Field Of The Invention
The invention relates generally to a method and device
for pesticide analysis on individual seeds. More particularly to methods and devices
for determining quantities of multiple pesticides extracted from seeds of crop plants.
Background Of The Invention
A reliable determination of pesticide quantities in agricultural
and environmental practices is very important. In the agricultural area, pesticides
are employed in a variety of ways including coating seeds to protect them against
many pests, including being attacked by insects or by soil borne diseases. It is
particularly important to control the quantity of pesticides coated on an individual
seed because too little pesticide will result in incomplete protection of the seed
and emerging plant while too much pesticide may have negative effects on the seed
and its germination.
The term "pesticide seed loading", therefore, refers to
the quantity of adhered pesticide on the seed.
The current art of quantitative pesticide determination
on seeds includes the chromatographic or fluorescence analysis of samples with large
numbers of seeds (several grams in weight) or extraction of the dye or pigment from
the surface of an individual seed followed by fluorescence analysis. Analysis of
larger quantities of seeds shows the average pesticide content of the seeds, however,
it does not allow the determination of how well each seed is protected on a single
seed basis. The known method of quantification of the dye from a single seed is
valid only if the dye content is directly correlated to the pesticide content. This
correlation is not always valid, especially in seeds treated with multiple active
ingredients having different physico-chemical properties (e.g., melting point, water
solubility, particle size). Also, application of this method is obviously limited
to seed treatments which include a fluorescent ingredient that can be extracted
from the seeds.
One of such methods is known from document US-A-5 900 944.
Current practice in chromatographic analysis of pesticides
from coatings of a large sample of seeds cannot be successfully translated to multiple
A.I. analysis of single seeds due to several key issues, such as:
- Classical methods of A.I. extraction utilized for gram quantities of seed gave
incomplete or erratic results at the trace levels required for single seeds.
- The typical manual solvent addition technique used for extracting the seeds
was not practical considering the large number of analyses required for a reliable
seed-to-seed distribution determination (typically 60 seeds or more).
- Difficulty is encountered when filtering the extract using a syringe equipped
with a syringe filter due to the low volume of extract available and the amount
retained on the filter.
- Very dilute extracts of the active ingredients from a single seed provided a
significantly sub-optimal matrix for reproducible chromatographic analysis.
The limitations of the art were overcome by the present
inventors through a number of experiments which ultimately provided successful results
with single seed analysis of commercially important seed treatment active ingredients
from crop seeds such as corn, cotton, wheat, soybean and canola.
With the amount of one or more pesticides loading on a
seed determined, a plot of the distribution of the seed loading for one or more
pesticides for a batch of seeds can be done for each pesticide.
The present invention, therefore, provides an indication
of how well seeds are treated with a seed treatment pesticidal composition.
Summary Of The Invention
According to the present invention, methods and devices
are provided for the quantitative analysis of trace levels of multiple pesticides
from a single seed.
Direct quantitative determination of multiple active ingredients
on a single seed has proved to be a significant challenge due to the extremely low
levels of the active ingredients on each individual seed and the difficulty to reproducibly
extract these trace quantities. For example, it is not unusual to have loadings
as low as 1 µg of each active ingredient on commercially treated seed.
Surprisingly, it has now been found that the methods of
high-pressure liquid chromatography or gas chromatography can be reproducibly applied
to analysis of multiple pesticides from a single seed by utilization of new seed
preparation, extraction, filtration and detection techniques.
Generally, the method comprises the steps of selecting
a subset of seeds, extracting the active ingredients from the seed using a suitable
extracting fluid based on the active ingredient and seed being extracted (with,
for example, by sonication or mechanical shaking), filtration, separation by HPLC
or GC, and detection.
