Immunochemical Technology for Environmental Applications

Chapter 25

Analysis of Hexazinone in Soil by EnzymeLinked Immunosorbent Assay 1

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Downloaded by UNIV MASSACHUSETTS AMHERST on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch025

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Rodney J . Bushway , Lynn E. Katz , Lewis B. Perkins , Anthony W. Reed , Titan S. Fan , and B. S. Young 3

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Department of Food Science and Human Nutrition, University of Maine, 5736 Holmes Hall, Orono, M E 04469-5736 Department of Civil and Environmental Engineering, University of Maine, 5706 Aubert Hall, Orono, M E 04469-5736 Millipore Corporation, 80 Ashby Road, P.O. Box 9125, Bedford, MA 01730-9125

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A tube enzyme immunoassay (EIA) procedure was developed for the determination of the triazine herbicide hexazinone in soil. The antibody was polyclonal and was prepared by employing metabolite A of hexazinone conjugated to bovine serum albumin as the immunogen. Hexazinone was extracted from soil by shaking with methanol-water 80/20 for 10 min and allowed to set overnight before reshaking for 5 min. Aliquots for EIA analysis were diluted in such a way as to always contain 8% methanol. Reproducibility results for both standards and samples were good. A correlation coefficient of 0.9562 was obtained for 78 soil samples analyzed by EIA vs. HPLC. Of the eight known metabolites of hexazinone, 7 were tested for cross-reactivity and 5 were shown to be cross-reactive. It is commonly known that pesticides applied on land surfaces can enter surface and groundwater either through surface runoff or infiltration processes. Indeed, non-point source contamination of ground and surface water due to these processes is well documented and the effects of migration of pesticides into surface and groundwater is both of regional and national interest (1-3). One pesticide of particular interest since it is a triazine is hexazinone. Many triazines have been found in groundwater and are considered environmental problems (1,3). Hexazinone (trade name Velpar) is primarily a contact herbicide that can be used as a foliar spray or applied directly to soils for weed control in blueberries, pineapple, sorghum, sugarcane, and forests. Recent studies have identified the presence of hexazinone in surface and groundwaters within blueberry areas of eastern, southern and western, Maine (4). A variety of degradative and non-degradative processes influence the extent of Velpar contamination, including biodegradation, photolysis and sorption (5-6). The importance of quantifying the extent of soil sorption has lead to the development of an EIA method for the determination of hexazinone in soil. Experimental Hexazinone and its Metabolites. Hexazinone (3-cyclo-hexyl-6-(dimethylarnino)-lmethyl-1.3.5-triazine-2,4-(lH,3H)-dione and the following 7 metabolites: metabolite A © 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS


