Fragmentation pathways of eight nitrogen-containing bisphosphonates (BPs) investigated by ESI-MSn in negative ion mode

Article abstract of DOI:10.1016/j.ijms.2010.07.017

Fragmentation pathways of eight nitrogen-containing bisphosphonates (BPs), including Pamidronate, Alendronate, and five corresponding acylated derivatives, and Risedronate, were investigated by electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry in negative ion mode. Characteristic fragment ions involved were formed by successive loss of water molecules, including α, β-unsaturated product ions formed by dehydration of the α-hydroxyl group and β-hydrogen, cyclic ions with P-O-P four-membered rings formed by dehydration of the two -OH groups attached to two phosphorus atoms, and cyclic phosphoramide ions with five- or six-membered rings formed by loss of water via an intramolecular nucleophilic substitution reaction, in which -NH₂ (or -NH-) group on γ or δ carbon acted as the nucleophile to attack phosphorus atom of PO group at the initial stage. Another notable characteristic product ion [HP₂O₅]^- at m/z 143, shown by all eight ESI-MS² spectra, is a diagnostic ion of bisphosphonate group, formed from the four-membered ring ion containing P-O-P. Some additional fragmentation ions were produced from chloroacetyl Alendronate, chloroacetyl Pamidronate, and Risedronate containing triple bond ions, formed by loss of water from the corresponding enol anions. The hydrogen/deuterium (H/D) exchange experiment, theoretical calculations, and the high-resolution mass spectrometry were appropriately employed to rationalize the proposed fragmentation pathways.

Full text of DOI:10.1016/j.ijms.2010.07.017

International Journal of Mass Spectrometry 295 (2010) 85–93
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
Fragmentation pathways of eight nitrogen-containing bisphosphonates (BPs)
investigated by ESI-MSn in negative ion mode
Zhibo Qua, Xiaolan Chena,∗, Chen Qub, Lingbo Qua,c, Jinwei Yuanc, Donghui Weia, Huina Lia,
Xiaoying Huanga, Yuqin Jianga, Yufen Zhaoa,d,∗
a Department of Chemistry, Zhengzhou University, Key Laboratory of Chemistry Biology and Organic Chemistry, Daxue Road No. 75, Zhengzhou, Henan 450052, China
b College of Chemistry, Nankai University, Tianjin 300071, China
c School of Chemistry & Chemical Engineering, Henan University of Technology, Zhengzhou 450001, China
d Department of Chemistry, Xiamen University, Xiamen 361005, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 May 2010
Received in revised form 10 July 2010
Accepted 12 July 2010
Available online 16 July 2010
Fragmentation pathways of eight nitrogen-containing bisphosphonates (BPs), including Pamidronate,
Alendronate, and five corresponding acylated derivatives, and Risedronate, were investigated by electro-
spray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry in negative
ion mode. Characteristic fragment ions involved were formed by successive loss of water molecules,
including ␣, ␤-unsaturated product ions formed by dehydration of the ␣-hydroxyl group and ␤-hydrogen,
cyclic ions with P–O–P four-membered rings formed by dehydration of the two –OH groups attached to
two phosphorus atoms, and cyclic phosphoramide ions with five- or six-membered rings formed by loss
of water via an intramolecular nucleophilic substitution reaction, in which –NH2 (or –NH–) group on ␥
or ␦ carbon acted as the nucleophile to attack phosphorus atom of P O group at the initial stage. Another
notable characteristic product ion [HP2O5]− at m/z 143, shown by all eight ESI-MS2 spectra, is a diag-
nostic ion of bisphosphonate group, formed from the four-membered ring ion containing P–O–P. Some
additional fragmentation ions were produced from chloroacetyl Alendronate, chloroacetyl Pamidronate,
and Risedronate containing triple bond ions, formed by loss of water from the corresponding enol anions.
The hydrogen/deuterium (H/D) exchange experiment, theoretical calculations, and the high-resolution
mass spectrometry were appropriately employed to rationalize the proposed fragmentation pathways.
Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
Keywords:
Nitrogen-containing bisphosphonate
ESI-MSn
Fragmentation pathway
Diagnostic ion
Theoretical calculation
1. Introduction
for calcium crystals, and a nitrogen atom-containing carbon chain
enhances their potency and determines their mechanism of action.
Bisphosphonates (BPs) or diphosphonates, are a class of drugs
that prevent the loss of bone mass, used to treat osteoporosis
and similar diseases. More recently, BPs have been used to reduce
fracture rates in children with osteogenesis imperfecta [1] and in
treatment of otosclerosis [2]. BP inhibits osteoclastic bone resorp-
tion both in vivo and in vitro [3,4]. The typical structure of BP is
generally similar to that of pyrophosphate. The only difference is
that the oxygen atom that binds the two phosphorus atoms in
pyrophosphate (P–O–P) is replaced by a carbon (P–C–P). The carbon
between the two phosphorous atoms is generally attached to two
additional side-chains in their molecules which are not present in
pyrophosphate. In general, there is a hydroxyl attached to the cen-
tral (geminal) carbon atom [5], which enhances the affinity of BPs
The structural features of BPs have led to the synthesis of a large
number of compounds with different properties [6–8]. The greatest
improvements in the biological activities of BPs have resulted from
the introduction of a basic nitrogen atom into the side chain on
the geminal carbon. Further jumps in the activity in bone resorp-
tion were obtained by transposing the amine group farther from the
“bone hook” or by alkylating the primary amine to a tertiary one [9].
The nitrogen-containing BPs are the latest and most potent addi-
tion to this family of BPs. They have high potency, and are specially
targeted to the osteoclast on bone with very low doses (5–10 mg
clinically) [10]. Nitrogen-containing BPs act on bone metabolism
by binding and blocking the enzyme farnesyl diphosphate syn-
thase (FPPS) in the HMG-CoA reductase pathway (also known as
the mevalonate pathway) [11].
Electrospray ionization multistage tandem mass spectrome-
try (ESI-MSn), a powerful tool for structure determination, has
been widely used in chemistry, biochemistry and pharmaceutical
research [12–14]. However, only a few methods based upon mass
spectrometric detection have been reported for BPs. For example,
∗
Corresponding authors at: Key Laboratory of Chemical Biology and Organic
Chemistry of Henan Province, Department of Chemistry, Zhengzhou University,
Zhengzhou 450052, China. Tel.: +86 371 67767051; fax: +86 371 67767051.
E-mail address: chenxl@zzu.edu.cn (X. Chen).
1387-3806/$ – see front matter Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.ijms.2010.07.017

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Z. Qu et al. / International Journal of Mass Spectrometry 295 (2010) 85–93
Scheme 1. Synthesis of nitrogen-containing BPs (A1–A8).
only the mass spectrometric (MS) behavior and fragmentation of
a relatively simple bisphosphonate of clodronate, in which there
are two chloro groups attached to the geminal carbon atom, were
reported by Risto Kostiainen’s group [15]. Electrospray ionization
mass spectrometric (ESI-MSn) fragmentation of the very impor-
tant nitrogen-containing BPs has not been reported in previous
literature. In the study described in this paper, eight nitrogen-
containing BPs were synthesized by reference to the literature [16]
and the fragmentation patterns were investigated by electrospray
ionization multistage tandem mass spectrometry (ESI-MSn) in neg-
ative ion mode in details. The hydrogen/deuterium (H/D) exchange
experiment, theoretical calculations, and the high-resolution mass
spectrometry were appropriately employed to rationalize the pro-
posed fragmentation pathways.
2.2. Mass spectrometric conditions
The ESI mass spectra of BPs were acquired on an Esquire 3000
ESI-MS with an ion trap mass spectrometer in negative ionization
mode (Bruker Daltonik Gmbh, Germany). The MSn spectra were
obtained by collision-induced dissociation (CID) with helium after
isolation of the appropriate precursor ions. Ionization of analysis
was carried out using the following setting of the ESI: nebulizer gas
flow 7 psi, dry gas 4.5 L/min, dry temperature 300 ◦C, spray voltage
4 kV. Samples were dissolved in CH3OH:H2O = 1:1 (v/v) and ionized
by electrospray ionization. Calibration of m/z was performed using
a standard ESI-tuning-mixture. Scan range was 50–500 m/z and
scan resolution was normal (13,000 m/z/s).
