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Thermospray liquid chromatographic—mass spectrometric method for the analysis of metribuzin and its metabolites
Journal of Chromatography, 438 (1988) 359-367 Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands CHROM. 20 300
THERMOSPRAY LIQUID CHROMATOGRAPHIC-MASS RIC METHOD FOR THE ANALYSIS OF METRIBUZIN OLITES
SPECTROMETAND ITS METAB-
C. E. PARKER* Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.) A. V. GEESON*, D. E. GAMES and E. D. RAMSEY Department of Chemistry, University College, P.O. Box 78, Card& CFI IXL (U.K.) E. 0. ABUSTEIT** and F. T. CORBIN Crop Science Department, North Carolina State University, Raleigh. NC 27695 (U.S.A.) and K. B. TOMER Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 f U.S.A.) (First received November lOth, 1987; revised manuscript received December 3Oth, 1987)
SUMMARY
A thermospray liquid chromatographic-mass spectrometric (TSP LC-MS) method has been developed for the analysis of the herbicide metribuzin and its three major metabolites in plant tissue. Metribuzin and its metabolites exhibited widely varying sensitivities in positive-ion TSP, with metribuzin being the most sensitive and deaminated diketo metribuzin being the least sensitive. All four compounds of interest were detected in an extract of a soybean plant which had been treated with metribuzin.
INTRODUCTION
Metribuzin [4-amino-6-(l,l-dimethyJethyl)-3-(methylthio)-l,2,4-triazin-5(4~one; BAY 943371 is a systemic herbicide which is widely used for control of broadleaf weeds in a variety of food crops, including soybeans, tomatoes, and potatoes. Deaminated metribuzin (DA), diketometribuzin (DK), and deaminated diketometribuzin (DADK) have been identified as metabolites of this herbicide in crops and soillV3. Recent studies indicate that DA may exist predominantly in the enol form at physiological pH4. Structures of metribuzin and the three major metabolites are shown in Fig. 1. * Present address: Central Research Establishment, Home Office Forensic Science, Aldermaston, Reading, Berkshire, U.K. ** Present address: Agronomy Department, College of Agriculture, Cairo University, El-Giza, Egypt.
360
C. E. PARKER ef al.
TSP LC-MS OF METRIBUZIN
AND ITS METABOLITES
361
Gas chromatography (GC)s-g with electron-capture detection (ECD)+*, flame photometric detection in the sulfur mode 5, Coulson conductivity detection*, and alkali flame ionization detection9, and thin-layer chromatography (TLC)8,1 O-l * have been used for the detection and quantitation of metribuzin and its metaholites in plant tissue and soil samples. While these techniques may be sensitive, they do not provide structural confirmation of the peak detected. In addition, GC-ECD requires extensive sample clean-up, and TLC has problems with the separation of all of the metabolites and with providing quantitative data. Recently, we reported a high-performance liquid chromatographic (HPLC)13 method for the identification and quantification of metribuzin and its metabolites. This aproach has the advantage of requiring minimal clean-up of samples before analysis. It is, however, still limited by the non-specific nature of UV detection. Development of a combined high-performance liquid chromatographic-mass spectrometric (LC-MS) method for metribuzin and its metabolites would be of considerable assistance in the confirmation of HPLC peak identities prior to quantitative studies, and should assist in the observation and identification of new metabolic products. While thermospray (TSP) LC-MS is a relatively new technique114-16, its potential for the analysis of pesticides and their metabolites has already been demonstrated”-la. We have also shown that metribuzinlg is amenable to analysis using direct liquid introduction. TSP LC-MS has the advantage that it is commercially available and works best with aqueous mobile phases at flow-rates of approximately 1 ml min-‘, thus enabling all of the eluent from the liquid chromatograph to be fed into the mass spectrometer ion source. In “classical” TSP (i.e., TSP without a filament), ionization occurs through the use of a buffer such as ammonium acetate, and results in spectra often containing abundant protonated molecular ion species (positive-ion TSP), or proton abstraction or acetate addition peaks (negative-ion TSP). We report here the results of our investigation into the development of a TSP LC-MS method for the determination of metribuzin and its metabolites in plant tissues. The application of the developed protocol to an extract of a metribuzin treated soybean plant is also reported. EXPERIMENTAL
