Application of the UHPLC method for separation and characterization of major photolytic degradation products of trazodone by LC-MS and NMR

Article abstract of DOI:10.1039/c8nj03545h

Drug degradation products are a type of drug impurity which affects the safety of the drug products. Trazodone (TZD) is an antidepressant drug subjected to forced degradation as per ICH embedded guidelines for predicting the drug degradation products. It undergoes degradation in acidic hydrolysis, peroxide induced oxidation and upon exposure to day light and results in the formation of ten degradation products (DPs). A UHPLC method was developed to separate TZD and its DPs using the stationary phase of an Acquity UPLC CSH C₁₈ column (100 × 2.1 mm, 1.7 μm) and a mobile phase of solvent-A (10 mM ammonium acetate, pH 8.5) and solvent-B (methanol) at a flow rate of 0.25 mL min^-1 in gradient elution. This method was transferred to a quadrupole time-of-flight tandem mass spectrometer (QTOF-MS/MS) for identification of the DPs. Very interestingly, four dimer DPs were found under photolytic degradation conditions and they are found to be isomers. The major isomer (DP-10) was isolated by using preparative HPLC and its structure was identified using proton and carbon NMR. Three N-oxide DPs were also observed and the site of N-oxidation was identified by using atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The developed UHPLC method was validated as described in the ICH prescribed guidelines and USP general chapter on method validation. The proposed validated stability indicating assay method can be used for identification and quantification of TZD and its DPs in a quality control lab for product release testing and stability studies of the drug in much less time with greater selectivity.

Full text of DOI:10.1039/c8nj03545h

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Application of UHPLC method for separation and characterization of major photolytic
degradation products of trazodone by LCꢀMS and NMR
a
a
a
a
Mohit Thummar , Prinesh N. Patel , Bhoopendra Singh Kushwah , S. Gananadhamu *
a
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and
Research (NIPER), Balanagar, Hyderabad, 500037, Telangana, India.
*Author for correspondence
Dr. S. Gananadhamu,
Department of Pharmaceutical Analysis
National Institute of Pharmaceutical Education and Research [NIPER], Hyderabadꢀ
500037, Telangana, India.
Eꢀmail: gana@niperhyd.ac.in

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Abstract
Drug degradation products are type of drug impurities which affects the safety of the drug
products. Trazodone (TZD) is an antidepressant drug subjected to forced degradation as per
ICH embedded guidelines for predicting the drug degradation products. It undergoes
degradation in acidic hydrolysis, peroxide induced oxidation and upon exposure to day light
and results in formation of ten degradation products (DPs). A UHPLC method was developed
to separate TZD and its DPs using the stationary phase of Acquity UPLC CSH C column
1
8
(100 x 2.1 mm, 1.7 µm) and mobile phase of solventꢀA (10 mM ammonium acetate, pH 8.5)
ꢀ1
and solventꢀB (Methanol) at a flow rate of 0.25 mL min in gradient elution. This method
was transferred to quadrupole timeꢀofꢀflight tandem mass spectrometer (QTOFꢀMS/MS) for
identification of DPs. Very interestingly, four dimer DPs were found in photolytic
degradation condition and they are found to be isomers. The major isomer (DPꢀ10) was
isolated by using preparative HPLC and its structure was identified using proton and carbon
NMR. Three Nꢀoxide DPs were also observed and the site of Nꢀoxidation was identified by
using atmospheric pressure chemical ionization mass spectrometry (APCIꢀMS). The
developed UHPLC method was validated as described in ICH prescribed guideline and USP
general chapter on method validation. The proposed validated stability indicating assay
method can be used for identification and quantification of TZD and its DPs in the quality
control lab for product release testing and stability studies of the drug in much lesser time
with greater selectivity.
Keywords: Trazodone (TZD); forced degradation; UHPLC; LCꢀESIꢀQTOFꢀMS/MS; NMR;
Dimer degradation products.

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Introduction
Trazodone (TZD) is a triazolopyridinone derivative antidepressant drug belongs to the class
1
of the serotonin antagonist (5HTꢀ2) and reuptake inhibitors (SRIs). TZD is chemically
described as 2ꢀ(3ꢀ(4ꢀ(3ꢀchlorophenyl)piperazinꢀ1ꢀyl)propyl)ꢀ[1,2,4]triazolo[4,3ꢀa]pyridinꢀ
3(2H)ꢀone hydrochloride. It is a multifunctional drug having dose dependent pharmacological
activities, at a lower dose it worked as a hypnotic due to blockage of 5HT2A, H ꢀhistamines
1
and α adrenergic receptors while at a higher dose it shows an antidepressant action because of
2
serotonin antagonist and reuptake inhibitor (SARI) activity. Different drug formulations of
TZD are commercially available for the treatment of major depression, generalized anxiety
3
ꢀ4
disorder, uniꢀpolar depression and insomnia.
Forced degradation study is very important in the initial stage of pharmaceutical drug
development process because it deals with the identification and characterization of possible
degradation products (DPs) which can originate from the drug substance and/or drug
5
ꢀ7
product. Hence, it is necessary to findꢀout the intrinsic stability of the drug as well as drug
degradation pathways. The forced degradation studies are also useful for self life
determination, designing of manufacturing process, formulation and packaging development.
Thus, various global regulatory bodies like international conference on harmonization (ICH)
guidelines emphasized to conduct the forced degradation and characterization of all the
degradation products (DPs) in an early stage of drug discovery and development process
because the presence of DPs may impose the risk of higher and unpredictable toxicity along
8
ꢀ9
with formulation related defects. Modern analytical techniques like UHPLCꢀESIꢀQTOFꢀ
MS/MS and NMR are very promising in impurity profiling areas to identify and characterize
1
0ꢀ14
all the types of impurities.
The literature shows stability indicating HPLC method
estimation of trazodone and/or with its metabolite in biological samples.
15ꢀ16
and bioꢀanalytical methods for
17ꢀ21
But no analytical

