U.S. Department of the Interior

U.S. Geological Survey

Techniques and Methods 5–A9

Methods of Analysis by the U.S. Geological Survey 

Organic Geochemistry Research Group—Determination 

of Dissolved Isoxaflutole and Its Sequential 

Degradation Products, Diketonitrile and Benzoic Acid, 

in Water Using Solid-Phase Extraction and Liquid 

Chromatography/ Tandem Mass Spectrometry

Methods of Analysis by the U.S. Geological 

Survey Organic Geochemistry Research 

Group—Determination of Dissolved 

Isoxaflutole and Its Sequential Degradation 

Products, Diketonitrile and Benzoic Acid, 

in Water Using Solid-Phase Extraction 

and Liquid Chromatography/Tandem Mass 

Spectrometry

By Michael T. Meyer, Edward A. Lee, and Elisabeth A. Scribner

Techniques and Methods 5–A9

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

DIRK KEMPTHORNE, Secretary

U.S. Geological Survey

Mark D. Myers, Director

U.S. Geological Survey, Reston, Virginia: 2007

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Suggested citation:

Meyer, M.T., Lee, E.A., and Scribner, E.A., 2007, Methods of analysis by the U.S. Geological Survey Organic Geochemistry Research Group—determination of dissolved isoxaflutole and its sequential degradation products, diketonitrile 

and benzoic acid, in water using solid-phase extraction and liquid chromatography/tandem mass spectrometry: U.S. 

Geological Survey Techniques and Methods, book 5, chap. A9, 13 p.

Cover: Photograph showing liquid chromatograph/mass spectrometer used in analysis of isoxaflutole and its sequential degradation products, diketonitrile and benzoic acid.
iii

Contents

Abstract............................................................................................................................................................. 1

Introduction....................................................................................................................................................... 1

Purpose and Scope ......................................................................................................................................... 2

Analytical Method............................................................................................................................................ 2

 1. Scope and Application............................................................................................................... 2

 2. Summary of Method................................................................................................................... 3

 3. Safety Precautions ..................................................................................................................... 3

 4. Apparatus and Instrumentation ............................................................................................... 3

 5. Reagents and Consumable Materials ..................................................................................... 4

 6. Standards Preparation Procedure........................................................................................... 4

 7. Solid-Phase Extraction Procedure .......................................................................................... 5

 8. Instrument Analysis Procedure................................................................................................ 5

 9. Quality Assurance and Quality Control (QA/QC).................................................................... 7

 10. Calculation of Results ................................................................................................................ 8

 11. Reporting of Data Results.......................................................................................................... 9

 12. Method Performance................................................................................................................. 9

Conclusions....................................................................................................................................................... 12

References Cited.............................................................................................................................................. 12

Figure

1. Diagram showing chemical structures of isoxaflutole and its sequential degradation 

products, diketonitrile and benzoic acid analogue................................................................... 2

Tables

1. U.S. Geological Survey method number, method code, parameter code, Organic 

Geochemistry Research Laboratory code, and molecular weight for isoxaflutole and 

its sequential degradation products, diketonitrile and benzoic acid analogue................... 3

2. Liquid chromatography/tandem mass spectrometry analytical sequence for use in 

determining isoxaflutole and its sequential degradation products, diketonitrile and benzoic 

acid, in water................................................................................................................................... 6

3. Retention times and multiple-reaction monitoring molecular (precursor)-ion and 

product-ion transitions for isoxaflutole, and its two sequential degradation products, and 

the internal and surrogate standards.......................................................................................... 8

4. Summary of mean solid-phase extraction recovery and relative standard deviation 

percentages in buffered reagent water for isoxaflutole, diketonitrile, benzoic acid, and 

the surrogate standard .................................................................................................................. 10

5. Summary of mean concentrations, standard deviations, and mean normalized 

percentage of theoretical spiked concentrations and relative percentage standard 

deviations for isoxaflutole, diketonitrile, benzoic acid, and the surrogate standard in 

four water matrices........................................................................................................................ 11
iv

6. Combined mean concentrations, standard deviations, and combined mean normalized 

percentage of theoretical spiked concentrations and relative percentage standard 

deviations for isoxaflutole, diketonitrile, benzoic acid, and the surrogate standard in 

four water matrices........................................................................................................................ 12

Conversion Factors, Abbreviated Water-Quality Units, 

and Other Abbreviations 

Multiply By To obtain

acre 4,047 square meter (m2)

kilopascal (kPa) 0.1450 pound-force per square inch (lb/in2)

liter (L) 0.2642 gallon (gal)

microliter (μL) 2.642 x 10-7 gallon (gal)

micrometer (μm) 3.937 x 10-5 inch (in.)

millimeter (mm) 0.03937 inch (in.)

nanogram (nm) 3.527 x 10-11 ounce (oz)

ounce, fluid (oz) 0.02957 liter (L)

gram (g) 0.002204 pound (lb)

Temperature can be converted to degrees Celsius ( ̊C) or degrees Fahrenheit ( ̊F) by the 

equations: 

 ̊C = 5/9 ( ̊F -32)

 ̊F = 9/5 ( ̊C) +32.

Abbreviated Water-Quality Units

gram (g)

liter per hour (L/hr)

microgram per liter (μg/L)

microgram per milliliter (μg/mL)

microliter per minute (μL/min)

milligram (mg)

milligram per milliliter (mg/mL)

milliliter (mL)

milliliter per minute (mL/min)

nanogram per microliter (ng/μL)
Other Abbreviations or Acronyms Used in This Report

± plus or minus

ACN acetonitrile

ACS American Chemical Society

BA benzoic acid

c-18 carbon-18

CAS Chemical Abstracts Service

cc cubic centimeter

CCV continuous calibration verification

COB carryover blank

DF dilution factor

DKN diketonitrile

ESI electrospray ionization

HLB hydrophilic-liophilic balance

HPLC high-performance liquid chromatography

ISTD internal standard

kPa kilopascal

LC/MS/MS liquid chromatography/tandem mass spectrometry

LD laboratory duplicate

MDL method detection limit

MRL method reporting level

MRM multiple-reaction monitoring

m/z mass-to-charge ratio

M-H molecular-hydrogen

OGRL Organic Geochemistry Research Laboratory

QA quality assurance

QC quality control

r

2 correlation coefficient

RF radio frequency

RRTc relative retention time

RT expected retention time

SD standard deviation

SPE solid-phase extraction

USEPA U.S. Environmental Protection Agency

USGS U.S. Geological Survey

v/v volume-to-volume

V voltage

WP water pollution
vi
Abstract

An analytical method for the determination of isoxaflutole and its sequential degradation products, diketonitrile and a 

benzoic acid analogue, in filtered water with varying matrices was developed by the U.S. Geological Survey Organic 

Geochemistry Research Group in Lawrence, Kansas. Four 

different water-sample matrices fortified at 0.02 and 

0.10 μg/L (micrograms per liter) are extracted by vacuum 

manifold solid-phase extraction and analyzed by liquid 

chromatography/tandem mass spectrometry using electrospray ionization in negative-ion mode with multiple-reaction 

monitoring (MRM). Analytical conditions for mass spectrometry detection are optimized, and quantitation is carried out 

using the following MRM molecular-hydrogen (precursor) 

ion and product (p) ion transition pairs: 357.9 (precursor), 

78.9 (p), and 277.6 (p) for isoxaflutole and diketonitrile, and 

267.0 (precursor), 159.0 (p), and 223.1 (p) for benzoic acid. 

