Isolation and Structural Elucidation of Novel Isomeric ......Citation: Neelakandan K, Ashok C,...

Open AccessResearch Article

Volume 4 • Issue 2 • 1000165ISSN:2155-9872 JABT, an open access journal

Keywords: Zolmitriptan; LCMS; HPLC; chromatography; Isolation;Isomeric; NMR

IntroductionHThe impurity profile of a drug substance is a critical parameter for

its safety assessment and is inherent to its manufacturing process. As per the ICH guidelines the impurities that exceed 0.1% in a drug must be identified prior to the clinical trials. Since the associated impurities are usually process related, they are invariably, structurally similar to the synthesized target drugs.

Zolmitriptan, chemically known as 4(S)-4-[3-(2-dimethylaminoethyl)-1H-5-indolylmethyl]-1,3-oxazolan-2-one be-longs to a group of medicines known as Serotonin 5-HT1D recep-tor agonist [1,2]. Zolmitriptan (ZOL) is well absorbed in liver and undergoes extensive metabolism to give major metabolites such as 4(S)-4-[3-(2-methylaminoethyl)-1H-5-indolyl-methyl]-1,3-ox-azolan-2-one (N-desmethylzolmitriptan; impurity A in USP draft 2011) and (4S)-4-[3-[2-(dimethyloxidoamino)ethyl]-1H-indol-5-yl]methyl-2-oxazolidinon(zolmitriptan N-oxide) [3]. Several HPLC methods for their determination have been reported. Quantification of zolmitriptan in human plasma using mass [4], coulometric [5] or fluorescence detection [6] is well described. LC methods are report-ed for the determination of zolmitriptan and its related substances known as (S)-4-(4-aminobenzyl)-1,3-oxazolidin-2-one (ZOL1), (2S)-2-amino-3-[3-(2-dimethylaminoethyl)-1H-5-indolyl]-propan-1-ol(RCB; impurity B in USP draft 2011), (S)-4-(4-hydrazinobenzyl)-1,3-oxazolidin-2-one (ZOL-hydrazine) and (4S)-4-[3-(2-aminoethyl)-1H-indol-5-ylmethyl]-1,3-oxazolidon-2-one (ZOL-amine) (Figure1) [7,8]. Another unknown impurity was isolated from zolmitriptan

tablets using preparative chromatography and characterized to be 4-4-[3-[2-(dimethyloxidoamino) ethyl]-1H-indol-5-yl] methyl-2-oxazolidinone by mass and NMR spectroscopy [9]. A new impuritywas identified, isolated, prepared and characterized as (4S,4S’)-4,4’-(2,2’-(4-(dimethylamino)butane-1,1-diyl)bis(3-(2-(dimethylamino)ethyl)-1H-indole-5,2-diyl))bis(methylene)di(oxazolidin-2-one) (ZOL-dimer) [10]. Many associated organic impurities may arise as a resultof side reactions or degradation of product during the manufacturingprocesses and or during storage of the drug substances. The criterionfor their acceptance up to certain limits are based on pharmaceuticalstudies or known safety data. As per regulatory guidelines, pharma-ceutical studies using a sample of isolated impurity can be consid-ered for safety assessment [11]. It is therefore essential to isolate andcharacterize unidentified impurities present in Active Pharmaceuti-cal Ingredients (APIs). Many synthetic protocols for Zolmitriptanhave been reported in literature [12-20]. The present work describesisolation, identification and characterization of the four isomeric

*Corresponding author: Neelakandan K, API Research Centre, Emcure Pharmaceutical Limited, Hinjawadi, Pune, 411057, India, Fax: +91 20 39821445; E-mail: [email protected]

Received March 11, 2013; Accepted April 05, 2013; Published April 08, 2013

Copyright: © 2013 Neelakandan K, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AbstractFour isomeric unknown impurities ranging from 0.08-0.12% were found in the purified sample of Zolmitriptan

during the batch analysis by gradient reverse phase ultra performance liquid chromatography (UPLC) and their molecular weights determined by liquid chromatography mass spectroscopy (LC-MS) analysis. Subsequently, all the four impurities were isolated by flash chromatography followed by semi-preparative HPLC and characterized by 1H NMR, 13C NMR, 1H-1H COSY, HMBC, HSQC, MS spectroscopy and HPLC. The structures for these four impurities were assigned to be following

Isomeric Impurity-1: 4-((3-(2-(dimethylamino)ethyl)-4-(2-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one,

Isomeric Impurity-2: 4-((3-(2-(dimethylamino)ethyl)-2-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one-,

Isomeric Impurity-3: 4-((3-(2-(dimethylamino)ethyl)-7-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one,

Isomeric Impurity-4: 4-((3-(2-(dimethylamino)ethyl)-6-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one

Isolation and characterization of impurities has helped us in improving the purity of API by removing these impurities using crystallization.

