Food Chemistry 172 (2015) 585595

Contents lists available at ScienceDirect

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

Analytical Methods

Development, validation and determination of multiclass pesticide

residues in cocoa beans using gas chromatography and liquid
chromatography tandem mass spectrometry
Badrul Hisyam Zainudin a,, Salsazali Salleh a, Rahmat Mohamed a, Ken Choy Yap b, Halimah Muhamad c
a
Analytical Services Laboratory, Chemistry and Technology Division, Malaysian Cocoa Board, Cocoa Innovative and Technology Centre, Lot 12621 Kawasan Perindustrian Nilai,
71800 Nilai, Negeri Sembilan, Malaysia
b
Advanced Chemistry Solutions, 43 Jalan Wangsa 1/2, Taman Wangsa Permai, 52200 Kuala Lumpur, Malaysia
c
Product Development and Advisory Services, Analytical and Quality Development Unit, Malaysian Palm Oil Board, No. 6 Persiaran Institusi, Bandar Baru Bangi, 43000
Kajang, Selangor, Malaysia

a r t i c l e

i n f o

Article history:
Received 28 May 2014
Received in revised form 18 September 2014
Accepted 21 September 2014
Available online 28 September 2014
Keywords:
Pesticide residues
LCMS/MS
GCMS/MS
Cocoa beans
Method validation

a b s t r a c t
An efcient and rapid method for the analysis of pesticide residues in cocoa beans using gas and liquid
chromatographytandem mass spectrometry was developed, validated and applied to imported and
domestic cocoa beans samples collected over 2 years from smallholders and Malaysian ports. The method
was based on solvent extraction method and covers 26 pesticides (insecticides, fungicides, and herbicides) of different chemical classes. The recoveries for all pesticides at 10 and 50 lg/kg were in the range
of 70120% with relative standard deviations of less than 20%. Good selectivity and sensitivity were
obtained with method limit of quantication of 10 lg/kg. The expanded uncertainty measurements were
in the range of 425%. Finally, the proposed method was successfully applied for the routine analysis of
pesticide residues in cocoa beans via a monitoring study where 10% of them was found positive for chlorpyrifos, ametryn and metalaxyl.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Currently, Malaysia is the largest cocoa processor in Asia and
ranks fth in the world with a volume of 293,000 tonnes in 2013
(International Cocoa Organization., 2014). Unfortunately, due to
the shortage of locally produced beans (2809 tonnes), Malaysia
had to import 311,608 tonnes of dried cocoa beans mainly from
Indonesia and Africa to meet the growing local grindings requirement (Malaysian Cocoa Board, 2014). Hence, food safety played
an important role in cocoa industry since the originality of the
agrochemicals used in the exporting countries is unknown.
The identication of pesticide residues in food with high fat
content such as cocoa beans is a difcult and challenging task since
the inherent complexity of the matrix could interfere in the determination and quantication of the targeted analyte of interests.
Among other constituents contained in cocoa beans are high
amount of fatty acids, fatty acid esters, phytosterols, tocopherols,
sugar, polyphenols, theobromine, and caffeine. It is well known
Corresponding author. Tel.: +60 6 7999593; fax: +60 6 7941910.
E-mail addresses: badrul@koko.gov.my (B.H. Zainudin), salsazali@koko.gov.my
(S. Salleh), rahmat@koko.gov.my (R. Mohamed), haruyapkc@gmail.com (K.C. Yap),
halimah@mpob.gov.my (H. Muhamad).
http://dx.doi.org/10.1016/j.foodchem.2014.09.123
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

that the main problem associated when dealing with these kinds
of matrices for analysis is that dirty extracts with even a small
amount of fats may disrupt the analytical column used in the
experiment and harm the analytical instrument ion sources and
detectors, and nally upsetting the correct analyte determination
through signal suppression and enhancement. Hence, the development of sensitive, selective and reproducible analytical method
and technique have always been a prerequisite for the achievement of high quality results in enforcement and monitoring programme (Pizzutti, de Kok, Hiemstra, Wickert, & Prestes, 2009).
While the number of publications on the determination of pesticide residues in vegetables, fruits and other foodstuffs were
extensive (van der Lee, van der Weg, Traag, & Mol, 2008), the number of papers dedicated to cocoa beans analysis is relatively limited
(Rodrguez, Permanyer, Grases, & Gonzlez, 1991; Hirahara et al.,
2005; Guan, Brewer, & Morgan, 2009; Frimpong et al., 2012a;
Paul, Lajide, Aiyesanmi, & Lacorte, 2012; Frimpong, Yeboah,
Fletcher, Pwamang, & Adomako, 2012b; Ademola & Gideon,
2012). Rodrguez et al. (1991) studied the identication and determination of some organophosphorus and organochlorine pesticides in cocoa beans by gas chromatography mass spectrometry
(GCMS) using Universal Trace Residue Extractor (UNITREX). In

