Author's Accepted Manuscript
LC, MSn and LC-MS/MS studies for the characterization of degradation products of amlodipine Ravi N. Tiwari, Nishit Shah, Vikas Bhalani, Anand Mahajan
PII: DOI: Reference:
S2095-1779(14)00065-3 http://dx.doi.org/10.1016/j.jpha.2014.07.005 JPHA230
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Journal of Pharmaceutical Analysis
Received date: Revised date: Accepted date:
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Cite this article as: Ravi N. Tiwari, Nishit Shah, Vikas Bhalani, Anand Mahajan, LC, MSn and LC-MS/MS studies for the characterization of degradation products of amlodipine, Journal of Pharmaceutical Analysis, http://dx.doi.org/10.1016/j. jpha.2014.07.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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LC, MSn and LC-MS/MS studies for the characterization of degradation products of
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amlodipine
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Ravi N. Tiwari*1, Nishit Shah1, Vikas Bhalani1, Anand Mahajan2
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1
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Technology Management Shirpur Dist. Dhule 425405 Maharashtra, India
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2
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India.
Department of Pharmaceutical Chemistry, SVKM’s NMIMS, School of Pharmacy and
Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmacy, Pune, Maharashtra,
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*Corresponding author.
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SVKM’s NMIMS,
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School of Pharmacy and Technology Management,
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Near Bank of Tapi River, Agra-Mumbai Road
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Babulde, Shirpur- 425405
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Dist. Dhule, Maharashtra
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India
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Tel.: +91-2563-286545; fax: +91-2563-286552
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E-mail address:
[email protected].
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Abstract
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In the present study, comprehensive stress testing of amlodipine (AM) was carried out according
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to International Conference on Harmonization (ICH) Q1A(R2) guideline. Amlodipine was
27
subjected to acidic, neutral and alkaline hydrolysis, oxidation, photolysis and thermal stress
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conditions. The drug showed instability in acidic and alkaline conditions, while remained stable
29
to neutral, oxidative, light and thermal stress. A total of nine degradation products (DPs) were
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formed from AM, which could be separated by the developed gradient LC method on a C18
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column. The products formed under various stress conditions were investigated by LC-MS/MS
32
analysis. The previously developed LC method was suitably modified for LC-MS/MS studies by
33
replacing phosphate buffer with ammonium acetate buffer of the same concentration (pH 5.0). A
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complete fragmentation pathway of the drug was first established to characterize all the
35
degradation products using LC-MS/MS and multi-stage mass (MSn) fragmentation studies. The
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obtained mass values were used to study elemental compositions, and the total information
37
helped in the identification of DPs, along with its degradation pathway.
38 39 40 41 42 43 44 45 46
Keywords: Amlodipine; LC–MS/MS; Characterization; Degradation pathway.
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1. Introduction
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The stability testing of drugs under different stress conditions is indispensable during the drug
49
development process. In addition, stability testing guidelines stated by International Conference
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on Harmonization (ICH) and other international agencies [1,2] require the reporting,
51
identification and characterization of degradation products (DPs). But DPs generated during
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storage may be in very low levels; hence, stress studies are suggested to generate them in higher
53
amounts [3]. Still sometimes it is very difficult to identify these DPs from the generated stressed
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mixture due to their lower amounts. Therefore, hyphenated techniques like LC-MS is currently
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extensively used for this purpose [4,5]. Amlodipine (AM) (Fig. 1) is chemically 2-[(2-
56
aminoethoxy) methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5 pyridinedicarboxylic acid 3-
57
ethyl 5-methyl ester. It acts as a calcium channel blocker and inhibits the transmembrane influx
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of calcium ions into vascular smooth muscles and cardiac muscle, hence, used as an
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antihypertensive for the treatment of angina.
60
A thorough literature revealed that there exist several reports on bioanalytical method
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development and pharmacokinetic studies of amlodipine [6,7]. A wide array of articles are
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reported on stability-indicating HPLC method for the estimation of single drug amlodipine in
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pure bulk samples, dosage forms as well as in combination with other drugs such as atorvastatin,
64
benazepril, perindopril, olmesartan, valsartan, hydrochlorthiazide, etc [8-16]. A few articles are
65
available on photostability studies of AM, where the thermal degradation kinetics are reported
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from 0 h to 108 h [17-20]. There exists report on isolation and characterization of three thermal
67
degradation products of AM, which were formed due to the intramolecular reactions and
68
cyclization [21]. Moreover, there exist few reports on isolation and characterization of process
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related impurities of AM and accelerated stability studies. From the above literatures, the
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reported masses and fragmentation pattern of all the degradation products/impurities of AM are
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quite different from our degradation products of AM formed under different stress conditions. In
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fact, the reported masses of impurities and their fragment ions were compared with the masses of
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DP’s proposed in this article and they all were found non-resembling with each other. Moreover,
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as per previous reports [22-26] the masses of six different process related impurities such as m/z
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538, 569, 406, 394, 408 and 422 were not matching with the masses of any of the DP’s of AM.