Isolation and Structural Elucidation of Novel Isomeric Process Related Impurities of ZolmitriptanNeelakandan K1,2*, Chaudhari Ashok1, Manikandan H2, Santosha N1, Prabhakaran B1 and Mukund Gurjar1 1API Research Centre, Emcure Pharmaceutical Limited, Hinjawadi, Pune, 411057, India2Department of chemistry, Annamalai University, Chidhambaram, India

Neelakandan et al. J Anal Bioanal Tech 2013, 4:2 DOI: 10.4172/2155-9872.1000165

Citation: Neelakandan K, Ashok C, Manikandan H, Santosha N, Prabhakaran B, et al. (2013) Isolation and Structural Elucidation of Novel Isomeric Process Related Impurities of Zolmitriptan. J Anal Bioanal Tech 4: 165. doi:10.4172/2155-9872.1000165

J Anal Bioanal Tech

Journal ofAnalytical & Bioanalytical TechniquesJo

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nalyt

ical & Bioanalytical Techniques

ISSN: 2155-9872

Citation: Neelakandan K, Ashok C, Manikandan H, Santosha N, Prabhakaran B, et al. (2013) Isolation and Structural Elucidation of Novel Isomeric

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process related impurities 4-((3-(2-(dimethylamino)ethyl)-4-(2-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one,4-((3-(2-(dimethylamino)ethyl)-2-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one-, 4-((3-(2-(dimeth-ylamino)ethyl)-7-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one and 4-((3-(2-(dimethylamino)ethyl)-7-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one which were found in the drug substance Zolmitriptan during manufacturing. Isolation and characterization of these impurities helped to improve the purity of the final API.

ExperimentalSamples and chemicals

The investigated sample of Zolmitriptan was synthesized in Emcure Pharmaceuticals Ltd, Hinjwadi Pune India. All the reagents were of

analytical grade unless stated otherwise. acetonitrile (HPLC grade), Milli-Q-grade water, Methanol HPLC grade.

Ultra performance liquid chromatography (UPLC)

A Waters Ultra performance liquid chromatography system equipped with UV detector having empower pro software system was used. The analysis was carried out on Acquity CSH phenyl-hexyl (2.1 mm×100 mm) 1.7 μ. by using mobile phase A (ammonium dihydrogen phosphate (2.72 gm), potassium dihydrogen phosphate (1.2 gm) and hexane-1-sulphonic acid sodium salt (1.0 gm) in 1.0 liter of HPLC grade water and pH was adjusted to 2.0 and mobile phase-B (homogeneous mixture of acetonitrile and methanol, 85:15 ratio) along with diluents such as acetonitrile and water in the ratio of 50:50. The LC gradient program was set as: time (min)/% solution B: 0/10, 3/10, 10/32, 11/32, 11.1/10 and 16/10. UV detection was carried out at 220 nm and flow rate was kept as 0.4 ml/min. This analytical method was able to detect

Isomeric Impurity-1

Isomeric Impurity-3

Isomeric impurity-2

Isomeric impurity-4

1

2

34

5

67

NH

N

NH

OO

8

91011

12

13

1415

1617 18

1920

21

Zolmitriptan

NH2

NH

OO

(Oxazolidine 2-one) NH

N

NH2

OH

ZOL2 (or) RCB

NH

NH

NH

OO

N-desmethylzolmitriptan

NH

N

NH

OO

O

N-Oxide

NH

NH2

NH

OO

ZOL-amine

NH

N

NH

OO

ONH

O

8

910

11

12

13

1415

16

1718

1920

21

2223

24

25

26

27

2829

3031

3233

7

1

2

34

5

6

34

NH

N

NH

OO

NH

OO

1

2

34

5

67

31

3233

8

91011

12

13

1415

16

17 18

192021

22

2324

25

2627

28 29

30

34

ONH

O

NH

N

NH

OO

26 2829 30

1

2

34

5

6

7

3132

33

8

91011

12

13

14

15

1617 18

192021

2223

24

25

27

34

NHO

O

NH

N

NH

OO

1

2

34

5

67

8

91011

12

13

1415

1617 18

19 20

21

22

2324

25

262728

2930

3132

33

34

NH

NH

OO NH2

ZOL-hydrazine or RCA

Figure 1: Structure of zolmitriptan and impurities.