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B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

another study, Hirahara et al. (2005) reported a validation of multiresidue screening method for the determination of 186 pesticides
in 11 agricultural products including cocoa beans using a combination of solvent extraction and solid phase extraction (SPE) clean-up
with mini column (SAX/PSA). Guan et al. (2009) published a new
approach to multiresidue pesticide determination in foods with
high fat content using disposable pipette extraction (DPX) and
determination with GCMS. Several papers also appeared on the
determination of organochlorine (Frimpong et al., 2012a; Paul
et al., 2012), synthetic pyrethroid (Frimpong et al., 2012b) and
organophosphorus (Ademola & Gideon, 2012) residues employing
a combination of solvent extraction and SPE clean-up.
Until now, there are very few studies on the method and level of
pesticide residues in cocoa beans. Most of the current methods discussed above involve high volume of extraction solvent, long sample preparation time, and required specialised materials or
instrumentation. Furthermore, some of the current methods are
focused only to certain classes of pesticides. Therefore, the need
for a fast, robust and efcient method for the determination of pesticide residues of different chemical classes in cocoa beans is evident. For this reason, the purpose of this work was to develop and
validate an efcient and rapid method for the analysis of different
classes of pesticide residues in cocoa beans. Finally, the optimised
method was then applied in a real sample monitoring programme
carried out on imported and domestic cocoa beans samples collected over two years from smallholders and Malaysian ports.
2. Material and methods
2.1. Reagents and materials
HPLC grade acetonitrile and reagent grade formic acid were
obtained from Merck (Darmstadt, Germany) while reagent grade
ammonium formate was obtained from SigmaAldrich (St. Louis,
USA). Water was puried through an Elga Purelab Option-Q system
(High Wycombe, UK). Two mL mini-centrifuge tube containing
150 mg MgSO4, 50 mg C18, and 50 mg primary secondary amine
(PSA) was purchased from Agilent Technologies (Palo Alto, USA).
Pesticide reference standards of all analytes were purchased
from Dr. Ehrenstorfer (Augsburg, Germany). Individual pesticide
stock solutions (1000 mg L 1) were prepared in acetonitrile and
kept at 20 C in the dark. Mixed intermediate standard solutions
(10 mg L 1 and 1 mg L 1) of multiple pesticides were prepared by
diluting an appropriate volume of each individual stock standard
solution in acetonitrile. All working solutions containing the target
pesticides were prepared freshly by dilutions of the intermediate
standard solution in acetonitrile and kept in scintillation vials at
4 C in the refrigerator.
2.2. Cocoa beans samples for fortication
Dried cocoa beans were obtained from Cocoa Research and
Development Centre, Jengka. The samples were used as blanks, fortied samples for recovery assays and matrix-matched standards
for calibration in the experiments. 10 g samples were weighed
and transferred into 50 mL screw cap centrifuge tubes and fortied
with 100 and 500 lL from the 1 mg L 1 intermediate standard
solution. The samples were then allowed to stand at room temperature until analysis to give nal spiking concentration levels of 10
and 50 lg/kg.
2.3. Extraction and clean-up procedure
Ten g of dried cocoa beans was weighed into a 50 mL screw cap
centrifuge tube. Then, 10 mL of acetonitrile was added and the
mixture was vigorously shaken manually for 1 min and another

1 min using vortex mixer. After that, the mixture was centrifuged
at 12,000 rpm for 5 min at 4 C. From this extract, 3 types of experiment were conducted to study the clean-up effect on the nal
extracts.
The rst experiment involves no clean-up step. One mL of the
supernatant was ltered through 0.2 lm PVDF lter into autosampler vial before analysis.
The second experiment involves hexane partitioning of the
extract. Two mL of the supernatant was taken out and mixed with
4 mL of n-hexane in a scintillation vial. The vial was vortex for 30 s
and aliquot of 1 mL acetonitrile layer was ltered through 0.2 lm
PVDF lter into autosampler vial before analysis.
The nal experiment consists of d-SPE clean-up using 150 mg
MgSO4, 50 mg C18 and 50 mg PSA as sorbents. One mL of the
supernatant was transferred into d-SPE tube. The tube was vortexed for 30 s. After centrifugation at 12,000 rpm for 5 min, an aliquot of 0.5 mL extract was ltered through 0.2 lm PVDF lter into
autosampler vial before analysis.
2.4. Instrumentation
2.4.1. Liquid chromatographytriple quadrupole mass spectrometry
analysis
LCMS/MS analysis was performed using a Perkin Elmer Flexar
FX-15 ultra-high performance liquid chromatography (UHPLC)
(Perkin Elmer, USA). It was equipped with a reversed-phase C18
analytical column of 50 mm  2.1 mm  1.9 lm particle size (Perkin Elmer, USA). The column oven temperature was set to 40 C
and the ow rate was 250 ll/min. Mobile phase A and B were
water and acetonitrile each containing 5 mM ammonium formate
and 0.1% formic acid respectively. The linear gradient programme
was set as follows: 10% B to 95% B from 05 min, followed by
2 min elution time before re-equilibration back to 10% B for 3 min.
The injection volume was 5 lL with a run time of 10 min. The
UHPLC was hyphenated to a triple quadrupole mass spectrometer
AB Sciex 3200 QTrap (Toronto, Canada) equipped with an electrospray ionisation interface set at positive mode. The interface heater
was held at the temperature of 550 C and an ion-spray (IS) voltage
of 5500 eV. The nebulising gas (GS1), heating gas (GS2) and curtain
gas pressures were set at 40, 40 and 10 psi, respectively during the
whole analysis. Puried nitrogen gas was used as collision and
spray gas. Analyst software version 1.5.2 was used for method
development, data acquisition and data processing.
2.4.2. Gas chromatographytriple quadrupole mass spectrometry
analysis
GCMS/MS analysis was performed using an Agilent 7890A GC
equipped with an Agilent 7693B autosampler and an Agilent 7000B
triple quadrupole mass spectrometry system (Agilent Technologies, Palo Alto, USA). HP-5MS 30 m  0.25 mm i.d.  0.25 lm lm
thickness was used for the chromatographic separation of the compounds. 1 lL injection volume was performed using a 7890A GC
multimode inlet system operated in a cold-splitless injection
mode. In this mode, injector temperature was ramped from 70 C
to 280 C at 900 C/min. He (99.999%) was used as carrier gas
and quenching gas at a ow rate of 1.2 mL/min (constant ow)
and 2.25 mL/min, respectively. Nitrogen (99.999%) was used as
the collision gas at a ow rate of 1.5 mL/min.
The initial oven temperature was 70 C, with an initial time of
2 min. The oven was heated to 150 C at 25 C/min, then to 200 C
at 3 C/min, followed by a nal ramp at 8 C/min to 280 C. The nal
temperature was held for 10 min and the total run time was
41.867 min. The mass spectrometer was operated in electron
impact ionisation (EI) mode. The temperatures of the transfer line,
ion source, quadrupole 1 and quadrupole 2 were 280 C, 300 C,

