Food Chemistry 162 (2014) 270276

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

Analytical Methods

Gas chromatography with ame photometric detection of 31

organophosphorus pesticide residues in Alpinia oxyphylla dried fruits
Xiangsheng Zhao a,b, Weijun Kong a, Jianhe Wei a,b, Meihua Yang a,b,
a
b

Institute of Medicinal Plant Development, Chinese Academy of Medicinal Science & Peking Union Medical College, Beijing 100193, China
Hainan Branch Institute of Medicinal Plant Development, Chinese Academy of Medicinal Sciences & Peking Union Medical College, Wanning 571533, China

a r t i c l e

i n f o

Article history:
Received 12 November 2013
Received in revised form 21 March 2014
Accepted 13 April 2014
Available online 24 April 2014
Keywords:
Organophosphorus pesticide
Alpinia oxyphylla
Multi-component determination
Gas chromatographyame photometric
detection
Gas chromatographytandem mass
spectrometry

a b s t r a c t
A simple, rapid and effective gas chromatographyame photometric detection method was established
for simultaneous multi-component determination of 31 organophosphorus pesticides (OPPs) residues in
Alpinia oxyphylla, which is widely consumed as a traditional medicine and food in China. Sample preparation was completed in a single step without any clean-up procedure. All pesticides expressed good
linear relationships between 0.004 and 1.0 lg/mL with correlation coefcients higher than 0.9973. The
method gave satisfactory recoveries for most pesticides. The limits of detection varied from 1 to 10 ng/
mL, and the limits of quantication (LOQs) were between 4 and 30 ng/mL. The proposed method was
successfully applied to 55 commercial samples purchased from ve different areas. Five pesticide residues were detected in four (7.27%) samples. The positive samples were conrmed by gas chromatography
with tandem mass spectrometry (GCMS/MS).
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Alpinia oxyphylla (Zingiberaceae) is a perennial herb that is
widely cultivated in southern China. The fruit of this herb is used
as a traditional Chinese medicine (Chinese Pharmacopeia
Commission, 2010), and is also widely consumed as a health food.
Many compounds, including sesquiterpenes, diterpenes, avonoids,
and diarylheptanoids, have been isolated from A. oxyphylla
(Morikawa, Matsuda, Toguchida, Ueda, & Yoshikawa, 2002;
Muraoka et al., 2001; Xie, Sun, Wang, & Ito, 2009; Xu, Tan, Zeng,
Han, & Peng, 2010; Xu et al., 2009). Some of these compounds
showed insecticidal activity (Miyazawa, Nakamura, & Ishikawa,
2000), signicant neuroprotective activity (Guan, Bao, Jiang, & An,
2006), and inhibition of nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages (Morikawa et al.,
2002; Muraoka et al., 2001). A. oxyphylla takes 23 years to reach
maturity, and during this period it can accumulate contaminants
from the environment or the pesticides directly applied. Therefore,
there is a risk of contamination from agricultural chemicals.
Organophosphorus pesticides (OPPs) are widely used in agriculture because they are inexpensive and effectively control crop pests
and diseases. The output of pesticides in China has appeared to
Corresponding author at: Institute of Medicinal Plant Development, Chinese
Academy of Medical Sciences & Peking Union Medical College, Beijing 100193,
China. Tel.: +86 10 57833277; fax: +86 10 62896288.
E-mail address: yangmeihua15@hotmail.com (M. Yang).
http://dx.doi.org/10.1016/j.foodchem.2014.04.060
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

