Advances in Materials Physics and Chemistry, 2011, 1, 1-5
doi:10.4236/ampc.2011.11001 Published Online June 2011 (http://www.SciRP.org/journal/ampc)
Copyright © 2011 SciRes. AMPC
Effectiveness of Selected Reaction Monitoring for rapid
assay of Cypermethrin Residue in Perilla Leaves
DATUM Solution Business Op erations, JEOL Ltd., Tokyo, Japan
Received May 20, 2011; revised June 14, 2011; accepted J u ne 25, 2011
Many kinds of pesticides have been developed and used to yield a good harvest but the residues in agricul-
tural products cause health problems. It is important to keep watch on these residues by using adequate
methods of analysis. Pretreatment such as gel permeation chromatography (GPC) or column chromatography
is often needed for the quantitative analysis of pesticide in agricultural products by conventional methods
such as gas chromatography/low resolving power mass spectrometry (GC/LRMS). However, these pretreat-
ments need a lot of work and take time. New methods saving the necessity of these pretreatments have been
desired. We have applied selected reaction monitoring (SRM) to quantitatively determine cypermethrin
residues in Perilla frutescens samples and compared the results with LRMS and HRMS in SIM mode. A
background peak caused by the matrix overlapped the cypermethrin peak in the analysis using LRMS. SRM
and HRMS in SIM mode provided chromatograms without matrix interference. The high selectivity of the
product ion (m/z 127) produced from precursor ion (m/z 163) isolated the target peaks from the matrix peaks
when using SRM. This method eliminates the pretreatment step, thus saving time and simplify ing the ana-
Keywords: Pesticide Residue, Cypermethrin, Perilla Frutescens, Selected Reaction Monitoring, high Re-
solving Power mass Spectrometry
Many kinds of pesticides have been used for the control
of harmful insects in agriculture. These have helped to
improve the productivity of crops, but these residues in
agricultural products have unfortunately posed a health
hazard to the consumers. As for mammalian neurotoxic-
ity, the serious damage to health by prolonged exposure
to cypermethrin was reported . Stricter consumer pro-
tection laws have been enacted for the control of pesti-
cides in Japan . The legal limit for cypermethrin in
Perilla frutescens leaves was set to be less than 6 g/ml.
This underscores the need for efficient analytical proce-
dures for pesticide residue detection in agricultural
products. Conventional methods are complicated and
time consuming, because the sample matrixes extracted
from agricultural products are usually very complex .
For example, methods using a gas chromatography/low
resolving power mass spectrometry (GC/LRMS) or a GC
with an electron capture detector (GC-ECD) need pre-
treatments such as gel permeation chromatography (GPC)
or column chromatography to remove matrix interfer-
ences [4-9]. A simpler method without the time consum-
ing clean-up and purification procedures is needed for
routine analysis. However, the method without pre-
treatment using a GC/LRMS suffers from lack of accu-
racy in quantitative analysis, because of the overlapping
of the matrix peaks.
In this study, high resolving power mass spectrometry
(HRMS) and selected reaction monitoring (SRM) were
applied. These methods can isolate target peaks from
matrix peaks saving pretreatments. Using these methods,
we analyzed cypermethrin added intentionally to the ex-
tracts of the leaves of Perilla frutescens and the results
were compared with that obtained by using LRMS.
2.1. Standards and Reagents
Cypermethrin (purity > 98%) was supplied from Dr.
2 N. NIIMURA
Ehrenstorfer GmbH (Germany). Acetone, n-hexane, an-
hydrous sodium sulfate and sodium chloride (pesticide
residue analysis grade) were supplied from Wako Pure
Chemical Industries (Japan).
The leaves of Perilla frutescens (20 g) were minced and
placed into a blender cup. Acetone (100 ml) was added into
the blender cup and these were homogenized at 10000 rpm
for 2 min. The homogenate was filtered through a filter
paper: No. 5A (Advantec Toyo, Japan). The residue was
once again homogenized with 100 ml of acetone at 10000
rpm for 2 min. The homogenate was filtered through No.
