Development of a stability-indicating UPLC method for terconazole and characterization of the acidic and oxidative degradation products by UPLC-Q-TOF/MS/MS and NMR

Article abstract of DOI:10.1039/c8nj00509e

Terconazole, a triazole antifungal drug, is used to treat infections in the form of a cream or suppositories. A simple, rapid, precise and accurate ultra high performance liquid chromatography method has been developed for the quantitative determination of the terconazole drug substance in the presence of process impurities and degradation products. The method showed adequate separation of terconazole, process impurities and degradation products on a CSH C₁₈ (100 × 2.1 mm, 1.7 μm) column. The mobile phase used was ammonium acetate (10 mM; pH 7.5) and acetonitrile was used as an organic modifier. Terconazole was subjected to forced degradation studies and found to be degraded in acid hydrolytic and oxidative conditions, whereas it was stable under basic, neutral, photolytic and thermal conditions. LC/electrospray ionization and/or LC/atmospheric pressure chemical ionization coupled with quadrupole time-of-flight mass spectrometry have been utilized for identification and structural characterization of the degradation products. The major oxidative degradation products were isolated by preparative HPLC and their structures were confirmed using ¹H NMR and ¹³C NMR. The probable mechanistic explanation is given for the formation of the degradation products. The method was validated in terms of specificity, linearity, accuracy, precision and robustness as per the International Conference on Harmonisation guidelines.

Full text of DOI:10.1039/c8nj00509e

View Article Online
View Journal
NJC
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: B. B. Chavan, L. P.
G., P. Radhakrishnanand, E. R. kosuri, P. D. Kalariya and M. V. N. K. Talluri, New J. Chem., 2018, DOI:
1
0.1039/C8NJ00509E.
Volume 40 Number
1
January 2016 Pages 1–846
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry
www.rsc.org/njc
A journal for new directions in chemistry
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

Page 1 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
Development of stability indicating UPLC method for terconazole
and characterization of acidic and oxidative degradation products
by UPLC-Q-TOF/MS/MS and NMR
a
Balasaheb B. Chavan , G. Lakshmi Prasanna , P.Radhakrishnanand , Eswara Raju Kosuri , Pradipbhai D.
a
b
b
a
Kalariya , M.V. N Kumar Talluri
a*
a
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education & Research,
IDPL R&D
Campus, Balanagar, Hyderabad- 500 037. India.
b
United States Pharmacopeia (USP) India Pvt. Ltd., Hyderabad-500078, Telangana, India
*a
Corresponding author: Dr. M.V.N. Kumar Talluri, Department of Pharmaceutical Analysis, National Institute of
Pharmaceutical Education & Research (NIPER), Hyderabad, India;
Tel: +91-40-23074750, Ext: 2012, Fax: +91-40-23073751.
E-mail: narendra.talluri@gmail.com, narendra@niperhyd.ac.in
1

New Journal of Chemistry
Page 2 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
Abstract
Terconazole, a triazole antifungal drug is used to treat infections in the form of cream or
suppositories. A simple, rapid, precise and accurate ultra high performance liquid
chromatography method has been developed for the quantitative determination of the terconazole
drug substance in the presence of process impurities and degradation products. The method
showed adequate separation of terconazole, process impurities and degradation products on the
CSH C (100 × 2.1 mm, 1.7 µm) column. The mobile phase used was ammonium acetate (10
1
8
mM; pH 7.5) and acetonitrile as organic modifier. Terconazole was subjected to forced
degradation studies and found to be degraded in acid hydrolytic and oxidative conditions
whereas; it was stable in basic, neutral, photolytic and thermal conditions. LC/electrospray
ionization or and LC/atmospheric pressure chemical ionization coupled with quadrupole time-of-
flight mass spectrometer has have been utilized for identification and structural characterization
of the degradation products. The major oxidative degradation products were isolated on
1
13
preparative HPLC and structures were confirmed using H NMR and C NMR. The probable
mechanistic explanation is given for the formation of degradation products. The method was
validated in terms of specificity, linearity, accuracy, precision and robustness as per International
Conference on Harmonisation guideline.
Keywords: terconazole; process impurities; forced degradation; degradation products;
characterization; UPLC-Q-TOF/MS-MS.
2

Page 3 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
Graphical abstract
3

New Journal of Chemistry
Page 4 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
Introduction
Terconazole (TER) 1-[4-[ [(2S,4S)-2-(2,4-Dichlorophenyl)-2-(1,2,4-triazol-1-ylmethyl)-1,3-
dioxolan-4-yl]methoxy]phenyl]-4-propan-2-yl-piperazine, is an antifungal drug with molecular
formula of C H Cl N O . TER is a triazole antifungal drug, primarily used for the fungal and
26
31
2
5
3
1
, 2
yeast infections in vagina in the form of vaginal cream or suppository. The drug may undergo
degradation which result in a loss of potency and initiate the possible adverse effect due to
formation of degradation product or impurities. Stability testing guidelines Q1A (R2) developed
by International Conference on Harmonization (ICH) involve identification of degradation
3
products, degradation pathways and intrinsic stability of drugs. Intrinsic stability of drugs is
determined by applying the stress under hydrolytic, oxidative, thermal and photolytic conditions
as described in ICH guideline, Q1A (R2). To ensure the quality and safety of the drug, the
identification and characterization of the degradation products (DPs) is of prime importance
which helps us to establish degradation pathways of the drug.
Based on the comprehensive literature search, it was observed that, a few reports are
4
available on TER like; micellar liquid chromatographic, HPTLC determination (from biological
5
matrix) method. Maria Castro et al. have investigated the enanatiomeric separation of
6
ketoconazole and terconazole. Nadia Bounoua et al. reviewed achiral and chiral separations of
7
antifungal drugs including TER by HPLC and CE. As per the best of our knowledge, no
information is exists in the literature about the stability indicating UPLC method for the
separation of TER from its PIs and DPs. Also there is no information available on the forced
degradation behavior of the drug. This dearth of information prompted us to develop a method
which can be useful for the separation of the drug from its PIs/DPs and characterization of DPs
using UPLC-ESI/MS/MS and NMR. Nowadays, liquid chromatography electrospray ionization
4