increase steadily in recent years, and the amount of OPPs used

accounted for more than 70% of the total pesticides used between
1980 and 2000 (Jin, Wang, Shao, & Jin, 2010). While pesticides
increase agricultural production, bioaccumulation through the food
chain can eventually become a risk to mammals because the majority of those pesticides act as acetylcholinesterase inhibitors (Banks &
Lein, 2012). Public health concerns around improper use of pesticides and poisoning have increased in recent years. To date, some
countries, regions, and international organizations have established
maximum residue limits (MRLs) for foodstuffs and medicinal plant
materials. The Codex Alimentarius Commission of the United
Nations Food and Agriculture Organization and the World Health
Organization have established international standards for foods
and spices (European Pharmacopoeia, 2010; Kosalec, Cvek, &
Tomic, 2009; World Health Organization (WHO), 2007). The United
States Pharmacopoeia and European Pharmacopoeia (EP) have
established MRLs for medicinal plant materials (e.g., 0.1 mg/kg for
chlorpyrifos-methyl, 2.0 mg/kg for ethion) (Kosalec et al., 2009;
World Health Organization (WHO), 2007). However, to date, a
MRL has not been set for A. oxyphylla in China, and there are few
reports on chemical contamination of this plant. The amount of
pesticides residues in dried fruit is relative higher than that in fresh
fruit, and more easily detected (Athanasopoulos, Pappas, Kyriakidis,
& Thanos, 2005). There is only one report on organochlorine
pesticide residues in A. oxyphylla (Zhang, Lai, Fu, Liu, & Liu, 2004).
Therefore, for consumer health and safety, it is very important to

X. Zhao et al. / Food Chemistry 162 (2014) 270276

establish a fast, accurate and sensitive analytical method for the

analysis of multiple OPPs in A. oxyphylla dried fruits.
Gas chromatography (GC) method is commonly used for OPP
analysis and has been coupled with different detection methods,
such as ame photometric detection (FPD) (Wong et al., 2007;
Bidari, Ganjali, Norouzi, Hosseini, & Assadi, 2011; Wong et al.,
2007), nitrogenphosphorous detection (Garca de Llasera &
Reyes-Reyes, 2009), and mass spectrometry (MS) (Dai, Ren, He, &
Huo, 2011; Wong et al., 2007).
Prior to the analysis, the sample preparation a crucial step in the
analysis of trace compounds. Commonly used sample preparation
methods include liquidliquid extraction and solid-phase extraction, and these require large volumes of organic solvents. Analysis
for solid matrices requires several steps, such as extraction, isolation, clean-up, and enrichment. These steps can greatly inuence
the reliability of the method. Analytical technology developments
have led to new techniques with fewer steps, low-time requirements, and use of lower amounts of solvents. These methods
include matrix solid-phase dispersion (MSPD) (Garca de Llasera
& Reyes-Reyes, 2009; Rodrigues, Caldas, & Primel 2010), solidphase micro-extraction (SPME) (Cai et al., 2006; Bidari et al.,
2011), stir-bar sorptive extraction (SBSE) (Ochiai et al., 2005),
Quick, Effective, Cheap, Effective, Rugged and Safe method
(QuEChERS) (Anastassiades, Lehotay, Stajnbaher, & Schenck,
2003), liquid-phase microextractions (LPME) (Jeannot & Cantwell,
1996), dispersive liquidliquid microextraction (DLLME) (Ho,
Tsoi, & Leung, 2013). The main pitfalls of sorbent-based extraction
methods (MSPD, SPME, SBSE) are the lack of exibility, or the
unideal coating selectivity and reproducibility for complex matrices (Wei, Ge, & Lv, 2009). While the solvent-based extraction
methods, such as QuEChERS, LPME, DLLME, increased the amount
of organic solvents used and the sample-pretreatment procedure.
In the present study, a multi-residue GCFPD method was
developed for the analysis of 31 organophosphorus pesticides
(OPPs) residues in 55 commercial dry A. oxyphylla fruit samples
purchased from ve different regions in China (Hainan, Guangdong, Guangxi, Yunnan, and Beijing). This method was simple
and fast with using a low volume of organic solvent, and all the
sample preparation procedures were completed in one step. No
signicant interference was encountered in this matrix, and the
positive reports were further conrmed by gas chromatography
with tandem mass spectrometry (GCMS/MS). To the best of our
knowledge, this is the rst report of simultaneous determination
of OPPs in A. oxyphylla dried fruits.

