Structural elucidation of degradation products of olaquindox under stressed conditions by accurate mass measurements using electrospray ionization hybrid ion trap/time-of-flight mass spectrometry

Article abstract of DOI:10.1016/j.ijms.2011.01.004

In this study, the stress degradation of olaquindox under conditions of hydrolysis (neutral, acidic and basic), oxidation and photolytic stress was investigated. In order to characterize each degradation product, we developed a rapid, sensitive and reliable high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF) method. The degradation products formed under different forced conditions were separated using an ODS-C18 column with gradient elution. Multiple scans of degradation products in MS and MS/MS modes and accurate mass measurements were performed through data-dependent acquisition. The structural elucidations of degradation products were performed by comparing the changes in the accurate molecular masses and fragment ions generated from precursor ions with those of parent drug. The present results showed that maximum degradation was observed in hydrolysis, especially in the acidic condition. The drug was also degraded significantly under photolytic conditions. A total of 12 degradation products of olaquindox were detected and characterized using the developed method. The main degradation product was formed by the complete cleavage of side chain to form 3-methyl-2-hydroxylquinoxaline-4-oxide. A degradation pathway of olaquindox was also tentatively proposed for the first time based on these characterized structures.

Full text of DOI:10.1016/j.ijms.2011.01.004

International Journal of Mass Spectrometry 303 (2011) 90–96
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
Structural elucidation of degradation products of olaquindox under stressed
conditions by accurate mass measurements using electrospray ionization hybrid
ion trap/time-of-flight mass spectrometry
Zhao-Ying Liua, Hua-Hai Zhangb, Xiao-Jun Chena, Xiao-Ni Zhoua, Leren Wanc, Zhi-Liang Suna,∗
a Hunan Engineering Research Center of Veterinary Drugs, College of Veterinary Medicine, Hunan Agricultural University, Furong District, Changsha, Hunan 410128, China
b College of Forestry, Northwest Agriculture and Forestry University, Yangling, Shanxi 712100, China
c Shimadzu International Trading (Shanghai) Co., Limited, Shanghai 200052, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, the stress degradation of olaquindox under conditions of hydrolysis (neutral, acidic and
basic), oxidation and photolytic stress was investigated. In order to characterize each degradation prod-
uct, we developed a rapid, sensitive and reliable high-performance liquid chromatography combined
with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF) method. The degradation prod-
ucts formed under different forced conditions were separated using an ODS-C18 column with gradient
elution. Multiple scans of degradation products in MS and MS/MS modes and accurate mass measure-
ments were performed through data-dependent acquisition. The structural elucidations of degradation
products were performed by comparing the changes in the accurate molecular masses and fragment ions
generated from precursor ions with those of parent drug. The present results showed that maximum
degradation was observed in hydrolysis, especially in the acidic condition. The drug was also degraded
significantly under photolytic conditions. A total of 12 degradation products of olaquindox were detected
and characterized using the developed method. The main degradation product was formed by the com-
plete cleavage of side chain to form 3-methyl-2-hydroxylquinoxaline-4-oxide. A degradation pathway
of olaquindox was also tentatively proposed for the first time based on these characterized structures.
© 2011 Elsevier B.V. All rights reserved.
Received 7 December 2010
Received in revised form 1 January 2011
Accepted 7 January 2011
Available online 14 January 2011
Keywords:
Olaquindox
LC/MS-IT-TOF
Degradation product
Photo-degradation
Mass spectrometry
1. Introduction
carried out to elucidate the stability characteristics of veterinary
drug substance and medicinal products [2]. Information on the sta-
The identification of drug degradation products plays an impor-
tant role in the drug discovery and development processes. It has
been well documented that drug products will undergo physico-
chemical degradation during manufacturing processes and storage
[1]. Stress testing of the drug substance can help identify the likely
degradation products, which can in turn help establish the degra-
dation pathway and the intrinsic stability of the molecule. The
degradation products usually arise from the ingredients used in
dosage formulation and/or in the process of formulation where
temperature, humidity and light may all play a part. To acceler-
ate drug development, various stress-testing protocols had been
designed to emulate stresses the compound may experience dur-
ing manufacturing and storage conditions. These methods expose
the drug to forced degradation conditions such as acid, base, oxida-
tion and exposure to light. The Veterinary International Conference
on Harmonization (VICH) guideline requires that stress testing be
bility of the drug substance is an integral part of the systematic
approach to stability evaluation.
