192 Merdassa et al.: Journal of AOAC International Vol. 98, No.
1, 2015
RESIDUES AND TRACE ELEMENTS
Development and Validation of One-Step Ultrasound-Assisted
Extraction for Simultaneous Determination of Multiclass
Fungicides in Soils
Yared Merdassa
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese
Academy of Sciences, PO Box 2871, Beijing 100085, China; and Addis Ababa University, Department of Chemistry, PO Box
1176, Addis Ababa, Ethiopia
Jing-fu Liu1
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese
Academy of Sciences, PO Box 2871, Beijing 100085, China
Negussie Megersa
Addis Ababa University, Department of Chemistry, PO Box 1176, Addis Ababa, Ethiopia
A rapid, efficient, and simple one-step ultrasound-
assisted extraction (UAE) method was developed
for the analysis of seven fungicides (cymoxanil,
metalaxyl, mandipropamid, folpet, chlorothalonil,
kresoxim-methyl, and famoxadone) in horticultural
soils. Analytes in the samples were determined
by HPLC with variable wavelength detection. Key
parameters that influence the UAE procedure
were optimized, such as the nature and volume of
extraction solvent, number of sonication steps, and
sonication time. The highest extraction efficiencies
in the range of 61.1−87.8% were obtained by using
only 7.5 mL of ethyl acetate–hexane (1 + 1, v/v) and
sonicating for 10 min. At 0.5 and 2.0 µg/g fortification
levels, satisfactory recoveries (>60%) with RSD <13%
were obtained for each analyte, except for folpet
(>52%). The method was linear over the range of
0.005 to 10 µg/g and the correlation coefficients (r2)
obtained ranged from 0.9955 to 0.9992. The LODs
(S/N = 3) varied from 0.0015 to 0.006 μg/g. The
proposed UAE procedure was compared to classical
extractions (shake-flask and Soxhlet extraction) and
showed satisfactory extraction efficiencies using
shorter time and smaller amounts of organic solvents,
thereby minimizing the costs of the analysis and the
disposal of waste solvent.
T
he use of pesticides is becoming indispensable in
agriculture to satisfy the food demands of the ever-growing
human population by way of increasing agricultural
outputs (1, 2). Fungicides are one of the various classes of
pesticides that are used for the protection of cereals, fruits, and
vegetables from fungal diseases (3). Only a small fraction of the
amount of pesticides applied reaches the target pest, and a larger
proportion may be found in the environmental compartments as
residues or transformation products, mainly in soil and water
bodies (3, 4). Soil is the principal reservoir of pesticides, thus
Received February 7, 2014. Accepted by AK March 27, 2014.
1
Corresponding author’s e-mail: jfliu@rcees.ac.cn
DOI: 10.5740/jaoacint.14-033
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representing the source from which residues can be released
to the atmosphere, groundwater, and living organisms (2). The
problem of pesticide contamination is currently of great social
and scientific concerns (5), and there is an increasing demand to
develop sensitive and selective methods for the determination of
different pesticides in soil.
Previously, quantitative extraction of pesticide residues from
complex soil matrixes was limited to the use of conventional
sample preparation methods such as Soxhlet extraction (6),
liquid–solid extraction (7), and solid-phase extraction (8), with
subsequent cleanup of the extracts. However, these methods
are generally time-consuming and usually use large amounts
of toxic organic solvents (3). To overcome these drawbacks,
alternative extraction approaches that reduce the use of toxic
solvents and long extraction requirements have been developed
and commercialized (2, 6). These methods include solid-phase
microextraction (1, 9, 10), supercritical fluid extraction (11, 12),
pressurized liquid extraction (13–17), microwave-assisted
extraction (18–23), and ultrasound-assisted extraction (UAE;
2–4, 24–36). A successful methodology based on UAE as an
efficient method of pesticide extraction from several solid
matrixes has been developed and reported by several authors
(2, 4, 26, 32, 33, 35).
The choice of analytical technique for detection of pesticides
is predominantly dependent on the chemical characteristics of
the analytes, mainly polarity. Pesticides with high log Kow are
nonpolar and are preferably analyzed by GC (2, 3, 6, 12, 21)
while polar pesticides are normally detected by LC (20, 31, 36).
For the quantification of fungicides in soil, UAE was successfully
combined with GC with electron capture detection (ECD) and
nitrogen phosphorus detection (NPD; 36) and LC-MS/MS (3).
To the best of our knowledge, UAE combined with HPLC with
variable wavelength detection (VWD) for the simultaneous
determination of multiclass fungicides in soil has not been
previously reported. Therefore, this research work was designed
to develop a simple, efficient, and low cost one-step UAE
procedure combined with HPLC-VWD that could be utilized
for quantitative and selective extraction of seven multiclass
fungicides from soil which have been used widely in Ethiopia
(37).
 Merdassa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 193

Table 1. Physicochemical properties of multiclass fungicides selected for study

Vapor pressure, Henry’s constant, logKowb, Solubility in water,
3
Fungicide Classa Structure mPa, 25°C Pa.m /mol, 20°C 25°C mg/L, 25°C

Cymoxanil Aliphatic H3C O N O 1.5 × 10−1c 3.8 × 10–5d 0.6c 1.0 × 103
nitrogen C C O

N C HN C

HN CH2 CH3

Metalaxyl Acylamino acid; H3C O O CH3 7.5 × 10−1 1.6 × 10–5 1.8 7.1 × 103c
anilide O C C CH2 O

CH N CH3

H3C

H3C

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Mandipropamid Amide O CH2 C CH
O CH3
9.4 × 10−4 1.2 × 10–21 3.2c 4.2
Cl CH

