Forced degradation studies of lansoprazole using LC-ESI HRMS and 1H-NMR experiments: in vitro toxicity evaluation of major degradation products

Article abstract of DOI:10.1002/jms.3949

Regulatory agencies from all over the world have set up stringent guidelines with regard to drug degradation products due to their toxic effects or carcinogenicity. Lansoprazole, a proton-pump inhibitor, was subjected to forced degradation studies as per ICH guidelines Q1A (R2). The drug was found to degrade under acidic, basic, neutral hydrolysis and oxidative stress conditions, whereas it was found to be stable under thermal and photolytic conditions. The chromatographic separation of the drug and its degradation products were achieved on a Hiber Purospher, C18 (250?×?4.6?mm, 5?μ) column using 10?mM ammonium acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0?ml/min. The eight degradation products (DP1–8) were identified and characterized by UPLC/ESI/HRMS with in-source CID experiments combined with accurate mass measurements. DP-1, DP-2 and DP-3 were formed in acidic, DP-4 in basic, DP-5 in neutral and DP-1, DP-6, DP-7 and DP-8 were in oxidation stress condition Among eight degradation products, five were hitherto unknown degradation products. In addition, one of the major degradation products, DP-2, was isolated by using semi preparative HPLC and other two, DP-6 and DP-7 were synthesized. The cytotoxic effect of these degradation products (DP-2, DP-6 and DP-7) were tested on normal human cells such as HEK 293 (embryonic kidney cells) and RWPE-1(normal prostate epithelial cells) by MTT assay. From the results of cytotoxicity, it was found that lansoprazole as well as its degradation products (DP-2, DP-6 and DP-7) were nontoxic up to 50-μM concentrations, and the latter showed slightly higher cytotoxicity when compared with that of lansoprazole. DNA binding studies using spectroscopic techniques indicate that DP-2, DP-6 and DP-7 molecules interact with ctDNA and may bind to its surface. Copyright

Full text of DOI:10.1002/jms.3949

1
Forced degradation studies of lansoprazole using LC-ESI HRMS and H-
NMR experiments: in vitro toxicity evaluation of major degradation
products
1
1
1
2
2
G. Shankar , Roshan M. Borkar , Suresh Udutha , Lalitha Guntuku , VGM Naidu ,
3
1
Narayana Nagesh and R. Srinivas *
1
National Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology,
Hyderabad, 500 007, India
2
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical
Education and Research, Balanagar, Hyderabad 500037, India
3
CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India
Keywords: Lansoprazole, degradation products, accurate mass measurements and in vitro
toxicity
*
Corresponding author
:srini@iict.res.in (R. Srinivas)
sragampeta@yahoo.co.in
:+91-40-27193122
Telephone Number
Fax Number
: +91-40-27193156
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jms.3949
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Abstract
Regulatory agencies from all over the world have set up stringent guidelines with regard to
drug degradation products due to their toxic effects or carcinogenicity. Lansoprazole, a
proton-pump inhibitor, was subjected to forced degradation studies as per ICH guidelines
Q1A (R2). The drug was found to degrade under acidic, basic, neutral hydrolysis and
oxidative stress conditions, whereas it was found to be stable under thermal and photolytic
conditions. The chromatographic separation of the drug and its degradation products were
achieved on a Hiber Purospher, C18 (250 X 4.6mm, 5µ) column using 10 mM ammonium
acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0
mL/min. The eight degradation products (DP1-8) were identified and characterized by
UPLC/ESI/HRMS with in-source CID experiments combined with accurate mass
measurements. DP-1, DP-2 and DP-3 were formed in acidic, DP-4 in basic, DP-5 in neutral
and DP-1, DP-6, DP-7 and DP-8 were in oxidation stress condition Among eight degradation
products, five were hitherto unknown degradation products. In addition, one of the major
degradation products, DP-2 was isolated by using semi preparative HPLC and other two, DP-
6
and DP-7 were synthesized. The cytotoxic effect of these degradation products (DP-2, DP-6
and DP-7) were tested on normal human cells such as HEK 293 (embryonic kidney cells) and
RWPE-1(normal prostate epithelial cells) by MTT assay. From the results of cytotoxicity, it
was found that lansoprazole as well as its degradation products (DP-2, DP-6 and DP-7) were
nontoxic up to 50 μM concentrations and the latter showed slightly higher cytotoxicity when
compared with that of lansoprazole. DNA binding studies using spectroscopic techniques
indicate that DP-2, DP-6 and DP-7 molecules interact with ctDNA and may bind to its
surface.
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1
.Introduction
Stress degradation studies are conducted on the drug substances and drug products under
[
1]
more severe conditions than accelerated stability studies.
All drug substances and drug
products have a tendency to degrade over the period of time under the influence of catalytic
external and internal factor such as heat, moisture, pH, oxidants, light, etc. The main purpose
of the stability studies is to establish the intrinsic stability of drug substance and to provide
more comprehensive information on the drug degradation pathway by using highly sensitive
[
2-4]
and sophisticated analytical tools.
These studies will help to evaluate the time up to which
any drug molecule or formulation would retain the quality safety, efficacy specification.
Characterization of the trace level degradation products has become possible with the help of
sophisticated analytical tools. Structure elucidation and evaluation of the toxicity of the
degradation product is important from safety point of view. Severe adverse effects of the drug
degradation products have been reported for the degradation product of tetracycline and
[5-6]
aminopencillins.
Recently, we have reported in vitro toxicity of degradation products of
[
7]
rabeprazole. It showed more than 50% of cell inhibition on HepG2 and PANC-1 cell lines.