2,4-dichlorophenoxyacetic acid-d3 is used as the internal 

standard, and alachlor ethanesulfonic acid-d5 is used as the 

surrogate standard.

 Compound detection limits and reporting levels are 

calculated using U.S. Environmental Protection Agency 

procedures. The mean solid-phase extraction recovery values 

ranged from 104 to 108 percent with relative standard deviation percentages ranging from 4.0 to 10.6 percent. The 

combined mean percentage concentration normalized to the 

theoretical spiked concentration of four water matrices analyzed eight times at 0.02 and 0.10 μg/L (seven times for the 

reagent-water matrix at 0.02 μg/L) ranged from approximately 

75 to 101 percent with relative standard deviation percentages 

ranging from approximately 3 to 26 percent for isoxaflutole, 

diketonitrile, and benzoic acid. The method detection limit 

(MDL) for isoxaflutole and diketonitrile is 0.003 μg/L and 

0.004 μg/L for benzoic acid. Method reporting levels (MRLs) 

are 0.011, 0.010, and 0.012 μg/L for isoxaflutole, diketonitrile, 

and benzoic acid, respectively. On the basis of the calculated 

MRLs and MDLs and evaluation of the signal-to-noise ratios 

for each compound, the MRLs and MDLs are set at 0.010 and 

0.003 μg/L, respectively, for all three compounds. 

Introduction

Isoxaflutole, a member of the benzoyl isoxazole family, 

is a low-application-rate preemergence herbicide for control 

of annual broadleaf weeds and some grasses in field corn and 

sugar cane (Pallet and others, 2001). Isoxaflutole, also known 

by its trade name Balance, degrades sequentially through 

hydrolysis to the biologically active diketonitrile (DKN) 

degradation product through the opening of the isoxazole ring 

which then degrades to a benzoic acid analogue (BA; Viviani 

and others, 1998) that is biologically inactive. Diketonitrile 

also is more water soluble than isoxaflutole, and therefore, 

its transport to surface or ground water is a concern (Lin and 

others, 2002).

For example, isoxaflutole is applied in Iowa at a rate that 

is 8 percent of the rate of atrazine application and 4 percent 

of the rate of metolachlor application and is only applied to 

24 percent of the number of acres to which atrazine is applied 

(U.S. Department of Agriculture, 2004). Thus, the frequent 

detection of the sequential herbicidally active degradation 

product of isoxaflutole, DKN, and BA was unexpected during 

a 2004 study to document the occurrence of these compounds 

and to compare their occurrence with that of more commonly 

measured herbicides such as atrazine and metolachlor (Scribner and others, 2006). Analytical results of 75 water samples 

collected in 2004 showed that isoxaflutole was detected in four 

of the samples collected during the post-planting (May-June) 

period. Diketonitrile was detected in 53 water samples, and 

Methods of Analysis by the U.S. Geological Survey 

Organic Geochemistry Research Group—Determination 

of Dissolved Isoxaflutole and Its Sequential Degradation 

Products, Diketonitrile and Benzoic Acid, in Water Using 

Solid-Phase Extraction and Liquid Chromatography/

Tandem Mass Spectrometry

By Michael T. Meyer, Edward A. Lee, and Elisabeth A. Scribner
benzoic acid was detected in 41 water samples collected from 

all three sampling periods. Metolachlor was the most frequently detected acetamide parent (59 of 60 samples) during 

all three sampling periods, and atrazine was the most detected 

triazine parent compound with 65 detections of 69 samples 

during all three sampling periods.

Purpose and Scope

The purpose of this report is to describe a new analytical 

method. The method described in this report was developed 

by the U.S. Geological Survey (USGS) Organic Geochemistry 

Research Laboratory (OGRL), Lawrence, Kansas, to analyze 

dissolved isoxaflutole [5-cyclopropyl-4-(2-methylsulfonyl4-(trifluoromethylbenzoyl) isoxazole] and its sequential 

degradation products (fig. 1), diketonitrile [1-(2-methylsulfonyl-4-trifluoromethylphenyl)-2-cyano-3-cyclopropyl propan1,3-dione], and benzoic acid analogue [2-methylsulfonyl4-(trifluoromethyl) benzoic acid], by solid-phase extraction 

(SPE) and liquid chromatography/tandem mass spectrometry 

(LC/MS/MS) using electrospray ionization in negative-ion 

mode. This report also provides method detection and reporting limits, mean extraction recoveries, and relative standard 

deviations and mean concentration of two spiked levels in four 

water matrices.

The method of analysis described in this report is 

assigned a USGS method number O–2140–07, USGS method 

code LCM47, and OGRL code LCIX. These unique codes 

represent the automated method of analysis as it is described 

in the report and can be used to identify the method. This 

method will contribute to an improved understanding of the 

occurrence, persistence, and transport of isoxaflutole and its 

degradation products in the environment.

Analytical Method

Organic Compounds and Parameter Codes: Isoxaflutole 

and its sequential degradation products, diketonitrile and 

benzoic acid, in filtered water using solid-phase extraction and 

liquid chromatography/mass spectrometry/mass spectrometry, USGS method number O–2140–07, USGS method code 

LCM47, and OGRL code LCIX.

1. Scope and Application

This method is suitable for the determination of low-level 

concentrations (in micrograms per liter) of isoxaflutole and its 

two sequential degradation products, DKN and BA, in water 

samples (table 1). Because suspended particulate matter is 

removed from the samples by filtration, the method is suitable only for filtered-phase compounds. The calibration range 

for the method is equivalent to concentrations from 0.005 to 

0.200 μg/L.

Figure 1. Chemical structures of isoxaflutole and its sequential 

degradation products, diketonitrile and benzoic acid analogue.

 Determination of Dissolved Isoxaflutole and Its Sequential Degradation Products, Diketonitrile and Benzoic Acid

C

N

O

Isoxaflutole [5-cyclopropyl-4-(2-methylsulfonyl-4-

(trifluoromethylbenzoyl) isoxazole] 

Diketonitrile of isoxaflutole [1-(2-methylsulfonyl-4-

trifluoromethylphenyl)-2-cyano-3-cyclopropyl propan-1, 

3-dione] 

Benzoic acid analogue [2-methylsulfonyl-4-

(trifluoromethyl) benzoic acid] 

SO2CH3

SO2CH3

F3C

F3C

SO2CH3

F3C

O

O

OH

O

O

N
2. Summary of Method 

Water samples are collected in the field by using the 

methods outlined in either Shelton (1994), Wilde and others 

(1998), or Webb and others (1999). Samples are then chilled 

immediately and shipped to the OGRL and refrigerated at 

approximately 4 oC until analysis.