2. Materials and methods

2.1. Materials and standards
All chemical reagents used were of residue analysis or HPLC
grade. Dichloromethane (DCM), n-hexane, toluene, and ethyl
acetate (EA) for residue analysis were purchased from Sinopharm
Chemical Reagent Co., Ltd (Beijing, China), while HPLC grade
acetone and acetonitrile (MeCN) were obtained from Sigma
Aldrich (St. Louis, MO, USA).
Opps (>98% purity), including methamidophos, trichlorfon,
dichlorvos, acephate, formothion, omethoate, monocrotophos, phorate, demeton, dimethoate, diazinon, disulfoton, phosphamidon,
parathion-methyl, chlorpyrifos-methyl, fenitrothion, malathion,
fenthion, parathion, chlorpyrifos, isocarbophos, isofenphos-methyl,
quinalphos, methidathion, ditalimfos, profenofos, ethion, triazophos, phosmet, azinphos-methyl, and phosalone, were purchased
from the Agriculture Environment Quality Supervision, Inspection
and Testing Center in Tianjin, China. The stock solution (100 mg/
kg) of each pesticide was prepared in acetone and stored at

271

20 C. The working solutions (0.0041.0 mg/kg) were prepared

by serial dilution of the stock solutions with acetone/ethyl acetate
(1:1, v/v) and stored in brown bottles at 20 C.
The dry, ripe A. oxyphylla fruits were purchased from pharmacies or markets in Hainan, Guangdong, Guangxi and Yunnan province, and Beijing city, China. These materials were identied by
Prof. Weiping Chen (Hainan Branch Institute of Medicinal Plant
Development, Chinese Academy of Medicinal Sciences, Wanning,
China). The obtained samples were stored at 20 C in a refrigerator and ground before analysis.
2.2. Apparatus
An Agilent 6890A GC (Agilent Technologies, Palo Alto, CA, USA)
was equipped with an Agilent 7683B autosampler and an FPD
operated in phosphorus mode. A DB-5 capillary column
(30.0 m  0.25 mm I. D., 0.25 lm lm thickness, Agilent Technologies, USA) was used. The injector and detector temperature were
both set at 250 C. The injection volume was 1.0 lL in splitless
mode. Nitrogen (P99.99%) was used as the carrier gas at 1.0 mL/
min and the make-up gas at 3.0 mL/min. Hydrogen and air were
used as the detector gases at 75.0 and 100.0 mL/min, respectively.
The column temperature program was as follows: the initial temperature was 60 C, held for 1 min; increased to 180 C at 20 C/
min, held for 10 min; increased to 200 C at 3 C/min, held for
10 min; then increased to 250 C at 5 C/min, held for 5 min. The
total run time was 49 min.
The GCMS/MS analysis was performed on an Agilent 7890A GC
(Agilent Technologies) coupled with an Agilent 7000A Triple Quad
mass detector (Agilent Technologies) operated in the electron
ionization mode and an Agilent 7693 autosampler. A 1.0 lL aliquot
of each sample was injected at an injector temperature of 260 C in
splitless mode, and separation was performed on an Agilent
DB-1701 column (30.0 m  0.25 mm I. D., 0.25 lm lm thickness,
Agilent Technologies, USA). The temperatures of the ion-source,
quadrupole, and transfer line were set at 250, 150 and 270 C,
respectively. Helium at a constant ow of 1.2 mL/min was used
as the carrier gas. The oven temperature program was as follows:
initial temperature, 80 C, held for 1 min; increased at 30 C/min
to 130 C; increased at 6 C/min to 210 C; and increased to
290 C at 10 C/min, held for 8 min. The total run time was 32 min.
2.3. Sample preparation
All the samples were crushed and passed through a 40-mesh
sieve. An aliquot of 0.5 g homogenized sample powder was accurately weighed and transferred into a 10-mL centrifuge tube for
analysis. Four milliliters of acetone/ethyl acetate (1: 1, v/v) were
added, followed by vortex mixing for 1 min and centrifugation
(1062.5g) for 5 min. The supernatant was transferred to a 25 mL
ask. The extract was concentrated to near dryness on a rotary vacuum evaporator (EYELA-N-1100D-WD, Tokyo, Japan) at 30 C. The
residue was re-dissolved in 1 mL of acetone/ethyl acetate (1:1, v/v)
and then ltered through a 0.22 lm PTFE membrane (Agela
Technology, Tianjin, China). The ltrate was transferred into an
autosampler vial for GCFPD analysis under the above-described
conditions.
3. Results and discussion
3.1. Optimization of sample preparation procedure
The inuence of extraction solvent, solvent volume and extraction time on the extraction efciency was rstly investigated.
GCFPD was used to optimize these extraction conditions. The