DNA binding studies indicated that the degradation products bind to the surface of double
stranded DNA and stabilize the DNA complex.
Due to toxic effect or carcinogenicity of degradant, stringent reporting has been setup by
several regulatory agencies from all over the world. Stability testing protocol was different
from region to region. It differs from one manufacture to another, and introduction of
International Conference on Harmonization (ICH) guideline brought protocols in a
harmonized way. ICH Q3 guideline includes identification and qualification thresholds of
[
8-9]
degradation products of drugs.
The guidelineQ1A (R2) highlighted the purposes of stress
testing and outlined a broad protocol. However, features were also covered in ICH guidelines
[
10]
[1]
[8]
[9]
[11]
Q1B , Q2 (R1) , Q3A , Q3B and Q5C . Other guidelines such as the World Health
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[12]
[13]
Organization
, United States Food and Drug Administration
, European Medicines
[
14]
Agency
mention stress-testing requirements. e.g., US, Indian and Chinese
pharmacopoeias, have recommended force degradation studies, whereas, British and
European pharmacopoeias have not mentioned anything related to stress studies. The
identified and characterized degradation products can be subjected to toxicity studies for risk
assessment of potentially genotoxic degradation products.
Lansoprazole, ((RS)-2-([3-methyl-4-(2, 2, 2-trifluoroethoxy) pyridine-2yl] methylsulfinyl)-
1
H benzo[d]imidazole) is a proton-pump inhibitor which inhibits stomach's production of
gastric acids. It is used to treat and prevent ulcer, Zollinger-Ellison syndrome and erosive
esophagitis. Few HPLC methods have been reported for the determination of lansoprazole in
[
15-16]
plasma
impurities
hydrolytic degradation products.
and quantification of (R)-(+) - and (S)-(-)-lansoprazole enantiomers and related
[17]
[18]
. Few studies have been reported on the characterization of acidic
and
[
19]
However, no study exists on systematic forced
degradation behavior of lansoprazole under the influence of stress conditions as per ICH
guidelines. Hence the purpose of this study is to identify and characterize the degradation
1
products of lansoprazole using LC/ESI/HRMS and H-NMR and to evaluate in vitro
toxicities of major degradation products. In the present study, among eight degradation
products a total of five hitherto unknown degradation products were identified and
[
18-19]
characterized. Degradation products, DP-1, DP-2 and DP-5 were reported earlier.
This
study may help to evaluate the quality of drug product which are on the edge of expiration.
Degradation products may have potential genotoxicity and leads to severe toxic effects.
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2
2
. Experimental
.1 Chemicals and reagents
Lansoprazole (LAN), dimethyl sulfoxide-d6 (DMSO-d6), methanol-d4, tris hydrochloride
Tris-HCl), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and
(
Deoxyribonucleic acid from calf thymus (ctDNA) was purchased from Sigma Aldrich,
India.HPLC Grade acetonitrile (ACN) and methanol (MeOH) were purchased from Merck,
India. HPLC grade water was prepared by filtrating through a Millipore Milli-Q- plus system
(Millipore, Milford, MA, USA). Ammonium acetate of HPLC grade, ethyl acetate and
sodium bicarbonate were purchased from Finar Chemicals Pvt. Ltd. (Ahmedabad, India). All
analytical grade reagents, formic acid, sodium hydroxide, hydrochloric acid, dichloromethane
(DCM)and 30% hydrogen peroxide were purchased from Merck (Mumbai, India).
2
2
.2 Instrumentation
.2.1. Liquid Chromatography-Mass spectrometry
Analysis was carried out on a U-HPLC instrument (Thermo Scientific Accela, Germany)
equipped with a quaternary pump, a de-gasser, a diode-array detector, an auto sampler and a
column compartment. Mass spectrometric analysis was carried out on an Orbitrap high-
resolution mass spectrometer (Exactive Thermo Scientific, Germany) equipped with a heated
electrospray ionization (ESI) source. The data acquisition was under the control of Xcalibur
software.
2
.2.2 Preparative HPLC
Isolation was carried out on a semi preparative HPLC instrument (GILSON, USA) equipped
with a binary pump (331 and 332), a de-gasser, a diode-array detector (157 UV-VIS), a liquid
handler (GX-241) and a column compartment. The data acquisition was under the control of
TRILUTION LC software.
2
.3. Stressed degradation conditions
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Forced degradation studies of lansoprazole were carried out on the bulk drug as per ICH
guidelines Q1A (R2). The drug solutions were prepared in 1.0mg/ml concentration for all the
stressed reactions. Acidic hydrolysis of the drug was conducted in 0.01N HCl at room
temperature for 15 min. Basic and neutral (H O: ACN, 50:50) hydrolysis were performed by
2
°
refluxing drug in 2N NaOH and water at 80 c for 72 h and 48 h, respectively. The oxidative
degradation study was conducted using 2% H O at room temperature for 30min. Photolytic
2
2
6
studies were performed by exposing solid and solution of the drug sample to 1.2×10 lux h of
2
fluorescent light and 200W h/m UV light in a photostability chamber. For thermal
degradation study, the drug sample was sealed in glass vials and kept in a thermostatic block
o
at 80 C for 1week. All stressed samples were kept in a refrigerator at 4°C until analysis.
2
.4. Sample preparation
All the degradation samples were collected and neutralized. The collected samples were
diluted ten times with the mobile phase and filtered through 0.22 µm membrane syringe filter
before LC/ESI/MS analysis.