The LC-MS experiments were performed using a Waters 6000A pump (Waters Assoc., Milford, MA, U.S.A.) connected to a 25 cm x 4.6 mm I.D. column packed with 5-pm particle size DuPont Zorbax (DuPont Instruments Analytical Division, Wilmington, DE, U.S.A.). The mobile phase used was methanol-water (60:40, v:v) containing 0.05 A4 ammonium acetate, at a flow-rate of 1.2 ml min-‘. The column was connected to a Finnigan MAT TSP LC/MS interface on a Finnigan/MAT 4500 quadrupole mass spectrometer (Finnigan/MAT, San Jose, CA, U.S.A.). Interface parameter were vaporizer temperature, 118”C, and jet temperature, 200°C. Mass spectra obtained over the mass range m/z 145-950, at a scan rate of 2 s per scan, were recorded and processed using a Finnigan/Incos 2300 data system (Finnigan/ MAT). Solvents for LC were redistilled using an all-glass apparatus before use. Metribuzin was obtained from the U.S. Environmental Protection Agency Pesticide Repository (USEPA, Research Triangle Park, NC, U.S.A.). Analytical standards of DA, DK, and DADK were a gift from Mobay Chemical Corp., Agricultural Chemicals Division (Kansas City, MO, U.S.A.).
C. E. PARKER
362
et al.
RESULTS AND DISCUSSION
For the LC-MS studies, the LC conditions which we had developed for HPLC-UV studies12 of metribuzin and its metabolites were modified by substitution of 0.05 M ammonium acetate for acetic acid as the mobile phase modifier. Initial LC-MS experiments were performed using solutions of individual compounds. Aliquots were injected into a stream of methanol-water (60:40), containing 0.05 M ammonium acetate. This solution was continuously fed into the thermospray ion source operated in the positive ion mode. The compounds exhibited widely barying molar sensitivities (based on the relative intensities of the base peaks in the spectra). Metribuzin is the most sensitive, followed by DK and DA. DADK gives a very weak spectrum (Table I). These sensitivity differences probably reflect differences in gasphase proton affinities of the metabolites 2o. The negative ion spectra contained [M - HI-and [M + OC(O)CH$ions oflow abundance. Since negative ion sensitivities were significantly lower than positive-ion sensitivities, and because high sensitivities were expected to be needed for the detection of these compounds at biologically significant levels, positive-ion conditions were employed in the remainder of the study. As DADK was the least sensitive compound in the positive-ion mode, TSP conditions were optimized for this analyte. Optimal sensitivity was obtained at 1lS”C, the highest temperature accessible with the Finnigan TSP interface used. Under these conditions, metribuzin and DA gave [M + H]+ ions at m/z 215 and 200, respectively, which were also the base peaks in their respective spectra (Fig. 1). In contrast, DK gave an [M + NH41 + ion at 202, as the base peaks in its spectrum. The spectrum of TABLE I TSD MASS SPECTRA OBTAINED BY LC-MS OF A STANDARD MIXTURE AND A PLANT EXTRACT SPIKED WITH A STANDARD MIXTURE OF METRIBUZIN (MZN) AND ITS THREE MAJOR METABOLITES Peak
Identity
Mol. wt.
Positive-ion TSP mass spectra [m/z (“/a rel. int.)] Standard mixture
Spiked sample
Rel. mol. sens.
A
DK
184
202 (100) 185 (2)
202 (100) 261 (lo), 185 (3)
70
B
DADK
169
247 (lOO), 246 (69), 187 (15)
247 (loo), 246 (67) 187 (28)
4
C
DA
199
200 217 273 259
200 217 254 273 399
D
MZN
214
215 (loo), 274 (3)
(loo), (13), (IO), (7)
(100) (44), (26), (14), (3)
300
215 (100) 274 (8)
1000
C. E. PARKER et al.
364
with metribuzin, but which had been spiked with all four compounds. The solution injected into the chromatograph contained 2.8 pg each of DA, DK, and metribuzin, and 5.6 pg of DADK. Table I summarizes the spectra obtained. The spectra obtained compare well with those obtained from standard mixtures. Some variation in retention time was observed, presumably due to interaction of the matrix with the column, but this presented no difficulty in the detection of the compounds of interest. No 188.1 B-
TIC
w
2b0
‘I&
I&
Sk
638
SCAH
2910
12m8,DADK
187.03 f 0.59
$ 187_ ...
