Anal. Chem. 1984, 56,2547-2552
2547
Chemical Ionization Mass Spectrometry of Isofenphos and Its Metabolites Thomas Cairns,* Emil G . Siegmund, and Rodney L. Bong Department of Health and Human Services, Food and Drug Administration, Office of Regulatory Affairs, 1521 West Pic0 Boulevard, Los Arigeles, California 90015
Gas chromatography/mass spectrometry technlques uslng chemlcal lonlzatlon have been employed to characterlze lsofenphos and three of Its metabolltes. The results have lndlcated some unusual fragmentatlon processes for such nitrogen contalnlng organophosphates In whlch Intramolecular hydrogen bondlng plays an Important role In stablllzatlon. Tandem use of deuterated methane and ammonla as reagent gases have permitted a comparatlve assessment of protonatlon and exchangeable hydrogens within many of the Important fragment Ions. Resultant structural lnformatlon has been employed to conflrm a resldue flndlng In alfalfa pellets at the low parts-per-mllllon level.
Organophosphorus insecticides have continued to replace the many popularly used organochlorine pesticides such as TDE [ l,l-dichloro-2,2-bis(chlorophenyl)ethane] (1) on the basis of less concern over both environmental contamination and susceptibility to degradation and/or metabolism. In the course of analyzing a wide variety of crops to protect the consumer from potentially harmful residues in the food supply, unknown analytical responses (UARs) are often encountered via element-selectiveGC detectors which contain phosphorus (2). The use of combined gas chromatography/mass spectrometry (GC/MS) to identify these possible organophosphorus compounds is necessary to achieve an unambiguous answer. This paper describes the report of an unusual residue finding on alfalfa pellets which was subsequently identified as isofenphos (1) (1-methylethyl 2 4 [ethoxy(l-methylethy1)amino]phosphinothioyl]oxy]benzoate) at the parts-per-million level. Such an occurrence stimulated a full scale mass spectrometric study of this particular type of nitrogen containing phosphate together with its oxygen analogue (2) and two known metabolites (3 and 4). The fragmentation processes
(3)
(4)
involved under chemical ionization (CI) conditions (both This article not subject to
methane and ammonia) have provided a hitherto unknown insight into the dominant prevailing characteristics of such molecules upon soft ionization. Detailed fragmentation pathways have been outlined based on additional studies with C2H4and N2H3as reagent gases to determine both origin of hydrogen transfers and exchangeable hydrogens in an attempt to confirm the proposed assignments. Such data from a detailed structural elucidation could then provide a predictive set of rules into the selection process of suitable ions for multiple ion detection experiments where low levels in crop residues would demand such an analytical approach be adopted. EXPERIMENTAL SECTION Apparatus. All spectra were obtained on a Finnigan Model 3300 quadrupole mass spectrometer equipped with a CI source and an INCOS Data System. Operating conditions for residue sample are as follows: 30 cm X 2 mm i.d. glass column packed with 2% DEGS on 80/100 mesh Chromosorb W; carrier gas as reagent gas, 30 mL m i d , column inlet 250 "C, column temperature 200 O C isothermal. Operating conditions for structural elucidation: 25 m SE54 capillary column, 50 to 250 O C at 10 "C min-l, source pressure 700 mtorr (adjusted to maximize the intensity of CHSf and NH4+at m / z 17 and 18, respectively). Sample Preparation. Sample (20 g) was extracted ( 3 , 4 )and then concentrated to 100 pL with dry nitrogen; 3 p L was injected into the GC/MS system. Reference Materials. Isofenphos (l),its oxygen analogue (Z), and two metabolites (3 and 4) were obtained from the Enviromental Protection Agency (EPA). DISCUSSION AND RESULTS The sample extract of alfalfa pellets was first examined by GC using the Hall electrolytic conductivity detector (HECD) and found to contain an unidentified organophosphorus compound at RR, = 2.12 (on DEGS), 1.36 (on OV-lOl), and 1.73 (on OV-225) [all relative to chlorpyrifos]. Lack of comparison GC retention data from our extensive data base (3) prompted immediate examination by GC/MS. The present in-house architectural approach to solving such UARs is deployment of CI to recognize the protonated molecular ion and cross reference with a data collection of pesticides sorted by molecular weight with available CI mass spectral data (5). Via this scenario, the suspected UAR was tentatively identified to be isofenphos (1). However, the importance placed on characterizing the molecule of interest before final determination is paramount to providing unambigious identification. For these reasons, isofenphos (1)and its family members (2, 3, and 4)were first obtained from EPA for detailed study. The metabolites were automatically included in this study since the opportunity of also observing them in the incurred residue was a real possibility. Organophosphorus compounds are often found to be thermally labile. Before a residue confirmation of isofenphos was attempted, a comparison of probe spectra and those obtained by GC/MS was made. The fragmentation patterns obtained indicated thermal degradation was not operative. Methane CI Spectra. With methane as reagent gas, all four compounds were examined concurrently via capillary gas
US. Copyright. Published 1984 by the American Chemical Society
2548
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
'001
245
..