Page 5 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
tandem mass spectrometry (LC-ESI/MS/MS) and NMR has been widely used for the
8
-10
identification and characterization of forced degradation products (DPs) and impurities.
So the endeavour of the present study was to (1) carry out stress degradation of the TER
under various stress conditions, (2) develop selective RP-UPLC method for TER, PIs and DPs,
(3) isolate the major oxidative DPs using preparative HPLC (4) characterize the degradation
products using either UPLC-ESI/MS/MS or UPLC-APCI/MS and NMR (5) validate the
1
1
developed method according to the ICH prescribed guideline.
Experimental
Drug and reagents
TER and PIs (Imp-1, Imp-2, Imp-3 and Imp-4) were obtained as gratis sample from Nosch labs
Pvt. Ltd, Hyderabad, India. Analytical reagent (AR) grade ammonium acetate, ammonium
formate (HPLC grade), formic acid (FA) and acetic acid (AA) were purchased from S.D. Fine
Chemicals (SDFCL) (Mumbai, India). Analytical reagent (AR) grade sodium hydroxide (NaOH)
and laboratory reagent (LR) grade hydrogen peroxide (H O ) was obtained from SDFCL
2
2
Mumbai, India. Hydrochloric acid (HCl) was purchased from FCL Ahmadabad, India. LC-MS
CHROMASOLV grade methanol (MeOH) and acetonitrile (ACN) were obtained from Sigma-
Aldrich (Bangalore, India). Water used for the present study was purified by the Milli Q
purification system (Millipore, Bedford, MA, USA). Tetramethylsilane (TMS) and CDCl were
3
purchased from Sigma-Aldrich (Bangalore, India) and Euriso-top (France), respectively.
Instrumentation
The chromatographic method development was carried out by using Acquity UPLC-H class
system from Waters (Milford, MA, USA). The instrument was equipped with bio-quaternary
5

New Journal of Chemistry
Page 6 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
gradient pump with in-line degasser, and flow through needle design integral sample manager
SM-FTN). A column compartment with temperature control and a photodiode array (PDA)
(
detector was used. The system was controlled by Empower-3 software. Thermal stability study
was performed in a dry air oven (Mack pharmatech Pvt. Ltd., Mumbai, India). Photostability
chamber (Mack equipment, Thane, India) was used for photo degradation study with temperature
range of 20 °C to 50 °C. The photo stability chamber (MK2-40P) was equipped with eight UV
and visible light tubes. All pH measurements were done on pH meter (Metrohm Schweiz AG,
7
80 pH meter, Germany). Other equipments used includes, a Power Sonic405 ultrasonicator
from Hwashin Technology Co., (Seoul, Korea) and Sartorius balance (CD 225 D, 22308105
Germany) was used to dissolve the samples and to weigh the samples, respectively.
UPLC-MS analysis of TER was performed using an Agilent 1200 infinity series UPLC
instrument (Agilent Technologies, USA) attached to a quadrupole time-of flight (Q-TOF) mass
spectrometer (Q-TOF LC-MS G6540B series, Agilent Technologies, USA) equipped with either
an electrospray ionization (ESI) source or an atmospheric pressure chemical ionization (APCI)
source. The data acquisition was under the control of Mass-Hunter Workstation software. The
typical operating source conditions for MS scan of TER, PIs and DPs in positive ESI mode were
optimized as, fragmentor voltage 144 V, capillary Voltage 3500 V, capillary temperature 250 °C,
-1
skimmer Voltage 65 V, drying gas (N ) pressure 40 psi (325 °C, 10 L min ), Scan range 100-
2
1
000, scan rate 5 spectra/sec. In APCI, the vaporizer temperature was 400 °C and the source
current was set at 3µA. All other source parameters were same as used in ESI. Collision-induced
dissociation (CID) experiments were conducted by keeping MS1 static, the precursor ion of
interest was selected using the quadrupole analyzer, and the product ions were analyzed using
6

Page 7 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
the TOF analyzer. Ultrahigh pure nitrogen gas was used as collision gas. All the spectra were
recorded under identical experimental conditions.
1 13
NMR experiments ( H and C) were carried out using 500 MHz NMR (AVANCE III
HD-500, Bruker, Switzerland) spectrometer. TMS and CDCl were used as an internal standard
3
and solvent for NMR experiments, respectively. The chemical shift values were reported on the δ
scale in ppm.
Stress degradation studies
-1
Stress degradation studies were carried out with TER drug substance (0.2 mg mL ) to identify
the possible DPs and validate the developed stability indicating analytical method. Specificity of
the developed method was conducted in the presence of its DPs. TER was subjected to forced
degradation studies under hydrolytic conditions like acidic (1N HCl), basic (3N NaOH) and
neutral (pure water) for 30 h, 4 days and 5 days, respectively at 80 °C. Oxidative studies were
carried out at room temperature in 0.6% hydrogen peroxide for 1 h. Thermal degradation was
performed by keeping the sample evenly spread in a petri plate for 7 days in hot air oven at 100
ºC. Photolytic stress studies were carried out by exposing a solid layer of TER evenly spread in
2
6
glass petri plate to UV (200Wh/m ) and fluorescent light (1.2×10 lux h) in photo stability
chamber for 7 days. Suitable controls were kept in photo stability chamber under the dark with
the aluminium foil wrapped for the conditions mentioned above. The optimized stress
degradation studies for all the conditions are given in Table S1.
Sample preparation
TER and its impurities were poorly soluble in the water, hence it was dissolved in the diluent
(water: ACN, 50:50 v/v). The stock solution of TER and each impurity were prepared in the
7