C
NH CH2 CH2 O
O
H2C C CH

Folpet Phthalimide O 2.1 × 10−2 7.8 × 10–3 3.0c 1.0c

N S Cl

Cl Cl
O

Chlorothalonil Aromatic C N
7.6 × 10−2 2.5 × 10–2e 2.9 8.1 × 10−1
Cl Cl

Cl C N

Cl

Kresoxim-methyl Methoxy­ O
CH3 2.3 × 10−3c 3.6 × 10–4 3.4 2.0c
iminoacetate O N
C
Strobilurin C H3C
H3C O

CH2 O

Famoxadone Dicarboximide; H3 C O
6.4 × 10−4c 4.6 × 10–3d 4.6 5.2 × 10−2c
oxazole O C C O

C N

O HN

a
http://www.alanwood.net/pesticides/class_fungicides.html.
 
b
Octanol–water partition coefficients.
 
c
20°C.
 
d
25°C.
 
e
pH = 7.
 

Experimental famoxadone (99.0%), and folpet (98.5%) were purchased from

Dr. Ehrenstorfer GmbH (Augsburg, Germany). Chlorothalonil
Instrumentation (99.7%) was obtained from AccuStandard, Inc. (New Haven,
CT). The structures and physicochemical properties of these
Chromatographic analysis was performed using an Agilent pesticides are listed in Table 1. Methanol, acetonitrile, acetone,
1200 HPLC system (Agilent Technologies, Santa Clara, CA) ethyl acetate, and hexane were HPLC grade solvents and were
equipped with quaternary pump, vacuum degasser, auto sampler, purchased from Fisher Scientific (a part of Thermo-Fisher
and variable wavelength detector. The analytes were separated Scientific, Waltham, MA). Other chemicals, such as sodium
on an Agilent TC-C18 column (250 × 4.6 mm id, particle size chloride (GR grade) and sodium sulfate anhydrous (AR grade),
5 µm). LC ChemStation Software (Rev. B.04.01) was used for were purchased from Sinopharm Chemical Reagent Co., Ltd
data acquisition and processing. (Shanghai, China). Reagent water was obtained from a MilliQ
water purification system (Millipore, Billerica, MA).
Chemicals and Reagents
Chromatographic Conditions
Analytical grade standards of cymoxanil (99.0%), metalaxyl
(99%), mandipropamid (99%), kresoxim-methyl (98.5%), The mobile phase delivery was performed at the flow rate
194 Merdassa et al.: Journal of AOAC International Vol. 98, No. 1, 2015
Figure 1. Effect of (A) nature of solvent and (B) number of
sonication steps on the UAE efficiency (n = 5). Error bar: RSD, %.
Abbreviations: ACE, acetone; EA, ethyl acetate; HEX, hexane; CMX,
cymoxanil; MTL, metalaxyl; MNP, mandipropamid; FLT, folpet; CLT,
chlorothalonil; KSM, kresoxim-methyl; FMX, famoxadone. Extraction
conditions: wt. of soil sample, 0.5 g; extraction solvent volume,
5 mL; sonication time, 15 min; and spiked concn level, 0.5 µg/g for
each fungicide.
of 1.0 mL/min in isocratic conditions. The mobile phase
composition was acetonitrile and water (60 + 40, v/v) and the
column temperature was maintained at 30°C. The detection
wavelength was programmed as follows: initially held at 232 nm
for cymoxanil; 220 nm (5 min) for metalaxyl; 229 nm (7 min) for
mandipropamid, folpet, and chlorothalonil; 225 nm (12 min) for
kresoxim-methyl; and finally at 229 nm (14 min) for famoxadone.
The injection volume was 20 µL, eluted for 18 min run time and
2 min post time. Peak area was used as an instrumental response
and comparison of the response. Under these chromatographic
conditions, baseline separation was maintained for all the target
analytes.
Preparation of Standard Solutions
A stock solution of each analyte (100 mg/L) was prepared by
dissolving 2.50 mg in a 25 mL volumetric flask with methanol.
All stock solutions were stable during the entire analysis and
stored in a refrigerator at 4°C when not in use. Working standard
solution (10 mg/L) was prepared by mixing 1 mL of the stock
solutions containing each fungicide in a 10 mL volumetric flask
and diluting to the final volume with methanol. A series of
calibration standards were prepared in methanol.
Sample Collection
Representative soil samples were collected from intensive
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Figure 2. Effect of (A) volume of solvent and (B) sonication time
on the UAE efficiency (n = 5). For error bar and abbreviations,
refer to Figure 1. Extraction conditions: wt. of soil sample, 0.5 g;
extraction solvent, ethyl acetate–hexane (1 + 1, v/v); and spiked
concn level, 0.5 µg/g for each fungicide.
horticulture areas around Teji river, Atsebela river, and Ziway
Lake in Ethiopia (21) to obtain a real environmental matrix for
method development and validation (36). The soil samples were
packed, air-dried, grounded, sieved, and stored following the
procedure in the previously published work (15). Preliminary
analysis was carried out, and none of the seven fungicides under
study was detected in the soil samples (35).
Preparation of a Fresh Spiked Soil Samples
Soil sample collected from Teji river area (T2) was used as
a blank soil sample throughout the optimization and validation
experiments (25). Freshly spiked soil samples at 0.5 µg/g
level (2) were prepared by adding 25 µL of 10 µg/mL standard
mixture of fungicides to 0.5 g of dry soil by using a 100 µL
microsyringe with a blunt needle (Shanghai Gaoge Industrial
and Trading Co., Ltd, Shanghai, China). Then, the samples
were allowed to dry after the solvent slowly evaporated at
room temperature. Finally, the fortified dry soil samples were
transferred to a 50 mL Erlenmeyer flask in which the ultrasonic
extractions were carried out (2).
UAE Procedure
UAE was performed in a KQ-600DE single-frequency
ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd,
Kunshan, China) at 80 kHz working frequency and 100 W
power. A 0.5 g portion of the soil sample and then 7.5 mL
of ethyl acetate–hexane (1 + 1, v/v) were placed in a 50 mL
Erlenmeyer flask. The contents of the flask were manually
 Merdassa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 195