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X. Zhao et al. / Food Chemistry 162 (2014) 270276

Fig. 1. Effects of different organic solvents on the extraction efciency (n = 5). 1: methamidophos; 2: trichlorphon + dichlorovos; 3: acephate; 4: formothion; 5: omethoate; 6:
monocrotophos; 7: phorate; 8: demeton; 9: dimethoate; 10: diazinon; 11: disulfoton; 12: phosphamidon; 13: parathion-methyl + chloropyrifos-methyl; 14: fenitrothion; 15:
malathion; 16: fenthion; 17: parathion + chloropyrif-os; 18: isocarbophos; 19: isofenphos-methyl; 20: quinalphos; 21: methidathion; 22: ditalimfos; 23: profenfos; 24:
ethion; 25: triazophos; 26: phosmet; 27: azinphose-methyl; 28: phosalone.

blank samples (0.5 g) were spiked with pesticides at concentration

level of 0.1 mg/kg for the 31 OPPs standard solution before the
extraction procedure. Six organic solvents (4 mL), including
acetone, ethyl acetate, acetonitrile/dichloromethane (1:1, v/v),
toluene/acetonitrile (1:3, v/v), ethyl acetate/n-hexane (4:1, v/v)
and acetone/ethyl acetate (1:1, v/v), were evaluated. Fig. 1 showed
the recoveries obtained with the different solvents in the extraction of the 31 OPPs. The recoveries with acetonitrile/dichloromethane (1:1, v/v), toluene/acetonitrile (1:3, v/v) and acetone/ethyl
acetate (1:1, v/v) were high, while those with the other three solvents were low. However, toluene and acetonitrile are expensive
and also harmful to the operator and the environment. Therefore,
acetone/ethyl acetate (1:1, v/v) was optimized as the extraction
solvent.
The inuence of the volume of the extraction solvent was investigated using different volumes (1, 2 and 4 mL) of acetone/ethyl
acetate (1:1, v/v). The results showed that the extraction efciency
with 4 mL of the extraction solvent was the best among the solvent
volumes tested. As for the extraction time (1, 5 and 10 min), the
extraction efciency has no signicant increase with the time
above 1 min and the recoveries were satisfactory for most pesticide at three spiking levels in the method validation experiment.
Therefore, the optimized extraction procedures for subsequent
experiments were as follows: the extraction solvent was acetone/
ethyl acetate (1:1, v/v) with the volume of 4.0 mL and the extraction time of 1 min.

pesticides at concentration levels of 0.05, 0.1 and 0.5 mg/kg were

prepared. Here, the ME was studied by comparing the responses
of the working standard solutions in solvent alone and in the pesticide-free sample spiked with the same concentration of standards, and was calculated using the following formula (Li, Chen,
Fan, & Pang, 2012):

Matrix effectME;% A2  A1  100=A1

where A1 is the average area of the pesticide standard in pure
solvent and A2 is the average area of the pesticide standard in the
extracts of the pesticide-free samples. When the values of ME are
between 20% and 20%, it is considered to be a mild signal suppression or enhancement effect; and when the values are between 50%
and 20% or 20% and 50%, it is considered to be of medium effect;
while, when the values are below 50% or above 50%, it is considered to be a strong effect of signal suppression or enhancement
(Kmellr et al., 2008). In the present study, most of the values of
ME were between 20% and 20% except demeton (20.01%) and
phosalone (20.34%) at the levels of 0.5 mg/kg, suggesting that
the analytes may have a similar response in both matrices. The
results showed a mild ME, which meant that there was no need
to use analyte protectants or masking agents to minimize ME.
Hence, calibration standard in solvent can be used for quantication
of pesticide residue in A. oxyphylla without the need of matrixmatching.
3.3. Validation of the GC method

3.2. Matrix effect

Matrix effect (ME) is one of the main factors that affect the
accuracy of analytical method in pesticides analysis. In order to
estimate the inuence of matrix components on the detection
response, two sets of standard solutions containing the 31

3.3.1. Linearity
The linearity of the method was tested over the concentration
range of 0.0041.0 mg/kg by diluting stock standard solution
(100 mg/kg) in acetone/ethyl acetate (1:1, v/v). Calibration plots
were obtained by injecting solutions at each concentration level