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method was found for identification and characterization of forced degradation products of
TZD. Hence, the authors have developed a UHPLC method for separation of all the
degradation products of the drug followed by characterization of their chemical structures
using UHPLCꢀESIꢀQTOFꢀMS/MS and NMR.
Experimental
Chemicals and Reagents
The drug was obtained as a gift sample from INTAS Pvt. Ltd. (Gujarat, India). Gradient
grade acetonitrile (ACN) and methanol (MeOH) were purchased from Merck Specialities Pvt.
Ltd. (Mumbai, India). HPLC Buffer gradeꢀAmmonium acetate and formic acid; Analytical
reagent gradeꢀsodium hydroxide (NaOH), hydrochloric acid (HCl) and hydrogen peroxide
(30% v/v) were purchased from fine chemicals Pvt. Ltd. (Ahmadabad, India). High purity
water was obtained by Millipore MilliꢀQ Plus water purification system (Millipore, Milford,
MA, USA).
Instrumentation
The Acquity UPLC HꢀClass system from Waters (Waters, Milford, MA, USA) composed of
a quaternary solvent manager, sample manager of flowꢀthrough needle design (SMꢀFTN),
temperature controlled multiple column compartments and PDA detector was used for
chromatographic method development. The separation system was controlled by Empowerꢀ3
software. LCꢀMS/MS characterization was carried out using an Agilent 1290 series LC
instrument (Agilent Technologies, USA) attached to a quadrupole – time of flight mass
analyzer (QꢀTOF, Agilent 6540, Agilent Technology, USA) and coupled with electrospray
ionization (ESI) and/or an atmospheric pressure chemical ionization (APCI) source. The mass
spectrometric data analysis was carried out using Mass Hunter workstation software. NMR
studies were performed using 500 MHz NMR (AVANCE III HDꢀ500, Bruker, Switzerland)
spectrometer. TMS (Trimethylsilane) was used as internal standard. A mixture of CDCl3

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with d6ꢀ DMSO and CDCl
3
were used as solvents for proton and carbon NMR, respectively.
A photo stability chamber (model no. OPSH S/Gꢀ16, Osworld science Pvt Ltd India)
21
consisting of UV and fluorescent lamps as described in ICHꢀQ1B guideline was used for
the photoꢀdegradation study. Hydrolytic and thermal degradation studies were performed
using high precision water bath (temperature control ± 1 ºC) and hot air oven (Oswarld
scientific Pvt Ltd. India), respectively. Samples were dissolved using an ultraꢀsonicator from
Power Sonicꢀ405 (Hwashin Technology Co., Seoul, Korea). A pH meter from pH tutor
(Eutech Instruments, Singapore) was used to measure pH of solution.
Optimized Chromatographic and Mass spectrometric Conditions
Optimum chromatographic separation of drug and DPs was achieved by using Acquity UPLC
CSH C18 (100 x 2.1 mm, 1.7 µm) column with gradient mobile phase of solventꢀA (10 mM
ꢀ1
ammonium acetate buffer, pH 8.5) and B (Methanol) at a flow rate of 0.25 mL min . The
gradient program was set as follows: (Time in min / % of solventꢀA): 0ꢀ0.5/60, 0.50ꢀ4.0/30,
4
.0ꢀ11.0/15, 11.0ꢀ11.50/60, 11.50ꢀ15.0/60. Obtained chromatograms were monitored at
wavelength of 252 nm. The column oven and auto sampler temperatures were maintained at
5 °C and 10 °C respectively. The sample injection volume of 1 µL was used in study. For
2
LCꢀESIꢀQTOFꢀMS/MS analysis, the source condition for ESI was optimized as follows: the
fragmentor voltage at 140 V, the capillary voltage at 3500 V and the skimmer voltage at 65
V. Nitrogen gas was used as the drying (325°C; 10 L/min) and nebulizing (40 psi) gas. In
collision induced dissociation (CID) experiment, keeping MS1 static, the quadrupole mass
analyzer was used for the precursor ion scanning and the TOF mass analyzer was used for the
product ions scanning. All the mass spectra were recorded under identical experimental
conditions and an average of 25 scans. In APCI ionization, source current and vaporizer
temperature were set at 3 ꢁA and 400 °C, respectively. All the remaining source parameters
were maintained same as in ESI.

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Forced degradation study
Acid hydrolysis (3N HCl for 24 h), basic hydroxylation (3N NaOH for 24 h) and neutral
hydrolysis (water for 48 h) were carried out by refluxing stressed solutions under heat at 70
°C. Oxidative degradation was carried out using 30% (w/v) hydrogen peroxide solution at
room temperature for 24 h. In photolytic degradation, solid and solution state drug samples
were kept in photoꢀstability chamber and irradiated with UVꢀlight and visible light of not less
2
22
than 200 Wh/m and 1.2 million lux hr, respectively as per ICH guidelines. A parallel set of
the drug samples were stored in dark at same temperature to serve as a control. Thermal
degradation of solid sample was performed by making 1 mm thickness layer of drug sample
and kept it at 90 °C for 7 days.
Sample preparation
ꢀ1
All stress degradation experiments were carried out on 1 mg mL of TZD solutions. After
obtaining sufficient degradation, 1 mL of stressed solution was taken and allowed to cool at
room temperature followed by neutralization and/or dilution with diluent (ACN: waterꢀ40: 60
v/v) to get assay concentration of 100 ppm and sonicated for 5 min. The final injection
solution was filtered through a 0.22 ꢁm whatmann filter paper before injected into the
chromatographic UHPLC system. All solutions were stored in an amber color flask at
approximately 4 °C temperature.
Method validation
The chromatographic method was validated as per ICH guideline Q2 (R1) and USP general
chapter on method validation for specificity, limit of detection (LOD), limit of quantification
2
3ꢀ24
(LOQ), linearity, accuracy, precision and robustness.
Preparative chromatographic conditions for isolation of DPꢀ10
Waters (Waters, M.A, USA) preparative HPLC system equipped with 515 binary HPLC
pumps and 2489 UV–visible detector was used for isolation of major degradation product

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(DPꢀ10). An optimized chromatographic condition for isolation includes stationary phase of
Waters Xbridge C18 column (250 × 19 mm, 5ꢁm, 130 Å) and isocratic mobile phase of 10
mM ammonium acetate buffer (pH 6.5) and Methanol (60:40 % v/v) at a flow rate of 12 mL
ꢀ1
min . The 80 % of DPꢀ10 peak was collected after each injection in preparative system. The
purity of isolated fraction was confirmed by reꢀinjecting into UHPLC system and it was
found to be more than 99 % pure. The isolated fraction was concentrated and dried using
rotary evaporator at 45 °C temperature under vacuum. The corresponding preparative
chromatogram is given in supplementary information (Fig. Sa).
Results and Discussion
Method Development and Optimization
The chromatographic conditions were optimized to attain well separation of drug and its DPs.
Systematic trials on chromatographic separation conditions were based on improving peak
shape and resolution between peaks of TZD and its DPs. Being a non polar drug, TZD
showed better selectivity and retention on C18 column. In initial trials on different column
chemistries suggested that Acquity UPLC CSH C18 (100 x 2.1 mm, 1.7 µm) column was
appropriate for method development. Isocratic and gradient trails using MeOH/ACN as an
organic phase and water as aqueous phase on above column shows insufficient separation of
DPꢀ7, DPꢀ8, DPꢀ9 and DPꢀ10. Hence, mass compatible buffers like ammonium formate and
ammonium acetate at pH ± 1.5 of drug pKa were tried. The gradient trials with MeOH/ACN
and buffers at acidic pH (1ꢀ5) shown coꢀelution (DPꢀ7, DPꢀ8 and DPꢀ9). While the trials with
buffers at basic pH (8ꢀ11) shows improved separation as compared to acidic buffers. Finally
wellꢀresolved degradation product peaks with a sharp and symmetrical drug peak was
obtained on stationary phase of Acquity UPLC CSH C18 (100 x 2.1 mm, 1.7 µm) column with
a mobile phase containing gradient mixture of solventꢀA (10 mM ammonium acetate buffer
pH 8.5) and B (Methanol) as: (Time in min / % of solventꢀA): 0ꢀ0.5/60, 0.50ꢀ4.0/30, 4.0ꢀ

