Mass

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Chapter 28

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ELISA and Liquid Chromatography/Mass Spectrometry/Mass Spectrometry Methods for Sulfentrazone and Its Acid Metabolite in Groundwater Samples Audrey W. Chen Agricultural Products Group, FMC Corporation, P.O. Box 8, Princeton, NJ 08543

Sulfentrazone represents a new class of herbicides (aryl triazolinones). It inhibits the protoporphyrinogen oxidase (PPO) in the plant chlorophyll biosynthetic pathway. The major metabolite identified in the water is sulfentrazone-3carboxylic acid (SCA). An ELISA (enzyme-linked immunosorbent assay) test kit has been used as an analytical screening tool for the water samples (including well, lysimeter, and other source water) from three groundwater studies. Compared to the conventional chemical assay, ELISA uses minimum chemicals (solvents and reagents) and is simpler and time- and cost-effective. LC/MS/MS (liquid chromatograph equipped with triple quadruple mass spectrometry) was used for the confirmation of all positive and partial negative residues. Method development and comparisons of precision, accuracy, cross-reactivity, recovery, and false negatives and positives for ELISA and LC/MS/MS are discussed herein.

© 2005 American Chemical Society In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

295

296

Introduction

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Sulfentrazone is a broad-spectrum, pre-emergent herbicide that provides good control over broadleaf weeds, grasses and sedges in crop fields and turf. The residue of interest in water includes the parent sulfentrazone and SCA (Figures 1 and 2).

Chemical name: Trade name: Common name: CAS No.:

N-[2,4-dichloro-5-[4-(difluoromethyl)-4,5-dihydro-3-methyl-5oxo-1 H-1,2,4-triazol-1 -yl]phenyl] methanesulfonamide Authority™ and Spartan® Sulfentrazone 122836-35-5

Figure I. Sulfentrazone Structure and Nomenclature

CHF

2

COOH

Chemical name:

CAS No.:

1 -[2,4-dichloro-5-(N-(methyl-sulfonyl)aminuteo)phenyl]-4difluoromethyl-4,5-dihydro-5-oxo-1 H-1,2,4-triazole-3carboxylic acid 134391-01-8

Figure 2. Sulfentrazone-3-carboxylic acid (SCA) Structure and Nomenclature

The ELISA (enzyme-linked immunosorbent assay) test kit for sulfentrazone and SCA residues in groundwater is developed with limit of quantitation (LOQ)

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

297

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and limit of detection (LOD) at 0.1 and 0.05 ppb, respectively. Polyclonal antibodies were generated by immunizing with a sulfentrazone crop metabolite (3-hydroxymethyl sulfentrazone, HMS). In order to provides better sensitivity and reproducible results, SCA is decarboxylated and converted to 3-desmethyl sulfentrazone (DMS) in acidic conditions with heat (i.e., reflux) prior to LC/MS/MS measurement. The LOQ and LOD for the analytes of interest by LC/MS/MS are at 0.1 and 0.02 ppb, respectively. The structures and chemical names for HMS and DMS are presented in Figures 3 and 4.

Chemical name: N-[2,4-dichloro-5-[4-(difluoromethyl)-4,5-dihydro-3 hydroxymethyl-5-oxo-1H-1,2,4-triazol-1 -yl]phenyl]methanesulfonamide CAS No.: 134390-99-1 Figure 5. HMS Structure and Nomenclature

Chemical name: N-[2,4-dichloro-5-[4-(difluoromethyl)-4,5-dihydro-5-oxo-1H1,2,4-triazol-1 -yl]phenyl]-methanesulfonamide CAS No.: 134391-02-9 Figure 4. DMS Structure and Nomenclature

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

298

ELISA Method

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Three immunogens, sulfentrazone conjugated with keyhole limpet hemocyanin (KLH), HMS with KLH, and SCA with ovalbumin (OVA) have been tested to detect sulfentrazone and SCA in water. Only the HMS conjugated with K L H would react with both analytes of interest with 100% inhibition (recognition and reactivity) for sulfentrazone and 50% inhibition for SCA (Table 1). Therefore, LOD and LOQ are 0.05 and 0.1 ppb and 0.1 and 0.2 ppb for sulfentrazone and SCA, respectively.

Table I. Comparison of Immunogen and Reactivity Recognition and Reactivity (%) SCA Sulfentrazone

Immunogen

Sulfentrazone-KLH 100 HMS-KLH" 100 SCA-OVA 0 a. The only immunogen that reacts with both analytes.