B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

180 C and 180 C respectively. Agilent MassHunter B.05.00 software was used for instrument control and data analysis.

2.5. Analytical method validation and performance criteria

Validation on the optimised analytical method for cocoa beans
was performed as described in Document No. SANCO/12495/
2011 (European Commission DG-SANCO., 2012). The method was
tested to assess for validation parameters and criteria in terms of
linearity, matrix effect, limit of quantication (LOQ), specicity,
accuracy, precision and robustness. The calibration curves were
plotted to obtain the linearity of the system at six calibration levels
ranging between 5 and 100 ng/mL. The reagent-only calibration
standards and matrix-matched calibration standards were used
to assess the matrix effects. The LOQ was set at the minimum concentration than can be quantied with acceptable accuracy and
precision. Specicity of the proposed method was assessed by analysing the response in both blank and control samples. The accuracy of the method was expressed in terms of average recoveries
of spiked blank matrix at 10 and 50 ng/g concentration levels. Precision of the method was represented as relative standard deviation (RSD%) of within-laboratory reproducibility analyses.
Robustness was assessed by making deliberate variations to the
method (duration of manual shaking, vortex shaking and centrifugation), and the subsequent effects on method performance (accuracy, precision) were investigated. Uncertainty measurement was
calculated individually for each pesticide following the guidance
of EURACHEM/CITAC Guide CG 4 (Ellison & Williams, 2012).

2.6. Real samples

Domestic cocoa beans samples for monitoring study were collected from local farmers, while imported beans were collected
from ports and comprised beans from Indonesia, Cameroon, Nigeria, Venezuela, Ghana, Ecuador and Papua New Guinea. A total of
132 samples were collected quarterly in 2012 and 2013 and stored
at 4 C until analysis. Each sample was analysed in duplicate and
adhered to the conrmation criteria as described in Document
No. SANCO/12495/2011 (European Commission DG-SANCO, 2012).

3. Results and discussion

3.1. Optimisation of LCMS/MS parameters
Initially, a Q1 scan of the mass spectra was recorded to select
the most abundant mass to charge ratio (m/z) ion using continuous
infusion of each pesticide directly into the MS using syringe pump
at a ow rate of 10 lL/min. In this study, the proton adduct [H+] of
the molecular ion was chosen as the precursor ion for all analytes.
Then, enhance product ion (EPI) scan was conducted to obtain the
product mass spectra of the precursor ion. The rst transition,
which corresponds to the most abundant product ion was used
for identication and quantication, while the second one for conrmation purpose. In order to obtain maximum sensitivity for the
identication and quantication of the analytes, manual optimisation of the declustering potential (DP), collision energy (CE),
entrance potential (EP) and collision exit potential (CXP) was performed for each analyte using 1 lg/mL solution of individual compounds in acetonitrile. Finally the presence of precursor and
product ions was investigated using the multiple reaction monitoring (MRM) experiments with a dwell time of 50 ms. The optimised
LCMS/MS parameters were summarised in Table 1.