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ꢀ1
1
1.0/15, 11.0ꢀ11.50/60, 11.50ꢀ15.0/60. The flow rate was set at 0.25 mL min . All the
chromatograms were processed and analyzed at wavelength of 252 nm.
Degradation behavior of TZD
TZD was degraded in the presence of acid hydrolytic, oxidative and photolytic stress
degradation conditions while it was found to be stable in remaining stress degradation
conditions. A total of ten DPs were observed and overlaid chromatogram was given in Fig. 1.
Minor degradation (2.8 %) of the drug was observed in acid hydrolysis to give rise of two
DPs (DPꢀ4 and DPꢀ6). Three DPs (DPꢀ1, DPꢀ2 and DPꢀ3) were obtained in peroxide stress
condition with percentage of degradation of 5.73 %. A solid sample of the drug was remained
stable in both UV and visible light while solution state sample was extensively degraded in
UVꢀlight (42.5 %) and five DPs were obtained (DPꢀ5 and DPꢀ7 to DPꢀ10) while one
degradation product (DPꢀ10, 1.5%) was observed under visible light.

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Fig. 1. UHPLCꢀPDA Chromatograms of [a] TZD (100 ppm), [b] Acid hydrolysis (3N HCl,
for 24 h), [c] Peroxide induced oxidation (30% w/v H for 24 h), [d] Solution sample
2
O
2
exposed to UV light and [e] Solution sample under visible light.
LC–MS/MS and NMR of TZD and DPs:
LCꢀESIꢀQTOFꢀMS/MS and NMR experiments were carried out to characterize the drug and
its degradation products. Most possible structures were proposed based on m/z values of
+
[
M+H] of precursor and product ions, fragmentation pattern of precursor ion and elemental
composition derived from high resolution mass spectrometric (HRꢀMS) data. Molecular
formulas corresponding to the observed m/z and to the calculated m/z with mass error in ppm
are given in Table 1.
Table 1. high resolution mass data corresponding to elemental composition of TZD and its
DPs
Molecular
Formula [M+H]
Observed
Calculated
Error
(ppm)
+
Description
m/z
m/z
+
TZD
C
19
H
23ClN
5
O
372.1583
372.1586
237.1153
219.124
0.81
ꢀ4.22
0.46
+
C H ClN
237.1163
219.1239
209.0844
194.0612
176.0820
154.0429
148.0507
135.0432
120.0554
93.0451
1
3
18
2
+
C
11
H
15
N
4
O
+
C H ClN
209.084
ꢀ1.91
ꢀ3.61
ꢀ1.14
ꢀ7.14
ꢀ1.35
ꢀ3.70
1.67
ꢀ4.30
ꢀ5.13
1.48
2.84
4.06
ꢀ3.58
3.86
2.74
1
1
14
2
C
10
H
11ClN
2
194.0605
176.0818
154.0418
148.0505
135.0427
120.0556
93.0447
+
C H N O
9
10
3
+
C
8
9
H ClN
+
C H N O
7
6
3
3
3
2
C
6
H
5
N
O
+
C H N
6
6
+
C
5
H
5
N
+
C H N
78.0342
78.0338
5
4
+
DPꢀ1
C
19
H23ClN
5
O3
404.1478
387.1446
369.1336
251.0955
233.0831
219.1234
210.0549
192.0459
176.0818
154.0424
148.0509
404.1484
387.1457
369.1351
251.0946
233.084
C H ClN O2
1
9
22
5
5
C
19
H
20ClN
O
+
C H ClN O
1
3
16
2
+
13
C H14ClN2
+
C H N O
219.124
1
1
15
4
C
10
H
11ClN
2
O
210.0554
192.0449
176.0818
154.0418
148.0505
2.38
ꢀ5.21
0.00
ꢀ3.90
ꢀ2.70
C H ClN
1
0
9
2
+
C
9
H
10
3
N O
+
C H ClN
8
9
+
C
7
H
6
N
3
O

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DOI: 10.1039/C8NJ03545H
+
C
C
C
6
5
5
H
H
H
6
6
4
N3O
N2
N
136.0511
94.0527
78.0335
136.0505
94.0525
78.0338
388.1535
370.1429
356.1273
235.0997
219.124
ꢀ4.41
ꢀ2.13
3.84
+
+
DPꢀ2
C H ClN O
388.1518
370.1432
356.1289
235.0988
219.1248
209.0848
205.1084
194.0615
176.0823
4.38
1
9
23
5
2
+
C
C
C
C
C
C
C
C
19
18
13
11
11
10
10
H
H
H
H
H
H
H
21ClN
19ClN
16ClN
O
14ClN2
5
O
ꢀ0.81
ꢀ4.49
3.83
ꢀ3.65
ꢀ3.83
0.00
+
5
O
+
2
+
15
N
4
+
209.084
+
13
N
4
O
205.1084
194.0605
176.0818
11ClN
2
ꢀ5.15
ꢀ2.84
+
9
8
7
H
10
N
3
O
+
C
C
H
H
8
6
N
3
O
162.0660
162.0662
1.23
+
N
3
O
148.0510
136.0507
120.0553
148.0505
136.0505
120.0556
ꢀ3.38
ꢀ1.47
2.50
+
C H N O
C H N
6
6
3
+
6
6
3
+
C H N
93.0441
93.0447
6.45
5
5
2
+
C H N
78.0344
388.1541
370.1410
78.0338
388.1535
370.1429
ꢀ7.69
ꢀ1.55
5.13
5
4
+
DPꢀ3
C H ClN O
C H ClN O
1
9
23
5
2
+
1
9
21
5
+
C
C
C
C
C
C
C
C
C
C
C
C
C
18
13
11
11
10
10
H
H
H
H
H
H
19ClN
16ClN
O
14ClN2
5
O
356.1266
235.0985
219.1246
209.0849
205.1093
194.0610
176.0820
162.0658
148.0511
136.0510
120.0559
93.0449
356.1273
235.0997
219.124
1.97
5.10
+
2
+
15
N
4
ꢀ2.74
ꢀ4.30
ꢀ4.39
ꢀ2.58
ꢀ1.14
2.47
+
209.084
+
13
N
4
O
205.1084
194.0605
176.0818
162.0662
148.0505
136.0505
120.0556
93.0447
78.0338
346.1429
219.124
176.0815
154.0418
148.0505
135.0427
120.0556
118.0651
93.0447
11ClN
2
+
9
8
7
6
6
5
5
H
10
N
3
O
+
+
+
H
H
H
H
H
H
8
6
6
6
5
4
N
3
3
3
3
2
O
O
N
N
N
N
N
ꢀ4.05
ꢀ3.68
ꢀ2.50
ꢀ2.15
ꢀ5.13
ꢀ5.20
ꢀ5.48
ꢀ3.41
ꢀ6.49
ꢀ4.73
ꢀ3.70
ꢀ3.33
ꢀ6.78
ꢀ2.15
ꢀ6.41
0.56
O
+
+
+
78.0342
+
DPꢀ4
C
17
H
21ClN
5
+
O
346.1447
219.1252
176.0821
154.0428
148.0512
135.0432
120.0560
118.0659
93.0449
C
C
C
C
C
C
C
C
C
11
H
15
N
4
O
+
9
8
7
6
6
8
5
5
H
10
N
3
O
+
H
H
H
H
H
H
H
9
6
5
6
8
5
4
ClN
+
N
N
N
N
N
N
3
3
3
O
O
+
+
+
2
+
78.0343
354.1923
219.1484
78.0338
354.1925
219.1492
+
DPꢀ5
C
C
19
H
24
N
5
O
O
2
+
13
H
19
N
2
3.65

