Hffb

SDCa

b

c

a

ARR1AA

KECLIV

1

dItbstawa

0d

Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 165– 175

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rn al h om epage: www.elsev ier .com/ locate / jpba

ighly efficient, selective, sensitive and stability indicating RP-HPLC–UV methodor the quantitative determination of potential impurities and characterization ofour novel impurities in eslicarbazepine acetate active pharmaceutical ingredienty LC/ESI-IT/MS/MS

aji Thomasa,∗, Amber Bharti a, Pawan Kumar Maddhesiaa, Sanjeev Shandilyaa, Ashutosh Agarwala,haramvirb, Sujay Biswasb, Vikas Bhansalb, Ashish Kumar Guptab, Praveen Kumar Tewarib,handra S. Mathelac

Jubilant Life Sciences Ltd., Analytical Research Department, R&D Centre, C-26, Sector – 59, Noida, Uttar Pradesh 201 301, IndiaJubilant Life Sciences Ltd., Chemical Research Department, R&D Centre, C-26, Sector – 59, Noida, Uttar Pradesh 201 301, IndiaDepartment of Chemistry, Kumaun University, D.S.B. Campus, Nainital, Uttarakhand 263 001, India

r t i c l e i n f o

rticle history:eceived 30 August 2011eceived in revised form9 November 2011ccepted 23 November 2011vailable online 1 December 2011

eywords:slicarbazepine acetateharacterizationC/MS/MS

a b s t r a c t

A novel, sensitive, selective and stability indicating LC–UV method was developed for the determi-nation of potential impurities of eslicarbazepine acetate. High performance liquid chromatographicinvestigation of eslicarbazepine acetate laboratory sample revealed the presence of several impuri-ties. Three impurities were characterized rapidly and four impurities were found to be unknown.The unknown impurities were identified by liquid chromatography coupled with electrospray ioniza-tion, ion trap mass spectrometry (LC/ESI-IT/MS/MS). Structural confirmation of these impurities wasunambiguously carried out by synthesis followed by characterization using nuclear magnetic reso-nance spectroscopy (NMR), infrared spectroscopy (FT–IR) and mass spectrometry (MS). Based on thespectroscopic, spectrometric and elemental analysis data unknown impurities were characterized as 5-acetyl-5,11-dihydro-10H-dibenzo [b,f]azepin-10-one, N-acetyl-5H-dibenzo[b,f]azepine-5-carboxamide,

mpurityalidation

5-acetyl-10,11-dihydro-5H-dibenzo[b,f]azepin-10-yl acetate and 5-acetyl-5H-dibenzo[b,f]azepin-10-ylacetate. The newly developed LC–UV method was validated according to ICH guidelines consideringeleven potential impurities and four new impurities to demonstrate specificity, precision, linearity, accu-racy and stability indicating nature of the method. The newly developed method was found to be highlyefficient, selective, sensitive and stability indicating. A plausible pathway for the formation of four newimpurities is proposed.

. Introduction

Eslicarbazepine acetate is chemically S-(−)-10-acetoxy-10,11-ihydro-5H-dibenzo[b,f] azepine-5-carboxamide acetate (Fig. 1a).

t is a second generation antiepileptic drug to oxcarbazepine and ahird generation drug to carbamazepine. It is a prodrug to eslicar-azepine (Fig. 1b) also an active metabolite of oxcarbazepine. Thetructural modification was made to improve efficacy, safety ando prevent formation of toxic epoxide metabolite. Eslicarbazepine

cetate is rapidly and extensively metabolized to eslicarbazepinehich is responsible for pharmacological activity [1–4]. It is used as

dd on therapy in refractory partial epilepsy and in bipolar disorder.

∗ Corresponding author. Tel.: +91 120 4362210; fax: +91 120 2580033.E-mail address: saji thomas@jubl.com (S. Thomas).

731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jpba.2011.11.024

© 2011 Elsevier B.V. All rights reserved.

A very few methods have been reported in literature for theanalysis of eslicarbazepine acetate drug substance. Validated chiralHPLC method for the estimation of (R)-enantiomer in eslicar-bazepine acetate has been reported recently [5]. Chromatographicconditions have been mentioned for the analysis of eslicarbazepineacetate but not fully disclosed [6]. LC methods were not reportedin any of the pharmacopoeias. Extensive literature survey revealsthat no reference exists for the quantitative determination of impu-rities by a stability indicating HPLC method and characterizationof unknown impurities of eslicarbazepine acetate drug substance.Hence it was felt necessary to develop an accurate, rapid, selectiveand sensitive stability indicating LC method for the determination

of eslicarbazepine acetate impurities.

Objective of the current study was to develop a stabilityindicating LC–UV method for the quantitative determination ofimpurities in eslicarbazepine acetate and characterization of four

166 S. Thomas et al. / Journal of Pharmaceutical and

N

O

H3C

O

OH2N

[1] [2]

[3] [4]

[5]

[6][7]

[8][9]

[10][11]

[12][13]

[14] [15]

[16]

[17]

[18]

N

HO

OH2N

(a) (b)

Fa

uaurPittwwsMmppta

2

2

sIcLMMgspbSmG

2

aPucsspeTd

ig. 1. (a) Eslicarbazepine acetate and (b) eslicarbazepine. Numbering has beenssigned for NMR characterization.

nknown impurities detected in several batches of eslicarbazepinecetate in the range of 0.09–0.13%. ICH guidelines indicate thatnknown impurities at or above 0.05% in the drug substanceequire identification [7] depending on the maximum daily dosage.resence of impurities in drug substance can have significantmpact on the quality, safety and efficacy of drug products. Fur-her characterization of impurities is also required to ascertainhat an impurity does not have genotoxic concern. Impuritiesere identified using LC–MS/MS and the proposed structuresere further unambiguously confirmed by independent synthe-

is followed by characterization using NMR and IR techniques.ethod was validated as per ICH guidelines [8]. A plausibleechanism for the formation of new impurities is also being pro-

osed in this paper. To the best of our knowledge this is the firstaper to report a stability indicating LC–UV method for the quanti-ative determination of impurities and the impurities characterizedre novel impurities in eslicarbazepine acetate.