Figure 3. Effect of (A) matrix moisture content and (B) salt addition
on the UAE efficiency (n = 5). For error bar and abbreviations,
refer to Figure 1. Extraction conditions: wt. of soil sample, 0.5 g;
extraction solvent, 7.5 mL ethyl acetate–hexane (1 + 1, v/v); and
spiked concn level, 0.5 µg/g for each fungicide.
shaken for 1 min and then exposed three times to UAE for
10 min each time (25). The internal volume of the ultrasonic
cleaning bath was 49 × 29 × 15 cm. The instrument temperature
was initially set at 30°C and did not exceed 45°C during any
experiment (31). After each extraction period, extracts were
collected in a vial containing 1.0 g of 400 mesh Cu powder and
manually shaken. The collected extracts were poured through a
funnel plugged with a GF/C glass microfiber filter (Whatman,
GE Healthcare Life Sciences, Maidstone, UK) overlaid by 2.0 g
anhydrous sodium sulfate, previously washed with 5 mL of
the same solvent. Finally, the Büchner funnel was thoroughly
rinsed three times (3 × 1 mL), with the extraction solvent. The
final combined extract (approximately 20 mL) was evaporated
to dryness under a gentle stream of nitrogen in a water bath
at 50°C, and no further cleanup was needed (2). The residues
were redissolved in 200 µL methanol, and 20 µL of the resulting
solution was injected into the HPLC-VWD system for analysis.
The amount of extracted fungicides was determined by HPLC,
and the mean recoveries were calculated (33).
Shake-Flask Extraction
A fortified soil sample (0.5 g, T2) at 0.5 µg/g level was
transferred into a 50 mL Erlenmeyer flask and 20 mL of ethyl
acetate–hexane (1 + 1, v/v) was added (26, 29, 32, 33). The
contents of the flask were shaken mechanically for 5 h using a
KS 501 digital shaker (IKA-Werke GmbH & Co. KG, Staufen,
Germany) at room temperature (25°C). The extracts were
filtered, evaporated to dryness, redissolved in methanol, and
injected into the HPLC-VWD system for analysis as described
above in UAE Procedure.
Soxhlet Extraction
A soil sample (0.5 g, T2) at 0.5 µg/g fortification level was
weighed into an extraction thimble and extracted for 5 h with
150 mL ethyl acetate–hexane (1 + 1, v/v) in an oil bath at 90°C
(26, 27, 32, 33). The extract was then filtered and concentrated
to about 5 mL using an IKARV10 rotary evaporator (IKA-Werke
GmbH & Co. KG) at 50°C under a pressure of 350 mbar at
100 rpm. Finally, the extracts were filtered, evaporated to dryness,
redissolved in methanol, and injected into the HPLC-VWD
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system for analysis as described above in UAE Procedure.
Cleanup of Extracts
Blank soil sample (T2, 0.5 g) was extracted using 7.5 mL ethyl
acetate–hexane (1 + 1, v/v) under optimum UAE conditions
followed by HPLC-VWD analysis. The chromatogram of the
blank sample was observed to be very clean, no interfering
compounds were noted in the extract, and therefore no further
cleanup was needed (24).
Results and Discussion
Optimization of UAE Conditions
In the present study, a simple UAE procedure was optimized
for quantitative enrichment of seven multiclass fungicides
in soil. The experiments were performed using soil samples
(T2) spiked with 0.5 μg/g of each analyte, and all results
were expressed as mean values of five replicates (n = 5;
24, 26, 31, 34). By changing the nature and volume of solvent,
number of sonication steps, and time of sonication (29, 33), the
UAE procedure was optimized, and extraction efficiency was
improved. Both solvent consumption and extraction time were
reduced (29).
Effect of nature of extraction solvent.—The nature of the
extraction solvent is a critical parameter in multiclass analysis
that can influence both the extraction efficiency and selectivity
in UAE (2, 24, 31). In the current study, solvent selection
was performed by evaluating the sonicating potential (24) of
acetone, ethyl acetate, acetone–hexane (1 + 1, v/v), and ethyl
acetate–hexane (1 + 1, v/v; 26). To this end, 0.5 g soil sample
(T2) at 0.5 µg/g spiked level was sonicated three times, for
15 min each time, using 5 mL of each solvent. Dichloromethane
was not tested in this study because of its toxicity (34).
The extraction solvent’s polarity should be compatible
with all the target analytes. Ethyl acetate has a vapor pressure
lower than acetone; as a result, the phenomenon of acoustic
cavitation in ethyl acetate is more violent (31). Hence, ethyl
acetate exhibited slightly better mean recoveries (49.1–82.1%)
than acetone (18.1–76.1%). Moreover, the findings of the
current work revealed that ethyl acetate had fewer co-extracted
compounds than acetone (31). To minimize the effect of soil
co-extractives on the determination of analytes, as well as to
improve recoveries (2), n-hexane was added to both solvents in
the ratio of 1 + 1, v/v. It was observed that the mean recoveries
for acetone–hexane (1 + 1, v/v) and ethyl acetate–hexane
196 Merdassa et al.: Journal of AOAC International Vol. 98, No. 1, 2015
Table 2. Analytical performance of the proposed UAE-HPLC-VWD method
Fungicide Linearity, µg/g y = ax ± ba Correlation coefficient, r²
Cymoxanil 0.005−10 y = 852.9x – 45.95
Metalaxyl 0.005−10 y = 220.4x – 13.52
Mandipropamid 0.02−10 y = 82.4x + 0.06
Folpet 0.01−10 y = 288.3x – 13.50
Chlorothalonil 0.005−10 y = 239.0x – 34.79
Kresoxim-methyl 0.02−10 y = 275.0x – 11.36
Famoxadone 0.005−10 y = 556.9x – 9.33
a
 a = Slope, b = intercept on the ordinate.
b
 
Numbers in brackets indicate the number of concentration points from which the calibration curves were prepared.
c
 
Repeatability at 0.5 µg/g spiked level.
d
Reproducibility at 0.5 µg/g spiked level.
 
