression (−92%<ME<-82%). Where, propiconazole displayed middle
effect (41%<ME<52%) for eggplant, celery and rape. Whereas tomato, cucumber showed
weaken signal than standard solution. As a result, calibration was performed for
218 J. CAO ET AL.

Table 3. Matrix-matched calibration, LOD and LOQ of the method.

Compound Matrix R2 Linear range (mg L−1) Matrix effect (%) LOQ (mg kg−1) LOD (mg kg−1)
Myclobutanil Acetonitrile 0.9917 0.01–1 - 0.0084 0.0025
Spinach 0.996 0.01–1 −9 0.0108 0.0032
Tomoto 0.9953 0.01–1 −25 0.0108 0.0032
Cucumber 0.9976 0.01–1 −3 0.0045 0.0014
Eggplant 0.992 0.01–1 −12 0.0101 0.0030
Celery 0.9993 0.01–1 −41 0.0108 0.0032
Lettuce 0.9969 0.01–1 −52 0.0108 0.0032
Pakchoi 0.9924 0.01–1 19 0.0048 0.0014
Rape 0.9951 0.01–1 −7 0.0100 0.0030
Romaine lettuce 0.9901 0.01–1 86 0.0107 0.0032
Simeconazole Acetonitrile 0.9981 0.01–1 - 0.0056 0.0017
Spinach 0.9976 0.01–1 105 0.0045 0.0014
Tomoto 0.9988 0.01–1 60.4 0.0055 0.0017
Cucumber 0.9932 0.01–1 67 0.0048 0.0014
Eggplant 0.9934 0.01–1 6 0.0029 0.0009
Celery 0.9928 0.01–1 78 0.0097 0.0029
Lettuce 0.9954 0.01–1 −15 0.0097 0.0029
Pakchoi 0.9959 0.01–1 26 0.0109 0.0033
Rape 0.993 0.01–1 26 0.0045 0.0014
Romaine lettuce 0.9937 0.01–1 53 0.0070 0.0021
Triadimenol Acetonitrile 0.9993 0.01–1 - 0.0058 0.0017
Spinach 0.9956 0.01–1 37 0.0037 0.0011
Tomato 0.9932 0.01–1 −3 0.0103 0.0031
Cucumber 0.9969 0.01–1 6 0.0052 0.0016
Eggplant 0.993 0.01–1 −15 0.0108 0.0032
Celery 0.9912 0.01–1 15 0.0052 0.0016
Lettuce 0.9916 0.01–1 10 0.0052 0.0016
Pakchoi 0.9975 0.01–1 −6 0.0033 0.0010
Rape 0.9987 0.01–1 23 0.0096 0.0029
Romaine lettuce 0.9987 0.01–1 11 0.0101 0.0030
Tebuconazole Acetonitrile 0.994 0.01–1 - 0.0076 0.0023
Spinach 0.9986 0.01–1 54 0.0074 0.0022
Tomato 0.9918 0.01–1 −19 0.0100 0.0030
Cucumber 0.9957 0.01–1 9 0.0076 0.0023
Eggplant 0.9973 0.01–1 −11 0.0055 0.0017
Celery 0.9934 0.01–1 13 0.0101 0.0030
Lettuce 0.9919 0.01–1 −16 0.0101 0.0030
Pakchoi 0.9956 0.01–1 37 0.0107 0.0032
Rape 0.9981 0.01–1 2 0.0037 0.0011
Romaine lettuce 0.9967 0.01–1 27 0.0105 0.0032
Flusilazole Acetonitrile 0.9991 0.01–1 - 0.0036 0.0011
Spinach 0.9985 0.01–1 −82 0.0017 0.0005
Tomato 0.9976 0.01–1 7 0.0100 0.0030
Cucumber 0.9991 0.01–1 −4 0.0029 0.0009
Eggplant 0.9924 0.01–1 −91 0.0041 0.0012
Celery 0.9978 0.01–1 −84 0.0057 0.0017
Lettuce 0.9905 0.01–1 −92 0.0057 0.0017
Pakchoi 0.9987 0.01–1 26 0.0026 0.0008
Rape 0.9995 0.01–1 −89 0.0047 0.0014
Romaine lettuce 0.9955 0.01–1 −86 0.0032 0.0010
Propiconazole Acetonitrile 0.9966 0.01–1 - 0.0084 0.0025
Spinach 0.9999 0.01–1 −42 0.0100 0.0030
Tomato 0.9991 0.01–1 −61 0.0100 0.0030
Cucumber 0.9995 0.01–1 −57 0.0101 0.0030
Eggplant 0.9947 0.01–1 41 0.0068 0.0020
Celery 0.9986 0.01–1 46 0.0101 0.0030
Lettuce 0.993 0.01–1 12 0.0101 0.0030
Pakchoi 0.9998 0.01–1 9 0.0077 0.0023
Rape 0.9967 0.01–1 52 0.0101 0.0030
Romaine lettuce 0.9965 0.01–1 11 0.0052 0.0016
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 219

myclobutanil, simeconazole, triadimenol, tebuconazole, flusilazole and propiconazole

using matrix-matched standards to eliminate the matrix effect to gain more accurate
and realistic results for all samples.