[3-(4-hydroxycyclohexyl)-6-(dimemylamino)-1 -methyl-1,3.5-triazine-2,4-( 1 H,3H)dione]; metabolite B [3-cyclohexyl-6-(methylarnino)-1 -methyl-1,3.5-triazine-2,4(lH,3H)-dione]; metabolite C [3-(4-hyaYoxycyclohexyl)-6-(methylarnino)-l-methyl1,3,5-triazine-2,4-( 1 H,3H)-dione]; metabolite D [3-cyclohexyl-1 -methyl-1,3,5triazine-2,4,6-(lH,3H,5H)-trione]; metabolite E [3-(4-hydroxy-cyclohexyl)-l-methyl1,3,5-triazine-2,4,6-( 1 H,3H,5H)-trione]; metabolite A-1 [3-(trans-2-hydroxycyclohexyl-6-(dimethylarnino)-1 -methyl-1,3,5-triazine-2,4-( 1 H,3H) dione]; and metaboUte 1 [3-(4-oxycyclohexyl)-6-(dirr«thylarnino)-1 -methyl-1,3,5-triazine-2,4(lH,3H)-dione] were gifts from E.I. DuPont de Nemours & Company, Experimental Station, Wilmington, DE. Structures of hexazinone and its metabolites are shown in Figure 1. Soil Samples. Soil samples were collected from blueberry fields from Florida and eastern, western and southern Maine. They were dried and sieved (20 mesh) before being extracted. Preparation of Hexazinone Standard for Immunoassay. A stock solution of hexazinone was prepared by weighing 20 mg of hexazinone into a 50 mL volumetric flask and bringing to volume with HPLC grade methanol. An intermediate standard solution was made by pipetting 50 | i L of stock solution into a 50 mL volumetric flask and bringing to volume with HPLC grade water. Working standards (0.11, 0.22, 0.44, 1.1, 2.2, 4.4, 8.8, and 17.6 ppb) were prepared by serially diluting the intermediate standard into HPLC grade water. Preparation of Hexazinone Standards for HPLC. The same hexazinone stock solution that was employed above for immunoassay was used for HPLC. An intermediate standard was prepared by pipetting 50 [iL of the stock standard into a 50 mL volumetric flask and bringing it to volume with methanol-acetonitrile-water (20:40:40, v/v/v). Working standards (12.5, 62.5, 312.5, 1562.5, and 7812.5 Tig/mL) of hexazinone were prepared by making serial dilutions of the intermediate standard with the methanol-acetonitrile-water solution. Production of Antisera and Immunogen. These procedures are described in a previous paper (4). Briefly, rabbits were injected intradermal and subcutaneous with the active ester of hexazinone hemisuccinate. The hexazinone hemisuccinate was prepared by refluxing hexazinone metabolite A and succinic anhydride in pyridine. Extraction of Hexazinone in Soil. One gram of soil was weighed into a 25 mL polypropylene bottle followed by the addition of 5 steel ball-bearings and 10 mL of 80:20 MeOH ACS grade/water. Samples were shaken by hand for 10 min and allowed to set overnight before shaking again for 5 min. A 100 uL aliquot was removed for ELISA and a 5 mL aliquot was taken for HPLC analysis. EIA Analysis of Hexazinone in Soil. A tube kit from Millipore Corp. (Bedford, MA) was employed for the analysis. The 100 fiL aliquot was added to 0.9 mL of HPLC grade water (This makes the sample 8% methanol). A 200 u,L aliquot of the sample and/or standards were added to no more than 10 EIA tubes followed by 200 \xL of enzyme conjugate. Each tube was mixed briefly by swirling. After a 20 min incubation at room temperature, the tubes were rinsed 4 times under tap water and blotted dry before the addition of 500 [iL of K-blue (Elisa Technologies, Lexington, K Y ) substrate to each tube. Tubes were incubated at room temperature for 10 min before 300 [iL of 1 N HC1 which stops the reaction and changes the color from blue to yellow. Absorbance of each tube was read at 450 nm using an EnviroGard tube reader. If samples needed to

In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


N

k

N(CH )

H Metabolite H

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N

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3

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N(CH )

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Hexazinone

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2

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C H

Metabolite A l

CH

^ N ^

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Figure 1. Structure of hexazinone and metabolites

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Metabolite D

CH

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*N(CH )2 CH Metabolite A

Xxi 3

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N 3

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Metabolite 1

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3

CH Metabolite E

CH Metabolite B

0,1


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be diluted to remain in the linearity range then they were diluted in an 8% methanol solution. In fact all standards and samples must be run in 8% methanol.


Quantitation of Hexazinone. Control tubes were run with each set of tubes to calculate %B values of standards and samples (absorbance at 450 nm of standard or sample/absorbance at 450 nm of negative control X 100). Standards were run at the beginning and end of each day with the average of both runs used to prepare the standard curve which was made by plotting %B versus the log of hexazinone concentration. Hexazinone levels in unknown water samples were interpolated from the standard curve. Cross-Reactivity of Hexazinone Metabolites. performed in 8% methanol.

