distinguishing between n-methylated imidazolinones using mass spectrometry

ORGANIC MASS SPECTROMETRY, VOL. 22, 342-347 (1987)

Distinguishing Between N-methylated Imidazolinones Using Mass Spectrometry

Stanley J. Cardaciotto, Patrick C. Mowery, Michael L. Thomson and Richard S. Wayne American Cyanamid Company, Agricultural Research Division, PO Box 400, Princeton, New Jersey 08540, USA

Imidazolin-4-ones are a new class of herbicides which contain both an amide and amino nitrogen in a five-membered ring. During the manufacturing process, it is possible to methylate either nitrogen. This presented an interesting analytical problem in distinguishing between the two N-methylated derivatives of ASSERT@ (a mixture of 6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl~m- and p-toluic acid methyl esters) a t low levels in a complex mixture. After developing a high-performance liquid chromatography method for separating and isolating components in the mixture, mass spectrometric techniques, which included electron impact (EI) and collisional activation decomposition (CAD), were used to distinguish between two isomeric N-methylated derivatives. The data from the EB and CAD experiments enabled amidomethyl and aminomethyl structures to be assigned. Accurate mass measurements and proton nuclear magnetic resonance spectroscopy were used to support the assignments.

thesis in plants. ASSERT is a selective herbicide for weeds in wheat and barley.' The registration of these INTRODUCTION herbicides requires development of analytical meth-

ASSERT@? (a mixture of 6-(4-isopropyl-4-methyl-5- odology for identifying by-products arising from the oxo-2-imidazolin-2-yl)-m- and p-toluic acid methyl manufacturing process. The preparation of ASSERT esters) is one of an important new class of selective involves an esterification of the acid precursor (1, Fig. post-emergence imidazolinone herbicides which are 1). Of particular analytical interest are compounds 3 potent inhibitors of branched-chain amino acid syn- and 4 (Fig. l ) , which may be generated during this

E s t e r i f i c a t i o n I h b

2 , ASSERT

+

3 , Amidomethyl 4, Aminomethyl

Figure 1. The preparation of ASSERT. (Reproduced by permission of American Cyanarnid Company)

? A S S E R p is a registered trademark of American Cyanarnid Company.

0030-493X/87/060342-06$05.00 @ 1987 by John Wiley & Sons, Ltd.

Received 13 February 1986 Accepted 11 December 1986

DISTINGUISHING BETWEEN N-METHYLATED IMIDAZOLINONES 343

Table 1. EI mass measurements for the amidomethyl and aminomethyl isomers'

Deviation from Elemental Measured mass theoretical mass (mmu)

composition Aminomethyl Amidomethyl Aminomethyl Amidomethyl

302.1 61 9

271.1477 260.1 182

243.1 533

190.0898

144.0458

116.0525 98.0974

302.1622 287.1423 271.1460 260.1 135 259.1059

228.0939 200.0930 190.0824 159.0701 144.0435 131.0744 1 16.0505

0.4 0.8

3.0 1.3 2.1 -2.6

-2.4 3.5

-2.7

4.0 -2.0

3.0 4.4 1.6

0.8 -1.4 1 .o

2.5 0.5 0.4

a Reproduced by permission of American Cyanamid Company

reaction. Their possible presence at 0.1% levels in ASSERT presented a challenging analytical problem. Mass spectrometry was used to study two impurities which were purported to be these isomers. Chemical ionization (CI) was used to enhance the molecular weight information, whereas electron impact (EI) and collisional activation decomposition (CAD)* were used

to study the fragmentation patterns for chemical struc- ture information. The CAD process involves the use of tandem mass spectrometry, in which the molecular ion and its fragments are resolved in a first mass analyser, and the fates of selected ions after collisions with neutral molecules are monitored by a second mass analyser. Exact mass measurements and proton nuclear magnetic resonance (NMR) spectroscopy were also used to support the structural assignments.

EXPERIMENTAL

Isolation of components

Components of interest were isolated from ASSERT using repetitive collections from a semi-preparative high-performance liquid chromatography (HPLC) column (Whatman 5p Partisil RAC I1 C8). Separation of the components was achieved using a mobile phase composition of 20% acetonitrile/water. The HPLC equipment consisted of an Altex model llOApump, a Rheodyne model 7120 sample injection valve and single wavelength detection at 254-nm with a LKB UVICORD- 5. The combined collections were evaporated to dryness by heating the flask in a water bath at 40 "C while purging the solution with nitrogen.