._ .
. . -.
A_... ._. . A
. _ de _ . CI
.
.. . .
. .
.
r
1 la@
ze
4m 13:29
2z
Fig. 3. Total ion current chromatogram and selected ion chromatograms which had been treated with metribuzin (MZN).
2%
ma 33ta
9znN WE
for an extract of a soybean plant
TSP LC-MS
OF METRIBUZIN
AND ITS METABOLITES
365
peaks corresponding in mass and retention time to the metribuzin metabolites were found in the unspiked extract. In order to determine whether this method could be used to detect biological levels of these compounds, we then examined an extract from a tetraploid soybean plant which had been treated with metribuzin and extracted according to procedures described in Abusteit et aZ.12. The plant was grown in half-strength Hoagland’s solution containing 0.05 ppm metribuzin, and was harvested after 8 days. An amount of 5 g (fresh weight) shoot issue was extracted with 100 ml methanol, and concentrated to 2 ml. The extract was placed on an XAD-2 column, washed with water, eluted with methanol, concentrated to 20 ~1, and injected into the mass spectrometer. Fig. 3 shows the TIC trace and reconstructed ion chromatograms obtained from this plant extract, and the spectra of peaks A-J are summarized in Table II. The retention times and comparison of the spectra with authentic metribuzin (Tables I and II) confirm the identity of peak J as metribuzin. Components H and I were resolved by mass chromatography, and comparison of retention time and spectra confirmed H as being DA. Peaks A and E gave spectra characteristic of DK and DADK, respectively. Preconcentrating the extract allowed the detection of many more components in the sample, but did not interfere with the detection of metribuzin and its metabolites. Accurate quantitation of these metabolites by TSP LC-MS would require co-injection of labeled standards 23. Based on a comparison of relative reconstructed ion chromatogram peak areas, one obtains estimates of 0.5, 18, 0.5, and 0.8 pg of DK, DADK, DA, and metribuzin in the plant. This corresponds to 0.2, 3.2,0.2, and 0.3 pm, respectively. The total of these estimated concentrations (3.9 ppm) is in reasonable agreement with the total concentration of metribuzin metabolites found by 14C accumulation, 4.1 ppm12. The predominance of the relatively non-phytotoxic metabolite DADK has in “Lee 68” soybeans. The tetraploid variety been observed by other researcherG under study is resistant to metribuzin, and the ability of a plant to metabolize me-
TABLE II TSP MASS SPECTRA
OBTAINED
BY LC-MS
OF TETRAPLOID
SOYBEAN
Peak
Tentative identljkation
Mol. wt.
Mass spectra [m/z. rel. int. (!%)I
A B C D E F G H I J
DK
184
DADK
169
DA
199
MZN
214
202 167 199 180 247 164 164 200 217 215
EXTRACT
(IOO), 185 (1) (lOO), 150 (9), 226 (5), 258 (25) (100), 170 (12), 258 (2) (loo), 163 (2) (IOO), 246 (65), 356 (21), 187 (30), 170 (23) (loo), 169 (42), 184 (85), 201 (48), 243 (15) (25), 169 (5), 184 (loo), 210 (50) (loo), 217 (26) (loo), 200 (4), 249 (8), 276 (8) (lOO), 274 (9)
C. E. PARKER
366
Fig. 4. Total ion current chromatogram ploid soybean extract.
and selected-ion chromatograms
et al.
for the DA region of the tetra-
tribuzin to non-toxic metabolites has been correlated with resistance to this herbicide21,22 While peaks, A, E, H, and J corresponded to the expected metribuzin metabolites, the other components present (B-D, F, G, and I) may be new metabolites of metribuzin, endogenous compounds from the plant, degradation products of these metabolites, or artifacts introduced during the extraction procedure. Further studies to determine the origin of these components are in progress. The value of LC-MS as a confirmatory technique for peak identification in HPLC is illustrated by the treated plant extract (Fig. 4), where one of the metribuzin metabolites (DA, peak H) is only partially resolved from another compound. Quantitative studies conducted without resolution of these compounds would lead to erroneous results. CONCLUSIONS
TSP LC-MS provides a useful method for the confirmation and identification of metribuzin and its metabolites in plant extracts. All of the metabolites could be detected in the extract from the treated plant, even without the further sensitivity enhancement obtainable by selected-ion monitoring. In conclusion, this study demonstrates that TSP LC-MS can be used for the analysis of metribuzin and its metabolites at agronomically significant levels.