The method according to the invention for determining the
single seed loading distribution (Gaussian and non-Gaussian) of one or more pesticides
on pesticidally treated seeds comprises the steps of:
- 1) Selecting a subset of seeds sufficient to determine said distribution;
- 2) Maintaining a seed from said subset in contact with an extracting fluid to
substantially selectively extract one or more pesticides from said seed to yield
a test sample, and optionally using one or more other extracting fluids to substantially
selectively extract one or more other pesticides from the seed to yield one or more
other test samples, and optionally then combine the test samples to yield a single
test sample;
- 3) Filtering the test sample containing the pesticide to substantially remove
undesired substances extracted from the seed;
- 4) Separating the one or more pesticides from other substances in the filtered
test sample by chromatography;
- 5) passing the one or more separated pesticides into a detector;
- 6) Detecting the signal generated by the pesticide at the detector;
- 7) Relating the amount of signal detected to a quantity of pesticide;
- 8) Repeating Steps 2-7 sequentially for each seed in said subset;
- 9) Determining the single seed loading distribution (both Gaussian and non-Gaussian)
for the pesticidally treated seeds based on the pesticide quantity determined for
each seed in the subset; and
- 10) Optionally, repeating Steps 4-9 to determine quantity and seed loading distribution
of another pesticide in the test sample.
The method is applicable to determination of a broad range
of pesticides when loaded on seeds of key agronomic crops such as corn, cotton,
wheat, soybean and canola. Suitable crop seeds to be analyzed in accordance with
the invention include conventional as well as genetically enhanced or engineered
varieties such as, for example, insect resistant (e.g., Bt.) as well as herbicide
and disease resistant varieties. Examples of suitable pesticides include azoxystrobin;
bitertanol; carboxin; cymoxanil; cyproconazole; cyprodinil; dichlofluamid; difenoconazole;
diniconazole; epoxiconazole; fenpiclonil; fludioxonil; fluquiconazole; flusilazole;
flutriafol; furalaxyl; guazatin; hexaconazole; hymexazol; imazalil; imibenconazole;
ipconazole; kresoxim-methyl; metalaxyl; R-metalaxyl; metconazole; myclobutanil;
oxadixyl; pefurazoate; penconazole; pencycuron; prochloraz; propiconazole; pyroquilone;
spiroxamin; tebuconazole; thiabendazole; tolifluamide; triazoxide; triadimefon;
triadimenol; triflumizole; triticonazole, uniconazole; abamectin; captan; spinosad;
emamectin; fipronil; thiacloprid; imidacloprid; thiamethoxam; tefluthrin and chlothianidin.
Especially preferred are abamectin; captan; emamectin; fipronil; thiacloprid; imidacloprid;
thiamethoxam; tefluthrin; chlothianidin; azoxystrobin; difenoconazole; fludioxonil;
flutriafol; ipconazole; metalaxyl; R-metalaxyl; myclobutanil; tebuconazole; and
thiabendazole.
Detailed Description Of The Invention
The present invention enables the reproducible quantitative
analysis of trace levels of multiple pesticides from a single seed. The method is
workable for pigment-free compositions as well as those containing a pigment (also
known as dye or colourant).
The present invention is described in more detail below:
Automation Device
Preferably, an automatic solvent addition device that can
reproducibly add low volumes of solvent to a large number of samples is required
because of the large volume of seeds being analyzed (e.g., 60 for each analysis).
The repetitive use of a manual pipette is not preferred due to excessive operator
exposure to the solvent and operator errors that can be introduced in such a repetitive
and tedious operation. A Hamilton Autodiluter outfitted with a 5 ml syringe was
successfully utilized for this purpose, but other devices may also be workable.
Solvent Choice
A solvent or a combination of solvents (used herein as
"extracting fluid") that will reproducibly extract mainly the active ingredients
from a single seed is identified. This combination can be different for the different
seeds and active ingredients being tested and is not purely based on the solubility
of the active ingredients. Generally, a seed is treated with a precise amount of
pesticide, and then experiments carried out to determine the extracting fluid that
gave the closest to the theoretical amount. For example, thiamethoxam on corn seeds
is preferably exposed to water in a pre-wetting or soaking step to effectively remove
thiamethoxam from the corn seed. After this wetting step the addition of acetonitrile
is used to complete the extraction. When extracting thiamethoxam from cotton seeds
a mixture of 50:50 water:acetonitrile can be used in one step with no soaking of
the seed. The com extraction scheme (involving soaking of seed in water and then
application of acetonitrile) was successfully used with no further testing to remove
fludioxonil, mefenoxam, imidacloprid and metalaxyl. However, the addition of acetone
was required for extraction of captan from corn seeds, due to its low water solubility.