In the laboratory, filtered water samples spiked with 

surrogate standards are passed through an Oasis hydrophilicliophilic balanced (HLB) cartridge (Waters Corporation, 

Milford, Massachusetts). The adsorbed compounds are eluted 

from the cartridge with 50:50 methanol and acetonitrile and 

spiked with an internal standard. The sample eluate is evaporated further under nitrogen.

The sample components are separated, identified, and 

quantified by injecting an aliquot of the concentrated sample 

extract into a LC/MS/MS with electrospray ionization (ESI) 

in negative-ion mode using multiple-reaction monitoring 

(MRM). Compounds separated on the LC are identified by 

comparing the retention times of the mass spectral signals 

against the retention times of standards analyzed under the 

same conditions used for the samples. Compounds are identified further by measuring pre-optimized MRM molecular 

(precursor) ion and product ion transitions. The concentration 

of each identified compound is calculated by determining the 

ratio of the MS area response produced by the quantitation 

daughter ion to the MS area response produced by the quantitation daughter ion of the internal standard.

3. Safety Precautions

The following safety precautions are followed:

3.1 All steps involving organic solvents and strong acids are 

performed under a well-vented fume hood.

3.2 Appropriate personal protective equipment is used during 

the handling of reagents and chemicals.

3.3 The electrospray waste exhaust and the vacuum pump 

exhaust are vented through a laboratory hood system.

3.4 Precautions are taken when handling columns or working 

with the spray chamber of the MS as temperatures are in 

excess of 300 oC. These areas are allowed to cool before 

touching them.

4. Apparatus and Instrumentation 

The following apparatus and instrumentation are used:

4.1 Analytical balance—capable of accurately weighing 

0.050 g + 0.0001 g.

4.2 Autopipettes—10- to 10,000-μL, variable-volume autopipettes with disposable plastic tips (Rainin, Woburn, 

Massachusetts).

4.3 Mechanical vortex mixer.

4.4 Test tube rack—40 holders for 10-mL conical bottom 

centrifuge tubes.

4.5 Vacuum manifold for SPE.

4.6 Cartridges—Oasis hydrophilic-liophilic balanced, 6 cc, 

500-mg, extraction cartridges (Waters Corporation, 

Milford, Massachusetts).

4.7 Automated solvent evaporator—Heat bath temperature 

maintained at 45 oC and the nitrogen gas pressure at 

103 kPa (Turbovap LV, Zymark, Inc., Hopkinton, 

Massachusetts).

4.8 Analytical column—Keystone Scientific 250- x 4.6-mm 

Betasil C-18 column with 5-μm packing (Bellefonte, 

Pennsylvania).

4.9 HPLC/MS benchtop system—Agilent (Wilmington, Delaware) model 1100 HPLC with autoinjector and MS detector and Waters (Milford, Massachusetts) Quattro Micro 

API triple-stage quadrupole mass spectrometer system 

using ESI mode. 

Analytical Method   

Table 1. U. S. Geological Survey method number, method code, parameter code, Organic Geochemistry Research Laboratory code, and molecular 

weight for isoxaflutole and its sequential degradation products, diketonitrile and benzoic acid analogue. 

 [--, not applicable; USGS, U.S. Geological Survey; OGRL, Organic Geochemistry Research Laboratory]

Compound

Chemical 

Abstract

Registry

number

Chemical

name

USGS

method 

number

USGS

method 

code

USGS

parameter

code

OGRL

code

Molecular

weight

Isoxaflutole 141112–29–0 [5-cyclopropyl-4-(2-methylsulfonyl-4-

(trifluoromethylbenzoyl) isoxazole]

O–2140–07 LCM47 62733 LCIX 359.32

Diketonitrile -- [1-(2-methylsulfonyl-4-trifluoromethylphenyl)-

2-cyano-3-cyclopropyl propan-1,3-dione]

O–2140–07 LCM47 63011 LCIX 359.32

Benzoic acid 

analogue

-- [2-methylsulfonyl-4-(trifluoromethyl) benzoic acid] O–2140–07 LCM47 62656 LCIX 268.21
4.10 HPLC system—computer with Agilent chemstation 

software.

4.11 Mass spectrometer system software—MassLynx software 

(Waters Corporation, Milford, Massachusetts) is used to 

operate the MS and acquire and store data. QuanLynx 

software (Waters Corporation, Milford, Massachusetts) is 

used for quantitation of compounds for the MS.

5. Reagents and Consumable Materials

The following reagents and consumable materials are 

used:

5.1 Analytical standards—solutions of isoxaflutole, diketonitrile, and benzoic acid (U.S. Environmental Protection 

Agency, Fort Meade, Maryland), the surrogate, and the 

internal standard (ISTD), and calibration curve.

5.2 Internal standard solution––The ISTD, 2,4-dichlorophenoxyacetic acid-d3

, is purchased from Cambridge Isotope 

Laboratory, Andover, Massachusetts.

5.3 Surrogate standard solution––The surrogate, alachlor 

ethanesulfonic acid (ESA)-d5, is provided by Monsanto 

(St. Louis, Missouri).

5.4 Reagent water—generated by purification of tapwater 

through activated charcoal filter and deionization with 

a high-purity, mixed-bed resin, followed by another 

activated charcoal filtration, and finally distillation in an 

autostill (Barnstead, Dubuque, Iowa).

5.5 Formic acid—chemical reagent, American Chemical Society (ACS) grade.

5.6 Glacial acetic acid—chemical reagent, ACS grade.

5.7 Humic acid––chemical reagent.

5.8 Water pollution (WP) minerals A and B

5.9 Solvents—

5.9.1 Acetonitrile, ACS and HPLC grade.

5.9.2 Methanol, ACS and HPLC grade.

5.10 Sample bottles—baked 4-oz amber glass bottles 

(Boston round) withTeflon-lined lids (VWR, West 

Chester, Pennsylvania).

5.11 Disposable plastic test tubes—(Fisher Scientific, 

Pittsburg, Pennsylvania).

5.12 Sample filters—nominal 0.7-μm glass-fiber filters 

(Gilson, Middleton, Wisconsin).

5.13 Plastic rack—40 holes for tubes.

5.14 SPE cartridges—Oasis HLB extraction, Prospekt 

(10-mm x 2-mm) cartridges (Waters, Milford, 

Massachusetts).

5.15 Gas for mass spectrometer—nitrogen; argon.

6. Standards Preparation Procedure

6.1 Primary standard solutions––Individual stock solutions 

(corrected for purity) of 1.0 mg/mL for isoxaflutole and 

its sequential degradation products, DKN and BA, are 

prepared by accurately weighing, to the nearest 0.0001 g, 

50 mg of the pure material into a 50-mL volumetric 

flask with acetonitrile (ACN). The solutions are stored at 

-10 oC.

6.2 Intermediate composite standard––30 mL of intermediate 

standard mix containing 12.3 ng/μL of isoxaflutole, DKN, 

and BA are prepared by diluting individual 1-mg/mL 

stock solutions into acetonitrile.

