INTRODUCTION
Atrazine belongs to a class of herbicides called sñtriazines. The triazine pesticides have been in use for the past few decades to control weeds in agricultural crops such as corn, soybean, sugar cane, etc. Due to the stability of the nitrogenñcarbon aromatic ring (the triazine structure) these compounds do not readily mineralize. Atrazine can be found in ground water in many of the areas of the east and midwest as well as in the Great Lakes at low ppt concentrations.
A water sample has been obtained from a stream below a major corn producing region of Ontario. Your objective is to analyze for Atrazine, a major corn-use herbicide, in this water. You will utilize the two techniques of ELISA and GC/MS to "measure" the atrazine in your sample. Be aware that other compounds might also be present in the water sample and please consider what these might be and what, if any, this knowledge influences your analysis.
Preliminary characterization indicates the expected Atrazine
concentration to be between 50 and 200 ppb. This information
is given because you'll need to decide how to "prepare"
the sample for ELISA and GC/MS analysis. Your standards for ELISA
analysis are 0, 0.1, 1.0, and 5.0 ppb while the standards for
GC/MS are nominally 2, 6, 12, and 20 ng/ml.
Thus you will need to "dilute" the sample for ELISA
analysis and extract, clean-up, and concentrate your sample for
GC/MS.
BACKGROUND ON IMMUNOASSAY
Antibody titers are raised in an experimental animal (most commonly mice and rabbits) in response to the antigen. Usually an effective antigen must be large and foreign to the animal immunized. As most pesticides are rather small molecules, they must be conjugated to a large protein molecule before they can be used as antigens. The pesticide may or may not have a functionality suitable for conjugation. If not, then a derivative of the pesticide must be synthesized.
All immunoassays are physical assays and are based on the Law of Mass Action. They combine the specificity and sensitivity of bioassays with the speed and precision of physical assays. The enzymeñlinked immunosorbent assay is based on the fact that antigen or antibody can be attached to a solidñphase support yet retain immunological activity, and either antigen or antibody can be linked to an enzyme while the complex retains immunological and enzymatic activity. The ELISA can be used to detect both antigen and antibody. There are many basic formats that can be used use for ELISA. We will be using a technique that involves antibody-coated magnetizable particles. A competition is set up between atrazine in the water sample and an enzyme-conjugated hapten (looks like atrazine) for the magnetizable antibodies. Following a set incubation period the antibodies are separated by applying a strong magnet to hold the the paramagnetic particles (with atrazine and labeled atrazine analog bound to the antibodies in proportion to their original concentration) in the tube to allow the unbound reagents to be decanted. The presence of atrazine is detected by adding the enzyme substrate (hydrogen peroxide) and the chromogen. The enzyme-labelled atrazine analog bound to the atrazine antibody catalyzes the conversion of the substrate/chromogen mixture to a colored product. Following a second incubation period the reaction is stopped and the color monitored by spectrophotometry. Since the labeled atrazine (conjugate) was in competition with the unlabeled atrazine (sample) for the antibody sites, the color developed is inversely proportional to the concentration of atrazine in the sample.
We will be using the magnetizable antibodies provided by Ohmicron,
as well as conventional GC analysis with Mass Spec detection,
to determine the concentration of Atrazine in a field water sample.
Try to observe the inherent strengths and weaknesses of each
technique. Also ask yourself whether there could be other compounds
you might be interested in "measuring" in the field
water. For example would you expect any atrazine degradation
products and if so what would they be?
PROCEDURE
I. Sample extraction: C18 extraction:
Take 2 new C18 cartridges, clean them by rinsing with two column
volumes each of ethyl acetate, methanol, then water. Do not let
the columns go dry, but do not add the next cleaning solvent until
the previous one is down to just above the packing level. You
may do this on the vacuum manifold. As the last column volume
of water is added, place the reservoirs on top of the columns
and add the triazine water sample to one cartridge and distilled
deionized water through the other until the total sample has passed
through the cartridges. Remove the cartridges from the vacuum
manifold and elute with 1 or 2 mL of ethyl acetate into a clean,
graduated test tube. Note: Check with the TA as to
the appropriate volume of sample to extract and the amount of
eluting solvent.
II. GC analysis
Using an instrument equipped with a mass spec detector, analyze the extracted samples for atrazine content. You should make a three point standard curve with 2 µl injections of the standards provided (1, 5, and 10 ng/µl). You should start making the standard curve while doing your sample extractions. Inject 2 µl portions of your extracts and determine the concentration of atrazine in your water sample. Again, are there any other possible atrazine degradation products that you might want to look for?
Check with the TA whether your volume of sample to be extracted is appropriate for GC/MS analysis. With this information proceed to the SPE sample preparation area and prepare two (you're to do replicate samples) SPE C-18 cartridges (note the size of the tube and sorbent mass, etc) by sequentially passing 2 column volumes of hexane, two of methanol, and two of water before begin to pull your sample through. Note: do not let the cartridge go dry during the solvent prep or sample extraction stages. After your samples have been drawn, by vacuum, through the SPE cartridges let them dry for a few minutes under vacuum after which transfer them to the centrifuge for further drying (5 minutes). The final step is to put exactly 1 ml of hexane (glass volumetric pipette) into the top of the cartridge and spin this in the centrifuge (1 min.). Clearly label both tubes for subsequent analysis on the GC/MS.
With your samples proceed to the Mass Spec facility where you'll
be under the tutelage of Dan Mathers. Your objective is to determine
the amount of atrazine and any other interesting compounds in
your sample using GC/MS in single ion monitoring mode (SIM).
This will require you to choose a suitable mass peak to monitor
during the running of samples and standards for each of your target
analytes. This information will be obtained from a mass spectrum
provided in the lab. After you obtain responses for the standards
and samples you can determine the concentrations back in your
original water sample using the external calibration method you've
used previously.
