Food Chemistry 229 (2017) 580–587

Contents lists available at ScienceDirect

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

Analytical Methods

Multiresidue method for the determination of pesticides in Oolong tea

using QuEChERS by gas chromatography-triple quadrupole tandem mass
spectrometry
Chia-Chang Wu
Veterans Affairs Council, Fushoushan Farm, Taichung Heping 42492, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: We propose a simple, rapid analytical method for determination of 89 pesticides in Oolong tea by GC/MS/
Received 29 March 2016 MS. Samples were extracted via QuEChERS. The limits of detection and quantification range of the 89 pes-
Received in revised form 2 September 2016 ticides were 1–25 lg L
1 and 10–50 lg L
1, respectively. Good separation was attained in less than
Accepted 16 February 2017
36 min. A wide linear range of 1–250 lg L
1 was observed with r2 values from 0.9955 to 0.9998.
Available online 17 February 2017
Pesticide-free tea powder spiked at 50 and 100 lg L
1. Recovery ranges of the 86 (50 lg L
1) and 83
(100 lg L
1) pesticides were from 60% to 120%. Relative standard deviations were less than 20%. The lab-
Keywords:
oratory proficiency test (FAPAS, 2014) shows satisfactory (|z| < 2) z-score values. The proposed monitor-
Oolong tea
QuEChERS
ing technique rapidly and efficiently screens, multiple pesticides in Oolong tea.
GC/MS/MS Ó 2017 Elsevier Ltd. All rights reserved.
Pesticide residues

1. Introduction in 2002, scientists developed a simplifying sample pretreatment

‘‘Food safety” has become the subject of popular interest in
recent years, especially with the endless stream of incidents
related to pesticide residue in foods or crops which have caused
consumer panic. In order to ensure consumer health, research
and development institutions in many countries have begun to
analyze pesticide residues in agricultural products and monitor
the safety of agricultural products (Luke & Doose, 1983). In Taiwan,
the Tea Research and Extension Station also conduct tests to eval-
uate the safe usage of tea pesticides (Wu, 2001).
Conventional sample extraction methods for pesticide residues
in agricultural products include solid-phase extraction (Economou,
Botitsi, Antoniou, & Tsipi, 2009; Wu, Chu, Wang, & Lur, 2009),
ultrasonic-assisted extraction (Wang & Curtis, 2006), accelerated
solvent extraction (Lehotay & Eller, 1995), soxhlet extraction
(Luke & Doose, 1983), and gel permeation chromatography (Tekel
& Hatrik, 1996).
However, the above methods required many amounts of sol-
vents and very labor intensive, while offering poor recovery rates.
In addition, they generate large amounts of waste may cause lead
to human toxicity and carcinogenicity, and treatment of the
wasted liquid is expensive and polluting. Thus, it is necessary to
develop low-solvent or solvent-free techniques for pretreatment
in pesticide analysis. At the European Pesticide Residue Workshop
E-mail address: wu0933036183@gmail.com
method for pesticide analysis in food called QuEChERS (pro-
nounced ‘‘Quichers”), which is an acronym for Quick, Easy, Cheap,
Effective, Rugged and Safe.
The above method is a fast-growing sample extraction and
cleanup procedure for analyzing pesticide residues in agricultural
products. Therefore, the QuEChERS method has been established
as the AOAC Official draft law and the United States Department
of Agriculture has carried out 20 kinds of pesticides and 3 kinds
of inspection bodies (grapes, lettuces, and citrus) for collaborative
study. These efforts have led to better results (Anastassiades,
Lehotay, Stajnbaher, & Schenck, 2003).
Moreover, the European Union has approved the QuEChERS
method as a standard method of analysis (European Standard EN
15662:2008., 2008). As for its analysis features, QuEChERS uses
an inspection body with little solvent extraction. Without multiple
extraction processes, a concentrator, constant volume or the usual
pesticide residue analysis steps, this method significantly reduces
processing time and increases the varieties of pesticide analysis.
The QuEChERS method has been widely used in the European
Union and the United States for pesticide residue analysis of veg-
etables and fruits (Elham, Fatemeh, & Mansour, 2016; Tesfa,
Abera, & Negussie, 2015).
Tea is a popular drink throughout the world, and in recent
years, consumers have become more and more concerned about
the presence of pesticide residues in tea, Therefore investigation
of pesticide residues in tea is an important issue (Shivani, Sood,