)
217
287
386
3 0
looi
"
'
4bo
1229
Flgure 1. Methane chemical ionization spectra: (A) isofenphos (1); (B) isofenphos oxygen analogue (2); (C) des-N-isopropyl isofenphos (3),and
(D)des-N-isopropyl isofenphos oxygen analogue (4). chromatography mass spectrometry (GC/MS) (Figure 1). In the case of the parent compound, isofenphos (l),the lack of a protonated molecular ion, [M HI+, would have normally precluded a facile identification if it were not for the appearance of the adduct ions at m/z 374 and 386 representing [M CzH5]* and [M C3H5]+,respectively. Predictably the corresponding oxygen analogue (2) demonstrated a parallel fragmentation pattern (Figure 1B)shifted down 16 daltons. The similarity in the spectra of isofenphos (1) and the desN-isopropyl isofenphos (3)) i.e., base peak at mlz 245, was strong evidence that this ion had a structure resulting from the complete loss of the nitrogen containing side chain from the phosphorus atom. Such an ion is visualized in Scheme I where the precursor to m / z 245 is depicted as m / z 287. To accommodate this ion value of m / z 245, the loss of the isopropyl group from the ester side chain would be necessary. The implications of these proposed ion structures deserves additional commentary. While the total absence of the [M H]+ ions is noticeable, the production of m / z 287 could proceed via an unstable [M H]+yielding an ion structure containing no protons from the reagent gas species with the actual site of the positive charge as yet undetermined. Additional loss of the isopropyl group from the ester group attached to the aromatic nucleus with a hydrogen rearrangement is then postulated to occur in the production of the stable ion at m / z 245. The unusual incidence of a hydrogen transfer from the isopropyl group to the ether oxygen can be stabilized through intramolecular hydrogen bonding with the second ether oxygen attached to the pentavalent phosphorus. Conditions seem optimum to permit such an arrangement via a six-membered ring formation. To test these postulations regarding the structures of these two ions (mlz 287 and 245), the compounds (1 and 3) were rerun using C2H4as reagent gas (Figure 2A,C). The observed ions once again occurred a t mlz 287 and 245, thus confirming that no reagent protons
+
+
+
Scheme I. Proposed Fragmentation Pathway for Isofenphos under Chemical Ionization Conditions
+
/
(jGizG]
(287)
/ G
O
H
and
dsl' /
CH3CH2O
+
I
I
OH k 0 H
OH '?+-OH
+
were involved. It would seem, therefore, that the preliminary assignments were indeed correct. Empirical hydrogen transfer from the side chain of the ester group to the ether oxygen is highly unlikely in that the normal fragmentation mechanism expected is via the McLafferty rearrangement, i.e., hydrogen transfer to the oxygen of the carbonyl group. While such a mechanism may well occur in these compounds, it is difficult to prove the actual sites involved since resonance structures involving the ether oxygen and the protonated oxygen of the carbonyl function might take place before ultimate stabilization via the proposed intramolecular hydrogen bonding.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 roo,
2549
241
!