0 50 100

HMS Charge of Ο (-H): -0.70 Rotational Barrier (N-C-C-O): 2.6 kcal/mol

SCA

ο

„CHF

2

Charges of Ο (=C) and Ο (-H): -0.55 and -0.68 Rotational Barrier (N-C-C-O): 7.3 kcal/mol

IT

Figured. N-C-C-O bonds in HMS and SCA

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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299 The charge densities of "O" in HMS (-0.70) and SCA (-0.68) are similar based on the high-level quantum mechanical calculations. However, the C-C bond of N-C-C-O in HMS can rotate freely at room temperature (< 3 kcal/mol), but such rotation is hindered in SCA. The degree of freedom of N-C-C-O in HMS might be the reason why only HMS-KLH could react with both analytes of interest. The Calculated charge density and rotational energy were provided by FMC Computational Sciences Team. The N-C-C-O bond angles in HMS and SCA are demonstrated below. The competitive inhibition ELISA 96-well format used in this program was developed by Strategic Diagnostics Inc. (SDI). The antibody will bind sulfentrazone either immobilized to the wells of the plate or the free analytes in sample solution. When sulfentrazone is present in the water sample and added to the plate with antibody, it competes with the immobilized sulfentrazone for a limited number of antibody binding sites. Since the same number of sulfentrazone molecules are immobolized on every test well and each well receives the same concentration of antibody (same number of antibody binding sites), a sample containing a low concentration of sulfentrazone will allow more antibody to bind to the immobilized sulfentrazone. The antibody bound to the plate can then be marked by a labeled secondary antibody. This marker, in the presence of substrate, results in a yellow solution and the concentration of analyte can then be measured by a spectrophotometric plate reader. If a sample contains a high concentration of sulfentrazone, less antibody will be available to bind to the immobilized sulfentrazone and ultimately result in a lighter or even colorless solution. This ELISA kit is sensitive and selective and allows reliable and rapid screening for a total concentration of sulfentrazone and SCA. The kit has been tested on several agrochemicals with similar chemical structures (e.g., carbofuran, carfentrazone-ethyl, alachlor, chlorosulfron, atrazine, trifluralin, metribuzin, imazethapyr, terbufos and chlorimuron ethyl) and none of them reacted with the kit [IC (concentration produces 50% inhibition of maximum signal) > 1000 ppb]. 50

LC/MS/MS Method The water sample is acidified (pH=l) and boiled one hour under reflux to convert SCA to DMS. The analytes of interest are then concentrated and separated using C and silica gel solid phase extraction (SPE) cartridges prior to the instrument analysis. Micromass Quattro L C triple quadrupole mass spectrometer is used for analyte concentration determination. The molecular ions monitored are 385 (MS) and 199 (MS/MS) for sulfentrazone and 371 (MS) 8

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

300 and 201 (MS/MS) for DMS. The LOD and LOQ are 0.02 and 0.1 ppb for both sulfentrazone and SCA (analyzed as DMS) in water. Comparing to ELISA, which determines a total concentration of the two analytes, LC/MS/MS measures individual analyte and provides better sensitivity than ELISA. Therefore, LC/MS/MS is commonly used for the confirmation of analyte residues. The example of chromatograms for sulfentrazone and DMS by LC/MS/MS are presented in Figure 6.

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100η 9.17 1868

sulfentrazone (2 pg/uL, 0.05 ppb) 30

•I"

I

100η

•%

8.85 1162

1

DMS(2pgML, 0.05 ppb)

• ι • • ι ι ! ι ι ι ι ι • • ι ι ! ι ι ι • | ι n ι ι ι ι ι ι, ι • ι ι , ι ι ι ι , , ι ι ι; ι ι ι ι ι ι ι ι » ι ι ι ι u ι • • ι ι ι ι ι • ι ι ι •• ι ι ι • ι I ι ii ι I Time 6.00 7.00 8.00 9.00 10.00 11.00 1200 13.00 14.00 15.00

Figure 6. sulfentrazone and DMS at 0.05 ppb by LC/MS/MS

Method Recoveries of Sulfentrazone and SCA in Water Water samples from the three groundwater studies (A, Β and C) were first screened for residues using ELISA method. The samples were then analyzed using LC/MS/MS when the residues were found at detectable levels or higher (£ 0.05 ppb). Also, a representative number of water samples with negative responses from ELISA were analyzed by LC/MS/MS. In general, a set of 30 (triplicate), 40 (duplicate) or 80 (singular) water samples can be analyzed on each ELISA 96-well plate and a set of 10-12 samples can be analyzed for conventional LC/MS/MS method. Both methods require about a day to perform a set of samples. When residues were expected to be detected in most of the water samples collected at certain times, only LC/MS/MS method was used for residue determination. Method recoveries of sulfentrazone and SCA in water from Study A by both methods are provided in Table 2. Both methods provide

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

301 reliable and reproducible results based on the respective accuracy (average recoveries) and precision (standard deviations).