. Experimental

.1. Materials and reagents

Sample of eslicarbazepine acetate API (batch No. SLB-crude) andtandards of Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7,mp-8, Imp-9, Imp-10 and Imp-11 were obtained from Chemi-al Research and Development Department, Jubilant Life Sciencesimited (Noida, India). Deionized water was prepared using ailli-Q plus water purification system from Millipore (Bedford,A, USA). HPLC grade methanol, acetonitrile, analytical reagent

rade potassium dihydrogen phosphate, ammonium bicarbonate,odium hydroxide, hydrochloric acid, hydrogen peroxide and orthohosphoric acid were purchased from Merck India Limited (Mum-ai, India). Dimethyl sulphoxide-d6 (for NMR) was purchased fromigma–Aldrich Corporation (St. Louis, MO, USA). Potassium bro-ide FT–IR grade was purchased from Merck KGaA (Darmstadt,ermany).

.2. High performance liquid chromatography

Samples were analysed on a Waters alliance 2690 sep-ration module equipped with 2487 UV detector and 2996DA detector for stress study (Waters corporation, MA, USA)sing a symmetry shield RP-8, (250 mm × 4.6 mm, 5 �m, Watersorporation, MA, USA). Mobile phase A consisted, 10 mM potas-ium dihydrogen phosphate adjusted to pH 5.0 ± 0.05 withodium hydroxide solution–acetonitrile (95:5, v/v) and mobile

hase B consisted acetonitrile–water (80:20, v/v) in gradi-nt mode (TminA:B) T070:30, T1565:35, T2050:50, T4030:70,5570:30, T6070:30. The flow rate was set to 1.0 mL/min withetector wavelength fixed at 215 nm. The injection volume

Biomedical Analysis 61 (2012) 165– 175

was 10 �L for a sample concentration of 400 �g/mL pre-pared in diluent (mobile phase A–acetonitrile, 50:50, v/v).Detector wavelength was fixed at 215 nm and the columntemperature was maintained at 35 ◦C throughout the analy-sis.

2.3. Liquid chromatography–tandem mass spectrometry(LC–MS/MS).

The equipment and chromatographic conditions used forLC–MS investigation were exactly same as described underSection 2.2. Mobile phase A consisted 10 mM ammoniumbicarbonate–acetonitrile (95:5, v/v) and mobile phase B consisted,acetonitrile–water (80:20, v/v) in gradient mode (TminA:B) T070:30,T1565:35, T2050:50, T4030:70, T5570:30, T6070:30.

The MS and MS/MS studies were performed on Thermo LCQ-Advantage and Xcalibur software (Thermo Electron, San Jose, CA,USA) using electrospray ionization source, atmospheric pressureionization (APCI) and ion trap mass spectrometer. The typicalelectrospray source conditions were: spray voltage, 5 kV, capil-lary voltage, 15–20 V, heated capillary temperature 250 ◦C, tubelens offset voltage 20 V, sheath gas (N2) pressure, 20 psi andhelium was used as damping gas. In the full scan MS2 mode,collision energies of 15–35 eV and isolation width of 5 amuwere used. The excitation time was 30 ms, the isolated ionswere then subjected to a supplementary AC signal to resonantlyexcite hence causing collision induced dissociation (CID). Thecollision energies of 15–35 eV and isolation width of 5 wereused.

2.4. Sample preparation for forced degradation studies

Stress degradation studies were performed as per ICH guide-lines Q1 (R2) to demonstrate the stability indicating nature andspecificity of the proposed method. About 20 mg each of esli-carbazepine acetate sample was transferred into four separate50 mL volumetric flasks and subjected to forced degradation studyunder acidic (0.5 N HCl, room temperature, 1 h), basic (0.015 NNaOH, room temperature, 2 min) and in water (80 ◦C for 3 h).The stressed samples of acid and base degradation were neutral-ized with NaOH and HCl respectively and made up to volumewith diluent. Oxidative degradation was carried out using 30%hydrogen peroxide (80 ◦C for 2 h). Solid state stability of the drugsubstance was carried out by (a) thermal degradation at 105 ◦Cfor 24 h and (b) photolytic degradation was performed by keep-ing 500 mg of each solid sample in two separate loss on dryingbottles (LOD, dark control and photolytic exposure) in photostability chamber model TP 0000090G (Thermo Lab equipmentsPvt. Ltd., Mumbai, India). Samples were exposed to get a min-imum exposure of 1.2 million lx hours for light and 200 W h/m2

for ultraviolet region. Samples were withdrawn at appropriatetimes and subjected to LC analysis using 400 �g/mL sample con-centration. Accelerated and long term stability studies on solidsamples were also carried to establish the retest period of drugsubstance to know the effect of storage conditions at differentatmospheric conditions in a stability chamber (Binder GmbH, Tut-tlingen, Germany).

2.5. NMR spectroscopy

1H and 13C NMR spectra were recorded at 399.957 MHz and100.432 MHz respectively, using a Bruker AVANCE 400 MHz spec-

trometer (Bruker, Fallanden, Switzerland) equipped with a 5 mmBBO probe and a z-gradient shim system. The 1H spectra wererecorded with 1 s pulse repetition time using 30◦ flip angle,while 13C spectra were recorded with power gated decoupling

S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 165– 175 167

Table 1Potential impurities of eslicarbamazepine acetate.