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X. Zhao et al. / Food Chemistry 162 (2014) 270276

Table 1
Validation parameters of the GC-FPD method.
Pesticides

Methamidophos
Trichlorphon + dichlorovos
Acephate
Formothion
Omethoate
Monocrotophos
Phorate
Demeton
Dimethoate
Diazinon
Disulfoton
Phosphamidon
Parathion-methyl + chloropyrifos-methyl
Fenitrothion
Malathion
Fenthion
Parathion + chloropyrifos
Isocarbophos
Isofenphos-methyl
Quinalphos
Methidathion
Ditalimfos
Profenofos
Ethion
Triazophos
Phosmet
Azinphose-methyl
Phosalone

Slope

Intercept

Average

SD

Average

SD

3616.9
4454.2
1698.7
2416.1
2051.5
1872.1
3842.7
1790.8
3997.3
3898
3453.1
1720.8
5429.7
3339.1
2379.7
2815.8
5125.7
2825.8
2043.5
2945.1
2272.7
836.37
2673.2
9531
1706.5
746.55
499.78
596.1

14.94
26.94
13.59
14.70
17.51
7.79
89.48
4.83
98.11
140.20
114.78
7.35
230.38
29.16
20.18
7.84
24.09
44.66
32.68
10.34
44.18
12.66
11.32
221.70
29.17
14.57
1.61
2.24

7.6163
104
86.49
104.07
74.632
54.325
32.792
24.876
23.72
65.784
84.184
34.494
116.8
64.334
63.759
56.229
173.11
61.512
99.666
53.738
39.437
34.011
7.95
266.37
2.9518
1.8704
1.4797
10.966

2.36
20.09
30.40
17.27
36.04
11.57
56.00
7.64
12.20
8.74
22.60
6.86
51.64
9.73
29.47
10.30
41.41
9.42
15.51
8.49
10.54
12.83
1.32
116.51
3.03
1.61
0.74
4.52

three times (six points). The reference calibration curve was

constructed by plotting the mean peak area ratio versus analyte
concentration. (Y = aX + b, where X is the concentration, Y is the
mean peak area). The linear range, slope, intercept and R2 are listed
in Table 1. As shown in Table 1, satisfactory correlation coefcients
for the 31 pesticides were obtained ranging from 0.9973 to 0.9999.
3.3.2. Limit of detection (LOD) and quantication (LOQ)
The standard stock solutions were further diluted with acetone/
ethyl acetate (1:1, v/v) to provide a series of solutions with appropriate concentrations for analysis. The LOD and LOQ for each
pesticide, which were determined by analyzing a series of diluted
solutions until the signal-to-noise ratios were about 3 and 10,
respectively, were between 0.001 and 0.01 mg/kg and between
0.004 and 0.03 mg/kg (Table 1), respectively.
3.3.3. Precision
The precision of the proposed method was assessed by the
study of intra- and inter-day repeatability. The repeatability of
the method was evaluated by six replicates of the above sample
pretreatment in one day, and the relative standard deviation
(RSD) of six values was calculated to determine the intra-day precision. The inter-day precision of one sample extraction solution
was determined on three successive days. The RSD values for the
precision study were 0.424.52% (intra-day precision) and 1.85
8.77% (inter-day precision) for the 31 OPPs, showing good precision of the proposed method.
3.3.4. Recovery
To investigate the extraction recoveries, blank samples spiked
at three levels (0.05, 0.1 and 0.5 mg/kg) were extracted under the
optimized conditions. Each treatment was performed in triplicate,
and the results were shown in Table 2. The recoveries for the 31
pesticides were between 57.6% (profenofos) and 104.7%
(acephate), and the RSDs were between 0.7% (fenitrothion) and
13.7% (fenthion). The results demonstrated that the recovery rates

R2

Linear range (mg/kg)

LOD (mg/kg)

LOQ (mg/kg)

0.9999
0.9998
0.9987
0.9976
0.9973
0.9980
0.9999
0.9990
0.9997
0.9993
0.9995
0.9993
0.9988
0.9997
0.9995
0.9993
0.9993
0.9994
0.9988
0.9997
0.9980
0.9997
0.9988
0.9989
0.9997
0.9995
0.9995
0.9977

