Anal. Chem. 1996, 68, 653-658
Microwave-Assisted Extraction Coupled with Gas Chromatography/Electron Capture Negative Chemical Ionization Mass Spectrometry for the Simplified Determination of Imidazolinone Herbicides in Soil at the ppb Level Steven J. Stout,* Adrian R. daCunha, and Darryl G. Allardice
American Cyanamid Company, Agricultural Products Research Division, P.O. Box 400, Princeton, New Jersey 08543-0400
The imidazolinones are a significant new class of low-userate, reduced-environmental-risk herbicides for protection of a wide variety of agricultural commodities. Because of their low rates of application, residues of the imidazolinones in soil need to be monitored at the low ppb level. Current methodology for this purpose involves an exhaustive base extraction followed by a laborious, time-consuming cleanup procedure to permit analysis at the 5 ppb level. Using imazethapyr, the most widely utilized member of the class, as a representative, we have achieved not only a limit of quantitation of 1 ppb but also a greatly simplified cleanup procedure by combining microwaveassisted extraction technology with gas chromatography/ electron capture negative chemical ionization mass spectrometry. On a variety of soil types covering a fortification range of 1-50 ppb, an average recovery of 92% with a standard deviation of 13% was achieved. Control soil extracts gave apparent residues of 90% at pH 10, where the yellow color of the fulvic acids became apparent. At pH 11, the recovery was 98%, but the dark brown color of the humic acids was evident (Figure 2). From these data, it appeared important to achieve a pH of greater than 7.0 in the final extract to maximize extractability of the imidazolinone. In fact, adsorption of imidazolinones to soil has been reported to increase significantly as the soil pH decreases to 6.17 The soil that provided the toughest test for extraction was the ARK soil. This soil has been reported to have one of the highest (17) Mangels, G. In ref 1, Chapter 16.
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Table 4. Recoverya (%) of Imazethapyr from Soil
soil
control (ppb)
1
fortification (ppb) 10
50
IND ARK NJ ND
0.17 0.15 0.18 0.13
102.6 96.9 94.2 81.6
112.7 92.6 96.6 84.8
100.3 78.1 88.6 77.0
a
Figure 2. Extracts of the Tippecanoe (IND) silt loam soil after MAE and centrifugation. From left to right, pH 7, 8, 9, 9.5 (1.0 M), 10, and 11. Except for pH 9.5, all other extracts are from 0.1 M NH4OAc. Table 3. Evaluation of NH4OAc/NH4OH Extractants on IND, ARK, and Aged Field-Treated Soilsa soilb
fortification (ppb)
pH (init)
concnc (M)
extractability (%)d
IND IND IND IND IND ARK ARK ARK ARK ARK ARK 090D 270D 270D 270D 270D 270D 270D
10 10 10 10 10 10 10 10 10 10 10 T T T T T T T
9.0 9.0 9.5 9.5 10 9.0 9.0 9.0 9.5 9.5 10 10 10 10 9.5 9.5 10 10
0.1 1.0 0.1 1.0 0.1 0.1 0.5 1.0 0.1 1.0 0.1 0.1 0.1 0.1 1.0 1.0 1.0 1.0
87 98 99 107 91 66 81 79 83 96 92 104 87 85 83 78 80 81
comments clear yellow yellow yellow amber clear yellow tint yellow tint light yellow more yellow yellow tint
above after C18 above after C18
a
Each result given for a field-treated sample represents the average of duplicate extractions. Conditions: TAP, 3 min; 125 °C. b IND, Tippecanoe silt loam; ARK, Sharkey clay loam; T, aged field-treated soil 90 and 270 days after application. c [NH4OAc]. d Extractability determined by comparing dpm/g from extraction to dpm/g from combustion (1868 at 090D and 689 at 270D).
adsorption coefficients for the imidazolinones.17 As shown in Table 2, recoveries with 0.1 M NH4OAc were unacceptably low at both pH 7 and 9. As with a simple H2O extraction, lengthening the extraction time to 10 min from 3 min gave no improvement. Increasing the concentration of NH4OAc increased the recovery but, as with increasing the pH to 10 at 0.1 M NH4OAc, gave more yellow color in the final extract. A final evaluation of NH4OAc/ NH4OH extractants from pH 9.0 to 10.0 with NH4OAc concentrations ranging from 0.1 to 1.0 M was conducted on the IND soil to check for color and on the ARK soil to check for extractability. As shown in Table 3, the net result was that pH 9.5 at 1.0 M gave results comparable to those with pH 10.0 at 0.1 M. The above conclusion was also reached following an evaluation of extractants on aged field-treated soils, the ultimate test of an extraction technique. As shown in Table 3, pH 9.5 at 1.0 M gave essentially the same results as pH 10.0 at 0.1 M. Even increasing the NH4OAc concentration to 1.0 M at pH 10.0 had no effect on the extractability. With these results indicating little additional advantage in trying to further fine-tune the extractant, the pH 10, 0.1 M NH4OAc/NH4OH extractant was selected for the remainder of the study. 656 Analytical Chemistry, Vol. 68, No. 4, February 15, 1996
The averages of duplicate samples processed through the procedure.