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Hence, the endeavor of our present study was to: (i) carry out the stress studies on AM under the
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ICH defined conditions; (ii) separate the degradation products by HPLC; and (iii) characterize
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and establish degradation pathway of all the degradation products with the help of LC–MS/MS.
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2. Experimental
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2.1. Drug and reagents
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Pure AM was obtained as gratis sample from Osaka Pharmaceuticals Pvt. Ltd. (Vadodara, India).
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Analytical reagent (AR) grade formic acid and sodium hydroxide (NaOH) were purchased from
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S.D. Fine-Chem Ltd. (Mumbai, India), hydrochloric acid (HCl) and HPLC grade methanol
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(MeOH) from Merck Specialities Pvt. Ltd. (Mumbai, India) and hydrogen peroxide (H2O2) from
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Qualigens Fine Chemicals Pvt. Ltd. (Mumbai, India). Ultra-pure water obtained from Millipore
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water purification system (Molsheim, France) was used throughout the studies..
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2.2. Equipment
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A high-performance liquid chromatography (HPLC) system from PerkinElmer (Shelton, CT,
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USA) was used for the LC studies, which consisted of an on-line degasser, a sample injector
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(Rheodyne sample loop 20 mL), a UV-visible detector (Series 200), a pump (Reciprocating,
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series 200), and a computer system loaded with Total Chrome Navigator (version 6.3.1)
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software. The LC-MS system was control by Xcalibur software (version 2.0) consisted of LCQ
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Fleet and TSQ Quantum Access with Surveyor Plus HPLC System (Thermo, San Jose, USA).
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Precision water baths equipped with MV controller (Thermostatic Classic Scientific India Ltd.
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Mumbai, India) 90 were used for stress studies. Degradation experiments in acid, base, and
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neutral conditions were performed using a dry-bath (Labline Sun Scientifics Ltd. New Delhi,
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India). The solid state thermal stress studies were carried out in dry-air oven (NSW Limited,
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New Delhi, India). Other equipments used were a pH meter (Labindia, Mumbai, India), a
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weighing balance (Shimadzu, AUX220, Kyoto, Japan), and a micro-pipette (Erba Biohit,
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Mannheim, Germany). In all studies, separations were achieved on a C-18 column (250 mm ×
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4.6 mm i.d., particle size 5 µm; Kromasil (Eka Chemicals AB, Bohus, Sweden). Photolytic
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studies were carried out in a photostability chamber (Thermolab, 95 Th-400G Mumbai, India),
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set at 40 ± 1 °C/75% ± 3% RH in accordance with option two of the ICH guideline Q1B [27].
106 107
2.3. Stress degradation studies
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Stress degradation studies were carried out on AM as per ICH Q1A(R2) prescribed stress
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conditions. As per ICH the stress degradation studies are conducted to determine stability of drug
110
substances or drug products by knowing degradation pathways to identify the likely degradation
111
products. Moreover, the guideline explicitly requires conduct of forced degradation studies under
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a variety of conditions like, wide range of pH, light, oxidation and dry heat. AM along with
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various stressors was exposed to different temperatures with the objective to achieve 15-20%
114
degradation and for this the stress conditions were systematically optimized in the initial stages.
115
AM was subjected to acidic (1 M HCl, 30 min, 80 °C), basic (1 M NaOH, 1 h, 80 °C), neutral
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(H2O, 2 h, 80 °C), oxidative (H2O2, 15%, 48 h) at room temperature, thermal (50 °C, 48 h) and
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photolytic (1.2 × 106, lx h of fluorescent light and 200 Wh/m2 UV-A light, 14 days) stress
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conditions.
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2.4. Sample preparation for HPLC and LC–MS analysis
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The stressed samples of AM collected during desired time intervals were adequately diluted 10
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times with water before injection into the HPLC. Moreover, the samples were filtered through a
123
0.22 µm membrane filter before making injections. A mixture containing all the degradation
124
products was also prepared for final HPLC separation and LC-MS analysis. In all 100 µg/mL
125
samples were prepared and injected, so as to adequately compare the percentage degradation
126
with the standard unstressed drug sample of same concentration.