New Journal of Chemistry
Page 8 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
-1
-1
diluent at the concentration of 1.0 mg mL and 0.2 mg mL , respectively. The sample solution
-1
-1
of the TER containing 500 µg mL and 200 µg mL were prepared from the stock solution for
-1
the related substances and assay determination, respectively. TER (500 µg mL ) spiked with
0
.1% of PIs i.e; Imp 1, methyl 2-(2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
yl)benzoate, Imp 2, (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
yl)methyl methanesulfonate, Imp 3, 4-(4-isopropylpiperazin-1-yl)phenol, Imp 4, 1-(4-
methoxyphenyl)piperazine (Fig. S1) was used throughout study. The stock solutions of TER and
PIs were diluted adequately to perform precision, accuracy, linearity, limit of detection (LOD),
limit of quantification (LOQ) and robustness. The stress samples were withdrawn at suitable time
intervals. Neutralization of the stressed samples of acid and base was done with suitable
strengths of NaOH and HCl, respectively. All the stressed samples were diluted with the diluent
-1
to the final concentration of 200 µg mL and filtered through nylon-66 membrane syringe filters
(0.22 µm) before analysis.
The sample solution of TER was prepared by mixing terconazole cream equivalent to
-
1
1
mg mL with the diluent. The solution was filtered through 0.22 µ nylon filter and clear
solution was diluted five times with diluent to perform assay of TER.
Method validation
The developed UPLC method was validated for assay and related substances as per ICH
1
1
-1
guideline. The accuracy of assay for TER (200 µg mL ) was calculated in triplicate at
-1
concentrations 100, 200 and 300 µg mL (50-150%) on drug substance and recoveries were
calculated for each sample. The recoveries of the PIs were evaluated at three concentration levels
-1
i.e; 0.05%, 0.1% and 0.15% of the nominal concentration of TER (500 µg mL ). The precision
-1
of the assay was determined by injecting six samples at assay concentration (200 µg mL ) and
8

Page 9 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
RSD (%) values of the results was calculated. The repeatability of the related substance (RS)
method was determined by injecting six replicates of TER spiked with 0.1% each of the PI. Intra-
-1
day precision was evaluated by injecting 500 µg mL TER (n=3) spiked with 0.1% of PI. The
similar experiments were carried out on different days by different analyst to determine inter-day
precision. The peak area RSD (%) was calculated for PIs. Linearity for the assay of TER was
-
analyzed at seven different concentrations ranging from 50, 100, 150, 200, 250, 300, 350 µg mL
1
.
Linearity test samples for the related substances were prepared by adequately diluting the stock
solution of the PIs. The linearity for all four PIs were determined at seven concentration levels
ranging from 50% to 200% (i.e; 0.05, 0.075, 0.1, 0.125, 0.15, 0.175 and 0.2%) with respect to
-
1
5
00 µg mL of TER. The peak area versus concentration of TER and PIs were treated by linear
regression analysis. The correlation coefficients, Y-intercepts and slopes of calibration curve
were determined. The LOD and LOQ of TER and PIs were determined at signal-to-noise ratio of
3
:1 and 10:1, respectively, by analyzing dilute solutions of known concentration. The robustness
study was conducted to determine the effect of small but deliberate change in optimized LC
-1
method. The factors evaluated were mobile phase flow rate by ±0.03 mL min , column oven
temperature by ±5 °C, mobile phase pH by ±0.2 units. The resolution between TER and all four
impurities were determined.
Isolation of DPs by preparative HPLC
The chromatographic method was transferred from UPLC to HPLC for isolation of the DPs. The
preparative isolation of the major oxidative degradation products were carried out on preparative
HPLC from Waters (Milford, MA, USA) equipped with 515 HPLC pumps and 2489 UV–visible
detector. The separation of DPs was carried out on Waters X bridge C18 column (250 × 19 mm
×
5 ꢀm) with ammonium acetate (pH 7.5; 10 mM) and ACN in gradient elution mode. The
9

New Journal of Chemistry
Page 10 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
gradient program was set as 0/10, 2/10, 10/60, 15/70, 17/10, 20/10 (T /%ACN). The flow rate
min
-1
and injection volume was 10 mL min and 1 mL, respectively. The detection wavelength was
fixed at 227 nm. The oxidative DPs (DP5, DP6 and DP7) were isolated for the structural
-
1
confirmation by NMR. The drug (2000 µg mL ) was allowed to degrade in 6% peroxide (H O )
2
2
solution for 4 h to get more percentage of the DPs. The fraction of the DPs were collected by
multiple injections into preparative HPLC and pooled together. Pooled fractions were
concentrated at 30 °C using rotary evaporator to remove ACN and further lyophilized to get the
solid compound. All the DPs were found to be pure with chromatographic purity ˃97%. The
solid form of the DPs were dissolved in CDCl and submitted for NMR analysis.
3
Results and Discussion
Optimization of Chromatographic separation
The objective of the present study was to develop stability indicating UPLC method to separate
TER from the PIs as well as from the DPs formed under stress conditions. For the method
development studies TER was spiked with PIs (Imp 1 to Imp 4). Simultaneously, forced
degradation samples were also analyzed in UPLC. The Acquity CSH C column (100 x 2.1 mm,
1
8
1
.7 µm) was found to be suitable for the purpose. The method development was initiated by
using 0.1 % FA and ACN, where Imp1 and Imp2 got separated from the TER as well as from
each other but the specified method does not produce adequate resolution of Imp 3 and Imp 4.
MeOH was used instead of ACN as an organic modifier; but there was no significant
improvement in the resolution between Imp3 and Imp4. Further, trials were conducted by using
different aqueous buffers like ammonium formate (10 mM), ammonium acetate (10 mM) with
organic modifiers like ACN and MeOH. By using ammonium formate (10 mM) with ACN, TER
1
0