High-resolution mass spectra were performed on a Micromass
Q-TOF MicroTM mass spectrometer with an ESI source (Waters,
Manchester, UK). Argon was used as the collision gas. Nitrogen was
used as the nebulizer and dissolvation gas. The ESI source was oper-
ated in negative ion mode with optimized conditions. The mass
spectra over the mass range 50–500 with a capillary voltage of
3.5 kV, a cone voltage of 25 V, a source temperature of 80 ◦C, a dis-
solvation temperature of 170 ◦C, a collision voltage of 7 V, a cone
gas flow of 50 L/h and a dissolvation gas flow of 350 L/h. During the
MS/MS experiments, the deprotonated A1 was chosen as precursor
ion and isolated in the quadruple.
2. Experimental
2.1. Chemicals and synthesis of bisphosphonates
Amino acids were purchased from Sino-American Biotechnol-
ogy Co. (Shanghai, China). Deuterated solvents D2O were from
Beijing Chemical Factory (Beijing, China). Other reagents were
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China).
2.3. Computational details
Experimental BPs were prepared by the method described
previously. Compounds containing a carboxylic functional group
(0.025 mol), including two amino acids and pyridin-3-yl-acetic
acid, were mixed with phosphorus acid (0.0375 mol) and then
stirred for 1 h at 90 ◦C. Phosphorus trichloride (0.05 mol) was added
dropwise to the suspension at 100 ◦C under stirring. After 4 h, the
solution was cooled down to room temperature. The residue was
hydrolyzed by adding 10 mL H2O, the solution decolorized by an
appropriate amount of activated carbon was added to 150 mL iso-
propanol, then white flocculent products were precipitated very
quickly. The crude product was filtrated and washed three times
with water. Compounds (A4–A6) were obtained. Place compound
A4 or A5 (10 mmol), acyl chloride (20 mmol), and 12 mL (3.5%)
NaOH in the flask and cool it to −10 ◦C in an ice-salt bath, then
stir for 6 h. Using (1 mol/L) HCl to adjust to pH = 1, then pour it
into 200 mL of ethanol. The white deposit appeared quickly and
was distilled. The residue was then purified by recrystallization
from CH3OH:H2O = 1:1 (v/v). The white crystals (A1, A2, A3, A7
and A8) were obtained in moderate yields (shown in the brackets,
Scheme 1). All of samples were synthesized as shown in Scheme 1.
The structrues were confirmed by 31P NMR, 1H NMR, 13C NMR and
ESI-MS.
To simplify the calculations, compound A1 was chosen as a
model. Using density functional theory, the geometries of the reac-
tant, intermediates and products in all reaction channels were
optimized at the B3LYP/6-31G (d, p) level. All theoretical calcula-
tions were performed using the GAUSSIAN03 [17] suite of software.
3. Results and discussion
ESI-MS spectra in most cases, were acquired in positive ion
mode to produce [M+Na]+ and [M+H]+ ions. However, four acidic
hydroxyl groups of BPs can be easily deprotonated to obtain the
conjugate anions in ionization process. Positive ionization by pro-
tonization was not observed in any case and higher sensitivity was
obtained by negative ionization. ESI-MSn spectra of compounds
A1–A8 in negative mode were studied in detail and the data are
shown in Table 1. The most representative fragmentation ways
were shown in Scheme 2. The main fragment ions derived from
[M−H]− ion were [M−H−H2O]−, [M−H−2H2O]−, [M−H−H3PO3]−
and [HP2O5]− ions. There were three ways to lose the first molecule
of water. The relatively stable ␣, ␤-unsaturated product ion (a1)

Table 1
Negative ion ESI-MSn of [M−H]− ions of compounds A1–A8 [m/z (relative abundance, %), m = MW − 1].
Com.
