1.0 INTRODUCTION
1.1 Scope of the Method
Analytical method GRM060.08A is suitable for the determination of Flumetralin
(CGA41065) (Figure 1) in soil. The limit of quantification (LOQ) of the method has been
established at 0.01 mg/kg (or 0.01 ppm, 10 ppb).
This method satisfies US EPA guideline OCSPP 850.6100 and EC Guidance Documents
SANCO/3029/99 rev 4 and SANCO/825/OO rev 8.1.
Additional clarification added to Section 8.3 in regards to LOQ.
Additional footnotes added to Tables 1&2.
1.2 Method Summary
10 g sub samples of soil are extracted with methanol: water (80/20 v/v). An aliquot of the
extract is diluted with aqueous sodium chloride and then partitioned into hexane: toluene
(50/50 v/v). Final determination is by GC-NICI-MSD.
The limit of quantification of the method is 0.01 mg/kg (0.01 ppm, 10 ppb).
2.0 MATERIALS AND APPARATUS
2.1 Apparatus
The recommended equipment and apparatus are listed in Appendix 1. Equipment with
equivalent performance specifications may be substituted.
2.2 Reagents
All solvents and other reagents must be of high purity, e.g. glass distilled/HPLC grade
solvents and analytical grade reagents. Particular care must be taken to avoid contamination
of the reagents used. Reagents of comparable purity may be substituted as long as acceptable
performance is demonstrated. A list of reagents used in this method along with details of
preparation of solutions is included in Appendix 2.
2.3 Preparation of Analytical Standard Solutions
It is recommended that the following precautions should be taken when weighing the
analytical materials.
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1. Ensure good ventilation.
2. Wear gloves and laboratory coat.
3. Prevent inhalation and contact with mouth.
4. Wash any contaminated area immediately.
2.3.1 Stock Solutions
Prepare individual 100 |j,g/mL stock solutions for flumetralin (CGA41065) by one of the
following methods.
Weigh out accurately, using a five figure balance, sufficient flumetralin (CGA41065)
analytical standard to give a 100 |ag/m L solution (>10 mg, after correction for purity) and
transfer into a "Class A" volumetric flask (100 mL). Dilute with an appropriate volume of
acetone to give a stock solution of exactly 100 |j,g/mL of flumetralin (CGA41065). Transfer
the standard into an amber bottle for storage.
Alternatively, the appropriate volume of acetone to add to a known amount of standard
material may be determined using the equation below. The standard concentration is
corrected for its chemical purity.
1000 = Unit conversion factor
In this case, the standard material is weighed directly into an appropriate storage vessel.
2.3.2 Fortification Solutions
Sample fortification solution containing flumetralin (CGA41065) should be prepared by
serial dilution in acetone. It is recommended that the following solutions are prepared: 10.0
Hg/mL, 1.0 |j,g/mL and 0.1 |j,g/mL.
2.3.3 Preparation of Calibration Standards for GC-MSD
No significant suppression or enhancement of the instrument response for flumetralin
(CGA41065) has been observed in the soil types tested using the procedures described in
Section 3 during method validation and non-matrix standards should normally be used for
calibration.
A calibration curve should be generated to quantify flumetralin (CGA41065) residues. At
least 5 standards ranging from 0.25 pg/|iL to 10.0 pg/|iL flumetralin (CGA41065) should be
x1000
C
P
V
w
c
Standard purity in decimal form (P(%)/100)
Volume of acetone required
Weight, in mg, of the solid analytical standard
Desired concentration of the final solution, (|j,g/mL)
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prepared in hexane: toluene (50/50 v/v). Recommended concentrations: 0.25 pg/|iL, 0.5
pg/|iL, 1.0 pg/|iL, 2.5 pg/|iL, 5.0 pg/|iL, and 10.0 pg/|iL
2.3.4 Standard Solution Storage and Expiration
All stock solutions should be stored in a refrigerator when not in use to prevent
decomposition and/or concentration of the standard. Standard solutions should be allowed to
equilibrate to room temperature prior to use.
An expiration date of one month for flumetralin (CGA41065) in acetone is recommended
unless additional data are generated to support a longer expiration date.
2.4 Safety Precautions and Hazards
The following information is included as an indication to the analyst of the nature and
hazards of the reagents used in this procedure. If in any doubt, consult the appropriate
MSDS or a monograph such as 'Hazards in the Chemical Laboratory', edited by S G Lux on,
The Chemical Society, London (Reference 1).
Solvent and Reagent Hazards
Toluene
Hexane
Acetone
Methanol
Harmful Vapour
~
~
~
~
Highly Flammable
~
~
~
Harmful by Skin Absorption
~
~
~
~
Irritant to respiratory system and eyes
~
V
~
~
OES Short Term (mg/m3)
384
N/A
3620
333
OES Long Term (mg/m3)
191
75
1210
266
N/A not known
Suitable personal protective equipment should be worn when handling chemicals and
reagents. The appropriate SDS should be consulted for each reagent and a local risk
assessment should be carried out. In all cases avoid breathing vapor. Avoid contact with
eyes and skin.