http://dx.doi.org/10.1016/j.foodchem.2017.02.081
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
 C.-C. Wu / Food Chemistry 229 (2017) 580–587
Kuma, Ravindranath, & Shanker, 2001; Wu, 2001). The Tea
Research and Extension Station is a governmental laboratory
which has conducted tests to evaluate the safe usage of pesticides
in Taiwan since 1985 (Wu et al., 2012). Starting in 1985, we have
developed methods by using solvent extraction and gas chro-
matography with electron-capture detector and flame-
phosphorus detector (GC/ECD/FPD), liquid chromatography with
fluorescence detector (LC/FLD) and liquid chromatography–triple
quadrupole tandem mass spectrometry (LC/MS/MS) to analyze
pesticide residues in fresh tea leaves, ready-made tea and tea
brews (Lin, Wu, & Ho, 2008; Wu, 2001, 2003; Wu, Chu, Wang, &
Lur, 2007; Wu, Li, Lin, & Chu, 2010; Wu et al., 2009).
The problem is that the matrix of tea is highly complex, because
tea contains protein, polyphenolic compounds, sugars, alkaloids,
pigments and other substances. During the solvent extraction pro-
cess for the analysis of pesticide residues, many complex chemical
substances are seized and co-extracted. This leads to interference
problems, and affects the detection limit. Extraction, vacuum con-
centration and purification treatments can be used to resolve the
matrix interference problems, but these methods increase the
amount of solvent used and the processing time.
Liquid and gas chromatography are applied for multi-residue
pesticide analysis for decades. Since people are getting more con-
cerned about food safety issue, the Government established lower
and lower tolerances in regulations. Therefore, it is necessary to
adopt liquid or gas chromatography-tandem mass spectrometry
detection to improve the selectivity and accuracy of results in
the qualitative and quantitative. For the past ten years, LC/MS/
MS workflow is well established, but GC/MS/MS is still developing.
The aim of this paper is to optimize and validate the QuEChERS
extraction method followed by GC/MS/MS simultaneous determi-
nation of pesticide residues in tea.
2. Experimental
2.1. Reagents, chemicals and materials
A total of 89 standard pesticides with purity ranging from 98.6%
to 99.9% were purchased from Accu Standard (New Haven, USA),
Chem Service Company (PA, USA), Dr. Ehrenstorfer Company
(Augsburg, Germany), Fluka Company (Steinheim, Germany),
Merck Companies (Germany), and Riedel de-Haën (Hannover, Ger-
many), Wako (Richmond, USA). All the solvents and chemicals
used in the experiment were analytical grade and were purchased
from Merck (Germany). Primary secondary amine (PSA), graphi-
tized carbon black (GCB), and SampliQ extraction tube (packed
with sodium acetate anhydrous, 1.5 g, and anhydrous magnesium
sulfate, 6 g, with 50 ml plastic centrifuge tubes) were purchased
from Agilent Company (CA, USA).
2.2. Extraction studies
We applied 4 g of Oolong tea powder added to a 50 mL cen-
trifuge tube with 16 mL distilled water and left to stand for
20 min. The samples were then extracted with 10 mL acetonitrile
(containing 1% acid), shook for 30 s, and put in a QuEChERS extrac-
tion package (packed with 1.5g anhydrous sodium acetate and 6g
anhydrous magnesium sulfate, Agilent SampliQ extraction tube,
AOAC Met (PN. 5982-5755) and shook for 1 min, with 3500 rpm
centrifugal force for 10 min. We then cleaned up the upper 4 mL
extraction solvent with a florisil cartridge (J. T. Baker, USA), and
evaporated to dryness with nitrogen gas. The residue was then dis-
solved in a 1 mL acetone- N-hexane (1/1 (v/v)) and passed through
0.2 lm membrane filters before GC/MS/MS injection (Fig. 1).
581
2.3. Analytical instruments and conditions
We applied gas chromatograph (Agilent Technologies 6890,
USA) with tandem mass spectrometry (Agilent GC/7000A GC/MS
triple quadrupole), equipped with a HP-5MS column (AgilentHP-
5MS,3000 mm  0.25 mm, 0.25 lm) for pesticides separation. The
gradient analysis of the gas chromatograph was as follows: tem-
perature 70 °C, maintain temperature rising rate of 25 °C /min to
150 °C for 2 min, temperature rising rate of 3 °C/min to 200 °C, a
temperature rising rate 8 °C/min, and an end temperature 280 °C,
maintained 10 min. Tandem mass spectrometer component opera-
tion conditions setting were as follows: the temperature ion source
was 230 °C, the quadrupole temperature was 150 °C, the mass
spectrometer and gas chromatograph in the line of the junction
(transfer line) temperatures were 280 °C, the ionization energy
was 70 eV, and the carry gas and all the gas in the colliding room
were helium. Using the multiple reaction monitoring mode
(MRM) to scan the ions, we set the retention time (RT), fragment
voltage and collision energy.
2.4. Calibration, limits of detection and quantification
A standard stock solution (1000 lg L
1) was prepared in ace-
tonitrile and stored at
20 °C. The working solutions required for
preparing a standard curve (1, 2.5, 5,10,25,50,100 and 250 lg
L
1) in pesticide-free tea powders for matrix-matched were
obtained. All measurements were performed in triplicate for each
level and 10 lL of solution was injected into the mass spectrome-
ter for analysis. Calibration curves were calculated by linear least-
squares regression using peak areas. Based on their concentrations,
x axis, peak area, and y axis, which were obtained by regression
analysis of standard inspection lines: y = a + bx, and a and b are
constant. The coefficient of determination of r2 must be >0.99.
2.5. Recovery studies
During recovery studies, the 4 g pesticide-free tea powder sam-
ples which spiked at 50 and 100 lg L
1 pesticide levels were used
for fortification analysis to evaluate the recovery (%) and determine
the relative standard deviation (RSD) of 89 pesticides. The recovery
assays were replicated three times.
3. Results and discussion
3.1. Optimization of GC/MS/MS conditions
In our study, a total of 89 pesticides used for common tea