Precursor ions
Fragment ions
a (m-18)
b (m-36)
c (m-82)
d
e (b-64)
f (d-64)
g (m-54)
h
i
j
372
354
336
290
143
354 (21)
336 (21)
336 (22)
290 (100)
290 (100)
290 (7)
143 (11)
143 (14)
272 (100)
127 (28)
A1
135 (100)
79 (100)
338
320
302
256
143
320 (57)
258 (49)
230 (69)
302 (6)
302 (30)
256 (100)
256 (100)
143 (5)
143 (7)
238 (100)
127 (64)
127 (33)
168 (14)
A2
A3
135 (100)
135 (100)
79 (100)
79 (9)
276
258
240
194
143
240 (33)
240 (61)
194 (100)
194 (100)
143 (22)
143 (31)
176 (14)
176 (48)
176 (100)
211 (14)
137 (7)
63 (28)
79 (100)
79 (12)
248
230
212
194
166
148
143
212 (100)
212 (95)
166(50)
143(19)
148 (42)
148 (19)
148 (19)
194 (72)
194 (100)
194 (100)
166 (10)
79 (100)
A4
148 (100)
79 (100)
79 (100)
63 (25)
63 (38)
234
216
198
152
143
216 (100)
264 (46)
292 (45)
198 (58)
198 (100)
152 (43)
152 (23)
143 (10)
134 (13)
134 (67)
134 (100)
79 (14)
127 (5)
180 (7)
A5
A6
79 (100)
282
264
246
200
143
246 (71)
246 (100)
200 (100)
200 (25)
143 (9)
143 (11)
127 (6)
127 (18)
127 (100)
182 (59)
63 (100)
79 (100)
79 (100)
310
292
274
228
210
192
143
274 (23)
274 (57)
228 (100)
228 (100)
143 (15)
143 (5)
210 (3)
210 (13)
210 (100)
192 (8)
256(8) 192 (6)
135 (3)
127 (16)
136 (40)
136 (29)
192 (100)
192 (6) 174(25) 131 (8)
A7
324
306
288
306 (16)
288 (8)
288 (33)
242 (100)
242 (100)
143 (17)
143 (52)
270 (11)
270 (38)
270 (26)
252 (15) 206 (10)
252 (24) 224 (10) 194 (14)
252 (97) 224 (100)
188 (60)
127 (10)
270
252
242
206
143
252 (100)
188 (100)
206 (100) 81(14) 121 (29)
121 (100)
A8
79 (2)
79 (100)

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Z. Qu et al. / International Journal of Mass Spectrometry 295 (2010) 85–93
Scheme 2. Main fragment pathways of nitrogen-containing BPs.
was produced by dehydration of the –OH at ␣ carbon atom and
the –H group at ␤ carbon atom (Path 1). The four-membered ring
ion containing P–O–P (a2) was produced by dehydration of the two
–OH groups attached to two phosphorous atoms separated by a
carbon atom (Path 2). The cyclic phosphoramide ion with a five- or
six-membered ring (a3) was produced by loss of water via a nucle-
ophilic addition–elimination mechanism, in which –NH2 (or –NH–)
group on ␥ (n = 2) or ␦ (n = 3) carbon acted as the nucleophile to
attack phosphorous atom of P O group at the initial stage (Path 3).
Supports for the three dehydrated ways resulted from further frag-
mentation pathway analyses about precursor ion [M−H−2H2O]−
as illustrated in Scheme 3 in the following. [HP2O5]− ion at m/z
143 (d) is the most characteristic product ion shown by all eight
ESI-MS2 spectra, formed spontaneously from the four-membered
ring ion containing P–O–P (a2) by loss of an aldehyde. Therefore,
[HP2O5]− ion at m/z 143 can hopefully be treated as a diagnos-
tic ion of bisphosphonate group. The further fragment ion at m/z
79 (f) can be formed from the ion at m/z 143 by losing HPO2. A7
and A8 ions showed some additional fragmentation pathways, aris-
ing from extra fragmentation of acyl parts, including a triple bond
ion derived from their enol ions by loss of a molecule of water
(Scheme 4). The ESI mass spectral fragmentation pathways of com-
pound A1, A6 and A8 are discussed as typical examples in details.