127 128
2.5. Separation studies
129
A concentration of 10 µg/mL of AM was scanned from 200 to 400 nm in the ultraviolet
130
spectrophotometer to select UV wavelength suitable for HPLC analysis. From the spectra, 240
131
nm wavelength was found to show maximum absorbance. Initially separation of AM was
132
attempted by varying relative ratio of methanol to phosphate buffer, along with the variation in
133
buffer pH. All the separations were carried out using C18 column. Separation studies were first
134
carried out on all reaction solutions individually, and then on a mixture of degraded drug
135
solutions.
136 137 138
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2.6. MS, MSn and LC-MS studies
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The fragmentation profile of the drug was established by carrying out mass spectral studies on
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AM, while multi-stage (MSn) mass studies were carried out up to MS6 to determine origin of
142
each fragment. AM was subjected to MS system at a concentration of 10 µg/mL prepared in
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methanol in positive electrospray ionization (ESI) mode in the mass range of 50-800 Da. High
144
purity nitrogen was used as the nebulizer and auxiliary gas. The interpretation data of fragments
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obtained in MS studies for amlodipine are listed in Table 1. A previously developed LC method
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was used to analyze degraded drug samples on LC-MS system, and provided the phosphate
147
buffer was replaced by 10 mM ammonium acetate buffer of pH 5.0. The mass parameters were
148
properly tuned to get high intensity peaks of molecular ions and daughter ions of degradation
149
products. The fragments of all DPs obtained in LC-MS studies along with best possible
150
molecular formula, ring plus double bonds (RDB) and chemical formula of major fragments are
151
shown in Table 2.
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3. Results and discussion
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3.1. HPLC analysis
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During the initial separation trials, methanol and water were adopted as a mobile phase on
156
different gradient LC modes, but the separation of drugs and degraded products was not
157
optimum, this may be due to the poor buffering capacity of water. Then in the later trials water
158
was replaced with phosphate buffer. Stronger organic modifier viz., acetonitrile was not utilized
159
during the HPLC analysis, since the methanol showed satisfactory resolution between drugs and
160
degraded products, hence it was kept unchanged. Logical modifications like change in pH and
161
gradient program were made to improve resolution between drug and degradation products and
162
also in between the degradation products. Finally an acceptable separation was achieved using
163
methanol and phosphate buffer (10 mM, pH 5.0) in a gradient mode (Tmin/A:B; T0/10:90;
164
T8/50:50; T13/60:40; T25/75:25; T27/10:90;T30/10:90) on C18 column at room temperature. The
165
detection wavelength was 240 nm, flow rate was 1 mL/min and injection volume was 20 µL.
166 167
3.2. Stress decomposition behavior
168
A total of nine DPs AM1-AM9 were formed from amlodipine during the stress degradation
169
study. Out of these AM2, AM3, AM5, AM6 and AM7 were formed as major DPs, while AM1,
170
AM4, AM8 and AM9 were minor DPs. Among all the DP’s AM1, AM6 and AM9 were formed
171
in both acidic and alkaline conditions, while the products AM2, AM3, AM4 and AM5 were
172
formed only under alkaline stress condition, and AM7 was the product of acidic stress. A total of
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16.41% degradation was observed in acid stress, while in alkaline stress 27.53% degradation was
174
recorded. The chromatogram showing separation of AM and all the DPs is shown in Fig. 2. The
175
degradation products of AM are denoted as AM1 to AM9 in accordance with the sequence in
176
which the peak appeared from left to right in the chromatogram. From the chromatogram AM
177
was found to be more susceptible to alkaline stress.
178 179
3.3. Mass fragmentation pattern of AM
180
A total of six fragments were formed from amlodipine during its MS studies. A multi-stage
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(MSn) mass fragmentation study was also carried out up to MS6, to determine the origin of each
182
fragment, which could help to propose the fragmentation pathway of AM. Line spectra of AM,
183
obtained in MS and MSn studies, are shown in Fig. 3. The fragment with m/z 431.23 was formed
184
as a potassium adduct, since its mass was ~28 dalton (Da) higher than the molecular ion peak of
185
AM (m/z 408.90). The fragments with m/z 392.23 was formed on loss of ammonia (NH3) from
186
AM, while in the subsequent step the fragment with m/z 392.23 on cleavage of methyl but-2-
187
enoate moiety resulted in the formation of m/z 294.13. There onwards a parallel pathway was
188
initiated from the fragment with m/z 294.13, here a direct loss of methoxy ethane entity led to
189
the formation of a daughter ion with m/z 238.10, while loss of 1-chloro-2-methyl benzene and
190
propionic acid moieties resulted in the formation of the last daughter ion with m/z 102.13.