2
2
.5. Analysis of stressed samples
.5.1. Method development
The LC method was optimized for the separation of the drug and its degradation products
using Hiber Purospher, C18 (250 X 4.6mm, 5µ) (Merck Lichro, Switzerland) column with a
mobile phase composed of 10 mM ammonium acetate(A) and ACN(B). Initially, several
gradient conditions were tried using MeOH/ACN with various pH buffers in different
proportion. Finally, the mobile phase with a combination of 10 mM ammonium acetate and
ACN gives better separation and good peak shape for lansoprazole and its degradation
products (DP 1-8). The linear gradient programme as follows: (Tmin / % proportion of
solvent B): 0-5/10, 5-7/30, 7-14/ 40, 14-20/45, 20-30/ 45, 30-35/ 50, 35-37/70, 37-38/10, 38-40/10. The
column temperature, flow rate, injection volume, and detector wavelength were at 30 °C, 1.0
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ml/min, 20.0 µl, and 285 nm, respectively. Samples analysis was performed in the ESI
positive mode in the mass range from 50 to 600.The typical operating source conditions for
MS scan were optimized as follows such as sheath gas flow rate 70; aux gas flow rate 32;
spray voltage 3.50 kV; capillary temperature 380 °C; capillary voltage 25.00 V; tube lens
voltage 100.0 V; skimmer voltage 18.00 V and heater or source temperature 300°C.
2
.5.2. In-source collision induced dissociation (CID) experiments
In-source fragmentation data of the drug and its degradation products were obtained by using
in-source CID at collision energy of 35 eV which was found to be suitable to obtain
reasonable fragmentation with reproducible signal intensity.
2
.5.3. Preparative HPLC method
The degradation product DP-2 was isolated by using semi preparative HPLC with Water’s X-
bridge Prep C18 (250 X 10mm, 5µ) (Waters Corporation, Milford, USA) column. The mobile
phase composed of 10 mM ammonium acetate (A) and ACN (B) in a gradient method. The
gradient elution method was set as follows. (T/ % B): 0-3/ 10. 3-5/ 35, 5-10/ 50, 10-18/ 70, 18-21
/
8
0, 21-22/ 10, 22-25/ 10. The flow rate, injection volume and detection wavelength were 8.0
mL/min, 500µL and 285 nm, respectively. Ethyl acetate was added to the isolated compound.
The mixture solution was kept in shaker for 15 min. The supernatant was evaporated on a
1
ScanVac speed Vacuum. The isolated sample was subjected to H-NMR study using
deuterated MeOH.
2
.5.4. Chemical synthesis
The degradation products DP-6 and DP-7 were synthesized by using lansoprazole as a
starting material and meta- chloroperoxybenzoic acid (m-CPBA) reagent. To a solution of
lansoprazole (369 mg, 1.0 mM) in DCM (3ml), m-CPBA (172mg, 1 mM for DP-6and 2 mM
o
for DP-7) was added at 0 C and the mixture was stirred overnight at room temperature. The
reaction mixture was quenched with sodium bicarbonate followed by extraction with ethyl
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acetate. After extraction, the residue was purified by using silica gel column chromatography
(DCM–MeOH, 9.5:0.5 %) to get the pure products as white solid. The samples were
1
characterized by H-NMR using deuterated methanol and a mixture of deuterated methanol
and chloroform for DP-6 and DP-7, respectively.
2
2
.6. In vitro toxicity evaluation
.6.1. In vitro 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay
(MTT
assay)
The cytotoxic effect of the compounds on normal human cells was determined by performing
MTT assay. Briefly, 5 * 10^3 cells of HEK-293 (Human embryonic kidney cells) and
RWPE-1(Human prostate epithelial cells) were seeded in each well of 96 well flat bottom
tissue culture plates and incubated overnight. Cells were treated with the designated
concentration of the degradation products (DP-2, DP-6 and DP-7) for 48h; subsequent to the
treatment, media was aspirated and 100 μl of MTT (0.5mg/mL) was added and incubated for
4
5
2
2
hr and later purple formazan crystals were dissolved in DMSO and absorbance was read at
70nm with a spectrophotometer (Spectramax M4, Molecular devices, USA).
.7. DNA binding studies
.7.1. UV-visible spectroscopy
UV-visible absorption spectra were recorded using Perkin Elmer ABI 35 Lambda
o
Spectrophotometer (Waltham, MA, USA) at 25 C. All the experiments were carried out in
polystyrene cuvettes to minimize binding of derivatives to the surface of the cuvettes. 50 μM
of lansoprazole and degradation products (DP-2, DP-6 and DP-7) stock solution was
prepared in DMSO and 25.0 μM of ctDNA in 100 mM Tris-HCl (pH 7.0). About 1 mL of
2
5.0 μM complex solutions was taken in a 1 cm path length cuvette and each time 5μl of
DNA was added. Absorption spectra were recorded in the range of 200 nm to 400 nm. All the
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solutions used were freshly prepared before commencing the experiment and titration was
carried out until saturation of absorbance occurs.
2
.7.2. Fluorescence titration
o
Fluorescence emission spectra were measured at 25 C using a Hitachi F7000
spectrofluormeter (Maryland, USA) using a 1 cm path length quartz cuvette. Quartz cuvettes
was thoroughly washed with distilled water and dilute nitric acid (approximately 0.1 N) to
minimize non-specific binding of the molecules to the surface of the cuvette. Throughout the
fluorescence experiment, the concentration of the lansoprazole and degradation products
(DP-2, DP-6 and DP-7) were kept constant (10 µM) and titrated with increasing
concentrations of ctDNA (multiples of 0.5 µM). Fluorescence spectra were recorded after
each addition of ctDNA to the fluorescent cuvette. The complexes were excited at 280nm
(lansoprazole), 325 nm (DP-2), 322 nm (DP-6), 278 nm (DP-7) and emission spectra for each
titration were recorded from 308 to 365 nm depending upon the molecule. Each spectrum
was recorded three times and the average of three scans was taken.