Page 11 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
and PIs were well separated with optimum resolution but when the same method was applied to
stress degradation samples, Imp3 and DP1 (acidic DP) were eluted at the same retention time and
oxidative DPs (DP6 and DP7) were not resolved. Ammonium formate and ammonium acetate
were tried at different pH values with organic modifiers (ACN and MeOH), so as to get best
separation of the analytes. Finally, TER, PIs and all the DPs were well separated with optimum
resolution with no interference from the excipients by using mobile phase composed of volatile
buffer (A) ammonium acetate (pH 7.5; 10 mM) and (B) ACN as organic modifier employing a
gradient program of 0/20, 4/40, 7/80, 10/80, 12/20, 14/20 (T /%ACN) for 14 min (Fig. S2a-e).
min
-1
Column temperature and mobile phase flow rate was maintained at 30 °C and 0.25 mL min
respectively. Injection volume was 1.0 µL. Prior to sample injection, column was equilibrated
with the mobile phase at the initial gradient composition for 10 min. Mobile phase was filtered
through 0.22 µm, Millipore Nylon-66 filter. All the samples were analyzed using a PDA detector
and chromatograms were extracted at 227 nm to detect PIs and the possible degradation peaks.
The same method was transferred for UPLC-MS studies by optimizing all the MS parameters as
discussed in experimental section.
Method validation
Specificity of the method was determined by estimating the purity angle and purity threshold of
TER and its PIs using a PDA detector. It was noticed that purity angle was lesser than purity
threshold for all PIs and TER in presence of the DPs, indicates the specificity of the method.
Accuracy of the developed method was determined by the recovery experiments in triplicate at
three levels. Recovery of TER and all four PIs was found between 98.09-100.73 and 93.04-
1
00.05, respectively (Table S2). The RSD (%) values for the repeatability of TER assay and PIs
(
Imp 1 to Imp 4) was found to be within 2.0 %. The RSD (%) results of intermediate precision
1
1

New Journal of Chemistry
Page 12 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
(intra-day and inter-day) for TER and PIs are less than 3% (Table S2). These results prove that
the method was precise. Linearity for TER and PIs was determined by plotting calibration curve
over tested range and linear regression analysis of peak area versus concentration gives good
linearity. Slope, intercept and correlation coefficient data was presented in Table S2. The LOD
and LOQ values for the TER and PIs (Imp 1 to Imp 4) are given in Table S2. Robustness of the
method was studied by deliberate change in chromatographic parameters. In all experiments
TER and PI peaks were found to be well resolved with resolution more than 2 and elution order
of the analytes remain unchanged. RSD (%) value for all the parameters was within the limits
indicates the robustness of method.
Degradation behavior of TER
The behavior of TERunder various stress degradation conditions were studied by employing
UPLC. The drug was found to be stable under basic, neutral, thermal and photolytic stress
conditions, while it was found to be degraded under acid hydrolytic (Fig. S2d) and oxidative
conditions (Fig. S2e). Total of seven DPs were identified and characterized by UPLC-
ESI/MS/MS and their elemental compositions are depicted in Table S3. The structures of
oxidative
DPs
were
confirmed
by
NMR
analysis.
The drug was refluxed in 0.5 N HCl for 8 h at 80 °C, where it was found to be stable.
Whereas four DPs (DP1 to DP4) were observed in 1N HCl refluxed at 80 °C for 30 h (Fig. S2d).
A total of four DPs (DP2, DP5 to DP7) were formed in presence of 0.6% H O after 1 h in the
2
2
dark room at 25 °C indicates that the drug is sensitive to oxidative condition (Fig. S2e). No
degradation products were formed in basic hydrolytic, neutral hydrolytic, thermal and photo
solid conditions.
1
2

Page 13 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
UPLC-Q-TOF/MS/MS study of TER and its DPs
+
ESI/MS/MS of TER [(M+H) , m/z 532]
+
The UPLC-ESI/MS/MS line spectrum of TER [M+H] ion (m/z 532) is shown in Fig. 1a. It
clearly showed product ions at m/z 490 (loss of C H from m/z 532), m/z 447 (loss of C H N
3
6
5
11
from 532), m/z 312 (loss of C H N O from m/z 532), m/z 277 (loss of C H Cl N O from 532),
3
1
3
20
2
10
7
2
m/z 259 (loss of H O from m/z 277), m/z 256 (loss of C H O from 312), m/z 235 (loss of C H
6
2
3
4
3
from m/z 277), m/z 219 (loss of C H O from m/z 277), m/z 218 (loss of NH from m/z 235), m/z
3
3
6
2
17 (loss of H O from m/z 235), m/z 205 (loss of C H O from m/z 277), m/z 192 (loss of C H N
2 3 4 2 5 11
from m/z 277), m/z 178 (loss of C H N from m/z 235), m/z 177 (loss of C H from m/z 219), m/z
3
7
3
7
1
63 (loss of C H from m/z 205), m/z 148 (loss of CH N from m/z 177), m/z 136 (loss of C H N
3 6 3 2 3
+
2
+
from m/z 177), m/z 111 (C H N ) and m/z 86 (C H N ). The elemental compositions of all
6
11
5
12
these ions have been confirmed by accurate mass measurements (Table 1). The formation of
abundant product ion m/z 277 can be explained by the loss of 1-(2,4-dichlorophenyl)-2-(1H-
1
,2,4-triazol-1-yl)ethanol from m/z 532 which indicates the presence of dichlorophenyl moiety
and triazole ring in TER. The formation of m/z 447 can be explained by the loss of
propylaziridine from the TER (m/z 532) which confirms the presence of piperazine moiety in
1
2
TER. Proposed mass fragmentation pathway of TER is depicted in Scheme 1.
1
3

New Journal of Chemistry
Page 14 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
+
Fig. 1 UPLC-ESI/MS/MS spectrum of [M + H] ions of (a) TER (m/z 532) at 25 eV, (b) DP1
(m/z 295) at 25 eV, (c) DP2 (m/z 330) at 25 eV, (d) DP3 (m/z 256) at 25 eV
1
4

