American Journal of Anal yt ical Chemistry, 2011, 2, 26-31
doi:10.4236/ajac.2011.228120 Published Online December 2011 (http://www.SciRP.org/journal/ajac)
Copyright © 2011 SciRes. AJAC
Development and Validation of QuEChERS Method for
Estimation of Propamocarb Residues in Tomato
(Lycopersicon esculentum Mill) and Soil
Sanjay Kumar Sahoo*, Raminderjit Singh Battu, Balwinder Singh
Pesticide Residue Analysis Laboratory, Department of Ent om ol o gy ,
Punjab Agricultural University, Ludhiana, India
E-mail: *sksahoo_2006@rediffmail.com
Received November 1, 2011; revised December 5, 2011; accepted Decem ber 16, 2011
Abstract
An easy, simple and efficient analytical method was standardized and validated for the estimation of residues
of propamocarb in tomato and soil. QuEChERS method included extraction of the sample with ethyl acetate
and cleanup by treatment with PSA and graphatised carbon. Final clear extracts of ethyl acetate were con-
centrated under vacuum to almost dryness and reconstituted into hexane. The residues of propamocarbwere
estimated using gas chromatograph-mass spectrometry (GC-MS). Propamocarb presented a distinct peak at
retention time of 8.962 min. Consistent recoveries of propamocarb ranging from 87 to 92 percent were ob-
served when samples were spiked at 0.10, 0.50 and 1.00 mg·kg–1 levels. The limit of quantification (LOQ) of
this method was determined to be 0.10 mg·kg–1.
Keywords: QuEChERS, Propamocarb, Tomato, Soil, GC-MS
1. Introduction
Propamocarb hydrochloride [propyl 3-(dimethylamino)
propylcarbamate hydrochoride] shown in Figure 1, a
systemic fungicide with protective action against phyco-
mycetous diseases (Pythium, Phytophthora spp.), is used
on a wide variety of mainly greenhouse vegetables [1].
Propamocarb was introduced into European markets for
control of oomycete pathogens in ornamental crops and
certain vegetables [2]. It has been widely used as a soil
drench against Phytophthora and Pythium diseases of
numerous crops [3-6]. More successfully, it has been used
to control potato late blight by Phytopthera infestans,
where metalaxyl-resistant populations presented cause a
serious problem [7,8]. Due to its lack of adverse effects
on beneficial microorganisms such as mycorrhizae and
Trichoderma species, propamocarb has been considered
as a good component of IPM programs [9,10].
Gas chromatographic determination of propamocarb in
agricultural products have appeared in the literature [11]
where the analyte was extracted with acetone-water and
cleaned up by liquid-liquid partition into diethyl ether
and analysed by GC. The object of this study has been
the development of a QuEChERS technique for fast, ac-
curate and direct determination of propamocarb in com-
mercial pesticides. The method can be used in the quan-
titative analysis of the analyte in different agricultural
products, also reducing as much as organic solvents ful-
filling the purposes to establish a wider acceptability of
the methodology.
2. Material and Methods
2.1. Standards and Reagents
The technical grade analytical standard of propamocarb
Figure 1. Structure of propamocarb.
S. K. SAHOO ET AL.27
(purity 96.8 %) was supplied by M/s Bayer CropScience
India Ltd., Mumbai, India and stored at –10˚C in a deep
freezer. Solvents like ethyl acetate and hexane were ob-
tained from E. Merck (India) Limited, Mumbai. Sodium
chloride was also obtained from E. Merck (India) Lim-
ited, Mumbai. Sodium sulfate anhydrous was from S. D.
Fine Chemicals, Mumbai. Activated anhydrous MgSO4
was also obtained from E. Merck (India) Limited, Mum-
bai. Primary Secondary Amine (PSA) Sorbent and gra-
phatised carbon decolorizing powder were obtained from
Sigma-Aldrich, Mumbai, India. All common solvents
were redistilled in all-glass apparatus before use. The
suitability of the solvents and other chemicals were en-
sured by running reagent blanks before actual analysis.
2.2. Apparatus
Estimation of propamocarb residues were carried out on
a GC (Shimadzu 2010) coupled with mass detector
(Fisons MD-800, quadrupole mass detector) equipped
with capillary column (Rtx-5 Sil MS, 30 m × 0.25 mm
i.d. × 0.25 µm film thickness). Rotary vacuum film
evaporator (Heidolph Labrota 4002) was supplied by
Heidolph, Germany was used for concentration of sam-
ple. A high speed homogenizer (Heidolph Silent Crusher
-M®) was used for homogenization of sample.