3.0 ANALYTICAL PROCEDURE
A summary of the method is included in flow-chart form in Appendix 4.
3.1 Sample Preparation
All samples should be prepared using an approved method of preparation to obtain a
homogeneous sample prior to analysis.
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3.2 Sample Fortification
In order to verify method performance and allow recovery corrections to be made (if
appropriate), fortified control samples should be included with each sample set. To each pre-
weighed control soil sample, fortify using flumetralin (CGA41065) in acetone using volumes
less than 1 mL. Let each sample stand for at least five minutes after fortification to allow the
spiking solution to soak into the matrix before proceeding with the extraction procedure. At
least one untreated control and two fortified control samples should be analysed with each
sample set.
3.3 Extraction
a) Weigh a representative amount of soil (10 g) into a 150 mL polypropylene bottle.
b) Add 100 mL methanol: water (80/20 v/v).
c) Place on a mechanical shaker and shake at 275 rpm or at a speed that visually agitates
sample for 2 hours.
d) Centrifuge at 3500 rpm for 5 minutes.
3.4 Liquid-liquid Partition
a) Transfer 15 mL of the extract into a polypropylene centrifuge tube (50 mL).
b) Add 15 mL aqueous saturated sodium chloride and 5 mL hexane: toluene (50/50 v/v).
c) Cap and place on a mechanical shaker and shake at 275 rpm for 10 minutes.
d) Centrifuge at 3500 rpm for 5 minutes.
e) Transfer 1.0 mL of the organic layer (upper) into a clean polypropylene centrifuge
tube (15 mL).
f) Dilute to 4 mL with hexane: toluene (50/50 v/v).
g) Further dilutions using hexane: toluene (50/50 v/v) can performed at this point if
instrument sensitivity permits.
h) Transfer final fraction to a suitable autosampler vial. Final determination is by GC-
NICI-MSD. The final sample concentration is 0.075 g/mL.
3.5 Time Required for Analysis
The methodology is normally performed with a batch of 12 samples. One person can
complete the analysis of 12 samples in 1 day (8 hour working period).
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3.6 Method Stopping Points
The analytical procedure can be stopped at various points for overnight and weekend breaks
unless otherwise specified in the analytical procedure. Acceptable method recoveries will
validate any work flow interruptions. Samples should be stored refrigerated in sealed
containers where the analysis cannot be completed in a single day.
4.0 FINAL DETERMINATION
The method has been developed for use on an Agilent 7890B GC with 5977B MSD. The
following instrumentation and conditions have been found to be suitable for this analysis.
Other instrumentation can also be used, though optimisation may be required to achieve the
desired separation and sensitivity. The operating manuals for the instruments should always
be consulted to ensure safe and optimum use.
4.1 Instrument Description
GC
Detector
Autosampler
Agilent 7890B
Agilent 5977B
Agilent 7693
4.2 Chromatography Conditions
Column
Injection Port
Carrier Gas
Injection Mode
Purge Time
Injection Volume
Injector Temperature
Transfer Line Temperature
Ion Source Temperature
Ouadrupole Temperature
HP-5MS (30.0m x 0.25mm x 0.25|im)
GooseNeck Carbofrit liner (Restek 20799-209.5)
Helium at 1.0 mL/min
Pulsed Splitless (pressure 30 psi)
1 minutes
2 |iL
250°C
280°C
150°C
150°C
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Oven Temperature Gradient
Steo
Rate PC/min)
Temperature
Time (min)
1
-
120
1
1
20
300
2
Under these conditions the retention time for flumetralin (CGA41065) is approximately 9.3
minutes.
4.3 Mass Spectrometer Conditions
Ionization Mode
Polarity
Calibration
Analyte
Target Ion
Qualifier 1
Qualifier 2
Ion Ratio
Chemical (NICI)
Negative
AutoTune
Flumetralin (CGA41065)
421 m/z
423 m/z
391 m/z
100:70:20
4.4 Confirmatory Procedures
Final determination by GC-MS with two qualifier ions is considered to be highly specific;
hence no further confirmatory conditions are included.
5.0 CALCULATION OF RESULTS
5.1 Multi-Point Calibration Procedure
Flumetralin (CGA41065) residues may be calculated in mg/kg for each sample as follows.
a) Prepare standard solutions over a concentration range appropriate to the expected
residues in the samples (30% LOQ to at least 20% above the highest fortified level as
a minimum). An appropriate number of different concentrations within this range
should be prepared (at least five).
b) Make an injection of each sample solution and measure the areas of the peaks
corresponding to flumetralin (CGA41065). Calibration standard solutions should be
interspersed throughout the analysis, bracketing the first and last samples analysed in
the run.