3.5. Precision and accuracy

The recoveries and RSDs of the six fungicides were carried out to certify the method
by spiking the blank vegetable samples (spinach, tomato, cucumber, eggplant, celery,
pakchoi, rape and romaine lettuce) at three levels (0.01,0.1 and 1 mg kg−1). For
recoveries experiment, we choose MWCNT (10 mg) which have the best purification
effect as a clean-up sorbent. The precision of the method was determined using
repeatability and reproducibility studies. Table 4 shows that the recoveries values
from 73.1% to 118.2%, and that all RSD values below 20% at three fortified concen-
tration levels. The results of the recoveries studies displayed that the method can
achieve satisfactory recoveries, precision and sensitivity for this six fungicide analysis
in different vegetables.

3.6. Application to real samples

The method was applied to actual samples, which were obtained from Xingfu markets in
Beijing (China) and treated with the sample preparation method described in Section
2.4. Ninety samples were analyzed with 10 samples for each matrix (spinach, tomato,
cucumber, celery, lettuce, rape, pakchoi, romaine lettuce and eggplant), and all the six
fungicides were below the LOQ (0.01 mg kg−1), which was much lower than the MRL
determined by the Codex Alimentarius Commission (0.2 mg kg−1 for tomato) and the
MRL determined by Japan (0.05 mg kg−1 for lettuce). The method can provide basic data
for the development of the MRL and related risk assessment work.

4. conclusion
In this study, nine sorbents were compared in the aspects of the pigment removal
and recoveries. The results showed that sorbent 5 has better efficiency in removing
high pigment content. A simple modified quick, easy, cheap, effective, rugged and
safe (QuEChERS) analytical method for the simultaneous detection of six triazole
fungicides (myclobutanil; simeconazole; triadimenol; tebuconazole; flusilazole; propi-
conazole) in vegetables (spinach, tomato, cucumber, celery, lettuce, rape, pakchoi
romaine lettuce and eggplant) has been developed. The six compounds were
extracted based on the QuEChERS procedure and detected using UPLC−MS/MS in
the ESI positive modes. All analyses were successfully separated with good specificity.
The recoveries ranged from 73.1% to 118.2% with RSDs below 20%, and LOQs for six
pesticides ranged from 0.0036 to 0.011 mg kg−1. The routine analysis of actual
samples confirmed the reliability and efficacy of this method in fruits vegetables.
 220

Table 4. Recoveries and RSD for the target compounds in nine matrices at three spiked levels.
Myclobutanil Simeconazole Triadimenol Tebuconazole Flusilazole Propiconazole
Spiked level
Compound (mg kg−1) Recovery (%) RSDr (%) Recovery (%) RSDr (%) Recovery (%) RSDr (%) Recovery (%) RSDr (%) Recovery (%) RSDr (%) Recovery (%) RSDr (%)
J. CAO ET AL.

