Negative ion electrospray mass spectrometry of aminomethylphosphonic acid and glyphosate:...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2004; 18: 37–43

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1264

Negative ion electrospray mass spectrometry of

aminomethylphosphonic acid and glyphosate:

elucidation of fragmentation mechanisms by multistage

mass spectrometry incorporating in-source deuterium

labelling

Lee Goodwin1, James R. Startin2, David M. Goodall1 and Brendan J. Keely1*1Chemistry Department, University of York, Heslington, York YO10 5DD, UK2Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK

Received 14 July 2003; Revised 11 October 2003; Accepted 11 October 2003

Glyphosate and its main metabolite, aminomethylphosphonic acid, introduced by direct infusion in2H2O, appear in negative ion electrospray mass spectrometry (ES-MS) as triply deuteriated [M–H]�

ions. Sites of deuterium residence and loss were established using the multistage (MSn) capabilites

of an ion trap mass spectrometer to assist in the determination of fragmentation mechanisms.

The study reveals specific mechanisms, common to each analyte, such as those involving a five-

membered transition state between the amine and phosphonate group, as well as analyte specific

transitions. Copyright # 2003 John Wiley & Sons, Ltd.

The analysis of glyphosate (GLYP) and its main metabolite,

aminomethylphosphonic acid (AMPA), presents an impor-

tant analytical challenge. Although GLYP is one of the

world’s most commonly used herbicides, its low volatility,

high water solubility and lack of a chromophore has

restricted methods of analysing the native (underivatised)

compound, with capillary electrophoresis/mass spectrome-

try (CE/MS) being the only reported technique allowing its

analysis at regulatory levels.1 Previously,2 we reported the

fragmentation pathways of GLYP and AMPA in MS2 and

MS3. The study provided a wealth of information on typical

MSn fragment ions produced from the analytes and showed

which were of greatest interest for monitoring by MSn. The

fragment ions due to loss of water and those derived from

the phosphonic acid group (phosphonate, H2PO3�; phospho-

rate, PO3�; phosphinate, PO2

�) were particularly abundant.

Although the study revealed transitions that increase the spe-

cificity of MS detection of the native analytes, it did not pro-

vide detailed information on the mechanisms of the

fragmentation reactions.

To gain an understanding of the gas-phase fragmentation

mechanisms, the analytes need to contain a label that can be

followed through the various MS transitions. One method

that has been useful in elucidating fragmentation mechan-

isms is the exchange of acidic hydrogen atoms for deuterium

atoms in solution,3–5 isotopically labelling the analytes in

specific, known positions. GLYP and AMPA are particularly

amenable to this type of analysis due to their ionisable groups

and aqueous solubility. Thus, solvation in 2H2O effects

proton/deuterium exchange at the ionisable groups. Here

we present a detailed account of the fragmentation mechan-

isms occurring to produce MSn product ions from GLYP and

AMPA using an ion trap instrument.

EXPERIMENTAL

Chemicals and preparation of solutionsAminomethylphosphonic acid, glyphosate and 2H2O (99.9%

pure) were obtained from Sigma (Poole, UK). Standard solu-

tions of both analytes (1 mg mL�1) were prepared in 2H2O.

SLR-grade dimethyl sulfoxide (DMSO) was obtained from

Fisher Scientific (Loughborough, UK). For measurement of

uptake of the deuterium label a 1 mg mL�1 solution of

GLYP was prepared in 50:50 (v/v) 2H2O/DMSO and the

solution was refluxed for 1 h.

Mass spectrometryAll analyses were performed using a Finnigan LCQ ion trap

mass spectrometer (ThermoFinnigan, Hemel Hempstead,

UK) operated in negative ion mode with a heated capillary

temperature of 2258C, spray voltage of 4.2 kV and sheath

gas flow (N2) of 20 arbitrary units. Auxiliary gas was not

used and an isolation width of 1 mass unit was set for efficient

trapping of precursor and product ions. Instrument control,

acquisition and data processing were performed using Navi-

gator software (version 1.2). The advanced controls, available

as a software patch, allowed MSn fragmentation parameters,

Copyright # 2003 John Wiley & Sons, Ltd.