The composition of the extracting fluid depends on the
type of seed, the solubility of the active ingredients applied to the seed and the
analytical method that will be used. In general a 50:50 acetonitrile: 0.1% acetic
acid mixture is used for HPLC analysis and methyl isobutyl ketone (MIBK) or acetone
containing an internal standard such as dimethyl phthalate is used for GC of many
common active ingredients off the majority of seeds. Exceptions are many, however,
and include corn seeds in general and the active ingredient of captan. One or more
extracting fluids may be necessary to effectively extract two or more active ingredients.
In some cases, the extracting fluid may extract from the
seed other substances than the pesticides. Examples include compounds that are soluble
in the extracting fluid, e.g., colourant, surfactant, emulifiers. These other substances
are separated from the pesticides in step 4 using chromatography.
Filtration Technique
The test sample (the solution after the extraction step)
has to be filtered to remove any undesired substances, such as pieces of seed and
fibrous material, before analysis by a chromatographic technique. Filtering a small
volume (e.g., one milliliter) of the test sample and still obtaining sufficient
volume for chromatographic analysis can be difficult. The 13 mm 0.45 micron Acrodisc
syringe filters typically used for this purpose retain between 0.5ml and 1.0 ml,
leaving an inadequate amount to fill the autosampler vial. A preferred solution
involved the use of a Whatmann Uniprep vial which is an autosampler vial with a
built-in filter.
Detection Sources
Types of detectors suitable for use in the present method
depend on the chromatographic method used. For example, for HPLC, an UV (ultraviolet)
detector, a conductivity detector, a Rl (Refractive index) detector, or a ELS (evaporative
light scattering) detector are examples of suitable detectors. Whereas for GC, a
FID (flame ionisation detector), an ECD (electron capture detector) or a TCD (thermal
conductivity detector) are suitable examples. A UV detector on HPLC and FID on GC
have been found to be suitable detectors for a broad range of pesticides.
The mechanism for the generation of the pesticide signal
in the detector depends on the type of detector used. For example, in the instance
a UV detector is used, the pesticide is illuminated with UV light and the amount
of UV light absorbed is measured; in the instance of a conductivity detector is
used, the conductivity of the pesticide in the solution is measured; in the instance
a RI detector is used, the pesticide is illuminated with beam of radiation light
and the resulting refractive index is monitored; and in the instance a FID detector
is used, the pesticide is ionized and the resultant increase in ionization current
is measured; and in the instance a ECD detector is used, the detector measures the
change in standing current due to the capture of electrons by the pesticide, the
ECD detector is especially useful for halogenated pesticides.
Signal Optimisation
Optimizing the signal to allow for detection of the active
ingredients, especially when multiple active ingredients were being determined from
one seed, is a major hurdle. The test sample could have a concentration about 0.0002
mg/ml because a single seed could be coated with 10 ppm of pesticide and one milliliter
of extracting fluid is used. A typical injection volume of 10 microliters into the
chromatograph would require detection of amounts as small as 0.2 nanograms of pesticide.
Normally when faced with trace level analysis, a chemist can increase the sample
size; since this method was not possible for a single seed, the sensitivity can
be improved with the following:
- It was found that most pesticide components can be detected at either 265 nm
or 230 nm wavelength. Therefore, use of a dual channel detector allows simultaneous
detection at both wavelengths thereby optimizing the detection signals.
- Further, an increase of the injection volume to 15 µl led to an in the
increase amount of pesticide detected.
- Use of a HPLC column with a smaller internal diameter and smaller particle size
(for example, a switch from a column having 150 mm x 4.6 mm diameter, and 5 micron
packing to a column with 100 mm x 3.0 mm diameter, and 3 micron packing) was found
to increase the sensitivity. Columns of diameter 50 mm x 2.1 mm, 3 micron packing
may also be employed in the present invention for highest sensitivity requirements.
Further improvement is possible by optimizing the elution
gradient to allow the desired peak (i.e., of the targeted active ingredient) to
elute in a clear region of the chromatogram and to shorten elution time to optimize
peak shape. This is generally achieved by varying the amount of aqueous eluent to
organic eluent.
Technical details for the novel single seed analysis method
are provided in the description below.
Reference solutions
Methods for preparing reference solution are known to a
skilled person.