2
.7.3. Circular Dichroism studies
DNA conformational studies were carried out on a JASCO 815 circular dichroism
spectropolarimeter (Jasco, Tokyo, Japan). Circular dichroism spectroscopic studies were
performed to study the change in DNA conformation brought by lansoprazole and
degradation products (DP-2, DP-6 and DP-7) on the interaction with ctDNA at micro molar
concentration range. A solution of ctDNA was prepared in 100mM Tris-HCl (pH 7.0). To 10
μM of ctDNA, about 10.0 μM and 20 μM (1:1 and 1:2 ratios of ctDNA: complex) of each
solution containing lansoprazole and degradation products (DP-2, DP-6 and DP-7) were
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added and CD spectra were recorded from 200 nm to 300 nm in a 1 mm path length cuvette.
The spectra were averaged over 3 scans.
2
.7.4. Viscosity studies
Viscosity experiments were conducted on Ostwald viscometer, immersed in a water bath
o
maintained at 25 C. Viscosity experiments were performed for lansoprazole and degradation
products (DP-2, DP-6 and DP-7) (15 µM), after mixing them with ctDNA solution (150
µM). Before mixing DNA and sample, viscosity measurements were performed with ctDNA
alone. Ethidium bromide (Et Br)- ctDNA complexes were considered as control. DNA
solution was prepared in 100 mM Tris-HCl (pH 7.0). The graph was drawn by plotting
1
/3
(
ŋ/ŋo) versus complex/ctDNA, where ŋ is the viscosity of ctDNA in the presence of
lansoprazole and degradation products and ŋo is the viscosity of ctDNA alone. Viscosity
[
21]
values were calculated according to the protocol mentioned by Tan et al.
3
3
. Results and discussion
.1. Degradation behaviour of the drug
The degradation behaviour of lansoprazole was analyzed under various stressed degradation
conditions. The optimized UPLC/MS method was used to identify the degradation products
of lansoprazole. The degradation products of lansoprazole were observed in acid, base,
neutral hydrolysis and oxidation. The drug was stable at thermal and photolytic degradation
conditions. A total of 8 degradation products, DP-1 to DP-8, were identified and
characterized by using UPLC/ESI/HRMS within-source CID experiments. The proposed
structures of degradation products of lansoprazole and their elemental compositions are given
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in scheme S1 (Supplementary Scheme S1, see supporting information) and table 1,
respectively.
3
.2.1. Hydrolysis
UPLC/ESI/HRMS analysis of lansoprazole (Figure 1) in acid hydrolytic condition showed
formation of three degradation products (DP-1, DP-2 and DP-3) (Figure 2(a)) whereas, base
(
Figure 2(b)) and neutral (Figure 2(c)) hydrolysis showed formation of DP-4 and DP-5,
respectively.
.2.2. Oxidation
The drug degraded significantly when subjected to 2% H O at room temperature for 30 min.
3
2
2
It showed formation of four degradation products (DP-1, DP-6, DP-7 and DP-8) (Figure
3
3
(a)).
.2.3. Thermal degradation
o
The exposures of the drug to 80 C for 1week did not show any significant degradation
product and it was to be stable under thermal condition (Figure 3(b))
3
.2.4. Photolytic degradation
Lansoprazole was found to be stable under UV and fluorescence conditions (Figure 3(c)).
3
3
.3. In-source CID
.3.1. Fragmentation of lansoprazole
The positive ion UPLC/ESI/HRMS spectrum of lansoprazole (Rt=20.45 min) shows an
+
abundant [M+H] ion at m/z 370.In-source CID spectrum shows abundant product ions at m/z
1
36 (loss of HCN from m/z 163) and low abundant ions at m/z 352. Formation of these ions
can be attributed to an ortho effect involving loss of H O. Other product ions are at m/z 252
2
(
loss of 1H –benzo[d]imidazole), m/z 234 (loss of C H N ), m/z 222 (loss of C H F O S), m/z
7 4 2 2 3 3 2
2
1
05 (loss of C H N OS), m/z 204 (loss of C H N OS), m/z 163(loss of C H F NO ), m/z
7 5 2 7 6 2 8 8 3 2
52(loss of 2,2,2-trifluoro ethanol(C H F O) from m/z 252), m/z 122 (loss of C H N OS,
2
3
3
7
4
2
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C H F ), m/z 119 (loss of C H F NO S), m/z 108 (loss of C H F N O S), m/z 107(loss of
2
3
3
9
8
3
2
9
5
3
2
2
C HF O from m/z 205), m/z 106(loss of C HF O, C H N OS) and m/z 122(loss of HCN from
2
3
2
3
7
6
2
m/z 119). (Scheme 1, Figure 4(a). While the product ions at m/z 252, m/z 204, m/z 152, m/z
22, m/z 108 and m/z 106 are characteristic of 3-methylpyridine skeleton, m/z 119 and 92 are
diagnostic of 1H-benzo[d]imidazole skeleton in lansoprazole. The loss of C H N and
1
7
6
2
C H N OS from protonated drug clearly indicates that the sulphur monoxide group forms a
7
6
2
bridge between benzimidazole and 3-methylpyridine moieties of lansoprazole. The elemental
compositions of all these ions have been confirmed by accurate mass measurements
(Supplementary Table S1, see Supporting Information).