6.3 Working standard mix––Using volumetric procedures, a 

5-mL, 0.123-ng/μL working standard mix is prepared 

from the intermediate standard mix by diluting it 1:100 

into 90/10 percent formic acid (1.1 percent, aqueous)/acetonitrile solution and then stored at -10 o

C. The working 

standard mix is prepared frequently.

6.4 Internal standard solution––The ISTD, 2,4-dichlorophenoxyacetic acid-d3

, is prepared, using volumetric procedures, at a concentration of 0.615 ng/μL in methanol from 

a 1-mg/mL stock solution.

6.5 Surrogate standard solution––The surrogate standard, 

alachlor ESA-d5, is prepared, using volumetric procedures, at a concentration of 0.615 ng/μL in methanol from 

a 1-mg/mL stock solution.

6.6 Preparation of reagent-water samples––Reagent-water 

blank and spike samples, including the calibration 

samples of 0.005, 0.010, 0.050, 0.100, and 0.200 μg/L, are 

prepared for extraction in 4-oz baked, amber glass bottles, 

by adding 75 mL of reagent water, 0.5 mL of 1-mg/mL 

aqueous humic acid solution (Sigma Aldrich, Allentown, 

Pennsylvania), and 0.25 mL of water pollution (WP) 

minerals A and B (Ultra Scientific, North Kingstown, 

Rhode Island). Spiked reagent-water (CCV, continuous 

calibration verification) and matrix-spiked samples are all 

spiked at 0.10 μg/L. The appropriate amount of working 

standard mix then is added to each reagent-water spike, 

and deionized water is added to bring the final volume of 

the calibration solutions to 123 mL.

6.7 Preparation of environmental samples—The filtered 

environmental samples are stored in 4-oz baked, amber 

glass bottles. If a sample bottle contained less than 123 

mL, reagent water is added to bring the volume of the 

   Determination of Dissolved Isoxaflutole and Its Sequential Degradation Products, Diketonitrile and Benzoic Acid
sample to 123 mL. The volume added is recorded, and the 

dilution factor is calculated. Samples selected for matrix 

spikes are prepared at concentrations of 0.10 μg/L by adding 100 μL of the working standard mix.

6.8 Adding solutions—All laboratory and environmental 

samples are prepared by adding 100 μL of 0.615-ng/μL 

surrogate and 100 μL of glacial acetic acid to all samples 

to help stabilize the isoxaflutole. The bottles then are 

capped and mixed well.

7. Solid-Phase Extraction Procedure

The SPE procedure requires a 40-position plastic testtube rack, a 12- or 24-position vacuum manifold, 10- or 

15-mL glass conical bottom test tubes, Oasis HLB SPE cartridges (6 mL, 500 mg), and a nitrogen evaporation apparatus 

with a hot-water bath. SPE preparation steps include the 

following:

7.1 Sample preparation––A full extraction set consists of 

60 environmental samples plus four laboratory duplicates, 

four environmental matrix spikes, two reagent-water 

(CCV) spikes, five reagent-water carryover blanks, and 

five reagent-water calibration standard samples (table 2).

7.2 Cartridge preparation––The appropriately labeled SPE 

cartridges are placed on a vacuum manifold and rinsed 

with 5 mL of 50:50 methanol and acetonitrile followed by 

5 mL of reagent water.

7.3 Loading sample to cartridges—SPE cartridge adapters are 

connected with tubing to the top of each SPE cartridge, 

and the end of the tubing placed into the sample bottles. 

Vacuum is applied, and the manifold adjusted to load 

sample onto cartridge in 15 to 20 minutes.

7.4 Rinsing cartridges—Cartridges are rinsed by adding 3 

mL of 1-percent formic acid (aqueous) under vacuum for 

1 minute.

7.5 Eluting compounds from the cartridge—Appropriately 

labeled 10-mL glass, conical bottomed tubes are placed in 

the vacuum manifold test-tube rack, making certain that 

the vacuum manifold valve stems are inside or directly 

above each test tube. Four milliliters (4 mL) of 50:50 

methanol/acetonitrile are added to each cartridge. A small 

amount of vacuum is applied to begin flow through the 

cartridge, and then the vacuum is turned off. After the 

solvent mixture drips through the cartridge, another 4 mL 

of 50:50 methanol/acetonitrile is passed through each 

cartridge. The test tubes are removed from the vacuum 

manifold. The second set of labeled test tubes are placed 

into the vacuum manifold rack. The SPE cartridges are 

eluted with two more 4-mL aliquots of 50:50 methanol 

and acetonitrile. The test tubes are removed from the 

vacuum manifold.

7.6 Spiking of internal standard—Add 100 μL of 0.615-ng/μL 

ISTD solution to the first set of test tubes and gently

vortex.

7.7 Evaporation—All samples in test tubes are placed in 

preheated 45 oC Turbovap and the lid closed. Nitrogen gas 

flow is turned on slowly to 15 lb/in2, and the sample eluates are evaporated. After all sample eluates have evaporated to 4 mL or less, the sample eluate from the second 

set of test tubes is combined with the sample eluates in 

the first set of test tubes. If volume is reduced to l mL 

or less in a test tube from the second set, 1 mL of 50:50 

methanol/acetonitrile is added to the test tube and gently 

vortexed before adding it to the appropriate test tube from 

the first set. Sample eluates are reduced to a final volume 

of 200 μL.

7.8 Transfer to vials—Sample eluates then are transferred 

to 11-mm, plastic autosampler vials with 200-μL glass 

inserts using glass Pasteur pipettes; a new pipette is used 

for each sample. The sample label is transferred from 

the sample test tube to the autosampler vial immediately 

before each sample is pipetted. The sample eluates then 

are stored in a freezer at –10 oC until analysis.

8. Instrument Analysis Procedure

Aliquots of the sample eluates are injected and the 

compounds separated on an Agilent 1100 model D series 

LC system with a Keystone 250- x 4.6-mm C-18 analytical 

column. The LC column is equilibrated with the mobile phase 

for 2 hours prior to analysis by running the LC method three 

times without an injection and then running the LC method at 

initial conditions for the remainder of the 2 hours. Mass spectral analysis was conducted using a Waters Quattro Micro API 

benchtop, triple quadrupole MS system with ESI in negativeion mode using MRM.

8.1 Sample analysis––The LC/MS/MS conditions for the 

analysis of isoxaflutole and its sequential degradation 

products are listed below.

8.1.1 LC conditions:

Injection volume––20 μL;

LC column oven conditions––50 oC.

8.1.2 LC mobile-phase:

A, 1.5-percent v/v glacial acetic acid in reagent 

water;

B, methanol.

Analytical Method   
Table 2. Liquid chromatography/tandem mass spectrometry analytical sequence for use in determining isoxaflutole and its sequential degradation products, diketonitrile and benzoic acid, in water.

[CCV, continuing calibration verification; COB, carryover blank; LD, laboratory duplicate; no., number; ppt, parts per trillion]

Analytical 

sequence Date

received Sample type Projectcode

Analytical 

sequence Date

received Sample type Project

Vial code

no.

Triathlon

no.

Vial

no.

Triathlon

no.