191
Finally the fragment with m/z 238.10 underwent cleavage of hydrogen chloride entity and
192
resulted in the formation of ion with m/z 208.14. The mass fragmentation pattern of amlodipine
193
is shown in Fig. 4.
194 195
3.4. Characterization of degradation products
196
The data obtained in MS, MSn and LC-MS/MS studies were systematically utilized for the
197
structure elucidation of degradation products of AM.
198
3.4.1. AM1 (m/z 363.08)
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As shown in Fig. 2, AM1 was formed as the first degradation product and the most polar DP
200
among all because of its immediate elution after the solvent front at 2.87 min of retention time.
201
LC-MS/MS line spectra of AM1 in Figure 5 showed the formation of fragments with m/z
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363.08, m/z 292.92, m/z 226.83, m/z 157.00 and m/z 112.92, and among these the m/z 363.08
203
and m/z 157.00 were formed as molecular ion and base ion peak, respectively, since 472.75
204
cannot be considered as molecular ion peak of AM1 because of its huge difference in the
205
molecular mass as compared to molecular mass of AM (m/z 409). Hence, AM1 was obtained
206
from the drug by the loss of hydrogen chloride entity of benzene ring and a methyl group from
207
the ethyl ester moiety at third position of 1,4-dihydropyridine ring. The fragmentation pattern of
208
AM1 is shown in Fig. 6.
209
3.4.2. AM2 (m/z 392.67)
210
A total of five line fragments were formed from AM2 viz., m/z 392.67, m/z 347.08, m/z 305.92,
211
m/z 158.92 and m/z 113.08. Among these the fragment with m/z 392.67 was the molecular ion
212
peak, while m/z 347.08 was the base peak. AM2 was formed on loss of ammonia (NH3) from
213
terminal position of the side chain attached at second position of 1,4-dihydropyridine ring. The
214
subsequent steps in the fragmentation pathway of AM2 is shown in Fig. 6, which involved
215
conversion of ester to aldehyde (m/z 347.08) intermediate, followed by formation of fragments
216
with m/z 305.08, m/z 158.11 and m/z 113.08, due to the losses such as ethanol, methyl formate,
217
chlorobenzene and methoxymethane entities. The impurity with m/z 392.2 reported by A.P.
218
Kumar et al [22] was formed during accelerated stability studies. Even though this impurity had
219
the same mass as that of AM-2, but showed different MS/MS fragmentation pattern, hence, the
220
proposed structure of AM-2 is different. The major ions formed during MS/MS of this impurity
221
(m/z 392.2) were m/z 360.2, 346.5, 318.1 and 286.2, while the fragment ions formed from AM-2
222
were m/z 347.08, 305.92, 158.92 and 113.08. The fragmentation pattern of AM2 is shown in Fig.
223
6.
224
3.4.3. AM3
225
The structure of AM3 degradation product was not elucidated, probably due to its poor
226
ionization behavior during LC-MS/MS analysis in both ESI positive and ESI negative ionization
227
modes.
228 229
230 231
3.4.4. AM4 (m/z 399.92)
232
As shown in Fig. 5, AM4 had the molecular ion peak of 399.92, with the base peak of 331.92.
233
The drug (AM) underwent loss of a methyl group from ethyl 1,4-dihydropyridine-3-carboxylate
234
and resulted in the formation of AM4. There onwards AM4 followed a parallel fragmentation
235
pathway involving loss of hydrogen chloride and acetate moiety to form ions with m/z 363.83
236
and m/z 331.92, respectively. A loss of another acetate moiety and a methyl group from m/z
237
331.08 resulted in the formation of fragment with m/z 263.08. The formation of other minor
238
fragments along with their structures and masses is depicted in Fig. 6.
239
3.4.5. AM5 (m/z 380.83)
240
A total of seven fragments were formed from AM5, where fragment with m/z 380.83 was
241
formed as base peak as well as molecular ion peak. A direct cleavage of terminal methamine
242
entity of the side chain attached to second position of 1,4-dihydropyridine ring, resulted in the
243
formation of AM5. Ion with m/z 302.25 was formed from AM5 on loss of hydrogen chloride and
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dimethyl ether entities. Ion with m/z 302.25 underwent loss of two ring protons and a methyl
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group attached to sixth position of 1,4-dihydropyridine ring and led to the formation of fragment
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with m/z 284.09. Subsequent losses such as acetate and methylene entity resulted in
247
dihydropyridine ring opening to form ion with m/z 222.00. The structures and masses of
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daughter ions with m/z 192.00, m/z 175.08 and m/z 142.92 formed in the same pathway are
249
shown in Fig. 6.