Cross-reactivity studies were

HPLC Operating Conditions. A Zorbax C18 column (stainless steel, 4.6 mm I.D. X 250 mm) (Phenomenex, Torrance, CA) was employed for the separation along with a mobile phase comprised of methanol-acetonitrile-water (20:40:40) at a flow rate of 1 niL/min using a Hewlett-Packard (HP) 1050 pump (Wilmington, DE). Injection (50 uX) were performed by a HP 1050 auto-injector. The samples were detected with a HP 1050 photodiode array detector set at 247 nm while a Vectra HP 486 Chem Station for windows was used to measure peak areas. Analysis of Hexazinone in Soil by HPLC. The 5 ml aliquot from the soil extract was added to 100 ml of HPLC grade water. This mixture was passed through an activated C18 Sep-Pak (Waters Associates, Milford, MA) (Activation was done by passing 5 mL of HPLC methanol through a Sep-Pak followed by 5 mL of HPLC water) at 5 rnL/min. After drying the Sep-Pak for 20 min under vacuum, it was eluted with 4 mL of a mixture of ethyl acetate-methyl tertiary butyl ether (20:80). The 4 mL were evaporated to dryness under nitrogen and the residue was dissolved in 1 mL of HPLC mobile phase. A 50 uL aliquot was injected into the HPLC. Results and Discussion A typical EIA standard curve is shown in Figure 2. The linear range was from 0.22 to 17.6 ppb with an IC50 (concentration of hexazinone at a %B value of 50) of 3.0 ppb. The lower limit of detection (LLD) for hexazinone in soil was deterrnined to be 25 ppb and the lower limit of quantitation (LQD) to be 50 ppb (7). All standards and samples including controls were made up in 8% methanol. This was done to quicken the EIA analysis. Otherwise an evaporation step would have been needed because methanol at 8% does have an inhibitory effect on immunoassay even though it is small. Since the inhibitory effect of the methanol is slight, diluting the methanol to obtain even a smaller decrease in inhibition makes it impossible to obtain the L L D of 25 ppb. Thus the inhibitory effect of the 8% methanol is balanced out by performing all tests in that concentration of methanol. The types of soils employed for spiking studies and aged soils analyzed come under 5 classes (loamy sand, loamy sand/sand, sand, sandy loam, and loam) based on texture (Table I). A l l but one soil type were acid which is normal for blueberry soils. The cross-reactivity of this antibody is very broad reacting with most major metabolites. Exact cross-reactivity values are given in Table II. The values given in


25. BUSHWAY ET AL.

Analysis of Hexazinone in Soil by ELISA

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100

Hexazinone Concentration (ppb) Figure 2. Hexazinone Standard Curve

Table II are based on compounds in 8% methanol and are less than what was given in a previous paper (4) where the compounds were dissolved in water. It appears that the cross-reactivity values for D and E did not change. This is not surprising since these two metabolites are not considered to be cross-reactive. Thus, metabolites A, A l , 1, B , and C demonstrate all the reactivity which is expected since they are the most structurally similar to hexazinone (Figure 1). This can be a problem if one wants to quantitate individual compounds and the others are present. However in a screening mode, in which more classical methods would be performed if the immunoassay was positive, total would be advantageous. Futhermore, some soils need extensive cleanup in order to quantify the metabolites of hexazinone, fractions could be collected and analyzed by EIA without performing the cleanup. Recently, by employing a G C method (8) we have shown that the blueberry soil samples contain only 2 metabolites (B and D). Metabolite D has no cross-reactivity and; therefore, will not interfere with hexazinone analysis by EIA. Metabolite B demonstrates sufficient cross-reactivity to yield interference with hexazinone analysis by EIA but it must be at a concentration of 0.5 ppm or higher before it can interfere with hexazinone quantitation by EIA at a 1/10 dilution which is the smallest dilution ever made. Our analysis of metabolite B in 50 blueberry soils from Maine only showed 2 had sufficient B concentration to interfere with hexazinone analysis. As with any analytical technique, precision within and between days is important. Reproducibility results for hexazinone standards and soil samples were good (Table III). For standards, the CVs ranged from 3.3 to 13% with all but one below 7% while actual field soils demonstrated CVs varying from 5.2 to 21% with an average of 13%. The accuracy of the EIA method was tested using fortified soil samples spiked at 25, 50, 100, 250, 1000, and 5000 ppb (Table IV). Recoveries were excellent ranging from 76 to 96% with a mean of 86%. Coefficients of variations ranged from 12 to 25% with only 1 above 17% and that was the 25 ppb spike which was at the L L D of the method.



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Table I. Characteristics of Soil Analyzed %OM %Silt CEC %Sand 0.5 84

Immunochemical Technology for Environmental Applications

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