2.3. Standard Solution
A standard stock solution of propamocarb (1 mg/mL)
was prepared in hexane. The standard solutions required
for preparing a calibration curve (0.10, 0.25, 0.50, 1.00,
1.50 and 2.00 µg/mL) were prepared from stock solution
by serial dilutions with hexane. All standard solutions
were stored at –4˚C before use.
3. QuEChERS Sample Preparation
The tomato and soil samples were prepared by following
QuEChERS method for the determination of propamo-
carb residues shown in Figure 2.
3.1. Tomato
A sub sample of 15 g tomato was weighed into a 50 mL
polypropylene tube and added 30 mL ethyl acetate. The
sample was homogenized using high speed homogenizer
(Heidolph Silent Crusher-M®) for 2 - 3 min at 14 -
15,000 rpm. Anhydrous sodium chloride (NaCl) 10 ± 0.1
g was added to homogenized sample for phase separation.
The contents were centrifuged at 25 - 3,000 rpm for 3
min. An aliquot of 15 mL ethyl acetate layer was trans-
ferred over 10 ± 0.1 g sodium sulfate (Na2SO4) in a test
Substrate (tomato)
Chop the sample and blended thoroughly in a high speed blender.
Weigh 15± 0.1 g samples into each centrifuge tube (50 mL)
Dispense 30 ± 0.1 mL ethyl acetate to centrifuge tube (50 mL), cap
well and shake
Homogenise the sample @ 15,000 rpm for 2 - 3 min
Add 10 g of sodium chloride and shake vigorously and centrifuge for 3
minutes @ 2,500 rpm and decant the organic layer
Add 5 - 10 g of anhydrous sodium sulfate and shake well (1)
Weigh 0.15 ± 0.01 g PSA sorbent and 0.90 ± 0.01 g anhydrous
magnesium sulfate and 0.05 ± 0.01 g graphatised carbon into 15 mL
centrifuge tubes for 6 ml extract
Transfer 6 mL extract from (1) to the centrifuge tube, cap the tube well
and vortex for 30 sec
Centrifuge the tubes for 1 min @ 2,500 rpm
Transfer 4 mL extract to the test tube
Evaporate at 40˚C to almost dryness and reconstitute the volume with
distilled hexane
Estimate residues on GCMS for propamocarb
Figure 2. Flow chart of exteaction and cleanup methodology
for propamocarb.
tube. The ethyl acetate extract subjected to cleanup by
dispersive solid phase extraction (DSPE). An aliquot of 6
ml acetonitrile was taken in a test tube containing 0.15 ±
0.01 g PSA sorbent, 0.90 ± 0.01 g anhydrous MgSO4 and
0.05 ± 0.01 g graphatised carbon and the content was
thoroughly mixed on vortex shaker. Again centrifuged at
25 - 3,000 rpm for 1 min. 4 mL aliquot of this ethyl ace-
tate extract was evaporated to dryness using low volume
evaporator to dryness using low volume evaporator at
40˚C. Volume was made with distilled hexane.
3.2. Soil
A sub sample of 10 g soil was weighed into a 50 mL
centrifuge tube and water was added before the initial
extraction to get a total of 10mL water and added 20 mL
ethyl acetate. The sample was homogenized using high
speed homogenizer (Heidolph Silent Crusher-M®) for 2 -
3 min at 14 - 15,000 rpm. Anhydrous sodium chloride
(NaCl) 3 ± 0.1 g was added to homogenized sample for
phase separation. The contents were centrifuged at 25 -
3000 rpm for 3 min. An aliquot of 15 mL ethyl acetate
layer was transferred over 10 ± 0.1 g sodium sulfate
(Na2SO4) in a test tube. The ethyl acetate extract sub-
jected to cleanup by dispersive solid phase extraction
(DSPE). An aliquot of 6 ml acetonitrile was taken in a
test tube containing 0.15 ± 0.01 g PSA sorbent, 0.90 ±
Copyright © 2011 SciRes. AJAC
S. K. SAHOO ET AL.
28
0.01 g anhydrous MgSO4 and 0.05 ± 0.01 g and the con-
tent was thoroughly mixed on vortex shaker. Again cen-
trifuged at 25 - 3000 rpm for 1 min. 4 mL aliquot of this
ethyl acetate extract was evaporated to dryness using low
volume evaporator to dryness using low volume evapo-
rator at 40˚C. Volume was made with distilled hexane.
