Immunochemical Technology for Environmental ... - ACS Publications

Chapter 22

Rapid Determination of Dioxins in Drinking Water by Enzyme Immunoassay 1

1

1

2

Downloaded by UNIV MASSACHUSETTS AMHERST on October 1, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch022

1

1

H. Wang , L. Wang , J. E. George III , G. K. Ward , J. J. Thoma , R. O. Harrison , and B. S. Young 2

1

Environmental Health Laboratories, 110 South Hill Street, South Bend, IN 46617-2702 Millipore Corporation, 80 Ashby Road, P.O. Box 9125, Bedford, MA 01730-9125 2

An enzyme immunoassay (EIA) was developed to rapidly analyze trace levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in water samples. Water samples were extracted by the solid phase extraction method (SPE) using 47 mm, C Empore extraction disks (3M). Dioxins were eluted from the disks with dichloromethane. The extracts were dissolved in methanol through a solvent exchange step. EnviroGard reagents and a microwell strip reader (Millipore) were used to perform the dioxin enzyme immunoassay. The working range of the dioxin enzyme immunoassay was found to be 15 pg/L to 100 pg/L in water. The precision and accuracy of EIA were determined by performing five replicates of reagent water spiked at a concentration of 25 pg/L. The recovery of the dioxin assay ranged from 74% to 122%, and % C V for five replicates was less than 15%. The accuracy of EIA results was also confirmed by ion trap GC/MS/MS (Varian). In general, EIA provides a relatively easy and cost effective means for measuring trace levels of dioxins in drinking water samples. 18

Dioxins, particularly 2,3,7,8-TCDD (Figure 1), have achieved great notoriety and evoked the greatest fears as probable human carcinogens. EPA has regulated 2,3,7,8-TCDD in drinking water with a maximum contamination level (MCL)of 30 pg/L. Dioxins in water and other sample matrices are usually determined by the EPA Method 1613 developed by the EPA Office of Science and Technology (/). This method, however, requires expensive instrumentation (HRGC/HRMS) and a highly trained analyst. A GC/EI/MS/MS technique has been developed for the ultra-trace detection of dioxins and furans (2-4). The sensitivity of this method is 1 pg of native analyte. High specificity for polychlorinated

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


272

IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

dibenzo-p-dioxins and furans (PCDD/F's) has been demonstrated by using an ion trap detector with MS/MS capability. When considering costs to monitor the dioxin contaminants in drinking water and assess the health risk, the analytical technology should be reasonably affordable by regional and large metropolitan public water systems and also available for widespread utility. Enzyme Immunoassays (EIAs) have been successfully used in the field and laboratory for the rapid determination of pesticides in environmental water and soil samples (5-9). Because enzyme immunoassay is based on an antibody coated plate and colorimeter to measure 2 2 enzymatic color development, it is a simple and quick method which requires very little training to run. In addition, the sample preparation is simpler and shorter due to the specificity and sensitivity of the dioxin antibody. Therefore, the dioxin enzyme immunoassay will significantly reduce the cost compared to classical EPA methods. A competitive inhibition enzyme immunoassay based on a mouse monoclonal antibody which is specific for 2,3,7,8-TCDD and related dioxin and furan congeners was developed (70). Specificity of this assay roughly parallels the toxicity of the dioxin and furan congeners which have been tested. The feasibility of the enzyme immunoassay as an analytical system for dioxins has been shown by repetition of standard curves having detection limits below 100 pg/tube. The goal of our research was to develop a method for rapid determination of low level 2,3,7,8-TCDD and 2,3,7,8-substituted dioxins in water using solid phase extraction (SPE) followed by a dioxin enzyme immunoassay. The specific objectives were the following: 1) determine the detection range and stability of the dioxin enzyme immunoassay, 2) demonstrate the extraction efficiency of SPE disks for extracting dioxins from water, 3) demonstrate the precision, accuracy and matrix interferences of the dioxin enzyme immunoassay, and 4) develop the quality control procedures. These studies would provide a very interesting bench mark for comparison to conventional methods and allow the EPA to further demonstrate the potential capability of enzyme immunoassay for the monitoring of ground water and surface water. Materials and Methods The stock solution of 2,3,7,8-TCDD was obtained from AccuStandard, Inc. A n independent quality control sample was obtained from ULTRA Scientific. The EnviroGard Dioxin plate kit was supplied by Millipore Corporation. A Varian Saturn IH GC/MS/MS equipped with a Varian large volume injector (LVI) was used to confirm the results of EIA. Safety. 2,3,7,8-TCDD has been found to be acnegenic, carcinogenic and teratogenic in laboratory animal studies. Disposable plastic gloves, lab coat, safety glasses should be used. Workers must be trained in the proper method of removing contaminated gloves and clothing without contacting the exterior surfaces. Primary solutions should be prepared in a hood.