*Correspondence to: B. J. Keely, Chemistry Department, Univer-sity of York, Heslington, York YO10 5DD, UK.E-mail: [email protected]/grant sponsors: University of York; Central ScienceLaboratory.

activation time (AT), activation amplitude (AA) and activa-

tion Q (AQ; Q represents the Mathieu parameter qz) to be con-

trolled. The method for optimisation of these parameters to

determine the most abundant MS2 and MS3 fragment ions

for each analyte has been described previously.6,7 The spectra

were acquired with the parameters optimised for these parti-

cular analytes.2 Samples were introduced by direct infusion

(10 mL min�1) using the syringe pump on the LCQ.

RESULTS AND DISCUSSION

Deuteriation of GLYP and AMPA in the aqueousand gaseous phasesThe negative ion ES-MS spectra of AMPA and GLYP

prepared individually in 2H2O display [M–H]� ions at m/z

values 3 Th greater than the native analytes (d3-AMPA¼m/

z 113, d3-GLYP¼m/z 171), demonstrating incorporation of

three deuterium atoms (Fig. 1). The spectra also reveal

the presence of small amounts of the doubly deuteriated

[M–H]� ions at 2 Th greater than in the native form. Thus,

even though there was a large excess of deuterium present,

protonation also occurred, probably by intermolecular trans-

fer or from adventitious moisture. As noted previously, the

signal intensity of native [M–H]� AMPA is much weaker

than that obtained from native GLYP, and results in higher

background noise.2 The degree of deuteriation in 2H2O solu-

tion at neutral pH is controlled by the pKa values of the acidic

and basic groups contained within the molecules. In aqueous

solution at pH 7, GLYP has an overall charge of �2 and

AMPA �1. Thus, the hydroxyl groups on the molecules

will be fully deprotonated and the labile hydrogens on the

amine groups will be replaced by deuterium (Fig. 2(a)). The

molecules are both detected in the gas phase in the �1 charge

state.2 Therefore, in the presence of 2H2O, GLYP binds

another 2Hþ ion during the electrospray process. Based on

the pKa values of the ionisable groups it is likely that the

site of this deuteriation in GLYP will be one of the oxygen

atoms on the phosphate group. Although the analytes exist

as zwitterions in the aqueous phase, previous work on related

molecules suggests that AMPA and GLYP are unlikely to

remain in their zwitterionic forms in the gas phase.8,9 Both

compounds are, therefore, likely to undergo internal deuter-

ium ion transfer to form structures with a single negative

charge (Figs. 2(b) and 2(c)). AMPA has only one possible

structural isomer formed by deuterium transfer from the

amine to the phosphonate group, while GLYP has two struc-

tural isomers due to there being two proximal oxygen atoms,

those of the carboxylate and the phosphonate groups.

MS2 fragmentation mechanisms of d3-AMPAThe MS2 spectrum of d3-AMPA (Fig. 3) reveals product ions at

m/z values of 83, 82, 81, 79 and 63. The MS2 spectrum of native

AMPA also contains the fragment ions at m/z 79 and 63,2 but

differs in the presence ofm/z 81 as base peak which is in lower

abundance relative to m/z 82 and 83 (base peak) in the MS2

spectrum of d3-AMPA. Thus, the ions at m/z 82 and 83 appear

to correspond to the same product ion as m/z 81 in the spec-

trum of native AMPA but containing one or two deuterium

atoms, respectively. The d3-AMPA product ions at m/z 79

and 63 are observed in similar abundance to native AMPA,

indicating that the three deuterium atoms associated with

d3-AMPA are lost in the neutral fragment. Rationalisation

of possible fragmentation mechanisms allows assignment

of the product ions as phosphorate (m/z 79) and phosphinate

(m/z 63), both of which contain only oxygen and phosphorus

(Figs. 4(a) and 4(b)). The lower abundance ofm/z 79 relative to

m/z 63 may be due to the requirement for a four-membered

transition state for fragmentation to occur.

The low abundance product ion at m/z 81 in the MS2

spectrum of d3-AMPA is believed to be different from that

formed from native AMPA, both from its difference in

relative abundance and from rationalisation of possible

structures for the deuteriated counterpart. Furthermore, m/

z 81 in the spectrum of d3-AMPA has a similar relative

abundance to m/z 80 in the MS2 spectrum of native AMPA,

suggesting that they are produced by the same mechanism.