S. No. Structure Mol Wt. IUPAC name Code Origin

1

N

O OH

223.23 Acridine-9-carboxylic acid Imp-1 Degradation

2N

HO

OH2N

254.28 (10S)-10-Hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine-5-carboxamide Imp-2 Process and degradation

3N

O

OH2N

252.27 10-Oxo-10,11-dihydro-5H-dibenzo[b,f]azepine-5-carboxamide Imp-3 Process

4 NH2NOC

236.27 Dibenzo[b,f]azepine-5-carboxamide Imp-4 Process

5 NH2NOC

238.28 10,11-Dihydro-dibenzo[b,f]azepine-5-carboxamide Imp-5 Process

6NC

OCOCH3

OH2N

294.30 10-Acetoxy-5H-dibenzo[b,f]azepine-5-carboxamide Imp-6 Process

7N

H3CO

H2NOC

266.29 10-Methoxy-dibenzo[b,f]azepine-5-carboxamide Imp-7 Process

8NH

HO

211.26 10,11-Dihydro-5H-dibenzo[b,f]azepin-10-ol Imp-8 Process

9NC

OCH3

OH3C

265.31 10-Methoxy-dibenzo[b,f]azepin-5-yl acetate Imp-9 Process

10NH

O

209.24 5,11-Dihydro-10-H-dibenzo [b,f]azepin-10-one Imp-10 Process

11NH

OCH3

223.27 10-Methoxy-5H-dibenzo[b,f]azepine Imp-11 Process

udsDs

sing 30◦ flip angle with repetition time of 2 s. Samples were

issolved in dimethyl sulfoxide-d6. The 1H and 13C chemicalhift values were reported on the ı scale in ppm relative toMSO-d6 (2.50 ppm). All spectra were recorded with sample

pinning.

2.6. FT–IR spectroscopy

The IR spectrum was recorded in the solid state as KBr powderdispersion using Nicolet FT–IR model AVTAR 370 (Thermo Elec-tron Scientific Instruments, Madison, WI, USA) with a DTGBS KBr

168 S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 165– 175

Imp-

2 - 6

.501

Imp-

3 - 1

0.78

9

Esl

icar

baze

pine

ace

tate

- 14

.334

Imp-

6 - 1

7.09

3

Imp-

A -

20.8

09

Imp-

B -

22.8

56Im

p-C

- 23

.775

Imp-

D -

25.7

09

V

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Mi0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

carbaz

dap

2

s558od1trsr

3

3

eisstwdgtmUmsa4wmhb

Fig. 2. Chromatogram of esli

etector. Data were collected between 400 and 4000 cm−1, with resolution of 4.0 cm−1. A total of 16 scans were obtained androcessed using the OMNIC software version 6.0.

.7. Preparation of stock solutions for method validation

A test preparation of 400 �g/mL of eslicarbazepine acetate APIample was prepared by dissolving in diluent (buffer–acetonitrile,0:50). A stock solution of impurities was prepared by dissolving

mg each of Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7, Imp-, Imp-9, Imp-10, Imp-11, Imp-A, Imp-B, Imp-C and Imp-D and 5 mgf eslicarbazepine acetate in diluent and made up to 25 mL withiluent. Transferred 5 mL of each individual stock solution into a00 mL volumetric flask and made up to volume with diluent. Fromhis stock solution standard solution of 0.60 �g/mL of each impu-ity and 0.60 �g/mL of eslicarbazepine acetate was prepared. Thistandard solution was also used for checking solution stability andobustness parameters.

. Results and discussion

.1. Method development

The main objective of method development was to achievefficient separation between eslicarbazepine acetate and knowmpurities (Table 1). The main difficulty was to obtain sufficientelectivity and sensitivity of impurities due to the similar chemicaltructure of eslicarbazepine and most of the impurities. The resolu-ion between eslicarbazepine acetate, Imp-4 and Imp-5 were poorhen different stationary phase viz; C 18, and phenyl were used inifferent mobile phases containing buffers like dipotassium hydro-en orthophosphate, ammonium acetate or combination of thesewo with pH ranging from 4 to 6 using organic modifiers such as

ethanol, acetonitrile and isopropyl alcohol in the mobile phases.se of symmetry shield-RP8 column, use of acetonitrile–waterixture as mobile phase-B and column temperature 35 ◦C, was

ignificant in achieving the desired resolution of eslicarbazepinecetate, Imp-4 and Imp-5. Optimum resolution between Imp-, Imp-5, and Imp-C, Imp-8 were achieved when pH of buffer

as adjusted to 5.0. After several trials for gradient profile, chro-atographic conditions were finalized as described under section

igh performance liquid chromatography. The LC–MS compati-le method was optimized using ammonium acetate bicarbonate

nutes

epine acetate crude sample.

buffer as other volatile buffers such as ammonium formate andammonium acetate could not give steady base line.

3.2. Identification of unknown impurities by LC–ESI/MS/MS

HPLC analysis of eslicarbazepine acetate (Fig. 2) revealed thepresence of seven impurities ranging from 0.09% to 0.13% consis-tently in several batches. RRTs of all known impurities (Table 1)were compared and found that four impurities at RRTs 1.45, 1.59,1.66 and 1.79 were unknown and marked as Imp-A, Imp-B, Imp-Cand Imp-D respectively. A LC–ESI/MS method as described in Sec-tion 2.4 was used to identify these impurities. Mass spectral datashowed protonated molecular ion peaks at m/z 252, m/z 279, m/z296 and m/z 294 for Imp-A, Imp-B, Imp-C and Imp-D respectively.Prior to characterization of impurities, LC–MS/MS data of eslicar-bazepine acetate was generated to understand the fragmentationpattern. The MS2 spectrum obtained for eslicarbazepine acetateparent ion at m/z 297 (Fig. 3a), showed a prominent peak at m/z237 (Fig. 3b) which can be attributed to the neutral loss of ethenone(CH2 C O, 42 amu) due to the formation of metastable ion at m/z255 followed by loss of water. The product ion at m/z 237 wasfurther subjected to MS3 analysis, which gave daughter ions atm/z 220 and m/z 194 (Fig. 3c). Formation of these product ionscan be attributed to the loss of ammonia (NH3, 17 amu) and imi-nomethanone (HN C O, 43 amu) as depicted in Fig. 4a.