587

3.2. Optimisation of GCMS/MS parameters

Optimisation of GCMS/MS was performed as previously
reported (Lozano et al., 2012). The MS/MS detection method was
optimised rstly with individual injections in full-scan mode of
each analyte at 1 lg/mL to obtain their retention times and to
select the optimal precursor ions. The most intense ion with the
highest m/z relationship was selected in most cases. In contrast
to LCMS/MS in which the proton adduct [H+] of the molecular
ion was chose as the precursor ion in all cases, the pseudo-molecular ion is hardly used as a precursor ion in GCMS/MS determination, due to the stronger ionisation occurring with electron impact.
Then, product ion scan was conducted with various collision energies, ranging from 10 to 40 V, to obtain the best product ions from
the selected precursor ions. The rst transition, which corresponds
to the most abundant product ion was used for quantication,
while the second one for identication purpose. An 8-time-segments MRM method was developed with a solvent delay of
7 min. Finally, once a segment was adjusted, dwell time was
increased for the less sensitive analytes by decreasing the dwell
time for analytes with enough signal in order to obtain sufcient
data points to perform acceptable and accurate quantication.
The optimised GCMS/MS parameters were summarised in
Table 2.
3.3. Optimisation of extraction and clean-up procedure
3.3.1. Effect of hexane partitioning
GC-ToF analysis of cocoa beans extracts revealed high amounts
of interfering co-extractives such as alkaloids, fatty acids esters,
phytosterols and tocopherols (data not shown). The total ion chromatogram showed a distinctly large quantity of interfering coextractives especially fatty acids such as propanoic, propenoic,
butanoic, octenoic, hexanoic, heptanoic and tetradecanoic acid. In
order to overcome this matrix interfering problem, the addition
of a very non-polar solvent such as hexane in the extraction step
has already been proved to be an efcient way to remove this kind
of compounds especially fatty acids and fatty acid esters in baby
food (Charlton & Jones, 2007) and later was successfully applied
to honeybees and pollens (Wiest et al., 2011). In another study,
liquidliquid extraction using hexane with the aid of 20% aqueous
sodium chloride (w/w) solution was able to signicantly reduce
the amount of matrix co-extracts in the clean-up step (Cajka
et al., 2012). Considering this effect, we also evaluated the possibility of purifying the crude acetonitrile extracts with the addition of
hexane to remove the matrix co-extracts. Unfortunately, the addition of hexane in the clean-up of the acetonitrile extracts did not
help much to the recoveries of some of the pesticides studied
(supplementary data). The differences in recoveries were
performed using t-test at 95% condence interval. Statistics
showed that signicant decrease in average recoveries (at 10 and
50 lg/kg) were observed when hexane partitioning was included
in the clean-up step. This can be explained by the higher tendency
of some of the pesticides to partition into the hexane layer. Since
the recoveries of the pesticides represent an important analytical
performance characteristics in the method validation, we
concluded that hexane partitioning was not very efcient in cocoa
beans matrices and hence rejected form the method development.
3.3.2. Effect of dispersive-SPE clean-up
In this study, the extract of the solvent extraction was subjected
to dispersive-SPE clean-up using 150 mg MgSO4, 50 mg C18 and
50 mg PSA as sorbents. Graphitised carbon black (GCB) was omitted from the dispersive-SPE since it is well known that GCB has
high afnity to planar pesticides (chlorothalonil). Anhydrous
MgSO4 was used to absorb micro quantities of water in the solvent

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B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

Table 1
LCMS/MS acquisition method parameters.
Analyte

Retention
time (min)

Q1 mass
(m/z)

Q3 mass
(m/z)

Declustering
potential DP (V)

Entrance
potential EP (V)

Collision
Energy CE (V)