Rape 0.01 82.9 4.3 99.5 5.5 106.8 11.3 115.0 10.1 94.5 6.9 111 5.8
0.1 98.3 4.8 101.0 0.4 107 3.8 97.5 2.8 113 2.9 98.1 4.0
1 105.3 3.2 100.0 3.3 94.7 6.2 95.6 9.8 97.5 2.9 103.3 3.6
Celery 0.01 94.7 5.3 88.6 1.6 80.7 7.9 93.4 5.3 78.1 2.3 104.5 5.0
0.1 104.2 11.2 111.5 14.0 113.4 12.2 105.5 15.5 116.3 14.7 105.2 11.9
1 95.8 1.8 81.4 4.0 99.8 3.5 91.8 3.2 90.3 2.6 94.4 3.2
Lettuce 0.01 97.0 6.7 102.3 4.2 88.5 3.8 101.3 4.9 94.3 13.3 90.9 10.7
0.1 104.9 3.3 100.7 3.0 104.4 3.1 106.1 4.9 98.7 4.6 94.1 4.0
1 102.9 5.9 107.9 7.6 94.3 6.0 110.1 8.5 102.4 8.8 103.5 9.8
Pakchoi 0.01 83.3 12.3 98.2 2.2 106.6 5.0 102.1 5.0 98.2 17.6 80.5 15.5
0.1 88.3 3.1 83.8 6.5 92.8 7.4 83.6 4.1 86.3 3.9 86.2 4.8
1 94.0 2.8 92.0 2.1 94.5 2.5 88.7 3.9 97.5 1.8 89.6 1.4
Spinach 0.01 93.2 3.3 104.3 9.6 95.0 5.8 95.9 15 114.5 12.1 98.7 10.5
0.1 101.7 7.3 109.6 7.1 102.8 11.8 99.8 3.8 114 6.2 102.5 3.4
1 98.7 12.5 96.6 13.7 103.2 4.7 92.6 12.4 109.6 16.8 94.9 8.8
Romaine lettuce 0.01 111.2 10.9 101.9 3.8 104.7 4.6 108.2 5.1 103.8 2.1 108.3 10.8
0.1 98.5 1.6 104.4 1.7 105.9 6.8 99.3 1.8 108 3.3 106.4 3.1
1 92.6 7.0 96.0 4.8 76.0 9.4 96.0 2.8 91.3 4.0 84.8 3.1
Tomato 0.01 105.0 18.0 99.0 9.0 100.3 10.6 102.2 9.3 103.4 7.6 95 8.4
0.1 115.1 12.9 104.6 16.5 100.2 9.8 109.9 10.2 105.7 12.3 96.5 13.4
1 89.7 4.0 91.1 6.7 95.7 5.5 88.9 4.3 98.3 2.4 92.8 3.4
Cucumber 0.01 98.8 7.0 86.4 3.4 80.8 10.6 103.9 8.6 107.8 6.6 104.6 8.0
0.1 114.1 3.3 104.5 4.9 94.3 6.9 110.5 7.5 106.9 2.4 118.2 1.5
1 111.3 5.9 89.0 5.7 92.9 11.2 83.9 5.1 109.7 7.0 73.1 4.1
Eggplant 0.01 94.9 10.5 109.7 6.8 90.5 6.4 96.3 8.1 79.4 5.5 83.7 7.1
0.1 100.6 5.3 82.8 7.0 93.1 12.0 102.7 5.0 94.4 5.8 108.2 3.1
1 84.3 5.3 85.1 4.8 88.6 4.6 80.0 2.8 86.3 4.1 80.4 2.2
RSD is intra-day precision (n = 5)
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 221

Acknowledgments
We gratefully acknowledge the financial support provided by the National Natural Science
Foundation of China (No. 31672057).

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Natural Science Foundation of China [31672057].