While, unfortunately, the ultimate goal of a clear final extract was not achieved, acceptable extractability of imazethapyr from soil with MAE was demonstrated at the required level using only a 3 min extraction, and a visually cleaner initial extract was obtained compared to that from the conventional extraction. This extractant evaluation also highlighted the conflicting nature of needing to have one extractant that spanned a range of soil types. While a “less harsh” extractant gave acceptable recoveries and clear extracts from NJ and IND soils, a “stronger” extractant was needed for the “more difficult” ARK soil. Even though the ARK soil extract was not very colorful under these conditions, the need to use the same extractant on the IND and NJ soils released more color from these soils. SPE Cleanup and GC/ECNCI. The imidazolinones can be methylated for GC/MS by using TMAH for an in situ methylation in the GC injector port. Previous work in our laboratory indicated an enhanced negative ion response compared to positive ion response from electron capture by the methylated imidazolinone with generation of solely the M-.16 While the enhancement was not as tremendous as that from heavily halogenated, nitrated, and/ or conjugated systems,18-21 it was still substantial. Therefore, we attempted to use the sensitivity and specificity of GC/ECNCI to ascertain if a simpler cleanup could be accomplished with the initially cleaner extract from MAE. We predicated our sample requirements around needing 10 pg of analyte on-column to achieve a reasonable GC/ECNCI response. To detect 1 ppb in soil, this parameter dictated that we process 10 g-equiv of soil after extraction into a final volume of 1 mL for injection of 1 µL. With the IND soil extract showing the most color and with the worst-looking extract usually posing the greatest challenge for cleanup, the IND soil was selected as the initial target for cleanup efforts. Since the CH3OH elution of a 500 mg C18 SPE cartridge gave ∼95% recovery of the analyte in our 14C extractability studies, our initial attempt at a simple cleanup procedure was to process 10 g-equiv of the soil extract in the same manner. While this procedure left a brown band at the top of the SPE sorbent bed, a noticeable light yellow color was present in the CH3OH eluent collected from the cartridge. While no particular interference peaks were detected at the retention time of the analyte in the chromatograms of control extracts, extracts fortified at 10 ppb gave recoveries of 400-700%, indicating a severe matrix enhancement of the response. Obviously, there were limits to the level of matrix coextractives that even the sensitivity and specificity of (18) Hunt, D. F.; Stafford, G. C.; Crow, F. W.; Russell, J. W. Anal. Chem. 1976, 48, 2098-2105. (19) Dougherty, R. C. Anal. Chem. 1981, 53, 625A-636A. (20) Stout, S. J.; Steller, W. A. Biomed. Mass Spectrom. 1984, 11, 207-210. (21) Stout, S. J.; Cardaciotto, S. J.; Millen, W. G. Biomed. Mass Spectrom. 1983, 10, 103-106.
A
B
Figure 3. Chromatograms from selected ion monitoring of (A) control IND extract and (B) 1 ppb fortified IND extract. Dimethylated imazethapyr elutes at 14:11.
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GC/ECNCI could tolerate. Further cleanup of these CH3OH eluents using the SCX cartridge procedure of the conventional method5 did allow us to achieve the first and foremost goal of the projectsquantifying imazethapyr at 1 ppb in soil. Unfortunately, while shortening the extraction time and eliminating large volumes of organic solvents per sample, the overall method was not inordinately simpler than the existing ones. However, during this phase of development, two important observations were made that resulted in a dramatic simplification of sample cleanup. First, we found that our initial estimate of negative ion response for the imidazolinones was overly conservative. By reducing the level of analyte to 1 pg on-column, we were able either to dilute the final extract 10-fold or, as we found more beneficial, to reduce the amount of soil extract processed 10-fold to 1 g-equiv. Second, we determined that CH2Cl2 could selectively elute the analyte from the C18 cartridge while leaving all of the color on the cartridge. Combining these two features with the initially cleaner extract from MAE enabled us to effect cleanup on a single, small (200 mg) C18 cartridge. Thus, we were able to achieve the second goal of the project. To validate the method, control soils were spiked with the appropriate imazethapyr standard solution and carried through the procedure. Recoveries were run in the fortification range of 1-50 ppb, and the results are given in Table 4. Overall, the recovery (expressed as the average (1 standard deviation) was 92 ( 13%. Control soils showed apparent imazethapyr residues of