Ametryn

4.36
4.36

228.0
228.0

186.0
96.0

36.0
36.0

10.0
10.0

25.0
35.0

7.0
4.0

Chlorpyrifos

5.64
5.64

350.0
350.0

198.0
97.0

21.0
21.0

10.0
10.0

25.0
41.0

7.0
6.0

Cinosulfuron

3.85
3.85

414.0
414.0

183.2
83.1

36.0
36.0

8.0
8.0

21.0
57.0

4.0
4.0

Cyproconazole

4.74
4.74

292.1
292.1

70.1
125.1

56.0
56.0

10.0
10.0

41.0
39.0

4.0
4.0

Difenoconazole

5.46
5.46

406.0
406.0

251.1
111.1

61.0
61.0

12.0
12.0

31.0
77.0

4.0
4.0

Dimethoate

3.11
3.11

230.0
230.0

199.0
125.0

16.0
11.0

10.0
10.0

13.0
29.0

7.0
6.0

Fluazifop-butyl

5.55
5.55

384.0
384.0

282.0
328.0

51.0
46.0

10.0
10.0

27.0
23.0

10.0
12.0

Isazofos

4.9
4.9

314.0
314.0

120.0
162.0

41.0
41.0

10.0
10.0

35.0
21.0

6.0
7.0

Isoprocarb

4.17
4.17

194.0
194.0

95.0
137.2

20.0
51.0

10.0
10.0

20.0
17.0

17.0
8.0

Metalaxyl

4.18
4.18

280.0
280.0

220.0
160.0

46.0
51.0

10.0
10.0

19.0
31.0

8.0
6.0

Oxadixyl

3.67
3.67

279.0
279.0

219.0
133.0

46.0
41.0

10.0
10.0

17.0
29.0

8.0
6.0

Propoxur

3.79
3.79

210.0
210.0

111.0
168.0

11.0
6.0

10.0
10.0

19.0
11.0

6.0
7.0

Quinalphos

5.04
5.04

299.0
299.0

147.0
163.0

31.0
21.0

10.0
10.0

29.0
29.0

6.0
7.0

Quizalofop-ethyl

5.41
5.41

373.0
373.0

299.0
271.0

71.0
76.0

10.0
10.0

25.0
33.0

10.0
10.0

Terbuthylazine

4.61
4.61

230.0
230.0

174.0
104.0

41.0
41.0

10.0
10.0

23.0
43.0

7.0
6.0

and by doing so, it will make the nal acetonitrile extracts less
polar and causing precipitation of certain polar matrix co-extracts.
PSA represents a weak ion exchanger which mainly removes sugars, fatty acids, organic acids, and some pigments, while C18 can
be used for the reduction of lipids and non-polar interferences
(Cajka et al., 2012). The recovery proles of the effect of dispersive-SPE clean-up were summarised in the bar chart as shown in
Fig. 1.
While the addition of C18 is quite benecial in reducing the
matrix interferences, the use of PSA could raise some concerns
especially to the recoveries of certain pesticides. Previous studies
reported the decreasing of recoveries of some pesticides that
attributed to the binding to PSA (Lozano et al., 2012;
Mayer-Helm, 2009; Muhamad, Zainudin, & Abu Bakar, 2012). In
this study, the most prominent effect of PSA was on the reduced
recovery rate (less than 20%) of cinosulfuron in LCMS/MS
compared to acetonitrile extract without clean-up. The possible
reason maybe because cinosulfuron contains acidic sulfonamide
group which may react with basic PSA sorbent that contains amino
groups. When PSA was omitted, the recoveries obtained improved
considerably. On the other hand, most of the LC amenable pesticides
studied gave acceptable recoveries and precision with or without
dispersive-SPE clean-up except for acephate and methidathion.
In contrary, GC amenable pesticides gave noticeable results
when dispersive-SPE were used in the clean-up step. From the
analysis, it was found that fatty acids (butanoic acid, decanoic acid,
heptanoic acid, hexanoic acid, and linoleic acid) and phytosterol
(stigmasterol) were reduced signicantly while alkaloids (caffeine
and theobromine) and tocopherol (c-tocopherol) did not affect

Collision cell exit

potential CXP (V)

much when dispersive-SPE was applied. As predicted from the

chromatograms (not shown), heavy interferences in the extracts
would result in the matrix enhancement effect of some pesticides.
Fig. 1(B) revealed that 25 out of 31 pesticides analysed using GC
MS/MS showed signal enhancement without the dispersive-SPE
clean-up and 13 of them gave recoveries of more than the acceptable value of 120%. This shows that the addition of PSA removed
some of the matrix compounds which caused signal enhancement
in GCMS/MS. In contrast, strong matrix suppression was observed
for ametryn when PSA was not present. Here, large matrix interferences at the analyte retention time is likely to be the cause, since
they can hinder the correct and accurate analyte integration and
quantitation.
Besides using the full scan chromatograms, clean-up efciency
of the method was also assessed through gravimetric measurements. In this study, the amount of co-extractives from the samples into the extracts were measured gravimetrically at each step
of acetonitrile extraction and dispersive-SPE clean-up. Vials of
the nal solution were weighed before the addition of extracts.
Then, each sample extracts obtained after acetonitrile extraction
and dispersive-SPE clean-up were dried via N-evaporator under a
hot water bath. The weight difference was recorded to estimate
the amount of co-extracted matrix in the initial and nal extracts.
From the results obtained, the amount of matrix co-extracted for
coca beans samples after acetonitrile extraction and dispersiveSPE clean-up was 3.3 0.6 mg/g (n = 3) and 1.3 0.3 mg/g (n = 3)
respectively, originated from 0.5 g of the sample. These values represented 0.3% and 0.1% of the sample mass. Therefore, solvent
extraction using acetonitrile was able to remove 99.7% of the

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B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

Table 2
GCMS/MS acquisition method parameters.
Analyte

Retention time (min)

Q1 mass (m/z)

Q3 mass (m/z)

Time segment

Collision Energy CE (V)

Dwell time (ms)

Acephate

8.10
8.10

136.0
142.0

94.0
96.0

10
5

10
10

Ametryn

17.90
17.90

227.0
227.0

58.1
170.1

10
10

5
5

Chlorothalonil

15.54
15.54

263.8
265.8

229.0
168.0

20
25

5
5

Chlorpyrifos

19.97
19.97

198.9
196.9

171.0
169.0

15
15

5
5

Cyuthrin (II)

32.23
32.23

162.9
162.9

90.9
127.0

15
5

4
4

Cyuthrin (III,IV)