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+
C
C
C
C
C
C
C
C
C
11
11
H
H
15
15
N
N
4
O
219.1235
219.1240
191.1179
176.0818
162.0913
148.0505
135.0427
120.0556
93.0447
78.0338
406.1196
271.0763
243.045
228.0216
219.124
188.0028
176.0818
148.0505
135.0427
120.0556
93.0447
78.0338
743.3099
372.1586
237.1153
219.124
209.084
176.0818
148.0505
135.0427
93.0447
78.0338
743.3099
372.1586
237.1153
219.124
209.084
176.0818
148.0505
135.0427
93.0447
78.0338
743.3099
372.1586
237.1153
219.124
209.084
2.28
4.19
0.00
4.32
4.05
4.44
5.00
6.45
2.56
ꢀ2.22
2.58
ꢀ3.29
2.63
ꢀ1.83
0.53
2.27
ꢀ6.08
4.44
ꢀ1.67
2.15
ꢀ6.41
2.42
5.37
ꢀ5.06
ꢀ3.65
2.87
ꢀ3.98
ꢀ4.73
5.18
3.22
ꢀ3.84
ꢀ1.48
2.42
3.80
5.48
ꢀ5.74
3.98
4.73
5.92
6.45
6.41
1.61
0.81
ꢀ1.27
5.93
3.83
+
2
O
191.1171
176.0818
162.0906
148.0499
135.0421
120.0550
93.0441
+
+
9
H
10
N
3
O
10
H
12NO
+
7
6
6
5
5
H
6
N
N
N
N
N
3
3
3
2
O
H
5
6
5
4
O
+
H
H
H
+
+
78.0336
+
DPꢀ6
C
19
H
22Cl
2
N
5
O
406.1205
271.0756
243.0458
228.0210
219.1244
188.0027
176.0814
148.0514
135.0421
120.0558
93.0445
+
C
C
C
C
C
C
C
C
C
C
C
13
11
10
11
H
H
H
H
8
17Cl
13Cl
10Cl
2
N
N
N
2
2
2
+
2
2
+
15
4
N O
+
8
9
7
6
6
5
5
H
2
Cl N
+
H
10
N
3
O
+
H
H
H
H
H
6
5
6
5
4
N
3
3
3
2
O
N
N
N
N
O
+
+
+
78.0343
+
+
+
DPꢀ7
DPꢀ8
DPꢀ9
C
38
H
45Cl
2
N
10
+
O
2
743.3081
372.1566
237.1165
219.1248
209.0834
176.0825
148.0512
135.0420
93.0444
C H ClN O
19
23
5
+
C
13
H
18ClN
2
+
C H N O
11
15
4
+
11 2
C H14ClN
+
C H N O
C
C H N O
C
C H N
C
C
C H ClN
C
C H ClN
C
C H N O
C
C H N
C
9
10
3
+
7
H
6
N
3
O
6
5
3
+
5
H
5
N
2
+
78.0341
5
4
38
H
45Cl
23ClN
2
N
10
+
O
2
743.3110
372.1577
237.1144
219.1228
209.0852
176.0811
148.0498
135.0419
93.0441
19
H
5
O
+
13
18
2
+
11
H
15
N
4
O
+
11
14
2
+
9
H
10
N
3
O
+
7
6
3
3
2
6
H
5
N
O
+
5
5
+
5
H
4
N
78.0333
C
38
H
45Cl
2
N
10
+
O
2
743.3087
372.1583
237.1156
219.1227
209.0832
C H ClN O
19
23
5
+
C
13
H
18ClN
2
+
C H N O
C H14ClN
11 2
11
15
4
+

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+
C
9
H
10
N
3
O
176.0812
176.0818
148.0505
93.0447
78.0338
743.3099
372.1586
237.1153
219.124
176.0818
148.0505
93.0447
78.0338
3.41
5.40
8.60
3.84
0.27
2.15
ꢀ0.42
9.13
ꢀ0.57
4.05
8.60
ꢀ3.84
+
C H N O
148.0497
93.0439
78.0335
743.3097
372.1578
237.1154
219.1220
176.0819
148.0499
93.0439
78.0341
7
5
5
6
5
4
3
+
C
C
C
H
H
N
N
2
+
+
DPꢀ10
38
19
H
H
45Cl
2 10 2
N O
+
C
23ClN
5
O
+
C H ClN
1
3
18
2
+
C
11
H
15
N
4
O
+
C H N O
9
10
3
+
C
7
H
6
N
3
O
+
C H N
C
5
5
2
+
5
H
4
N
Further major degradation product (DPꢀ10) was isolated and structure was confirmed using
NMR experiments. The proton and carbon NMR spectra were given in supplementary
information (Fig. Sb to Fig. Se). The proposed structures of protonated DPs are shown in Fig.
2.
Fig. 2. Chemical structures of protonated TZD and its DPs.
MS/MS of TZD (Rt. 7.2 min)
To elucidate the fragmentation behavior of TZD, the ESIꢀMS/MS spectrum (Fig. 3a) of its
+
[
M+H] (m/z 372.1583) was examined. The spectrum shows product ions at m/z 237, m/z
219, m/z 209, m/z 194, 176, m/z 154, m/z 148, m/z 135, m/z 120, m/z 93 and m/z 78 (Scheme

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1
4
). Product ions at m/z 237 and m/z 135 can confirm the basic skeleton of 1ꢀ(3ꢀchlorophenyl)ꢀ
ꢀpropylpiperazine and [1,2,4]triazolo[4,3ꢀa]pyridinꢀ3(2H)ꢀone of the drug. The elemental
compositions of all the proposed fragment ions have been confirmed by accurate mass
measurements and given in Table 1.