Besides the common fragmentation pathways mentioned above,
the different fragmentation pathways induced by the difference
among the structures of the BPs would also be presented and ana-
lyzed in the following using three typically related examples of
compound A1, A6 and A8, based on the fact that the similar fragmen-
tation pathways were shown by Compounds A1–A5 and the similar
fragmentation pathways by Compounds A7 and A8, as concluded in
Table 1.
The ESI-MSn fragmentation of deprotonated A1 at m/z 372
(Fig. 1(1)) is summarized in Scheme 3. The ESI-MS2 of the ion
at m/z 372 produced the product ion at m/z 354, corresponding
to [M−H−H2O]−, assigned as a1, a2, and a3 ions based on three
different dehydrated ways. Ion at m/z 336 was corresponding to
[M−H−2H2O]−, assigned as b1 and b2 ions in accordance with fur-
ther dehydration of a1, a2, and a3 ions. The product ion c at m/z
290 was corresponding to [M−H−H3PO3]−. The most characteris-
tic product ion d concerning the biphosphonate part at m/z 143
was formed by losing an aldehyde C10H10ClNO2 from the cyclic ion
a2. The existing of m/z 143 supports the Path 2 and the structure
of product ions, based on the fragmentation pathways shown in
Scheme 2 and 3. ESI-MS3 spectrum (Fig. 1(2)) of the ion at m/z 354,
corresponding to [M−H−H2O]− was shown in Fig. 1(2). Three ions
at m/z 336, m/z 290 and m/z 143 were produced from the ion at m/z
354. The product ions c at m/z 290 and d at m/z 143, were produced
by Path 2 from fragmentation of the ion a2 at m/z 354 by losing
HPO2 and an aldehyde C10H10ClNO2, respectively. The product ion
b1 at m/z 336 can be formed by losing water from the ion a1 at m/z

Z. Qu et al. / International Journal of Mass Spectrometry 295 (2010) 85–93
89
Scheme 3. Proposed fragment pathways of deprotonated A1. The values next to the arrows (in kJ/mol) represent the relative Gibbs free energies (ꢀG) for each individual
fragmentation reaction.
and e, from the corresponding ESI-MS3 spectra, respectively, were
additional ions besides the common fragmentation ions shown in
Scheme 2.
354 by Path 1 or from the ion a2 at m/z 354 by Path 2, meanwhile ion
2 at m/z 336 can be formed by successive losing water from the ion
3 at m/z 354 as shown in Scheme 3. ESI-MS3 spectrum of the ion at
b
a
m/z 336 was shown in Fig. 1(3). Two ions at m/z 272, m/z 127 were
produced from the precursor ion at m/z 336. It is especially worth
mentioning that ion e can be reasonably formed by loss of HPO2
not only from ion b1 produced by Path 1 and 2, but also from the
ion b2 produced by Path 3, giving an important support for the Path
1 and 3. Moreover the easy formation of cyclic phosphoramide ion
with a five- or six-membered ring, corresponding to the first water
loss ion a3, admittedly results from the extraordinary stability of
a five- or six-membered ring structure. The existing of m/z 143
mentioned in our manuscript supports the Path 2 and especially
supports the structure of product ion a2 with four-membered ring.
To a great extent, the ␥- or ␦-cyclic phosphoramide ion a3 with a
five- or six-membered ring should be favorably formed under the
mass spectral condition, based on the premise that product ion a2
with four-membered ring can be formed in this circumstance. The
ion i at m/z 127, corresponding to the low-intensity of peak of ESI-
MS3 spectrum, was reasonably produced from the ion b1 by loss
of a molecule C10H8ClNO2, giving an assistant support for Path 1.
The ESI-MS3 spectrum of the ion at m/z 290 was shown in Fig. 1(4).
The ion with P–O bond five-member ring at m/z 135 was assigned
as h produced by loss of a molecular of p-chlorobenzamide. The
ESI-MS3 spectrum of the ion at m/z 143 was shown in Fig. 1(5). The
ion at m/z 79 was assigned as f formed by losing HPO2 from the
parent ion at m/z 143. It is also worth mentioning that the ions i, h
The analysis showed that the main fragment pathways of com-
pound A8 were in good agreement with the characteristic fragment
pathways of nitrogen-containing BPs. Besides similar fragmenta-
tion patterns shown by compound A1–5, Compounds A7 and A8
displayed additional fragmentation pathways owing to the acyl
group of the two molecules. Herein compound A8 was discussed
as an example (Scheme 4). Ions at m/z 306 and at m/z 288 were
corresponding to [M−H−H2O]− and [M−H−2H2O]−, respectively.