TSP LCLMS OF METRIBUZIN
AND ITS METABOLITES
367
ACKNOWLEDGEMENTS
A.V.G. and E.D.R. thank the Agricultural and Food Research Council and the Science and Engineering Research Council, respectively, for financial support. The Cardiff group also thank the S.E.R.C. for financial assistance in the purchase of mass spectral equipment and the Royal Society for funds for the purchase of the thermospray source. REFERENCES 1 R. R. Gronberg. D. R. Flint, H. R. Shaw and R. A. Robinson. Report No. 29800. Res. and Dev. Dept., Mobay Corp., Agricultural Chemicals Division, Stillwell. KA. 1971. 2 D. D. Church, R. R. Gronberg and D. R. Flint, unpublished data, 1972. 3 B. E. Paper and M. J. Zabik, J. Agric. Food Chem., 20 (1972) 72. 4 P. W. Albro, C. E. Parker, G. D. Marbury, 0. Hernandez and F. T. Corbin, Appl. Spectrosc.. 38 (1984) 556. 5 G. R. B. Webster, S. R. MacDonald and L. P. Sarna, J. Agric. Food Chem., 23 (1975) 74. 6 J. S. Thornton and C. W. Stanley, J. Agric. Food Chem., 25 (1977) 380. 7 H. W. Hilton, N. S. Nomura, W. L. Yauger, Jr. and S. S. Kameda, J. Agric. Food Chem., 25 (1974) 578. 8 A. E. Smith and R. E. Wilkerson, Physiol. Plant., 32 (1974) 253. 9 G. R. B. Webster and G. Reimer, Pestic. Sci., 7 (1976) 292. 10 M. A. Maun and W. J. McLeod, J. Plant Sci., 58 (1978) 485. 11 D. W. Britton, F. T. Corbin, D. P. Schmitt, J R. Bradley, Jr. and J. W. Van Dwyn, Proc. South. Weed
Sci. Sot., 35 (1982) 367. 12 E. 0. Abusteit, F. T. Corbin, D. I?. Schmitt, J W. Burton, A. D. Worsham and L. Thompson, Jr., Weed Sti., 33 (1985) 618. 13 C. E. Parker, G. H. Degen, E. 0. Abusteit and F. T. Corbin, J. Liq. Chromatogr., 6 (1983) 725. 14 C. R. Blakeley, M. J. McAdams and M. L. Vestal, J. Chromafogr., 158 (1978) 261. 15 C. R. Blakeley, J. J. Carmody and M. L. Vestal, J. Am. Chem. Sot., 102 (1980) 5931. 16 C. R. Bldkeley and M L. Vestal, Anal. Chem., 55 (1983) 750. 17 R. D. Voyksner, J. T. Bursey and E. D. Pellizzari, Anal. Chem., 56 (1984) 1507. 18 R. D. Voyksner and C. A. Haney, Anal. Chem., 57 (1985) 991. 19 C. E. Parker, C. A. Haney, D. J. Harvan and J. R. Hass, J. Chromatogr., 242 (1982) 77. 20 M. M. Bursey, C. E. Parker, R. W. Smith and S. J. Gaskell, Anal. Chem., 57 (1985) 2597. 21 T. G. Hargroder and R. L. Rogers, Weed Sci., 22 (1974) 238. 22 B. L. Mangeot, F. E. Slife and C. E. Rieck, Weed Sci., 27 (1979) 267. 23 S. J. Gaskell, K. Rollins, R. W. Smith and C. E. Parker, Biomed. Environ. Mass Spectrom., 14 (1987)
717.