Usually, reference solutions are prepared using reference
material (i.e., of the targeted active ingredient) of known purity. In general multiple
weights of approximately 0.1 grams is transferred to a 100 ml volumetric flask or
a 2 oz. bottle. The weight is recorded from an analytical balance to four decimal
places. The volumetric flask is filled to volume with an appropriate solvent (selected
based on the solubility of the reference material with the most common solvent being
acetonitrile for HPLC analysis and MIBK or acetone containing an internal standard
such as dimethyl phthalate for GC analysis). The solution is sonicated or manually
shaken until all the reference material is dissolved. This serves as a stock solution.
An appropriate amount of this stock solution is serially
diluted using a volumetric pipette into another volumetric flask to prepare the
standard solution. For analysis of multiple active ingredients a stock solution
for each reference material is made, then all are combined by adding the appropriate
amount by volumetric pipette into a fresh volumetric flask to prepare a combined
standard solution.
The standard solution is prepared by adding a defined amount
of stock solution to a second volumetric flask and then filling it to volume with
the extracting fluid used to extract the pesticide from the seed. Again, the solution
is sonicated or manually shaken to ensure homogeneity. For consistency with the
sample preparation below the solution is preferably filtered using a disposable
syringe and filter as its transferred into an autosampler vial. Typically this would
be a 0.45 micron Acrodisc filter. The type of filter depends on the solvent used.
Single seed preparation
A set of seeds is taken from the pesticidally treated seeds
that would be representative of them. The number of seeds can vary, but, for statistical
purposes, sixty or more are preferred.
Sixty individual seeds, for example, are transferred into
separate scintillation vials using care not to disturb the seed coating (in general
forceps or a scapula are used). The weight of each seed is recorded using an analytical
balance. The weight is recorded to four decimal places.
The suitable extracting fluid is added using, for example,
a 5 ml Hamilton autodiluter. The desired amount of extracting fluid is precisely
added to each seed. To this end a volumetric pipette operated manually is not recommended.
In addition the precision of the autodiluter should be tested prior to its use to
ensure it performs adequately.
The amount of the extracting fluid used should be sufficient
such that any manipulation (e.g., filtration) of the test sample thereafter would
leave enough test sample to carry out the chromatographic analysis.
The volume of extracting fluid added is typically 1-4 ml
depending on the size of the seed. The fluid should cover the seed completely to
ensure substantial extraction of the pesticide, preferably, 90, more preferably
95, especially 97, %, advantageously complete extraction, yet should be minimized
to yield the most concentrated solution. The mixture is preferably sonicated followed
if needed by mechanical shaking for a time adequate to substantially extract the
active ingredients off the seed and into the extracting fluid and yield the test
sample. This time has to be determined experimentally for each combination of seed
plus active ingredient by assaying the final solution and evaluating for full recovery
of the theoretical amount of each active ingredient. Typically 30 minutes of sonication
followed by 15 minutes of mechanical shaking is sufficient time. The test sample
is then filtered, for example, into an autosampler vial, preferably using a disposable
syringe and a syringe filter (for example, a 0.45 micron Acrodisc filter) unless
the total volume of the test sample is 1 ml or less. In that case a Whatmann Uniprep
vial, for example, with built-in filter can be used for the autosampler vial to
prevent solvent loss within the filter. The filters are chosen based on the fluid
being used for extraction.
Instrumental Analysis
The test sample in the autosampler vials are injected into
the instrument using a volume of typically 15 microliters for an HPLC run and 3
microliters for a GC run. An autosampler is preferred because manual injections
are not precise enough for the analysis. Separation of the active ingredients is
achieved via HPLC or GC.
Suitable HPLC columns include Nucleosil C18, Prism RP,
Inertsil ODS-3, Lichrospher NH2, Discovery C18 and many others. The packing material
is selected based on the physical properties of the active ingredients being evaluated.
Suitable GC columns include DB-1, DB-5, DB-1701 and many
others. Again the column is selected based on the active ingredients being determined.
Appropriate parameters for consideration in HPLC include
the length, internal diameter and particle size of the columns. In general a 15
cm or less column is used preferably with 5 micron particles or less and an internal
diameter of 4.6 mm or less. In the cases of small seeds or seeds treated with low
amounts of active ingredient it is desirable to evaluate the column choice to ensure
the smallest peak can be detected.