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+
Fig. 3. ESIꢀQꢀTOFꢀMS/MS Spectra of [M+H] of (a) TZD, (b) DPꢀ1, (c) DPꢀ2 and (d) DPꢀ3.

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+
Scheme 1. Proposed fragmentation pathway of [M+H] of TZD and DPꢀ6 to DPꢀ10.
MS/MS of degradation products
DPꢀ1 (Rt. 1.6 min)
+
DPꢀ1 was obtained in oxidative stress degradation. The ESIꢀMS/MS spectrum of [M+H] of
DPꢀ1 shows precursor ion peak at m/z 404.1502 Da (Fig. 3b) which has 32 Da more
molecular mass than the drug suggests the presence of two oxygen atoms in DPꢀ1 structure.
Further it was fragmented to give product ions at m/z 387, m/z 369, m/z 251, m/z 233, m/z
2
19, m/z 210, m/z 192, m/z 176, m/z 154, m/z 148, m/z 136, m/z 94 and m/z 78 (Scheme 2).
Common fragment ions at m/z 219, m/z 179, m/z 148, m/z 135 and m/z 78 compared to drug
and consecutive loss of OH (m/z 404 to m/z 387) and H O (m/z 387 to m/z 369) indicates DPꢀ
is Nꢀoxide degradation product and formed by Nꢀoxidation on the both nitrogen of the
piperazine ring of TZD. Based on the above data DPꢀ1 is identified as 1ꢀ(3ꢀchlorophenyl)ꢀ4ꢀ
3ꢀ(3ꢀoxoꢀ[1,2,4]triazolo[4,3ꢀa]pyridinꢀ2(3H)ꢀyl)propyl)piperazine 1,4ꢀdioxide, with
chemical formula of C19 and mass error of 1.48 ppm.
2
1
(
H
5 3
23ClN O

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+
Scheme 2. Proposed fragmentation pathway of [M+H] DPꢀ1.
DPꢀ2 (Rt. 3.1 min) and DPꢀ3 (Rt. 4.5 min)
The DPꢀ2 and DPꢀ3 were formed under oxidative stress degradation show 16 Da more
molecular mass when compared to drug suggests the presence of oxygen atom in their
+
structures. The ESIꢀMS/MS spectrum of [M+H] of DPꢀ2 (m/z 388.1518, (Fig. 3c)) and DPꢀ3
(m/z 388.1451, (Fig. 3d)) shows same fragmentation pattern of precursor ions and similar
accurate masses of precursor and product ions. Further these precursor ions were fragmented
and gave product ions at m/z 370 (loss of water), m/z 356 (Meisenheimer rearrangement, loss
of CH O), m/z 235, m/z 219, m/z 209, m/z 205, m/z 194, m/z 176, m/z 162, m/z 148, m/z 136,
4
m/z 120, m/z 93 and m/z 78 (Scheme 3). Based on the obtained product ions and
Meisenheimer rearrangement clearly indicates both DPs were Nꢀoxide degradation products
which formed by Nꢀoxidation on the each nitrogen of the piperazine ring. The sample was
analyzed in full scan APCIꢀMS mode to determine the site of Nꢀoxidation. The APCIꢀMS
spectrum of DPꢀ2 and DPꢀ3 shows common product ions at m/z 372 because of thermal
deoxygenation in the APCI source (Fig. 4). In addition, APCIꢀMS spectrum of DPꢀ3 (Fig. 4b)
shows product ions at m/z 212 and m/z 195 because of Meisenheimer rearrangement which
proves DPꢀ3 formed by Nꢀoxidation on the nitrogen attached to the propyl chain while
absence of these characteristic product ions in APCIꢀMS spectrum of DPꢀ2 (Fig. 4a) proves
that it was formed by Nꢀoxidation on nitrogen attached to the chlorophenyl ring of drug.
Based on the mass fragmentation data DPꢀ2 and DPꢀ3 are identified as 1ꢀ(3ꢀchlorophenyl)ꢀ4

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+
Scheme 3. Proposed fragmentation pathway of [MꢀH] of DPꢀ2 and DPꢀ3.
+
Fig. 4. APCIꢀMS spectra of [M+H] of (a) DPꢀ2 and DPꢀ3.

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ꢀ(3ꢀ(3ꢀoxoꢀ[1,2,4]triazolo[4,3ꢀa]pyridinꢀ2(3H)ꢀyl)propyl)piperazine 1ꢀoxide and 4ꢀ(3ꢀ
chlorophenyl)ꢀ1ꢀ(3ꢀ(3ꢀoxoꢀ[1,2,4]triazolo[4,3ꢀa]pyridinꢀ2(3H)ꢀyl)propyl)piperazine 1ꢀoxide,
respectively. The DPꢀ2 and DPꢀ3 shows same elemental compositions of C19
mass error of 4.38 and ꢀ1.55 ppm, respectively.
DPꢀ4 (Rt. 5.7 min)
5 2
H23ClN O and
The ESIꢀMS/MS spectrum of protonated DPꢀ4 shows precursor ion peak at m/z 346.1447 Da
Fig. 5a). The mass difference of 26.0136 Da compare to drug indicates DPꢀ4 formed by loss
(
of ethylene group from the drug. Further it was fragmented to give product ions at m/z 219,
m/z 176, m/z 154, m/z 148, m/z 135, m/z 120, m/z 118, m/z 93 and m/z 78 (Scheme 4). Based
on common product ions at m/z 219, m/z 176, m/z 154, m/z 148, m/z 135, m/z 120, m/z 93 and
m/z 78, while absence of product ions at m/z 237 and m/z 209 compared to drug proves DPꢀ4
formed by loss of ethylene group from piperazine ring of drug molecule. Based on HRꢀMS
data DPꢀ4 was identified as 2ꢀ(3ꢀ((2ꢀ((3ꢀchlorophenyl)amino)ethyl)amino)propyl)ꢀ
[
1,2,4]triazolo[4,3ꢀa]pyridinꢀ3(2H)ꢀone, contain chemical formula of C H ClN O with
17 20 5
calculated mass error of ꢀ5.20 ppm.
DPꢀ5 (Rt. 6.9 min)
The ESIꢀMS/MS spectrum of protonated photolytic degradation product shows precursor ion
peak at 354.1923 (Fig. 5b). Further DPꢀ5 fragmented to obtain product ions at m/z 219 (m/z
219.1484 and m/z 219.1235), m/z 191, m/z 176, m/z 162, m/z 148, m/z 135, m/z 120, m/z 93
and m/z 78 (Scheme 4). The precursor ion peak at m/z 354 clearly shows absence of M+2
isotopic peak of chlorine atom (zoomed portion of Fig. 5b) and 18 Da less molecular mass
than the drug suggest the presence of ꢀOH in DPꢀ5 structure. MS/MS spectrum of DPꢀ5
shows common fragment ions as that of TZD at m/z 219.1235, m/z 176, m/z 148, m/z 135, m/z
120, m/z 93 and m/z 78. The fragment ion at m/z 219.1235 indicates the intact portion of
[
1,2,4] triazolo[4,3ꢀa]pyridine and ruled out the presence of –OH at these portion. The