4. Estimation of Propamocarb Residues
The estimation of propamocarb residues was done by
GC-MS. Helium was used as a carrier gas with flow rate
of 1 ml–1. The injector temperature was maintained at
260˚C, Interface and ion source temperatures were
maintained at 220˚C and 200˚C, respectively. Injection
volume was 1µl in split less mode. Detector voltage was
maintained at 0.9 KV in SIM mode. The samples were
injected and confirmed on electron ionization (EI) mode.
The compounds were identified based on m/z ratio of
total ion chromatograph (TIC) and fragmentations of se-
lective ions monitoring (SIM) compared with fragmenta-
tions of different mass numbers obtained with standard
propamocarb. The compounds were identified both in
total scan and SIM mode based on m/z ratio. The sensi-
tivity of the instrument increased in SIM mode as the
most abundant fragment ions, characteristics of the ana-
lyte, were counted. The mass spectra of standard pro-
pamocarb showed the most abundant ions at m/z 72, 129,
143, 188 and base peak at 58. These ions values were
compared with the tomato and soil samples spiked with
propamocarb and tomato samples collected from treated
plots for quantitation and confirmation of propamocarb
residues.
5. Results and Discussions
5.1. GCMS Chromatograms of Propamocarb
GCMS detection has proven to be a good for propamo-
carb determination because no derivation step is needed.
Under the chosen conditions, propamocarb showed a
retention time of 8.962 min. The mass spectra of stan-
dard propamocarb showed the most abundant ions at
m/z 72, 129, 143, 188 and base peak at 58 shown in
Figure 3.
5.2. Validation of the Method
As the quantitative determination of propamocarb in to-
mato and soil is directly related to the evaluation and
interpretation of data, a reliable method is required which
is reproducible and can be applicable to different com-
modities. The method was fully validated according to
bio analytical method recommendations described in the
Food and Drug Administration (FDA) guidelines in
terms of selectivity, linearity, precision (repeatability),
precision (reproducibility), and accuracy for both detec-
tion systems [12,13].
5.3. Linearity
For most chromatographic procedures a linear relation is
observed between detector response (y) and analyte con-
centration (x). This can be expressed as a linear regres-
sion equation: y = a + bx. The parameters obtained by
the selected chromatographic conditions for propamo-
carb correspond to: y = 27005x + 11414, R2 = 0.999 was
shown in Figure 4. The linearity of a method is a meas-
ure of range within which the results are directly, or by a
well defined mathematical transformation, proportional
to the concentration of analyte in samples within a given
range [14].
5.4. Limit of Detection and Limit of Quantitation
The limit of detection (LOD) is the lowest concentration
of analyte detectable by an analytical method and is ex-
pressed in concentration units. The limit of quantification
(LOQ) is the lowest solute concentration that can be de-
termined with acceptable precision and accuracy, under
the stated experimental conditions. It is also expressed in
concentration units. The residues of propamocarb were
estimated by comparison of peak height of standard and
that of the unknown samples run under identical condi-
tions. Fairly good response i.e. about 10% of full scale
defection was observed by injecting 0.2 ng of propamo-
carb. When tomato sample (15 g) was processed and 4
mL aliquot was concentrated to a final volume 2 mL
from which 2 µL was injected (equivalent to 2 mg sam-
ple) into the instrument, there was no base line noise. In
case of soil samples (10 g), was processed and 4 mL ali-
quot was concentrated to a final volume 2 mL from
which 2 µL was injected (equivalent to 2 mg sample)
into the instrument, there was no base line noise. There-
fore, the limit of quantification (LOQ) was observed to
be 0.1 mg·kg–1 for both the substrates. The limit of de-
tection (LOD) was determined as the concentration hav-
ing peak area three times higher in relation to the noise
of the base line at the retention time of the peak of inter-
est. Therefore, the LOD was calculated to be 0.03 mg·kg–1
for propamocarb.
