Acs analytical method validation for the determination of md by lc-msms

pubs.acs.org/JAFCPublished on Web 07/22/2010© 2010 American Chemical Society
J. Agric. Food Chem. 2010, 58, 8911–8917 8911
DOI:10.1021/jf1008346
Analytical Method Validation for the Determination of
Meptyldinocap As 2,4-Dinitrooctylphenol Metabolite in Mango
and Soil Using LC-MS/MS and Dissipation Study of the
Fungicide in Indian Mango Field Ecosystem
SUDEB MANDAL,†
BAPPADITYA KANRAR,‡
SAKTIPADA DAS,†
AND
ANJAN BHATTACHARYYA*,‡
†
Department of Chemistry, Kalyani University, Kalyani-741235, WB, India, and ‡
Department of
Agricultural Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-741252, WB, India
An analytical method for the quantitative determination of meptyldinocap (2,4-DNOPC) as 2,4-
dinitrooctylphenyl (2,4-DNOP) in mango and soil was developed as well as validated using liquid
chromatography tandem mass spectrometry (LC-MS/MS). The method comprised an extraction
with an acetone:methanol:4 N HCl (100:10:5, v/v/v) mixture followed by hydrolytic conversion of
parent 2,4-DNOPC to the corresponding phenol metabolite (2,4-DNOP), and cleanup was done
by liquid:liquid partition using ethyl acetate. Final quantitation was performed by LC-MS/MS of
2,4-DNOP with negative electrospray ionization using gradient elution. The method was validated at
concentrations ranging from 0.025 to 2 μg/g, and the limit of quantification (LOQ) of meptyldinocap
in mango and soil samples was 0.025 μg/g. The recovery of meptyldinocap from mango and soil
sample was found to be 93-98% spiked at different levels with analyte, and the relative standard
deviation for repeatability (RSDr) and reproducibility (RSDR) were acceptable (2-6%). The method
was rugged as evident from a low measurement uncertainty at 0.05 μg/g. In order to evaluate
its safety use in India a multilocational field dissipation study on meptyldinocap in mango was
conducted by following the proposed analytical method.
KEYWORDS: Meptyldinocap; method validation; dissipation; LC-MS/MS; mango; soil
INTRODUCTION
Mango (Mangifera indica L.) is one of the most important
tropical fruits worldwide in terms of production and consumer
acceptance (1). India is the largest producer of mango (10.8
million tonnes) in the world, accounting for about 41% of world
output in 2004 (2). Powdery mildew is an important disease
in most of the mango growing areas of the world causing a
considerable crop loss. Powdery mildew caused by Oidium
mangiferae Berthet is widely distributed and can be a major foliar
disease of field-grown mango trees (3), on which it can infect
leaves, bloom clusters and young fruits.
Dinocap is a mixture of six dinitrooctylphenyl crotonate (2,4-
DNOPC) isomers (both ortho and para methylheptyl, ethylhexyl,
and propylpentyl crotonate isomers (4) (Figure 1). The new
meptyldinocap, an enhanced offering of the single 2,4-DNOPC
methylheptyl isomer, is going to be introduced in the Indian
market by Dow AgroSciences to replace dinocap. Meptyldinocap
has an improved toxicological profile relative to the older dino-
cap. It is a novel powdery mildew (Erysiphe necator) fungi-
cide which shows protectant and postinfective activities. It was
primarily to be used for the control of powdery mildew in
grapevine, but is also being developed for use on cucurbits and
strawberries (5). The efficacy of meptyldinocap against grape
powdery mildew is the same as that of dinocap (6). The maximum
residue limit (MRL) of meptyldinocap (sum of 2,4-DNOPC and
2,4-DNOP expressed as meptyldinocap) was 0.05 μg/g in man-
goes set by the European Union (EC-No 839/2008) (7).