Olaquindox,
N-(2-hydroxyethyl)-3-methyl-2-quinoxalincar-
boxamide-1,4-dioxide, is one of the quinoxaline-N,N-dioxides
that is used as medicinal feed additives for the prevention of pig
dysentery and bacterial enteritis. Olaquindox is often responsible
for induced photoallergic contact dermatitis and persistent light
reaction in a pig breeder [3,4]. Detailed investigation of the iden-
tification of the photolysis products of this drug has not yet been
carried on. The degradation behaviors of olaquindox in manure
and environmentally relevant matrices, such as soil water and
sewage sludge water, have been investigated, but the degradation
products remain to identification [5,6].
LC–MS/MS has become the technique of choice in the degrada-
tion product studies due to its selectivity, sensitivity, and speed
of analysis [7–9]. There have been a number of reports in the
literature that applied LC–MS/MS for characterization of degra-
dation products. In 2000, Wu [10] reviewed the application of
LC–MS/MS in the analysis of drug degradation products in pharma-
ceutical formulations under various stress conditions (oxidation,
∗
Corresponding author. Tel.: +86 731 84635229; fax: +86 731 84673618.
E-mail address: sunzhiliang1965@yahoo.com.cn (Z.-L. Sun).
1387-3806/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijms.2011.01.004

Z.-Y. Liu et al. / International Journal of Mass Spectrometry 303 (2011) 90–96
91
hydrolysis, dimerization and adduct formation with excipients).
Exact mass measurements and elemental composition assignment
are essential for the characterization of small molecules. Accurate
mass measurement of the product ions facilitates the struc-
tural elucidation of new or unknown compounds [11]. Currently,
liquid chromatography combined with hybrid ion trap/time-of-
flight mass spectrometry (LC/MS-IT-TOF) provides high sensitivity
and accuracy of the TOF analyzer with over 10,000 resolving
power at m/z 1000. Moreover, multiple scans of metabolites in
MS and MS2 modes and accurate mass measurements can be
automatically performed simultaneously through data-dependent
acquisition. Although LC/MS-IT-TOF has rapidly developed as one
of the most effective techniques for the determination of unknown
compounds, its application to drug degradation product char-
acterization is much less routine than for the drug metabolite
characterization [12–14].
Therefore, the purpose of the present study was to perform
stress studies on olaquindox in order to evaluate its inherent stabil-
ity. To achieve this goal, we developed an LC/MS-IT-TOF method to
characterize the degradation products of olaquindox in solutions.
On the basis of accurate MS2 spectra and elemental compositions of
degradation products, we proposed their chemical structures. This
work demonstrates that the use of LC/MS-IT-TOF approach appears
to be rapid, efficient and reliable in structural characterization of
degradation products.
acid) and mobile phase B (acetonitrile). The gradient was as follows:
0–5 min, a linear gradient from 2% B to 5% B; 5–15 min, a linear gra-
dient to 30% B; 15–18 min, a linear gradient to 100% B; 18–23 min,
100% B; 23–23.1 min, a linear gradient back to 2% B. The whole
analysis took 25 min. The injection volume was 20 ␮L, the flow rate
was 0.2 mL/min, and the PDA-detection was performed from 200 to
400 nm. The sample chamber in the autosampler was maintained
at 4 ◦C, while the column was set at 40 ◦C.
The MS system consisted of a hybrid IT/TOF mass spectrometer
(Shimadzu Corp., Kyoto, Japan) equipped with an electrospray ion-
ization source. The total effluent from the detector was transferred
directly to the hybrid IT/TOF mass spectrometer without splitting.
The mass spectrometer was operated in the positive mode. Mass
spectroscopy analyses were carried out on full-scan MS with a
mass range of 100–400 Da and data-dependent MS/MS acquisi-
tion on the suspected precursor ions. Liquid nitrogen was used as
the nebulizing gas at a flow rate of 1.5 L min−1. The capillary and
skimmer voltages were set at 4.5 kV and 1.6 kV, respectively. The
CDL and heat block temperatures were both maintained at 200 ◦C.
The MS2 spectra were produced using CID of the selected precur-
sor ions using argon as collision gas with relative energy of 50%.
The ion accumulation time was set at 50 ms, the precursor ion
isolation width at 1 Th. External mass calibration was carried out
prior to data acquisition using direct infusion of a reference stan-
dard from 50 to 1000 Da. The reference standard was consisted of
0.25 mL L−1 trifluoroacetic acid and 0.1 g L−1 sodium hydrate. The
flow rate of the infusion pump was 5 ␮L min−1. All calculated mass
error was less than 5 ppm after mass calibration with the reference
standard.