6
.3.2. In-source CID of degradation products
+
DP-1 ([M+H] , m/z 322)
The degradation product, DP-1 was formed in acid hydrolysis and oxidation conditions. The
+
elemental composition of its [M+H] ions with a mass difference of 48 u compared to that of
protonated drug indicates that it is formed by the loss ‘SO’ from the protonated drug. The in-
+
source CID spectrum of [M+H] ion (m/z 322) of DP-1 (Rt = 14.7 min) shows highly
abundance product ions at m/z 222 (loss of 2,2,2-trifluoroethanol) and low abundance ion at
m/z 307 (loss of CH radical), m/z 239 (loss of C H F ), m/z 238 (loss of 1,1,1-
3
2
2 3
triflouroethane) and m/z 224 (loss of C HF O). These ions clearly indicate the absence of
2
3
‘
SO’ group in DP-1 when compared to lansoprazole. (Supplementary Scheme S2 and
Figure 4(b), see Supporting Information). Based on these data, DP-1 was identified as 2-
3-methyl-2-mythylene-4-(2,2,2-trifluoroethoxy) pyridin-1(2H)-yl)-1H-benzo[d]imidazole.
The elemental compositions of all these ions were confirmed by accurate mass measurements
(
(supplementary Table S2, see Supporting Information).
+
DP-2 ([M+H] , m/z 354)
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+
The positive ion UPLC/ESI/HRMS and in-source CID spectrum of [M+H] ions (m/z 354) of
DP-2 (Rt = 33.85 min), shows characteristic product ions at m/z 321 (loss of SH radical), m/z
2
36 (loss of 1H-benzo[d]imidazole), m/z 204 (loss of C H N S) and m/z 163 (loss of
7 6 2
C H F O, C H N from m/z 354) for the absence of an oxygen atom in DP-2 as compared to
2
3
3
6
5
lansoprazole. (Supplementary Scheme S3 and Figure 4(c), see Supporting Information).
The elemental compositions of DP-2 and its product ions have been confirmed by accurate
mass measurements (Supplementary Table S2, see Supporting Information). Based on
these data DP-2 was identified as 2-((3-(4-(2, 2, 2-trifluoroethoxy) pyridin-2-yl) methylthio)-
1
H-benzo[d]imidazole.
+
DP-3 ([M+H] , m/z 352)
+
The in-source CID spectrum of [M+H] ions (m/z 352) of DP-3 (Rt = 23.31 min) shows
abundant product ions at m/z 238 (loss of C H F O) and low abundance product ions at m/z
3
5 3
3
1
06 (loss of CH S), at m/z 268 (loss of 1,1,1-triflouroethane), m/z 252 (loss of C H F O), m/z
2 2 3 3
04 (loss of C H N S from m/z 252) (Supplementary Scheme S4 and Figure 4(d), see
7
4
2
Supporting Information). The mass difference of 18 u between the drug and DP-3 clearly
indicates that it is formed by loss H O from the protonated drug. The elemental compositions
2
of DP-3 and its product ions have been confirmed by accurate mass measurements
(Supplementary Table S3, see Supporting Information). Based on all these a structure of
DP-3 has been proposed which is shown in scheme S4.
+
DP-4 ([M+H] , m/z 302)
+
Figure 5(a) shows in-source CID spectrum of [M+H] ions (m/z 322) of DP-4 (Rt = 14.6
min) which was formed in base hydrolysis condition. The spectrum shows highly abundant
product ions at m/z 136 (loss of C H N OS, m/z 137 (loss of C H N OS), m/z 122 (loss of
7
6
2
7
5
2
C H N OS), and low abundance ions at m/z 284 (loss of H2O), m/z 184 (loss of 1H-
8
8
2
benzo[d]imidazole), m/z 167 (loss of OH radical from m/z 184), m/z 166 (loss of H O from
2
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m/z 184) and m/z 152 (loss of CH OH from m/z 184) (Supplementary Scheme S5 and
3
Figure 5(a), see Supporting Information). The peaks at m/z 284 and 166 are diagnostic ions
for the presence of the free –OH group in DP-4. The elemental compositions of DP-4 and
its product ions have been confirmed by accurate mass measurements (Supplementary
Table S3, see Supporting Information). Based on these data, DP-4 was identified as (Z)-1-
(1H-benzo[d]imidazol-2-yl)-4-methoxy-3-methylpyridin-2(1H)-yiidene) methyl) sulfanol.
+
DP-5 ([M+H] , m/z 272)
+
The in-source CID spectrum of [M+H] ion (m/z 272) of DP-5 (Rt = 11.65 min) shows
characteristic highly abundant product ions at m/z 119 (loss of C H NOS), m/z 154 (loss of
7
7
1
H-benzo[d]imidazole) and low abundance ion at m/z 239 (loss of SH radical)
(Supplementary Scheme S6 and Figure 5 (b), see Supporting Information). The peak at
m/z 239 is diagnostic ion for the presence of the –S=O group in between benzo imidazole and
methylprridine ring of DP-5. The elemental compositions of DP-5 and its product ions have
been confirmed by accurate mass measurements (Supplementary Table S4, see Supporting
Information). Based on these data, DP-5 was identified as (2-((3-methylpyridine-2-yl)
methyl sulfinyl)-1H-benzo[d]imidazole.
