Application Note AN-004
Last Revised: 12/4/98

  Analysis of PCDD/Fs in soil at 10 ppb using immunoassay specific extraction and cleanup

Contents of This Application Note

A.

Summary of this Application Note

B.

Materials Required for this Analysis

C.

Summary of Sample Preparation and Immunoassay Analysis

D.

Detailed Sample Preparation Procedure

E.

Interpretation of Immunoassay Results
F. Validation Data Supporting this Method

A.

Summary of this Application Note

This Application Note describes how to use an immunoassay specific extraction and cleanup method to prepare soil samples for screening analysis at 10 ppb using the CAPE Technologies High Performance Dioxin/Furan Immunoassay Kit.

The materials required for the analysis are listed in section B. The procedure is summarized below in section C and is described in detail in section D. Information on interpretation of results is given in section E. Validation data supporting this method are given in section F. The procedure described in this Application Note eliminates most of the time and effort required for sample extraction and most of the extract cleanup required for GC-MS analysis. This Application Note is intended to be used in conjunction with the Dioxin/Furan Immunoassay Kit Insert (IN-DF1) and the Sample Preparation Kit (SP1).

 

B.

Materials Required for this Analysis
1.

DF1-60 Immunoassay Kit and items specified in section G of the kit insert (IN-DF1)

2. SP1-12 Sample Preparation Kit and items specified in section D of the kit insert (IN-SP1)
3. solution of 7% SO3 in conc. H2SO4 (1.5 mL per sample)
4. hexane, HPLC or residue grade (approx. 4 mL per sample)
5. glass pasteur pipets
6. pipets for items 3 and 4 above

 

C.

Summary of Sample Preparation and Immunoassay Analysis
1. Add sodium sulfate to soil sample and mix. Add dimethylformamide (DMF) and extract by shaking 2 hours. Remove the supernatant DMF extract.
2. Treat an aliquot of the DMF extract with 7% SO3 in conc. H2SO4, then extract 3 times with hexane.
3. Exchange hexane extract to methanol using the procedure described in the kit insert IN-DF1.
4. Perform the immunoassay procedure as described in the kit insert IN-DF1.
5. Interpret the immunoassay results as described in section D of this Application Note.


D.

Detailed Sample Preparation Procedure
1. Use CAPE Technologies Sample Preparation Kit (SP1). Using wooden spatula from SP1 kit, mix sample thoroughly and weigh 5 g into 40 mL extraction vial from SP1 kit.
2. Add 15-20 g anhydrous sodium sulfate to extraction vial and mix with wooden spatula until sample is free flowing. Add 3 steel mixing balls from the SP1 kit, then 15 mL dimethylformamide (DMF). Read and follow precautions and other instructions in SP1 kit insert (IN-SP1). Cap vials tightly and extract by shaking 2 hours at 350 rpm on orbital platform shaker. Extraction vials should lay flat on their sides for maximum agitation.
3. Centrifuge at 2000 x g for 10-15 minutes and remove a portion of the supernatant DMF extract to a 4 mL vial from SP1 kit. The concentration of soil matrix in the extract will be 0.33 mg soil equivalent per µL.
4. For screening analysis of soil at 10 ppb, it is necessary to use 1 mg of sample equivalent for each immunoassay tube. The following procedure allows analysis using single or duplicate EIA tubes with minimal leftover sample. Use 9 µL of extract, which is equivalent to 3 mg of original sample. This will ultimately be reconstituted in 30 µL, allowing either one or two 10 µL aliquots to be removed for immunoassay analysis. It is also possible to use a larger volume of extract to allow significant leftover sample. However, the maximum amount of DMF which can be introduced into the oxidation protocol below is 100 µL. In either case, the amount of extract delivered to each immunoassay tube should be equivalent to 3 µL of the original DMF extract (1 mg of the original soil sample).
5. Place 9 µL of DMF extract into a 4 mL vial from SP1 kit..
6. Add 1 mL of hexane, then 1.5 mL of 7% SO3 in concentrated H2SO4. Cap vial and mix vigorously for at least 2 minutes. Centrifuge to separate phases completely (5 minutes or less at 1000 to 5000 x g).
7.

Remove as much hexane supernatant as possible without disturbing the lower layer.