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product ions at m/z 219.1484 and m/z 191 are characteristic ions that proved DPꢀ5 formed by
replacement of chlorine on phenyl ring with hydroxyl group in the presence of UVꢀlight.
Based on the observed data DPꢀ5 was identified as 2ꢀ(3ꢀ(4ꢀ(3ꢀhydroxyphenyl)piperazinꢀ1ꢀ
yl)propyl)ꢀ[1,2,4]triazolo[4,3ꢀa]pyridinꢀ3(2H)ꢀone, having elemental composition of
C
19
H
23
N
5
O
2
with calculated mass error of 0.56 ppm.
+
Fig. 5. ESIꢀQꢀTOFꢀMS/MS Spectra of [M+H] of (a) DPꢀ4, (b) DPꢀ5 and (c) DPꢀ6.

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+
Scheme 4. Proposed fragmentation pathway of [MꢀH] of DPꢀ4 and DPꢀ5.
DPꢀ6 (Rt. 8.3 min)
The ESIꢀMS/MS of protonated DPꢀ6 shows precursor ion at m/z 406.1205 Da (Fig. 5c).
Further it was fragmented to obtain product ions at m/z 271, m/z 243, m/z 228, m/z 219, m/z
188, m/z 176, m/z 148, m/z 135, m/z 120, m/z 93 and m/z 78 (Scheme 1). DPꢀ6 shows
common fragment ions at m/z 219, m/z 176, m/z 148, m/z 135, m/z 120, m/z 93 and m/z 78 to
the drug molecule. The increase in mass of 34 Da when compared to drug indicates the
presence of another chlorine atom in chemical structure of DPꢀ6. While product ions at m/z
271, m/z 243, m/z 228 and m/z 188 shows 34 units increased in mass when compared to drug
product ions at m/z 237, m/z 209, m/z 194 and m/z 154, respectively proves chlorination of
chlorophenyl ring of drug. Further double chlorine pattern was observed in mass spectrum of
drug precursor ion peak and product ions peak at m/z 271 and m/z 243 proves chlorination of
drug (zoomed portion of Fig. 5c). Based on these characteristic features the DPꢀ6 was
identified as 2ꢀ(3ꢀ(4ꢀ(3,5ꢀdichlorophenyl)piperazinꢀ1ꢀyl)propyl)ꢀ[1,2,4]triazolo[4,3ꢀa]pyridinꢀ
2 5
3(2H)ꢀone, having elemental composition of C19H21Cl N O with calculated mass error of ꢀ
2.22 ppm.

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DPꢀ7 (Rt. 10.2 min), 8 (Rt. 11.0 min), 9 (Rt. 11.4 min) and 10 (Rt. 11.8 min)
+
The ESIꢀMS/MS spectrums of [M+H] of DPꢀ7, DPꢀ8, DPꢀ9 and DPꢀ10 shows precursor ions
peak at m/z 743.3081, m/z 743.3110, m/z 743.3087 and m/z 743.3097, respectively (Fig. 6a,
6b, 6c and 6d). Further these DPs were fragmented and the same product ions were obtained
at m/z 372, m/z 237, m/z 219, m/z 209, 176, m/z 148, m/z 135, m/z 93, and m/z 78 (Scheme 1).
Similar fragmentation pattern with similar accurate masses for precursor and product ions
proves that these are the isomeric degradation products having double molecular mass of
2 10 2
TZD (dimers). All these DPs show same elemental composition of a C38H45Cl N O and
calculated mass error at 2.42, ꢀ1.48, 1.61 and 0.27 ppm for DPꢀ7, DPꢀ8, DPꢀ9 and DPꢀ10,
respectively. DPꢀ7, DPꢀ8 and DPꢀ9 were observed in small quantity and hence these dimers
could not be isolated for structure elucidation. The major dimer (DPꢀ10) was isolated by
1
preparative HPLC and the DPꢀ10 structure was proposed by proton and carbon NMR. H and
1
3
C NMR spectrums of DPꢀ10 shows presence of two piperazine ring (15, 16, 18, 19, 15’,
16’, 18’,and 19’), two propyl chain (11, 12, 13, 11’, 12’ and 13’) and two chlorophenyl ring
(21, 22, 23, 24, 25, 21’, 22’, 23’, 24’ and 25’) compare to TZD (Table 2). Also it shows that
the two drug molecules were joined at the pyridine rings to form cyclobutyl ring bridge
(2,2’,3,3’ in Table 2) which proves DPꢀ10 structure. Further, the cyclobutyl ring bridge
1
shows δ values of H NMR at 2.51 ppm (2H, atoms of 2 and 2’) and 2.44 ppm (2H, atoms of
3
and 3’) while δ values of 13C NMR shows peaks at 33.24 ppm and 36.18 ppm (for atoms 2
and 2’); 41.62 ppm and 46.57 ppm (for atoms of 3 and 3’) proves two drug molecules were
joined from the position of 2 and 3 of the pyridine ring of TZD but not from positions of 4
and 5 because presence of nitrogen at position 6 can do deshielding and increase the δ values.

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Fig. 6. ESIꢀQꢀTOFꢀMS/MS Spectra of [M+H]+ of (a) DPꢀ7, (b) DPꢀ8, (c) DPꢀ9 and DPꢀ10.