5A filter paper. The filtrate was concentrated to <30 ml by
using a rotary evaporator: RE 801 (Yamato Scientific, Ja-
pan) at 40℃ of bath temperature.
The sodium chloride aqueous solution (10%, 100 ml)
and n-hexane (100 ml) were added to the extract. The
mixture was shaken vigorously for 5 min and n-hexane
(100 ml) was added to the parted aqueous layer. Repeat-
ing this procedure, the mixture was extracted two times.
The extract was added with anhydrous sodium sulfate
(20 g) and concentrated to 5 ml.
The standard solution of cypermethrin (Mm 415.0742 u)
diluted with acetone at 100 g/ml was used to assign the
peaks in mass chromatogram (m/z 163). Aliquots of cy-
permethrin was added to the extracts of the leaves of
Perilla frutescens at 1 g/ml and measured using HRMS
2.4. GC/MS Condition
GC/MS was carried out with an Agilent 7890A gas
chromatograph (Agilent Technologies, USA) and a two-
sector mass spectrometer composed of a magnetic sector
and an electric sector: JMS-GCmateⅡ(JEOL, Japan). A
HP-5MS fused-silica capillary column (0.25 mm i.d. ×
30 m, Agilent Technologies, USA) was used for separa-
tion. Each sample (1 l) was injected into a GC injector
at 250℃ under splitless condition. The GC oven was
programmed at a constant temperature increase of 10℃
/min from 50℃ to 200℃ after holding for 1.5 min at 50
℃ followed by 5℃/min increase from 200℃ to 300℃.
Electron ionization with 70 eV of ionization energy and
210℃ of ion source temperature was adopted on all MS
analyses. LRMS in scan mode was carried out with a
mass resolving power of 500 and a scan range of m/z
50-500 to analyze the standard solution of cypermethrin
diluted with acetone (100 g/ml). HRMS in scan mode
was carried out with a mass resolving power of 3500 and
a scan range of m/z 140 - 185 to analyze the accurate
mass of the fragment ion m/z 163 detected as the base
peak in the LRMS. Perfluorokerosene (PFK) was simul-
taneously analyzed as an internal reference to correct a
mass drift. LRMS in selected ion monitoring (SIM)
mode was carried out monitoring m/z 163.0076 with a
mass resolving power of 500. HRMS applied SIM mode
was carried out monitoring m/z 163.0076 with a mass
resolving power of 3500. SRM was also performed with
the instrument, which is the BE geometry mass spec-
trometer, with the dissociation occurring in reaction re-
gion, prior to the magnetic sector [10,11]. The monitored
reaction was m/z 163 → 127, which was determined by
applying a linked scan MS at constant B/E of m/z 163 (B
and E are magnetic and electric sector field strength,
3. Results and Discussion
3.1. LRMS in scan Mode (Peak Assignment)
The chromatogram of the cypermethrin standard solution
(100 g/ml) obtained by GC/LRMS in scan mode is
shown in Figure 1. Four peaks (peak # 1-4) attributed to
the isomers of cypermethrin were detected at retention
times of 30.87, 31.06, 31.20 and 31.28 min. The ratios of
respective peak areas were 26.2, 29.1, 20.2 and 24.5 %.
The 4 isomers of cypermethrin present very similar mass
spectra and in Figure 2 is shown the mass spectrum of
peak #1 as an example. Figure 3 shows the assignment
of the molecular ion and the base peak ion into the mass
spectrum. The molecular ion of cypermethrin was de-
tected at m/z 415, the isotopic molecular ion attributed to
37Cl corresponds to m/z 417, the base peak [12C7
35Cl2]+was detected at m/z 163 and the isotopic fragment
35Cl37Cl]+ was detected at m/z 165 showing
about 64 % peak intensity of the base peak. Cyperme-
thrin isomers were reported to be detected as four peaks
. These previous reports support the results in this
3.2. HRMS in scan Mode
Using HLMS in scan mode, the accurately measured
mass of the fragment ion [12C7
as 163.0072 u. The calculated mass of the fragment ion
was 163.0076 u. The difference between the measured
mass and the calculated mass was 0.0004 u.