1 1 5 ppt Standard 32 1 CCV 1 ppt

2 2 10 ppt Standard 33 2 COB C

3 3 50 ppt Standard 34 3 Sample 1 Set C

4 4 100 ppt Standard 35 4 Sample 2 Set C

5 5 200 ppt Standard 36 5 Sample 3 Set C

6 6 COB A 37 6 Sample 4 Set C

7 7 Sample 1 Set A 38 7 Sample 5 Set C

8 8 Sample 2 Set A 39 8 Sample 6 Set C

9 9 Sample 3 Set A 40 9 Sample 7 Set C

10 10 Sample 4 Set A 41 10 Sample 8 Set C

11 11 Sample 5 Set A 42 11 Sample 9 Set C

12 12 Sample 6 Set A 43 12 Sample 10 Set C

13 13 Sample 7 Set A 44 13 Sample 1 Set C LD

14 14 Sample 8 Set A 45 14 Sample 10 Set C Spike

15 15 Sample 9 Set A 46 15 COB D

16 16 Sample 10 Set A 47 16 Sample 1 Set D

17 17 Sample 1 Set A LD 48 17 Sample 2 Set D

18 18 Sample 10 Set A Spike 49 18 Sample 3 Set D

19 19 COB B 50 19 Sample 4 Set D

20 20 Sample 1 Set B 51 20 Sample 5 Set D

21 21 Sample 2 Set B 52 21 Sample 6 Set D

22 22 Sample 3 Set B 53 22 Sample 7 Set D

23 23 Sample 4 Set B 54 23 Sample 8 Set D

24 24 Sample 5 Set B 55 24 Sample 9 Set D

25 25 Sample 6 Set B 56 25 Sample 10 Set D

26 26 Sample 7 Set B 57 26 Sample 1 Set D LD

27 27 Sample 8 Set B 58 27 Sample 10 Set D Spike

28 28 Sample 9 Set B 59 28 CCV 1 ppt

29 29 Sample 10 Set A 60 29 COB E

30 30 Sample 1 Set B LD 61 30

31 31 Sample 10 Set B Spike 62 31

32 32

   Determination of Dissolved Isoxaflutole and Its Sequential Degradation Products, Diketonitrile and Benzoic Acid
8.1.3 Gradient––

Time (min) Mobile-phase 

A (percent) 

Mobilephase B 

(percent)

0.00 90 10

1.00 90 10

1.01 60 40

10.00 20 80

16.00 20 80

8.1.4 Flow rate––0.40 mL/min.

8.1.5 MS/MS source parameters:

MS ionization mode––ESI in negative-ion mode;

Capillary voltage––2,000 V;

Extractor voltage––1 V;

Radio frequency (RF) lens voltage––0.1 V;

Source temperature––100 oC;

Desolvation temperature––350 oC;

Cone gas flow––40 L/hr; and

Desolvation gas flow––540 L/hr.

8.1.6 Quadrupole 1 parameters:

Low mass (LM 1) resolution––13;

High mass (HM 1) resolution––13; and

Ion energy––1.0.

8.1.7 Collision cell parameters:

Entrance—3 and Exit—3. 

8.1.8 Quadrupole 2 parameters:

Low mass (LM 2) resolution—15;

High mass (HM 2) resolution—15;

Ion energy––2.0; and

Photo multiplier––650 V.

8.1.9 Data acquisition and processing––The data are 

acquired using Waters MassLynx and quantified 

using the Waters QuanLynx data analysis program.

8.2 Liquid chromatograph (LC) performance evaluation––

Background absorbance readings, peak shape, and system 

pressure are used to evaluate LC performance. Background absorbance readings should remain stable and low 

and indicate that the LC column is equilibrated with the 

mobile-phase flow. If peak shape deteriorates, the columns may need to be cleaned or replaced. If the pressure 

reading is high, there may be a clog in the mobile-phase 

flow path or the column compartment thermostat may not 

have reached the required temperature.

8.3 Mass spectrometer (MS) performance evaluation––For 

the MS, a static, scanning, and scan-speed compensation 

mass calibration of the quadrupoles is performed every 

6 months unless mass “drift” is identified as a problem. 

The MRM transition parameters (cone voltage and collision energy) needed to maximize the response of the 

molecular-hydrogen (M-H, precursor)- and product-ion 

transitions for each compound are listed in table 3. The 

tune and MRM parameters are optimized by infusing 

50-mg/mL solutions of each individual compound into 

the mobile phase with a “T” fitting at 10 μL/min using 

a syringe pump. The MS tune parameters for the source 

and quadrupole 1 are adjusted to optimize the response of 

the M-H ion for isoxaflutole, DKN, BA, and the internal 

standard and surrogate standard and the collision cell, 

and quadrupole 2 parameters are adjusted to identify and 

optimize response of the daughter ions.

The MS performance is evaluated prior to each sample 

run by injecting 20 μL of the 0.123-ng/μL working standard 

mix and assessing product-ion abundances and ratios. If the 

abundances are low, the general tune parameters and compound specific tune parameters are reoptimized.

9. Quality Assurance and Quality Control 

(QA/QC)

The quality-control program consists of primarily internal 

checks on precision and accuracy of analytical results. Laboratory quality-control data from continuous calibration verification (CCV), laboratory blank and spike samples, and internal 

and surrogate standards are used by the analyst to determine 

if corrective actions are needed. Field sampling errors also are 

determined by the analyst.

9.1 Field sampling––Accuracy of the handling of samples in 

the field is monitored when field blanks and field replicates are included for analysis by the laboratory. Each 

sample is handled separately for proper data determination by the analyst.

9.2 Continuous calibration verification (CCV)––The CCV 

solutions, which are reagent-water spike samples, are used 

to monitor the method stability in comparison to the initial 

calibration curve. The CCV control limits are established 

at ± 25 percent of the expected concentration for each 

compound. If a CCV fails the QC criteria, the affected 

samples are reanalyzed. The CCV results are compiled for 

long-term method recovery performance used for creating 

control limits and charts. If the recovery of a compound is 

not within control limits, the source of the problem must 

be identified and corrected before continuing the procedure.

9.3 Internal standards––Internal standards are added to correct 

quantitative results for slight differences in extract volume 

as well as compensate for differences in the injected 

sample volume. They also are used to monitor instrument 

conditions, such as extract injection errors, retention time 

shifts, or instrument abnormalities or malfunctions.

Analytical Method   
9.4 Laboratory blank––The laboratory blank (carryover blank, 

COB) is reagent water prepared to monitor the entire 

sample preparation and analytical procedure for possible 

laboratory contamination. The blank is acceptable when a 

compound is undetected or detected at five times less than 

the method reporting limit (MRL). On the basis of data 

from this method, there are no interferences in the laboratory blanks.

9.5 Laboratory matrix spike––The laboratory matrix spike is 

an aliquot of an environmental sample to which known 

quantities of the method analytes are added in the laboratory. The laboratory matrix spike is analyzed exactly 

like a sample, and its purpose is to measure recovery by 

determining whether the sample matrix contributes bias 

to the analytical results and, therefore, determines to what 

degree the method is successful in analyzing the target 

analytes. The background concentrations of the analytes 

in the sample matrix must be determined in a separate 

aliquot, and the measured values in the laboratory matrix 

spike corrected for background concentrations.