Several analytical methods are available for determining dino-
cap in foods, and the majority are classical methods based on gas
chromatography (GC) techniques with electron-capture detector
(GC/ECD) that had been used for the determination of trace
amounts of dinocap in wine (8). Identification and qualified
estimation of dinocap was done using GC/IR and GC/MS
(9) as well as using HPLC (10, 11). A procedure for high-
performance liquid chromatographic (HPLC) determination of
four active ingredients of dinocap in crops was done by Duxiang
et al. (12). Two improved methods for the determination of
dinocap in apples, strawberries and cucumbers using GC and
spectrophotomety and qualitative confirmation ofthe presenceof
dinocap are performed by thin-layer chromatography (TLC)
(13). The LC-UV methods often have low sensitivity and lack
of sufficient selectivity toward endogenous compounds, which
absorb at the same wavelength. Christer Jansson et al. (14)
presented ethyl acetate extraction and determination by means
of LC-MS/MS in a multiresidue method. But there is no reliable
validated LC-MS/MS method for measuring trace levels of
meptyldinocap where the fungicide is determined as a derivative
*Corresponding author. Tel: þ91-33- 25827139 (R), þ91-9433007139
(M). Fax: þ91-33-25828460. E-mail: anjan_84@rediffmail.com.

8912 J. Agric. Food Chem., Vol. 58, No. 16, 2010 Mandal et al.
(2,4-DNOP) with accuracy, precision, and repeatability in food
and soil samples. The proposed analytical method in this paper is
comparable to other methods described in Federal Register/
Vol. 74, No. 69/Monday, April 13, 2009 (15), but improvements
have been made regarding sample preparation and other crucial
points.
Although the consumption of mango is widespread, there is no
report dealing with the methodological approach for the analysis
of meptyldinocap in mango. In particular, there is a lack of data
regarding the disappearance in-field of meptyldinocap on mango
for determination of the most realistic PHI (preharvest interval)
to ensure the safe use of this fungicide.
The primary aim of this work was to develop an analytical
method for the determination of meptyldinocap (2,4-DNOPC) as
2,4-DNOP by optimizing the preparation of sample and optimiz-
ing LC-MS/MS conditions as well as validate the method in
mango and soil samples. Moreover, the developed and validated
method was used to study its dissipation and residue (at harvest)
of meptyldinocap in the mango field ecosystem under the Indian
tropical climate with a view to ensure human and environmental
safety.
MATERIALS AND METHODS
Apparatus. An Alliance 2695 separations module (Waters, Milford,
MA) was coupled to a Micromass Quattro Micro triple-quadrupole mass
spectrometer (Micromass, Manchester, U.K.). MassLynx V4.1 software
was used for instrument control and QuanLynx for data analysis. A
homogenizer (Polytron, PT-3100, Switzerland), pH meter (CL 46 type;
Toshniwal Instruments, Pvt, Ltd., India) and rotary-vacuum-evaporator
of Eyela (SB-1000, Japan) were used in this study. The shaker (ZHWY-
200D; Zhicheng, China) and the centrifuge were from Beckman Coulter
(Avanti J-30I, USA) and were used for sample preparation.
Chemicals. An analytical standard of meptyldinocap (2,4-DNOPC,
99.23% pure) was procured from M/S Dow AgroSciences India Pvt. Ltd.,
Mumbai. An analytical standard of 2,4-DNOP (99.00% pure) was
prepared in our laboratory. Pesticide residue analysis grade acetone,
acetonitrile, n-hexane, methanol, and ethyl acetate were purchased from
Rankem (India). Sodium sulfate (anhydrous) and sodium hydroxide were
purchased from Qualigen (India). Hydrochloric acid, acetic acid and
ammonium acetate were purchased from Emerck (India). Purified water
was prepared by using a Milli-Q (Millipore, Bedford, MA) water purifica-
tion system.
Preparation of 2,4-DNOP Metabolite. An amount of 10 mg of 2,4-
DNOPC was dissolved in 25 mL of methanol in a 100 mL round-bottom
flask, and 4 mL of 4 (N) NaOH was added to it. The flask was heated at
60 °C temperature inan ultrasonic bath for 1 h. Aftercooling of the round-
bottom flask,thepHofthereactionmixture was neutralizedbyadditionof
concentrated HCl dropwise and the methanol solvent was evaporated in a
rotary vacuum evaporator at 40 °C. Then the reaction mixture was
partioned with (50 þ 50) mL of ethyl acetate which was concentrated by
rotary-vacuum-evaporator and purified by column chromatography. The
purity (99.00%) was confirmed by IR, LC-MS/MS and NMR spectros-
copy.
Standard Solutions. Stock solutions of 2,4-DNOPC and 2,4-DNOP
standards were prepared by weighing 10 ( 0.02 mg in volumetric flasks
(certified “A” class) and dissolving each in 100 mL of methanol. Five levels
of calibration concentration containing 0.01, 0.025, 0.05, 0.1, and 0.5 μg/
mL of 2,4-DNOP were obtained by serial dilution of 2,4-DNOP stock
solution with methanol. Three levels of fortification concentration con-
taining 0.025, 0.05, and 0.1 μg/mL were prepared by serial dilution of
2,4-DNOPC stock solution with methanol required for recovery study.