(1 + 1 v/v) significantly improved and were in the range of
22.1–77.5% and 55–79.4%, respectively. The cleanest extracts
were obtained using ethyl acetate–hexane (1 + 1, v/v) with better
repeatability. Figure 1A summarizes the recovery results of the
seven fungicides obtained with different extraction solvents.
Information can be found in the literature regarding the
selection of the best extraction solvent for UAE of pesticides
(2, 4, 24–36). However, direct comparisons between the solvents
may be difficult because the nature of target compounds,
variation in the nature of matrixes, and experimental conditions
are different (24). In the present study, only partial comparisons
between metalaxyl and chlorothalonil could be made.
Accordingly, Sánchez-Brunete et al. (3) reported that ethyl
acetate was better than methanol for UAE of chlorothalonil
in soil. On the other hand, Chitescu et al. (36) indicated that
methanol–citric acid (0.2 M; 50 + 50, v/v) was a preferred
solvent mixture for the UAE of metalaxyl in soil. In general, the
degree of partitioning between the soil and the solvent depended
on the polarity of both the solvent and the analyte (35). These
observations are explained by the fact that larger recoveries were
obtained for the medium polar compounds when the solvents
of medium polarity were used. Therefore, ethyl acetate–hexane
(1 + 1, v/v) was the solvent mixture of choice as this mixture
also exhibited improved recoveries. Thus, extraction solvent of
this latter composition was selected for further experiments.
Effect of number of sonication steps.—The number of
sonication steps is another parameter that needs to be optimized
in UAE. In this procedure, 0.5 g soil sample with 0.5 µg/g
fortification level for each fungicide was extracted for 15 min
with 7.5 mL of ethyl acetate–hexane (1 + 1, v/v). Figure 1B
shows the effect of the number of sonication steps on the UAE
of the seven multiclass fungicides in the soil sample. The
results indicated that with up to three sonication steps, the mean
recoveries increased from 20.9–67.2% (1 step) to 55.0–79.4%
(3 steps) for all compounds. As the number of sonication steps
increased from one to three, the instrument temperature, initially
set at 30°C, gradually increased. Under elevated temperature,
the diffusivity of the solvent into the internal parts of the matrix
increased, which further resulted in enhanced desorption of
the components from active sites of the matrix. When four
sonication steps were conducted, the mean recoveries for
mandipropamid, kresoxim-methyl, and famoxadone increased
further (70.8–81.2%) while the remainder of the fungicides
Repeatabilityc Reproducibilityd
LOD, µg/g LOQ, µg/g RSD, % %,RSD
b
0.9986[10] 0.0015 0.005 4.4 6.1
0.9980[10] 0.0015 0.005 7.0 6.0
0.9980[10] 0.006 0.02 9.7 7.0
0.9992[10] 0.003 0.01 12 11
0.9955[10] 0.0015 0.005 7.6 8.3
0.9983[10] 0.006 0.02 3.2 2.7
0.9987[10] 0.0015 0.005 4.4 3.5
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decreased significantly (43.6–50.2%), probably due to the
degradation of the compounds (33). Therefore, three sonication
steps provided optimum extraction efficiency and were selected
for the further study. Direct comparisons between this study
and other similar works found in the literature could not be
made because no other extraction studies had a similar number
of multiclass fungicides nor did the fungicides studied have
similar basic physical and chemical properties, such as polarity.
Effect of volume of solvent.—The major purpose of this
experimental procedure was to determine the minimum solvent
volume required for optimum extraction efficiency (24) with the
lowest LODs (35). For this purpose, 0.5 g soil sample at 0.5 µg/g
spiked level was extracted using UAE. Three sonication steps
(15 min) used 2.5 mL (ratio of soil–solvent 1 + 5, w/v), 5 mL