Similarly for GC it is recommended to use a capillary column
and not a wide-bore column to ensure narrow peak shape of the smallest peaks. Detection
of the active ingredients is generally done with UV for the HPLC analysis, selected
a wavelength optimized for the active ingredients being determined based on their
UV response. In general either 265 nm or 230 nm has been shown to be suitable for
most active ingredients studied.
Quantification
Quantification of the amount of each active ingredient
is done by measuring the amount of each ingredient seen by the detector, in general
by measuring the area of the peak seen. This area is compared to that of the reference
solution using in general external calibration for HPLC and internal calibration
for GC. An average calibration factor is preferably calculated based on multiple
injections of the reference solutions.
The single seed loading distribution of a sub-set of seeds
may be carried out on more than one instrument (chromatographic column & detector)
so that the results are available faster, for example, a set of 30 seeds on one
instrument and another set of 30 seeds on another instrument, and then results consolidated.
EXAMPLES
Commercial products for seed treatment are mixed with water
and optionally a colorant at , laboratory scale according to label instructions.
Seed treatment is performed with a Hege 1I Seed Treater (Hege Equipment, Inc., 13915
W. 53rd Street N., Colwich KS). One kilogram of seed are added for each trial. The
rotation speed of the Hege is set at 60 rpm. Once the correct rotational speed is
achieved, the slurry is added through a syringe over 5 seconds, followed by a 30
second mixing period. Seeds are allowed to air dry for 24 hours prior to analysis.
Example 1- Analysis of imidacloprid, metalaxyl and captan from corn seeds.
A batch of corn seed is treated with imidacloprid, metalaxyl
and captan (applied as Gaucho®, Allegiance® and Captan® 400, respectively)
with a target concentration of 500 ppm imidacloprid, 20 ppm metalaxyl, and 460 ppm
captan. The samples are analyzed as described below and the resultant seed-to-seed
distribution data for this treatment are shown in Tables 1, 2 and 3 for imidacloprid,
metalaxyl and captan, respectively.
Eluent preparation (0.1 % acetic acid in water): 1.8 ml
of glacial acetic acid is added to 1800 ml of deionized water. Stir well, filter
and degas prior to use.
Part A imidacloprid and metalaxyl standard and sample preparation
Stock preparation for imidacloprid and metalaxyl: Accurately
weigh in duplicate (standard A and B) 0.0400- 0.0500 g of imidacloprid primary standard
and 0.0300- 0.0400 g of metalaxyl primary standard into separate 2-ounce bottles.
Add 50 mL of a 50:50 mixture of acetonitile: deionized water. Sonicate for 30 minutes
and mechanically shake for one hour.
Standard preparation for imidacloprid and metalaxyl: Add
by pipette 5 mL of imidacloprid stock solution and 5 mL of Metalaxyl stock solution
to a 250 mL volumetric flask. Add all A weights to one flask and label A, add all
B weights to a separate flask and label B. Fill each flask to volume with 50:50
water: acetonitrile. Invert several times to mix.
Individual corn seed sample preparation for determination
of imidacloprid and metalaxyl: Transfer one corn seed into a scintillation vial.
Add 2.0 mL of deionized water. Allow to stand undisturbed for 30 minutes. Add 2.0
mL acetonitrile. Sonicate 30 minutes and mechanically shake for one hour. Filter
with a 0.45 micron syringe filter prior to analysis.
Part B Captan standard and sample preparation
Stock preparation: Accurately weight in duplicate (standard
A and B) 0.0900- 0.1100 g of captan primary standard into a 2-ounce bottle. Add
50 ml acetone. Sonicate for 30 minutes and mechanically shake for one hour.
Standard preparation for captan: Add by pipette 5 ml of
stock solution into a 250 ml volumetric flask. Fill volume with acetone. Invert
several times to mix.
Individual corn seed sample preparation for determination
of captan: Transfer one corn seed into a scintillation vial. Add 4.0 mL of acetone.
Sonicate 30 minutes and mechanically shake for one hour. Filter with a 0.45 micron
syringe filter prior to analysis.
INSTRUMENTATION
Perkin Elmer Series 200 LC pump or equivalent.