32.38
32.38

162.9
162.9

90.9
127.0

15
5

4
4

k-Cyhalothrin

30.35
30.35

181.1
197.0

152.0
141.0

25
10

10
10

Cypermethrin

32.77
32.77

163.1
181.2

127.1
152.1

5
25

4
4

Cyproconazole

25.28
25.28

222.0
139.0

125.1
111.0

15
15

5
5

Deltamethrin

35.50
35.50

250.7
181.0

172.0
152.1

5
25

5
5

Difenoconazole I

34.77
34.77

322.8
324.8

264.8
266.8

15
15

5
5

Difenoconazole II

34.90
34.90

322.8
324.8

264.8
266.8

15
15

5
5

Dimethoate

13.39
13.39

86.9
92.9

46.0
63.0

15
10

20
20

Endosulphan I

23.22
23.22

241.0
207.0

206.0
172.0

15
15

5
5

Endosulphan II

25.53
25.53

207.0
241.0

172.0
206.0

15
15

5
5

Endosulphan sulphate

27.00
27.00

271.9
273.8

237.0
238.9

15
15

5
5

Fenvalerate I

34.02
34.02

167.0
181.0

125.1
152.1

5
20

5
5

Fenvalerate II

34.40
34.40

167.0
181.0

125.1
152.1

5
25

5
5

Fluazifop-butyl

25.64
25.64

281.9
281.9

91.0
238.0

20
20

5
5

Isazofos

15.79
15.79

161.0
161.0

119.1
146.0

5
5

5
5

Isoprocarb

9.69
9.69

121.0
136.0

77.1
121.1

20
10

10
10

Metalaxyl

18.09
18.09

234.0
234.0

146.1
174.1

20
10

5
5

Methidathion

22.90
22.90

144.9
144.9

85.0
58.1

5
15

5
5

Oxadixyl

26.17
26.17

163.0
132.0

132.1
117.1

5
15

5
5

Permethrin I

31.29
31.29

163.0
183.1

127.0
168.1

5
10

10
10

Permethrin II

31.47
31.47

162.9
182.9

127.1
168.1

5
10

10
10

Propoxur

10.98
10.98

110.0
152.0

63.0
110.0

25
10

10
10

Quinalphos

22.32
22.32

146.0
146.0

118.0
91.0

10
30

5
5

Quizalofop-ethyl

32.73
32.73

371.8
163.0

298.9
100.0

10
20

4
4

Terbuthylazine

14.53
14.53

228.9
214.0

173.1
71.0

5
20

10
10

Triadimenol

22.34
22.34

168.0
128.0

70.0
65.0

10
25

5
5

590

B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

Fig. 1. Comparison of percentage recoveries at 10 lg/kg (n = 20) spiking level between acetonitrile extracts and acetonitrile extracts + d-SPE clean-up using (A) LCMS/MS
and (B) GCMS/MS.

Table 3
Average recovery (%), RSD (%), method LOQ (lg/kg), measurement uncertainty (%) and MRLs obtained by acetonitrile extraction and d-SPE clean-up of cocoa beans samples, spiked at 10 and 50 lg/kg, and analysed by LCMS/MS and
GCMS/MS.
Analyte

MRLa (lg/kg)

LCMS/MS

10 lg/kg (n = 20)

50 lg/kg (n = 14)

Rec (%)

RSD (%)

Rec (%)

RSD (%)

106.7
91.7
101.1
105.0

106.1
104.3
107.0
117.1
96.6
118.7
110.6
108.5
105.9
84.0
89.0
97.3
113.4
109.0
98.9
102.8
101.1
95.6
109.4
98.5
98.3
95.6
107.3
105.5
104.3
92.3
105.8

12
4
12
7

9
11
10
5
3
8
10
3
7
14
9
7
7
11
3
4
6
5
6
4
11
6
8
6
3
2
6

103.1
86.4
119.3
94.0

94.6
113.6
104.5
104.7
92.2
106.2
100.5
99.2
101.9
83.2
83.9
92.4
107.8
104.8
94.5
98.8
95.2
90.4
103.5
93.7
88.3
86.5
101.9
98.3
95.8
87.1
101.4

18
6
6
6

17
12
7
4
3
11
2
2
5
9
6
8
6
3
5
3
8
5
5
5
7
4
13
6
4
3
7

LOQ (lg/kg)

10
10
10
10

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

Measurement
uncertainty (%)

10 lg/kg (n = 20)

50 lg/kg (n = 14)

Rec (%)

RSD (%)

Rec (%)

RSD (%)

18
5
17
12

21
16
10
16
5
19
8
6
8
23
11
8
13
12
5
6
7
6
8
7
9
7
10
7
6
4
7

50.0
80.1

88.5
102.1

96.0

93.5

82.9

84.2
80.6
81.6
90.3
83.5
86.1

85.4
95.9
78.4
85.8

107
7

20
10

7
7
6
7
29
11

9
10
14
7

41.9
82.2

84.0
95.1

89.6

92.7

79.4

83.0
81.0
82.0
87.3
84.1
84.5

79.2
86.8
80.9
82.5

52
5

7
7

5
7
4
4
33
5

14
5
5
4

LOQ (lg/kg)

Measurement
uncertainty (%)

n.q
10

10
10

10

10

10

10
10
10
10
n.q
10

10
10
10
10

n.q
10

25
10

13

7
12

8
9
9
11
n.q
14

18
12
21
11

200
200
50
50
100
100
100
100
50
100
50
100
100
100
100
100
100
50
50
100
50
100
200
100
1000

50
100
100
500
200

B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

Acephate
Ametryn
Chlorothalonil
Chlorpyrifos
Cinosulfuron
Cyuthrin (II)
Cyuthrin (III,IV)
k-Cyhalothrin
Cypermethrin
Cyproconazole
Deltamethrin
Difenoconazole I
Difenoconazole II
Dimethoate
Endosulphan I
Endosulphan II
Endosulphan Sulphate
Fenvalerate I
Fenvalerate II
Fluazifop-butyl
Isazofos
Isoprocarb
Metalaxyl
Methidathion
Oxadixyl
Permethrin I
Permethrin II
Propoxur
Quinalphos
Quizalofopethyl
Terbuthylazine
Triadimenol

GCMS/MS

n.q.: not qualifying for quantitation criteria.

a
Food Regulations (2010).