250
3.4.6. AM6 (m/z 393.00)
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The mass of AM6 was found to be m/z 393.00, which suggests loss of a methyl group from AM
252
along with the charge migration from terminal amine to sixth carbon of 1,4-dihydropyridine ring.
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AM6 in its subsequent steps lost ethyl carboxylate entity to form daughter ion with m/z 306.42,
254
which on ring opening and cleavage of larger fragment of around 216 Da resulted in the
255
formation of ion with m/z 90.92. On the other hand, fragment with m/z 306.42 on loss of
256
methoxyethane and hydrogen chloride lead to the formation of fragment ion with m/z 218.17,
257
which followed a minor parallel pathway with the formation of daughter ions with m/z 158.75
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and m/z 112.75 on loss of methylcarboxylate and benzene moieties, respectively. The proposed
259
structures of all the fragments of AM6 are shown in Fig. 7.
260
3.4.7. AM7 (m/z 380.75)
261
The ester functional group at third position of 1,4-dihydropyridine underwent hydrolysis with the
262
loss of ethyl group and resulted in the formation of AM7 with m/z 380.75. The loss of 2-
263
aminoethanol from second position of 1,4-dihydropyridine along with two ring protons resulted
264
in the formation of fragment with m/z 352.83, which on loss of a methyl group led to the
265
formation of a fragment with m/z 335.92. Then m/z 335.09 followed a parallel fragmentation to
266
form ion with m/z 230.83 on loss of hydrogen chloride and ethylcarboxylate entities, while ion
267
of m/z 163.17 was formed on loss of chlorobenzene, methylcarboxylate and two ring protons.
268
The proposed structures of all the fragments of AM7 are shown in Fig. 7.
269
3.4.8. AM8 (m/z 407.17)
270
As shown in the Fig. 5, AM8 had the mass of m/z 407.13, which indicates around mass of 2 Da
271
lesser (around two protons) than the mass of drug (AM). AM8 would be the result of one of the
272
main degradation pathways of amlodipine such as oxidative aromatization of dihydropyridine
273
fragment to the pyridine moiety, which takes place in solution and solid states as well as under
274
photolytic conditions [28]. One of the degradation products reported by Damale et al [29] had
275
the same mass of m/z 407 formed under oxidative and acidic stress conditions. The first fragment
276
of AM8 had the mass of m/z 286.08 formed on losses of ammonia, ethylcarboxylate and
277
hydrogen chloride. Then the fragment with m/z 286.08 on loss of methylcarboxylate resulted in
278
the formation of ion with m/z 231.25, which in its subsequent step lost ring nitrogen, methyl
279
group and a benzene entity to form daughter ion with m/z 123.92. The proposed structures of all
280
the fragments of AM8 are shown in Fig. 7.
281
3.4.9. AM9 (m/z 395.00)
282
A direct cleavage of methyl group from sixth position of 1,4-dihydropyridine ring of the drug
283
(AM) resulted in the formation of AM9 with m/z 395.00. There onwards AM9 followed a
284
parallel fragmentation pathway. In its subsequent steps loss of methanamine and o-demethylation
285
of the side chain resulted in the formation of fragments with m/z 366.75 and m/z 350.08
286
respectively. On the other hand, AM9 on loss of ethylcarboxylate and ammonia from the side
287
chain resulted in the formation of ion with m/z 302.17. The same degradation product with m/z
288
395 was reported by Damale et al [29] was formed under alkaline stress, while in our case this
289
DP was formed in acidic and alkaline conditions. The proposed structures of all the fragments of
290
AM9 are shown in Fig. 7.
291 292
3.5. Degradation pathway of AM
293
Based on the data obtained from the line spectra of LC-MS/MS studies of each degradation
294
product, a final fragmentation pathway of AM was established. The degradation pathway of
295
amlodipine is shown in Fig. 8 along with proposed structures and masses of all the DPs.