plants, vegetables, fruits and rice were selected for investigation,
such as organ phosphorus pesticides, organic chlorine pesticides,
organic nitrogen pesticides, heterocyclic compounds, and synthetic
insecticides were used to establish the MRM for GC/MS/MS. Table 1
summarizes the optimized GC/MS/MS MRM acquisition parame-
ters of the targeted pesticides. We established a parent ion scan
mode, with a scanning range from 50 to 550 (m/z). Next, we
selected the precursor ion of 89 pesticides for the second collision
of ions with different voltages. Then we selected a signal value and
the highest percentage collision value of the two ions and voltages.
Finally, we determined the highest value for the quantitative ion,
followed by the qualitative ion.
The qualitative ion collision voltage range for the 89 pesticides
was 5–35 (eV) and the quantitative ion collision cover range was
5–40 (eV). In order to avoid RT variation of the pesticides, we
added pesticides to the tea matrix, and then changed the GC/MS/
MS pressure to stabilize the retention time of the chromatography
of the target compounds, and ensure the retention times of the 89
582 C.-C. Wu / Food Chemistry 229 (2017) 580–587
Fig. 1. The QuEChERS extracted procedure and analyzed by GC/MS/MS.
pesticides were within the allowed range (± 0.05 s). As Table 1
indicates, the analysis chromatogram of the first material which
appeared was propoxur with an RT of 10.36 min, qualitative ion
of 110.00 > 63.00 (m/z), collision voltage of 30 (eV), quantitative
ion of 110.00 > 64.00 (m/z), and collisions voltage of 20 (eV). The
latest material, Deltamethrin, occurred with an RT of 35.94 min,
qualitative ion of 253.00 > 93.00 (m/z), collision voltage of 22
(eV), quantitative ion of 253.00 > 174.00 (m/z), and collisions volt-
age of 10 (eV).
Fig. 2 showed chromatograms of the separation for 89 analyses
in standard solution of pesticides. Working on the stated model of
multiple reactions for monitoring mass spectrometry analysis of
pesticides, we obtained a good separation in a relatively short time
(42 min), without interference with the matrix. In this study, we
also selected the two transition ions to be detected. The selected
ions contained 1 precursor (1 IP) and 2 precursors (1.5x2 = 3IPs),
with a total of 4 IPs received, in line with the EU 2002/657/EC
(Commission Decision, 2002) analysis of the specification. Thus,
we can be fairly certain that this method is effective in identifying
pesticide residues in tea samples (European Commission. Docu-
ment No SANCO/2007/1331, 2007).
3.2. Matrix-matched calibration curve
In order to reduce the effect of the matrix on quantitative deter-
mination, and improve the accuracy of real samples of concentra-
tion, we added a series of working solutions ranging from 5-
250 lg L
1 in pesticide-free Oolong tea powders to create a
matrix-matched calibration curve. With the accurate preparation
of the known purity standards of pesticide products, the pesticides
were prepared with acetone/N-hexane (1/1, v/v) in 10 lg L
1 as a
stock solution. Avoiding light, the samples were stored at
20 °C.
We then took a suitable amount of a standard liquid mixture,
and gradually diluted the working solution at a low concentration
to obtain the working solution. We used acetone /N-hexane (1/1, v/
v) diluted to 1, 2.5, 5, 10, 25, 50, 100 and 250 lg L
1 as a mixed
standard solution.
Calibration curves were calculated by linear least-squares
regression using peak areas. Based on their concentrations, the x
axes, peak areas and y axes were obtained by regression analysis
of standard inspection lines: y = a + bx, and a and b are constant
for the intercept and the slope, respectably. The linear concentra-
tions of the 89 pesticides range from 1 to 250 lg L
1.
The results of the linear correlation coefficient (r2) are shown in
Table 2. The r2 of 89 pesticides ranged from 0.995–0.999, except
bifenox (r2 = 0.985). In this study, the r2 of bifenox was less than
0.99, it might be due to that bifenox (C14H9Cl2NO5) contain rela-
tively polar groups are susceptible to matrix effect. Table 2 shows
that the analysis of pesticide residues in tea by the GC/MS/MS
obtained good qualitative and quantitative analysis of the result
Table 3.
3.3. Limits of detection (LODs) and limits of quantification (LOQs)
To prepare the homogeneous pesticide-free Oolong tea pow-
ders, we added a mixed standard solution of pesticides containing
1–100 lg L
1 in the inspection sample. Instrumental analyses of
the target compounds were conducted 3 and 10 times to obtain
the signal to noise ratio for the LODs and LOQs, respectively
(SANCO/10476/2003, 2004). The LODs and LOQs were calculated
using the following relations: LOD = X + 3 s, LOQ = X + 10 s, in
which, ‘‘X” is the mean concentration of spiked sample blank val-
ues, and ‘‘s” is the sample standard deviation.
The 89 pesticides mixed using the standard solution were
added to pesticide-free Oolong tea powders. This was repeated 3
times with different concentrations. Results showed that for the
89 pesticides, the LODs and LOQs ranged from 1 to 25 lg L
1and
10 to 50 lg L
1, respectively. Table 2 shows 70 pesticides had LODs
at 1 lg L
1, accounting for a total of 78.65%. The LODs of cyfluthrin
and diniconazole were 25 and 10 lg L
1, respectively. As for the
LOQs, cyfluthrin had 50 lg L
1, but for b-endosulfan and dinicona-
zole, the LOQs were 20 lg L
1. The LOQs of 86 pesticides were at
10 lg L
1, making up a total of 96.63%.
It is quite conceivable that the LOQs of most of the pesticides
reach up to 10 lg L
1, and that the qualitative ion and quantitative
ion ratio meet the identification requirements of the European
Union (SANCO/10476/2003, 2004).
The results show that using GC/MS/MS to analyze the pesticide
residues in tea produced good quantitative detection limit findings.
In addition, the LODs of the analysis data of quantitative limits are
 C.-C. Wu / Food Chemistry 229 (2017) 580–587 583