Field Experiment. A multilocation field study was conducted in
mango fields in four different locations with different variety in India,
viz., (I) Bidhan Chandra Krishi Viswavidyalaya, Mohanpur (BCKV),
West Bengal (Local), (II) Konkan Krishi Vidyapeeth (KKV), Dapoli,
Maharashtra (Alphonso), (III) G. B. Pant University of Agricultura &
Technology (GBPUAT), Pantnagar, Uttarakhand (Dessheri), and (IV)
Acharya N. G. Ranga Agricultural University (ANGRAU), Hyderabad
(M. Vikarabad) on mango during December, 2007, to June, 2008. A plot
size of 1 Plant/Trt was selected for the control and each treatment of the
fungicide under study, and the average agesofthe mango treeswere 23-30
years. Meptyldinocap (35% EC) was applied to mango at the rate of 270
gai/ha (T1) and 540 gai/ha (T2) along with untreated control (T3). Each
treatment including control was replicated thrice in a randomized block
Table 1. Monthly Meteorological Data during the Experimental Period
temp (°C) rel humidity (%)
trial location month and year max min max min rainfall (mm)
I Jan 2008 25.5 12.3 94.7 50.2 60.5
Feb 2008 26.8 13.9 94.7 46.3 27.8
March 2008 33.7 21.9 91.9 46.9 16.2
April 2008 37.5 21.3 93.4 49.4 40.8
II Jan 2008 32.8 16.9 90.2 58.4 0.0
Feb 2008 30.6 18.6 85.6 62.6 0.0
March 2008 32.9 22.1 89.7 65.1 51.4
April 2008 33.6 25.4 84.3 68.3 0.0
III March 2008 31.5 10.2 85.8 25.8 1.8
April 2008 33.4 13.6 66.7 28.0 46.6
May 2008 35.5 18.8 70.0 36.5 63.2
June 2008 31.4 23.8 92.3 72.2 301.5
IV Dec 2007 27.8 9.2 82.7 75.8 0.0
Jan 2008 29.4 9.6 54.8 48.6 0.0
Feb 2008 30.4 16.2 67.5 55.9 0.0
March 2008 32.1 19.0 75.7 68.5 0.0
Figure 1. Chemical structure of six isomers of dinocap: A = 2,4-dinitro-
6-(1-methylheptyl)phenyl crotonate or meptyldinocap or 2,4-DNOPC; B =
2,4-dinitro-6-(1-ethylhexyl)phenyl crotonate; C = 2,4-dinitro-6-(1-propyl-
pentyl)phenyl crotonate; D = 2,6-dinitro-4-(1-methylheptyl)phenyl croto-
nate; E = 2,6-dinitro-4-(1-ethylhexyl)phenyl crotonate; F = 2,6-dinitro-4-(1-
propylpentyl)phenyl crotonate).
Figure 2. Conversion of 2,4-DNOPC to 2,4-DNOP.

Article J. Agric. Food Chem., Vol. 58, No. 16, 2010 8913
design (RBD). Two sprays were given at a 15 day interval. Mango samples
were randomly collected at 0 (3 h after application), 1, 3, 5, 7, and 15
(at harvest) days after second application (DAA). Soil samples were
randomly collected at 15 days (at harvest) after the second application.
The samples were transported to the laboratory in dry ice bags and kept
at -20 °C until analysis in order to avoid any degradation of residues
between sampling and analysis. Meteorological data including rainfall,
humidity, and temperature were recorded using a field weather station
during the period of field experiments and are given in Table 1.
Sample Extraction. Chopped mango sample (10 g) was macerated
with 50 mL of acetone:methanol:4 N HCl (100:10:5; v/v/v) using a
Polytron homogenizer, and the homogenate was centrifuged. The super-
natant was transferred to a clean bottle via a glass wool. An aliquot
(30 mL) of acetone:methanol:4 N HCl (100:10:5; v/v/v) was added to the
residuum and macerated as previously. The sample was centrifuged, and
the supernatantswerecombined. The totalvolumewas adjustedto100 mL
using the extraction solvent. An aliquot (20 mL) of extract was transferred
to a round-bottom flask and reduced to a low volume (<1 mL) by rotary
evaporation at 35 °C.