ORCID
Abdul Kaium http://orcid.org/0000-0003-1952-8617

References
[1] P. Bowyer and D.W. Denning, Pest Manag. Sci. 70, 173 (2014). doi:10.1002/ps.3567.
[2] M. Kahle, I.J. Buerge, A. Hauser, M.D. Müller and T. Poiger, Environ. Sci. Technol. 42, 7193
(2008). doi:10.1021/es8009309.
[3] A.C. Vieira, M.G. Santos and E.C. Figueiredo, Int. J. Environ. An. Ch. 97, 29 (2017). doi:10.1080/
03067319.2016.1272679.
[4] Z. Kong, F. Dong, J. Xu, X. Liu, C. Zhang, J. Li, Y. Li, X. Chen, W. Shan and Y. Zheng, Food
Control 23, 54 (2012). doi:10.1016/j.foodcont.2011.06.010.
[5] G.P. Munkvold, C.A. Martinson, J.M. Shriver and P.M. Dixon, Phytopathology 91, 477 (2001).
doi:10.1094/PHYTO.2001.91.5.477.
[6] B. Yan, F. Ye and D. Gao, Pest Manag. Sci. 71, 65 (2015). doi:10.1002/ps.3763.
[7] D.E. Groth, Crop Prot. 27, 1125 (2008). doi:10.1016/j.cropro.2008.01.010.
[8] M.B. Klix, J.-A. Verreet and M. Beyer, Crop Prot. 26, 683 (2007). doi:10.1016/j.cropro.2006.06.006.
[9] M. Reuveni and D. Sheglov, Crop Prot. 21, 951 (2002). doi:10.1016/S0261-2194(02)00073-X.
[10] S. Elmholt, Pest Manag. Sci. 34, 139 (1992). doi:10.1002/ps.2780340208.
[11] J.R. Vogel, M.S. Majewski and P.D. Capel, J. Environ. Qual. 37, 1101 (2008). doi:10.2134/
jeq2007.0079.
[12] R.M. González-Rodríguez, R. Rial-Otero, B. Cancho-Grande and J. Simal-Gándara,
J. Chromatogr. A. s 1196–1197, 100 (2008). doi:10.1016/j.chroma.2008.02.087.
[13] C.G. Zambonin, A. Cilenti and F. Palmisano, J. Chromatogr. A. 967, 255 (2002).
[14] Y. Zhang, Y. Zhang, J. Nie, B. Jiao and Q. Zhao, Food Anal. Method 9, 3509 (2016). doi:10.1007/
s12161-016-0548-9.
[15] M. Rezaee, Y. Assadi, M.R.M. Hosseini, E. Aghaee, F. Ahmadi and S. Berijani, J. Chromatogr. A.
1116, 1 (2006). doi:10.1016/j.chroma.2006.03.007.
[16] S. Navarro, A. Barba, G. Navarro, N. Vela and J. Oliva, J. Chromatogr. A. 882, 221 (2000).
doi:10.1016/S0021-9673(00)00337-X.
[17] R. Jeannot, H. Sabik, E. Sauvard and E. Genin, J. Chromatogr. A. 879, 51 (2000). doi:10.1016/
S0021-9673(00)00098-4.
[18] J.B. Baugros, B. Giroud, G. Dessalces, M.F. Grenier-Loustalot and C. Cren-Olivé, Anal. Chim.
Acta. 607, 191 (2008). doi:10.1016/j.aca.2007.11.036.
[19] L. Wang, X. Zang, Q. Chang, G. Zhang, C. Wang and Z. Wang, Food Anal. Method 7, 318
(2013). doi:10.1007/s12161-013-9629-1.
222 J. CAO ET AL.