Process Related Impurities of Zolmitriptan. J Anal Bioanal Tech 4: 167. doi:10.4172/2155-9872.1000167

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the process related impurities with good resolution.

Liquid Chromatography-Mass Spectroscopy (LC-MS)

The LC-MS analysis was carried out on YMC-Triart C18 (4.6 mm×150 mm) 3 µ or equivalent by using mobile phase A (0.01 M ammonium bicarbonate in HPLC grade water), mobile phase B (methanol and HPLC grade water, 90:10 ratio) with flow rate 1.0 ml/min. The LC gradient program was set as: time (min)/, % solution B:

0/10, 15/35, 30/40, 60/70, 61/10 and 70/10. The column temperature was maintained at 27°C and the detection was monitored at a wavelength of 225 nm. The mass spectral analysis of the impurities was carried out on a triple quadruple mass spectrometer (MDS Sciex model API 2000) performed in the positive (+ve) ion mode with electron spray ionization technique (ESI) interface using following conditions: declustering potential at 40 V, entrance potential at 10 Volts, focusing

Zolmitriptan

Isomeric impurity-1

Isomeric impurity-2

Isomeric impurity-3

Isomeric impurity-4

11 10 9 8 7 6 5 4 3 2 1 ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

Figure 3a: An overlaid 1H NMR of Zolmitriptan, Isomeric impurity-1, Isomeric impurity-2, Isomeric impurity-3, Isomeric impurity-4.


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NH

N

NH

OO

NH2

NH

OO

NH

NH

OO NH2

ON

O

Zolmitriptan

ZOL-1ZOL-hydrazine

NaNO2/HCl

SnCl2/HCl WaterIPA,HCL

.HCl

Conc. HCl

75- 80°C

Figure 2: Synthetic route for the preparation of zolmitriptan.


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potential at 325 Volts, curtain gas 20 liter per minute, ion spray voltage 4500 Volts and temperature 450°C.

Preparative liquid chromatography

A waters auto purification system equipped with binary gradient module (Waters 2545), system fluidics organizer (waters SFO), photodiode array detector (Waters 2998) and a sample manager (Waters 2767) with Mass lynx data handling system was used along with Modcol column hard ware (50×700 mm) packed with diasogel C-18, 10 μm silica gel having bed length 300 mm was employed for

the isolation of impurities. Mobile phase-A (0.5% (v/v) acetic acid in water) and Mobile phase-B (acetonitrile) was chosen with flow rate at 25 ml/min and UV detection was carried out at 254 nm. The gradient program was as follows. Time (min)/A (v/v):B (v/v), T0.01/100:00, T10.0/100:00, T11.0/90:10, T25/90:10, T26.00/80:20, T40.0/80:20, T41.00/75:25, T55.0/75:25, T56.0/70:30, T70/70:30, T71.0/50:50, T80.0/50:50.

Flash chromatography

A grace Revelers flash chromatographic system equipped with

160 140 120 100 80 60 40 20 ppm

160 140 120 100 80 60 40 20 ppm

160 140 120 100 80 60 40 20 ppm

160 140 120 100 80 60 40 20 ppm

160 140 120 100 80 60 40 20 ppm

Zolmitriptan

Isomeric impurity-1

Isomeric impurity-2

Isomeric impurity-3

Isomeric impurity-4

Figure 3b: An overlaid 13C NMR of Zolmitriptan, Isomeric impurity-1, Isomeric impurity-2, Isomeric impurity-3, Isomeric impurity-4.

160 140 120 100 80 60 40 20 ppm

160 140 120 100 80 60 40 20 ppm

160 140 120 100 80 60 40 20 ppm

160 140 120 100 80 60 40 20 ppm

160 140 120 100 80 60 40 20 ppm

Zolmitriptan

Isomeric impurity-1

Isomeric impurity-2

Isomeric impurity-3

Isomeric impurity-4

Figure 3c: An overlaid DEPT NMR of Zolmitriptan, Isomeric impurity-1, Isomeric impurity-2, Isomeric impurity-3, Isomeric impurity-4.