Page 15 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
Table 1 High resolution mass spectrometry (HRMS) data of product ions of protonated
terconazole (TER) and its degradation products
Molecular formula
Calculated m/z Observed m/z Error (ppm)
+
TER
C H Cl N O
532.1877
490.1407
447.0985
312.0301
277.1911
259.1805
256.0039
235.1441
219.1492
218.1176
217.1335
205.1699
192.1019
178.0863
177.1022
163.1230
148.0757
136.0757
111.0917
86.0964
532.1875
490.1400
447.0973
312.0300
277.1908
259.1799
256.0032
235.1437
219.1488
218.1176
217.1328
205.1693
192.1013
178.0862
177.1017
163.1222
148.0755
136.0753
111.0916
86.0966
-0.38
-1.43
-2.68
-0.32
-1.08
-2.31
-2.73
-1.7
26
32
2
5
3
3
3
2
+
+
+
C H Cl N O
23
26
2
5
C H Cl N O
21
21
2
4
C H Cl N O
13
12
2
3
+
C H N O
2
16
25
2
+
C H N O
23
16
2
+
C H Cl N O
1
0
8
2
3
+
+
+
+
C H N O
23
14
2
C H N O
19
-1.83
0.00
13
2
C H NO
2
13
16
C H N O
17
-3.22
-2.92
-3.12
-0.56
-2.82
-4.90
-1.35
-2.94
-0.90
2.32
13
2
+
2
C H N
21
1
3
+
+
C H NO
14
11
2
C H NO
12
10
2
+
C H N
17
11
2
+
C H N
15
10
2
+
+
C H NO
10
9
C H NO
8
10
+
C H N2
6
11
+
C H N
5 12
+
DP1
C H N O
27
295.2016
277.1911
259.1805
253.1547
236.1281
210.1125
196.0968
177.1022
162.0913
148.0757
136.0757
86.0964
295.2014
277.1931
259.1802
253.154
-0.68
7.22
16
2
3
+
C H N O
2
1
6
25
2
+
C H N O
23
-1.16
-2.77
-1.27
-0.48
-3.06
0.00
16
2
+
C H N O
2
1
3
21
3
+
C H NO
18
236.1278
210.1124
196.0962
177.1022
162.0907
148.0755
136.0757
86.0967
13
3
3
3
+
+
+
C H NO
16
11
C H NO
14
10
C H N O
13
10
2
+
C H NO
12
-3.70
-1.35
0.00
10
+
C H NO
9
10
+
C H NO
8
10
+
C H N
5 12
3.48
+
DP2
C H Cl N O
3
330.0407
256.0039
186.9712
158.9763
330.0404
256.0036
186.9704
158.9759
-2.34
-1.17
-4.28
-2.52
13
14
2
3
+
C H Cl N O
3
10
8
2
+
C H Cl O
8
5
2
+
C H Cl
5
7
2
1
5

New Journal of Chemistry
Page 16 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
+
C H N
3
82.0400
256.0039
186.9712
158.9763
83.0491
82.0398
256.0034
186.9713
158.9762
83.0486
-2.44
-1.95
0.53
3
4
+
DP3
DP4
C H Cl N O
10 8 2 3
+
C H Cl O
8
5
2
+
C H Cl
7
-0.63
-6.02
-0.99
-1.79
-2.85
-3.20
-2.73
-4.69
5
2
+
C H N
3
3
4
+
+
+
+
C H Cl N O
506.1720
447.0985
421.0829
312.0301
256.0039
192.1019
506.1715
447.0977
421.0817
312.0291
256.0032
192.1010
24
30
2
5
3
3
3
2
C H Cl N O
21
21
2
4
C H Cl N O
19
19
2
4
C H Cl N O
13
12
2
3
+
C H Cl N O
2
10
8
3
+
C H NO
2
11
14
+
C H NO
12
174.0913
162.0913
148.0757
134.0600
122.0600
86.0964
174.0905
162.0902
148.0750
134.0596
122.0593
86.0963
-4.60
-6.79
-4.73
-2.98
-5.73
-1.16
-6.56
-0.73
-1.83
-2.27
0.38
11
+
C H NO
12
10
+
C H NO
9
10
+
C H NO
8
8
+
C H NO
7
8
+
C H N
5 12
+
DP5
C H Cl N O
564.1775
547.1748
546.1669
528.1564
522.1306
505.1278
485.1016
446.0907
312.0301
256.0039
98.0964
564.1738
547.1744
546.1659
528.1552
522.1308
505.1263
485.1012
446.0900
312.0304
256.0031
98.096
26
32
2
5
5
4
4
3
5
4
3
3
2
•+
C H Cl N O
26
31
2
5
+
+
+
+
C H Cl N O
26
30
2
5
C H Cl N O
26
28
2
5
C H Cl N O
23
26
2
5
C H Cl N O
-2.97
-0.82
-1.57
0.96
23
25
2
5
•+
C H Cl N O
23
21
2
5
•+
C H Cl N O
21
20
2
4
+
C H Cl N O
13
12
2
3
+
C H Cl N O
8
-3.12
-4.08
2.32
10
2
3
+
C H N
6
12
+
C H N
5 12
86.0964
86.0966
+
DP6
C H Cl N O
548.1826
531.1798
530.1720
516.1564
488.1251
487.1172
461.1142
446.0907
312.0301
108.0556
548.1825
531.1790
530.1713
516.1568
488.1234
487.1169
461.1132
446.0902
312.0297
108.0552
-0.18
-1.51
-1.32
0.77
26
32
2
5
4
3
3
3
3
3
3
3
2
•+
C H Cl N O
26
31
2
5
+
+
+
C H Cl N O
26
30
2
5
C H Cl N O
25
28
2
5
C H Cl N O
-3.48
-0.62
-2.17
-1.12
-1.28
-3.70
23
24
2
5
•+
C H Cl N O
23
23
2
5
+
C H Cl N O
22
23
2
4
•+
C H Cl N O
21
20
2
4
+
C H Cl N O
+
13
12
2
3
C H N
3
5
6
1
6