0.011
0.0041
0.0161
0.0051
0.0061
0.021
0.0061
0.0071
0.0181
0.0071
0.0181
0.031
0.031
0.011
0.0251
0.0151
0.0251
0.031
0.0151
0.021
0.0251
0.021
0.031
0.011
0.0151
0.0251
0.031
0.021

0.003
0.001
0.005
0.002
0.002
0.007
0.002
0.003
0.006
0.003
0.006
0.009
0.01
0.004
0.008
0.003
0.008
0.008
0.005
0.006
0.008
0.007
0.009
0.003
0.005
0.008
0.01
0.007

0.01
0.004
0.016
0.005
0.006
0.02
0.006
0.007
0.018
0.007
0.018
0.03
0.03
0.01
0.025
0.015
0.025
0.03
0.015
0.02
0.025
0.02
0.03
0.01
0.015
0.025
0.03
0.02

for the 31 pesticides were within the acceptable range (70110%),

except for omethoate, parathion-methyl, chlorpyrifos-methyl, isocarbophos, methidathion, profenofos and ethion.
3.3.5. Comparison of the proposed method with other methods for
OPPs analysis
To evaluate the analytical performance of the proposed method,
a comprehensive comparison of the proposed method with other
reported OPPs analytical methods using SPE (Mao, Wan, Yan,
Shen, & Wei, 2012; Wan, Mao, Yan, Shen, & Wu, 2010), ASE (Du
et al., 2012; Jia, Mao, Chen, Wang, & Ji, 2010), SFE (Zuin,
Yariwake, & Bicchi, 2003), DLLME (Ho, Tsoi, and Leung, 2013)
and modied QuEChERs (Chen et al., 2012) applied to the herbs
was studied. Table S1 listed the comparison of the volume of
organics, extraction time, LOQ, recovery and residue levels
required for the development method with other reported methods. The proposed method showed good repeatability, linear range
and recoveries for most pesticides. Compared with them, less
reagents and time consume was an outstanding advantage of the
presented method. The LOQs achieved from the developed method
were better than or comparable with the others when using the
FPD detector. In addition, the sample preparation process for the
proposed method is simple, cost-effective and easy. These results
revealed that the proposed method could be used for the pesticides
residues analysis in the fruit of A. oxyphylla.
3.4. Analysis of real samples
Optimal conditions for the determination of OPPs in A. oxyphylla
were investigated. Fig. 2 showed the GCFPD chromatograms of
the 31 pesticide standards. A total of 55 batches of samples
collected from ve different places were assayed for OPPs using
the proposed method. All the samples were run in triplicate and
the results were shown in Table 3. Positive samples were conrmed by GCMS/MS. Seven OPPs were detected in eight (14.5%)
of the samples. Among them, residues level in four samples

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X. Zhao et al. / Food Chemistry 162 (2014) 270276

Table 2
Recoveries and the relative standard deviations obtained after spiking A. Oxyphylla samples (n = 3) with 31 pesticides at three concentration levels.
Pesticides

Methamidophos
Trichlorphon + dichlorovos
Acephate
Formothion
Omethoate
Monocrotophos
Phorate
Demeton
Dimethoate
Diazinon
Disulfoton
Phosphamidon
Parathion-methyl + chloropyrifos-methyl
Fenitrothion
Malathion
Fenthion
Parathion + chloropyrifos
Isocarbophos
Isofenphos-methyl
Quinalphos
Methidathion
Ditalimfos
Profenofos
Ethion
Triazophos
Phosmet
Azinphose methy
Phosalone

Mean recovery RSD (%)

Average

0.05 (mg/kg)

0.1 (mg/kg)

0.5 (mg/kg)

99.6 1.6
108.3 5.2
111.0 2.8
100.6 1.8
71.1 1.6
101.4 10.1
88.5 5.4
92.8 5.4
100.3 8.3
102.3 12.2
71.8 7.0
73.2 2.9
70.1 3.6
87.6 11.4
87.7 4.4
100.5 9.8
98.1 13.2
72.7 19.3
112.4 3.0
89.2 6.9
71.7 3.7
105.8 6.7
66.3 2.6
62.7 5.1
89.4 3.0
95.6 3.5
87.4 3.8
85.7 2.2