The protonated molecular ion of Imp-A was obtained at m/z252 and M + Na at m/z 274 (Fig. 3d). MS2 analysis showed a majorfragment at m/z 210 (Fig. 3e) due to the neutral loss of ethenone(CH2 C O, 42 amu). MS3 analysis of the precursor ion at m/z 210gave fragment at m/z 182 (Fig. 3f) due to the loss of carbon monox-ide (CO, 28 amu). A critical inspection of molecular ion along withthe fragmentation pattern in both MS2 and MS3 spectrum indi-cated that Imp-A contains an N-acetyl functional group and thestructure for Imp-A can be proposed as 5-acetyl-5,11-dihydro-10H-dibenzo[b,f]azepin-10-one. A plausible fragmentation patternshowing the formation of these daughter ions is depicted in Fig. 4b.

Imp-B showed protonated molecular ion peak at m/z 279 andM + Na at m/z 301 (Fig. 3g). The MS2 analysis of Imp-B showeddaughter ions at m/z 237 and m/z 220 (Fig. 3h) which can beattributed due to the loss of ethenone and ammonia respectively.

The MS3 spectrum showed a major fragment at m/z 194 was due tothe neutral loss of iminomethanone (NH C O, 43 amu). Based onthe LC–MS/MS data the structure of this impurity was proposedas N-acetyl-5H-dibenzo[b,f] azepine-5-carboxamide. A plausible

S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 165– 175 169

IMP-A #56 RT : 0.61 AV: 1 SB: 25 1T: + c ESI Full ms [ 50 .00-500 .00]

200m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

252 .09

274.02

296.93224 .14

IMP-A #111 RT : 1.52 AV: 1 NL:T: + c ESI Full ms 2 25 2.00@35 .00 [ ...

200 400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

210.14

252.15

180.20 465 .99269.90

IMP-A #135 RT : 2.09 AV: 1 NL:T: + c ESI Full ms 3 25 2.00@35 .00 2 ...

200 400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

182.15

210.00159.87 362.53

Imp-B. #48 RT : 0.84 AV: 1 SB: 304T: + c ESI Full ms [ 50 .00-100 0.00]

200 400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

301 .07

279.02

306.75220.11

Imp-B #20 9 RT : 2.92 AV: 1 NL:T: + c ESI Full ms 3 27 9.00@32 .00 2 ...

100 200 300m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

194.22

237 .07

192.01 286.30

Imp-C #28 RT : 0. 36 AV: 1 SB : 53T: + c ESI Full ms [ 50 .00-500 .00]

200 40 0m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

312.77

295.87

382.70236.04153.17

Imp-C #128 RT : 1.67 AV: 1 SB: 43T: + c ESI Full ms 2 29 6.00@25 .00 [ ...

200 400m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

236 .03

194.23

99.68 238.91 415.75

Imp-C #189 RT : 2.69 AV: 1 SB: 43T: + c ESI Full ms 3 29 6.00@25 .00 2 ...

200 400m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

194 .20

131.53 227.22 350.59

Imp-D #16 RT: 0. 26 AV : 1 SB : 59T: + c ES I Full ms [ 50 .00 -100 0.00 ]

200 400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

294.0 3

316.0 7166.1 7

361.0 8

Imp-D #137 RT : 2.44 AV: 1 NL :T: + c ESI Full ms 2 29 4.00@30 .00 [ ...

200 400m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

252.03

294.00

210.10310.37135.08

Imp-D #196 RT : 3.73 AV: 1 NL :T: + c ESI Full ms 3 29 4.00@32 .00 2 ...

200 400m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

210.11

252.14180.20 315.97

ESLICAR BAZEPINE AC ETATE #560T: + c ESI Full ms [ 50 .00-500 .00]

200 40 0m/z

0

20

40

60

80

100R

elat

ive

Abun

danc

e296.93

73.99 442.02193.46

ESLICARBAZEP INE ACET ATE. . #T: + c ESI Full ms2 297.00@35.00 [ ...

200 400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

237.06

194.20 247.89 377.84

ESLICARBAZEP INE ACET ATE. . #T: + c ESI Full ms3 297.00@35.00 2 ...

100 200 300m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

194.21

219.93192.20 243.41

Imp-B #130 RT: 1.6 1 AV : 1 NL:T: + c ES I Full ms2 27 9.00@ 32.0 0 [ .. .

100 200m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

194.2 1

219.9 1

237.0 6180.1 6125. 08

(a)

(d)

(g)

(j)

(m) (n) (o)

(k) (l)

(h) (i)

(e) (f)

(b) (c)

Fig. 3. (a) Mass spectra eslicarbazepine acetate showing [M + H] (at m/z 297), (b) MS2 spectrum of m/z 297, (c) MS3 spectrum of product ion at m/z 237, (d) Mass spectra ofImp-A showing [M + Na] (at m/z 274) and [M + H] (at m/z 252), (e)MS2 spectrum of m/z 252, (f) MS3 spectrum of product ion at m/z 210, (g) Mass spectra of Imp-B showing[M + Na] (at m/z 301) and [M + H] (at m/z 279), (h) MS2 spectrum of m/z 279, (i) MS3 spectrum of product ion at m/z 237, (j) Mass spectra of Imp-C showing [M + NH4] (at m/z313) and [M + H] (at m/z 296), (k) MS2 spectrum of m/z 296, (l) MS3 spectrum of product ion at m/z 236, (m) Mass spectra of Imp-D showing [M + Na] (at m/z 316) and [M + H](at m/z 294), (n) MS2 spectrum of m/z 294 and (o) MS3 spectrum of product ion at m/z 252.