Page 17 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
+
C H N
12
98.0964
548.1826
531.1798
530.1720
516.1564
488.1251
487.1172
461.1142
446.0907
312.0301
108.0556
98.0964
98.0960
548.1821
531.1787
530.1720
516.1562
488.1233
487.1165
461.1128
446.0904
312.0298
108.0556
98.0966
-4.08
-0.91
-2.07
0.00
6
+
DP7
C H Cl N O
26 32 2 5
4
3
3
3
3
3
3
3
2
•+
C H Cl N O
26
31
2
5
+
+
+
C H Cl N O
26
30
2
5
C H Cl N O
-0.39
-3.69
-1.44
-3.04
-0.67
-0.96
0.00
25
28
2
5
C H Cl N O
23
24
2
5
•+
C H Cl N O
23
23
2
5
+
C H Cl N O
22
23
2
4
•+
C H Cl N O
21
20
2
4
+
C H Cl N O
+
13
12
2
3
C H N
3
5
6
+
C H N
6 12
2.04
Scheme 1 Proposed ESI/MS/MS fragmentation pathway of protonated terconazole (TER)
1
7

New Journal of Chemistry
Page 18 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
MS/MS of DPs
+
MS/MS of DP1 [(M+H) , m/z 295]
The degradation product DP1 was eluted at retention time (Rt) of 1.6 min. in acidic condition
+
+
with molecular formula C H N O . The ESI/MS/MS spectrum (Fig. 1b) of [M+H] ion (m/z
16
27
2
3
2
95) showed abundant product ions at m/z 210 and m/z 136 formed by the loss of C H N from
2 5
m/z 253 and loss of C H from m/z 162, respectively. Other product ions include m/z 277, m/z
2
2
2
59, m/z 253, m/z 236, m/z 196, m/z 177, m/z 162, m/z 148, and m/z 86 (Scheme 2). The presence
+
of two nitrogen atoms in the molecular formula (C H N O ) and formation of characteristic
1
6
27
2
3
fragment ion at m/z 86, indicates that the DP 1 was formed by the hydrolytic cleavage of carbon
oxygen bonds by removal of 1-(2,4-dichlorophenethyl)-1H-1,2,4-triazole moiety. The
consecutive water loss to form fragment ions at m/z 277 and m/z 259 from DP1 (m/z 295) and
m/z 277, respectively confirms the presence of two aliphatic alcohol moieties in the structure of
DP1. Absence of characteristic isotopic pattern of two chlorine (ratio of 9:6:1) in the molecular
ion of DP1 indicates that dichlorobenzene moiety is removed during hydrolysis. Further the
presence of structure indicative fragment ions at m/z 253, m/z 236, m/z 210 and m/z 196, confirm
the DP 1 was 3-(4-(4-isopropylpiperazin-1-yl)phenoxy)propane-1,2-diol. A probable mechanism
for the formation of DP1 is given in Scheme S1.
1
8

Page 19 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
Scheme 2 Proposed ESI/MS/MS fragmentation pathway of protonated degradation product DP1,
DP2 and DP3
+
MS/MS of DP2 [(M+H) , m/z 330]
The DP2 was observed in acidic and oxidative condition and eluted at Rt of 6.0 min. with its
+
M+H] peak at m/z 330 (Fig. 1c). The MS/MS spectrum of DP2 shows highly abundant product
[
ions at m/z 158 formed by decarbonylation (CO) from m/z 186 and m/z 256 formed by the loss of
prop-1-ene-1,2-diol from m/z 330. Other product ions include m/z 186, m/z 82 (Scheme 2). The
presence of characteristic product ion peak at m/z 82 (due to methyl triazole) and m/z 158 (due to
1
9

New Journal of Chemistry
Page 20 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
methyl dichlorobenzene) proved that the structure of DP2 was (2-((1H-1,2,4-triazol-1-
yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methanol. The formation of DP1 and DP2
involves the acid catalyzed nucleophilic substitution reaction. A probable mechanism for the
1
3
formation of DP2 is given in Scheme S2.
+
MS/MS of DP3 [(M+H) , m/z 256]
+
The acidic degradation product DP3 was eluted at Rt of 6.2 min. with its [M+H] peak at m/z 256
(
Fig. 1d). The MS/MS spectrum shows product ions at m/z 186 (loss of C H N from m/z 256),
2
3
3
m/z 158 (loss of CO from m/z 186), m/z 83 (loss of C H Cl N from m/z 256) (Scheme 2). The
5
3
2
3
absence of characteristic product ions peak at m/z 86 (due to isopropylaziridine was present in
MS/MS of the drug) indicates that piperazine moiety was removed from the drug during
hydrolysis. Probable mechanism for the formation of DP3 is provided in Scheme S1.
+
MS/MS of DP4 [(M+H) , m/z 506]
+
Figure 2a displays the ESI/MS/MS spectrum of DP4 which shows [M+H] at m/z 506 with
+
3
+
3
elemental composition C H Cl N O , two carbon atoms less than the TER (C H Cl N O )
5
2
4
30
2
5
26 32
2
which indicates the elimination of two carbon atoms which in turn indicates that DP4 could be
formed by the hydrolytic cleavage of the piperazine ring which leads to elimination of ethene
moiety. MS/MS spectrum of DP4 displays the structure indicative fragment ions at m/z 447 and
m/z 421 formed by the loss of propan-2-amine and -C H N from m/z 506, respectively. Other
5
11
fragment ions include m/z 312, m/z 256, m/z 192, m/z 174, m/z 162, m/z 148, m/z 134, m/z 122
and m/z 86 (Scheme 3). All the fragment ions and molecular formula were consistent that DP4
was
N1-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-
dioxolayl)methoxy)phenyl)-N2-isopropylethane-1,2-diamine. A probable mechanism for the
formation of DP4 is shown in Scheme S3.
2
0

Page 21 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
+
Fig. 2 UPLC-ESI/MS/MS spectrum of [M + H] ions of (a) DP4 (m/z 506) at 30 eV, (b) DP5
(m/z 564) at 25 eV, (c) DP6 (m/z 548) at 25 eV and (d) DP7 (m/z 548) at 25 eV
2
1