2. Experimental
2.1. Chemicals
Data acquisition and processing were carried out using the
LC/MS solution version 3.41 software supplied with the instrument.
Any mass numbers corresponding to particular elemental compo-
sitions were also calculated by the formula predictor, and would
generate more than one formula proposed by the software. There-
fore, an accuracy error threshold of 20 ppm was set as a limit
to the calculation of possible elemental compositions. The other
following conditions for calculating elemental compositions were
taken into consideration: the upper limits on the number of C, H,
O, N, F atoms, C/H ratios, nitrogen rule and range of double-bond-
equivalent (DBE).
Olaquindox (99.8%) was obtained from China Institute of Veteri-
nary Drug Control (Beijing, China). The stock solution of olaquindox
was prepared by dissolving the compound in water at the concen-
tration of 200 mg L−1. HPLC-grade acetonitrile was purchased from
Fisher Chemicals Co. (NJ, USA). Water was freshly prepared with the
Millipore water purification system (MA, USA). All other chemicals
and reagents were of the highest analytical grade available.
2.2. Stress studies
The stress studies were carried out under the conditions of
hydrolysis, oxidation and photolysis. For all of the solution stability
studies, the samples were prepared by mixing a dilute solution of
olaquindox (200 mg L−1) with various media (1:1; v/v). Acidic and
alkaline hydrolysis were carried out in 0.1 N HCl and 0.1 N NaOH,
respectively, whereas neutral hydrolysis was performed in water.
All the hydrolytic studies were conducted at 80 ◦C for 12 h. The
oxidative study was carried out in 30% H2O2 at room temperature
for 12 h. The photostability testing was carried out by exposing the
solution of drug in water to UV fluorescent light (208 W h/m2) at
room temperature for 12 h. All the stressed samples were with-
drawn at suitable time intervals and diluted 10 times with water
before injection into the LC/MS-IT-TOF.
3. Results and discussion
3.1. Degradation behaviors of olaquindox
The forced degradation samples of olaquindox (neutral, acidic,
basic, oxidative and photolytic) were analyzed by LC/MS-IT-TOF to
identify the degradation products. The representative HPLC chro-
matograms of olaquindox at each of the above stress conditions
are shown in Fig. 1. Compared with the control (Fig. 1A), sev-
eral peaks appeared in the chromatograms of the stressed samples
(Fig. 1B–F). It indicated that olaquindox underwent degradation,
and the highest concentration of degradation products was found
in the acid-stressed sample (Fig. 1C). After 12 h exposure to the
hydrolytic medias (neutral, acidic and basic) at 80 ◦C, nine, ten and
four degradation products were formed under the neutral, acidic,
basic conditions, respectively. However, four and seven degrada-
tion products were formed under the oxidative and photolytic
conditions, respectively.
We assumed that they were degradation products of olaquindox
because of comparison of the incubation sample with the standard
solution, as well as the agreement between the accurate mass mea-
surement in MS spectra and predicted formula calculation within
10 ppm. The predicted elemental compositions, measured accurate
masses and exact masses, the mass errors, and the major fragment
ions of the degradation products are indicated in Table 1. The exact
2.3. LC/MS-IT-TOF analysis
For the characterization of olaquindox and its degradation
products, hybrid IT/TOF mass spectrometry coupled with a high-
performance liquid chromatography system was used (Shimadzu
Corp., Kyoto, Japan). The liquid chromatography system (Shi-
madzu) was equipped with a solvent delivery pump (LC-20AD), an
autosampler (SIL-20AC), a DGU-20A3 degasser, a photodiode array
detector (SPD-M20A), a communication base module (CBM-20A)
and a column oven (CTO-20AC). The separation was performed
on an ODS-C18 column (150 mm × 2.0 mm I.D.; particle size 5 ␮m)
using a gradient elution consisting of mobile phase A (0.1% formic

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Z.-Y. Liu et al. / International Journal of Mass Spectrometry 303 (2011) 90–96
Fig. 1. LC–UV chromatograms of olaquindox standard solution (A), and the stressed olaquindox by H2O for 12 h (B); 0.1 N HCl for 12 h (C); 0.1 N NaOH for 24 h (D); 30% H2O2
for 12 h (E); as well as UV fluorescent light at room temperature for 12 h (F).
and measured masses agree to within less than 10 ppm, providing
support for the proposed elemental compositions of the degrada-
tion products. The accurate extracted mass chromatograms of the
degradation products of olaquindox under various conditions are
shown in Fig. 2.
mine the empirical formula for the unknown degradation products.