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c) Generate calibration curve parameters using an appropriate regression package.
d) The following equation can be rearranged and used to calculate residues as follows:
y = mx + c
Where y is the instrument response value, x is the standard concentration, m is the
gradient of the line of best fit ("X-variable 1" in MS Excel) and c is the intercept
value. An example of this equation generated using the experimental values of m and
c should be included in the raw data, as should the "R-Squared" value for the
regression.
Re-arrangement for x gives
m
e) Calculate the flumetralin (CGA41065) residue in the sample, expressed as mg/kg, as
follows
Analyte found (//g/mL)
Residue (mg/kg) = ¦
Sample conc. (g/mL)
Where analyte found (ng/mL) is calculated from the standard calibration curve and
sample conc. is the final sample concentration in g/mL.
If residues need to be corrected for average percentage recovery e.g. for storage stability
studies, then the equation below should be used.
_ Residuex 100 . „ .
Corrected Residue = (mg/kg)
Average percentage Recovery
5.2 Single-Point Calibration Procedure
Flumetralin (CGA41065) residues may be calculated in mg/kg for each sample using a mean
standard response from each of the injections bracketing the sample as follows.
a) Make repeated injections of a standard containing flumetralin (CGA41065) at an
appropriate concentration into the GC-MSD operated under conditions as described in
Section 4. When a consistent response is obtained, measure the peak areas obtained
for flumetralin (CGA41065).
b) Make an injection of each sample solution and measure the areas of the peak
corresponding to flumetralin (CGA41065).
c) Re-inject the standard solution after a maximum of five injections of sample
solutions.
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d) Calculate the flumetralin (CGA41065) residue in the sample, expressed as mg/kg
using a mean standard response from each of the injections bracketing the sample as
follows.
„ . , , ,, . PK area (SA) Standard Cone.
Residue (mg/kg) = x
PK area (STD) Sample Cone.
PK area (SA) = Peak response for sample
PK area (STD) = Average peak response for bracketing standards
Standard Cone. = Concentration of standard (ng/mL)
Sample Cone. = Sample concentration (g/mL)
If residues need to be corrected for average percentage recovery e.g. for storage stability
studies, then the equation below should be used.
_ , _ . , Residue xl 00 . „ .
Corrected Residue = (mg/kg)
Average percentage Recovery
Although single point calibration may be used to quantify residues it is recommended that a
calibration curve is generated with each analytical run to demonstrate the linearity of
instrument response (Reference 3).
6.0 CONTROL AND RECOVERY SAMPLES
Control samples should be analysed with each set of samples to verify that the sample used to
prepare recovery samples is free from contamination. A minimum of one control should be
analysed with each batch of samples.
At least two recovery samples (control samples accurately fortified with known amounts of
flumetralin (CGA41065) in acetone) should also be analysed alongside each set of samples.
Provided the recovery values are acceptable they may be used to correct any residues found.
The fortification levels should be appropriate to the residue levels expected.
Recovery efficiency is generally considered acceptable when the mean values are between
70% and 110% and with a relative standard deviation of <20%.
Where the method is used for monitoring purposes, control and recovery samples are not
required where suitable control samples are not available.
7.0 SPECIFICITY
It is recommended that reagent blank samples be included in a sample set if contamination is
suspected.
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7.1 Matrix Effect
No significant interference arising from the matrices tested has been observed.
7.2 Reagent and Solvent Interference
Using high purity solvents and reagents no interference has been found.
7.3 Labware Interference
This method uses mainly disposable labware. All reusable glassware should be detergent
washed and then rinsed with HPLC grade methanol, acetone or acetonitrile prior to use.
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APPENDIX 3 GC-MS Tuning Procedure
Calibration of Instrument
The instalment must be mass calibrated on a regular basis. Perform instrument auto tune of
compound specific tune using specific calibration masses.
Tuning Instrument for flumetralin
Determine ionization mode and detection (EI or CI).
Perform scan of expected masses. Determine target ion and qualifier ions. Target plus two
qualifiers above 100 amu are recommended.
For flumetralin, in negative ion chemical ionization mode, the deprotonated molecular ion
generated is selected {m/z 421) as the target ion. The two most sensitive qualifier ions (m/z
423 and m/z 391) are then selected for confirmation.
Daughter Ion
m/z
Structure
423
37CI isotope
391
Loss of H2O
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APPENDIX 4 Method Flow Chart
Weigh 10 g of soil into a 150 mL plastic (Nalgene)
Add 100 mL methanol: water (80:20 v/v) and shake 1 hour
4,
Centrifuge (3500 rpm)
4,
Perform LLP with salt using 15 mL extract and 5 mL toluene: hexane (50:50 v/v)
4,
Centrifuge (3500 rpm)
4,
Dilute 1 mL supernatant (organic phase) to a final volume of 4 mL using toluene: hexane
(50:50 v/v)
Transfer 1 mL to autosampler vial and analyse by GC-NICI-MSD
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