(ratio of soil–solvent 1 + 10, w/v), 7.5 mL (ratio of soil–solvent
1 + 15, w/v), and 10 mL (ratio of soil–solvent 1 + 20, w/v) of
ethyl acetate–hexane (1 + 1, v/v). The results showed that the
mean recoveries of all the analytes increased significantly when
the volume of ethyl acetate–hexane (1 + 1, v/v) increased from
5 (55.0–79.4%) to 7.5 mL (50.4−86.1%), except for folpet and
chlorothalonil. Mean recoveries of these latter analytes slightly
decreased. For all the analytes, the mean recoveries decreased
when the volume increased from 7.5 to 10 mL (35.4–78.5%).
Moreover, when the volume decreased from 5 to 2.5 mL, the
mean recoveries decreased for all analytes (31.1–80.0%) and
significantly increased for mandipropamid and famoxadone.
Therefore, in this study, only 7.5 mL ethyl acetate–hexane
(1 + 1, v/v) was found to exhibit maximum efficiency and was
used for the UAE of target fungicides in soil. The effect of
changing the solvent volume can also be seen in Figure 2A.
For UAE of chlorothalonil in soil, Sánchez-Brunete et al. (3)
reported that 4 mL of ethyl acetate was optimum, while
Chitescu et al. (36) reported 15 mL methanol–citric acid (0.2 M;
50 + 50, v/v) was optimum for the UAE of metalaxyl in soil.
Effect of sonication time.—In this optimization procedure,
sonication time that yields the optimum extraction efficiency
was studied (24). For this reason, 0.5 g soil samples at 0.5 µg/g
fortification level was carried out at three sonication steps for
5, 10, 15, 20, and 25 min using 7.5 mL of ethyl acetate–hexane
(1 + 1, v/v). The results indicated that the mean recoveries
increased for all the analytes when the sonication time was
decreased from 15 (50.4–86.1%) to 10 min (61.1–87.8%),
except for cymoxanil, metalaxyl, and mandipropamid. Mean
Table 3. Application of the proposed UAE-HPLC-VWD method to dry field soil samples (n = 5)
Cymoxanil Metalaxyl Mandipropamid Folpet Chlorothalonil Kresoxim-methyl Famoxadone
c d
Soil Spike, Detected, R, RSD , Detected, R, RSD, Detected, R, RSD, Detected, R, RSD, Detected, R, RSD, Detected, R, RSD, Detected, R, RSD,
sample µg/g µg/g % % µg/g % % µg/g % % µg/g % % µg/g % % µg/g % % µg/g % %
a
T1 0 NDb — — ND — — ND — — ND — — ND — — ND — — ND — —
0.5 0.3 59.1 7.2 0.3 53.3 12 0.4 76.1 6.2 0.3 56.9 5.7 0.3 58.1 8.5 0.4 74.5 4.4 0.3 68.8 1.6
2.0 1.3 67.0 2.9 1.4 71.2 7.7 1.9 97.0 3.4 1.3 66.0 4.2 1.7 86.7 3.9 2.1 105 7.3 1.6 82.2 6.9
a
T2 0 ND — — ND — — ND — — ND — — ND — — ND — — ND — —
0.5 0.3 66.9 1.8 0.4 72.8 3.9 0.4 76.8 3.9 0.3 61.1 7.4 0.3 64.6 6.4 0.4 87.8 2.3 0.4 78.2 3.2
2.0 1.6 78.2 12 1.7 85.5 9.4 2.0 100 4.6 1.2 59.7 8.1 1.7 83.7 2.9 2.0 100 2.7 1.7 84.7 1.5
e
A1 0 ND — — ND — — ND — — ND — — ND — — ND — — ND — —
0.5 0.3 68.5 6.3 0.3 60.0 8.2 0.4 85.5 4.6 0.3 56.1 11 0.3 60.8 4.1 0.4 84.6 4.7 0.4 82.7 5.2
2.0 1.4 71.5 3.1 1.4 70.9 3.6 2.0 98.1 1.2 1.2 58.1 6.2 1.7 84.2 6.7 2.1 107 2.2 1. 9 94.3 1.9
e
A2 0 ND — — ND — — ND — — ND — — ND — — ND — — ND — —
0.5 0.4 75.0 8.5 0.3 63.9 8.0 0.4 85.7 7.9 0.3 57.9 12 0.3 66.0 2.3 0.4 87.1 4.6 0.4 85.8 2.9
2.0 1.5 74.0 3.9 1.4 72.5 2.0 2.0 99.0 5.9 1.1 52.9 11 1.7 83.8 6.2 2.1 106 3.9 1.7 87.0 1.8
f
Z1 0 ND — — ND — — ND — — ND — — ND — — ND — — ND — —
0.5 0.3 57.7 8.2 0.3 66.6 6.7 0.4 87.0 1.9 0.3 56.5 8.9 0.3 51.6 9.0 0.5 91.9 1.8 0.4 86.8 2.2
2.0 1.7 83.8 12 1.8 88.7 10 2.1 105 12 1.2 57.8 5.8 1.8 92.5 12 2.3 113 12 2.1 103 13
f
Z2 0 ND — — ND — — ND — — ND — — ND — — ND — — ND — —
0.5 0.3 61.1 3.4 0.3 68.1 8.4 0.4 87.6 4.6 0.3 55.6 5.3 0.3 53.1 6.1 0.5 91.7 6.6 0.4 85.7 4.9
2.0 1.6 81.4 5.2 1.7 85.4 0.4 2.0 97.7 1.0 1.2 58.7 11 1.7 85.5 12 2.1 105 2.5 1.9 94.1 1.9
a
 Teji river area soil samples.
b
 ND = Not detected.
c
R = Recovery.
d
 RSD = Relative standard deviation.
e
 Atsebela river area soil samples.
f
 Ziway lake area soil samples.
Merdassa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 197

