LC, MSn and LC-MS/MS studies for the ...

4 downloads 1934 Views 766KB Size Report
Jun 25, 2014 - Technology Management Shirpur Dist. Dhule 425405 Maharashtra, India. 6. 2. Department of Pharmaceutical Chemistry, Sinhgad Institute of ...
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

To appear in:

Journal of Pharmaceutical Analysis

Received date: Revised date: Accepted date:

12 March 2014 25 June 2014 7 July 2014

www.elsevier.com/locate/jpa www.sciencedirect.com

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.



LC, MSn and LC-MS/MS studies for the characterization of degradation products of



amlodipine

3  4 

Ravi N. Tiwari*1, Nishit Shah1, Vikas Bhalani1, Anand Mahajan2



1



Technology Management Shirpur Dist. Dhule 425405 Maharashtra, India



2



India.

Department of Pharmaceutical Chemistry, SVKM’s NMIMS, School of Pharmacy and

Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmacy, Pune, Maharashtra,

9  10  11  12 

*Corresponding author.

13  14 

SVKM’s NMIMS,

15 

School of Pharmacy and Technology Management,

16 

Near Bank of Tapi River, Agra-Mumbai Road

17 

Babulde, Shirpur- 425405

18 

Dist. Dhule, Maharashtra

19 

India

20 

Tel.: +91-2563-286545; fax: +91-2563-286552

21 

E-mail address: [email protected].

22  23 

24 

Abstract

25 

In the present study, comprehensive stress testing of amlodipine (AM) was carried out according

26 

to International Conference on Harmonization (ICH) Q1A(R2) guideline. Amlodipine was

27 

subjected to acidic, neutral and alkaline hydrolysis, oxidation, photolysis and thermal stress

28 

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

30 

formed from AM, which could be separated by the developed gradient LC method on a C18

31 

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

34 

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

36 

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.

47 

1. Introduction

48 

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

50 

on Harmonization (ICH) and other international agencies [1,2] require the reporting,

51 

identification and characterization of degradation products (DPs). But DPs generated during

52 

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

54 

mixture due to their lower amounts. Therefore, hyphenated techniques like LC-MS is currently

55 

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

58 

of calcium ions into vascular smooth muscles and cardiac muscle, hence, used as an

59 

antihypertensive for the treatment of angina.

60 

A thorough literature revealed that there exist several reports on bioanalytical method

61 

development and pharmacokinetic studies of amlodipine [6,7]. A wide array of articles are

62 

reported on stability-indicating HPLC method for the estimation of single drug amlodipine in

63 

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

66 

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

69 

related impurities of AM and accelerated stability studies. From the above literatures, the

70 

reported masses and fragmentation pattern of all the degradation products/impurities of AM are

71 

quite different from our degradation products of AM formed under different stress conditions. In

72 

fact, the reported masses of impurities and their fragment ions were compared with the masses of

73 

DP’s proposed in this article and they all were found non-resembling with each other. Moreover,

74 

as per previous reports [22-26] the masses of six different process related impurities such as m/z

75 

538, 569, 406, 394, 408 and 422 were not matching with the masses of any of the DP’s of AM.

76 

Hence, the endeavor of our present study was to: (i) carry out the stress studies on AM under the

77 

ICH defined conditions; (ii) separate the degradation products by HPLC; and (iii) characterize

78 

and establish degradation pathway of all the degradation products with the help of LC–MS/MS.

79  80 

2. Experimental

81 

2.1. Drug and reagents

82 

Pure AM was obtained as gratis sample from Osaka Pharmaceuticals Pvt. Ltd. (Vadodara, India).

83 

Analytical reagent (AR) grade formic acid and sodium hydroxide (NaOH) were purchased from

84 

S.D. Fine-Chem Ltd. (Mumbai, India), hydrochloric acid (HCl) and HPLC grade methanol

85 

(MeOH) from Merck Specialities Pvt. Ltd. (Mumbai, India) and hydrogen peroxide (H2O2) from

86 

Qualigens Fine Chemicals Pvt. Ltd. (Mumbai, India). Ultra-pure water obtained from Millipore

87 

water purification system (Molsheim, France) was used throughout the studies..

88  89 

2.2. Equipment

90 

A high-performance liquid chromatography (HPLC) system from PerkinElmer (Shelton, CT,

91 

USA) was used for the LC studies, which consisted of an on-line degasser, a sample injector

92 

(Rheodyne sample loop 20 mL), a UV-visible detector (Series 200), a pump (Reciprocating,

93 

series 200), and a computer system loaded with Total Chrome Navigator (version 6.3.1)

94 

software. The LC-MS system was control by Xcalibur software (version 2.0) consisted of LCQ

95 

Fleet and TSQ Quantum Access with Surveyor Plus HPLC System (Thermo, San Jose, USA).

96 

Precision water baths equipped with MV controller (Thermostatic Classic Scientific India Ltd.