Table 2. Method Recovery (%) of Sulfentrazone and SCA in Water from Study A No. of Analvsis

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Analvte

Fortification Level ÎDDD)

Recovery Ranee

Average ± Std. Dev.

ELISA (Contract Labf Sulfentrazone 18

0.06-2.0

79-159

120±24

ELISA ÎFMC) Sulfentrazone SCA

0.1 0.2

65-115 66-124

85±13 96±13

25 25

b

LC/MSD iFMC) Sulfentrazone 154 62-138 0.1-25.0 SCA 154 61-140 0.1-25.0 a. From the ELISA kit validation report by SDI. b. Including LC/MS and LC/MS/MS

91±12 87±14

Result Comparison of ELISA and LC/MS/MS for Sulfentrazone and SCA in Water For Study A there were 521 water samples analyzed by both ELISA and LC/MS/MS methods. These samples were initially screened using ELISA and resulted in 19 positive samples (residues > 0.05 ppb). These 19 samples along with 128 negative samples (no detectable resiudes) were further analyzed by LC/MS/MS for confirmation. Of the 19 positive samples by ELISA, 10 samples contained positive residues (> 0.02 ppb) by LC/MS/MS. The remaining 9 positive samples by ELISA were considered false-positives. Of the 128 negative samples by ELISA, 7 samples contained positive residues (> 0.02 ppb) by LC/MS/MS. These samples represented false-negatives by ELISA method. The negative and positive results using both methods are presented in Table 3.

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

302 Table 3. Comparison of Positive and Negative Residues by ELISA and LC/MS/MS in Study A

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ELISA Event

DAT'

1 5 6 7 8 9 10 11 12 13 15

-1 7 14 30 60 90 120 150 180 210 270

Negative 54 17 45 52 53 52 52 52 33 23 17

Total 502 Days After Treatment

a.

LC/MSD

Positive

Negative

Positive

3 1 0 2 2 3 3 2 2 1 0

11 10 16 10 18 12 15 18 9 11 1

0 0 0 0 1 4 2 8 1 1 0

19

131

17

The overall residue results by ELISA and LC/MS/MS for Study A correlated rather well, except for the 7 false-negative samples by ELISA. The residue levels for those 7 samples were between the LC/MS/MS LOD (0.02 ppb) and LOQ (0.1 ppb) and therefore, could not be detected by ELISA. The overall negative and positive results by both methods for three groundwater studies are also provided in Table 4.

Table 4. Comparison of Positive and Negative Residues by ELISA and LC/MS/MS in Three Groundwater Studies No. of Samples Analyzed

False Positive

False Negative

A (95 ) Β (85 ) C (75 )

502 283 330

9 10 21

7 11 1

Total

1115

40

19

Study No. th

th

th

a.

a

Percentile of soil vulnerability

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

303 A total of 1115 water samples were analyzed by ELISA for three groundwater studies and only 40 of them were false-positives (3.6%) and 19 samples (1.7%) were false-negatives after confirmation by LC/MS/MS. Since all the positive residues from ELISA would require confirmation by LC/MS/MS, the false-positives from ELISA are not of concern. In addition, the residues of the false-negatives from ELISA are insignificant because these rsidues are less than 0.05 ppb.

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Conclusion A total of-15 work days would be needed to analyze the 1115 water samples by ELISA based on the 80 singular sample on each 96-well plate. Whereas for LC/MS/MS, a total of-115 work days would be required to analyze the same amount of samples based on the 10-12 samples per assay set. In addition, ELISA uses and generates minimum chemicals and waste(solvents and reagents) and is simpler and time-and cost-effective compared to the conventional chemical method. The ELISA kit for sulfentrazone and SCA is, therefore, a reliable and effective analytical tool for screening water samples. It is particularly useful in the beginning and at the end of each groundwater study, when detectable residues are not expected.

Acknowledgements The author gratefully thanks D. Baffuto, J. Carroll, J.F. Culligan, R. Jones, D.J. Letinski, E.M. McCoy, R.T. Morris and M . Xiong for their help with sample preparation, analysis and report and L.R. Young for calculation of charge density and rotational energy.

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.