591

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B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

matrix co-extracted, while the additional dispersive-SPE clean-up

removed another 0.1%.
3.4. Method validation
GCMS/MS analysis with the clean-up step and LCMS/MS
analysis without the clean-up step were chose as the nal
method for validation study. Good linearity was achieved for
most of the pesticides studied using LCMS/MS and GCMS/MS
with correlation coefcients better than 0.990. However,
acephate and methidathion gave R values below 0.990. As previously mentioned in Section 3.3.2, both analytes gave rather low
signal intensity at lower concentration levels with high baseline
noise when using LCMS/MS. Fortunately, both pesticides gave
good linearity with R values of more than 0.990 when using
GCMS/MS. The observed effect of an increase or decrease in

detector response of pesticides in matrix extracts compared with

the same pesticides present in just pure solvent was studied in
order to assess matrix effect of cocoa beans extracts. In this
study, only minor matrix effects were observed for LCMS/MS
analysis. As for GCMS/MS, same good results were also achieved
after clean-up step with dispersive-SPE was introduced.
However, matrix effects are well known to be variable with time
and condition of instrument used. Therefore, quantication using
matrix-matched standards was opted rather than pure solvent
standards.
Table 3 displays the method performance and validation
parameters for acetonitrile extraction and dispersive-SPE cleanup of cocoa beans employing LCMS/MS and GCMS/MS. Limit of
quantication (LOQ) values were determined as the lowest concentration of the analyte that has been validated with acceptable
accuracy (70120%) and precision (RSD < 20%) by applying the

Fig. 2. LCMS/MS extracted ion chromatogram (34 MRM transitions) of (A) blank cocoa beans extract and (B) spiked cocoa beans at 10 lg/kg.

B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

complete analytical method. In this study, LOQ for all pesticides

studied were set at 10 lg/kg for both LCMS/MS and GCMS/MS
analysis since it was the lowest spiking level with acceptable accuracy and precision. This value is lower than national MRLs (Food
Regulations, 2010). Therefore, it can be concluded that both LC
MS/MS and GCMS/MS method are sensitive enough to quantify
all pesticides studied in cocoa beans.
Selectivity and specicity of the instruments are dened as the
ability of the extraction, the clean-up, the separation and the
detection method to discriminate between the analyte and other
compounds, and at the same time capable of providing signals that
effectively identify the analyte (European Commission DG-SANCO,
2012). In order to do that, the selectivity of the analytical method
in this work was determined by comparing the chromatograms of
a blank matrix solution with the spiked matrix solution (Figs. 2 and
3). Since multiple reaction monitoring (MRM) analysis was applied
in this study using LCMS/MS and GCMS/MS, we can see that the
analytes of interest were well separated from the other components present in the extracts and hence allowed the differentiation
and quantication of the analytes. In the meantime, the method
specicity was demonstrated using retention time matching and
ion ratios between the analyte in the sample extract and calibration standard. The tolerance for retention time is 0.5% for GC
and 2.5% for LC. Meanwhile, maximum permitted tolerance for
relative ion ratio is 20%. This showed that the analytical method
and subsequent detectors used are highly selective and highly
specic.
Duration of extraction that consists of shaking, vortex mix, and
centrifuge time may signicantly affect the efciency of extraction.
The effect of these parameters was checked with robustness/
ruggedness test and the optimised conditions should be kept
constant as far as possible. During the initial study, variations in
the extraction parameters previously mentioned generally had
little effect on the mean recoveries and RSDs. This showed that
the method was adequately robust to be successfully applied by
inexperienced technicians.

593

As demonstrated in Table 3, the recoveries of the majority of the

analytes at 10 and 50 lg/kg were ranged between 78.4102.1% for
LCMS/MS and 83.2119.3% for GCMS/MS with RSDs below 20%
for most cases. Since acephate gave unacceptable recovery values
and RSDs, it was deemed to be not quantiable and omitted from
the LCMS/MS analysis. The same thing goes to methidathion,
which obtained acceptable recoveries, but poor precision (>20%).
However, it is fascinating to know that both pesticides gave acceptable recoveries and RSDs when GCMS/MS was used. One would
think that acephate should give better results in LC, but we had
to resort to adding it to the list of analytes in GCMS/MS in overall
monitoring programme. The change in mobile phase or nal
extracts solution could rectify this problem in LC analysis though.
From the 26 pesticides studied, 25 can be quantied using GCMS/
MS, whereas LCMS/MS only managed to cover 15 pesticides. The
rest of the pesticides that can only be analysed using GCMS/MS
mostly belong to organochlorine and pyrethroid group. Pyrethroid
pesticides are known to pose difculties in multiresidue analysis
due to their highly non-polar nature and lower signal intensities
(Koesukwiwat, Lehotay, Miao, & Leepipatpiboon, 2010). The only
pesticide that cannot be analysed using GCMS/MS is cinosulfuron.
3.5. Estimation of measurement uncertainty
In the presented study, the bottom-up approach was used for
estimation of combined standard uncertainty. By using this
approach, it was found that uncertainty of extraction which comprises two components (i) repeatability of extraction and (ii)
uncertainty of extraction recovery, were shown to represent the
main source of combined standard uncertainty. On the other hand,
uncertainties associated with calibration (uncertainties of weighing or diluting standards, uncertainties of purity of standards) were
not so important. The relative expanded uncertainty was then calculated by using the coverage factor k = 2 at 95% condence level.
Combined standard uncertainties associated with the described
analytical method ranged for individual compounds from 7% to

Fig. 3. GCMS/MS extracted ion chromatogram (62 MRM transitions) of (A) blank cocoa beans extract and (B) spiked cocoa beans at 10 lg/kg.

594

B.H. Zainudin et al. / Food Chemistry 172 (2015) 585595

25% for LCMS/MS and 4% to 23% for GCMS/MS as shown in Table 3.