170 S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 165– 175

N

O

O

NH2O

N

CH3O

O

N

O

O

NH2O

N

CH3O

O

H

H

MS2

CH2=C=O

MS2

N

HO

NH2O

CH2=C=O

H

NH

O

MS2

H2O

H

N

NH2O

MS3

H

CO

NH=C=O

NH3

MS3

MS3

NH

H2C

NH2

NCO

m/z 194

m/z 220

[M+H] = 297Eslicarbazepine acetate

[M+H] = 252

(a)

Imp-A m/z 210

m/z 255 m/z 237

(b)

(c)

(d)

(e)

m/z 182

N

NHO

O

N

NHO

O

H

MS2

CH2=C=ON

NH3O

NHCO

NH3

N

O

NH2

Imp-B [M+H] = 279 m/z 237

m/z 220

m/z 194

MS3

MS3

MS2

NH2

m/z 194

CN

O

H3C

O

H HH

N

O

O

CH3O

N

O

O

CH3O

MS2 MS3

NH

O

N

HO

CH3O

N

O

O

CH3O

N

O

O

CH3O

MS2

N

CH3O

MS3

NH2

m/z 236 m/z 194[M+H] = 296Imp-C

H

CH2=C=O

CH2 CH=C=O 2=C=O

H

N

HO

CH3O

MS2

H2OCH2=C=O

m/z 254

H

(a) esl

fi

m

Imp-D [M+H] = 294

Fig. 4. Plausible pathway showing the formation of product ions of

ragmentation pattern showing the formation of these daughterons is depicted in Fig. 4c.

The full scan spectra of impurity-C showed its protonatedolecular ion at m/z 296 and M + NH4 at m/z 313 (Fig. 3j). The

m/z 252 m/z 210

icarbazepine acetate, (b) Imp-A, (c) Imp-B, (d) Imp-C and (e) Imp-D.

MS2 analysis of impurity-B showed daughter ion peak at m/z 236(Fig. 3k) which was formed due to the loss of water (H2O, 18 amu)from the metastable product ion at m/z 254 formed due to the lossof ethenone (CH2 C O, 42 amu) from the parent ion. MS3 analysis

S. Thom

as et

al. /

Journal of

Pharmaceutical

and Biom

edical A

nalysis 61 (2012) 165– 175

171

Table 2Method validation summary report.

Parameter Imp-1 Imp-2 Imp-3 Eslicarbazepineacetate

Imp-4 Imp-5 Imp-6 Imp-7 Imp-A Imp-B Imp-C Imp-8 Imp-D Imp-9 Imp-10 Imp-11

System suitabilityRT 2.70 6.49 10.80 14.35 15.80 16.37 17.12 20.18 20.82 22.87 23.79 24.14 25.72 27.86 30.23 39.22RRT 0.19 0.45 0.75 1.00 1.10 1.14 1.19 1.41 1.45 1.59 1.66 1.68 1.79 1.94 2.11 2.73Rs – 21.25 15.92 10.45 3.85 1.47 1.89 8.12 1.84 6.67 2.94 1.11 5.84 7.64 7.63 25.36

Linearityr 0.9993 0.9995 0.9994 0.9991 0.9994 0.9994 0.9994 0.9994 0.9994 0.9994 0.9993 0.9994 0.9995 0.9994 0.9995 0.9994Slope 145,376 202,214 205,407 176,494 294,124 232,870 258,461 271,544 207,675 293,899 171,602 272,262 261,456 288,292 220,009 273,401Intercept −237 −227 −277 226 −271 −489 −267 −7.92 70.29 −76 −192 −330 −268 −375 −20 −788

Detection limit (�g/mL) 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020Quantitation limit (�g/mL) 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060Precision (QL)

% RSD (n = 6)0.52 1.77 1.71 2.33 0.94 0.86 0.70 2.79 2.31 0.95 2.05 1.14 0.65 1.92 1.29 2.17

Repeatability (intra day)% RSD (n = 6)

2.86 2.86 2.67 NA 3.33 3.10 2.67 3.31 3.57 2.70 5.33 2.86 3.60 2.86 3.00 1.02

Intermediate precision (inter day)% RSD (n = 12)

5.33 2.14 2.00 NA 5.33 3.57 3.13 4.67 3.33 4.00 4.01 2.14 3.32 3.00 5.03 3.33

Accuracy at QL level (n = 3)Amount added (�g/mL) 0.060 0.060 0.060 0.060 0.061 0.060 0.060 0.061 0.062 0.060 0.061 0.062 0.061 0.061 0.062 0.062Amount recovered (�g/mL) 0.057 0.061 0.061 0.061 0.060 0.059 0.062 0.063 0.063 0.058 0.060 0.060 0.058 0.058 0.058 0.062% Recovery 95.00 101.67 101.67 101.67 98.36 98.33 103.33 103.28 101.61 96.67 98.36 96.77 95.08 95.08 93.55 100.00% RSD 0.00 0.00 0.00 0.32 4.33 0.00 0.00 4.22 0.64 0.00 0.00 0.00 0.00 4.33 0.00 0.00

Accuracy at 100% level (n = 3)Amount added (�g/mL) 0.600 0.592 0.600 0.612 0.596 0.600 0.596 0.600 0.600 0.596 0.568 0.596 0.596 0.592 0.596 0.596Amount recovered (�g/mL) 0.564 0.564 0.588 0.632 0.580 0.580 0.564 0.580 0.576 0.588 0.560 0.560 0.576 0.568 0.584 0.572% Recovery 94.00 95.27 98.00 103.27 97.32 96.67 94.63 96.67 96.00 98.66 98.59 93.96 96.64 95.95 97.99 95.97% RSD 0.44 0.43 0.38 1.21 0.02 0.41 0.45 0.43 0.00 0.41 0.82 0.79 0.37 0.42 1.82 0.41

Accuracy at 150% level (n = 3)Amount added (�g/mL) 0.900 0.888 0.900 0.916 0.892 0.900 0.892 0.900 0.896 0.892 0.852 0.892 0.888 0.888 0.896 0.892Amount recovered (�g/mL) 0.856 0.844 0.888 0.912 0.876 0.876 0.848 0.872 0.860 0.884 0.844 0.844 0.868 0.852 0.872 0.864% Recovery 95.11 95.05 98.67 99.56 98.21 97.33 95.07 96.89 95.98 99.10 99.06 94.62 97.75 95.95 97.32 96.86% RSD 0.95 0.78 0.51 0.70 0.68 0.89 0.68 0.43 0.46 0.77 0.97 0.90 0.51 0.68 0.68 0.91

n, number of determinations; RT, retention time; RRT, relative retention time; Rs, USP resolution; r, correlation coefficient.