9.6 Surrogate standards––Surrogate standards are compounds 

similar in physical and chemical properties to the method 

compounds but are not expected to be present in the 

environment. They are added to each environmental and 

QA/QC sample and used to monitor matrix effects and 

overall method performance. Their recoveries are not used 

to correct compound concentrations in environmental 

samples.

9.7 Instrumental analysis quality control––A sample analytical 

sequence used for this method is listed in table 2. Sample 

extracts are analyzed in an instrument sequence to provide 

additional information to facilitate corrective actions that 

might be required if performance criteria are not met.

10. Calculation of Results

Before quantitative results are reported, each compound 

first needs to meet qualitative criteria.

10.1 Qualitative Identification—Identification and quantitation 

of compounds are performed on the raw data files using 

MassLynx with the Quan-Lynx data analysis package. 

A compound is not identified correctly unless the correct molecular (precursor)- to product-ion transitions are 

detected, the relative ratio of the confirming to quantitation daughter ion is within ± 25 percent of the average 

ratio obtained from the calibration samples, and the 

relative retention time is within 5 percent of the expected 

retention time.

10.2 Quantitation—The concentration of a compound is 

calculated according to the calibration curve used to 

establish the best fit between the calibration points after 

the compound has passed qualitative criteria. Linearregressed five-point standard curves are constructed from 

the calibration standards. The correlation coefficient for 

each standard curve has to be greater than or equal to 0.99 

to be accepted.

10.3 Calculations—If a selected compound has passed the 

qualitative identification criteria, the concentration in the 

sample is calculated as follows:

C = ((Ac/Ai) (m) + y) (DF), in micrograms per liter, (1)

where

C = concentration of the selected compound in 

the sample, in micrograms per liter;

Ac = area of peak of the quantitation ion for the 

selected compound;

Ai = area of peak of the quantitation ion for the 

Table 3. Retention times and multiple-reaction monitoring molecular (precursor)-ion and product-ion transitions for isoxaflutole, its two 

sequential degradation products, and the internal and surrogate standards.

[ISTD, internal standard; M-H, molecular-hydrogen; m/z, mass-to-charge ratio; V, volts; --, no data]

Compound

Retention

time

(minutes)

Molecular

(precursor) ion

M-H

(m/z)

Cone

(V)

Quantitation

product

ion

(m/z)

Collision

energy

(V)

Confirming

product

ion

(m/z)

Collision

energy

(V)

Herbicide and degradation products 

Isoxaflutole 12.84 357.9 25 78.9 13 277.6 13

Diketonitrile 9.92 357.9 25 78.9 13 277.6 13

Benzoic acid 9.44 267.0 19 159.0 15 223.1 15

Internal standard

ISTD, 2,4-dichlorophenoxyacetic acid-d3 13.36 222.0 20 164.0 12 -- --

Surrogate standard

Alachlor ethanesulfonic acid (ESA)-d5, 11.28 319.0 40 121.0 26 -- --

   Determination of Dissolved Isoxaflutole and Its Sequential Degradation Products, Diketonitrile and Benzoic Acid
internal standard;

m = slope of calibration curve using extracted 

standards between the selected compound 

and the internal standard from the original 

calibration data;

y = intercept of calibration curve between 

the selected compound and the internal 

standard from the original calibration data; 

and

DF = dilution factor calculated using equation 4.

The internal standard is used as a retention-time reference 

and for quantitation. The relative retention time (RRTc) is calculated for each selected compound in the calibration samples 

or in a sample as follows:

RRTc = RTc /RTi , (2)

where

RTc = uncorrected retention time of the selected 

compound, and

RTi = uncorrected retention time of the internal 

standard.

 See table 2 for retention times, molecular (precursor)- 

and quantitation- and confirming product-ion transition pairs. 

The expected retention time (RT) of the peak of the selected 

compound needs to be within ± 5 percent of the expected 

retention time on the basis of the RRTc. The expected retention 

time is calculated as follows:

RT = (RRTc )(RTi ), (3)

where

RT = expected retention time of the selected 

compound,

RRTc = relative retention time of the selected 

compound, and

RTi = uncorrected retention time of the internal 

standard.

The dilution factor (DF) of the processed sample is calculated using equation 4:

DF = (123/123-Vnp) (123/123-Va), (4)

where

DF = dilution factor,

Vnp = volume not pumped = milliliters not 

pumped through the SPE column, and

Va = volume added = millilliters of distilled 

water added to a sample that contained less 

than 123 mL.

The DF is incorporated into the calculation for determining final concentrations of samples. Initial calibration data are 

acceptable if the correlation coefficient (r2) value for all curves 

is greater than or equal to 0.990 for all compounds. A quadratic formula is used for curve fitting. A complete extracted 

calibration curve is included within each instrument sequence.

11. Reporting of Data Results

Isoxaflutole, DKN, and BA are reported in concentrations 

ranging from 0.003 to 0.20 μg/L. If the concentration is greater 

than 0.250 μg/L, a portion of the original sample is diluted 

appropriately with reagent water and reanalyzed.

12. Method Performance

Method performance was evaluated for SPE recovery 

using 0.10- and 0.20-μg/L reagent-water spikes. Method 

performance also was evaluated at two different spiked levels 

(0.02 and 0.10 μg/L) for four sample matrices analyzed over 

different days. The four sample matrices consisted of reagent 

water; a ground-water sample collected from a well in Sedgwick County, Kansas; a surface-water sample from Poison 

Creek near Grandview, Idaho; and a surface-water sample 

from the spillway below Clinton Lake in Kansas. All samples 

were filtered through a nominal 0.7-μm glass fiber filter and 

stored at 4 °C. Method detection limit (MDL) and method 

reporting level (MRL) were determined using multiple 

0.010-μg/L reagent-water spikes.

12.1 Method recovery––The recovery of isoxaflutole, 

DKN, and BA from the SPE cartridges was determined 

by comparing eight spiked reagent-water samples (two at 

0.02 μg/L and six at 0.10 μg/L) to theoretical 100-percent 

recovery solutions (table 4). The theoretical 100-percent 

recovery solutions were prepared by adding the same 

volumes of working standard, surrogate standard, and 

internal standard mixes to test tubes with the SPE eluting 

solvent as they were spiked into the 0.10- and 0.20-μg/L 

reagent-water samples prepared for SPE. The spiked 

theoretical 100-percent recovery solutions and the SPEextracted samples eluated then were evaporated to 200 μL 

and stored at -10 oC until analysis. Table 4 shows that the 

mean SPE recoveries varied from 104 to 108 percent for 

isoxaflutole, DKN, and BA with relative standard deviation percentages of 4.0 to 10.6 percent.

 12.2 Matrix performance––To evaluate general method 

performance, 123-mL sample aliquots of each of the four 

samples matrices were spiked with isoxaflutole, DKN, 

and BA at concentrations of 0.02 and 0.10 μg/L and 

analyzed on eight different days so that comparison of 

different matrices and concentrations included bias from 

day-to-day variation. In addition, unspiked samples of 

each matrix were extracted and analyzed to determine 

background concentrations of isoxaflutole, DKN, and 

BA. All subsamples were analyzed at the USGS OGRL 

using one LC/MS/MS system. Eight subset sample sets 

were spiked during February 23 through March 5, 2004. 