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

22. WANG ET AL.

Rapid Determination ofDioxins in Drinking Water 273

Extraction. Two liters of water sample were extracted using a 47 mm, C i Empore extraction disk (3M). Dioxins were eluted with dichloromethane and the eluant was dried with sodium sulfate. The extract was evaporated to dryness under a nitrogen stream. The residue was redissolved by the addition of 100 uL of methanol with 5-15 seconds of vigorous mixing. The dioxin standards were also treated through the solvent exchange step.


8

Procedure of EIA. EnviroGard reagents and microwell strip reader (Millipore Corporation) were used to perform the dioxin enzyme immunoassay. Mouse antibodies which are specific for 2,3,7,8-TCDD and related congeners were immobilized on the walls of plastic microwells. The strip format was first planned to allow for the placement of the negative control, four calibration standards, samples, and quality control samples. One hundred fifty microliters of reagent water was added to a microwell, and then 50 uL of standards or prepared sample extracts in methanol was mixed with reagent water in the microwell. The microplate was covered and incubated for 30 minutes at room temperature. Following a washing step, the well was then incubated with a 200 uL aliquot of competitor-HRP conjugate. After 30 minute incubation, the unbound conjugate was washed away with reagent water and 100 uL of substrate was added to result in the formation of blue color. After 30 minutes, 100 uL stop solution was added to each well to stop the color development The optical density in each well was measured by the microwell strip reader at a wavelength of 450 nm. The concentration of 2,3,7,8-TCDD was determined by relating the absorbance response to that of standards. GC/MS/MS Confirmation. The extracted analytes were separated by injecting a aliquot of the concentrated extract to a high resolution fused silica capillary column and identified and quantitated by Ion Trap MS/MS. Reference product ion spectrum and retention time for dioxins are obtained by the measurement of calibration standards under the same GC/MS/MS conditions used for sample analysis. Results and Discussion Working range and reproducibility. We first evaluated the linear working range of the standard curve and then demonstrated the reproducibility of standard curves by generating five standard curves and calculating the coefficient of variation of these five observations. The standards were run in duplicate, and the absorbance values were converted to %Bo by dividing the absorbance of a non-zero standard by the absorbance value of the negative control. The working range of the assay was from 15 pg/L to 100 pg/L (Figure 2). The stability of the dioxin standard curve was evaluated by comparing the standard curves generated over six months. The reproducibility of the dioxin standard curves is presented in Table I. The percent coefficient of variation (%CV) was less than 15% and indicates good reproducibility.



274


Figure 1. Structure of 2,3,7,8-TCDD.

\

\>

\>

V

f e

\*

T>

Log [2,3,7,8-TCDD, pg/L] Figure 2. Standard curve of 2,3,7,8-TCDD. Each point represents the mean of five replicate determinations.