97 

Mumbai, India) 90 were used for stress studies. Degradation experiments in acid, base, and

98 

neutral conditions were performed using a dry-bath (Labline Sun Scientifics Ltd. New Delhi,

99 

India). The solid state thermal stress studies were carried out in dry-air oven (NSW Limited,

100 

New Delhi, India). Other equipments used were a pH meter (Labindia, Mumbai, India), a

101 

weighing balance (Shimadzu, AUX220, Kyoto, Japan), and a micro-pipette (Erba Biohit,

102 

Mannheim, Germany). In all studies, separations were achieved on a C-18 column (250 mm ×

103 

4.6 mm i.d., particle size 5 µm; Kromasil (Eka Chemicals AB, Bohus, Sweden). Photolytic

104 

studies were carried out in a photostability chamber (Thermolab, 95 Th-400G Mumbai, India),

105 

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

108 

Stress degradation studies were carried out on AM as per ICH Q1A(R2) prescribed stress

109 

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

112 

a variety of conditions like, wide range of pH, light, oxidation and dry heat. AM along with

113 

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

116 

(H2O, 2 h, 80 °C), oxidative (H2O2, 15%, 48 h) at room temperature, thermal (50 °C, 48 h) and

117 

photolytic (1.2 × 106, lx h of fluorescent light and 200 Wh/m2 UV-A light, 14 days) stress

118 

conditions.

119  120 

2.4. Sample preparation for HPLC and LC–MS analysis

121 

The stressed samples of AM collected during desired time intervals were adequately diluted 10

122 

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 

139 

2.6. MS, MSn and LC-MS studies

140 

The fragmentation profile of the drug was established by carrying out mass spectral studies on

141 

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

143 

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

145 

obtained in MS studies for amlodipine are listed in Table 1. A previously developed LC method

146 

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.

152  153 

3. Results and discussion

154 

3.1. HPLC analysis

155 

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

173 

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

181 

(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)

199 

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

202 

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

244 

dimethyl ether entities. Ion with m/z 302.25 underwent loss of two ring protons and a methyl

245 

group attached to sixth position of 1,4-dihydropyridine ring and led to the formation of fragment

246 

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

248 

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)

251 

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.

253 

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

258 

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 

5. References 1. ICH, Stability testing of new drug substances and products Q1A(R2), in: International Conference on Harmonisation, IFPMA, Geneva, 2003. 2. WHO, Stability Testing of Active Pharmaceutical Ingredients and Pharmaceutical Products, World Health Organization, Geneva, 2007. 3. M. Bakshi, S. Singh, Development of validated stability-indicating assay methods critical review, J. Pharm. Biomed. Anal. 28 (2002) 1011–1040. 4. S. Görög, The importance and the challenges of impurity profiling in modern pharmaceutical analysis, TrAC. 25 (2006) 755–757.

321 

5. T. Murakami, N. Fukutsu, J. Kondo, et al., Application of liquid chromatography-two-

322 

dimensional nuclear magnetic resonance spectroscopy using pre-concentration column

323 

trapping and liquid chromatography–mass spectrometry for the identification of

324 

degradation products in stressed commercial amlodipine maleate tablets, J. Chromatogr.

325 

A, 1181 (2008) 67–76.

326 

6. A. Zarghi, S.M. Foroutan, A. Shafaati, et al., Validated HPLC method for determination

327 

of Amlodipine in human plasma and its application to pharmacokinetic studies. Farmaco.

328 

60 (2005) 789-792.

329 

7. Y. Ma, F. Qin, X. Sun, et al., Determination and pharmacokinetic study of amlodipine in

330 

human plasma by ultra performance liquid chromatography–electrospray ionization mass

331 

spectrometry. J. Pharm. Biomed. Anal. 43 (2007) 1540-1545.

332 

8. B.G. Chaudhari, N.M. Patel, P.B. Sham, Stability indicating RP-HPLC for simultaneous

333 

determination of Atorvastatin Calcium and Amlodipine Besylate from their combination

334 

drug products. Chem. Pharm. Bull. 55 (2007) 241-246.

335 

9. K.R. Naidu, U.N. Kale, M.S. Shingare, Stability indicating RP-HPLC method for

336 

simultaneous determination of amlodipine and benzapril hydrochloride from their

337 

combination drug product. J. Pharm. Biomed. Anal. 39 (2005) 147-155.

338  339 

10. M.C. Nahata, R.S. Morosco, T.F. Hipple, Stability of amlodipine besylate in two liquid dosage forms. J. Am. Pharm. Assoc. 39 (1999) 375-377.

340 

11. M. Gumustas, S.A. Ozkan, A validated stability-indicating RP-LC method for the

341 

simultaneous determination of amlodipine and perindopril in tablet dosage form and their

342 

stress degradation behavior under ICH-recommended stress conditions. J. AOAC Int. 96

343 

(2013) 751-757.

344 

12. R.A. Shaalan, T.S. Belal, F.A. El Yazbi, et al., Validated stability-indicating HPLC-DAD

345 

method of analysis for the antihypertensive triple mixture of amlodipine besylate,

346 

valsartan and hydrochlorothiazide in their tablets. Arabian J. Chem. (2013), Article in

347 

Press.

348 

13. K.R. Patil, V.P. Rane, J.N. Sangshetti, et al., Stability indicating LC method for the

349 

simultaneous determination of amlodipine and olmesartan in dosage form. J. Chromatogr.

350 

Sci. 48 (2010) 601-606.

351 

14. A.R. Fakhari, S. Nojavan, S. Haghgoo, et al., Development of a stability-indicating CE

352 

assay for the determination of amlodipine enantiomers in commerical tablets.

353 

Electrophoresis. 29 (2008) 4583-4592.

354 

15. A. Mohammadi, N. Rezanour, M. Ansari Dogaheh, et al., A stability-indicating high

355 

performance liquid chromatographic (HPLC) assay for the simultaneous determination of

356 

atorvastatin and amlodipine in commercial tablets. J. Chromatogr. B: Anal. Technol.

357 

Biomed. Life Sci. 846 (2007) 215-221.

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 

574 (2006) 273-283.

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+),