THERMOSPRAY LIQUID CHROMATOGRAPHIC-MASS RIC METHOD FOR THE ANALYSIS OF METRIBUZIN OLITES
SPECTROMETAND ITS METAB-
C. E. PARKER* Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.) A. V. GEESON*, D. E. GAMES and E. D. RAMSEY Department of Chemistry, University College, P.O. Box 78, Card& CFI IXL (U.K.) E. 0. ABUSTEIT** and F. T. CORBIN Crop Science Department, North Carolina State University, Raleigh. NC 27695 (U.S.A.) and K. B. TOMER Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 f U.S.A.) (First received November lOth, 1987; revised manuscript received December 3Oth, 1987)
SUMMARY
A thermospray liquid chromatographic-mass spectrometric (TSP LC-MS) method has been developed for the analysis of the herbicide metribuzin and its three major metabolites in plant tissue. Metribuzin and its metabolites exhibited widely varying sensitivities in positive-ion TSP, with metribuzin being the most sensitive and deaminated diketo metribuzin being the least sensitive. All four compounds of interest were detected in an extract of a soybean plant which had been treated with metribuzin.
INTRODUCTION
Metribuzin [4-amino-6-(l,l-dimethyJethyl)-3-(methylthio)-l,2,4-triazin-5(4~one; BAY 943371 is a systemic herbicide which is widely used for control of broadleaf weeds in a variety of food crops, including soybeans, tomatoes, and potatoes. Deaminated metribuzin (DA), diketometribuzin (DK), and deaminated diketometribuzin (DADK) have been identified as metabolites of this herbicide in crops and soillV3. Recent studies indicate that DA may exist predominantly in the enol form at physiological pH4. Structures of metribuzin and the three major metabolites are shown in Fig. 1. * Present address: Central Research Establishment, Home Office Forensic Science, Aldermaston, Reading, Berkshire, U.K. ** Present address: Agronomy Department, College of Agriculture, Cairo University, El-Giza, Egypt.
360
C. E. PARKER ef al.
TSP LC-MS OF METRIBUZIN
AND ITS METABOLITES
361
Gas chromatography (GC)s-g with electron-capture detection (ECD)+*, flame photometric detection in the sulfur mode 5, Coulson conductivity detection*, and alkali flame ionization detection9, and thin-layer chromatography (TLC)8,1 O-l * have been used for the detection and quantitation of metribuzin and its metaholites in plant tissue and soil samples. While these techniques may be sensitive, they do not provide structural confirmation of the peak detected. In addition, GC-ECD requires extensive sample clean-up, and TLC has problems with the separation of all of the metabolites and with providing quantitative data. Recently, we reported a high-performance liquid chromatographic (HPLC)13 method for the identification and quantification of metribuzin and its metabolites. This aproach has the advantage of requiring minimal clean-up of samples before analysis. It is, however, still limited by the non-specific nature of UV detection. Development of a combined high-performance liquid chromatographic-mass spectrometric (LC-MS) method for metribuzin and its metabolites would be of considerable assistance in the confirmation of HPLC peak identities prior to quantitative studies, and should assist in the observation and identification of new metabolic products. While thermospray (TSP) LC-MS is a relatively new technique114-16, its potential for the analysis of pesticides and their metabolites has already been demonstrated”-la. We have also shown that metribuzinlg is amenable to analysis using direct liquid introduction. TSP LC-MS has the advantage that it is commercially available and works best with aqueous mobile phases at flow-rates of approximately 1 ml min-‘, thus enabling all of the eluent from the liquid chromatograph to be fed into the mass spectrometer ion source. In “classical” TSP (i.e., TSP without a filament), ionization occurs through the use of a buffer such as ammonium acetate, and results in spectra often containing abundant protonated molecular ion species (positive-ion TSP), or proton abstraction or acetate addition peaks (negative-ion TSP). We report here the results of our investigation into the development of a TSP LC-MS method for the determination of metribuzin and its metabolites in plant tissues. The application of the developed protocol to an extract of a metribuzin treated soybean plant is also reported. EXPERIMENTAL
The LC-MS experiments were performed using a Waters 6000A pump (Waters Assoc., Milford, MA, U.S.A.) connected to a 25 cm x 4.6 mm I.D. column packed with 5-pm particle size DuPont Zorbax (DuPont Instruments Analytical Division, Wilmington, DE, U.S.A.). The mobile phase used was methanol-water (60:40, v:v) containing 0.05 A4 ammonium acetate, at a flow-rate of 1.2 ml min-‘. The column was connected to a Finnigan MAT TSP LC/MS interface on a Finnigan/MAT 4500 quadrupole mass spectrometer (Finnigan/MAT, San Jose, CA, U.S.A.). Interface parameter were vaporizer temperature, 118”C, and jet temperature, 200°C. Mass spectra obtained over the mass range m/z 145-950, at a scan rate of 2 s per scan, were recorded and processed using a Finnigan/Incos 2300 data system (Finnigan/ MAT). Solvents for LC were redistilled using an all-glass apparatus before use. Metribuzin was obtained from the U.S. Environmental Protection Agency Pesticide Repository (USEPA, Research Triangle Park, NC, U.S.A.). Analytical standards of DA, DK, and DADK were a gift from Mobay Chemical Corp., Agricultural Chemicals Division (Kansas City, MO, U.S.A.).