Soilsamples(10g) were taken ina 250mL conical flask,and then50mL
of acetone:methanol:4 N HCl (100:10:5; v/v/v) was added to the flask. The
samples were then kept for two hours and shaken in a shaker (ZHWY-
200D) for one hour. Then the extracts were filtered in a Buchner funnel
using 50 mL of the same extracting solvent (2 Â 25 mL) for washing. The
total volume was adjusted using the extraction solvent to 100 mL. An
aliquot (4mL) ofthe extractwas transferredto a flaskand reduced to a low
volume (<1 mL) by rotary evaporation at 35 °C.
The extracted soil and mango samples were then transferred to a
graduated tube with 20 mL of ultrapure water, 1 N HCl (2 mL) and
10 mL of hexane:ethyl acetate (50:50, v/v). Then it was shaken vigorously,
and the phases were allowed to settle. The upper organic phase was
transferred into a round-bottom flask, and the extraction was re-
peated with a further two aliquots (10 mL) of the hexane:ethyl acetate
mixture. The combined organic phase was collected in the same round-
bottom flask. The solvent was removed by rotary vacuum evaporator
at 35 °C.
Conversion of crotonates to phenol (Figure 2) was done by taking
aliquots of methanol (10 mL), 5 (N) sodium hydroxide (5 mL) in the
concentrated round-bottom flask. The flask was then ultrasonicated and
the sample transferred to a graduated tube. The sample was placed in an
ultrasonic water bath set at 60 °C for 60 min.
Cleanup and Analysis. The sample was removed from the water bath,
the remaining methanol was evaporated in a rotary vacuum evaporator,
and the sample was allowed to cool at room temperature. Aliquots of
water (8 mL), concentrated hydrochloric acid (3 mL), and ethyl acetate
(10 mL) were then added in a separatory funnel. The sample was shaken
and the upper ethyl acetate phase was transferred through a glass funnel
containing 75 g of sodium sulfate into a round-bottom flask. The partition
was repeated with (10 þ 10) mL of ethyl acetate, and the combined organic
phase was evaporated to dryness in a rotary vacuum evaporator at 35 °C.
The sample was reconstituted in 10 mL of methanol:water (70:30; v/v)
containing 0.1% glacial acetic acid for final quantification by LC-MS/
MS analysis.
According tothe SANCO/10684/2009(16) guidelinea calibration curve
was constructed by plotting 0.01-0.1 ppm of 2,4-DNOP standards
injected versus peak area. From the calibration curve the concentra-
tion of 2,4-DNOP in samples was determined. Consequently the concen-
tration of meptyldinocap (2,4-DNOPC) was calculated by the following
equation:
C2,4-DNOPC ¼
MW2,4-DNOPC
MW2,4-DNOP
Â C2,4-DNOP ¼ 1:23C2,4-DNOP
The PHI, i.e. the time period (in days) required for dissipation of the initial
residue depositstobelow the maximumresidue limit (MRL) for first-order
kinetics, was determined by the equation
PHI ¼ ½logðinterceptÞ - logðMRLÞŠ=ðslope of first-order equationÞ
LC-MS/MS analysis. HPLC Conditions. Optimization of chro-
matographic separation was performed on a Waters 2695 liquid chroma-
tograph (Milford, MA) equipped with a quaternary solvent delivery
system, an autosampler and a Waters 2996 DAD detector. Different
columns were assayed for 2,4-DNOP analysis. A reverse phase column
SymmetryC18, 5 μm, 2.1 Â 100 mm, showed an excellent retention for 2,4-
DNOP (retention times0.99 ( 0.06 min). The first mobile phase assayed
was composed of 50:50 acetonitrile:water with 10 mM ammonium acetate.
The second mobile phase assayed was 50:50 acetonitrile:water with 10 mM
ammonium acetate and added with 0.1% of acetic acid. The third mobile
phase assayed was 50:50 acetonitrile:water with 10 mM ammonium
acetate and 0.1% formic acid. The mobile phase containing acetic acid
significantly improves the response of 2,4-DNOP (m/z 295.14) about 1.35-
fold compared to formic acid, and 2.1 times higher than the mobile phase
with only ammonium acetate. Acetic acid was selected as acidic modifier.