The product ion at m/z 242 was corresponding to [M−H−H3PO3]−.
The diagnostic ion at m/z 143 was also observed. The product ion at
m/z 79 was formed by losing HPO2 from the parent ion at m/z 143.
In addition, the ion peaks at m/z 270 and m/z 252, corresponding
to [M−H−3H2O]−, assigned as g (keto ion) and k (enol ion), and
[M−H−4H2O]−, assigned as l, were observed. The ion n at m/z 188
was reasonably produced from the ion l, the ion l from the enol ion
k at m/z 270, the keto ion g at m/z 270 from the ion b2 at m/z 288,
and the ion b2 at m/z 288 from the ion a3 at m/z 306 following Path
3. Again, the existing of ions n, l, k, g strongly support the Path 3 and
the structure of the product ions. Some other additional ions were
formed as shown in Table. 1 and Scheme 4, including the ion at m/z
81 formed by loss of C7H9ClO2 from the ion at m/z 242, the ion at
m/z 206 formed from the ion at m/z 242 by losing a molecule of HCl
and the ion at m/z 121 formed by losing a molecule of C4H7NO from
the ion at m/z 206. The existence of the enol ion (k) was supported

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Z. Qu et al. / International Journal of Mass Spectrometry 295 (2010) 85–93
Scheme 4. Proposed fragmentation pathways of compound A8.
by further hydrogen/deuterium (H/D) exchange experiment as the
following.
Gaussian 03 software. It is considered that in CID-ESI-MS process,
the energy that is transferred to the precursor ion owing to the col-
lision activation can even surpass the activation barrier for some
specific fragmentation reactions. The difference between the initial
and final energy states can be used to understand the mechanism,
and it is not necessary to consider the energies of any transitions
state species [19]. Therefore, the relative Gibbs free energies of
compound A1, which represent four different pathways a, b, c, and
d, respectively, were calculated. The deprotonated compound A1
was assumed as 0.00 kJ/mol and the data of calculation have been
shown in Scheme 3. The formation of the ion a3 from the depro-
tonated molecule A is an endergonic process, as evidenced by the
relative energies of A (0.00 kJ/mol) and a3 (50.90 kJ/mol). Moreover,
the ion a3 can easily transform to b2 (0.25 kJ/mol) via path c. Fur-
thermore, the ion a1 (8.32 kJ/mol) is the most easily transformed
from the ion A by losing one water molecule, so the path a is a more
thermodynamically favored process, thus, the ion A does not eas-
ily transform to the ion a2 (88.79 kJ/mol). The ion b1 (1.13 kJ/mol)
can easily be formed from the ion a2 via path b, however, the for-
mation of the ion d from the precursor ion a2 need 61.36 kJ/mol
via path d, that is why the relative abundances of the ion d is
relatively lower than other ions. As concerned as above, all the the-
oretical calculations are in good agreement with the experimental
results. Noteworthy, the results of theoretical calculations success-
fully demonstrated that their fragment ions can be rationalized on
the basis of the relative Gibbs free energies.
Compound A8 was dissolved in the D2O and shaken for 1 h to
ensure the active hydrogens were exchanged as much as possible.
Then the solution was analyzed by ESI tandem mass spectrome-
try in negative mode. The corresponding ESI-MS4 spectrum of the
ion around 270 of compound A8 was shown in Fig. 2. The peak at
m/z 271 should be corresponded to the deuterated enol or keto
ion, and the peak at m/z 270 should be corresponded to the non-
deuterated keto or enol ion. The deuterated enol or keto ion must
be formed through an equilibrium established between the enol
and keto forms. The deuterium experiment could give a support
for the existence of its enol form. The existence of its enol form at
m/z 270 can favorably result in the formation of the ion at m/z 252
by dehydration.