New Journal of Chemistry
Page 22 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
Scheme 3 Proposed ESI/MS/MS fragmentation pathway of protonated degradation product DP4
+
MS/MS of DP5 [(M+H) , m/z 564]
+
The Figure 2b shows the ESI-MS/MS spectrum of DP5 [M+H] ion peak at m/z at 564 (Rt = 5.4
min). The mass difference between DP5 (m/z 564) and protonated drug (m/z 532) is 32 Da,
indicates DP5 was formed by the addition of two oxygen atoms in the drug moiety. The mass
+
spectrum of [M+H] DP5 display the product ions at m/z 547 (loss of OH from m/z 564), m/z 546
(loss of H O from m/z 564), m/z 528 (loss of 2H O from m/z 564), m/z 522 (loss of C H from
2 2 3 6
m/z 564), m/z 505 (loss of C H from m/z 547), m/z 485 (loss of C H from m/z 528), m/z 446
3
6
3
7
(loss of C HN from m/z 485), m/z 312 (loss of C H N O from m/z 528), m/z 256 (loss of
2 13 16 2
2
2

Page 23 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
C H O from m/z 312), m/z 98 and m/z 86 (Scheme 4). The MS/MS spectrum shows
3
4
+
instantaneous loss of OH radical leads to formation of [MH-OH] ion (m/z 547) which indicate
1
4
the possibility of N-oxide degradation product . The fragment ions at m/z 546 and m/z 528
could be formed by consecutive loss of water from the DP5. The formation of characteristic
product ion of m/z 522 indicates the oxidation of the piperazine moiety. The formation of
daughter ions at m/z 522 can be explained by the meisenheimer rearrangement followed by loss
of propene. It can be observed that N-oxide instantly loses the OH radical giving rise to [MH-
+
OH] ions (m/z 547) in the MS/MS spectrum of m/z DP5. Based on the above data DP5 could
•
15
1 13
be N, N-dioxide of the TER. To confirm the formation of N, N-dioxide H and C NMR
1
13
experiments were carried out. The data from NMR experiments ( H and C NMR) revealed that
the difference between TER and DP5 are specific to the isopropyl group and piperazine ring
1
Fig. 3). The number of proton signals in the H NMR TER (Fig. S3) and H NMR of DP5 are
1
(
similar which excludes the possibility of hydroxylation and confirm that DP5 was formed by N,
1
N-dioxidation. The H NMR signals of DP5 (Fig. S4) for protons at H1/H3, H5/H9, and H6/H8
1
was shifted to downfield region as compared to H NMR signals of the drug (Table 2).
1
3
13
Furthermore, compared to the C NMR signals of TER (Fig. S5) the C signals of DP5
assigned as C5/C9 and C6/C8 are significantly deshielded (Fig. S6 and Table 3). Based on
above data the structure of the DP5 was characterized as 1-(4-((2-((1H-1,2,4-triazol-1-
yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)-4-isopropylpiperazine
1
,4-dioxide. The probable mechanism for formation of DP5 is shown in Scheme S4.
2
3

New Journal of Chemistry
Page 24 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
Scheme 4 Proposed ESI/MS/MS fragmentation pathway of protonated degradation product DP5
Fig. 3 Structures of TER, DP5, DP6 and DP7 with numbering for NMR values
2
4

Page 25 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
1
Table 2 H NMR assignments for terconazole and DPs
a
Position
Terconazole
DP5
DP6
DP7
δH (ppm), Multiplicity
δH (ppm), Multiplicity
δH (ppm), Multiplicity
δH (ppm), Multiplicity
1
/3
1.15 (d, 6H)
4.35 (m, 1H)
1.51 (d, 6H)
1.21 (d, 6H)
4.38 (m, 1H)
1.47 (d, 6H)
4.36 (m, 1H)
2
4.39 (m, 1H)
3.45 (t, 4H)
3.81 (m, 4H)
-
5
5
a/9a
e/9e
2.92 (d, 2H)
3.52 (d, 2H)
3.33 (d, 2H)
3.41 (td, 2H)
2
.78 (t, 4H)
6
a/8a
3.18 (t, 4H)
-
3.82 (m, 2H)
3.55 (m, 2H)
6.92 (d, 2H)
7.27 (d, 2H)
2.98 (dd, 1H)
3.3 (s, 1H)
3.67 (t, 4H)
-
6
1
1
b/8b
1/36
2/35
6.77 (d, 2H)
6.89 (d, 2H)
2.81 (m, 1H)
3.49 (dt, 1H)
3.91 (dd, 1H)
3.8 (dt, 2H)
4.8 (dd, 2H)
8.2 (s, 1H)
7.89 (s, 1H)
7.47 (d, 1H)
7.25 (dd, 1H)
7.56 (d, 1H)
6.94 (d, 2H)
7.27 (d, 2H)
3.56 (dd, 1H)
3.94 (dd, 1H)
4.89 (t, 1H)
4.49 (m, 2H)
4.81 (m, 2H)
8.22 (s, 1H)
7.91 (s, 1H)
7.58 (d, 1H)
7.48 (dd, 1H)
7.96 (d, 1H)
6.79 (d, 2H)
6.92 (d, 2H)
3.89 (dd, 1H)
3.51 (dd, 1H)
3.92 (dd, 1H)
3.8 (dt, 2H)
4.8 (dd, 2H)
8.2 (s, 1H)
7.89 (s, 1H)
7.47 (d, 1H)
7.25 (dd, 1H)
7.57 (d, 1H)
1
5a
1
5b
1
6
7
3.93 (dd, 1H )
4.11 (s, 2H)
4.8 (dd, 2H)
8.2 (s, 1H)
1
2
0a
2
2
4
0
3
4
2
7.91 (s, 1H)
7.59 (d, 1H)
7.48 (dd, 1H)
7.90 (d, 1H)
3
3
3
S= singlet; d= doublet; t= triplet; m= multiplet; dd= doublet of doublets; dt= doublet of triplet
numbering as given in Fig. 3
a
2
5