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quaternary gradient module, UV-VIS detector. Grace make flash cartridge having 80 gm silica and 40 micron practical size was employed for the isolation of the impurities. Mobile phase-A consists of dichloromethane and Mobile phase-B consists of 0.1% triethyl amine in methanol. Flow rate was kept at 35 ml/min and UV detection was carried out at 225 nm. The gradient program was as follows. Time (min)/A (v/v): B (v/v), T0.01/100:00, T10.0/100:00, T11.0/95:05, T21/95:05, T22.00/92:08, T32.0/92:08, T33.00/90:10, T43.0/90:10, T44.0/85:15, T54.00/85:15, T55.0/80:20, T65.00/80:20, T66.0/75:25, T76.0/75:25, T77/70:30, T87.0/70:30.

NMR spectroscopy

The NMR experiments were performed on Varian NMR spectrometer at 400 MHZ. The 1H chemical shift values were reported on the δ scale in ppm, relative to TMS (δ=0) as internal standard, 13C NMR, DEPT, HMBC and HSQC spectra were also recorded.

Isolation of impurities

2.0 liter of mother liquor obtained after Zolmitriptan purification

was concentrated on rotavapour to obtain a thick residue. 8.0 gm of this residue was loaded on flash chromatography using the conditions mentioned in the section Flash chromatography. Eluted fractions from flash chromatography were monitored by HPLC. Zolmitriptan was completely separated from the isomeric impurities. The enriched fraction of the isomeric impurities was further purified by semi-preparative HPLC.

In case of each impurity, fractions having more than 90% HPLC purity were combined, concentrated on rotavapour and extracted with dichloromethane. All the four impurities were analyzed by HPLC, 1H NMR, 13C NMR, and MS.

Results and Discussion Detection of the impurities

During the process development of Zolmitriptan (Figure 2 Scheme), four unknown impurities were found in the range of 0.08-0.15% by reverse phase liquid chromatography. The sample was subjected to LC-MS using the conditions mentioned in section Liquid Chromatography-Mass Spectroscopy (LC-MS). The LC-MS study revealed that all the four impurities were having the same mass. These were enriched by flash chromatography up to 10% and purified by preparative HPLC, using the conditions described in the section NMR Spectroscopy. For each impurity, fractions having purity more than 90% were combined, concentrated under reduced pressure and extracted with dichloromethane. These impurities were analyzed by HPLC, 1H NMR, 13C NMR, DEPT, HMBC, HSQC and MS.

Structure elucidation of the process impurities

The ESI-MS (positive ion) spectrum of the impurities showed a protonated ion at m/z 462.9 and zolmitriptan showed at m/z 288 amu. The difference of the mass between zolmitriptan and impurities was 175 amu, which indicated the presence of additional benzyl oxazolidinone moiety. The benzyl oxazolidinone could be attached anywhere on the indole ring to form number of isomers. As the MS/MS fragmentation pattern of the impurities could not give information regarding the possible structure of the impurities, the NMR experiments were carried out to confirm the structures. A comparison of 1H NMR spectra of the

Zolomitriptan Related Substances By UPLC

AU

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

0.020

0.018

0.016

0.014

0.012

0.010

0.008

0.006

0.004

0.002

0.000

Isom

eric

impu

rity

- 4 -

11.8

75

Isom

eric

impu

rity

- 1 -

10.9

67

Rel

Com

pd-D

- 1

- 10.

004

Rel

Com

p-d

- 4.

999

Rel

Com

p-B

- 3

.210

N-O

xide

- 6.

347

Oxa

zolid

ine-

2-on

e -

1.31

1

Zolm

itrip

tan

- 5.2

52

Isom

eric

impu

rity

- 2 -

12.3

94Is

omer

ic im

purit

y - 3

- 12

.655

Figure 4a: An UPLC chromatogram of mixture of all the impurities.


AU

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

0.010

0.008

0.006

0.004

0.002

0.000

Isom

eric

impu

rity

- 4 -

11.8

75

Isom

eric

impu

rity

- 1 -

10.9

67

Isom

eric

impu

rity

- 2 -

12.3

94Is

omer

ic im

purit

y - 3

- 12

.655

Figure 4b: UPLC chromatogram of Zolmitriptan API.