108.7 6.1
100.5 5.6
94.4 2.5
96.5 6.6
68.2 7.6
99.9 6.3
80.3 8.9
83.1 7.2
89.0 6.0
90.4 4.7
89.0 3.7
79.5 3.3
65.2 6.4
87.9 5.9
88.7 8.7
80.2 8.9
77.7 1.5
69.1 7.1
97.8 7.8
85.4 5.5
69.7 3.3
94.4 4.0
52.7 6.2
69.9 4.3
85.3 1.9
93.2 3.5
90.4 4.7
86.7 3.7

105.8 8.3
87.9 2.9
108.8 5.9
95.2 1.4
65.0 6.1
96.4 6.1
86.7 7.0
80.8 6.8
86.7 6.0
92.4 2.5
93.5 1.2
77.5 10.0
73.5 6.0
86.8 6.9
82.6 7.1
79.6 2.1
82.4 4.1
64.2 8.1
98.8 9.8
83.1 3.7
64.7 1.8
87.7 12.7
53.8 7.4
63.3 5.8
90.3 11.1
90.9 9.7
87.9 2.7
73.8 8.6

104.7 4.4
98.9 10.4
104.7 8.6
97.4 2.9
68.1 4.5
99.2 2.6
85.2 5.1
85.6 7.4
92.0 7.9
95.0 6.7
84.8 13.5
76.7 4.2
69.6 6.0
87.4 0.7
86.3 3.8
86.8 13.7
86.1 10.7
68.7 6.2
103.0 7.9
85.9 3.6
68.7 5.2
96.0 9.5
57.6 13.1
65.3 6.1
88.3 3.1
93.2 2.5
88.6 1.8
82.1 8.7

Fig. 2. GCFPD chromatograms of blank sample (a) and standard solution (b). 1: methamidophos; 2 + 3: trichlorphon + dichlorovos; 4: acephate; 5: formothion; 6:
omethoate; 7: monocr-otophos; 8: phorate; 9: demeton; 10: dimethoate; 11: diazinon; 12: disulfoton; 13: phosphamidon; 14 + 15: parathion-methyl + chloropyrifosmethyl; 16: fenitrothion; 17: malathion; 18: fenthion; 19 + 20: parathion + chloropyrifos; 21: isocarbophos; 22: isofenphos-methyl; 23: quinalphos; 24: methidathion; 25:
ditali-mfos; 26: profenfos; 27: ethion; 28: triazophos; 29: phosmet; 30: azinphose-methyl; 31: phosalone.

exceeded the LOQs. Demeton and formothion were found in two of

the samples, with concentrations less than their LOQs. The
concentrations of ve OPPs (dimethoate, monocrotophos, phosphamidon, fenitrothion and omethoate) in four of the samples
were above the LOQ, with values ranging from 42.4 (dimethoate)
to 138.2 (fenitrothion) lg/kg. Previous studies have reported MRLs
for fenitrothion and formothion of 0.5 mg/kg in medicinal plant

materials (European Pharmacopoeia, 2010) and 0.02 mg/kg in food

(Ministry Ministry of Health & Welfare of Japan., 2005), respectively. In the present study, the residual levels of fenitrothion
(138.2 lg/kg) and formothion (<LOQ) in A. oxyphylla were much
lower than the MRLs.
Because of high toxicity, monocrotophos, demeton and phosphamidon has been banned for use in fruit, vegetables, tea, and

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X. Zhao et al. / Food Chemistry 162 (2014) 270276

Table 3
Pesticide residues found in the A. oxyphylla samples collected from different regions in China.
Sample no.

Collected place

Pesticides found

Content (lg/kg)

Sample no.