172 S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 165– 175

N

O

[10]

[1] [2]

[3] [4]

[5]

[6][7]

[8][9]

[11][12]

[13]

[14]

[15] [16]

CH3O

N

[1] [2]

[3] [4]

[5]

[6][7]

[8][9]

[10][11]

[12]

[13]

[14] [15]

NHO

O

[16][17]

N

O

O

H3C[1] [2]

[3] [4]

[5] [6]

[7]

[8][9]

[10][11]

[12][13]

[14]

[15]

[16] [17]

CH3O

N

O

O

H3C[1] [2]

[3] [4]

[5] [6]

[7]

[8][9]

[10][11]

[12][13]

[14]

[15]

[16] [17]

CH3O

(a) (b) (c) (d)

Imp-

g4s1t

mDt4tBtmi

3a

IatmbDcbL

3

atWoN2(111

3

rsrwofa

[18]

Fig. 5. New impurities of eslicarbazepine acetate, (a) Imp-A, (b) Imp-B, (c)

ave fragment at m/z 194 due to the loss of ethenone (CH2 C O,2 amu) (Fig. 3l). Based on LC–MS/MS analysis this impurity wasuspected to be 5-acetyl-10,11-dihydro-5H-dibenzo[b,f] azepin-0-yl acetate (Fig. 4d). A plausible fragmentation pattern showinghe formation of these daughter ions is depicted in Fig. 4d.

The full scan spectra of impurity-D showed its protonatedolecular ion at m/z 294 (Fig. 3m). The MS2 analysis of impurity-

showed daughter ion peak at m/z 252 which can be attributedo the neutral loss of ethenone from O-acetyl moiety (CH2 C O,2 amu). The MS3 analysis of the precursor ion of m/z 210 due tohe loss of ethenone from N-acetyl moiety (CH2 C O, 42 amu).ased on LC–MS/MS analysis this impurity was suspected to behe 5-acetyl-5H-dibenzo[b,f]azepin-10-yl acetate. Proposed frag-

entation pattern showing the formation of these daughter ionss depicted in Fig. 4e.

.3. Synthesis and structural elucidation of Imp-A, Imp-B, Imp-Cnd Imp-D

Based on the proposed structure of impurities by LC–MS/MS,mp-A, Imp-B, Imp-C and Imp-D were independently synthesizednd used for further structural confirmation by NMR and FT–IRechniques (Fig. 5). Synthesized impurities were analysed by HPLC

ethod as described under Section 2.2 and the purity was found toe 95.2%, 97.6%, 98.2% and 97.9% for Imp-A, Imp-B, Imp-C and Imp-

respectively. The MS2 spectra obtained for synthesized authenticompounds of impurities using direct infusion mode was found toe exactly same as MS2 spectra of impurities obtained from on-lineC–MS/MS analysis.

.3.1. Synthesis of Imp-A10-Methoxy-5H-dibenzo[b,f]azepine is acetylated using acetic

nhydride in presence of triethylamine and toluene as solvento obtain 1-(10-methoxy-5H-dibenzo[b,f]azepin-5-yl)ethanone.

hich is hydrolysed using 1 N HCl in toluene medium tobtain 5-acetyl-5,11-dihydro-10H-dibenzo[b,f]azepin-10-one. 1HMR (400 MHz, DMSO-d6): 2.01 (br s, 3H, 10), 3.80–3.84 (br s, 2H,), 7.35–7.48, 7.66–7.94 (m, 8H, 5, 7, 12, 14, 4, 6, 13, 15). 13C-NMR100 MHz, DMSO-d6): 23.40 (10), 48.88 (2), 120.75, 122.24, 128.51,29.88, 130.34, 130.59 (7, 12, 5, 14, 6, 13, 4, 15), 135.12 (3, 16),41.34, 142.88 (8, 11), 169.41 (9), 192.78 (1). IR; C O str. (1678.64,666.74), C C str. (1597.28, 1489.52), C H str. (2872.19, 2963.94).

.3.2. Synthesis of Imp-BA suspension of eslicarbazepine acetate (2 g) and zinc chlo-

ide in acetic anhydride (30 mL) was heated to 80–85 ◦C andtirred at this temperature for 16 h. After reaction completion, theeaction mixture was concentrated under vacuum. The residue

as diluted in a mixture of dichloromethane and water. The

rganic layer was washed with 5% sodium bicarbonate solutionollowed by concentration of aqueous layer under vacuum to get

crude mass which was purified by column chromatography

[18] [18]

C and (d) Imp-D. Numbering has been assigned for NMR characterization.

to get N-acetyl-5H-dibenzo[b,f]azepine-5-carboxamide. 1H-NMR(400 MHz, DMSO-d6): 2.44 (s, 3H, 18), 6.98 (br s, 2H, 1, 2), 7.17(br s, 1H, 16), 7.42–7.51 (m, 8H, 4, 5, 6, 7, 11, 12, 13, 14). 13C-NMR(100 MHz, DMSO-d6): 24.74 (18), 129.96 (1, 2, 4, 5, 6, 7, 11, 12, 13,14, 3, 15), 151.59 (8, 10), 172.99 (9, 17). IR; >C O str. (1711.69,1670.83), C C str. (1482.86), N H str. (3272.64).

3.3.3. Synthesis of Imp-C5-Acetyl-5,11-dihydro-10H-dibenzo[b,f]azepin-10-one was

reduced using sodium borohydride in methanol to obtain 1-(10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)ethanone.Which on acetylation using acetic anhydride, in presence ofdimethyl pyridine, triethylamine using dichloromethane assolvent to obtain 5-acetyl-10,11-dihydro-5H-dibenzo[b,f]azepin-10-yl acetate. 1H-NMR (400 MHz, DMSO-d6): 2.03, 2.09 (s, 3H × 2,1, 18), 3.07–3.20, 3.55–3.58 (m, 2H, 4), 6.00 6.07 (br s, 1H, 3),7.12–7.45 (m, 8H, 6, 7, 8, 9, 13, 14, 15, 16). 13C-NMR (100 MHz,DMSO-d6): 21.21, 22.94 (1, 18), 35.93 (4), 71.89 (3), 127.39, 128.57,128.66, 128.70, 130.93, 131.25, 131.93 (7, 15, 9, 13, 6, 14, 16, 8),134.14, 140.46 (5, 17), 142.34, 142.82 (10, 12), 170.38, 170.58 (2,11). IR; >C O str. (1674.35, 1735.55), C C str. (1602.36, 1490.76).