Table 1
The optimized GC/MS/MS MRM acquisition parameters of the targeted pesticides.

Pesticide Rention time (RT) Quantification Qualification

MRM transition (m/z) Collision energy (eV) MRM transition (m/z) Collision energy (eV)
Alachlor 17.02 160.05 > 130.00 30 160.05 > 131.10 10
Benfluralin 11.72 291.70 > 206.30 15 291.70 > 264.10 15
Bifenox 29.21 311.00 > 279.30 10 311.00 > 216.30 20
Bifenthrin 28.84 180.80 > 165.10 20 180.80 > 166.10 20
Bromophos methyl 20.08 331.00 > 316.00 20 331.00 > 286.00 20
Bromophos-ethyl 22.55 358.70 > 303.00 15 358.70 > 331.00 5
Bromopropylate 28.65 341.00 > 183.00 20 341.00 > 185.00 20
Bupirimate 24.80 273.00 > 108.00 15 273.00 > 193.00 5
Butachlor 23.22 175.80 > 146.10 20 237.00 > 160.00 5
Butralin 20.20 265.90 > 190.20 10 265.90 > 220.20 10
Carbophenothion 26.66 153.00 > 96.90 10 157.00 > 121.00 25
Chinomethionat 21.98 234.00 > 206.00 10 206.00 > 148.00 15
Chlorfenapyr 25.29 247.00 > 227.00 15 247.00 > 197.00 20
Chlorpyrifos 19.23 198.70 > 171.00 30 198.70 > 97.90 30
Chlorpyriphos-methyl 16.61 286.00 > 93.00 20 286.00 > 270.90 20
Chlozolinate 21.39 188.10 > 147.10 15 330.80 > 259.10 5
Cyanofenphos 26.83 168.90 > 77.00 30 168.90 > 141.00 5
Cyfluthrin 32.43 163.00 > 91.00 15 163.00 > 127.00 5
Cyhalothrin 30.39 197.00 > 161.00 10 181.00 > 152.00 5
Cypermethrin 33.03 162.70 > 91.00 25 180.70 > 152.10 25
Deltamethrin 35.94 253.00 > 93.00 22 253.00 > 174.00 10
Diazinon 14.45 136.80 > 84.00 10 136.80 > 54.10 30
Dicofol 19.25 138.90 > 111.00 30 249.8 > 139.0 15
Dieldrin 23.88 263.00 > 193.00 30 263.00 > 191.00 30
Diniconazole 25.59 268.00 > 232.00 15 270.00 > 234.00 15
Dinitramine 14.84 305.00 > 216.00 10 305.00 > 244.00 15
Dioxabenzofos 11.47 215.80 > 201.10 10 215.80 > 138.10 10
Disulfoton 14.54 274.00 > 88.00 5 186.00 > 142.00 5
Ditalimfos 23.24 129.80 > 102.10 25 129.80 > 75.10 25
EPN 28.68 157.00 > 77.10 25 157.00 > 110.00 15
Ethion 21.98 230.70 > 175.00 5 230.70 > 203.10 5
Ethoprophos 10.76 157.80 > 114.00 5 157.80 > 96.90 5
Etrimfos 15.16 292.00 > 181.00 5 292.00 > 153.00 20
Fenazaquin 29.14 160.00 > 145.20 5 145.00 > 115.20 20
Fenitrothion 18.08 277.10 > 109.00 20 277.00 > 260.00 5
Fenpropathrin 29.00 181.00 > 152.00 25 265.00 > 210.00 15
Fenthion 19.12 278.00 > 109.00 10 278.00 > 125.00 15
Fenvalerate 34.60 167.00 > 125.00 10 225.00 > 119.00 15
Fipronil 21.90 367.00 > 213.00 30 367.00 > 228.00 30
Flucythrinate 33.35 199.00 > 107.00 30 199.00 > 157.00 5
Fluvalinate 34.88 250.00 > 200.00 22 252.00 > 200.00 18
Fonofos 13.89 246.00 > 109.10 15 246.00 > 81.10 30
Halfenprox 32.81 263.00 > 169.00 25 263.00 > 235.00 15
Haloxyfop-methyl 22.87 375.00 > 91.00 30 288.00 > 180.00 20
Heptachlor 16.78 272.00 > 237.00 20 274.00 > 239.00 20
Heptachlor Epoxide 20.72 352.90 > 262.90 10 352.90 > 281.90 15
Iprobenfos 15.35 204.00 > 91.10 10 90.90 > 39.10 25
Iprodione 28.46 313.60 > 56.10 10 313.60 > 245.10 10
Isazofos 15.02 118.80 > 76.00 15 118.80 > 84.10 15
Isofenphos 21.60 213.00 > 121.05 15 255.00 > 121.00 25
Kresoxim-methyl 24.89 206.00 > 116.00 5 206.00 > 131.00 10
Malathion 18.78 126.90 > 99.00 15 126.90 > 71.10 15
Methiocarb 18.09 168.00 > 153.00 10 153.00 > 109.00 10
Metolachlor 18.91 162.15 > 133.10 15 162.15 > 132.10 25
Metribuzin 16.30 198.05 > 82.10 20 198.05 > 89.00 15
Mirex 29.85 271.60 > 237.00 5 271.60 > 234.90 20
Oxadiazon 24.43 174.90 > 112.00 15 301.90 > 175.00 13
p,p’-DDE 24.05 246.00 > 176.10 30 248.00 > 176.00 30
Parathion 19.28 291.00 > 109.00 10 291.10 > 81.00 40
Parathion-methyl 16.61 263.00 > 109.10 15 263.00 > 79.10 30
Penconazole 21.05 248.00 > 157.00 25 248.00 > 192.00 15
Pendimethalin 21.00 252.10 > 162.10 10 252.10 > 161.20 20
Permethrin 31.53 183.00 > 153.00 20 183.00 > 168.00 20
Phenthoate 21.71 274.00 > 121.00 10 274.00 > 125.00 20
Phorate 11.95 121.00 > 65.00 10 260.00 > 75.20 5
Pirimiphos-ethyl 20.65 167.80 > 100.10 20 167.80 > 69.20 20
Pirimiphos-methyl 18.29 290.10 > 125.00 25 305.00 > 180.00 5
Pretilachlor 24.13 161.80 > 132.10 15 161.80 > 147.20 15
Procymidone 21.99 283.00 > 96.00 10 283.00 > 255.00 10
Profenofos 23.94 208.00 > 63.10 35 337.00 > 267.00 10
Propaphos 22.69 219.70 > 125.00 30 219.70 > 140.20 5
Propoxur 10.36 110.00 > 63.00 30 110.00 > 64.00 20
Prothiofos 23.78 267.00 > 239.00 5 309.00 > 239.00 15