22. WANGETAL.

Rapid Determination ofDioxins in Drinking Water


Extraction efficiency. The solid phase extraction method was established to achieve concentration factors required to meet the detection limit of 15 pg/L in water. The dioxin extraction efficiency was tested by extracting seven replicates of water samples at the spiked concentration of 30 pg/L which is the M C L for 2,3,7,8-TCDD set by USEPA. The concentrations of 2,3,7,8-TCDD in these extracts were determined by GC/MS/MS. The recovery and % C V of the extraction are presented in Table II. This study demonstrates that recoveries between 70% and 85% with a % C V of less than 7.5% can be achieved using our extraction procedure. Table I. The Stability of Dioxin Calibration Curve

_ Obs.#

Month

1 2 3 4 5 Mean SD. %CV

1 2 3 4 6

(15pg/L)

(25 pg/L)

77.4 77.2 74.2 75.4 83.4 77.5 3.56 4.59

55.9 65.3 65.1 59.1 68.7 64.6 3.47 5.37

(50pg/L) 34.2 39.8 41.8 31.9 42.5 39.0 4.22 10.8

(100 pg/L) 20.1 21.2 27.0 18.8 25.8 23.2 3.33 14.3

Table II. Recovery of 2,3,7,8 - TCDD Extraction as Determined by GC/MS/MS (Spiked Concentration: 30 pg/L in Reagent Water) Obs.#

Cal. Cone. pg/L 1 2 3 4 5 6 7 Mean(n=7) SD. (n=7) % C V (n=7)

25.7 21.4 24.6 20.9 22.9 22.2 21.0 22.3 1.73 7.74

% Recovery 85.7 71.3 81.9 69.6 76.2 74.0 69.9 75.5 5.75 7.74


21S


276


Precision and Accuracy. Five replicates of reagent water spiked with certified standards within the working range were tested to evaluate the precision and accuracy of this assay. Table III shows the validation results of the EIA by performing five replicates of reagent water spiked at a concentration of 25 pg/L. The recovery of the dioxin assay ranged from 74% to 122%, and % C V for five replicates was less than 15%. The results presented in Table III illustrate that the precision and accuracy of this assay were very good. However, the recoveries of some spiked samples were higher than 100%. These false positive results might be caused by residual methylene chloride in the extracts because methylene chloride may kill the antibody and reduce absorbance vaule.

Table in. Precision and Accuracy of Dioxin Enzyme Immunoassay (Spiked Concentration: 25 pg/L in Reagent Water) Cal. Cone. pg/L Rep-1 Rep-2 Rep-3 Rep-4 Rep-5 Mean (n=5) SD. (n=5) % C V (n=5)

18.6 23.8 30.4 23.7 26.4 24.6 3.53 14.4

% Recovery 74.3 95.1 121.7 95.0 105.5 98.3 14.1 14.4

Drinking water samples from different water sources and matrix spikes were also tested to demonstrate the accuracy and the matrix effects of this method. The drinking water samples were obtained from municipal water sources including ground water, surface water and bottled water. The results of dioxin enzyme immunoassay were compared with the GC/MS/MS method on all the samples in order to evaluate accuracy, matrix effects, and method performance. Before spiking with dioxin, the seven water samples were analyzed by GC/MS/MS in order to demonstrate that no dioxin residue was present The analysis of EIA for these samples showed that the response of the samples were similar to that of the negative control, thus the water matrix did not affect this assay. The accuracy of EIA was also tested by analyzing these samples spiked at the final concentration of 30 pg/L of 2,3,7,8-TCDD. The spiked samples were tested by EIA and GC/MS/MS. The results were summarized in Table IV. Compared with the GC/MS/MS results, EIA was consistent with the results obtained by GC/MS/MS indicating that the enzyme immunoassay has good correlation with GC/MS/MS for dioxin analysis. The slightly higher recovery of EIA than GC/MS/MS might be caused by some solvent residues and sample matrix interferences.


22. WANG ET AL.

Rapid Determination of Dioxins in Drinking Water


The specificity of the dioxin antibody has been shown to be primarily directed toward selected tetra- and pentachlorodibenzodioxins, with reduced recognition of the corresponding furans (Table V). This antibody is specific for 2,3,7,8-TCDD and 2,3,7,8-substituted dioxins and furans but detection levels are different for different congeners. Further studies are underway to evaluate the interferences in the dioxin enzyme immunoassay.

Table IV. Comparison of EIA with GC/MS/MS (Spiked Concentration: 30 pg/L) Sample #

Matrix

1 2 3 4 5 6 7

GW SW BW BW BW BW BW

GC/MS/MS Location EIA (States)

Immunochemical Technology for Environmental ... - ACS Publications

Recommend Documents