The initial step in elucidating structures of degradation prod-
ucts of olaquindox is to understand the fragmentation pattern
of the drug substance. The detailed mass spectrometry analysis
of the fragmentation pattern of olaquindox provides a basis for
assessing structural assignment for the degradation products. The
detailed fragmentation pattern of olaquindox was reported in pre-
vious publication [12,15]. In this study, the comparison of the
fragmentation pattern of the degradation products with that of
olaquindox assigned the location of the medication. Mass accuracy
measurements allowed the assignment of the molecular formula
of fragment ions.
3.2. Structural characterization of degradation products
Identification of degradation products was performed by hybrid
ion trap/time-of-flight mass spectrometer that allows collision
induced dissociation and accurate mass measurements on both
fragment ions and precursors. This enables the user to deter-

Z.-Y. Liu et al. / International Journal of Mass Spectrometry 303 (2011) 90–96
93
Table 1
The retention times (RT), measured accurate masses, predicated elemental compositions, theoretical masses, double bond equivalents (DBE), mass errors and major fragment
ions of degradation products of olaquindox.
Degradation
products
RT (min)
Measured
mass (Da)
Elemental
DBE
Theoretical
mass (Da)
Error (ppm)
Major fragment ion
compositions ([M+H])+
+
Olaquindox
5.5
264.0971
C
12H14N3O4
8
264.0979
−3.03
264.0840, 229.0793, 221.0534,
212.0791, 160.0612
230.0898, 161.0679, 143.0599
230.0898, 213.0896, 205.0607
246.0829, 177.0629, 160.0610
246.0816, 221.0561, 177.0634,
160.0583
+
+
+
+
1
2
3
4
11.6
13.6
7.5
248.1025
248.1026
264.0969
264.0961
C12H14N3O3
8
8
8
8
248.1030
248.1030
264.0979
264.0979
−2.02
−1.61
−3.79
−6.82
C
C
C
12H14N3O3
12H14N3O4
12H14N3O4
10.1
+
+
5
6
7
8
12.6
14.4
13.1
18.3
17.5
14.6
16.4
14.1
266.1118
282.1065
177.0649
177.0651
222.0858
248.1025
161.0693
193.0591
C12H16N3O4
C12H16N3O5
C9H9N2O2
8
7
7
7
7
8
7
8
266.1135
282.1084
177.0659
177.0659
222.0873
248.1030
161.0709
193.0608
−6.39
−6.74
−5.65
−4.25
−6.75
−2.02
−9.93
−8.81
206.0700
177.0646, 135.0533
160.0608, 132.0652
135.0515
135.0523
187.0486, 1161.0676, 159.0529
133.0740
+
+
C9H9N2O2
+
+
9
C
C
10H12N3O3
12H14N3O3
10
11
12
C9H93N2O+
+
C9H9N2O3
176.0559, 159.0515, 148.0544
3.2.1. Compounds 1 and 2
3.2.4. Compound 6
Compounds 1 and 2 were eluted at retention times of 11.6
and 13.6 min, respectively. Both compounds showed a protonated
molecule at m/z 248 and a loss of 16 Da (m/z 264–248) compared
with olaquindox, suggesting that they were reduced degradation
products of olaquindox. The difference between compounds 1 and
2 lay in the position of the N → O group reduction. Based on the
accurate MS/MS spectra and in comparison to a previous report
[12], compounds 1 and 2 were identified as 4-desoxyolaquindox
and 1-desoxyolaquindox, respectively.
Compound 6 was eluted at a retention time of 14.4 min and had
a measured elemental composition of C12H16N3O5 ([M+H]+ ion at
m/z 282). Compound 6 had 18 Da higher than that of olaquindox,
suggested that they were the oxidized products via the addition
of a water molecule. The elemental compositions of the fragment
ions at m/z 177.0646 and 135.0533 were C9H9N2O2 (predicted
177.0659 Da) and C7H7N2O (predicted 135.0553 Da), respectively,
according to the formula predictor software, indicating that C2H2O
was lost from m/z 177 to form m/z 135. This indicated the oxygen
atom of compound 6 was located on the methyl group of quinoxa-
line ring.