The mobile phase was composed of (A) a mixture of 0.01 M ammonium
acetate with 0.1% acetic acid in water:acetonitrile (80:20 v:v) and (B) 0.1%
Figure 3. Comparison of extraction capabilities of different solvent
systems (50 mL) in the final method from mango and soil (10 g) spiked
at 0.1 μg/g . Error bars signify standard deviation (n = 6).
Figure 4. Effect of pH of the extracting solvent (acetone:methanol:4 N
HCl;100:10:5; v/v/v) for meptyldinocap (2,4-DNOPC) extraction from
mango and soil samples.
Figure 5. Extraction capabilities of blending over shaking, sonicating and
vortexing of meptyldinocap after 2 h of spiking at 0.1 μg/g in soil sample
using 50 mL of acetone:methanol:4 N HCl (100:10:5; v/v/v) as extracting
solvent. Error bars signify standard deviation (n = 6).

acetic acid in acetonitrile (v/v). 75% solvent B was used initially; then, its
concentration was changed from 75% to 100% in 3 min, keeping these
conditions for 1 min. Finally, the program is switched to 75% B in 6 min
and kept for at least 5 min before starting a new analysis at a flow rate of
0.3 mL/min.
MS/MS Conditions. A Micromass (Manchester, U.K.) Quattro Micro
triple-quadrupole massspectrometerequippedwithanelectrospraysource
was used for detection and quantification. The optimized MS instrument
parameters obtained by the tuning were as follows: capillary voltage,
1.20 kV; cone voltage, 46 V; source temperature, 120 °C; desolvation
temperature, 350 °C; desolvation gas flow, 650 L h-1
nitrogen; cone gas
flow, 25 L h-1
; argon collision gas (argon) pressure to 3.5 e-3
psi for
MS/MS. The collision energy for each monitored transition was 13 eV in
MRM mode. In the MRM transitions the dwell and interscan times were
0.3 and 0.1 s, respectively. The LC-MS/MS run time was 6 min per
sample.
MS/MS Transitions. The collision energy for each monitored transi-
tion was optimized in MRM mode. The transitions monitored for 2,4-
DNOP were m/z 295.14 > 193.42 at 29 V, 295.14 > 134.50 at 40 V, and
295.14 > 208.9 at 46 V. The MRM mode of the degradation patterns m/z
295.14 > 193.42 was used for quantification, and 295.14 > 134.50 and
295.14 > 163.1 were used for confirmation, respectively. MS conditions
were optimized by observing responses for loop injections of 20 μL of 2,4-
DNOP standard (10 μg/mL) in sample matrices. A dwell time of 100 ms
per transition was used. 2, 4-DNOP was detected using electrospray
ionization in the negative ion mode.
Method Validation. The analytical method was validated as per the
single laboratory validation approach (17). The performance of the
method was evaluated considering different validation parameters that
include the following items.
Calibration Range. The calibration curves of 2,4-DNOP in pure solvent
and matrix were obtained by plotting the peak area against the concentra-
tion of the corresponding calibration standards at five calibration levels
ranging between 0.01 and 0.5 μg/mL.
Sensitivity. The limit of detection (LOD) was determined by consider-
ing a signal-to-noise ratio (S/N) of 3 with reference to the background
noise obtained from blank sample, whereas the limits of quantification
(LOQ) were determined by considering a S/N of 10 using matrix matched
standards.
Precision. Method precision was checked in terms of intraday repeat-
ability and interday reproducibility at three concentration levels (0.025,
0.05, and 0.1 μg/g). The intraday precision study was carried out by the
injectionofthesamestandardsolutionfiveconsecutive times (n = 5)inthe
same day under the same conditions. The interday precision was carried
out for three successive days using the same solution. The tested products
were mango and soil.
Accuracy-Recovery Experiments. Accuracy was evaluated through
recovery study by spiking untreated mango and soil samples (10 g)
in triplicate with meptyldinocap separately at three concentration
levels (25, 50, and 100 μg/g of 2,4-DNOPC). The mixture was extrac-
ted, derivatized, cleaned up and analyzed using the method mentioned
above.