Additionally, a Students t test was used to determine whether the
mean recovery is signicantly different from 100%. From the results
obtained, it was found that the calculated t values were greater than
the critical value tcrit, hence recoveries obtained in the validation
data were signicantly different from 100%. In this case, it was recommended to include the recovery in the calculation of the results.
3.6. Real samples monitoring
The effectiveness of the developed method was applied to routine monitoring analysis of imported and domestic cocoa beans
samples collected from 2012 to 2013 from smallholders and
Malaysian ports. 132 dried cocoa beans samples were analysed
using GCMS/MS and conrmed using LCMS/MS method. Among
those samples were from local smallholders (Malaysia), Indonesia,
Cameroon, Nigeria, Venezuela, Ghana, Ecuador and Papua New
Guinea. During the monitoring programme, most of the residue
levels detected are within the 110 lg/kg. The results are consistent with the study conducted by Pizzutti et al. (2009), where this
could be explained by the fact samples were mostly taken from big
shipments, usually representing large, mixed lots or consignments.
Overall, there were 3 different pesticides found in cocoa beans with
concentration of more than 10 lg/kg. Chlorpyrifos, ametryn and
metalaxyl were detected in 14 positive samples, ranged from 10
to 200 lg/kg. The most prevalent compound was chlorpyrifos,
which was found in 9 samples. It was found that some samples
contained residues around MRL (50 lg/kg) and two samples
exceeded the MRL, with concentrations of 149 and 200 lg/kg. In
Japan, several consignments of cocoa beans have been denied entry
into the country since the new legislation on MRLs came into effect
in May 2006 by the Japanese Ministry of Health, Labour and Welfare (MHLW) (Ministry of Health, Labour and Welfare, http://
www.mhlw.go.jp/english/topics/foodsafety/positivelist060228/
introduc-tion.html). According to the progress report from the
International Cocoa Organization (ICCO) regarding on the action
programme of pesticide residues, the most notable active ingredients detected included pirimiphos-methyl, chlorpyrifos and
2,4-D (ICCO Progress Report, http://www.icco.org/about-us/international-cocoa-agreements/cat_view/30-related-documents/34-pestsand-diseases.html). Data form Chocolate and Cocoa Association of
Japan (CCAJ) showed that from 2006 to 2010, 51 cases of chlorpyrifos residues violation occurred in Japan for imported cocoa beans
with minimum and maximum concentration of 60 lg/kg and
1760 lg/kg, respectively (Kaminaga, 2011). On the other hand, in
this study, residues detected for ametryn and metalaxyl were well
below the national MRLs. From the monitoring results, it can be
concluded that in order to reduce consumers risk to pesticide residues in Malaysia, frequent monitoring programme should be
undertaken to cocoa beans samples especially for imported beans
since the originality of the agrochemicals used in the exporting
countries is unknown.
4. Conclusions
According to Malaysian Food Act and Regulations (Food
Regulations, 2010), there are a total of 34 pesticides allowed to
be use in cocoa plantation with national MRLs regulated. Out of
these 34 pesticides, 26 pesticides were covered in this study. The
rest of the pesticides were either belong to difcult pesticides
(dithiocarbamate, fosetyl ammonium, MSMA) or highly polar pesticides (glufosinate, glyphosate, paraquat), hence require specic
single residue method (SRM). Reliable, efcient and rapid sample
extraction and clean-up technique based on acetonitrile extraction
and dispersive-SPE was successfully developed and validated to

simultaneously analyse 26 pesticides of different chemical classes

in cocoa beans. The diversity of the selected pesticides highlights
the need of applying both gas and liquid chromatography hyphenated to triple quadrupole mass spectrometry for consistent and
reliable monitoring programme. The analytical method gave satisfactory recoveries with good precision for most of the pesticides
studied. However, there are some problematic pesticides such as
acephate and methidathion, which gave poor recoveries and precision using LCMS/MS. Nevertheless, the problem was resolved by
using GCMS/MS instead. Therefore, a clear conclusion can be
made that both LC amenable and GC amenable pesticides react differently to dispersive-SPE clean-up. In LCMS/MS, matrix interferences were not prominent and the addition of PSA in the clean-up
step would result in the loss of cinosulfuron. Meanwhile, PSA
played a critical role in GCMS/MS especially in terms of matrix
effects. According to Lehotay et al. (2010), GCMS matrix effects
were more dependent on the condition of the instrument than
on the method or matrix. They also stated that a combination of
matrix enhancement for pesticides susceptible to degradation on
active sites occurs in GC at the same time as matrix diminishment
effects due to build-up of non-volatile materials in the inlet. For
this reason, dispersive-SPE clean-up is imperative in GCMS/MS
analysis, while acetonitrile extraction without dispersive-SPE
clean-up is the method of choice for LCMS/MS analysis in this
study. The method was applied to 132 cocoa beans samples via a
monitoring programme from 2012 to 2013 and 10% of them was
found positive for chlorpyrifos, ametryn and metalaxyl.
Acknowledgements
The authors would like to thank the Malaysian Cocoa Board
(MCB) for nancially supporting this work and the Director General of the MCB for permission to publish this paper. The authors
are also highly indebted to the Regulatory and Quality Control
Division for providing the monitoring samples for analysis.
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.
09.123.
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