Perkin Elmer LC 235 Diode Array detector or equivalent.
Hewlett-Packard Series 1050 autosampler or equivalent
Hamilton MicroLab 1000 autodiluter or equivalent capable of delivering 25.00 ±
0.05 mL aliquots and 2.00 ± 0.05 mL aliquots.
LC Column - Prism RP 150 mm column with 4.6 mm internal diameter and 5 micron particle
size. Analytical balance with accuracy of ± 0.1 mg.
INSTRUMENT CONDITIONS
Detection: UV detection at 265 nm with 5 nm bandwidth and
simultaneously UV detection at 230 nm with 5 nm bandwidth
Injection Volume: 10 µl
Flow: 1.0 ml/min
Column Temperature: 35°C
Run Time: Approximately 30 minutes
Gradient program (linear):
Time [minutes]
0.1 % acetic acid [%]
acetonitrile [%]
0
85
15
5
85
15
15
25
75
18
25
75
21
85
15
25
85
15
Expected Retention
Times:
Component
Retention time [Minutes]
Imidacloprid
10.4
Metalaxyl
17.1
Captan
19.5
TABLE 1
IMIDICLOPRID RESULTS
Percent of Averaqe
Number of Seeds
0-4%
-
5-15%
-
16-25%
-
26-35%
-
36-45%
-
46-55%
-
56-65%
1
66-75%
7
76-85%
8
86-95%
11
96-105%
11
106-115%
10
116-125%
6
126-135%
2
136-145%
3
146-155%
1
156-165%
-
166-175%
-
176-185%
-
186--195%
-
196-205%
-
206-215%
-
216-225%
-
226-235%
-
236-245%
-
246-250%
-
(Average =421 ppm, Target = 500 ppm)
TABLE 2
METALAXYL RESULTS
Percent of Averaqe
Number of Seeds
0-4%
-
5-15%
-
16-25%
2
26-35%
6
36-45%
7
46-55%
3
56-65%
3
66-75%
6
76-85%
1
86-95%
3
96-105%
4
106-115%
2
116-125%
4
126-135%
1
136-145%
3
146-155%
5
156-165%
1
166-175%
1
176-185%
3
186--195%
-
196-205%
1
206-215%
-
216-225%
1
226-235%
2
236-245%
1
246-250%
-
(Average = 21 ppm, Target = 20 ppm)
TABLE 3
CAPTAN RESULTS
Percent of Averaqe
Number of Seeds
0-4%
-
5-15%
-
16-25%
-
26-35%
-
36-45%
-
46-55%
-
56-65%
2
66-75%
6
76-85%
16
86-95%
11
96-105%
5
106-115%
7
116-125%
4
126-135%
5
136-145%
-
146-155%
-
156-165%
3
166-175%
-
176-185%
-
186--195%
-
196-205%
-
206-215%
-
216-225%
-
226-235%
-
236-245%
-
246-250%
-
(Average = 626 ppm, Target = 460 ppm)
Example 2- Analysis of thiamethoxam, fludioxonil and mefenoxam and myclobutanil
from cotton seeds.
A batch of cotton seed is treated with thiamethoxam (applied
as Cruiser® 5FS), fludioxonil (applied as Maxim® 4FS), mefenoxam (applied
as Apron XL® LS) and myclobutanil (applied as Systhane® WSP) with a target
concentration of 3000 ppm, 25 ppm, 75 ppm, and 210 ppm respectively. Tables 4-7
show the seed-to-seed distribution data for this treatment.
Eluent preparation (0.1% acetic acid in water): 1.8 ml
of glacial acetic acid is added to 1800 ml of deionized water. Stir well, filter
and degas prior to use.
Stock preparation: Accurately weigh in duplicate (standard
A and B) 0.0900- 0.1100 g of thiamethoxam primary standard, 0.0450- 0.0550 g of
fludioxonil primary standard, 0.0450- 0.0550 g mefenoxam primary standard and 0.0450-
0.0550 g of myclobutanil primary standard into separate 2-ounce bottles. Add 50
mL 50:50 acetonitrile: deionized water. Sonicate for 30 minutes and mechanically
shake for one hour.