Matrix Effect. The matrix effect (ME) was assessed by employing
matrix-matchedstandards. The slopeof the calibration graph based on the
matrix matched standards of mango and soil sample was compared with
the slope of the pure solvent based calibration graph. A higher slope of the
matrix calibration indicates matrix induced signal enhancement, whereas,
a lower slope represents signal suppression. The matrix effect (ME %) was
evaluated by the following equation:
ME, % ¼
ðpeak area of matrix standard- peak area of solvent standardÞ Â 100
peak area of solvent standard
Measurement Uncertainty. Sources and quantification of the global
uncertainty for the applied method were evaluted for meptyldinocap at the
level of 0.05 μg/g as per the statistical procedure of the EURACHEM/
CITAC Guide CG 4 (18) in the same way as reported by Banerjee
et al. (19). Five individual sources of uncertainty were taken into account,
viz., uncertainty associated with the calibration graph (U1), day wise
uncertainty associated with precision (U2), analyst wise uncertainty
associated with precision (U3), daywise uncertainty associated with
accuracy/bias (U4), and analystwise uncertainty associated with accu-
racy/bias (U5). The global uncertainty (U) was calculated as
U ¼ ðU1
2
þU2
2
þU3
2
þU4
2
þU5
2
Þ1=2
and was reported as expanded uncertainty, which is twice the value of the
global uncertainty.
RESULTS AND DISCUSSION
Optimization of Sample Preparation. The pH of the extracting
solvent plays an important role in controlling the degree of
extraction of the target molecule from mango and soil sample.
Optimization of the solvent pH was done from pH 2 to 9. Figure 3
shows that atpHvaluesbetween 3 and 4 anoptimal efficiency had
been reached, and a pH value of 3-4 was chosen for the
experiments.
For the selection of extraction solvent nine organic solvents
and solvent mixtures, viz., acetonitrile, ethyl acetate, methanol,
acetone, methanol þ acetone (1:1; v/v), acetone þ methanol (1:1;
v/v), acetone þ methanol (2:1; v/v), acetone þ methanol (5:1;
v/v), acetone þ methanol (10:1; v/v) and acetone þ methanol
(15:1; v/v) at pH 3-4 were evaluated for their extraction
efficiency. From this study combining the pH effect it is clearly
revealed that a mixture of acetone:methanol:4 N HCl (10:2:1; v/v/
v) gave higher recovery percentage than other solvent or solvent
mixtures used for extraction (Figure 4).
Different approaches have been described for soil sample
extraction, such as shaking, vortexing, blending and ultrasonica-
tion. To check for effectiveness of the extraction, the same sample
was incubated at different extraction times. From our results it is
revealed that shaking gave better recovery for the fungicide
compared to vortexing, blending and ultrasonication based
methods (Figure 5). It was observed that 1 h gave higher fungicide
extraction and that prolonged time did not exert additional
advantages.
Extraction of free meptyldinocap (2,4-DNOPC) is not possible
because it is likely to be converted to 2,4-DNOP. Hence
the parent 2,4-DNOPC must be derivatized to its metabolite
2,4-DNOP (Figure 2). The hydrolyzed sample must be methanol
free to get optimum recovery. The analyte might be lost in the
methanol-water phase in the liquid-liquid partition step with
Table 2. Linearity Range, Limit of Quantification (LOQ), Recovery (Rec), Relative Standard Daviation for Repeatability (RSDr), Relative Standard Daviation for
Reproducibility (RSDR) and Matrix Effect (ME) of Meptyldinocap from Mango and Soil
substrate linear range (μg/g) spiked level (μg/g) R 2
RSDr (%) RSDR (%) Rec (%) MEa
(%) LOQ (μg/g)
mango 0.025-2 0.025 0.9851 5.5 5.9 93.8 -19 0.025
0.05 0.9751 4.8 4.3 95.1 -17
0.1 0.9826 4.1 3.9 94.6 -12
soil 0.025-3 0.025 0.9912 5.2 4.2 97.6 -17 0.025
0.05 0.9862 3.4 3.8 96.8 -14
0.1 0.9754 2.2 2.6 98.2 -10
a
Minus sign designates induced signal suppression.

ethyl acetate. The method is based on the rapid hydrolysis of
dinocap by means of ethanolamine, but it is a toxic, costly viscous
liquid. So we have used sodium hydroxide for hydrolysis pur-
pose instead of ethanolamine. After hydrolysis of analyte to
2,4-DNOP from 2,4-DNOPC using NaOH solution, it was
neutralized prior to liquid-liquid partition to get maximum
recovery. For the development of a sensitive method to determine
the presence of pesticides with a 2,4-dinitrophenolic structure, it is
crucial to know whether the compounds are present in the free
phenolic or in the dissociated phenolate form, which depends on
the appropriate selection of pH (20). The results show that the
recovery of the compounds after the hydrolysis step increased
when it was neutralized at pH around 7.5.