The relatively higher activity attained to data has been with
the nitrogen-containing heterocyclic BP, Risedronate [18]. The ESI-
MSn spectra of Risedronate (A6) showed fewer fragmentation peaks
in comparison with the other seven BPs containing long chain.
Most representative fragmentation patterns of seven BPs men-
tioned above were also found in that of Risedronate, as shown in
Scheme 5. Ions at m/z 264 and at m/z 246 were corresponding to
[M−H−H2O]− and [M−H−2H2O]−, respectively. The product ion at
m/z 290 was corresponding to [M−H−H3PO3]−. The most charac-
teristic product ion at m/z 143 was also observed from the cyclic
ion at m/z 246. Additionally, a specific ion at m/z 182 was produced
by elimination of water from the enolate anion at m/z 200.
In order to investigate the reason for the above different frag-
mentation pathways of losing water molecules, the structures and
energies of those deprotonated ions were optimized and calculated
by density functional theory at the B3LYP/6-31G (d, p) level using
To certify the proposed structure of the ions produced from the
parent ion [MA1−H]−, the mass-to-charge ratios of the fragment
ions were measured using the ESI-Q-TOF (Table 2). The correla-
tion between the theoretical and measured values supports the
proposal fragmentation of compound A1.

Z. Qu et al. / International Journal of Mass Spectrometry 295 (2010) 85–93
91
Fig. 1. (1) ESI-MS2 of [M−H]− at m/z 372 of compound A1. (2) ESI-MS3 of the ion at m/z 354. (3) ESI-MS3 of the ion at m/z 336. (4) ESI-MS3 of the ion at m/z 290. (5) ESI-MS3
of the ion at m/z 143.

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Z. Qu et al. / International Journal of Mass Spectrometry 295 (2010) 85–93
Fig. 2. Expanded ESI-MS4 spectrum of the ion at m/z 270 of deuterated compound A8.
Scheme 5. Proposed fragmentation pathways of compound A6.
Table 2
High-resolution mass spectral data for the main ions of A1 (Obtained with ESI-MS2 in the negative ion mode).
Ion species
Theoretical mass (m/z)
Measured mass (m/z)
Relative error (ppm)
[M−H]−
371.9810
353.9705
335.9599
289.9991
142.9305
371.9801
353.9693
335.9607
290.0004
142.9312
2.4
2.2
2.3
4.4
4.8
[M−H−H2O]−
[M−H−H2O−H2O]−
[M−H−H3PO3]−
[M−H−H2O−C10H10ClNO2]−
4. Conclusions
chloroacetyl Alendronate, chloroacetyl Pamidronate and Rise-
dronate showed some additional fragmentation ions, containing
triple bond ions owing to elimination of water from their enol
anions. The hydrogen/deuterium (H/D) exchange experiment, the-
oretical calculations and the high-resolution mass spectrometry
(Q-TOF) were appropriately employed to rationalize the pro-
posed fragmentation pathways. These observations may have some
potential applications in the structural elucidation and interpre-
tation of mass spectra of nitrogen-containing bisphosphonate
derivatives.
In conclusion, the negative ion electrospray ionization mass
spectral fragmentation pathways of BPs, including seven nitrogen-
containing side chain BPs and one nitrogen-containing heterocyclic
BP, were analyzed by multistage mass spectrometry. The abun-
dant fragment ions were observed by successive loss one or two
molecules of water. The most representative fragmentation ions
derived from [M−H]− ion were [M−H−H2O]−, [M−H−2H2O]−,
[M−H−H3PO3]− and [HP2O5]− ions. [HP2O5]− ion at m/z 143
is the most characteristic product ion shown by all eight ESI-
MS2 spectra, formed spontaneously from the four-membered
ring ion with P–O–P by loss of an aldehyde. [HP2O5]− ion at
m/z 143 can hopefully be treated as a diagnostic ion of bispho-
sphonate group. Besides typical fragment ions of all the BPs,
Acknowledgements
The authors would like to thank the National Natural Science
Foundation of China (20472076 and 20972130) and Henan Aca-

Z. Qu et al. / International Journal of Mass Spectrometry 295 (2010) 85–93
93
demic Foundation of Science and Technology (No. 0512001400) for
their financial support.
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