This can be rationalised as homolytic cleavage of the C–P

bond (Fig. 4(c)), leading to protonated and deuteriated

phosphorate ion production from native and d3-AMPA,

respectively. The absence of m/z 80 for d3-AMPA is consistent

with the proposed mechanism for charge rearrangement

from the zwitterions (Fig. 2(b)) whereby a labile deuterium

ion is transferred from the amine group to the phosphonate

group as AMPA enters the gas phase. The alternative

mechanism where the phosphonate group picks up a proton

Figure 1. Mass spectra showing [M–H]� ion of d3-AMPA

(m/z 113) and d3-GLYP (m/z 171).

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 37–43

38 L. Goodwin et al.

from the neighbouring CH2 group is unlikely to be significant

due to the strength of the C–H bond.

The m/z 81 product ion formed from native AMPA is

consistent with a phosphorate group that contains two

hydrogen atoms. The presence of fragment ions at m/z 82

and 83 in the d3-AMPA spectrum indicates that more than one

mechanism occurs, though that producing m/z 83 is domi-

nant. The formation of m/z 83 from d3-AMPA can be

rationalised via a five-membered transition state whereby

deuterium is transferred from the amine group to the singly

deuteriated phosphonate group to produce a doubly deu-

teriated phosphorate (Fig. 4(d)). The m/z 82 fragment ion, at 1

mass unit lower, is rationalised as the singly protonated,

singly deuteriated phosphorate. For this ion to exist a proton

must be transferred to the phosphonate group during

fragmentation. The proton transfer most likely occurs from

the neighbouring CH2 group via a four membered transition

state (Fig. 4(e)).

MS2 and MS3 fragmentation mechanisms ofd3-glyphosateMS2 analysis of native (protonated) GLYP revealed fragment

ions at m/z 150, 124, 110 and 81, with m/z 150 as base peak and

the other ions having relative abundances between 5 and

20%.2 By contrast, MS2 analysis of d3-GLYP revealed ions at

m/z values 1–3 Th greater, corresponding to fragment

ions containing 1–3 deuterium atoms (Fig. 5). The MS2 base

peak for native GLYP (m/z 150) arises from loss of water. MS2

analysis of d3-GLYP reveals abundant product ions atm/z 151,

152 and 153, corresponding to losses of 2H2O, 2HOH and

H2O, respectively (Fig. 5). The presence of three ions suggests

that several mechanisms lead to elimination of water. The

presence of different sites from which water loss is possible

makes mechanistic assignment difficult from this data alone.

MS3 analysis of the m/z 151, 152 and 153 ions (Figs. 6(a)–6(c))

provides further insights into the mechanisms.

The simplest mechanism for loss of 2H2O from d3-GLYP

[M–H]�, giving m/z 151, involves loss from the isomer with a

doubly deuteriated phosphonate group (cf. Fig. 2(c)) via 2H

transfer to produce a phosphorate group (Fig. 7(a)). Another

possible mechanism for loss of 2H2O from d3-GLYP is from

the isomer possessing deuterium in both the carboxyl and

phosphonate groups, where deuterium transfer occurs from

Figure 3. MS2 spectrum of d3-AMPA from m/z 113.

Figure 2. (a) Structures of d3-AMPA and d3-GLYP at neutral pH in 2H2O and (b, c) their

rearrangement to neutralise two charges in the gas phase.

Fragmentation of aminomethylphosphonic acid and glyphosate 39


the carboxyl group to the phosphonate group, producing

phosphorate and carboxylate groups (Fig. 7(b)). These

mechanisms alone cannot explain the MS3 fragment ion at

m/z 79 (PO3�) from m/z 151 (Fig. 6(a)). Formation of this ion

requires that the oxygen lost in the MS2 transition comes from

the carboxyl group. The most likely mechanism involves a

similar transition state to that proposed in Fig. 7(b), but with

fragmentation occurring by deuterium transfer from the

phosphonate group to the carboxyl oxygen. The rearrange-

ment produces a six-membered ring intermediate that can be

further fragmented to produce m/z 79 in MS3 (Fig. 7(c)).

Fragmentation of the six-membered ring intermediate also

accounts for the MS3 fragment ion at m/z 123 (Fig. 7(d)) by

elimination of CO.