AU

Minutes

0.010

0.008

0.006

0.004

0.002

0.000

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Impu

rity

- 1 -

10.9

57

Isom

eric

impu

rity

- 4 -

11.8

85Is

omer

ic im

purit

y - 2

- 12

.399

Isom

eric

impu

rity

- 3 -

12.6

65

Figure 4c: UPLC chromatogram of Zolmitriptan API spiked with Isomeric impurity-1, impurity-2, impurity-3 and impurity-4.


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impurities and Zolmitriptan indicated the presence of 9 additional protons in each case. Three were in aromatic region, four in aliphatic region and one additional exchangeable proton was found. This proved that additional benzyl oxazolidinone moiety substituted on indole was present in these impurities. 13C NMR spectra of impurities showed that 10 additional carbons were present in the impurities as compared to zolmitriptan. Out of the 10 carbon six were aromatic, three were aliphatic and one was carbonyl. DEPT spectral analysis of the impurities showed the presence of six CH2, nine CH and two CH3

carbons. The position of the substitution was confirmed by comparing the 1HNMR and 13CNMR spectra of Zolmitriptan and corresponding isomeric Impurities. The assignment of protons was confirmed by 1H-1H COSY experiment and the attachments of the protons and carbons were confirmed by HSQC and HMBC experiments in case of each impurity.

Structural elucidation of Impurity-1: In the 1H NMR spectrum of Impurity-1, H-2 exhibited a doublet (7.05 ppm, J=1.6 Hz), where as H-6 (7.29 ppm, J=8.3 Hz) and H-7 (7.1 ppm, J=8.3 Hz) appeared

Position* Zolmitriptan Isomeric Peak-1 Isomeric Peak-2 Isomeric Peak-3 Isomeric Peak-41H, (ppm)(Hz)

13C DEPT 1H, (ppm) (Hz)


13C DEPT 1H, (ppm)(Hz)


13C DEPT

1 1H, 10.68 (brs) - - 1H, 10.87 (s) - - 1H, 11.06 (s) - - 1H, 10.58 (s) - - 1H, 10.72 (s) - -2 1H, 7.10

(dj=2.08)122.97 CH 1H, 7.00-7.06

(m)122.64 CH - 134.28 Q 1H, 7.10 (d) 122.76 CH 1H, 7.15

(d)123.19 CH

3 - 112.72 Q - 112.95 Q - 109.45 Q - 112.92 Q - 112.42 Q4 1H, 7.34

(s)119.13 CH - 123.7 Q 1H, 7.38-7.40

(m)118.9 CH 1H, 7.36-7.40

(m)118.19 CH 1H, 7.44 (s) 119.48 CH

5 - 126.17 Q - 135.26 Q - 128.96 Q - 128.5 Q - 135.08 Q6 1H, 6.91-6.93

(dd)122.86 CH 1H, 7.00-7.06

(m)123.96 CH 1H, 6.97

(d)123.15 CH 1H, 7.00

(s)123.54 CH - 123.83 Q

7 1H, 7.24 (d,j=8.24)

111.61 CH 1H,7.20-7.34 (m)

110.82 CH 1H, 7.27 (d)

111.02 CH - 124.59 Q 1H, 7.09 (s) 112.48 CH

8 - 135.39 Q - 135.62 Q - 134.96 Q - 135.42 Q - 135.08 Q9 - 127.79 Q - 125.56 Q - 126.35 Q - 126.61 Q - 126.76 Q10 4H, 2.75-2.91

(m)40.75 CH2 2H, 2.52-2.93

(m)40.35 CH2 2H, 2.79-2.97

(m)40.54 CH2 2H, 2.80-2.95

(m)40.41 CH2 2H, 2.70-3.01

(m)40.12 CH2

11 3H, 4.00-4.249 (m)

53.37 CH 1H, 3.79-4.39 (m)

53.39 CH 1H, 4.02-4.33 (m)

53.09 CH 1H, 4.04-4.34 (m)

53 CH 1H, 3.69-4.34 (m)

52.47 CH

12 3H, 4.00-4.24 (m)