Standard preparation: Add by pipette 40 mL of thiamethoxam
stock solution, 3 mL of mefenoxam stock solution, 1 mL of fludioxonil stock solution
and 8 ml myclobutanil stock solution to a 250 mL volumetric flask. Add all A weights
to one flask and label A, add all B weights to a separate flask and label B. Fill
each flask to volume with 50:50 water: acetonitrile. Invert several times to mix.
Individual cotton seed sample preparation: Transfer one
cotton seed into a scintillation vial. Add 3.0 mL of 50:50 acetontrile: deionized
water. Sonicate 30 minutes and mechanically shake for one hour. Filter with a 0.45
micron syringe filter prior to analysis.
INSTRUMENTATION
Perkin Elmer Series 410 LC pump or equivalent.
Perkin Elmer LC 235 Diode Array detector or equivalent.
Hewlett-Packard Series 1050 autosampler or equivalent
Hamilton MicroLab 1000 autodiluter or equivalent capable of delivering 25.00 ±
0.05 mL aliquots and 3.00 ± 0.05 mL aliquots.
LC Column - Prism RP 150 mm column with 4.6 mm internal diameter and 5 micron particle
size. Analytical balance with accuracy of ± 0.1 mg.
INSTRUMENT CONDITIONS
Detection: UV detection at 265 nm with 5 nm bandwidth and
simultaneously UV detection at 230 nm with 5 nm bandwidth
Injection Volume: 10 µl
Flow: 1.0 ml/min
Column Temperature:35°C
Run Time: Approximately 30 minutes
Gradient program (linear):
Time [minutes]
0.1 % acetic acid [%]
Acetonitrile [%]
0
85
15
5
85
15
20
25
75
23
25
75
26
85
15
30
85
15
Expected Retention
Times:
Component
Retention time [Minutes]
thiamethoxam
5.9
mefenoxam
16.6
myclobutanil
19.4
fludioxonil
20.2
TABLE 4
Thiamethoxam results
Percent of Averaqe
Number of Seeds
0-4%
-
5-15%
-
16-25%
-
26-35%
-
36-45%
-
46-55%
-
56-65%
1
66-75%
4
76-85%
18
86-95%
12
96-105%
4
106-115%
6
116-125%
5
126-135%
3
136-145%
4
146-155%
2
156-165%
-
166-175%
-
176-185%
-
186--195%
-
196-205%
1
206-215%
-
216-225%
-
226-235%
-
236-245%
-
246-250%
-
(Average = 2457 ppm, Target = 3000 ppm)
TABLE 5
Mefenoxam results
Percent of Average
Number of Seeds
0-4%
-
5-15%
-
16-25%
-
26-35%
-
36-45%
-
46-55%
2
56-65%
8
66-75%
10
76-85%
13
86-95%
7
96-105%
2
106-115%
3
116-125%
6
126-135%
3
136-145%
2
146-155%
1
156-165%
-
166-175%
-
176-185%
2
186--195%
-
196-205%
-
206-215%
-
216-225%
-
226-235%
-
236-245%
-
246-250%
-
>250%
1
(Average = 73 ppm, Target = 75 ppm)
TABLE 6
Fludioxonil results
Percent of Average
Number of Seeds
0-4%
-
5-15%
-
16-25%
-
26-35%
-
36-45%
-
46-55%
-
56-65%
1
66-75%
9
76-85%
12
86-95%
11
96-105%
7
106-115%
3
116-125%
7
126-135%
4
136-145%
4
146-155%
1
156-165%
-
166-175%
-
176-185%
-
186--195%
1
196-205%
-
206-215%
-
216-225%
-
226-235%
2
236-245%
-
246-250%
-
(Average = 21 ppm, Target = 25 ppm)
TABLE 7
Myclobutanil results
Percent of Average
Number of Seeds
0-4%
-
5-15%
-
16-25%
-
26-35%
-
36-45%
-
46-55%
-
56-65%
1
66-75%
7
76-85%
15
86-95%
12
96-105%
4
106-115%
7
116-125%
2
126-135%
5
136-145%
4
146-155%
2
156-165%
-
166-175%
-
176-185%
1
186-195%
-
196-205%
-
206-215%
-
216-225%
-
226-235%
-
236-245%
-
246-250%
-
(Average = 196 ppm, Target = 200 ppm)