+
DP-6 ([M+H] , m/z 386)
The degradation product DP-6 at m/z386 formed under oxidation condition. The mass
difference between the protonated drug (m/z 370) and DP-6 (m/z 386) is 16 Da which
suggests the addition of ‘O’ atom to the degradant. The positive ion in-source CID spectrum
+
of [M+H] ion of DP-6 (Rt = 23.13 min), shows characteristic product ions at m/z 119 (loss
of C H F NO S), m/z 366 (loss of HF), m/z 322 (loss of SO ), m/z 310 (loss of benzyne), m/z
9
8
3
3
2
2
68 (loss of C H N ), and m/z 222 (loss of C H F O) (Supplementary Scheme S7 and
7 4 2 7 7 3
Figure 5(c), see Supporting Information). The loss of SO from DP-6 yields m/z 322
2
which is diagnostic for the presence of sulphur dioxide group. In line with this, consecutive
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loss of 1H-benzo[d]imidazole and SO clearly indicates that SO forms bridge between benzo
2
2
imidazole and 3-methylpyridine. The elemental compositions of DP-6 and its product ions
have been confirmed by accurate mass measurements (Supplementary Table S4, see
Supporting Information). Based on these data, DP-6 was identified as 2-((3-methyl-4-
(2,2,2-trifluoroethoxy) pyridin-2-yl) methyl sulfonyl)-1H-benzo[d] imidazole.
+
DP-7 ([M+H] , m/z 402)
The degradation product, DP-7 was formed under oxidation condition. The mass difference
between the protonated drug (m/z 370) and DP-7 (m/z 402) is 32 u which suggests the
addition of two oxygen atoms to the degradant. The positive ion in-source CID MS/MS
+
spectrum of [M+H] ions (m/z 402) of DP-7(Rt = 11.12 min) shows characteristic product
ions at m/z 322 (loss of SO ), m/z 384 (loss of H O), m/z 268 (loss of 1H-benzo[d]imidazole
3
2
and oxygen), m/z 222 (loss of C H N O S), m/z 204 (loss of H O from m/z 222), and m/z 119
7
4
2
2
2
(
loss of C H F NO S). (Supplementary Scheme S8 and Figure 5(d), see Supporting
8 8 3 4
Information). The peak at m/z 322 is a diagnostic ion for the presence of SO group and also
2
indicate that it forms bridge between imidazole ring 2,3-dimethyl-4-(2,2,2-trifluoroethoxy)
pyridine moiety. Product ion at m/z 384 and m/z 119 clearly indicates that oxidation occur at
nitrogen of pyridine ring. The elemental compositions of DP-7 and its product ions have been
confirmed by accurate mass measurements (Supplementary Table S5, see Supporting
Information). Based on these data, DP-7 was identified as 2-((1H-benzo[d]imidazol-2-
ylsulfonyl) methyl)-3-(4-methyl)-4-(2,2,2-trifluoroethoxy) pyridine 1-oxide.
+
DP-8 ([M+H] , m/z 338)
+
In-source CID spectrum of [M+H] ion of DP-8 (m/z 338) (Rt = 27.93 min) shows
diagnostic product ions m/z 322 (loss of oxygen), m/z 254 (loss of 1,1,1-triflouroethane), m/z
1
63(loss of C H N from m/z 254), (Supplementary Scheme S9 and Figure 5(e), see
6 5
‘
’
Supporting Information) for the presence of O group. The product ion at m/z 163 clearly
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indicates that oxidation occur at nitrogen of benzo imidazole ring. The elemental
compositions of DP-8 and its product ions have been confirmed by accurate mass
measurements (Supplementary Table S5, see Supporting Information). Based on these
data, DP-8 was identified as 2-(3-methyl-2-methylene-4-(2, 2, 2-trifluoroethoxy) pyridine-
1
(2H)-yl)-1H-benzo[d]imidazole 3-oxide.
6
.4 Isolation of DP-2
The degradation product DP-2(Rt=14.2) was isolated by injecting 500 µL (30 mg/mL) of
acidic degradation solution into the semi preparative HPLC. The isolated compound was
1
concentrated for H-NMR study (Supplementary figure S1, see Supporting Information).
1
The H NMR data of DP-2 was found to be compatible with the proposed structure (Scheme
S3).
1
H NMR (500 MHz, CD OD) δ 8.21 – 8.08 (m, 1H), 7.48 – 7.26 (m, 2H), 7.15 – 7.07 (m,
3
2
H), 6.98 – 6.82 (m, 1H), 4.59 (dd, J = 16.5, 8.3 Hz, 3H), 4.53 (s, 2H), 2.17 (s, 3H).
7
. Procedure for synthesis of DP-6 and DP-7
To synthesize the DP-6 and DP-7, 1 mM and 2 mM m-CPBA (172mg) was added separately
to each of the two solutions of lansoprazole (369 mg, 1 mM) in 3mL of DCM, respectively.
The reaction mixture was stirred at room temperature for overnight. After the reaction was
completed, the reaction mixture was quenched with NaHCO followed by extraction with
3
ethyl acetate (15 mL) and the organic layer was separated. The organic layer was dried over
on anhydrous magnesium sulfate and concentrated under vacuum to get dry product. The
residue was purified by silica gel column chromatography (DCM–MeOH, 9.5:0.5 %) to
afford pure products DP-6 and DP-7 as a white solid. Pure solid compounds were subjected
1
to H NMR experiment study using deuterated solvents CD OD and CDCl + CD OD
3
3
3
1
mixture for DP-6 and DP-7, respectively. The H NMR data confirmed the DP-6 and DP-7
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(Supplementary figure S2, figure S3, Scheme S7 and Scheme S8, see Supporting
Information).
1
DP-6: H NMR (400 MHz, CD OD) δ 7.94 (d, J = 5.7 Hz, 1H), 7.58 (s, 2H), 7.36 – 7.29 (m,
3
2
H), 6.89 (d, J = 5.7 Hz, 1H), 4.95 – 4.78 (m, 2H), 4.58 (q, J = 8.3 Hz, 2H), 2.17 (s, 3H).