Table 2. Summary of mass spectral data for the amidomethyl isomer"

lsobutane Electron chemical impact ionization

m / z RI (%) m l z RI (%) m / z 260

302 2 303 100 287 0.5 271 3 260 78 260 5 260 259 58 243 2 228 12 228 < 0.5 228 200 3 198 3 190 100 190 0.5 176 5 176 < 0.5 176 174 38 174 162 3 159 12 159 147 8 147 144 25 131 38 131 < 0.5 131 118 11 116 20 105 16 103 7 91 21 89 31 79 9 77 10 65 14 63 13 55 13 41 58


CAD experiments Daughters of Parents of

m / z 259 m / r 190 m/z 190

302

259 259

190 190 190

174 174

159 147 144 131 118 116 105 103 91 89 79 77 65 63

100

90

80

70

60

98

30.00 4 0

[MI’’ 2 {9

2 2 8 \ 302 190

L II I

(a ) Amido isomer

I I. I I I ,,, I .I/ 1.1 I I l l I I 1

13 1

I

1

(3

100

90

80

70

6? 60 r v) C 0,

c .-

.- 50 al > 0 .- c

4c a

3c

2c

1c

L

6 0 80

30

260

259

200 220 240 260 280 300 315.00 rn/z

( b ) Amino isomer (41

30.00 4 0 60 8 0 100 120 140 160 180 2 0 0 220 240 260 280 300 315.00 m/z

Figure 2. Eil mass spectra of (a) the amidomethyl isomer (3) and (b) the aminomethyl isomer (4). (Reproduced by permission of American Cyanamid Company).


a , m h 260 (78%)

-CH30H i 1 +'

3, m h 302

1 -'C3H7

m / ! 259 (58%) b, m h 228 (12%)

1 + *

c"p --'OCH3 I

I m h 159 (12%) m h 190 base peak

'CH3

m h 131 (38%)

Scheme 1. Amidomethyl isomer. (Reproduced by permission of American Cyanamid Company).

Spectral analysis

Low-resolution CI, EI and CAD mass spectral data were acquired on a Finnigan 4500 Triple Stage Quadrupole (TSQ) instrument using isobutane for CI and 70-eV for EI. Samples were introduced using the solid probe. The high-resolution EI spectra were obtained using 70-eV and a resolution of 8000 on a Kratos MS-50 instrument. Proton NMR data were obtained using a Bruker CXP 300 NMR spectrometer operating at 300-MHz, or a Varian FT 80 at 80-MHz.

RESULTS AND DISCUSSION

The molecular weight information (from CI) and elemental composition (from high-resolution EI) obtained on two HPLC isolates from ASSERT suggest methylated compounds. An [M+ H]+ ion was generated for both isolated samples at m l z 303 (100%). Accurate mass measurements by EI for these compounds (Table 1) give m l z 302.1622 and 302.1619, which differ by less than 10-ppm from the theoretical elemental composition

346 S. J . CARDACIOTTO E T A L .

(C1,HZ2NO3). Their EI spectra (Figs 2(a) and (b)) are quite complex and have different fragmentation patterns. For example, the spectra have different base peaks ( m / z 190 and 56). The EI spectra may provide a means for identifying the isomers as 3 and 4 (Fig. 1).

In order to interpret the EI spectra, both CAD daughter and parent ion experiments were carried out. Daugh- ter ion experiments were performed on m / z 260 and 259 for each compound and on m l z 190 for the amidomethyl isomer. Parent ion spectra were obtained on the base peaks at m / z 190 and 56. Both the daughter and parent ion spectra were generated at collision ener- gies in the range 10-30-eV, using argon at 2.5 x Torr as the collision gas. This was sufficient to provide a high cross-section for dissociation and to optimize chemical in f~rmat ion .~ In the CAD experiments, it was found that the intensities for most of the lower-mass ions increased with an increase in the collision energy. Fur- thermore, several competitive fragmentation routes are open to both of the N-methylated compounds. Plausible mechanisms for the observed fragmentation patterns, together with the percentage of the EI base peak for the relevant ions, are depicted in Schemes 1 and 2.

4, r n h 302

j/ --C3H7

r n h 259 (8%)

CH 3 -k<-€H3

r n h 56 base peak

+

Scheme 2. Aminomethyl isomer. (Reproduced by permission of American Cyanamid Company).

The CAD experiments (Table 2) on the putative amidomethyl isomer support Scheme 1. The m / z 190 base peak appears to arise from m / z 302 and 259, but not from a or b. In Table 2, ion b is shown to be a daughter of ion a, but the ion responsible for the base peak is not. The fragment ions at m l z 131 and 159 are the daughters of both m / z 260 and 190.