New Journal of Chemistry
Page 26 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
13
Table 3 C NMR assignments for terconazole and DPs
a
Position
Terconazole
DP5
DP6
DP7
δC (ppm)
δC (ppm)
δC (ppm)
δC (ppm)
1
/3
18.40
54.94
16.42
55.45
18.18
55.10
16.57
45.39
2
5
/9
/8
48.70
62.46
43.44
70.57
6
50.41
70.79
66.68
58.85
1
0
146.13
115.23
118.24
152.37
67.67
145.37
115.08
121.75
158.78
67.18
146.15
114.92
121.83
158.53
67.18
144.92
115.36
119.03
153.24
67.38
1
1
1/36
2/35
1
1
1
1
1
3
5
6
7
9
74.73
74.35
74.37
74.68
67.47
67.46
67.39
67.60
107.62
53.64
107.78
53.53
107.77
53.54
107.62
53.63
2
2
2
2
2
3
3
3
3
0
2
4
7
8
0
1
3
4
144.90
151.39
134.09
133.14
131.43
136.05
127.25
129.63
144.95
151.29
133.84
133.14
131.51
136.20
127.32
129.60
144.95
151.30
133.85
133.15
131.52
136.21
127.33
129.61
144.59
151.23
133.14
134.01
131.45
136.10
127.27
129.62
a
numbering as given in Fig. 3
+
MS/MS of DP6 and DP7 [(M+H) , m/z 548] (Isomeric N-oxides)
+
The ESI/MS/MS spectra of DP6 (Rt = 6.4 min) and DP7 (Rt = 6.8 min) shows the [M+H] ion
peak at m/z 548, formed under oxidation condition. A mass difference of 16 Da between
protonated drug (m/z 532) and both protonated DPs (DP6 and DP7) suggest the addition of
oxygen in presence of hydrogen peroxide to TER. The formation of fragment ions at m/z 531 and
m/z 530 with the loss of -OH and H O from m/z 548 respectively, indicating DP6 and DP7 could
2
1
be the N-oxides of TER. Figure 2c and d shows the ESI-MS/MS spectrum of DP6 and DP7.
4
2
6

Page 27 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
The ESI/MS/MS spectrum displays highly abundant product ion at m/z 516, which could be
formed by the insertion of the oxygen into the piperazine ring of DP6 and DP7 to form an
1
intermediate and subsequent loss of methanol from the intermediate. The MS/MS spectra also
5
display low abundance similar product ions at m/z 488, m/z 487, m/z 461, m/z 446, m/z 312, m/z
1
08 and m/z 98 (Scheme 5). To demonstrate the presence of N-oxides and to define the position
of oxidation, APCI-MS experiments were conducted in scan mode. The formation of fragment
ion at m/z 532 in full scan APCI-MS, considering thermally induced de-oxygenation in the
16
vaporizer of the APCI source, confirms the formation of N-oxide. Further APCI spectrum of
DP7 also displays characteristic fragment ion at m/z 506 derived from Meisenheimer
17
rearrangement, on the contrary DP6 did not show such characteristic ion (Fig. 4a, b).
Scheme 5 Proposed ESI/MS/MS fragmentation pathway of protonated degradation product (DP6
and DP7)
2
7

New Journal of Chemistry
Page 28 of 31
View Article Online
DOI: 10.1039/C8NJ00509E
Fig. 4 Full scan UPLC-APCI/MS spectrum of (a) DP6 (m/z 548) and (b) DP7 (m/z 548)
However, to confirm the positions of N-oxides in DP6 and DP7 based on data provided
1
13
by mass spectrometric experiments, NMR ( H and C NMR) experiments were performed. The
possibility of hydroxylation can be ruled out as the numbers of proton signals are similar to that
of drug. Based on the NMR data of both the DPs the oxidation was expected to be on piperazine
ring of the TER as there were no notable differences in the NMR signals of any other moieties.
1
According to the H NMR signals of DP6 (Fig. S7) significant deshielding was observed for the
protons at H5/H9 and H6/H8 whereas no change in the delta values of H1/H3 protons was
noticed which concludes that DP6 was formed by the oxidation of the nitrogen atom attached to
2
8

Page 29 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ00509E
the phenyl ring (N7) (Table 2). The proton signals for the H5/H9 and H6/H8 were split into two
1
signals. Additionally, C spectrum of DP6 shows considerable downfield shift for the C6/C8
3
while C5/C9 which is β-carbons of N-7 has slightly shifted upfield (Fig. S8 and Table 3).
1
In case of DP7, the H NMR spectrum shows significant downfield shift for the protons
at H1/H3 and H5/H9 along with H6/H8 which confirms N-4 was oxidized to form DP7 (Fig. S9).
1
3
The protons signals at H5/H9 were split into two resonances. The C NMR spectrum of DP7
displays expected downfield shift for C5/C9 and C6/C8 as compared to the drug whereas
shielding effect was observed for the C2 and C1/C3 (Fig. S10 and Table 3). All this data proves
that DP6 and DP7 are oxidative DPs and formed by the oxidation of nitrogen where phenyl (N7)
and isopropyl group (N4) is attached, respectively. Based on above discussion, probable
structures for DP6 and DP7 could be given as 1-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-
dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)-4-isopropylpiperazine 1-oxide, and 4-(4-
((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4 yl)methoxy)phenyl)-
1
-isopropylpiperazine 1-oxide, respectively. The elemental composition of DPs and its product
ions were confirmed by accurate mass measurement. A probable mechanism of formation of
DP6 and DP7 is shown in Scheme S4.
Conclusion
A simple validated stability indicating UPLC method was developed for quantitative
determination of TER and PIs in drug substance and drug product. In addition, stress degradation
behavior of TER was studied according to ICH guidelines. Acid hydrolysis of TER yields four
DPs (DP1, DP2, DP3, and DP4) and four DPs (DP2, DP5, DP6 and DP7) were formed in
oxidative stress condition. The exposure of the drug to oxidative stress conditions resulted in the
generation of isomeric N-oxides, DP6 and DP7. The present study shows the application of
2
9