296 297 298
299
4. Conclusions
300
Stress degradation studies were carried out on amlodipine as per mentioned ICH guidelines,
301
which provided information on the degradation behavior of AM under the acidic, basic, neutral,
302
oxidative, photolytic and thermal stress conditions. It was revealed from HPLC analysis that a
303
total of nine degradation products were formed from AM, among these AM3 remained
304
unidentified probably due to its poor ionization behavior, while all the remaining DPs were
305
successfully characterized with the help of LC-MS/MS analysis. The above stated study was able
306
to explore various useful information which is not yet reported in the literatures on amlodipine
307
such as: (a) sensitivity of AM to different stress conditions, (b) total number of degradation
308
products of AM along with the nature of DPs, (c) mass fragmentation pathway of AM as well as
309
its DPs, (d) MSn study of the drug to determine origin of each mass fragment, (e) degradation
310
pathway of the drug.
311 312 313 314 315 316 317 318 319 320
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358 359 360 361
16. B. Marciniec, M. Ogrodowczyk, Thermal stability of 1,4-dihydropyridine derivatives in solid state. Acta Pol. Pharm. 63 (2006) 477-484. 17. G. Ragno, E. Cione, A. Garofalo, et al., Design and monitoring of photostability systems for amlodipine dosage forms. Int. J. Pharm. 265 (2003) 125-132.
362
18. Y. Kawabe, H. Nakamura, S. Suzuki, et al., Photochemical stabilities of some
363
dihydropyridine calcium-channel blockers in powdered pharmaceutical tablets. J. Pharm.
364
Biomed. Anal. 47 (2008) 618-624.
365 366
19. G. Ragno, A. Garofalo, C. Vetuschi, Photodegradation monitoring of amlodipine by derivative spectrophotometry. J. Pharm. Biomed. Anal. 27 (2002) 19-24.
367
20. R.J.V. Eranki, G. Inti, V. Jayaraman, et al., New stability indicating method for
368
quantification of impurities in amlodipine and benazepril capsules by validated HPLC,
369
Am. J. Anal. Chem. 4 (2013) 715-724.
370 371
21. A.S.L. Devi, Y.S. Rao, M. Satish, et al., Structure elucidation of thermal degradation products of amlodipine, Magn. Reson. Chem. 45 (2007) 688-691.
372
22. G.V.R. Reddy, A.P. Kumar, B.V. Reddy, et al., Separation, identification and structural
373
elucidation of a new impurity in the drug substance of amlodipine maleate using LC-
374
MS/MS, NMR and IR, Croat. Chem. Acta. 83 (2010) 443-449.
375
23. British Pharmacopoeia, British Pharmacopoeial Commission, London, 2010, p 138-140.
376
24. P. Sudhakar, M. Nirmala, J. Moses Babu, et al., Identification and characterization of
377 378 379
potential impurities of amlodipine maleate. J. Pharm. Biomed. Anal. 40 (2006) 605-613. 25. G. Ananchenko, J. Novakovic, J. Lewis. Amlodipine Besylate, Profiles Drug Subst. Excip. Relat. Methodol. 37 (2012) 31-77.
380
26. B. Suchanova, L. Sispera, V. Wsol, Liquid chromatography-tandem mass spectrometry in
381
chiral study of amlodipine biotransformation in rat hepatocytes. Anal. Chem. Acta 573-
382
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383 384 385 386
27. ICH, Stability testing: photostability testing of new drug substances and productsQ1B, in: International Conference on Harmonisation, IFPMA, Geneva, 1996. 28. M.M.M. Sadeghi, H.R. Memarian, A.R. Momeni, Aromatization of 1,4-dihydropyridines with free radical reagents. J. Sci. Islam. Repub. Iran 12 (2001) 141-143.
387
29. S. Damale, D. Bhandarkar, S. Raju, et al., Characterization of products formed by forced
388
degradation of Amlodipine Besylate using LC/MS/MS. Shimadzu Excellence in Science,
389
ASMS 2013-MP06-112.
390 391
Figure Captions:
392
Fig. 1. Structure of amlodipine.
393 394
Fig. 2. Chromatogram showing separation of degradation products of amlodipine (AM) in the mixture of stress sample. A: acid; B: base; N: neutral; RT: retention time.
395
Fig. 3. Line spectra of amlodipine obtained in MS and MSn studies.
396
Fig. 4. Mass fragmentation pattern of amlodipine.
397
Fig. 5. Line spectra of degradation products of amlodipine obtained in LC-MS/MS studies.
398 399
Fig. 6. Degradation products of amlodipine formed under acidic and alkaline stress conditions (AM-1, AM-2, AM-4 and AM-5).