3.2.2. Compounds 3 and 4
Compounds 3 and 4 were eluted at retention times of 7.5 min
and 10.1 min, respectively. Both compounds showed a protonated
molecule at m/z 264, 16 Da higher than that of m/z 248, suggest-
ing that they were oxidation product of compounds 1 or 2. The
compound eluting at 5.5 min possessed the same retention time,
protonated molecule ion (m/z 264) and MS2 spectrum as authentic
olaquindox. Therefore, it was identified as unchanged olaquindox.
The MS2 spectrum of compound 3 showed fragment ions at
m/z 246 and 177 which are all 16 Da higher than fragment ions
at m/z 230 and 161 of compound 1, respectively, indicating that
an oxygen atom had occurred on the quinoxaline ring. However,
The MS2 spectrum of compound 4 showed fragment ions at m/z
246 and 221 were all 16 Da higher than fragment ions at m/z
230 and 205 of compound 2, respectively, indicating that it was
an oxidation product of compound 2. The presences of ions at
m/z 177 and 160 of compounds 3 and 4 showed that the oxygen
was located on the quinoxlaline ring. Considering the chemical
structure of olaquindox and the oxidation environment, we pro-
posed the oxygen atom had occurred on the methyl group of
quinoxaline ring. Therefore, compounds 3 and 4 were identified
as 3-hydroxymethyl-4-desoxyolaquindox and 3-hydroxymethyl-
1-desoxyolaquindox, respectively.
3.2.5. Compounds 7 and 8
Compounds 7 and 8 were eluted at the retention times of
13.1 and 18.3 min, respectively. Both compounds showed simi-
lar protonated molecule at m/z 177.0649 and m/z 177.0651 and
the same predicated elemental composition of C9H9N2O2. Com-
pound 7 fragmented to yield abundant product ion at m/z 160
corresponding to the loss of OH radical from m/z 177, suggesting
an N → O group of compound 7 was existed on the quinoxaline
ring. The loss of CO from the ion m/z 160 leaded to fragment
ion at m/z 132, which indicated the presence of phenol within
this molecule. In addition, the N → O group reduction at position
1 is relatively easy with respect to the electronic effect, because
reduction of the N → O group at position 4 is hindered by the
adjacent methyl group. Therefore, compound 7 was identified as
3-methyl-2-hydroxylquinoxaline-4-oxide. However, the MS2 spec-
trum of compound 8 showed fragment ion at m/z 135 was in
accordance with that from compound 6, indicating that oxygen
atom had occurred on the methyl group of quinoxaline ring. There-
fore, compound 8 was identified as 3-hydroxymethylquinoxaline-
4-oxide.
3.2.6. Compound 9
3.2.3. Compound 5
Compound 9 was eluted at a retention time of 17.5 min and had
a measured elemental composition of C10H12N3O3 ([M+H]+ ion at
m/z 222). The elemental composition of the fragment ion at m/z
135.0526 was C7H7N2O (predicted 135.0553 Da), according to the
formula predictor software, indicating that C3H5NO2 was lost from
m/z 222 to form m/z 135. The MS2 spectrum of compound 9 showed
fragment ion at m/z 135 was in accordance with that from com-
pound 6, indicating that oxygen atom had occurred on the methyl
group of quinoxaline ring. Therefore, compound 9 was identified as
3-hydroxymethyl-2-quinoxalinecarboxamide-4-oxide.
Compound 5 was eluted at a retention time of 12.6 min and had
a measured elemental composition of C12H16N3O4 ([M+H]+ ion at
m/z 266). Compound 5 had 2 Da higher than that of olaquindox, sug-
gested that they were the hydrogenation product. The elemental
composition of the fragment ion at m/z 206.0700 was C10H10N2O3
(predicted 206.0686 Da), according to the formula predictor soft-
ware, indicating that C2H6NO was lost from m/z 266 to form m/z
206. This indicated the two hydrogen atoms of compound 5 were
located on the N → O group to form N-hydroxyl.

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Z.-Y. Liu et al. / International Journal of Mass Spectrometry 303 (2011) 90–96
Fig. 2. The accurate extracted mass chromatogram (EIC) of degradation products of olaquindox under the stressed by H2O for 12 h (A); 0.1 N HCl for 12 h (B); 0.1 N NaOH for
24 h (C); 30% H2O2 for 12 h (D); as well as UV fluorescent light at room temperature for 12 h (E).