1
DP-7: H NMR (300 MHz, CD OD+CDCl ) δ 7.87-7.94 (m, 1H), 7.55-7.64 (m, 2H), 7.26 –
3
3
7
7
7
.36 (m, 2H), 6.97-6.99 (d, J = 7.3 Hz, 1H), 4.50 – 4.59 (m, 2H), 3.26 (s, 2H), 2.30 (s, 3H).
. Mechanism for the formation of degradation products
.1. Hydrolytic Degradation products
Formation of DP-1 and DP-3 can be explained by abstraction of an acidic proton by ‘N’ of
the imidazole ring followed by nucleophilic attack of pyridine and cleavage of C-S bond to
form an intermediate with free S-OH group. The consecutive losses of ‘SO’ and H O
2
molecules can lead to the formation of DP-1 and DP-3, respectively (Scheme 2). Formation
of DP-2 can be explained by removal of oxygen from the drug which may involve a radical
[
21]
cation mechanism
(Scheme 2). DP-4 can be formed by a mechanism involving a
nucleophilic attack by hydroxide anion on trifluoro carbon followed by loss of CF OH
3
(
Scheme 2). Formation of DP-5 can be explained by hydrolysis, leading to O-CH CF bond
2 3
cleavage followed by loss of C HF O (Scheme 3).
2
3
7
.2. Oxidative Degradation products
The oxidative degradation product DP-6 can be explained by a nucleophilic attack of
peroxide on sulphur of the drug followed by H O loss to form DP-6. The intermolecular
2
nucleophilic attack of ‘N’ of pyridine on imidazole carbon of DP-6 and cleavage of C-S bond
to form an intermediate with the free SO H group followed by loss H SO to form DP-8. The
2
2
2
DP-7 is the N-oxide of DP-6 (Scheme 3).
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8
. MTT assay
Degradation products DP-2, DP-6 and DP-7 were evaluated for toxicity on HEK-293 and
RWPE-1 cell lines. Lansoprazole has exerted 3.1% loss of viability at 100 µm concentration
whereas DP-2, DP-6 and DP-7 have displayed 20.8, 46.5, 12 % loss of viability in RWPE1
cell line. Similarly, lansoprazole has exerted 8.5% loss of viability at 100 µm concentration.
DP-2 DP-6 and DP-7 has displayed 19.7, 39.1, 12 % loss of viability in HEK293 cells
(Figure 6).
9
. DNA binding studies
Lansoprazole, DP-2, DP-6 and DP-7 have shown maximum absorption at around 272, 283
and 285 nm. On addition of equal aliquots of ctDNA, the ligand absorption peaks exhibited
hyperchromicity accompanied by a slight blue shift. It is known that hypochromicity along
with bathochromic shift (red shift) is considered as an evidence of intercalative mode of
binding. The extent of hypochromicity of the absorption band can be taken as a measure of
[
21]
intercalative binding strength.
the interaction between the electronic states of the complex and the DNA bases.
UV-visible titration data, the LAN molecules ctDNA binding constants were found to be in
The absorption band hypochromicity is usually attributed to
[
22]
From
4
-1
the range of ≈ 2 x 10 M . The hyperchromic effect may also be due to the electrostatic
interaction between lansoprazole, DP-2, DP-6, DP-7 molecules and the phosphates in the
[23]
backbone at the periphery of the double helix ctDNA.
Hyperchromisity of ligand
absorption bands on addition of ctDNA, indicate that the lansoprazole, DP-2, DP-6, DP-7
molecules interact with DNA and they may bind electrostatically to the surface of ctDNA.
But only with UV-Visible titration data alone, is not possible to understand the mode of
complex molecules binding to DNA. The representative UV-visible spectra obtained on
interaction of complex with ctDNA were shown in Figure 7 (a).
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Fluorescence titration is yet another valuable technique for understanding the binding mode
of small molecules with DNA and to study the electronic environment around the DNA-
[
24]
complex at comparatively lower concentrations.
Since the complexes are fluorescent, their
interaction with DNA can be monitored at low concentration. Lansoprazole, DP-2, DP-6 and
DP-7 emission spectra shows a prominent peak in the range from 308 nm to 368 nm. On
addition of equal increments of ctDNA to the solution of the drug and DP-2, DP-6 and DP-7,
the fluorescence emission intensity has increased gradually, which is consistent with the non-
intercalative mode of binding such as electrostatic binding mode (surface binding) that leads
to effective protection of the drug, DP-2, DP-6 and DP-7 by DNA. The hydrophobic
environment inside the DNA helix reduces the drug, DP-2, DP-6 and DP-7 accessibility to
the solvent molecules. As a result, the complex mobility gets restricted at binding site,
leading to a decrease of the vibrational modes of relaxation. This will result in enhancement
[25]
of emission intensity or higher emission intensity.
The representative fluorescence spectra
are shown in Figure 7(b).
In order to understand the effect of the drug, DP-2, DP-6 and DP-7 on ctDNA conformation,
circular dichroism studies were performed. The CD spectra of ctDNA exhibited positive and
negative bands at 275 nm and 245 nm respectively. At an average all the drug, DP-2, DP-6
and DP-7demonstrated around 10-12 % hypochromicity of positive CD band on interaction
with CT DNA. On addition of 10 μM the drug, DP-2, DP-6 and DP-7 molecules to ctDNA
(at 1:1 ratio), the positive CD band showed hypochromicity, indicating unwinding of ctDNA
on interaction with them [25]. Further, on doubling the concentration (at 1:2 ratio), the
positive band intensity increased slightly. Hyperchromicity of the positive CD band is an
indication of stabilization of the DNA-ligand complex upon further interaction between the
[
26]
bound drug, DP-2, DP-6 and DP-7.