However, one subset of the reagent water was lost during analysis leaving only seven subsets of the 0.02-μg/L 

spiked reagent water.

Analytical Method   
Table 5 shows the mean concentration and standard deviation for isoxaflutole, DKN, BA, and the surrogate standard 

for each of the four sample matrices, and table 6 shows the 

combined mean and standard deviation of these compounds 

for the four matrices. These data show that the mean concentration among the sample matrices for isoxalfutole, DKN, and 

BA varied from 0.011 to 0.021 μg/L and 0.051 to 0.10 μg/L 

for the 0.02- and 0.10-μg/L spiked samples, respectively 

(table 5). The combined mean of concentrations of isoxalfutole, DKN, and BA for all four matrices varied from 0.016 to 

0.021 μg/L and 0.075 to 0.10 μg/L for the 0.02- and 0.10-μg/L 

spiked samples, respectively (table 6). The calculated mean 

and combined mean concentrations of the surrogate standards 

are close to 0.50 μg/L across all matrices.

Tables 5 and 6 also show the mean and combined mean 

concentrations, respectively, normalized as a percentage of the 

theoretical spiked concentrations. The data in table 5 show that 

the mean concentrations for isoxaflutole and DKN were 80 to 

102 percent of the theoretical spiked concentrations in the 

ground- and reagent-water samples and were 70 to 76 percent 

of the theoretical spiked concentrations in the Poison Creek 

surface-water sample and 51 to 59 percent of the theoretical 

spiked concentrations for the Clinton Lake samples. Benzoic 

acid calculated concentrations are 94 to 106 percent of the 

theoretical spiked concentrations for all four water matrices. 

Table 6 shows that the combined mean for isoxaflutole and 

DKN was 75 to 81 percent of the theoretical spiked concentration with relative standard deviations ranging from 3 to 

26 percent, whereas the combined mean for BA ranged from 

100 to 101 percent of the theoretical spiked concentation with 

a relative standard deviation ranging from 3 to 5 percent. The 

combined mean concentration of the surrogate standard ranged 

from 99 to 101 percent with relative standard deviations less 

than 2 percent.

The data in table 5 show that within sample sets that the 

precision was good for isoxaflutole, DKN, and BA with relative standard deviations generally less than 10 percent at both 

the 0.02- and 0.10-μg/L spike levels. The data in table 6 show 

that isoxaflutole and DKN exhibited more variation in concentration among matrices than BA or the surrogate standard.

12.3 Method detection limit (MDL) and reporting level 

(MRL)––A laboratory standard normally contains only the 

compounds of interest that do not interfere with and even 

might enhance the performance of the analytical instrument. Under these circumstances, the analytical system 

provides the minimum concentration that can be identified, measured, and reported with a 99-percent confidence 

that the compound concentration is greater than zero, 

while minimizing uncertainty due to matrix effects, which 

is referred to as the method detection limit (MDL). On 

the other hand, an environmental sample may not only 

contain the compounds of interest in relatively smaller 

concentrations but also many nontargeted compounds 

that can interfere with the sample analysis. Any deviation 

from the ideal laboratory sample results are accounted 

for in the method reporting level (MRL), which is the 

corrected concentration reportable for that sample under 

these conditions. The MRL is always equal to or greater 

than the MDL.

MDLs are determined according to procedures outlined 

by the U.S. Environmental Protection Agency (1992). Eight 

replicate samples of buffered reagent water are spiked at 

0.010 μg/L with the working standard mix. Each set of duplicate samples was analyzed on different days during February 

2004 so that day-to-day variation is included in the results.

The MDL was calculated using the following equation:

(5)

where

SD = standard deviation of replicate 

analysis, in micrograms 

per liter, at the spiked 

concentration;

 = Student’s t-value for the 99-

percent confidence level with 

n-1 degrees of freedom (U.S. 

Environmental Protection 

Agency, 1992); and

n = number of replicate analyses.

The calculated MDL is 0.003 μg/L for isoxaflutole and 

diketonitrile and 0.004 μg/L for benzoic acid. According to the 

U.S. Environmental Protection Agency procedure, the spiked 

concentrations should be no more than five times the estimated 

MDL. The spiked concentrations are within five times of the 

MDL. The MRL is calculated by multiplying the SD of the 

0.010-μg/L reagent-water spikes by 10 (U.S. Environmental Protection Agency, 1992). The calculated MRL is 0.011 

for isoxaflutole, 0.010 for diketonitrile, and 0.012 μg/L for 

benzoic acid. On the basis of calculated MDLs and MRLs and 

evaluation of the signal-to-noise ratios for each compound, 

the MRLs and MDLs are set at 0.010 and 0.003 μg/L, respectively, for all three compounds.

10 Determination of Dissolved Isoxaflutole and Its Sequential Degradation Products, Diketonitrile and Benzoic Acid

Table 4. Summary of mean solid-phase extraction recovery and 

relative standard deviation percentages in buffered reagent water for 

isoxaflutole, diketonitrile, benzoic acid, and the surrogate standard.

Compound

Mean 

solid-phase 

extraction

recovery

(percent)

Relative

standard 

deviation

(percent)

Isoxaflutole 104 10.6

Diketonitrile 108 7.0

Benzoic acid 105 4.0

Surrogate standard

Alachlor ethanesulfonic acid (ESA)-d3, 101 9.0

MDL SD n t = −  − ∂ =  

 ( ) ( , 1 1 0 9. ) 9 ,

t  ( , n − − ∂ = . )  

 1 1 0 99
Table 5. Summary of mean concentrations, standard deviations, and mean normalized percentage of theoretical spiked concentrations and relative 

percentage standard deviations for isoxaflutole, diketonitrile, benzoic acid, and the surrogate standard in four water matrices. 

[μg/L, microgram per liter; n, number of samples]

Reagent water

Compound

Spiked at 0.02 μg/L (n=7) Spiked at 0.10 μg/L (n=8)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Isoxaflutole 0.020 0.0008 101.3 4.10 0.097 0.0038 97.2 3.88

Diketonitrile .020 .0013 101.7 6.24 .100 .0073 100.1 7.35

Benzoic acid .020 .0015 102.0 7.42 .099 .0035 99.8 3.49

Surrogate standard .490 .0290 98.6 5.81 .500 .0244 99.4 4.91

Ground water, Sedgwick County, Kansas

Compound

Spiked at 0.02 μg/L (n=8) Spiked at 0.10 μg/L (n=8)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Isoxaflutole 0.017 0.0015 80.5 8.88 0.081 0.0071 81.6 8.71

Diketonitrile .018 .0011 90.2 6.31 .090 .0030 89.7 3.37

Benzoic acid .021 .0014 101.2 6.94 .104 .0024 104.4 2.28

Surrogate standard .500 .0240 99.7 4.87 .500 .0350 100.6 6.94

Surface water, Poison Creek near Grandview, Idaho

Compound

Spiked at 0.02 μg/L (n=8) Spiked at 0.10 μg/L (n=8)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Isoxaflutole 0.014 0.0083 69.6 6.10 0.071 0.0055 71 7.74