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198 Merdassa et al.: Journal of AOAC International Vol. 98, No. 1, 2015
Table 4. Comparison of UAE, Shake-flask, and Soxhlet for
the extractions of fungicides at 0.5 µg/g spiked levels for
each compound
Recovery, % [MD (%)]a
Fungicide Shake-flask Soxhlet
Cymoxanil 47.7 (4.8) 57.2 (7.4)
Metalaxyl 43.2 (6.5) 81.8 (11.6)
Mandipropamid 69.6 (9.5) 99.0 (12.0)
Folpet 52.4 (4.2) 48.1 (11.8)
Chlorothalonil 62.1 (11.4) 71.2 (12.5)
Kresoxim-methyl 88.0 (4.0) 96.8 (5.5)
Famoxadone 73.9 (4.2) 77.4 (13.4)
a
 MD = Mean deviation for n = 5.
recoveries slightly decreased for these latter analytes. Mean
recoveries were noted to decrease when the sonication time
rose from 15 to 20 min (50.9–83.8%), except for folpet and
famoxadone, which increased slightly.
When sonication time was increased from 20 to 25 min
(44.8–83.2%) there was no influence on the mean recoveries
of cymoxanil, metalaxyl, kresoxim-methyl, and famoxadone.
However, recovery was observed to significantly increase for
mandipropamid and chlorothalonil and decrease for folpet.
When the time for each sonication step was decreased from 15 to
10 min, extraction efficiencies increased, and hence, 10 min was
selected for further study. Figure 2B shows an average recovery
obtained when UAE was performed at different sonication
times. In comparison, the 10 min sonication time used in the
present study was 15 min less than that reported by Sánchez-
Brunete et al. (3) for chlorothalonil, and 30 min less than that
reported by Chitescu et al. (36) for UAE of metalaxyl in soil.
Effect of matrix moisture content.—Soil moisture content
may affect the efficiency of pesticide extraction and needs to be
evaluated (3). The degree of partitioning of semivolatile organic
compounds between the soil and the water is generally low, and
the addition of hydrophilic solvents may enhance the degree to
which the analytes may be released from the soil matrix (4). In
this study, the effect of matrix moisture content on the extraction
efficiency of the fungicides was evaluated using spiked soil
sample at 0.5 µg/g and adjusting the soil moisture content to
5, 10, 15, and 20% (ratio of water–soil, v/w) followed by UAE
using 7.5 mL ethyl acetate–hexane (1 + 1, v/v) three times,
for 10 min each time. The results in Figure 3A show that only
two of the target fungicides, mandipropamid and famoxadone,
exhibited improved mean recovery up to 10 and 15% water,
respectively. The remainder of the fungicides showed a
decrease in the mean recovery. When soil moisture content
was increased, recoveries were not enhanced for most of the
analytes, and hence, no water was added during the subsequent
experiments. Similar observations were reported by Sánchez-
Brunete et al. (3) in which the recovery of the fungicides from
some soils may be affected when the soil samples with low
water contents were analyzed. Lambropoulou and Albanis (4)
reported that the responses obtained from fungicides analyses,
with addition of different amounts of water, were increased up
to 30 mL (ratio of soil–water 1 + 6).
Effect of salt addition.—To further improve the extraction
capability of the UAE technique, the effect of adding salt to the
sample was studied by treating the soil sample with 5–20% NaCl
(ratio of salt-soil, w/w). UAE was performed three times, for
10 min each time, using a soil sample at 0.5 µg/g spiked level
and 7.5 mL of ethyl acetate–hexane (1 + 1, v/v). The results in
Figure 3B indicate that the salting-out effect expected to occur
UAE
with this strategy did not improve the extraction recoveries.
66.9 (1.8) Therefore, the above UAE optimum conditions exhibited reliable
72.8 (3.9) extraction capabilities and no further optimization would be
76.8 (3.9) needed. A similar observation was reported by Goncalves and
61.1 (7.4) Alpendurada (2).
64.6 (6.4)
87.8 (2.3) Method Performance
78.2 (3.2)
The practical application of the proposed method was
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evaluated for linearity, LODs, LOQs, repeatability, and
reproducibility (2), and results are shown in Table 2.
Linearity, repeatability, and reproducibility.—Linearity
of the analytical method developed was evaluated at
10 concentration levels from 0.01 to 10 µg/g. The method was
linear over the range of 0.005–10 µg/g for cymoxanil, metalaxyl,
chlorothalonil, and famoxadone; 0.01–10 µg/g for folpet; and
0.02–10 µg/g for mandipropamid and kresoxim-methyl. The
2
linear regression coefficient (r ) was greater than 0.9955 for
the analyte concentration range tested (Table 2). The precision
of the technique was also evaluated in terms of repeatability
(within-day RSD) and reproducibility (between-days RSD; 2).
For repeatability, five replicates of soil samples spiked at a
concentration of 0.5 µg/g were analyzed on the same day and by
the same analyst. For reproducibility, five replicates of the soil
samples spiked at the same concentration level were analyzed on
three nonconsecutive days by the same analyst using the same
HPLC-VWD instrument. Repeatability and reproducibility
ranged from 3.2−11.6%, RSD (average 6.8%) to 2.7−10.6%,
RSD (average 6.3%) for all the analytes. These results confirmed
robustness of the developed method (Table 2). The precision