Validation of the Proposed Method. The linearity in the res-
ponse was studied using matrix-matched calibration solutions
prepared by spiking 2,4-DNOP at five concentration levels,
ranging from 0.005 to 5 μg/mL in the mango and soil samples.
The calibration curves were obtained by plotting the peak area
against the concentration of the corresponding calibration stan-
dards in all matrices. Good linearity was observed in the studied
range with R2
values higher than 0.97. The mean recovery
percentages and the associated standard deviation are given in
Table 2. It can be seen that the mean recoveries ranged from 93.8
to 95.1% for mango and from 97.6 to 98.2% for soil and the
relative standard deviation (RSD) values obtained from run-to-
run (RSDr) and day-to-day (RSDR) precision are summarized
(2-6%) in Table 3. From the results obtained, the developed
method was found to be precise (21) for quantitative purposes.
An estimated value of LOD was 0.01 μg/mL, whereas LOQ
values were in the range from 0.02 to 0.25 μg/mL. The results are
summarized in Table 2. However, calculated LODs and LOQs
were at least 2 orders of magnitude lower than the established
MRLsfor mango,indicating that the proposedmethodissuitable
for quantification of meptyldinocap in mango.
No interfering endogenous compound peaks were observed at
the retention time of 2,4-DNOP in chromatograms obtained
from control blank food (mango and soil) samples (Figure 2). It
was therefore expected that assay of food samples by use of this
method would not be prevented by interfering peaks. Represen-
tative chromatograms obtained from extracted control and
2,4-DNOP spiked food samples are shown in Figure 2, from
which it is apparent that the retention time of 2,4-DNOP was
approximately 0.99 min (Figure 6).
The uncertainty values for meptyldinocap are reported as %
relativestandard deviation in Table 3. Calculated U valuesranged
below 4% for mango and soil matrices. The global uncertainty of
meptyldinocap in different matrices varied below 10% (U < 4)
and had lower uncertainties associated with precision (<0.3%
for both U2 and U3) as well as the accuracy/bias (<3% for both
U4 and U5). This suggests that the method gave repeatable and
reliable results for this fungicide, without any major loss of
residues during sample preparation.
Result of Field Sample Analysis and Safety Evaluation. The
residue of meptyldinocap in/on mango after its second applica-
tion at 15 day interval at the rate of 270 gai/ha (T1) and 540 gai/ha
(T2) in four locations in India is presented in Tables 4 and 5. The
results of this experiment showed that meptyldinocap dissipated
with gradual and continuous deterioration after application. The
dissipation trend of meptyldinocap in mango followed first order
kinetics. The possible routes of meptyldinocap dissipation and
transformation in the environment include plant species, climatic
conditions and biotransformation via soil microorganisms on soil
and photo conversion to simpler products on plant surfaces. The
initial deposit of meptyldinocap in mango of four locations was
Figure 6. Mass chromatographic profiles of product ion [m/z 295.14 f 193.42 (b)] for 2,4-DNOP from mango (A1) and soil (B1) matrices and
chromatograms obtained from control mango (A2) and soil (B2) sample monitored at m/z 295.14 f 193.42 of 2,4-DNOP.
Table 3. Individual and Global Uncertaintiesa
for Triasulfuron in Mango and
Soil Expressed as % Relative Standard Deviation
matrices U1 U2 U3 U4 U5 U 2U
mango 2.2 0.24 0.26 2.2 2.2 3.83 7.65
soil 1.8 0.28 0.25 2.6 1.7 3.61 7.22
a
Calculated at 50 ng/g.

found in the range of 1.31-1.45 μg/g for recommended dose (T1)
and 1.69-2.02 μg/g for double the recommended dose (T2)
respectively. The residues declined with time, and 64-86%
dissipation appeared on the fifth DAA.
The dissipation of meptyldinocap was slightly faster at recom-
mended dose(270 gai/ha) (T1/2 = 2.22-2.66day) thanthe double
the recommended dose (540 gai/ha) (T1/2 = 2.67-3.40 day).
Among the locations, dissipation of meptyldinocap in mango
was faster at ANGRAU [T1/2 = 2.66 day (T1); 2.67 day (T2)]
followed by BCKV [T1/2 = 2.61 day (T1); 3.19 day (T2)], KKV
[T1/2=2.66day (T1); 3.21day (T2)] and GBPUAT (T1/2=2.22day
(T1); 3.40 day (T2)]. The difference could be attributed to weather
conditions and the growth characteristics of different varieties of
mango grown at these locations. Though meptyldinocap was
applied on mango, part of the spray material falls on the field soil.