140 I
379
2?7
140 120
I
100-
245
218
lool
1140
z 0
I"""
500
Flgure 2. Deuterated methane chemical ionization mass spectra: (A) isofenphos (1); (6)isofenphos oxygen analogue (2); (C) des-N-isopropyl isofenphos (3), and (D) des-N-isopropyl isofenphos oxygen analogue (4).
Scheme 11. Proposed Fragmentation Pathway for Isofenphos Oxygen Analogue under Chemical Ionization Conditions COOCH(CHd2
)-(
(m/t)
/ / f
m/z 229
1
Similar arguments hold for the oxygen analogue of isofenphos (2) (Figure 1B and Figure 2B) with regards to the proposed structure for m/z 229 (Scheme 11). Other prominent ions in the methane CI mass spectra of isofenphos and its metabolites also merit discussion, namely, m/z 166,139, and 121. Production of the ion at m/z 166 can be visualized as occurring directly from the [M H]+ via cleavage of the P-0 bond and resulting in two ions dependent on the residual charge on either side of the original bond cleavage (Scheme I). While m/z 181 is not present in the spectra of isofenphos and its analogues (Figure l), the appearance of m / z 139 is clearly noticeable. This particular ion can be derived from m / z 181 via a McLafferty rearrangement process occurring at the ester side chain, thus inferring the
+
site of protonation in m / z 181 was the ether oxygen atom. Harrison et al. (6) had already demonstrated that protonation of carboxylic acids corresponding to the structure in Scheme I for m/z 139 is more stable in the gas phase than ions where the hydroxyl group is protonated. The additonal stability afforded by intramolecular hydrogen bonding to the adjacent hydroxyl group should assist greatly in the overall resonance stability of this ion. If such a structure for m/z 139 was indeed correct, then the use of C2H4 as reagent gas should reveal a 1 dalton shift (Figure 2). In all four compounds under study, a 1 dalton shift was observed, i.e., protonation site was the ether oxygen in m / z 181 prior to rearrangement. Production of m/z 121 can then be attributed to a loss of water from m/z 139. While the studies with C2H4 as reagent gas did not illustrate the expected strong shift of 1 dalton for this ion (Figure 2), the evidence clearly suggests the possibility of such a shift since m / z 122 is clearly present as a companion ion to mlz 121. Having reported the major fragmentation pathways of isofenphos (1) and its analogues (2, 3, and 4), the behavior of these compounds is highly unusual in many respects. First, the appearance of m / z 245 or m/z 229 as the base peak can be attributed to the fact that intramolecular hydrogen bonding can occur from the carboxylic acid proton to the 0-P bond location thereby lending resonance stabilization. Perhaps the most obvious difference in comparing the methane CI spectrum of isofenphos (1) (Figure 1A) and des-N-isopropyl isofenphos (3) (Figure IC) is that the base peak occurs at mlz 287 instead of mlz 245. While m / z 245 is still a prominent ion, the great increase in relative abundance of mlz 287 must be attributed to the much lower internal energy transmitted via the initial protonation in the production of [M + HI+. It would appear that a McLafferty rearrmgement is responsible for the production of the carboxylic acid grouping via hydrogen
2550
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 100;
260
1287
181
-0 loo]
181)
400
Flgure 3. Ammonia chemical Ionization mass spectra: (A) isofenDhos (1); (B) isofenphos oxygen analogue (2); (C) des-N-isopropyl isofenphos (3), and (D) des-N-isopropyl lsofenphos oxygen analogue (4).