Loss of 2HOH from d3-GLYP gives m/z 152 as base peak in

MS2, indicating it to be the most important mechanism of

water loss from m/z 171. This loss is rationalised as occurring

via a four-membered transition state whereby a proton from

the neighbouring CH2 group is transferred to the –O2H of the

carboxyl group, introducing a double bond into the backbone

of the molecule (Fig. 8(a)). The MS2 product ion can fragment

further to givem/z 63 in a manner analogous to that illustrated

in Fig. 4(a). It is not possible to rationalise the MS3 product

ions at m/z 79 and 124 as originating via such MS2

intermediates (Fig. 6(b)). An explanation for the formation

of these product ions comes from the MS2 ion at m/z 153. This

ion was unexpected as it is due to loss of H2O, the most likely

site of this being from the hydroxyl of the carboxyl or

phosphonate. Given that all of the hydroxyls contain

deuterium, at least one deuterium atom should be lost. The

presence of all three deuterium atoms in the ion at m/z 153

indicates that rearrangement of hydrogen and deuterium

atoms has occurred. One mechanism that can account for this

is internal hydrogen transfer via a six-membered transition

state (Fig. 8(b)). The isomer with the negative charge residing

on the phosphonate group can be expected to abstract a

hydrogen atom from the CH2 group adjacent to the carboxyl

group. The reverse reaction of this hydrogen exchange has an

equal probability of transferring deuterium or hydrogen into

the backbone of the molecule. Similarly, the isomer with the

negative charge residing on the carboxylate group can be

expected to abstract a hydrogen atom from the CH2 group

Figure 4. Mechanisms of d3-AMPA MS2 fragmentation.

Figure 5. MS2 spectrum of d3-GLYP from m/z 171.



Figure 6. MS3 spectra of d3-GLYP from (a) m/z 151, (b) m/z 152, (c) m/z 153 and (d)

m/z 127 precursor ions.

Figure 7. Mechanisms of d3-GLYP MS3 fragmentation via the m/z 151 intermediate ion.



adjacent to the phosphonate group, but the reverse reaction

will always transfer hydrogen.

Hydrogen exchange is not a significant process while the

ion is solvated in 2H2O, as is apparent from the uptake of only

two atoms of deuterium. The absence of GLYP ions with

masses 4–7 Th higher than the native form indicates that

deuterium is not incorporated into the carbon skeleton of the

ion. Hydrogen exchange is also unlikely to occur in the ion

trap, where the temperature is insufficient to facilitate the

reaction. Thus, the most likely place that hydrogen transfer

could occur is in the MS heated capillary which is maintained

at 2258C. To investigate the temperature dependence of the

reaction a high boiling point solution, 50:50 (v/v) 2H2O/

DMSO, of GLYP was refluxed for 1 h. The spectrum of GLYP

after reflux displayed an increase in m/z 172 to 20% relative

abundance compared with 3% before reflux (Fig. 9). Thus,

hydrogen transfer from the CH group to the carboxylate or

phosphinate group is facilitated by high temperature, and

deuterium atoms from the solvent replaced the labile

hydrogen atoms. The reverse reaction incorporated deuter-

ium into the backbone of the molecule, increasing the m/z

value. The mechanisms illustrated in Figs. 7(b) and 7(c) can,

therefore, yield the MS2 product ions at m/z 152 if the

phosphonate group has undergone hydrogen transfer and

deuterium has been transferred in the reverse reaction. The

mechanisms can also yield m/z 153 if two hydrogen transfer

cycles have occurred, replacing both hydrogen atoms by

deuterium. Similarly, the H2O loss involving the phosphinate

group (cf. Fig. 8(a)) to yield m/z 153 is possible when a

hydrogen atom resides on the phosphonate group.