3.3. LRMS in SIM Mode
The chromatogram of perilla leaves extract spiked with
cypermethrin at 1 g/ml is shown in Figure 4. A back-
ground peak caused by the matrix overlapped with the
Copyright © 2011 SciRes. AMPC
Copyright © 2011 SciRes. AMPC
3.4. HRMS in SIM mode
cypermethrin peak #1. The two retention times were al-
most the same, so that it was difficult to separate these
two peaks modifying the GC condition. The peak area
ratios of peak #1-4 were 40.0, 22.7, 18.5 and 18.8%, re-
spectively in these conditions. The peak area ratios were
significantly different from those of the standard solution;
hence HRMS or SRM will be needed for reliable quanti-
The chromatogram of perilla leaves spiked with cyper-
methrin at 1 g/ml is shown in Figure 5. The peak area
ratios of peak #1-4 were 26.3, 29.0, 20.4 and 24.3% re-
spectively. These ratios were very close to the ones in the
standard solution shown in Figure 1. These results re-
vealed that the background peak caused by the matrix did
Figure 1. Chromatogram of the cypermethrin standard solution (100 g/ml) obtained by GC/LRMS in scan mode. 1. Cyper-
methrin-1; 2. Cypermethrin-2; 3. Cypermethrin-3; 4. Cypermethrin-4.
Figure 2. Mass spectrum of the peak # 1 shown in Figure 1.
Figure 3. Assignment of the molecular ion (m/z 415) and the base peak ion (m/z 163).
Figure 4. Chromatogram of perilla leav es extract spiked wi th cypermethr in at 1 µg/ml obtained by GC/LRMS in SIM mode.
4 N. NIIMURA
Figure 5. Chromatogram of perilla leav es extract spiked wi th cypermethr in at 1 µg/ml obtained by GC/HRMS in SIM mode.
Figure 6. Linked scan mass spectrum at constant B/E of m/z 163.
Figure 7. Chromatogram of perilla leaves extract spiked with cypermethrin at 1 g/ml analyzed by SRM.
not overlap with any peaks of cypermethrin. High selec-
tivity of the fragment ion (m/z 163.0076) obtained by
using HRMS in SIM mode isolated the target peaks from
the matrix peaks. This method thus gave reliable results
for quantitative analysis.
A linked scan MS at constant B/E was performed to de-
termine the efficient reaction for SRM. Figure 6 shows
the linked scan mass spectrum at constant B/E of m/z 163.
The precursor ion was detected at m/z 163 and the product
ion was detected at m/z 127. According to this result, we
decided the reaction for SRM as m/z 163 → 127. Figure 7
shows the chromatogram of perilla leaves extract spiked
with cypermethrin at 1 g/ml analyzed by SRM. The peak
area ratios of peak #1-4 were 26.0, 29.3, 20.5 and 24.2 %
respectively. These ratios were similar to the result of the
standard solution shown in Figure 1 and the HRMS in
SIM mode shown in Figure 5. The background peak
caused by the matrix did not overlap any peaks of cyper-
methrin in the same way as the result of HRMS in SIM
mode. The high selectivity of the product ion (m/z 127)
produced from precursor ion (m/z 163) isolated the target
peaks from the matrix peaks. This method thus gave reli-
able results for quantitative analysis.
The SRM process employed in this work led to the isola-
tion of cypermethrin peaks from the matrix without the
GPC or column chromatography pretreatments. The peak
area ratios of the cypermethrin mass chromatogram (m/z
163 → 127) are similar to those obtained for the standard
solution and HRMS in SIM mode. SRM may thus have
potential application for rapid assay of other pesticide
residues common in agricultural products. That is the
subject of our ongoing investigation.
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