68.5 CH2 2H, 3.79-4.39 (m)

68.37 CH2 2H, 4.02-4.33 (m)

67.97 CH2 2H, 4.04-4.34 (m)

68.04 CH2 2H, 3.69-4.34 (m)

68.18 CH2

14 - 159-26 -C=O - 158.47 -C=O - 158.59 -C=O - 158.58 -C=O - 158.45 -C=O16 1H, 7.75 (s) - - 1H, 7.52 (s) - - -1H, 7.76

(s)- - 1H, 7.80 (s) - - 1H, 7.80(s) - -

17 2H, 2.48-2.52 (m)

- - 2H, 1.80-2.07 (m)

23.96 CH2 2H, 2.79-2.97 (m)

22.7 2H, 2.80-2.95 (m)

22.98 2H, 2.70-3.01 (m)

23.01 CH2

18 4H, 2.75-2.91 (m)

- - 2H, 1.80-2.07 (m)

61.38 CH2 2H, 2.50-2.53 (m)

59.89 2H, 2.53 (t) 59.85 2H, 2.54 (t) 59.99 CH2

20 6H, 2.21(s)

- - 3H, 1.80-2.07 (m)

44.65 CH3 3H, 2.23 (s)

45.12 3H, 2.23 (s) 45.12 3H, 2.24 (s) 45.12 CH3

21 6H, 2.21 (s)

- - 3H, 1.80-2.07 (m)

44.65 CH3 3H, 2.23 (s)

45.12 3H, 2.23 (s) 45.12 3H, 2.24 (s) 45.12 CH3

22 - - - - 138.06 Q 1H, 7.38-7.40 (m)

129.75 CH 1H, 7.36-7.40 (m)

129.83 CH 1H, 7.31 (d) 129.15 Q

23 - - - 1H, 7.20-7.34 (m)

129-84 CH 1H, 7.56 (d) 127.48 CH 1H, 7.59 (d) 128.21 CH 1H, 7.25 (d) 129.4 CH

24 - - - 1H, 7.20-7.34 (m)

128.27 CH - 135.65 Q - 137.05 Q - 140.69 CH

25 - - - 1H, 7.20-7.34 (m)

128.87 CH 1H, 7.56 (d) 127.48 CH 1H, 7.59 (d) 128.21 CH 1H, 7.25 (d) 129.4 Q

26 - - - 1H, 7.20-7.34 (m)

130.32 CH 1H, 7.38-7.40 (m)

129.75 CH 1H, 7.36-7.40 (m)

129.83 CH 1H, 7.31 (d) 129.15 CH

27 - - - - 133.48 Q - 131.24 Q - 132.25 Q - 134.61 CH28 - - - 2H, 2.52-2.93

(m)37.53 2H, 2.79-2.97

(m)39.92 CH2 2H, 2.80-2.95

(m)39.92 CH2 2H, 2.70-3.01

(m)38.3 CH2

29 - - - 1H, 3.79-4.39 (m)

52.85 CH 1H, 4.02-4.33 (m)

52.36 CH 1H, 4.04-4.34 (m)

52.53 CH 1H, 3.69-4.34 (m)

52.36 CH

30 - - - 2H, 3.79-4.39 (m)

68.49 CH2 2H, 4.02-4.33 (m)

68.03 CH2 2H, 4.04-4.34 (m)

68.04 CH2 2H, 3.69-4.34 (m)

68.18 CH2

32 - - - - 158.79 -C=O - -158.64 -C=O - 158.69 -C=O - 158.74 -C=O34 - - - 1H, 7.67 (s) - - -1H, 7.83 (s) - - 1H, 7.82 (s) - - 1H, 7.54 (s) - -

s: singlet; d: doublet; brs: broad singlet; J: coupling constant; Q: quaternary carbon.aRefer structures for numbering (Figure 1)

Table 1: 1H, 13C NMR, and DEPT data of Zolmitriptan, Impurity-1, Impurity-2, Impurity-3, Impurity-4.


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CH2 CH2

CH2 CH2

CH3 CH3

CH3 CH3

CH2


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as mutually coupled doublets and H-4 was absent. This indicated that the benzyl oxazolidinone was substituted on C-4. Also a complex splitting pattern was observed for H-22, H-23, H-25 and H-26 indicating presence of o-di-subtituted phenyl group. In 13C NMR spectrum of zolmitriptan C-4(CH) resonated at 119 ppm which was absent in Impurity-1. Also the chemical shift of C-22, C-23, C-25 and C-26 confirmed that benzyl oxazolidine was ortho-sustituted on C-4. From the above spectral information, the structure of the impurity was confirmed as 4-((3-(2-(dimethylamino)ethyl)-4-(2-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl) oxazolidin-2-one.