3.2.7. Compound 10
age to form the fragment ions at m/z 187 and 161. The elemental
compositions of the fragment ions at m/z 187.0486 and 159.0529
were C10H7N2O2 (predicted 187.0502 Da) and C9H7N2O (predicted
159.0513 Da), respectively, according to the formula predictor soft-
ware, indicating that CO was lost from m/z 187 to form m/z 159.
Therefore, the oxygen atom of compound 10 was located on the
methyl group of quinoxaline ring.
Compound 10 was eluted at a retention time of 14.6 min and
had a measured elemental composition of C12H14N3O2 ([M+H]+ ion
at m/z 248). Compound 10 had 16 Da lower than that of olaquin-
dox, suggested that it was the reduced products of parent drug.
No fragment ion at m/z 230 was observed in the MS2 spectrum
of compound 9, indicating the side chain was vulnerable to cleav-

Z.-Y. Liu et al. / International Journal of Mass Spectrometry 303 (2011) 90–96
95
Fig. 3. The proposed degradation pathway of olaquindox.
3.2.8. Compound 11
dation leading to the generation of hydroxylation compounds. The
oxidation of the N-oxides in soil has already been described for
quinoxaline N-oxides by Zhang and Huang [17]. Olaquindox also
readily underwent cleavage of side chain under hydrolytic and pho-
tolytic conditions to form compounds 7 and 12. The cleavage of
side chain of quinoxaline might accord with the mechanism pro-
posed for other drugs such as ciprofloxacin [18]. Based on these
results, the schematic representation of the degradation pathways
is tentatively proposed as shown in Fig. 3.
Compound 11 was eluted at a retention time of 16.4 min and had
a measured elemental composition of C9H9N2O ([M+H]+ ion at m/z
161). It fragments to yield product ion at m/z 133 corresponding to
the loss of CO from m/z 161, suggesting that the presence of phenol
within this molecule. Therefore, compound 11 was identified as
3-methyl-2-hydroxylquinoxaline.
3.2.9. Compound 12
Compound 12 was eluted at a retention time of 14.1 min and had
a measured elemental composition of C9H8N2O3 ([M+H]+ ion at m/z
193). The fragment ion at m/z 176 indicated the loss of an OH radical
from m/z 193, and a further loss of OH to form the fragment ion at
m/z 159, indicating the two N → O group were existed on the com-
pound 12. The MS2 spectrum of compound 12 showed fragment
ions at m/z 176 and 148 were all 16 Da higher than fragment ions at
m/z 160 and 132 of compound 7, respectively. Therefore, compound
12 was identified as 3-methyl-2-hydroxylquinoxaline-1,4-dioxide.
4. Conclusion
In the present study, we demonstrated the application of liq-
uid chromatography combined with hybrid ion trap/time-of-flight
mass spectrometry for the detection and structural characteriza-
tion of degradation products of olaquindox. The chromatographic
method we described here can resolve almost degradation products
from the parent as well as from each other under various differ-
ent conditions. Olaquindox underwent most degradation under
the acidic conditions to 10 products. The degradation was mild
under photolytic conditions, forming a total of seven products, but
in small quantities. All degradation products were characterized
with the help of the accurate mass measurements of fragment
ions and precursors. A comprehensive degradation pathway of
olaquindox was tentatively outlined. It was also found that N-oxide
radicalintermediates andhydroxyl radicalswere formed during the
degradation process. Hence, the study of degradation products of
olaquindox may give insight onto the mechanisms leading to the
phototoxicity and the photoallergic reaction of the drug.
3.3. Degradation pathways of olaquindox
Under various conditions, N → O group of olaquindox was
reduced to form the compounds 1 and 2 via the N-oxide radical
intermediate according to a mechanism proposed by Inbaraj et al.
[16]. The hydroxyl radical was also produced during the formation
of compounds 1 and 2. Thus, the phototoxicity and the photoal-
lergic reaction of the drug could be explained by the formation
of a radical intermediate and hydroxyl radical that could readily
react with DNA. The N-oxide radicals could undergo further oxi-

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Z.-Y. Liu et al. / International Journal of Mass Spectrometry 303 (2011) 90–96
Acknowledgment
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[12] Z. Liu, L. Huang, M. Dai, D. Chen, Y. Wang, Y. Tao, Z. Yuan, Metabolism
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by high-performance liquid chromatography combined with ion trap/time-
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1009–1016.
[13] Z. Liu, L. Huang, M. Dai, D. Chen, Y. Tao, Y. Wang, Z. Yuan, Metabolism of
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This work is supported financially by Major Projects of Science
and Technology of Hunan Province (2009FJ1005).
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