CD studies indicate that the drug, DP-2, DP-6 and
DP-7 interact with DNA and bring small changes in DNA conformation. It was found that,
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the extent of change in DNA conformation is more with DP-7 compared to other molecules.
The negative CD band intensities reduced in case of all the complexes, but maximum change
was noticed with DP-7, emphasizing the role of these molecules in effecting the DNA helix.
The CD spectra obtained with the drug, DP-2, DP-6 and DP-7 and ctDNA was shown in
Figure 7(c).
The spectroscopic studies indicate that the drug, DP-2, DP-6 and DP-7 interact well with
DNA. In order to understand the nature of interaction of the drug, DP-2, DP-6 and DP-7 with
double helical ctDNA, viscosity studies were carried out. Relative viscosity of DNA
increases when the molecules intercalate with DNA. The enhancement of viscosity is due to
increase in the axial length of DNA helix on molecule’s intercalation. Hence, the
[27]
intercalation of a compound results in an increase of the viscosity of DNA solutions
whereas, the reduction in the relative viscosity is typically observed with covalent DNA
[
28]
binding.
Viscosity does not change or show very less change when molecules bind to the
[
29]
surface of DNA.
On complex intercalation, the axial length of DNA helix must increase to accommodate the
binding complex molecules, which leads to the increase of DNA solution viscosity. Itis
noticed that the viscosity of DNA solution increased slightly on addition of the drug, , DP-2,
DP-6 and DP-7.Minor positive or negative changes in DNA solution viscosity are observed
[
29]
when binding occurs in the DNA grooves
As shown in Figure 7 (d), on the interaction of
the drug, DP-2, DP-6 and DP-7 molecules with DNA exhibited a slight increase in the
[
30]
viscosity, which is not as pronounced as observed for classical intercalators
and are
[
31]
consistent with substrates that bind to ctDNA through a groove-binding mode. Among all
the DPs, DP-2, DP-6 and DP-7, change in viscosity was more in case of DP-7 whereas, with
the others DPs, change DNA viscosity was comparatively low, may due to lesser interaction
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with ctDNA. But on the other hand, when 15 µM of Et Br was added to ctDNA, the viscosity
of DNA has increased continuously. This is due to intercalation of Et Br molecules with
1
/3
ctDNA. The graph plotted between (ŋ/ŋ ) and complex/ctDNA was shown in Figure 7(d).
o
Spectroscopic and viscosity studies indicate that the the drug, DP-2, DP-6 and DP-7
moleculesinteract well with DNA and they exhibit groove binding mode of interaction with
DNA.
8
. Conclusion
Forced degradation studies of lansoprazole, carried out as per ICH guide lines, gave rise to a
total of 8 degradation products. Five of these DPs are hitherto unknown. The major
degradation products, DP-2 was isolated by semi preparative HPLC and DP-6 and DP-7 were
synthesized and characterized by 1H-NMR. The cytotoxic effect of degradation products was
tested on normal human cells such as HEK 293 (embryonic kidney cells) and RWPE-1
(normal prostate epithelial cells) by MTT assay. From the results of cytotoxicity, it was found
that lansoprazole as well as its degradation products (DP-2, DP-6 and DP-7) were nontoxic
up to 50 μM concentration and overall it was found that the degradation products showed
slightly higher cytotoxicity when compared with that of the cytotoxicity of lansoprazole. In
order to understand the nature of binding and interaction of DP-2, DP-6 and DP-7 molecules
with double helical ctDNA, spectroscopic and viscosity studies were performed. These
studies indicate that DP-2, DP-6 and DP-7 molecules interact with ctDNA and they may bind
to its surface.
Acknowledgement
The authors thank Dr. S Chandrasekhar, Director, IICT, Hyderabad for facilities and their
cooperation. Financial support was provided partially by CMET (CSC0110) and AARF
(CSC0406) projects. G.S is thankful to U.G.C, New Delhi, for awarding Junior Research
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Fellowship. R.M.B and S. U are thankful to CSIR, New Delhi, for awarding Senior Research
Fellowship and Junior Research Fellowship, respectively.
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Scheme 1: Proposed fragmentation pathway of the protonated drug (m/z 370)
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Scheme 2: Probable mechanism for the formation of degradation products (DP-1, DP-2, DP-
, and DP-4) under acid and base hydrolysis conditions
3
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Scheme 3: Probable mechanism for the formation of degradation products (DP-5, DP-6, DP-
and DP-8) under neutral hydrolysis and oxidation conditions
7
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Table 1: UPLC/ESI/HRMS data of the drug and its degradation products
Degradation Retention Proposed molecular
Error
(ppm)
+
Observed(m/z) Calculated (m/z)
products
time(min)
formula [M+H]
+
LAN
20.45
C H F N O S
370.08320
370.08316
-0.10
16
15
3
3
2
+
DP-1
DP-2
DP-3
DP-4
DP-5
DP-6
DP-7
DP-8
14.41
33.85
33.14
14.54
11.65
11.05
23.13
27.94
C H F N O
322.11568
354.08791
352.07328
302.09642
272.08487
386.07834
402.07002
338.11234
322.11617
354.08824
352.07259
302.09577
272.08521
386.07807
402.07299
338.11109
1.52
0.93
-1.95
-2.10
1.24
0.69
7.38
-3.69
16
15
3
3
+
C H F N OS
16
15
3
3
+
C H F N OS
1
6
13
3
3
+
C H N O S
1
5
16
3
2
+
C H N OS
1
4
14
3
+
+
C H F N O S
C H F N O S
16
15
3
3
3
16
15
3
3
4
+
C H F N O
2
16
15
3
3
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Figure 1. UPLC-ESI-MS TICs of lansoprazole
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