The loss of 44 Da from d3-GLYP to give an MS2 product ion

at m/z 127 is most likely due to loss of CO2. The appearance of

a single ion at m/z 127 shows that it contains the three

deuterium atoms (cf. m/z 124 from native GLYP), confirming

loss of CO2. Little information about the structure of the

product ion at m/z 127, or the fragmentation mechanism

leading to its formation, can be gained in MS2. Based on the

possible isomers of d3-GLYP, it appears that the form in

which the carboxylate group bears the negative charge is the

most likely precursor for m/z 127 (Fig. 10(a)), there being no

simple mechanism for its production from the isomer bearing

the negative charge on the phosphonate group. To gain

meaningful information on the m/z 171! 127 fragmentation

pathway the ion at m/z 127 was fragmented in MS3. The m/z

171! 127 MS3 spectrum reveals ions at m/z 63, 79, 81, 82 and

83 (Fig. 6(d)). Production ofm/z 63 and 79 undoubtedly occurs

via similar mechanisms (Figs. 10(b) and 10(c)) to those

described previously. The fragment ions at m/z 81, 82 and 83

are attributed to the phosphorate ion containing zero, one or

two deuterium atoms. The fragment ion at m/z 81 could be

produced by homolytic cleavage of the C–P bond, with

deuterium/hydrogen transfer explaining the formation ofm/

z 80 by the same mechanism. Notably, native GLYP produced

a low abundance ion at m/z 80 attributed to this homolytic

cleavage, implying that a minor contribution tom/z 81 may be

due to this mechanism. The m/z 127 ion is very similar in

structure to AMPA, the difference being a methyl group

attached to the amine. It is feasible that deuterium transfer

Figure 8. Mechanisms of (a) d3-GLYP MS2 fragmentation to

m/z 152 and (b) internal hydrogen transfer.

Figure 9. Spectra of GLYP in 1:1 2H2O:DMSO before and

after 1 h reflux.



from the amine group to the phosphonate group via a five-

membered transition state yields the fragment ions at m/z 83

and 82 (Fig. 10(d)). The major product of this mechanism

should be m/z 83, as an internal hydrogen transfer is required

to produce m/z 82. Formation of m/z 82 as base peak can be

rationalised via a mechanism analogous to that for produc-

tion of m/z 82 from d3-AMPA (Fig. 4(e)), where a proton is

transferred from the CH2 group adjacent to the phosphinate

group, eliminating dimethylimine (Fig. 10(e)). An internal

hydrogen transfer prior to this fragmentation explains the

reasonably high abundance of m/z 81.

CONCLUSIONS

The study reveals how mechanisms that yield different isoba-

ric product ions can be investigated using deuterium labelling.

The MSn analysis ofd3-GLYP andd3-AMPA provides informa-

tion on the mechanisms of the MSn fragmentation pathways

described previously,2 and demonstrates that several mechan-

isms can occur to yield the same fragment ion. An important

factor highlighted by the study is the need to consider internal

hydrogen transfer occurring in the gas phase, leading to pro-

duct ions with different degrees of isotopic labelling being

formed via the same mechanism. The mechanisms for charge

rearrangement in the gas phase and fragmentation involving a

five-membered transition state are analogous for both ana-

lytes. The analytes also undergo similar mechanisms for

the elimination of water and for the production of deuteriated

phosphonate ((2H/H)2PO3�), phosphorate (PO3

�) and

phosphinate (PO2�). A major difference between GLYP and

AMPA is the ability of GLYP to form closed ring structures

and eliminate the carboxylate group through sequential loss

of water and CO. The approach described for determining

the different fragmentation mechanisms is much simpler

and more cost effective than using analytes with isotopically

labelled carbon, nitrogen and oxygen and is suitable for a

wide range of small molecules.

AcknowledgementsWe thank the University of York and Central Science Labora-

tory (CSL) for support for a studentship (LG) and Dr. Victor

Chechik for helpful discussions.

REFERENCES

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3. Bell AJ, Despeyroux D, Murrell J, Watts P. Int. J. MassSpectrom. 1997; 165: 533.

4. Wan KX, Gross J, Hillenkamp F, Gross ML. J. Am. Soc. MassSpectrom. 2001; 12: 193.

5. Barr JD, Bell AJ, Konn DO, Murrell J, Timperley CM, WatersMJ, Watts P. Phys. Chem. Chem. Phys. 2002; 4: 2200.

6. Evans CS, Startin JR, Goodall DM, Keely BJ. Rapid Commun.Mass Spectrom. 2000; 14: 112.

7. Evans CS, Startin JR, Goodall DM, Keely BJ. Rapid Commun.Mass Spectrom. 2001; 15: 699.

8. Latajka Z, Ratajczak H, Scheiner S, Barycki J. J. Mol. Struct.1991; 235: 417.

9. Ding Y, Krogh-Jespersen K. Chem. Phys. Lett. 1992; 199: 261.

Figure 10. Mechanisms of d3-GLYP MS3 fragmentation via the m/z 127 intermediate ion.



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