(continued on next page)

584 C.-C. Wu / Food Chemistry 229 (2017) 580–587

Table 1 (continued)

Pesticide Rention time (RT) Quantification Qualification

MRM transition (m/z) Collision energy (eV) MRM transition (m/z) Collision energy (eV)
Pyrazophos 30.70 221.00 > 193.00 10 232.00 > 204.00 10
Pyridaben 31.56 147.00 > 117.00 20 147.00 > 132.00 10
Pyriproxyfen 29.89 136.00 > 78.00 25 136.00 > 96.00 15
Quinalphos 21.67 146.00 > 118.00 10 157.00 > 129.00 15
Quizalofop-ethyl 32.98 372.00 > 299.00 5 299.00 > 255.00 15
Tetradifon 29.42 354.00 > 159.00 10 356.00 > 159.00 10
Tetramethrin 28.75 164.00 > 77.00 25 164.00 > 107.00 25
Triazophos 26.48 161.00 > 106.00 10 161.00 > 134.00 5
Triflumizole 22.36 206.00 > 179.20 10 287.00 > 218.00 15
Trifluralin 11.64 305.90 > 43.20 15 305.90 > 264.10 10
Vinclozolin 16.65 212.00 > 145.00 15 212.00 > 172.00 25
a-BHC 12.10 182.70 > 146.70 15 182.70 > 145.20 15
a-chlordane (cis) 22.84 272.00 > 237.00 20 372.70 > 266.10 25
a-Endosulfan 22.63 238.80 > 204.00 15 195.00 > 159.00 5
b-chlordane (trans) 22.05 372.70 > 266.10 25 372.70 > 264.10 25
b-Endosulfan 25.19 119.90 > 102.30 5 119.90 > 85.20 25

Fig. 2. Chromatograms for: A: blank tea sample; B: tea sample spiked with 89 pesticides at 100 mg L
1.

lower than the maximum residue limits (MRLs) of Taiwan, Japan,
or the European Union, with maximum residue limits ranging from
1 to 10%. Therefore, the established GC/MS/MS analysis method is
worth using for routine analysis of pesticide residues in tea.
3.4. Evaluation of the QuEChERS method
The QuEChERS extraction method is quite mature when it
comes to the inspection of vegetables and fruit, but due to the high
complexity of the tea matrix, many complex chemical substances
are extracted at the same time, causing interference and pesticides
analysis, and seriously affecting the detection limits.
Most QuEChERS extraction methods are sufficient for LC/MS/MS
analysis, but few papers focused on GC/MS/MS analysis in order to
reduce the matrix interference. Thus, in this study, we used applied
the above approach for the clean-up and the concentration pro-
cesses. To ensure the accuracy and the precision of experimental
data, at the time of each analysis process, we added a solvent blank
and recovery analysis. The solvent blank showed no signal interfer-
ence, indicating that the analysis process is pollution-free.
In the recovery experiments, we added different concentrations
of mixed standard agents to pesticide-free Oolong tea powder, at
concentrations of 50,100 lg L
1. At each concentration level, three
analyses were performed.
According to the results, with a 50 lg L
1concentration in the
tea sample, a total of 86 kinds of pesticides showed recovery
results ranging from 60% to 120%, 3 kinds of pesticides had recov-
ery values between 50% and 60%. Cyfluthrin had a recovery value of
134.5%, with an RSD value of 5.8%. Heptachlor had a recovery value
of 57.8%, with an RSD value of 20.68%. And mirex had a recovery
value of 57.8%, with an RSD value of 8%.
When the concentration was 100lg L
1 in the tea sample, a
total of 83 kinds of pesticides had recovery values from 60% to
120%, 1 kind of pesticide had a recovery value between 120% and
130%, and 5 kinds of pesticides showed recovery values between
50% and 60%. For bifenox, the recovery value was 129.4%, with
an RSD of 8%. For heptachlor, the recovery value with 53.2%, with
an RSD of 2.5%. Mirex had a recovery value of 52.7%, with an RSD
of 8%. Propoxur showed a recovery value of 57.4%, with an RSD
of 6.7%. Parathion-methyl had a recovery value of 57.7%, with an
RSD of 7.2%.
Results indicated that under three concentrations, the recovery
values of the tea samples ranged from 53 to 129.4%, with all RSD
values being lower than 20.86%. According to the current EU
 C.-C. Wu / Food Chemistry 229 (2017) 580–587 585

Table 2
Linearity, calibration curves, limits of detection (LODs), and limits of quantification (LOQs) for the 89 pesticides in the GC/MS/MS under study.