transfer from the isopropyl grouping to the carbonyl oxygen before an intramolecular hydrogen bonding situation can take place. On the other hand the protonation of the ether oxygen in the m/z 181 ion leads to the trihydroxy structure (mlz 139) (Scheme I) whereby the diol grouping is preferred to the carboxylic acid grouping resulting in perhaps two intramolecular hydrogen bonds for stabilization. Ammonia CI Spectra. Having established the main avenues of fragmentation, isofenphos ( 1 ) and its metabolites (2, 3, and 4) were then reexamined by using ammonia as the reagent gas (Figure 3). In general, such gross differences between methane and ammonia spectra have been previously reported (7). As originally verified by C2H4,the ion at m / z 287 (Scheme I) does not experience a shift when N2H3 is employed as reagent gas (Figure 4A,C). In the case of the oxygen analogue of isofenphos (2), the ion at mlz 287 (Figure 3B) does experience a 3 dalton shift when.deuteratedammonia is used (Figure 4B). While this ion represents a McLafferty rearrangement at the ester side chain on the aromatic ring, a 3 dalton shift can easily be explained by the structure having three exchangeable protons available (two hydroxyl protons and one secondary amine hydrogen). Indeed the protonated molecular ion at m/z 330 (Figure 3B) also gave indication that the NH proton was exchangeable under CI conditions [2 dalton shift with N%13(Figure 4B)]. The utilization of studies with deuterated ammonia to locate and verify exchangeable protons offers additional proof to the original assignments under methane ionization (Schemes I and 11). Under deuterated methane conditions the ion at m / z 245 (1 and 2) did not undergo a shift, while under deuterated ammonia conditions a 1 dalton shift was observed indicating one exchangeable proton on ionization but not from the reagent gas. Clearly the tandem use of deuterated methane and ammonia gases can distinguish reagent gas protonation from subsequent
exchangeable hydrogens. Even in the case of the oxygen analogue of isofenphos (2), the ion at mlz 229 (Figure 3B) did undergo a 1dalton shift at m / z 230 (Figure 4B) confirming the intramolecularly hydrogen bonded structure depicted in Scheme 11. One ion prevalent in the spectra of all four compounds under NH, conditions was the m / z 181 ion (Figure 3). The 2 dalton shift detected when deuterated ammonia was employed confirmed the structural identification illustrated in Scheme I [one proton from the reagent gas species and one exchangeable]. However, the two exchangeable hydrogens situation existing in the ion at m / z 121, when examined revealed only a 1 dalton shift. Clearly both hydroxyl hydrogens are not equivalent in this particular structure and the protonated carboxylic acid structure might well be more realistic to fit the data for this ion. Residue Confirmation. Translation of a residue finding of an “UAR” on HECD to GC/MS can sometimes be troublesome due to the lack of sensitivity of the mass spectrometer in the total ion monitoring mode as compared to an element-sensitive GC detector. In this particular case history, however, armed with the major ions of isofenphos derived from the structural study described above (mlz 245,287, and 374 under methane CI),the total ion chromatogram (Figure 5) yielded a primary indication via mass chromatograms that isofenphos was present. To ensure the unambiguous nature of such proof, the sample was rerun with four ions (mlz 121, 166, 245, and 374) in the selected ion detector mode (Figure 6). As can be clearly seen, the ion ratios at the correct retention time are an exact match of an authentic reference material. Lack of signal intensity at mlz 245 at a different retention time expected for the des-N-isopropyl isofenphos ruled out the presence of this metabolite. While signals for m/z 121 might be suggestive of the presence of the other two metabolites, further examination of the sample extract yielded
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
2551
287
1000 ,
P
B
A
:: 183
245 214 l " ' " ' " ' ~ '
200
100
"""'
300
1831
loo]
lo01
1287
183
*0
...,,...,,
rrrT-n.,,,,
. . , , . , ,
m/z
200
. , , , , , . . , , . , ,
, ,o. , .-,
,
300
lgure 4. Deuterated ammonia chemical ionization mass spectra: (A) isofenphos (1); (B) isofenphos oxygen analogue (2); (C) des-N-isopropyl ,ofenphos (3), and (D) des-N-isopropyl isofenphos oxygen analogue (4). Reconstructed Ion Chromatogram
1
: m/z 166
J/\-
A
Mass Chromatograms
I .M.
A , eA * + % ?
m/z 374
h
,A
Figure 5. Total ion chromatogram (TIC) of extract of alfalfa pellets via GC/MS using methane as reagent gas wlth resultant extracted mass chromatograms for the Ions at rnlz 245, 287, and 374.
negative results. The total lack of the presence of oxygen analogue of isofenphos was surprising since previous workers (8)had demonstrated the degradation in soil (and persistence) of isofenphos to its oxygen analogue under field conditions. In the control of D.floralis in rutabagas, the average residue found at harvest was 0.03 to 0.07 ppm for isofenphos and from