The residues of meptyldinocap in mango were found below its
LOQ of 0.025 μg/g, after 7 days irrespective of dose and location.
At harvest (15 DAA), the residues of meptyldinocap in mango
and soil samples were found to be lower than the European
Union’s maximum residue limit (MRL) of 0.05 μg/g in mango
(Table 5). So it is suggested that a waiting period (PHI) of 15 days
should be done at the recommended dose before consumption of
mangoes, as it will be safe for the consumer’s health.
ACKNOWLEDGMENT
We gratefully acknowledge M/S Dow AgroSciences, Mumbai,
India, for providing analytical standard. We are also thankful
to Export testing Laboratory (ETL), Bidhan Chandra Krishi
Viswavidyalaya, Mohanpur (BCKV), West Bengal, India, for
providing instruments.
LITERATURE CITED
(1) Food and Agriculture Organization. FAOSTAT database collec-
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United Nations. Available from http://faostat.fao.org, 2005.
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Table 4. Dissipation of Meptyldinocap Residues in Mango in Four Locationsa
amt of meptyldinocap recovered ( SD (μg g-1
) (% dissipation)
trial location DAA T1 (270 gai ha-1
) T2 (540 gai ha-1
)
BCKV 0 1.32 ( 0.06 (;) 1.72 ( 0.01 (;)
1 0.81 ( 0.02 (38.38) 1.41 ( 0.02 (17.83)
3 0.55 ( 0.02 (58.08) 0.83 ( 0.06 (51.74)
5 0.32 ( 0.03 (75.76) 0.60 ( 0.14 (64.92)
7 BDL BDL
regression equation Y = 3.0793 - 0.1153X Y = 3.2349 - 0.0944X
half life (t1/2) 2.61 days 3.19 days
waiting period (PHI) 15.52 19.18
KKV 0 1.31 ( 0.04 (;) 1.86 ( 0.07 (;)
1 0.87 ( 0.04 (53.55) 1.56 ( 0.03 (53.55)
3 0.57 ( 0.02 (69.50) 1.12 ( 0.05 (69.50)
5 0.34 ( 0.03 (81.91) 0.61 ( 0.06 (81.91)
7 BDL BDL
GBPUAT 0 1.45 ( 0.02 (;) 2.02 ( 0.02 (;)
1 0.90 ( 0.07 (52.30) 1.61 ( 0.05 (49.37)
3 0.57 ( 0.06 (69.50) 1.24 ( 0.02 (61.01)
5 0.28 ( 0.05 (85.28) 0.71 ( 0.07 (77.57)
7 BDL BDL
AGRAU 0 1.27 ( 0.10 (;) 1.69 ( 0.01 (;)
1 0.84 ( 0.05 (55.14) 1.41 ( 0.03 (55.77)
3 0.59 ( 0.01 (68.79) 0.93 ( 0.05 (70.65)
5 0.32 ( 0.03 (83.16) 0.43 ( 0.01 (86.58)
7 BDL BDL
a
SD, standard deviation; average of three replicates. DAA, days after application. BDL: below detectable level. BCKV, Bidhan Chandra Krishi Viswavidyalaya. KKV, Konkan
Krishi Vidyapeeth. GBPUAT, G. B. Pant University of Agricultura & Technology. ANGRAU, Acharya N. G. Ranga Agricultural University.
Table 5. Harvest Residue of Meptyldinocap in Mango and Mango Cropped
Soil in Four Locationsa
amt of meptyldinocap recovered ( SD (μg g-1
)
(% dissipation)
trial location DAA substrate T1 (270 gai ha-1
) T2 (540 gai ha-1
)
I 15 mango BDL BDL
15 soil BDL BDL
II 15 mango BDL BDL
15 soil BDL BDL
III 15 mango BDL BDL
15 soil BDL BDL
IV 15 mango BDL BDL
15 soil BDL BDL
a
SD, standard deviation; average of three replicates. DAA, days after applica-
tion. BDL, below detectable levels.

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Received for review March 1, 2010. Revised manuscript received July 8,
2010. Accepted July 12, 2010. We gratefully acknowledge M/S Dow
AgroSciences, Mumbai, India, for financial support.

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