Pesticide Linear range (lg L
1) r2 Parameters of linearity LOD (lg L
1) LOQ (lg L
1)
Slope Intercept
Alachlor 1–250 0.9988 2.067  101
11.240 2.50 10
Benfluralin 1–250 0.9969 1.810  101
67.402 1.00 10
Bifenox 1–250 0.9847 6.336  101 53.758 1.00 10
Bifenthrin 1–250 0.9991 2.295  102 197.071 1.00 10
Bromophos methyl 1–250 0.9993 4.193  101
12.539 1.00 10
Bromophos-ethyl 1–250 0.9992 4.258  101
28.679 1.00 10
Bromopropylate 1–250 0.9991 6.789  101 3.894 1.00 10
Bupirimate 1–250 0.9994 2.461  101
18.688 1.00 10
Butachlor 1–250 0.9998 1.502  101 14.858 1.00 10
Butralin 1–250 0.9955 1.875  101
81.548 1.00 10
Carbophenothion 1–250 0.9987 4.610  101
28.815 5.00 10
Chinomethionat 1–250 0.9988 8.972  101
129.562 1.00 10
Chlorfenapyr 1–250 0.9992 2.782  101 12.088 2.50 10
Chlorpyrifos 1–250 0.9995 1.032  101 90.515 1.00 10
Chlorpyriphos-methyl 1–250 0.9995 3.585  101
29.181 1.00 10
Chlozolinate 1–250 0.9987 1.573  101
17.670 1.00 10
Cyanofenphos 1–250 0.9993 5.192  101
22.042 1.00 10
Cyfluthrin 1–250 0.9989 6.111  101
20.900 25.00 50
Cyhalothrin 1–250 0.9983 1.787  101
34.421 1.00 10
Cypermethrin 1–250 0.9979 1.624  102 66.712 1.00 10
Deltamethrin 1–250 0.9967 3.132  101
80.828 1.00 10
Diazinon 1–250 0.9993 3.690  101
41.066 1.00 10
Dicofol 1–250 0.9994 3.066  101
8.515 1.00 10
Dieldrin 1–250 0.9995 7.589  101 1.747 1.00 10
Diniconazole 1–250 0.9994 3.687  101
35.041 10.00 20
Dinitramine 1–250 0.9947 1.833  101
21.468 1.00 10
Dioxabenzofos 1–250 0.9991 5.669  101
44.559 2.50 10
Disulfoton 1–250 0.9974 1.045  101
28.307 1.00 10
Ditalimfos 1–250 0.9995 5.673  101
12.512 2.50 10
EPN 1–250 0.9978 4.395  101
92.002 1.00 10
Ethion 1–250 0.9992 4.189  101
24.249 1.00 10
Ethoprophos 1–250 0.9992 3.900  101
30.891 1.00 10
Etrimfos 1–250 0.9992 3.884  101
50.426 1.00 10
Fenazaquin 1–250 0.9991 1.552  102
90.507 1.00 10
Fenitrothion 1–250 0.9970 2.394  101
62.374 1.00 10
Fenpropathrin 1–250 0.9991 3.311  101 20.992 1.00 10
Fenthion 1–250 0.9995 4.088  101
5.009 1.00 10
Fenvalerate 1–250 0.9988 1.798  102
327.376 1.00 10
Fipronil 1–250 0.9993 3.344  101
39.888 5.00 10
Flucythrinate 1–250 0.9988 7.909  101
155.402 1.00 10
Fluvalinate 1–250 0.9970 2.482  101
93.885 2.50 10
Fonofos 1–250 0.9993 6.261  101
60.808 1.00 10
Halfenprox 1–250 0.9981 1.788  101
44.842 1.00 10
Haloxyfop-methyl 1–250 0.9988 1.361  101
19.564 1.00 10
Heptachlor 1–250 0.9984 3.338  101
54.099 1.00 10
Heptachlor Epoxide 1–250 0.9992 1.506  101
22.763 2.50 10
Iprobenfos 1–250 0.9992 1.246  102
188.196 5.00 10
Iprodione 1–250 0.9979 8.718  101
25.826 1.00 10
Isazofos 1–250 0.9991 1.374  101
11.686 1.00 10
Isofenphos 1–250 0.9991 9.433  101
87.245 1.00 10
Kresoxim-methyl 1–250 0.9991 2.754  101
30.209 1.00 10
Malathion 1–250 0.9996 3.355  101
7.801 1.00 10
Methiocarb 1–250 0.9984 6.447  101
107.577 1.00 10
Metolachlor 1–250 0.9991 1.079  102
69.252 1.00 10
Metribuzin 1–250 0.9991 3.656  101
52.230 1.00 10
Mirex 1–250 0.9992 4.326  101
60.324 1.00 10
Oxadiazon 1–250 0.9994 8.834  101
25.943 1.00 10
p,p’-DDE 1–250 0.9994 8.146  101
58.117 1.00 10
Parathion 1–250 0.9958 3.104  101
129.426 1.00 10
Parathion-methyl 1–250 0.9965 4.896  101
156.463 2.50 10
Penconazole 1–250 0.9990 5.159  101
61.826 1.00 10
Pendimethalin 1–250 0.9955 2.450  101
94.184 1.00 10
Permethrin 1–250 0.9978 3.031  101 7.303 2.50 10
Phenthoate 1–250 0.9991 4.792  101
45.491 1.00 10
Phorate 1–250 0.9986 5.649  101
10.149 1.00 10
Pirimiphos-ethyl 1–250 0.9996 7.996  101
10.272 2.50 10
Pirimiphos-methyl 1–250 0.9993 2.535  101
26.639 1.00 10
Pretilachlor 1–250 0.9996 4.518  101 8.432 1.00 10
Procymidone 1–250 0.9994 4.311  101 16.199 1.00 10
Profenofos 1–250 0.9982 1.234  101
17.928 1.00 10
Propaphos 1–250 0.9992 5.775  101
51.683 1.00 10
Propoxur 1–250 0.9988 1.368  102
19.891 1.00 10
Prothiofos 1–250 0.9990 4.264  101
23.570 1.00 10

(continued on next page)

586 C.-C. Wu / Food Chemistry 229 (2017) 580–587

Table 2 (continued)