(%, RSD) can be considered very good; a repeatability of less
than 12% was obtained for all the analytes (2).
LODs and LOQs.—The sensitivity of the technique
was evaluated by determining the LODs whereby spiked
concentrations of the analytes were decreased until a S/N of 3 was
obtained (2, 35). LOQs give a S/N of 10 and were derived from
the LODs (2). Accordingly, the LODs determined were 0.0015
µg/g for cymoxanil, metalaxyl, chlorothalonil, and famoxadone;
0.003 µg/g for folpet; and 0.006 µg/g for mandipropamid and
kresoxim-methyl (Table 2). Generally, sensitivity of the method
depends on both the physicochemical properties of the analytes
(mainly the polarity), which determine extraction efficiency,
and the chromatographic signal in the VWD (35). For both
reasons, medium polar analytes with maximum absorbance
wavelength selected for each analyte gave the best LODs. For
determination of fungicides in the soil by GC, with NPD/ECD,
Sánchez-Brunete et al. (3) reported that the method detection
limits ranged from 0.002 to 0.01 µg/g for chlorothalonil and
the other multiclass fungicides. On the other hand, LODs lower
than 0.05 µg/g for ultra-HPLC (UHPLC)-MS determination of
metalaxyl and other class fungicides in soil was also reported
by Chitescu et al. (36). Therefore, LODs for metalaxyl and
 Merdassa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 199
Figure 4. HPLC-VWD chromatogram obtained from (A) blank soil
sample and spiked soil samples at (B) 0.5 µg/g and (C) 2.0 µg/g after
UAE. For abbreviations, refer to Figure 1.
chlorothalonil obtained in the present study (0.0015 µg/g) were
lower than those reported by the above authors.
Application of the Proposed UAE Method
To evaluate the applicability of the proposed UAE method,
the recovery for each target analyte in six horticultural field soil
samples was determined under the optimum conditions, and
results are displayed in Table 3.
In general, satisfactory recoveries in the range of 51.6 to
106.6% were obtained with %, RSD between 0.4 and 13.1%.
Even though recoveries >60% were obtained for the majority
of the fungicides studied, recoveries <60% were obtained for
folpet. For chlorothalonil, Sánchez-Brunete et al. (3) combined
UAE with GC-ECD and reported maximum recovery of 100%
with RSD, 0.9% (n = 5) in soil (10% water content), which
is relatively higher than the maximum recovery of 79.0%
obtained in the present study. Chitescu et al. (36) combined
UAE with UHPLC-MS and reported maximum recovery
of 87% for metalaxyl in soil, which is comparable with this
study’s maximum recovery of 86.1%. Figure 4 shows a typical
UAE-HPLC-VWD chromatogram obtained for all fungicides
studied in soil.
Comparison Study
Recovery and RSD by conventional extraction (shake-flask
and Soxhlet) versus the UAE technique for the extraction of
seven multiclass fungicides in soil at 0.5 µg/g fortification
levels are presented in Table 4. When extracted by shake-flask,
recoveries in the range of 43.2–88.0% with a mean deviation
of 4.0–9.5% were obtained, while Soxhlet extraction gave
recoveries in the range of 48.1–99.0% with a mean deviation of
5.5–13.4%. Recoveries were higher for the fungicides extracted
by UAE than those obtained by either shake-flask or Soxhlet
extraction for cymoxanil, folpet, and famoxadone. The results
of the comparison study revealed UAE to be more efficient than
shake-flask extraction for all fungicides, except for metalaxyl,
mandipropamid, chlorothalonil, and kresoxim-methyl. Thus,
the proposed UAE has several advantages over the conventional
shake-flask or Soxhlet extraction methods. Moreover, UAE is
more rapid (only 10 min/sample) than conventional shake-
flask or Soxhlet extraction methods (5 h), and the solvent
consumption for UAE (only 7.5 mL) is significantly lower than
shake-flask (20 mL) or Soxhlet extraction (150 mL; 26, 33).
In addition, it was noted that the extracts from sonication can
be chromatographed without further cleanup, and consequently,
the analysis time was considerably reduced. Therefore, the
proposed UAE procedure is rapid and utilizes less organic
solvent, making it cost-effective and environmentally friendly
for the extraction of multiclass fungicide residues in soil.
Conclusions
A simple and rapid one-step multiresidue method using
UAE was developed for the analysis of fungicides in soil. The
method allows excellent LODs from 0.0015 to 0.006 µg/g
using only 0.5 g of soil. Compared with conventional extraction
procedures, which use large amounts of solvents, UAE only
uses 7.5 mL ethyl acetate–hexane (1 + 1, v/v). Repeatability
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and reproducibility mean % RSD values were less than 12%
for all the investigated analytes. In addition, the method is very
simple to perform and requires much less sample handling
than the conventional methods (35). Because of its sensitivity,
robustness, and simplicity, the developed method is a powerful
tool for routine QC of multiclass fungicides in soil samples.
Acknowledgment
This work was supported by the National Natural Science
Foundation of China (21025729, 21321004). Author Merdassa
greatly acknowledges Jimma University for providing financial
support for his Ph.D. study at Addis Ababa University.
References