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Table 2. NMR chemical shift assignments for TZD and DPꢀ10
TZD
DP-10
2
3' 24'
26'Cl 2
2'
1'
25'
0' 17'
2
2
1
8'
N
16'
19'
1
0'
N14'
15'
O
1
2'
5
3
'
6
' N
'
13'
7
'
9
4'
3'
N
1
1'
8
'N 1'
8
N
2
'
2
1
7
11
N
9
N
13
14
N
4
6
12
5
15
O
1
0
16
19
N17
18
2
1
20
22
Cl 26
2
5
23
24
1
13
1
13
Atom
H ppm
C ppm
Atom position
H ppm
C ppm
position
1
2
3
-
141.92 (s)
123.69 (s)
110.96 (s)
1, 1’
2, 2’
3 , 3’
-
139.68 (s) and 137.05 (s)
33.25 (s) and 36.18 (s)
41.62 (s) and 46.57 (s)
7.09 (1H, d)
7.20 (1H, t)
2.51 (2H, dd)
2.44 (2H, t)
4
5
6
9
7.13 (1H, dd)
7.75 (1H, d)
-
130.46 (d)
115.37 (s)
-
4, 4’
5.20 (2H, m)
106.32 (s)
114.05 (d)
-
5, 5’
6.11 (2H, m)
6, 6’
-
-
-
148.67 (s)
55.09 (s)
23.34 (s)
43.24 (s)
9, 9’
149.89 (s),
55.13 (d)
1
1
1
1
2
3
4.17 (2H, t)
2.10 (2H, s)
3.73 (2H, m)
11, 11’
12, 12’
13, 13’
15, 16, 18, 19,
15’, 16’, 18’ and 2.74 (t, 8H)
19’
3.6-4.05 (4H, m)
1.89 (4H, m)
25.41 (d)
3.6-4.05 (4H, m)
3.20 (dd, 8H) and
43.44 (d)
1
1
5, 16, 18, 3.64 (4H, t),
46.39 (s), 50.64
(s) and 51.68 (s)
48.11 (s) and 52.63 (d)
9
3.20 (2H, m) and
3
-
.02 (2H, d)
2
2
2
2
2
2
0
1
2
3
4
5
150.23 (s)
117.32 (s)
135.20 (s)
121.64 (s)
115.11 (s)
130.46 (d)
20, 20’
21, 21’
22, 22’
23, 23’
24, 24’
25, 25’
-
151.94 (s) and 151.41 (s)
115.56 (s) and 120.32 (s)
134.97 (s) and 130.33 (s)
119.64 (d)
6.93 (1H, d)
-
6.85 (2H,m)
-
6.81 (1H,d)
6.53 (1H, t)
6.93 (1H, d)
6.78 (2H, m)
6.78 (2H, m)
7.14 (2H, m)
115.97 (d)
130.11 (d)
Degradation pathway of the drug
The chemical structure of drug contains triazoloꢀpyridine ring, propyl chain, piperazine ring
and chlorophenyl ring. Literature shows that the piperazine ring is more prone to be oxidized
10
to obtain Nꢀoxide degradation products . In oxidative stress degradation condition, by attack
of H on tertiary nitrogens of piperazine ring DPꢀ1 to DPꢀ3 were formed as Nꢀoxides. DPꢀ1
2
O
2

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was formed by Nꢀoxidation on the both nitrogen of the piperazine ring of TZD. The DPꢀ2
was formed by Nꢀoxidation on the nitrogen attached to the chlorophenyl ring of drug and DPꢀ
3
was formed by Nꢀoxidation on the nitrogen attached to the propyl chain of drug. The DPꢀ4
1
0
was formed in acidic hydrolysis by loss of ethylene group from the piperazine ring of TZD .
DPꢀ5 was formed by loss of chlorine atom from chlorophenyl ring followed by hydroxylation
in the aqueous environment under UV light. DPꢀ6 was formed by electrophilic substitution of
25
chlorine on chlorophenyl ring of TZD under acidic stress condition . The DPꢀ7, DPꢀ8, DPꢀ9
and DPꢀ10 were isomeric dimer degradation products of drug. They were formed by joining
of two drug molecules at the pyridine rings to form cyclobutyl ring bridge. Method
validation
Stability indicating UHPLC assay method was validated for specificity, limit of detection,
limit of quantification, linearity, accuracy, precision and robustness according to ICHꢀ
2
3ꢀ24
guideline Q2 (R1) and USP general chapter for method validation.
Specificity
The specificity of the developed method was demonstrated by checking peak purity of
chromatographic peaks using photodiode array detector (PDA) and additionally proved by
the mass detector. All chromatographic peaks (TZD and DPs) were well resolved from each
other and purity angle values were found to be within purity threshold values which
unambiguously prove the specificity of the developed method.
Limit of Detection (LOD) and Limit of Quantification (LOQ)
ꢀ1
Different concentration of drug standard solutions at 0.05, 0.1, 1.0, 1.5 and 2 µg mL were
injected to find the LOD and LOQ concentration values of the drug. Based on observed
ꢀ1
signalꢀtoꢀnoise (S/N) ratios, LOD and LOQ concentrations were found to be 0.035 µg mL
ꢀ1
and 0.11 µg mL , respectively. Replicated injections (n=5) were analyzed to confirm
obtained LOD and LOD concentrations of the drug.

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Linearity
The linearity of UHPLC method was determined on five different concentrations (80ꢀ120 ꢁg
ꢀ1
mL ) of TZD working standard solutions in triplicate (n=3). Plotted standard calibration
curve shows splendid relationship between concentrations of drug on xꢀaxis and respective
mean peak areas on yꢀaxis. The obtained result shows acceptable values of determination
2
coefficient (r ) of 0.999 and linear regression equation of 13551 x ꢀ 18300. Hence, the
developed method shows linear response over performed concentration range.
Accuracy
Accuracy of the method was determined by spiking three known concentrations of standard
drug solutions into stressed solution in triplicate (n=3). The recovery of spiked drug was
measured from difference peak areas of fortified and unfortified stressed samples. The
recovery and % RSD of TZD was was found to be 99.83 to 100.15 % (Table 3) and below 1
percentage, respectively proves the accuracy of proposed UHPLC method.
Precision
Intraꢀ and interꢀday precisions were determined using three different concentrations of 80,
ꢀ1
1
00 and 120 µg mL , on the same day (n= 3) and consecutive days (n= 3). The observed
values of % RSD for intra and interꢀday precisions were found to be below 1% which proves
developed UHPLC method was sufficiently precise (Table 3).
Table 3. Accuracy and Precision data for TZD
Concentration
TZD added (ꢁg
mL )
Concentration TZD
obtained
Intraꢀday precision
(n=3)
Interꢀday precision
(n=3)
ꢀ1
%
Recover
y
Mean TZD concentration
ꢀ1
ꢀ1
(
µg mL ) ± SD;
RSD
(µg mL ) ± SD; %RSD
%
8
1
1
0
79.86 ± 0.37; 0.47
100.04 ± 0.31; 0.31
120.18 ± 0.15; 0.12
99.83
79.75 ± 0.42; 0.53
79.86 ± 0.45; 0.56
100.10 ± 0.35; 0.35
120.4 ± 0.26; 0.21
00
20
100.04
100.15
100.03 ± 0.32; 0.32
120.26 ± 0.20; 0.17