C. E. PARKER
362
et al.
RESULTS AND DISCUSSION
For the LC-MS studies, the LC conditions which we had developed for HPLC-UV studies12 of metribuzin and its metabolites were modified by substitution of 0.05 M ammonium acetate for acetic acid as the mobile phase modifier. Initial LC-MS experiments were performed using solutions of individual compounds. Aliquots were injected into a stream of methanol-water (60:40), containing 0.05 M ammonium acetate. This solution was continuously fed into the thermospray ion source operated in the positive ion mode. The compounds exhibited widely barying molar sensitivities (based on the relative intensities of the base peaks in the spectra). Metribuzin is the most sensitive, followed by DK and DA. DADK gives a very weak spectrum (Table I). These sensitivity differences probably reflect differences in gasphase proton affinities of the metabolites 2o. The negative ion spectra contained [M - HI-and [M + OC(O)CH$ions oflow abundance. Since negative ion sensitivities were significantly lower than positive-ion sensitivities, and because high sensitivities were expected to be needed for the detection of these compounds at biologically significant levels, positive-ion conditions were employed in the remainder of the study. As DADK was the least sensitive compound in the positive-ion mode, TSP conditions were optimized for this analyte. Optimal sensitivity was obtained at 1lS”C, the highest temperature accessible with the Finnigan TSP interface used. Under these conditions, metribuzin and DA gave [M + H]+ ions at m/z 215 and 200, respectively, which were also the base peaks in their respective spectra (Fig. 1). In contrast, DK gave an [M + NH41 + ion at 202, as the base peaks in its spectrum. The spectrum of TABLE I TSD MASS SPECTRA OBTAINED BY LC-MS OF A STANDARD MIXTURE AND A PLANT EXTRACT SPIKED WITH A STANDARD MIXTURE OF METRIBUZIN (MZN) AND ITS THREE MAJOR METABOLITES Peak
Identity
Mol. wt.
Positive-ion TSP mass spectra [m/z (“/a rel. int.)] Standard mixture
Spiked sample
Rel. mol. sens.
A
DK
184
202 (100) 185 (2)
202 (100) 261 (lo), 185 (3)
70
B
DADK
169
247 (lOO), 246 (69), 187 (15)
247 (loo), 246 (67) 187 (28)
4
C
DA
199
200 217 273 259
200 217 254 273 399
D
MZN
214
215 (loo), 274 (3)
(loo), (13), (IO), (7)
(100) (44), (26), (14), (3)
300
215 (100) 274 (8)
1000
C. E. PARKER et al.
364
with metribuzin, but which had been spiked with all four compounds. The solution injected into the chromatograph contained 2.8 pg each of DA, DK, and metribuzin, and 5.6 pg of DADK. Table I summarizes the spectra obtained. The spectra obtained compare well with those obtained from standard mixtures. Some variation in retention time was observed, presumably due to interaction of the matrix with the column, but this presented no difficulty in the detection of the compounds of interest. No 188.1 B-
TIC
w
2b0
‘I&
I&
Sk
638
SCAH
2910
12m8,DADK
187.03 f 0.59
$ 187_ ...
._ .
. . -.
A_... ._. . A
. _ de _ . CI
.
.. . .
. .
.
r
1 la@
ze
4m 13:29
2z
Fig. 3. Total ion current chromatogram and selected ion chromatograms which had been treated with metribuzin (MZN).