400 401
Fig. 7. Degradation products of amlodipine formed under acidic and alkaline stress conditions (AM-6, AM-7 AM-8 and AM-9).
402
Fig. 8. Degradation pathway of amlodipine.
403 404 405 406 407 408 409 410 411 412 413 414 415 416
Table Captions: Table 1 Interpretation of MS data of fragments of amlodipine. Table 2 LC-MS/MS data of DPs of amlodipine (AM) along with their possible molecular formulae and major fragments.
Cl H3COOC
COOC2H5
O
417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432
H3C
N H
Fig. 1. Structure of amlodipine.
NH2
5
6
AM
3 7 2 1
8
9
4
433 Peaks RT (min) Stress conditions
434 435 436 437 438 439 440 441 442 443 444 445 446 447 448
AM1 2.87 A B
AM2 7.67 B
AM3 8.94 B
AM4 10.12 B
AM5 10.65 B
AM6 11.46 A B
AM7 15.19 A
AM8 25.01 B
AM9 26.32 A B
Fig. 2. Chromatogram showing separation of degradation products of amlodipine (AM) and in the mixture of stress sample. A: acid; B: base; N: neutral; RT: retention time.
MS-1
MS-2
1 MS-3
MS-4
MS-5 4
3
6
5
2
K+ Adduct
MS-6
449 450 451 452 453
Fig. 3. Line spectra of amlodipine obtained in MS and MSn studies.
Cl H3COOC
COOC2H5
O H3C
N H
NH3
Amlodipine 409.15
Cl H3COOC
COOC2H5
O H3C
N H
392.12
Cl COOC2H5
O H2N
294.08
O H3N Cl
102.09
COOC2H5
H2N
238.06
COOC2H5
454 455 456
H3N
208.13
Fig. 4. Mass fragmentation pattern of amlodipine.
AM-2 AM-1
461 2 458 463 AM-4
AM-5
464 465 466 467 468 469 470
467 AM M-6
472 3 473 4 474 5 475 6 476 7 477 8 478 9 479 0
476
AM-7
AM-88
A AM-9
481 478 479 480 481 482 483 484
Fig. 5. Line spectra of o degradatioon products of amlodipinne obtained in LC-MS/M MS studies.
H3COOC
COOCH3
H3C
N H2
292.15 H3COOC
COOCH3
O H3C
N H2
NH2
H3COOC
H3 C
H3COOC
OOC
AM-1 363.19 H3 C HC
N
226.08
N H2
112.05
157.10
CHO Cl H3COOC
COOC2H5
H3COOC
CHO
Cl
Cl
H3COOC
CHO
CHO
O H3 N
O H3 C
N H
H3 C
H3 C
N H
AM-2 392.12
H3 N
113.08
158.11
O
347.09
CH3
N H2
305.08
Cl H3COOC
COOC2H5 Cl O
H3 C
N H
Cl
NH3 H3COOC
Amlodipine 409.15
O N H
H3C
Cl
NH2
263.09
O N
OHC
NH2
331.08 H3COOC
CH3
COO
OHC
106.04 O H 3C
N H
CH
O NH3
H3 N H3COOC
399.16 AM4
O
158.11
CH3
COO
H3N
90.09
O H3C
N H
NH3
363.19
Cl H3COOC
COOC2H5
COOC2H5
H3COOC
COOC2H5
COOC2H5
O H3 C
N H2
AM-5 380.12
485 486 487 488 489
COOH
COOH H3COOC
175.07
H2N H3C
N H2
302.13
N
284.09
H3 N
192.10
222.14
COOC2H5
H3N
142.08
Fig. 6. Degradation products of amlodipine formed under acidic and alkaline stress conditions (AM-1, AM-2, AM-4 and AM-5).
H3COOC NH2
Cl
Cl
158.09 H3COOC
COOC2H5
H3COOC
OHC N H
218.11
O
O N H
N H
NH2
N H
O
306.08
AM-6 393.12
112.07
H 3N
90.09
H3COOC Cl
Cl H3COOC
H3COOC
COOH
Cl
COOC2H5
H3COOC
COOC2H5 N H2
Cl H3COOC
H 3C
COOC2H5
N H2
H 3C
NH2
N H2
CH3
N H2
AM-7 380.11
COOC2H5
CH3
335.09
352.13
N O H 3C
N H
CH3
230.11
O
CH2
163.06 NH3
Amlodipine 409.15
Cl H3COOC
COOC2H5
H3COOC O
O H3C
N
O NH3
H 3C
N H
AM-8 407.13
H3C
N H
286.14
231.16
Cl Cl H3COOC
123.08
O
H3COOC
Cl
COOC2H5
H2COOC
COOC2H5
H3COOC
H2COOC
COOC2H5 O
N H
N H
N H
366.11
NH3
OH
OH
N H2
O
244.09
350.07
AM-9 395.13
Cl H3COOC
H3COOC
O
490 491 492 493 494 495 496 497
N
N
302.05
226.08
CH2
Fig. 7. Degradation products of amlodipine formed under acidic and alkaline stress conditions (AM-6, AM-7 AM-8 and AM-9).