[20] M.M. Radišić, T.M. Vasiljević, N.N. Dujaković and M.D. Laušević, Food Anal. Method 6, 648
(2013). doi:10.1007/s12161-012-9448-9.
[21] A. Juan-García, Y. Picó and G. Font, J. Chromatogr. A 1073, 229 (2005). doi:10.1016/j.
chroma.2004.09.028.
[22] C. Almeida and J.M. Nogueira, J. Chromatogr. A. 1265, 7 (2012). doi:10.1016/j.chroma.2012.09.047.
[23] R.B. Schäfer, R. Mueller, W. Brack, K.D. Wenzel, G. Streck, W. Ruck and M. Liess, Chemosphere
70, 1952 (2008). doi:10.1016/j.chemosphere.2007.09.058.
[24] J.G. Martins, A.A. Chávez, S.M. Waliszewski, A.C. Cruz and M.M.G. Fabila, Chemosphere 92,
233 (2013). doi:10.1016/j.chemosphere.2013.04.008.
[25] M. Anastassiades, S.J. Lehotay, D. Stajnbaher and F.J. Schenck, J. AOAC. Int. 86, 412 (2003).
[26] S.W. Lee, J.H. Choi, S.K. Cho, H.A. Yu, A.M. Abd ElAty and J.H. Shim, J. Chromatogr. A. 1218,
4366 (2011). doi:10.1016/j.chroma.2011.05.021.
[27] F. Dong, X. Chen, X. Liu, J. Xu, Y. Li, W. Shan and Y. Zheng, J. Chromatogr. A. 1262, 98 (2012).
doi:10.1016/j.chroma.2012.08.100.
[28] A. Sadowska-Rociek, M. Surma and E. Cieślik, B Environ. Contam. Tox. 90, 508 (2013).
doi:10.1007/s00128-012-0951-x.
[29] L.D.C. Cabrera, M.L. Martins, E.G. Primel, O.D. Prestes, M.B. Adaime and R. Zanella. Sci.
Chromatogr. 4, 227. (2012).
[30] T.D. Nguyen, E.M. Han, M.S. Seo, S.R. Kim, M.Y. Yun, D.M. Lee and G.H. Lee, Anal. Chim.
Acta. 619, 67 (2008). doi:10.1016/j.aca.2008.03.031.
[31] X.W. Dou, X. Chu, W. Kong, Y. Yang and M. Yang, RSC. Adv. 5, 86163 (2015). doi:10.1039/
C4RA14244F.
[32] L.D.C. Cabrera, S.S. Caldas, O.D. Prestes, E.G. Primel and R. Zanella, J. Sep. Sci. 39, 1945 (2016).
doi:10.1002/jssc.201501204.
[33] U. Koesukwiwat, S.J. Lehotay, S. Miao and N. Leepipatpiboon, J. Chromatogr. A. 1217, 6692
(2010). doi:10.1016/j.chroma.2010.05.012.
[34] Z. Mei-Ai, F. Ya-Nan, Z. Yong-Zhe and K. Jeong-Han, J. Agric. Food Chem. 62, 11449 (2014).
doi:10.1021/jf504570b.
[35] T. Kiljanekl, J. Chromatogr. A. 1435, S0021967316300012 (2016).
[36] S. Walorczyk, Talanta 120, 106 (2014). doi:10.1016/j.talanta.2013.11.070.
[37] F. Yu, L. Chen, L. Pan, B. Hu and H. Liu, J. Sep. Sci. 38, 1894 (2015). doi:10.1002/jssc.201500148.
[38] P. Zhao, L. Wang, L. Zhou, F. Zhang, S. Kang and C. Pan, J. Chromatogr. A. 1225, 17 (2012).
doi:10.1016/j.chroma.2011.12.070.
[39] M. Inyang and E. Dickenson, Chemosphere 134, 232 (2015). doi:10.1016/j.chemosphere.2015.03.072.
[40] N. Karakoyun, S. Kubilay, N. Aktas, O. Turhan, M. Kasimoglu, S. Yilmaz and N. Sahiner,
Desalination 280, 319 (2011). doi:10.1016/j.desal.2011.07.014.
[41] S. Ye, G. Zeng, H. Wu, Z. Chang, J. Dai, L. Jie, J. Yu, X. Ren, H. Yi and C. Min, Crit. Rev.
Biotechnol. 37, 1 (2017). doi:10.1080/07388551.2017.1304357.
[42] S. Ye, G. Zeng, H. Wu, C. Zhang, J. Liang, J. Dai, Z. Liu, W. Xiong, J. Wan and P. Xu, Crit. Rev.
Env. Sci. Tec. 47, 00 (2017). doi:10.1080/10643389.2017.1386951.
[43] B.S. Sproat, Purification of Oligomers (US, 2003) (accessed 8 february 2019).https://patents.
google.com/patent/US6620926B2/en
[44] K.Y. Ren, W.L. Zhang, S.R. Cao, X.I. Cun-Xian, G.M. Wang and Z.Q. Zhou, Chinese J. Anal
Chem 2017.
[45] N. Volpi, F. Galeotti, B. Yang and R.J. Linhardt, Nat Protoc 9, 541 (2014). doi:10.1038/
nprot.2014.026.
[46] F. Guozhen, M. Guang, H. Jinxing, Z. Chao, Q. Kun and W. Shuo, J. Agr. Food Chem. 57, 3040
(2009). doi:10.1021/jf803913q.
[47] X. Hou, S.R. Lei, S.T. Qiu, L.A. Guo, S.G. Yi and W. Liu, Food Chem 153, 121 (2014).
doi:10.1016/j.foodchem.2013.12.031.
[48] M.B. Ahmed, J.L. Zhou, H.H. Ngo and W. Guo, Biomass. Bioenerg 84, 76 (2016). doi:10.1016/j.
biombioe.2015.11.002.
[49] D. Angın, T.E. Köse and U. Selengil, Appl. Surf. Sci. 280, 705 (2013). doi:10.1016/j.apsusc.2013.05.046.
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 223

[50] A.W.A.K.W. Ghani, A. Mohd, H.Y. Taufiq-Yap, U. Rashid and H.A. Al-Muhtaseb, Ind. Crop
Prod. 44, 18 (2013). doi:10.1016/j.indcrop.2012.10.017.
[51] D. Dan, M. Wang, J. Zhang, C. Jie, H. Tu and A. Zhang, Electrochem. Comm. 10, 85 (2008).
doi:10.1016/j.elecom.2007.11.005.
[52] P. Payá, M. Anastassiades, D. Mack, I. Sigalova, B. Tasdelen, J. Oliva and A. Barba, Anal. Bional.
Chem. 389, 1697 (2007). doi:10.1007/s00216-007-1610-7.
[53] N. Komasawa, R. Ueki, Y. Kaminoh and S.I. Nishi, J. Chromatogr. A. 1217, 2548 (2010).
doi:10.1016/j.chroma.2010.01.044.
[54] M. Li, X. Liu, F. Dong, J. Xu, Z. Kong, Y. Li and Y. Zheng, J. Chromatogr. A. 1300, 95 (2013).
doi:10.1016/j.chroma.2013.05.052.