Caution: Do not allow the lower phase to contaminate the hexane sample. Any oxidizer which contaminates the sample at this point will be carried through into the immunoassay, possibly leading to invalid results.

Place the recovered hexane in a small round bottom glass tube, such as 10 x 75 mm, or a small conical vial. Either shape permits efficient recovery of the small reconstitution volume in step 11.
8. Repeat the hexane addition, mixing, centrifugation, and supernatant removal of steps 6 and 7 twice more for a total of 3 hexane extractions. It is not necessary to add more 7% SO3 in concentrated H2SO4 after the first extraction.
9. Add an aliquot of methanol + 100 ppm Triton X-100 (see immunoassay kit insert IN-DF1, section I.1) to the hexane sample. The aliquot volume should be the same as the planned reconstitution volume (30 µL or larger volume if so chosen).
10. Evaporate the hexane samples at room temperature under a gentle stream of nitrogen as described in the immunoassay kit insert IN-DF1, section I.
11. Refer now to section I.6 of the immunoassay kit insert (IN-DF1). Redissolve the sample by adding methanol to give the same volume as methanol-Triton in step 9 above. Perform this step only after the EIA tubes have been prepared for standard and sample addition according to the immunoassay kit insert IN-DF1, section J, 1-4.
12. Perform the immunoassay procedure as described in the immunoassay kit insert IN-DF1, section J, 5-11.


E.

Interpretation of Immunoassay Results
1. Calculate each optical density (OD) reading as a percent of the negative control OD reading (%NC). Refer to the immunoassay kit insert IN-DF1, section D (Table 1), for comparison of standard results to acceptable ranges. If your standards are not within these ranges, your results may be invalid.
2. Samples which have %NC values greater than standard 2 (10 pg/tube) contain less than 10 ppb TEQ in the original sample. Remember that there is an inverse relationship between OD and concentration. Less color means a higher concentration of PCDD/Fs.
3. Samples which have %NC values less than standard 2 (10 pg/tube) contain more than 10 ppb TEQ in the original sample.
4. Samples which have %NC values the same as standard 2 (10 pg/tube) contain approximately 10 ppb TEQ in the original sample. Consult the immunoassay kit insert IN-DF1, Table 3, for guidance in deriving additional screening information from your results.
5. For suggestions on incorporation of these results into an immunoassay based screening program, consult CAPE Technologies Technical Note TN-002.
6. Advanced analysts may wish to use the CAPE Technologies High Performance Dioxin/Furan Immunoassay Kit to produce quantitative results. To understand the requirements and limitations of this approach, read CAPE Technologies Technical Note TN-004.


F.

Validation Data Supporting this Method
Validation Data Supporting this Method
Soil samples were analyzed by conventional GC-MS. Subsamples of the same 18 soil samples were extracted separately, without mass-labeled internal standards. These samples were analyzed by immunoassay using the protocol described in section C. Two different comparisons of the results from the two methods are given in the following figures. Separate subsamples of the same soils were extracted with hexane:acetone (1:1) and the non-volatile residue was weighed to determine oil content. The mean oil content for 9 of these 18 samples was 3.5% (range 0.2 to 7.2) [expanded data to be provided later] and did not correlate with TEQ. These results clearly establish the ability of the CAPE Technologies High Performance Dioxin/Furan Immunoassay Kit to analyze TEQ in soil samples using an immunoassay specific sample extraction and cleanup.

 

Figure 1. Correlation between quantitative immunoassay analysis and TEQ as determined by high resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS) for 18 soil samples. The calculated regression line is shown with 99% confidence limits. These results clearly establish the ability of the immunoassay to measure TEQ at ppb levels in soil samples using an immunoassay specific sample extraction and cleanup.

 

Figure 1 to be Added Later

 

Figure 2. Quantitative EIA results for 18 oxidized DMF soil extracts plotted in screening format. Figure 1 data were plotted and overlaid with lines indicating 1) the TEQ value chosen as a screening level (10 ppb vertical line) and 2) the EIA response at which the screening decision would be made (6 ppb horizontal line). The number of results in each quadrant is indicated on the plot. These results clearly establish the ability of the CAPE Technologies High Performance Dioxin/Furan Immunoassay Kit to screen soil samples at 10 ppb TEQ using an immunoassay specific sample extraction and cleanup.

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