(1) Navalón, A., Prieto, A., Araujo, L., & Luis Vı́lchez, J. (2002) J.
Chromatogr. A 946, 239−245. http://dx.doi.org/10.1016/S0021-
9673(01)01523-0
(2) Goncalves, C., & Alpendurada, M.F. (2005) Talanta 65,
1179−1189. http://dx.doi.org/10.1016/j.talanta.2004.08.057
(3) Sánchez-Brunete, C., Miguel, E., & Tadeo, J.L. (2002) J.
Chromatogr. A 976, 319−327. http://dx.doi.org/10.1016/S0021-
9673(02)00947-0
(4) Lambropoulou, D.A., & Albanis, T.A. (2004) Anal. Chim. Acta
514, 125−130. http://dx.doi.org/10.1016/j.aca.2004.03.035
(5) Hou, L., & Lee, H.K. (2004) J. Chromatogr. A 1038, 37−42.
http://dx.doi.org/10.1016/j.chroma.2004.03.012
(6) Manirakiza, P., Covaci, A., Andries, S., & Schepens, P.
(2001) Int. J. Environ. Anal. Chem. 81, 25−39. http://dx.doi.
org/10.1080/03067310108044356
(7) Djurovic, R., Djordjevic, T., Radivojevic, L., Santric, L., &
Gajic-Umiljendic, J. (2012) Pesticidi i fitomedicina 27, 239−244
(8) Redondo, M.J., Ruiz, M.J., Boluda, R., & Font, G. (1996) J.
Chromatogr. A 719, 69−76. http://dx.doi.org/10.1016/0021-
9673(95)00399-1
(9) Bouaid, A., Ramos, L., Gonzalez, M.J., Fernández, P., &
Cámara, C. (2001) J. Chromatogr. A 939, 13−21. http://dx.doi.
org/10.1016/S0021-9673(01)01332-2
(10) Beltran, J., López, F.J., & Hernández, F. (2000) J. Chromatogr.
A 885, 389−404. http://dx.doi.org/10.1016/S0021-
9673(00)00142-4
(11) Kreuzig, R., Koinecke, A., & Bahadir, M. (2000) J. Biochem.
Biophys. Methods 43, 403−409. http://dx.doi.org/10.1016/
S0165-022X(00)00056-7
(12) Gonçalves, C., Carvalho, J.J., Azenha, M.A., & Alpendurada,
M.F. (2006) J. Chromatogr. A 1110, 6−14. http://dx.doi.
org/10.1016/j.chroma.2006.01.089
(13) Schantz, M. (2006) Anal. Bioanal. Chem. 386, 1043−1047.
http://dx.doi.org/10.1007/s00216-006-0648-2
200 Merdassa et al.: Journal of AOAC International Vol. 98, No. 1, 2015
(14) Hussen, A., Westbom, R., Megersa, N., Mathiasson, L., &
Bjorklund, E. (2007) J. Chromatogr. A 1152, 247−253. http://
dx.doi.org/10.1016/j.chroma.2007.02.076
(15) Hussen, A., Westbom, R., Megersa, N., Mathiasson, L., &
Bjorklund, E. (2006) J. Chromatogr. A 1103, 202−210. http://
dx.doi.org/10.1016/j.chroma.2005.11.025
(16) Concha-Graña, E., Turnes-Carou, M.I., Muniategui-Lorenzo, S.,
López-Mahía, P., Fernández-Fernández, E., & Prada-Rodríguez,
D. (2004) J. Chromatogr. A 1047, 147−155. http://dx.doi.
org/10.1016/j.chroma.2004.06.114
(17) Björklund, E., Nilsson, T., & Bøwadt, S. (2000) TrAC Trends.
Anal. Chem. 19, 434−445
(18) Wang, W., Meng, B., Lu, X., Liu, Y., & Tao, S. (2007)
Anal. Chim. Acta 602, 211−222. http://dx.doi.org/10.1016/j.
aca.2007.09.023
(19) Silgoner, I., Krska, R., Lombas, E., Gans, O., Rosenberg, E.,
& Grasserbauer, M. (1998) Fresenius J. Anal. Chem. 362,
120−124. http://dx.doi.org/10.1007/s002160051043
(20) Moreno, D.V., Ferrera, Z.S., & Rodríguez, J.J.S. (2006)
Anal. Chim. Acta 571, 51−57. http://dx.doi.org/10.1016/j.
aca.2006.04.046
(21) Merdassa, Y., Liu, J.-F., & Megersa, N. (2013) Talanta 114,
227−234. http://dx.doi.org/10.1016/j.talanta.2013.04.035
(22) Fuentes, E., Baez, M.E., & Labra, R. (2007) J. Chromatogr. A
1169, 40−46. http://dx.doi.org/10.1016/j.chroma.2007.08.064
(23) de Andréa, M.M., Papini, S., & Nakagawa, L.E. (2001)
J. Environ. Sci. Health, Part B 36, 87−93. http://dx.doi.
org/10.1081/PFC-100000919
(24) Vagi, M.C., Petsas, A.S., Kostopoulou, M.N., Karamanoli,
M.K., & Lekkas, T.D. (2007) Desalination 210, 146−156. http://
dx.doi.org/10.1016/j.desal.2006.06.020
(25) Gonçalves, C., & Alpendurada, M.F. (2005) Talanta 65,
1179−1189. http://dx.doi.org/10.1016/j.talanta.2004.08.057
(26) Tor, A., Aydin, M.E., & Özcan, S. (2006) Anal. Chim. Acta 559,
173−180. http://dx.doi.org/10.1016/j.aca.2005.11.078
(27) Shen, J., & Shao, X. (2005) Anal. Bioanal. Chem. 383,
1003−1008. http://dx.doi.org/10.1007/s00216-005-0078-6
(28) Rodríguez-Gonzalo, E., Carabias-Martínez, R., Cruz, E.M.,
Domínguez-Álvarez, J.J., & Hernández-Méndez, J. (2009) J.
Sep. Sci. 32, 575−584. http://dx.doi.org/10.1002/jssc.200800538
(29) Rezić, I., Horvat, A.J.M., Babić, S., & Kaštelan-Macan, M.
(2005) Ultrason. Sonochem. 12, 477−481. http://dx.doi.
org/10.1016/j.ultsonch.2004.07.004
(30) Pena, A., Ruano, F., & Mingorance, M.D. (2006) Anal. Bioanal.
Chem. 385, 918−925. http://dx.doi.org/10.1007/s00216-006-
0449-7
(31) Pan, J., Xia, X.X., & Liang, J. (2008) Ultrason. Sonochem. 15,
25−32. http://dx.doi.org/10.1016/j.ultsonch.2007.06.005
Downloaded from https://academic.oup.com/jaoac/article/98/1/192/5654411 by guest on 06 July 2023
(32) Ozcan, S., Tor, A., & Aydin, M.E. (2009) Anal. Chim. Acta 640,
52−57. http://dx.doi.org/10.1016/j.aca.2009.03.030
(33) Babić, S., Petrović, M., & Kaštelan-Macan, M. (1998) J.
Chromatogr. A 823, 3–9. http://dx.doi.org/10.1016/S0021-
9673(98)00301-X
(34) Aydin, M.E., Ozcan, S., & Tor, A. (2007) CLEAN – Soil, Air,
Water 35, 660−668. http://dx.doi.org/10.1002/clen.200700049
(35) Martínez-Parreño, M., Llorca-Pórcel, J., & Valor, I. (2008)
J. Sep. Sci. 31, 3620−3629. http://dx.doi.org/10.1002/
jssc.200800355
(36) Chitescu, C.L., Oosterink, E., de Jong, J., & Stolker, A.A.M.
(2012) Talanta 88, 653−662. http://dx.doi.org/10.1016/j.
talanta.2011.11.054
(37) List of Registered Pesticides (2011) Animal and Plant
Regulatory Directorate, Ministry of Agriculture, Addis Ababa,
Ethiopia