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Robustness
Robustness study was carried out on the drug degradation samples by making deliberate
ꢀ1
changes to method variables like flow rate (0.05 mL min ), column oven temperature (
±5
°C), mobile phase pH (±0.3 unit) and organic percentage (±2 %). Obtained peak areas were
used to calculate % RSD and it was found to be below 1 %. Other assay parameters like
resolution between critical pair of DPꢀ8, DPꢀ9 and DPꢀ10, tailing factor and USP plate count
of drug peak were found to be within the limits which proved the robustness of the developed
method.
Conclusion
The degradation behavior of Trazodone under various stress degradation conditions was
studied. The drug was found to be stable under basic hydrolysis, neutral hydrolysis and dry
heat while it was degraded under acid hydrolysis, oxidative stress and photolytic
degradations. A total of ten DPs were obtained and were well separated using developed
UHPLC method. The DPs were characterized using UHPLCꢀESIꢀQTOFꢀMS/MS
experiments. The site of Nꢀoxidation in Nꢀoxide degradation products (DPꢀ2 and DPꢀ3) was
confirmed by using APCIꢀMS. The major degradation product (DPꢀ10, dimer of drug) was
isolated by preparative HPLC and further structure was confirmed by using proton and
carbon NMR. Formation of interesting DPs such, Nꢀoxides in oxidative stress degradation
(DPꢀ1 to DPꢀ3), loss of an ethylene group from the piperazine ring in acidic hydrolysis (DPꢀ
4), substitution of chlorine of the chloroꢀphenyl ring by hydroxyl group in the presence of
UVꢀlight (DPꢀ5), chlorination of the chlorophenyl ring in acidic hydrolysis (DPꢀ6) and
formation of dimers of TZD in the presence of UVꢀlight (DPꢀ7 to DPꢀ10) were observed. The
proposed method was validated as per ICH guidelines. The given method can be used in
routine analysis and stability study of drug in quality control laboratories.

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Conflicts of interest
Authors have declared no conflicts of interest.
Acknowledgements
The authors are very pleased to the National Institute of Pharmaceutical Education &
Research [NIPER], Hyderabad, and the Ministry of Chemicals and Fertilizers, New Delhi,
India, for providing a facility and research fellowship.
References
1. G. J. Marek, C. J. McDougle, L. H. Price, L. S. Seiden, Psychopharmacology, 1992,
109, 2ꢀ11.
2
3
. J. C. Ballenger, Prim Care Companion J Clin Psychiatry, 2000, 2, 71ꢀ79.
. A. Fagiolini, A. Comandini, M.C. Dell’Osso, S. Kasper, CNS Drugs, 2012, 26, 1033ꢀ
1049.
4
. FDA Approved Drug Products, available from: http://www.accessdata.fda.
gov/scripts/cder/drugsatfda/index.cfm.
5
. S. Singh, M. Junwal, G. Modhe, H. Tiwari, M. Kurmi, N. Parashar and P. Sidduri,
Trend in Ana. Chem., 2013, 49, 71ꢀ88.
6
. N. R. Ramisetti and R. Kuntamukkala, New J.Chem., 2014, 38, 3050ꢀ3061.
. M. Thummar, P. N. Patel, A. L. Petkar, D. Swain, R. Srinivas, S. Gananadhamu,
Rapid Commu. Mass Spectr., 2017, 31, 771ꢀ781.
7
8. ICH guideline, Q1A (R2) Stability Testing of New Drug Substances and Products,
International Conference on Harmonisation, IFPMA, Geneva, Switzerland, 2003.
9. WHO, Draft Stability Testing of Active Pharmaceutical Ingredients and
Pharmaceutical Products, World Health Organization, Geneva, 2007.
10. B.B. Chavan, G.L. Prasanna, P. Radhakrishnanand, E.R. Kosuri, P.D. Kalariya,
M.V.N. K. Talluri, New J.Chem., 2018, 42, 10761ꢀ10773.

New Journal of Chemistry
Page 28 of 29
View Article Online
DOI: 10.1039/C8NJ03545H
1
1
1
1
1
1
1
1
1
2
2
2
1. R. Rapolu, A. K. Pandey, Ch. K. Raju, K. Ghosh, K. Srinivas, A. Awasthi, G. N.
Sameer and K. V. Surendranath, J. Pharm. Biomed. Anal., 2015, 107, 488–494.
2. S. K. Pravinchandra, M. Kurmi, S. Kumara and S. Singh, New J. Chem., 2015, 39,
9679ꢀ9692.
3. M. Thummar, P. N. Patel, G. Samanthula and R. Srinivas, Rapid Commu. Mass
Spectr., 2017, 31, 1813ꢀ1824.
4. K.R. Chintalapati, J.V. Shanmukha Kumar, S.K.G. Palusa, New J. Chem., 2018, 42,
634ꢀ646.
5. A.E. El Gindy, M. Farouk, O. Abd El Aziz, E.A. Abdullah, J. of Applied Sciences
Research, 2009, 5, 2028ꢀ2034.
6. S. Ghosh, S. Venkatesh, B.V.V. Ravikumar, International J. PharmTech Research,
2015, 7, 204ꢀ211.
7. S. CardaꢀBroch, M.T. GilꢀAgusti, L. MonferrerꢀPons, J.S. EsteveꢀRomero, J.
Chromatogr. A, 2007, 1156, 254ꢀ258.
8. X. Kang, C. Pan, Q. Xu, Y. Yao, Y. Wang, D. Qi, Z. Gu, Anal. Chim. Acta, 2007,
587, 75ꢀ81.
9. O. Andriollo, C. LartigueꢀMattei, J.L. Chabard, H. Bargnoux, J. Petit, J.A. Berger, J.
Chromatogr., 1992, 575, 301ꢀ305.
0. B.N. Patel, N. Sharma, M. Sanyal, P.S. Shrivastav, J. Chromatogr. B, 2008, 871, 44–
5
4.
1. L. Mercolini, C. Colliva, M. Amore, S. Fanali, M.A. Raggi, J. Pharm. Biomed. Anal.,
008, 47, 882–887.
2
2. ICH Guideline, Photostability Testing of New Drug Substances and Products, Q1B,
IFPMA, Geneva, Switzerland, (1996).

Page 29 of 29
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View Article Online
DOI: 10.1039/C8NJ03545H
2
2
2
3. ICH guideline, Q2 (R1): Validation of Analytical Procedures: Text and Methodology,
International Conference on Harmonization IFPMA, Geneva, Switzerland, (2005).
4. US pharmacopeia, Validation of compendia methods, section <1223>, United States
Pharmacopeal Convention, Rockville, MD, (2013) 979.
5. V. Dhiman, D K. Singh, M. K. Ladumor, S. Singh, J. Seperation Science, 2017, 40,
4530ꢀ4540.
Graphical Abstract