NH3
102.05
498
H3COOC
Cl H3COOC
COOCH3 Cl
COOC2H5
O H 3C
O N H
NH3
N H2
NH2
H3COOC
COOC2H5
AM-1 363.19 O H 3C
AM-9 395.13
N H
AM-2 392.12 Cl H3COOC
Cl
COOC2H5
Cl H3COOC
N
O
NH3
H 3C
O
AM-8 407.13
H3C
N H
NH3
N H
NH3
AM-4 399.16
Amlodipine 409.15
Cl H3COOC
Cl H3COOC
COOC2H5
COOH Cl
O H 3C
O H3C
N H2
H3COOC
N H2
COOC2H5
AM-5 380.12
NH2 O
AM-7 380.11
499 500 501 502 503 504 505 506 507 508 509 510 511
CH3
COO
COOC2H5
O H 3C
H3COOC
N H
NH2
AM-6 393.12
Fig. 8. Degradation pathway of amlodipine.
Peak Experimental No. mass 1 2 3 4 5 6
408.90 392.23 294.13 238.10 208.14 102.13
Best possible molecular formulae C20H26N2O5Cl+ C20H23NO5Cl+ C15H17NO3Cl+ C12H13NO2Cl+ C12H18NO2+ C5H12NO+
RDB
8.5 9.5 7.5 6.5 4.5 0.5
Possible parent fragment
Difference Possible losses from parent ion corresponding to difference
1 2 3 4 2
16.67 98.10 56.03 29.96 290.10
512 513 514
Table 1 Interpretation of MS data of fragments of amlodipine. 515 RDB: ring plus double bonds 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534
NH3 C5H6O2 C3H4O HCl C15H11O4Cl
Table 2 LC-MS/MS data of DPs of amlodipine (AM) along with their possible molecular formulae and major fragments. DPs
AM-1
Experimental Best possible mass molecular formula 363.08 C19H27N2O5+
AM-2
392.67
C20H23NO5Cl+
9.5
AM-4
399.92
C19H28N2O5Cl+
7.5
AM-5
380.83
C19H23NO5Cl+
9.5
AM-6
393.00
C19H22N2O5Cl+
9.5
AM-7
380.75
C18H21N2O5Cl+
9.5
AM-8
407.17
C20H24N2O5Cl+
10.5
AM-9
395.00
C19H24N2O5Cl+
9.5
535 536
RDB: ring plus double bonds
RDB
8.5
Major fragments (chemical formula) 292.92 (C16H22NO4+), 226.83 (C14H12NO2+), 157.00 (C8H15NO2+), 112.92 (C6H8O2+) 347.08 (C18H18NO4Cl+), 305.92 (C16H16NO3Cl+), 158.92 (C8H16NO2+), 113.08 (C6H11NO+) 363.83 (C19H27N2O5+), 331.92 ( C17H16N2O3Cl+), 263.08 (C14H16N2OCl+), 158.58 (C8H16NO2+), 106.92 (C7H6O+), 90.75 (C4H12NO+) 302.25 (C17H20NO4+), 284.83 (C16H14NO4+), 222.00 (C13H20NO2+), 192.00 (C11H14NO2+), 175.08 (C11H11O2+), 142.92 (C7H12NO2+) 306.42 (C16H17NO3Cl+), 218.17 (C13H16NO2+), 158.75 (C11H12N+), 112.75 (C6H10NO+), 90.92 (C4H12NO+) 352.83 ( C18H13NO4Cl+), 335.92 (C17H18NO4Cl+), 230.83 (C14H16NO2+), 163.17 (C9H9NO2+), 286.08 (C17H20NO3+), 231.25 (C15H21NO+), 123.92 (C8H11O+), 366.75 (C18H21NO5Cl+), 350.08 (C17H17NO5Cl+), 302.17 (C16H13NO3Cl+), 244.17 (C14H14NO3+), 226.25 (C14H12NO2+), 102.25 (C4H8NO2+),