The fragmentation scheme revealed by the CAD experiment on the second (aminomethyl) isomer (Table 3) are more complex, but support a pattern such as that in Scheme 2. The m / z 259 and 260 fragment ions appear in Scheme 2 and Scheme 1, but have different structures. The ions in Scheme 2 give rise to the N-methyl base peak at m / z 56, as opposed to one at m / z 190. Thus, the driving force in each case appears to be consistent with ionization at the N-methyl, sp3 nitrogen4

To confirm the mass spectral assignments, additional quantities of each sample sufficient for proton NMR analysis were isolated. Analyses were conducted using proton NMR spectroscopy at 80 and 300-MHz. The spectrum of each compound was identical to that of ASSERT with one additional N-substituted methyl group. The spectrum of the amidomethyl isomer is consistent with the mass spectral data. This is indicated by the expected single N-methyl resonance found at 2.8- ppm. The proton spectra of the aminomethyl isomer in CDC13 have several broadened alkyl resonances. Trace quantities of DCl present in CDCl, are expected to cause line broadening in the spectrum of the amine because the two resulting diastereomeric quaternary ammonium salts are in dynamic equilibrium. This is particularly true in this experiment, since only micromolar quantities of the aminomethyl compound are available. To verify this interpretation, the solvent was evaporated and replaced with CDzCl2 which is less acidic than CDC13. The resulting 80-MHz spectrum shows sharp lines with the N-methyl resonance at 2.85-ppm.

SUMMARY AND CONCLUSIONS

The molecular weight information from CI and structural information obtained using EI, CAD and com- plementary NMR experiments indicate that the isolates are N-methylated positional isomers. In addition, CAD experiments (daughter and parent ions) were found to be very useful in sorting out the complex EI fragmentation patterns. These mass spectral experiments allowed the isomers to be distinguished by assigning the methyl groups to the respective nitrogens in the imidazolinone ring. As a consequence, mass spectrometry and NMR spectroscopy showed that either nitrogen can be methylated. The combined data allow rational structural assignments for each isomer. Since the fragmentation mechanisms are better understood, they can be applied to spectra of similar compounds. The data illustrate how CAD can be used as a routine tool for providing rational structural assignments to effluents from an HPLC or GLC separation. Similar techniques were used recently by Holtzmann et al.’ to identify isomeric imidazolin-2- ones. In their study, the fragmentation patterns were


Table 3. Summary of mass spectral data for the aminomethyl isomer"

m1.z

302 287 271 260 259 245 243 230 228 227 21 7 204 190 176 174 162 160 144 130 116 112 105 99 98 89 a4 77 69 56 42 41

Electron impact

RI (%]

2 2 1

13 8 0.5 9 5 4 2 0.5 3 2 4 4 5 5

14 1 5

12 3 5

10 8 4 2 7

100 13 29

lsobutane chemical ionization

m l z RI (%)

303 100

260 0.5

243 < 0.5 230 < 0.5

204 < 0.5

112 < 0.5

98 < 0.5

CAD experiments Daughters of Parents of

m / z 260 m / z 259 m1.z 56 m / z 259

302 302 287

260 260 260 259 259

245 245

230 228 228 227 227 21 7 21 7

- - - 176 174 162 160 144 144 130 130 116 116

105 105 112

99

89 84 77

56 56 42


derived from urea derivatives. In the imidazolinones studied in these laboratories, the patterns appear to be governed by the fragmentations characteristic of amino and amido nitrogens.

Acknowledgements

We are very pleased to thank William Millen and Ted Chang for performing the high-resolution mass spectrometric experiments.

REFERENCES

1. M. Los, in Pesticide Synthesis Through Rational Approaches, ed. by P. S. Magee, G. K. Kohn and J. J. Menn, pp. 29-44. American Chemical Society, Washington, DC (1984).

2. F. W. McLafieity, Science 214, 280 (1981). 3. C. A. Boitnott, J. R. B. Slayback and U. Steiner, Optimization of

Instrument Parameters for Collision Activated Decomposition (CAD) Experiments for a Triple Stage Quadrupole (TSQ)

GC/ MS/ MS/ DS, Finnigan Topic 8160. Finnigan-Mat. Sunnyvale, California (1981).

4. Q. N. Porter and J. Baldas, Mass Spectrometry of Heterocyclic Compounds, Chapt. 12. Wiley, New York (1971).

5. G. Holtzman, B. Krieg, H. Lautenschlager and P. Konieczny, J. Heterocycl. Chem. 16, 983 (1 979).

distinguishing between n-methylated imidazolinones using mass spectrometry

Documents