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
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 37–43
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.
40 L. Goodwin et al.
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 37–43
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.
Fragmentation of aminomethylphosphonic acid and glyphosate 41
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 37–43
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.
42 L. Goodwin et al.
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 37–43
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.
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Figure 10. Mechanisms of d3-GLYP MS3 fragmentation via the m/z 127 intermediate ion.
Fragmentation of aminomethylphosphonic acid and glyphosate 43
Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 37–43
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