III. ELISA
Check your suggested dilution factor with the TA prior to proceeding with sample analysis by ELISA; typically this will be 100 to 1. With this information obtain a sample of the field water and proceed with the dilution. You will want three replicates of your sample so do each dilution 3x with separate volumetric flasks; you may use the sample glass volumetric pipet.
Label the test tubes as follows TT#1: 0 ppb; TT#2: 0.1 ppb; TT#3: 1.0 ppb; TT#4: 5.0 ppb; TT#5: Control 3.0 ppb; TT#6: SR 1;TT#7: SR 2;TT#8: SR 3. SR refers to "sample replicate".
Then:
1). Add 200 mL of the appropriate standard, control, or sample.
2). Add 250 mL of Atrazine Enzyme Conjugate to each tube.
3). Mix the Atrazine Antibody Coupled Paramagnetic Particles thoroughly and add 500 mL to each tube.
4). Vortex for 1 to 2 seconds while minimizing any foaming.
5). Incubate for 15 minutes at room temperature.
6). Separate in the Magnetic Separation Rack for two minutes.
7). Decant and gently blot all test tubes in a consistent manner.
8). Add 1 mL of Washing Solution to each tube and allow them to remain in the magnetic separation unit for two minutes.
9). Decant and gently blot all tubes briefly in a consistent manner.
10). Repeat steps 8 and 9 an additional time.
11). Remove the rack from the separator and add 500 mL of Color Solution to each tube.
12). Vortex for 1 to 2 seconds minimizing foaming.
13). Incubate for 20 minutes at room temperature.
14). Add 500 mL of Stopping Solution to each tube.
15). Add 1 mL Washing Solution to a clean test tube. Use as blank in Step 16.
16). Read results at 450 nm within 15 minutes after adding the
Stopping Solution.
The photometer you'll be using to measure the absorbance of your
standards and samples is the mobile unit RPA-III RaPID Analyzer
System. Your TA will instruct you how to use this instrument.
After you obtain your data for standards, control, and sample
you are then able to generate a standard curve in order to determine
the concentration of "atrazine" in the original field
water sample.
REPORT
Present all data in table form. Enclose representative total ion chromatograms and mass spec of both standard and sample injection. Graph the GC-MS standard curve for atrazine and estimate the unknown concentration of the samples from the graph. Include your ELISA printout data. Indicate which data points were used to determine water concentration.
Compare the GC data with that of the ELISA, discuss both the strengths
and weaknesses of these analytical techniques, specifically with
regard to sensitivity, accuracy, precision and background noise.
Also compare the procedures and results of the two different
extraction procedures in terms of ease, relative recovery (extraction
efficiency), and background noise levels. Would the same method
be preferred in all situations? Are there some situations where
a method with poorer recoveries would have advantages over one
with more accurate results? What is actually being measured in
both techniques?
Data:
1. Calculate the concentrations of all atrazine constituents
from your GC/MS results and report your data in a table. Show
your calculations and include an example of a the std curve, and
a typical chromatogram from std and a sample.
2. Calculate the concentration of atrazine from your ELISA
method and report your data in the above table. Include the result
for the control sample. Also include your calculations and your
standard curve.
Questions:
1. What are the strengths of the GC/MS method? What are
the advantages of SIMs over a total ion chromatogram (TIC)? What
are the weaknesses of GC/MS?
2. What are the strengths of ELISA? What are the advantages
of ELISA over GC/MS? The disadvantages?
3. Draw a "cartoon" of the ELISA steps clearly
highlighting with each "panel" the steps involved in
carrying out the assay and showing the roles of the analyte, the
atrazine enzyme conjugate, the ironed out antibodies, the wash
solution, the color solution, etc.
4. When would you use GC/MS? When would you specifically use
ELISA?
BACKGROUND REFERENCES
Gee S.J., Miyamoto, T., Buster D., and Hammock B.D. 1988. Development of An Enzyme Immunosorbent Assay for the Analysis of the Thiocarbamate Herbicide Molinate In Water. J Agric Food Chem 36:863ñ870.
Goodrow M.H. et al. Hapten Synthesis, Antibody Development, and Competitive Enzyme Immunoassay for sñTriazine Herbicides. J Agric Food Chem (submitted manuscript).
Hammock B.D. and Mumma R.O. Potential of Immunochemical Technology for Pesticide Analysis. In: "Pesticide Analytical Methodology", ACS Symposium Series 136, ACS, Washington D.C., pp 321ñ352 (1980).
Huber R. Structural Basis for AntigenñAntibody Recognition. Science 233, August 1986.
Jung F. et al. 1989.Use of Immunochemical Techniques for the Analysis of Pesticides. Pesticide Science 26:303ñ317.
Milstein C. From Antibody Structure to Immunological Diversification of Immune Response. Science 231, March 1986.
Seiber J.N., McChesney M.M., Sanders P.F., and Woodrow J.E. 1986. Chemosphere 15:127ñ138.
Van Emon J., Hammock B.D., and Seiber J.N. 1986. Enzymeñlinked Immunosorbent Assay for Paraquat and Its Application to Exposure Analysis. Anal Chem 58:1866.
Meulenberg, E.P., W.H. Mulder, and P.G. Stoks. 1995. Immunoassays for Pesticides. Environ. Sci. Technol. 29:553-561.
Thurman, E.M., D.A. Goolsby, M.T. Meyer, M.S. Mills, M.L. Pomes,
and D.W. Kolpin. 1992. A Reconnaissance Study of Hericides
and Their Metabolites in Surface Water of the Midwestern United
States Using Immunoassay and Gas Chromatography/Mass Spectrometry.
Environ. Sci. Technol. 26:2440-2447.