3.3.4. Synthesis of Imp-D1-(10-Hydroxy-dibenzo[b,f]azepin-5-yl)-ethanone is acety-

lated using acetic anhydride in presence of triethylamine anddimethylamino pyridine using dichloromethane as solvent toobtain 5-acetyl-5H-dibenzo[b,f]azepin-10-yl acetate which isrecrystallised from isopropyl alcohol to obtain pure Imp-D. 1H-NMR (400 MHz, DMSO-d6): 1.80, 2.31 (s, 3H × 2, 1, 18), 6.96 (s, 1H,4), 7.34–7.69 (m, 8H, 6, 7, 8, 9, 13, 14, 15, 16). 13C-NMR (100 MHz,DMSO-d6): 21.22, 22.28 (1, 18), 120.41 (4), 125.65–132.46 (6,7, 8, 9, 13, 14, 15, 16), 140.66 (5, 17), 147.29 (3, 10, 12), 169.46,170.10 (2, 11). IR; >C O str. (1760.73, 1766.74), C C str. (1593.19,1486.95), C H str. (3060.16).

3.4. Formation of Imp-A, Imp-C and Imp-D

During the synthesis of eslicarbazepine acetate, unreactedmethoxy iminostilbene present in intermediate Ia (methoxy car-bamazepine) undergo acid hydrolysis resulting in the formation ofintermediate IIIa and IVa (Keto-enol) which on further acetylationleads to the formation of Imp-A, Imp-C and Imp-D. The presenceof carbamazepine in oxcarbazepine was found to contribute theformation of Imp-B. The synthetic scheme of plausible formationof Imp-B, Imp-A, Imp-C and Imp-D is depicted in Fig. 6b and crespectively.

3.5. Results of forced degradation and identification of major

degradant impurities

Eslicarbazepine acetate was found to be susceptible to acidic,alkaline and oxidative stress conditions and mild degradation was

S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 165– 175 173

NH

DMAP

NHO

N

TolueneBenzoic acid

Acetic anhydrideTriethylamine

DicholromethanePyridine

Imp-B

Carbamazepine

(c)

NH2O

N

CH3O

NH

H3CO

NH

O

NH

HO

DMAP

DMAP

CH3O

O

H3C

N

O

CH3O

N

OToluene

Acetic anhydrideTriethylamine

Dicholromethane

Methanol

Sodium borohydride

Pyridine

Acetic anhydrideTriethylamine

DicholromethanePyridine

Imp-A

IIIa

IIIb

Imp-C

(b)

NH

HO

DMAP

CH3O

O

H3C

N

OAcetic anhydrideTriethylamine

DicholromethanePyridine

Imp-D

NH

H3CO

N

NH2O

H3CO

N

NH2O

O

Methoxy iminostilbene

TolueneSodium cyanateBenzoic acid Toluene

Dil. HCl

Ia Ib

(a)

NH2O

O

H3C

N

O

DMAP

N

NH2O

HO

N

NH2O

HOAcetic anhydrideTriethylamine

Dicholromethane

Resolution

Pyridine

Eslicarbazepine acetate LicarbazepineEslicarbazepine

MethanolSodium borohydride

Methoxy iminostilbene

Iminostilbene

Dil. HCl

Sodium cyanate

IVa

Fig. 6. (a) Scheme for the synthesis of eslicarbazepine acetate, (b) plausible scheme for the formation of Imp-B and (c) plausible scheme for the formation of Imp-A, Imp-Ca

oheeib

nd Imp-D.

bserved in aqueous hydrolysis. In acidic alkaline and oxidativeydrolysis, major impurity formed at RRT 0.45 was identified as

slicarbazepine (Imp-2) formed due to the hydrolysis of acetyl moi-ty in the drug. Solid state stress study confirmed that molecules stable in thermal, photolytic conditions. Samples from threeatches were packed in double polyethylene bags, then packaged

in HDPE bottles and kept for stability studies under long-term(25± 2 ◦C, 60 ± 5% RH) and accelerated conditions (40± 2 ◦C, 75 ± 5%

RH) for three months. HPLC analysis at regular intervals (1, 2,3 months) demonstrated satisfactory resistance to degradationunder solid state conditions. From the degradation studies and peakpurity test results derived from PDA detector it was confirmed that

174 S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 165– 175

F s: (a) a( rbazep

tnd

4

eapdVIBccv

ig. 7. (A) Typical chromatogram of eslicarbazepine acetate under stress conditione) thermal degradation and (f) photolytic degradation. (B) Chromatogram of eslica

he spectral purity of eslicarbazepine acetate peak was homoge-ous thus confirmed the stability indicating power of the newlyeveloped method.

. Method validation

The newly developed method was validated for sensitivity, lin-arity, precision and accuracy, robustness and system suitabilityccording to ICH guidelines. The peak capacity of the method foreaks eluting up to 40 min gradient time was found to be about 211emonstrating the high efficiency of the newly developed method.alidation study was carried out for Imp-1, Imp-2, Imp-3, Imp-4,

mp-5, Imp-6, Imp-7, Imp-8, Imp-9, Imp-10, Imp-11, Imp-A, Imp-

, Imp-C and Imp-D. The system suitability and selectivity werehecked by injecting 400 �g/mL of eslicarbazepine acetate solutionontaining 0.6 �g/mL of all impurities monitored throughout thealidation. Method validation results are summarized in Table 2.

cid hydrolysis, (b) base hydrolysis, (c) oxidative degradation, (d) water hydrolysis,ine spiked with impurities.