Structural elucidation of Impurity-2: In the 1H NMR spectrum of Impurity-2, H-6 appeared as a double doublet (6.97 ppm, J=8.3, Hz), H-7 as a doublet (7.26 ppm, J=8.3 Hz), H-4 as a singlet (7.37 ppm) and H-2 was absent. This indicated that the benzyl oxazolidine was substituted on C-2. A typical p-di-substituted phenyl pattern was observed with ortho coupling between H-22, C-23 and H-25, H-26 (7.38 and 7.56 ppm, J=8.1 Hz). In 13C NMR spectra of Zolmitriptan, C-2(CH) appeared at 122.86 ppm which was absent in the Impurity-2, also the C-22, C-23, C-25 and C-26 were showing a typical p-di-substituted pattern. This data confirmed that benzyl oxazolidine was substituted on C-2, and the substitution was at para position. From the above spectral information, the structure of the impurity was confirmed as 4-((3-(2-(dimethylamino) ethyl)-2-(4-((oxazolidin-4-yl) methyl) phenyl)-1H-indol-5-yl) methyl) oxazolidin-2-one.

Structural elucidation of Impurity-3: In the 1H NMR spectrum of Impurity-3, H-2 appeared as a doublet (7.1 ppm, J=2 Hz) where as H-6 (6.98 ppm) and H-4 (7.36 ppm) appeared as singlets and H-7 was absent. This indicated that the benzyl oxazolidine was substituted on C-7. Also, a typical p-di-subtituted pehyl pattern was observed with ortho coupling between H-22, H-23 and H-25, H-26 (7.38 and 7.59 ppm, J=7.8 Hz). A comparison with 13C NMR spectra of Zolmitriptan indicated that the C-7(CH) at 111.61 ppm was absent in Impurity-3. Also, the C-22, C-23, C-25 and C-26 were showing a typical p-di-substituted phenyl pattern. This confirmed that benzyl oxazolidine was substituted on C-7 and the substitution was at para position. From the above spectra information, the structure of the impurity was confirmed as 4-((3-(2-(dimethylamino)ethyl)-7-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one.

Structural elucidation of Impurity-4: In the 1H NMR spectrum of Impurity-4, H-2 appeared as a doublet (7.15 ppm, J=1.6 Hz) while H-7 (7.10 ppm) and H-4 (7.43 ppm) appeared as singlets and H-6 was found to be absent. This indicated that the benzyl oxazolidine was substituted on C-4. A typical p-di-substituted phenyl pattern was observed with ortho coupling between H-22, H-23 and H-25, H-26 (7.23 and 7.30 ppm, J=7.9 Hz). In 13C NMR spectra of Zolmitriptan, C-6(CH) signal at 122.97 ppm was found to be absent in the spectrum of Impurity-4. Also, the C-22, C-23, C-25 and C-26 were showing a typical p-di-substituted phenyl pattern. This confirmed that benzyl oxazolidine was substituted on C-7 and the substitution was at para position. From the above spectral information, the structure of the impurity was confirmed as 4-((3-(2-(dimethylamino)ethyl)-6-(4-((oxazolidin-4-yl)methyl) phenyl)-1H-indol-5-yl)methyl)oxazolidin-2-one.

The 1H and 13C NMR chemical shift values of zolmitriptan, isomeric impurity-1, isomeric impurity-2, isomeric impurity-3 and impurity-4 are given in table 1. An overlaid 1H, 13C NMR and DEPT spectra’s of zolmitriptan, isomeric impurity-1, isomeric impurity-2, isomeric impurity-3 and impurity-4 were presented in figures 3a-3c. An UPLC

chromatogram of zolmitriptan API and zolmitriptan API spiked with isomeric impurity-1, impuity-2, impurity-3 and impurity-4 are

ConclusionIn conclusion, we have successfully identified impurities which

were detected during the batch analysis of final API. Subsequent experiments have enabled us to identify the critical parameters to control of the impurities below the acceptable level at laboratory scale well as commercial scale.

Acknowledgment

The author gratefully acknowledges the management of the Emcure Pharma-ceuticals Ltd for providing the facility/instruments for the research work. Authors would also like to thank colleagues of Analytical Research Department (ARD) for their support.

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