Pesticide Linear range (lg L
1) r2 Parameters of linearity LOD (lg L
1) LOQ (lg L
1)
Slope Intercept
Pyrazophos 1–250 0.9994 8.016  101
23.818 1.00 10
Pyridaben 1–250 0.9993 1.319  102
69.734 1.00 10
Pyriproxyfen 1–250 0.9993 6.638  101 28.629 1.00 10
Quinalphos 1–250 0.9991 1.251  102
153.969 2.50 10
Quizalofop-ethyl 1–250 0.9993 3.766  101
37.413 1.00 10
Tetradifon 1–250 0.9998 1.149  101
4.720 1.00 10
Tetramethrin 1–250 0.9988 1.009  102 40.456 2.50 10
Triazophos 1–250 0.9995 3.069  101 19.696 1.00 10
Triflumizole 1–250 0.9982 2.345  101
18.026 2.50 10
Trifluralin 1–250 0.9973 1.875  101
57.267 1.00 10
Vinclozolin 1–250 0.9997 7.502  101
5.501 2.50 10
a-BHC 1–250 0.9993 2.125  101
17.007 1.00 10
a-chlordane (cis) 1–250 0.9993 1.021  101
7.651 1.00 10
a-Endosulfan 1–250 0.9994 6.540  101
13.312 2.50 10
b-chlordane (trans) 1–250 0.9991 1.358  101
17.034 1.00 10
b-Endosulfan 1–250 0.9987 3.471  101
4.684 1.00 20

The limits of detection (LODs) and quantification (LOQs) in this procedure were defined as the concentrations of each of the pesticides in the tea samples (expressed as (lg
L
1) that gave signals of 3 and 10 times the noise, respectively.

Table 3 (continued)
Table 3
Recoveries of spiking at different concentrations of carbonated pesticides in the tea Pesticide Spiked level (lg L
1)
samples.
50 100
Pesticide Spiked level (lg L
1)
Heptachlor Epoxide 78.8 (13.9) 77.4 (3.6)
50 100 Iprobenfos 74.6 (16.1) 74.2 (8.7)
Iprodione 69.6 (12.7) 67.5 (14.8)
Alachlor 76.1 (16.8) 77.0 (6.5)
Isazofos 82.5 (17.3) 81.3 (7.6)
Benfluralin 68.3 (17.1) 63.6(0.6)
Isofenphos 85.5 (14.8) 85.6 (4.4)
Bifenox 111.3 (26.6) 129.4 (8.0)
Kresoxim-methyl 82.6 (15.9) 81.8 (7.7)
Bifenthrin 78.2 (7.4) 77.9(1.1)
Malathion 78.0 (17.9) 77.3 (6.1)
Bromophos methyl 67.2 (16.1) 66.7 (3.6)
Methiocarb 63.6 (15.3) 55.4 (9.1)
Bromophos-ethyl 75.6 (12.5) 77.6(0.5)
Metolachlor 78.5 (17.4) 79.4 (7.9)
Bromopropylate 81.6 (13.9) 81.8(3.4)
Metribuzin 64.2 (11.2) 62.5 (7.5)
Bupirimate 73.7 (13.9) 75.1 (8.1)
Mirex 57.8 (8.0) 53.7 (8.0)
Butachlor 84.4 (16.3) 83.4 (5.1)
Oxadiazon 86.2 (16.3) 87.7 (5.2)
Butralin 74.9 (7.6) 71.3 (5.0)
p,p’-DDE 71.5 (10.6) 70.6 (3.5)
Carbophenothion 78.7 (13.2) 79.5 (2.4)
Parathion 77.3 (12.2) 70.8 (6.6)
Chinomethionat 65.7 (16.7) 64.7 (4.6)
Parathion-methyl 63.5 (13.5) 57.7 (7.2)
Chlorfenapyr 86.8 (15.4) 85.7 (7.8)
Penconazole 64.9 (17.4) 63.4 (10.0)
Chlorpyrifos 76.4 (21.3) 79.9 (6.0)
Pendimethalin 74.1 (12.9) 71.3 (2.7)
Chlorpyriphos-methyl 72.7 (18.5) 71.6 (5.0)
Permethrin 76.3 (8.5) 75.8 (1.6)
Chlozolinate 80.6 (13.9) 81.8 (3.0)
Phenthoate 80.3 (14.1) 79.7 (6.4)
Cyanofenphos 80.0 (15.1) 81.4 (5.2)
Phorate 68.8 (21.2) 65.4 (2.7)
Cyfluthrin 134.5 (5.8) 82.6 (5.5)
Pirimiphos-ethyl 82.4 (8.8) 84.5 (4.5)
Cyhalothrin 78.8 (11.4) 79.6 (4.7)
Pirimiphos-methyl 80.9 (17.1) 82.8 (6.5)
Cypermethrin 72.8 (7.2) 74.1 (3.7)
Pretilachlor 77.7 (13.4) 81.8 (7.4)
Deltamethrin 70.1 (7.4) 66.2 (8.4)
Procymidone 81.6 (17.3) 83.0 (5.7)
Diazinon 64.7 (26.9) 60.6 (3.7)
Profenofos 72.0 (15.6) 69.0 (8.6)
Dicofol 71.7 (8.6) 75.0 (1.9)
Propaphos 70.2 (17.6) 70.0 (10.1)
Dieldrin 75.8 (14.8) 78.8 (3.9)
Propoxur 60.5 (14.0) 57.4 (6.7)
Diniconazole 66.9 (20.4) 66.8 (9.2)
Prothiofos 74.7 (12.5) 74.9 (1.4)
Dinitramine 95.8 (8.0) 82.6 (8.6)
Pyrazophos 69.6 (15.2) 71.9 (7.8)
Dioxabenzofos 69.4 (19.9) 66.4 (6.2)
Pyridaben 74.8 (13.6) 75.9 (5.2)
Disulfoton 65.7 (15.0) 65.1 (3.2)
Pyriproxyfen 80.4 (13.9) 80.9 (2.3)
Ditalimfos 73.09 (13.2) 74.6 (6.1)
Quinalphos 79.6 (16.8) 77.1 (5.3)
EPN 76.9 (10.6) 73.6 (6.5)
Quizalofop-ethyl 75.3 (16.9) 77.2 (7.2)
Ethion 83.1 (16.5) 82.8 (4.2)
Tetradifon 77.2 (13.5) 77.6 (5.6)
Ethoprophos 72.0 (21.3) 71.9 (6.5)
Tetramethrin 78.0 (16.2) 79.9 (6.3)
Etrimfos 79.8 (15.5) 78.5 (3.9)
Triazophos 67.4 (17.1) 67.6 (7.2)
Fenazaquin 66.6 (21.0) 73.9 (8.0)
Triflumizole 68.2 (20.2) 67.1 (8.9)
Fenitrothion 67.8 (13.3) 64.3 (5.1)
Trifluralin 68.5 (16.5) 64.5 (1.3)
Fenpropathrin 85.7 (14.8) 83.4 (4.6)
Vinclozolin 81.7 (16.6) 84.0 (5.8)
Fenthion 75.1 (15.8) 77.7 (4.6)
a-BHC 69.6 (20.5) 67.7 (3.6)
Fenvalerate 77.4 (11.7) 75.7 (3.3)
a-chlordane (cis) 72.1 (13.1) 71.5 (0.2)
Fipronil 84.4 (15.6) 79.3 (3.6)
a-Endosulfan 76.9 (10.2) 79.9 (5.3)
Flucythrinate 78.6 (13.1) 80.1 (4.5)
b-chlordane (trans) 73.0 (11.0) 71.1 (2.8)
Fluvalinate 77.6 (12.9) 75.0 (4.0)
b-Endosulfan 68.8 (14.2) 74.9 (9.0)
Fonofos 75.0 (18.0) 74.3 (3.1)
Halfenprox 71.4 (6.6) 70.0 (2.1) a
Recovery (%) = 100%  (Level of pesticides in spiked sample level of pesticides in
Haloxyfop-methyl 84.6 (18.9) 87.4 (2.7) blank sample)/Level of pesticides in spiked tea sample.
Heptachlor 57.8 (20.0) 58.2 (2.5) b
Relative standard deviation (RSD).
 C.-C. Wu / Food Chemistry 229 (2017) 580–587
guidelines for quality control in pesticide residue analysis
(SANCO/10232/2006, 2006), the recovery obtained during routine
analysis should be within 60–140%. From the results obtained,
the QuEChERS extraction method can be considered acceptable
for residue analysis in tea samples.
To confirm the accuracy of this method, we evaluated it exter-
nally by participating in the laboratories proficiency test (PT No.
19162) for pesticide residue analysis of tea organized by FAPAS
(http://fapas.com/proficiency-testing-schemes/fapas) in 2014. The
target compounds, including bifenthrin, chlorpyrifos, cyhalothrin,
p,p0 -DDE, covered by the proficiency test were properly identified
and the respective z-score values obtained were
0.7, 0.1,
0.2,