Collected place

Pesticides found

Content (lg/kg)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan

ND
Dimethoate
Monocrotophos
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Demeton, omethoate
ND
ND
ND
ND
ND
ND
ND
ND
Phosphamidon, fenitrothion

29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51

Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Guangdong
Guangdong
Guangdong
Guangdong
Guangdong
Guangdong
Guangxi
Guangxi
Yunnan
Yunnan
Beijing
Beijing
Beijing

ND
ND
ND
ND
ND
ND
ND
ND
Omethoate
ND
ND
ND
ND
ND
ND
Formothion
ND
Formothion
ND
ND
ND
ND
ND

ND
ND
ND
ND
ND
ND
ND
ND
63.2
ND
ND
ND
ND
ND
ND
<LOQ
ND
<LOQ
ND
ND
ND
ND
ND

24
25
26
27
28

Hainan
Hainan
Hainan
Hainan
Hainan

ND
ND
ND
ND
Demeton

ND
42.4
58.6
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
<LOQ
ND
ND
ND
ND
ND
ND
ND
ND
84.4
138.2
ND
ND
ND
ND
<LOQ

52
53
54
55

Beijing
Beijing
Beijing
Beijing

ND
ND
ND
ND

ND
ND
ND
ND

ND = not detected.

Table 4
The parameters for ve pesticides by GCMS/MS.
Pesticides

Omethoate
Monocrotophos
Dimethoate
Phosphamidon
Fenitrothion
a
b
c

RTa (min)

15.115
16.943
17.187
18.591
18.995

Qualier 1

Qualier 2

Qualier 3

Transitionb (m/z)

CEc (eV)

Transitionb (m/z)

CEc (eV)

Transitionb (m/z)

CEc (eV)

155.9 ? 110
192 ? 127
124.9 ? 79
226.9 ? 127
277 ? 260.1

5
10
10
5
5

155.9 ? 79
127 ? 109
92.9 ? 63
127 ? 109
125.1 ? 79

20
10
10
10
5

109.9 ? 79
127 ? 95
86.9 ? 46
127 ? 95
125.1 ? 47

15
15
15
15
15

RT: retention time.

Transition: precursor ion ? product ion.
CE: collision energy.

herbs according to the Notice of the Ministry of Agriculture of

China in 2002 (The The Notice of the Ministry of Agriculture of
China, 2002). However, these pesticides were detected in three
fruit samples (Nos. 3, 14 and 23). In addition, dimethoate and omethoate were detected individually in three samples (Nos. 2, 14 and
37), and their concentrations were lower than the MRLs (European
Pharmacopoeia, 2010). Furthermore, two samples (Nos. 14 and 23)
contained more than one pesticide residue. These results suggested
that good agriculture practices should be emphasized for Chinese
herbal medicines (State Food and Drug Administration, 2003),
and MRLs for OPPs in A. oxyphylla needed to be established
urgently to ensure product quality and safety.
The contaminated samples were mainly collected from Hainan
Island, which is located in a tropical zone at 18100 20100 N latitude and 108370 111030 E longitude. The climate is warm with
plentiful rain and high humidity. Therefore, during the growth
and storage processes, A. oxyphylla fruit may be more sensitive to
pests, diseases and mildew, and require more pesticide applications than they grew in drier regions. The contaminated A. oxyphylla fruit is a potential health risk for consumers. Therefore, it is
necessary to set up control measures and guidance for the proper
use of pesticides.

3.5. GCMS/MS conrmation

Tandem mass spectrometry is a powerful technique that has
excellent sensitivity and selectivity. Recent reports on identication and conrmation issues have mainly focused on MS techniques (Lehotay et al., 2008). To avoid false-positive results,
GCMS/MS was used to conrm the positive results detected in
the present study. The GCMS/MS parameters were optimized
with ve pesticide standards (omethoate, monocrotophos, dimethoate, phosphamidon and fenitrothion). The retention times, selective ions, and collision energies of each analyte were listed in
Table 4.
4. Conclusion
Fruit of A. oxyphylla is used as not only an important traditional
Chinese medicine, but also a health food. In the present study, A. oxyphylla fruit samples were pretreated in a single step to reduce the
time for samples preparation. Compared with other techniques,
low sample size and solvent were required. The optimized method
was successfully applied to 55 commercial samples, and OPPs were

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X. Zhao et al. / Food Chemistry 162 (2014) 270276

detected in four of these samples. This method can be used to determine OPPs in A. oxyphylla for consumer health and safety.
Acknowledgments
This work was supported by the Modernization of Traditional
Chinese Medicine of Hainan Province (Grant No. 2010ZY011),
Major Science and Technology Project of Hainan Province (Grant
No. ZDZX2013008), the Fundamental Research Funds for Central
Public Welfare Research Institutes (Grant No. 2011HNB04).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2014.
04.060.
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