Diketonitrile .015 .0155 74 10.20 .078 .0056 76.5 7.18

Benzoic acid .021 .0115 103 5.59 .106 .0026 106.3 2.48

Surrogate standard .490 .0560 99 11.42 .500 .0440 99.7 8.89

Surface water, Clinton Lake, Lawrence, Kansas

Compound

Spiked at 0.02 μg/L (n=8) Spiked at 0.10 μg/L (n=8)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Isoxaflutole 0.012 0.0015 59.3 12.70 0.051 0.0049 50.6 9.64

Diketonitrile .011 .0011 55.3 9.70 .052 .0042 52.4 8.00

Benzoic acid .019 .0015 95 7.64 .094 .0068 94.1 7.19

Surrogate standard .50 .0330 99 6.55 .510 .0360 102.7 6.91

Analytical Method 11
Conclusions

This method provides for routine analysis of isoxaflutole 

and its sequential degradation products, diketonitrile (DKN) 

and benzoic acid analogue (BA), in filtered ground- and surface-water samples. The method demonstrates that solid-phase 

extraction (SPE) coupled with liquid chromatography/tandem 

mass spectrometry (LC/MS/MS) can be used to analyze water 

for these compounds.

The analytical method showed good precision, with 

generally less than 10 percent relative standard deviation 

within a water matrix. Analysis of BA and the surrogate standard showed good accuracy and precision within and among 

matrices. For isoxaflutole and DKN, the mean calculated 

concentrations were within 30 percent of the theoretical spiked 

concentrations for three of the sample matrices and were 

within 50 percent of the theoretical spiked concentration for 

the surface-water sample from Clinton Lake, Kansas.

Compound detection limits (MDLs) and reporting levels 

(MRLs) were calculated using U.S. Environmental Protection 

Agency procedures. The MDL for isoxaflutole and diketonitrile is 0.003 μg/L and 0.004 μg/L for benzoic acid. MRLs are 

0.011, 0.010, and 0.012 μg/L for isoxaflutole, diketonitrile, 

and benzoic acid, respectively. On the basis of calculated 

MDLs and MRLs and evaluation of the signal-to-noise ratios 

for each compound, the MRLs and MDLs are set at 0.010 and 

0.003 μg/L, respectively, for all three compounds.

Information about the fate and transport of isoxaflutole 

and its two sequential degradation products, DKN and BA, in 

water can be acquired from the analysis of ground water and 

surface water. The method was tested and successfully used to 

analyze 75 samples from 10 Iowa streams between March and 

September 2004. This method will contribute to an improved 

understanding of the occurrence, persistence, and transport 

of isoxaflutole and its sequential degradation products in the 

environment.

References Cited

Lin, C.H., Lerch, R.N., Thurman, E.M., Garrett, H.E., and 

George, M.F., 2002, Determination of isoxaflutole (Balance) and its metabolites in water using solid-phase extraction followed by high-performance liquid chromatography 

with ultraviolet or mass spectrometry: Journal of Agriculture and Food Chemistry, v. 50, p. 5816–5824.

Pallet, K.E., Cramp, S.M., Little, J.P., Veerasekaran, P., 

Crudace, A.J., and Slater, A.E., 2001, Isoxaflutole—the 

background to its discovery and the basis of its herbicidal 

properties: Pesticide Management Science, v. 57, p. 133.

Shelton, L.R., 1994, Field guide for collection and processing stream-water samples for the National Water-Quality 

Assessment Program: U.S. Geological Survey Open-File 

Report 94–455, 42 p.

Scribner, E.A., Meyer, M.T., and Kalkhoff, S.J., 2006, Occurrence of isoxaflutole, acetamide, and triazine herbicides 

and their degradation products in 10 Iowa rivers draining to 

the Mississippi and Missouri Rivers, 2004: U.S. Geological 

Survey Scientific Investigations Report 2006–5169, 84 p.

U.S. Department of Agriculture, 2004, National Agricultural 

Statistics Service (NASS) agriculture chemical use database: Information available on the Web, accessed February 6, 2006, at http://www.pestmanagement.info/nass/

U.S. Environmental Protection Agency, 1992, Guidelines 

establishing test procedures for the analysis of pollutants 

(appendix B, part 136, Definition and procedures for the 

determination of the method detection limit): U.S. Code of 

Federal Regulations, Title 40, revised as of July 1, 1992, 

p. 565–567.

Table 6. Combined mean concentrations, standard deviations, and combined mean normalized percentage of theoretical spiked concentrations and 

relative percentage standard deviations for isoxaflutole, diketonitrile, benzoic acid, and the surrogate standard in four water matrices. 

[μg/L, microgram per liter; n, number of matrices]

Compound

Spiked at 0.02 μg/L (n=4) Spiked at 0.10 μg/L (n=4)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Mean

concentration

(μg/L)

Standard

deviation

(μg/L)

Mean

normalized

concentration

(percent)

Relative

standard

deviation

(percent)

Isoxaflutole 0.016 0.0037 78 23 0.075 0.0196 75 26

Diketonitrile .016 .0040 81 25 .080 .0204 80 26

Benzoic acid .021 .0007 100 3.3 .101 .0054 101 5.4

Surrogate standard .500 .0037 99 .74 .500 .0080 101 1.5

12  Determination of Dissolved Isoxaflutole and Its Sequential Degradation Products, Diketonitrile and Benzoic Acid
Viviani, F., Little, J.P., and Pallett, K.E., 1998, The mode of 

action of isoxaflutole II—characterization of the inhibition 

of carrot 4-hydroxyphenylpyruvate dioxygenase by diketonitrile derivative of isoxaflutole: Pesticide Biochemistry and 

Physiology, v. 62, no. 2, p. 125–134.

Webb, W.E., Radtke, D.B., and Iwatsubo, R.T., 1999, Surface-water sampling collection methods at flowing-water 

and still-water sites: U.S. Geological Survey Techniques in 

Water-Resources Investigations, book 9, chap. A4, section 4.1, p. 23–59.

Wilde, F.D., Radtke, D.B., Gibs, Jacob, and Iwatsubo, R.T., 

1998, National field manual for the collection of waterquality data—preparations for water sampling: U.S. Geological Survey Techniques of Water-Resources Investigations, book 9, chap. A1–A5 [variously paged].

References Cited 13
Prepared by Lawrence Publishing Service Center.

Edited by Lanna Combs.

Graphics by Jeff Hartley and Mike Kemppainen.

Layout and design by Kristi Hartley.

For more information concerning the research described in this report, 

contact:

U.S. Geological Survey

4821 Quail Crest Place

Lawrence, KS 66049

(785) 842-9909

http://ks.water.usgs.gov
Printed on recycled pape

r

Meyer and others—Determination of Dissolved Isoxaflutole and Its Sequential Degradation Products, Diketonitrile and Benzoic Acid—Techniques and Methods 5–A9