4.1. Sensitivity

Sensitivity was determined by establishing the detection limit(DL) and quantitation limit (QL) for all impurities and eslicar-bazepine acetate by injecting a series of dilute solutions withknown concentrations. The limit of detection and the limit of quan-titation were about 0.005% and 0.015% of analyte concentrationi.e. 400 �g/mL respectively. The relative standard deviation for QLconcentration for all impurities was below 3%.

4.2. Linearity and range

A linearity test solution for related substance method was pre-pared by diluting the impurity stock solution to the required

concentrations. The solutions were prepared at six concentra-tion levels. From QL to 150% of the permitted maximum level ofthe impurity (i.e. 0.06 �g/mL, 0.3 �g/mL, 0.45 �g/mL, 0.60 �g/mL,0.75 �g/mL, 0.90 �g/mL) was subjected to linear regression

al and

aorrTipw(

4

bcPtoofiowlsa

4

drfltwt2r

4

isafobsd

[

[

[

[

[

S. Thomas et al. / Journal of Pharmaceutic

nalysis with the least squares method. Calibration equationbtained from regression analysis was used to calculate the cor-esponding predicted responses. The residuals and sum of theesidual squares were calculated from the predicted responses.he correlation coefficient obtained was greater than 0.999 for allmpurities. The result showed an excellent correlation between theeak and concentration of all impurities. The range of the methodas from 0.06 �g/mL to 0.90 �g/mL of the analyte concentration

400 �g/mL).

.3. Precision and accuracy

The precision of the related substances method was checkedy injecting six individual preparations of (400 �g/mL) esli-arbazepine acetate spiked with 0.60 �g/mL each impuritiesercentage RSD for peak areas of each impurity was calculated andhe study was also determined by performing the same proceduresn a different day (inter-day precision). The intermediate precisionf the method was also evaluated by a different analyst and dif-erent instrument in the same laboratory. % RSD of areas of eachmpurity was within 6.0, confirming good precision at low levelf the developed analytical method. The accuracy of the methodas evaluated in triplicate at QL, 100% level (0.6 �g/mL) and 150%

evel (0.90 �g/mL). The percentage recovery of all impurities in drugubstance has been calculated. Chromatogram of eslicarbazepinecetate spiked with fifteen impurities was depicted in Fig. 7B.

.4. Robustness

To determine the robustness of the method, experimental con-itions were deliberately changed. Close observation of analysisesults of deliberately changed chromatographic conditions viz;ow rate (1.0 ± 0.1 mL/min), pH (3 ± 0.2), mobile phase composi-ion (mobile phase A; ± 10% acetonitrile, mobile phase B; ± 10%ater) and column temperature (35 ± 2 ◦C) revealed that resolu-

ion between eslicarbazepine acetate and Imp-4 was greater than.0 and no significant change in relative retention time for all impu-ities in spiked sample illustrating the robustness of the method.

.5. Solution stability and mobile phase stability

The solution stability of eslicarbazepine acetate and its relatedmpurities was carried out by leaving both spiked and unspikedample solutions in tightly capped HPLC vials at 25 ◦C for 24 h inn auto sampler. Content of each impurity was determined againstreshly prepared standard solution. No significant changes were

bserved in the content of any of the impurities. The solution sta-ility and mobile phase stability experiment data confirms that theample solutions and mobile phases used during related substanceetermination were stable for at least 24 h and 72 h, respectively.

[

[[

Biomedical Analysis 61 (2012) 165– 175 175

5. Conclusion

In this study, four unknown impurities in eslicarbazepineacetate drug substance were identified using LC–UV andLC–ESI/IT–MS techniques using a newly developed HPLC method.Proposed structures of these impurities were confirmed byindependent synthesis followed by structural elucidation usingNMR, FT–IR and MS techniques. Imp-A was characterized as5-acetyl-5,11-dihydro-10H-dibenzo[b,f]azepin-10-one. Imp-B wascharacterized as N-acetyl-5H-dibenzo[b,f]azepine-5-carboxamide.Imp-C was characterized as 5-acetyl-10,11-dihydro-5H-dibenzo[b,f]azepin-10-yl acetate and Imp-D was characterized as 5-acetyl-5H-dibenzo[b,f]azepin-10-yl acetate. Major impurity formedduring acidic and alkaline stress conditions was identified aseslicarbazepine. The LC–UV method was validated as per ICHguidelines. Newly developed HPLC method was found to be sim-ple, sensitive, selective, cost effective and stability indicating.Detection limit for impurities was found to be as low as 0.005%and was found to have excellent resolution for fifteen impuri-ties indicating high sensitivity and selectivity of the validatedmethod.

Acknowledgements

The authors are thankful to the management of JubilantLife Sciences Limited for providing necessary facilities. Authorswould like to thank Mr. Saroj Paul, Dr. Subhash ChandraJoshi, Dr. Hawaldar Maurya, Ms. Samreen Siddiqui and Mr.Sandeep Kanwar for their co-operation in carrying out thiswork.

References

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2] G. Alves, A. Fortuna, J. Sousa, R. Direito, A. Almeida, M. Rocha, A. Falcao, P.Soares-da-Silva, Enantioselective assay for therapeutic drug monitoring of esli-carbazepine acetate: no interference with carbamazepine and its metabolites,Ther. Drug Monit. 32 (2010) 512–516.

3] A. Fortuna, J. Sousa, G. Alves, A. Falcao, P. Soares-da-Silva, Development andvalidation of an HPLC–UV method for the simultaneous quantification of carba-mazepine, oxcarbazepine, eslicarbazepine acetate and their main metabolites inhuman plasma, Anal. Bioanal. Chem. 397 (2010) 1605–1615.

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5] M.K. Mone, K.B. Chandrasekhar, Development of liquid chromatographic enan-tiomer separation methods and validation for the estimation of (R)-enantiomerin eslicarbazepine acetate, J. Pharm. Biomed. Anal. 54 (2011) 248–251.

6] K.J. Prabahar, P.B. Kulkarni, S.B. Sawant, N.D. Limbekar, Glenmark generics lim-ited, United States patent 0173893 A1 (2010).

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