0.1,
1.5, respectively. The z-score values obtained were satisfac-
tory (|z| < 2), clearly showing good quantitative data were consis-
tently obtained by the proposed method.
4. Conclusions
The modified QuEChERS method has been evaluated for Oolong
tea sample preparation. We also optimized the operation parame-
ters and evaluated the performance characteristics of GC/MS/MS
for the analysis of 89 pesticides. From an analytical point of view,
we compared the tea sample pesticide residue extraction method
by modifying CNS 13570-2 liquid extraction method in our labora-
tory (Wu, 2003; Wu et al., 2009). Considering the use of reagents
and equipment, tea sample pesticide residues extraction method
by modifying CNS 13570-2 method has a high cost, requires
between 10 min to process a sample and require >200 mL amounts
of expensive organic solvents. The development of the modified
QuEChERS extraction method requires between 5 min processing
a sample and requiring less than 20 mL amounts of organic sol-
vents. Compared with traditional method the modified QuEChERS
method has proven to be fast, easy to conduct, and uses only small
quantities of reagents. It is therefore more environment-friendly
than the conventional methods. The results showed quite a good
analytical performance in terms of the repeatability, and recovery
achieved in the tea matrices. QuEChERS combined with GC/MS/MS
can be considered as a rapid and efficient monitoring technique for
screening multiple pesticides in tea. The proposed monitoring
technique provides the most effective approach to meet regulatory
needs.
Future work may include optimizing and validate the QuE-
ChERS extraction method followed by LC/MS/MS simultaneous
determination of polar, no-volatile and thermally unstable pesti-
cide residues in tea. Finding and confirming nontargeted pesticides
using GC/MS/MS and LC/quadrupole-time-of-flight (LC/Q-TOF/MS).
The application of the method for measuring pesticides in tea sam-
ples (Oolong tea, black tea, white tea and green tea) collected from
different places in Taiwan will also be planned. In addition, there
should be an investigation of the risk assessment of tea infusions.
Acknowledgment
This research was funded by a grant from the Council of Agri-
culture, Taiwan (project: 100AS-6.2.1-TS-T1). Analysis by Zheng-
Wai Lin, Chih Yu Lo, Ming Hua Tsai, Xu Fang Wang, Xu Jing Wang
and Yu-Ju Huang of Tea Research and Extension Station.
587
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