5.5. Precision
5.5.1. Precision (Repeatability)
Precision (repeatability) reflects the variation in results
hen repetitive analyses are made on the same condi- w
Copyright © 2011 SciRes. AJAC
S. K. SAHOO ET AL.
Copyright © 2011 SciRes. AJAC
29
(a)
(b)
Figure 3. GC-MS chromatograms of (a) Standard propamocarb and (b) Fortified tomato sample and its mass ions.
tions. The numerical value used is the relative standard
deviation for repeatability (RSDr). Repeatability of the
developed analysis method was determined by adding
propamocarb in different concentrations to blank sam-
ples. The within-batch recovery and repeatability (RSDr)
of spiked samples at the levels of 1.00, 0.50 and 0.10
mg·kg–1 for propamocarb are summarized in Table 1.
The precision (repeatability) in tomato range from 2.22%
S. K. SAHOO ET AL.
30
Figure 4. Calibration curve of different concentrations of
propamocarb.
Table 1. Recovery of propamocarb in tomato and soil (n =
6).
Propamocarb
Substrates
Level of
fortification
(mg·kg–1) Recovery % aSD bRSDr %
1.00 91.67 4.16 4.54
0.50 90.00 2.00 2.22
Tomato
0.10 87.33 2.52 2.89
1.00 92.35 2.57 2.78
0.50 86.67 2.08 2.40
Soil
0.10 86.67 1.15 1.33
aSD = Standard deviation; bRSDr = Relative standard deviation for repeat-
ability.
to 4.54% for propamocarb, in soil ranging from 1.3% to
2.78% for propamocarb. The results are fairly good for
the concentration levels investigated.
5.5.2. Precision (Reproducibility)
Precision (reproducibility) is the degree of agreement
obtained by the analysis of the same sample under vari-
ous test conditions. The usually numerical value used is
the relative standard deviation for reproducibility (RSDR).
The reproducibility of this analytical method was deter-
mined by analyzing spiked samples under various test
conditions (different analysts, different instruments and
different days). The between-batch recoveries and re-
producibility (RSDR) investigated at several levels are
given in Table 2. The RSDR values for propamocarb
residues in tomato and soil were very good and well
within 15% at all concentrations.
5.6. Accuracy
The accuracy of an analytical method is the agreement
between the true value of analyte in the sample and the
value obtained by analysis. Accuracy is usually ex-
Table 2. Recovery and RSD values obtained from analyses
of samples spiked with propamocarb.
Propamocarb
Sample Day
Recovery % aRSDr % bRSDR %
1 87.33 2.89
2 91.04 3.11
Tomato
3 86.29 2.65
7.88
1 86.67 1.33
2 88.72 3.64 Soil
3 93.12 2.37
9.87
aRSDr = Relative standard deviation for repeatability; bRSDR = Relative
standard deviation for reproducibility.
pressed as the recovery by the assay of known, added
amounts of analyte [14]. The recovery tests were carried
out on three replicates at each spike level. Results are the
average from three injections. The average recoveries
obtained for propamocarb at all concentrations and con-
ditions investigated (Tables 1 and 2) were determined as
87.85% and 87.52% in tomato and soil, respectively, for
propamocarb, which are very satisfactory.
6. Application of the Method to Real
Samples
Initially, the representative samples of tomato and soil
were extracted in acetonitrile, but the recoveries were
observed to be very poor. A representative 15 g sample
of tomato was spiked with propamocarb at 0.10, 0.50 and
1.00 mg·kg–1 level, extracted with ethyl acetate. The re-
sults were astonishing as the percent recoveries ranged
from 86.67 to 92.35. The same method was applied to
tomato and soil the results were found to be excellent.
The percent recoveries of propamocarb from different
vegetable substrates spiked at 0.10, 0.50 and 1.00 mg·kg–1
are reported in Table 1. Each value is the mean ± stan-
dard deviation of six replicate determinations. The re-
sults were encouraging and suggested that the method
could be extended to more substrates. Moreover, it is
simple, efficient, and easy to adopt in laboratories en-
gaged in pesticide residue analysis.
7. Conclusions
We used a highly sensitive, easy, less time consuming
and cost effective method for quantifying pesticide resi-
dues in tomato and soil samples. However, as the extrac-
tion was done with ethyl acetate, the method can be
suitable apply for quantifying propamocarb in other ag-
Copyright © 2011 SciRes. AJAC
S. K. SAHOO ET AL.
Copyright © 2011 SciRes. AJAC
31
ricultural products and also for other pesticides simulta-
neously as incase propamocarb is an active component in
the formulation mixtures of Infinto.
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