2%
ma 33ta
9znN WE
for an extract of a soybean plant
TSP LC-MS
OF METRIBUZIN
AND ITS METABOLITES
365
peaks corresponding in mass and retention time to the metribuzin metabolites were found in the unspiked extract. In order to determine whether this method could be used to detect biological levels of these compounds, we then examined an extract from a tetraploid soybean plant which had been treated with metribuzin and extracted according to procedures described in Abusteit et aZ.12. The plant was grown in half-strength Hoagland’s solution containing 0.05 ppm metribuzin, and was harvested after 8 days. An amount of 5 g (fresh weight) shoot issue was extracted with 100 ml methanol, and concentrated to 2 ml. The extract was placed on an XAD-2 column, washed with water, eluted with methanol, concentrated to 20 ~1, and injected into the mass spectrometer. Fig. 3 shows the TIC trace and reconstructed ion chromatograms obtained from this plant extract, and the spectra of peaks A-J are summarized in Table II. The retention times and comparison of the spectra with authentic metribuzin (Tables I and II) confirm the identity of peak J as metribuzin. Components H and I were resolved by mass chromatography, and comparison of retention time and spectra confirmed H as being DA. Peaks A and E gave spectra characteristic of DK and DADK, respectively. Preconcentrating the extract allowed the detection of many more components in the sample, but did not interfere with the detection of metribuzin and its metabolites. Accurate quantitation of these metabolites by TSP LC-MS would require co-injection of labeled standards 23. Based on a comparison of relative reconstructed ion chromatogram peak areas, one obtains estimates of 0.5, 18, 0.5, and 0.8 pg of DK, DADK, DA, and metribuzin in the plant. This corresponds to 0.2, 3.2,0.2, and 0.3 pm, respectively. The total of these estimated concentrations (3.9 ppm) is in reasonable agreement with the total concentration of metribuzin metabolites found by 14C accumulation, 4.1 ppm12. The predominance of the relatively non-phytotoxic metabolite DADK has in “Lee 68” soybeans. The tetraploid variety been observed by other researcherG under study is resistant to metribuzin, and the ability of a plant to metabolize me-
TABLE II TSP MASS SPECTRA
OBTAINED
BY LC-MS
OF TETRAPLOID
SOYBEAN
Peak
Tentative identljkation
Mol. wt.
Mass spectra [m/z. rel. int. (!%)I
A B C D E F G H I J
DK
184
DADK
169
DA
199
MZN
214
202 167 199 180 247 164 164 200 217 215
EXTRACT
(IOO), 185 (1) (lOO), 150 (9), 226 (5), 258 (25) (100), 170 (12), 258 (2) (loo), 163 (2) (IOO), 246 (65), 356 (21), 187 (30), 170 (23) (loo), 169 (42), 184 (85), 201 (48), 243 (15) (25), 169 (5), 184 (loo), 210 (50) (loo), 217 (26) (loo), 200 (4), 249 (8), 276 (8) (lOO), 274 (9)
C. E. PARKER
366
Fig. 4. Total ion current chromatogram ploid soybean extract.
and selected-ion chromatograms
et al.
for the DA region of the tetra-
tribuzin to non-toxic metabolites has been correlated with resistance to this herbicide21,22 While peaks, A, E, H, and J corresponded to the expected metribuzin metabolites, the other components present (B-D, F, G, and I) may be new metabolites of metribuzin, endogenous compounds from the plant, degradation products of these metabolites, or artifacts introduced during the extraction procedure. Further studies to determine the origin of these components are in progress. The value of LC-MS as a confirmatory technique for peak identification in HPLC is illustrated by the treated plant extract (Fig. 4), where one of the metribuzin metabolites (DA, peak H) is only partially resolved from another compound. Quantitative studies conducted without resolution of these compounds would lead to erroneous results. CONCLUSIONS
TSP LC-MS provides a useful method for the confirmation and identification of metribuzin and its metabolites in plant extracts. All of the metabolites could be detected in the extract from the treated plant, even without the further sensitivity enhancement obtainable by selected-ion monitoring. In conclusion, this study demonstrates that TSP LC-MS can be used for the analysis of metribuzin and its metabolites at agronomically significant levels.
TSP LCLMS OF METRIBUZIN
AND ITS METABOLITES
367
ACKNOWLEDGEMENTS
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