Isolation and Structure Characterization of related impurity in Olanzapine key starting material by LC/ESI-MS and NMR

 

Jagadeesh Narkedimilli^1,2*, Vishweshwar Vavilala¹, Sandeep Mohanty¹,Saravanan Manvalan¹, Jayashree Anireddy²

¹Dr. Reddy’s Laboratories Limited, API Plant, Bollaram-III, Plot No 116, IDA Bollaram, Medak District, Hyderabad 502325, Telangana, India.

²Centre for Chemical Sciences and Technology, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad, Kukatpally, Hyderabad 500085, Telangana, India.

*Corresponding Author E-mail: jagadeeshn@drreddys.com

Olanzapine key starting material, 5-methyl-2-((2-nitrophenyl)amino)thiophene-3-carbonitrile (3), is a key starting material of Olanzapine. During the impurity profile study an unknown peak was detected by HPLC and MS methods. Identification, isolation and structural characterization in olanzapine key starting material is described in this article. The impurity was isolated by preparative HPLC, Based on the complete spectral analysis, MS, 1D NMR (¹H and ¹³C) and 2D NMR (COSY, HSQC and HMBC) the chemical structure of unknown impurity was characterized as 2-(bis(2-nitrophenyl)amino)-5-methylthiophene-3-carbonitrile.

 

 

INTRODUCTION:

Olanzapine (1), chemically known as 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5] benzodiazepine is an antipsychotic drug that belongs to the thienobenzodiazepine class. Olanzapine is one of the most widely used atypical antipsychotics. It is effective against both positive and negative symptoms of schizophrenia and is often used in the treatment of patients who are non-responders to classical neuroleptics, such as butyrophenones and phenothiazines and in comparison it has the advantage of not causing extrapyramidal side effects as classical neuroleptics do. USFDA has approved the use of olanzapine also for the treatment of acute mania¹.

 

To commercialize an active pharmaceutical ingredient (API), it is mandatory for the manufacturer to identify and characterize all the unknown impurities. For impurities in new drug substances, according to International Conference on Harmonization (ICH) guidelines for a maximum daily dose ≤2 g/day of a drug substance, the reporting and identification thresholds are 0.05% and 0.10%, respectively², and need for the overall assessment of manufacturing processes in terms of the final product quality is of paramount importance to the company. To achieve this, understanding of the overall manufacturing process, quality assessment of raw materials and reagents used in the process and detection, identification, quantification and control of single maximum unknown impurities at each and every step of the process is critical for delivering an API of high quality. In this context, a comprehensive study was taken for impurity profile study of Olanzapine key starting material of laboratory batch, identification and characterization of unknown impurity so that it will help to minimization at a level of NMT 0.1 % at the final product can be worked out for delivering an API of high quality³.

 

To the best of our knowledge, no earlier reports have been discussed on this impurity profile study of Olanzapine key starting material.

 

A through literature survey reveals a variety of synthetic methods^4-5(Figure 1), analytical methods^6-24 of quantitative determination of olanzapine and its related substances, mass spectrophotometric methods and also simultaneous methods for the determination of olanzapine. Other journals^25-33 have been reviewed related to characterization.

 

 

Figure 1: Olanzapine (1) manufacturing process.

 

MATERIAL AND METHODS:

Chemical and reagents:

The investigated samples of Olanzapine key starting materials were obtained from synthetic R&D laboratory of Dr. Reddy’s Laboratories Ltd., CTO-IV, Hyderabad, India. HPLC grade methanol and acetonitrile were obtained from Merck, Mumbai, India. AR grade monobasic sodium phosphate salt, tetrabutyl ammonium hydrogen sulfate and sodium hydroxide were obtained from Merck, Mumbai, India. Dimethylsulphoxide-d₆ was purchased from Aldrich Chemical Co., USA. Water used for preparing mobile phase was purified using Millipore (MA 01821, USA) water purification system.

 

High performance liquid chromatography (Analytical):

A Waters Model Alliance 2695 separation module (Waters Corporation, Milford, MA, USA) equipped with a waters 2998 photo diode array detector was used. Data was processed through Waters empower software. An isocratic analytical method of Solvent A and Solvent B in the ratio of 40: 60 (v/v) with Kromasil C18 column, 250 x 4.6mm i.d., 5mm particle size (AkzoNobel, Sweden) with a mobile phase consisting of solvent A: Dissolved 2.1842 gr of sodium dihydrogen orthophosphate in 400 ml of milli-q-water (0.035M). Adjusted the pH to 6.8 with diluted sodium hydroxide solution filter through 0.45µm filter and degas and solvent B: Mixture of methanol and acetonitrile in the ratio of (1: 1). Column temperature was maintained at ambient condition. Flow rate was kept at 1.5 mL min^-1 and the column eluent was monitored at 250 nm.

 

Preparative liquid chromatography:

An Agilent 1200 preparative chromatography system equipped with Agilent G1315D photo diode array UV detector, fraction collector model Agilent G1346B and Rheodyne Injector Model 2260A with 1.8 ml loop was used. A 250 x 21.2 mm i.d column packed with 10µm Kromasil C18 (AkzoNobel, Sweden) was employed for separation with a mobile phase consisting of water and acetonitrile in the ratio of 30:70 (v/v) with UV detection at 250 nm at a flow rate of 15.0 mL min^-1. The data was recorded using Agilent Chemstation software.

 

Liquid chromatography-mass spectrometry:

An Agilent 1200 series LC system coupled to a quadrupole mass spectrometer (Agilent LC/MS model 6120, Agilent Technologies Inc., Santa Clara, CA, USA) with multimode source which contains both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) and ionization was done in positive mode. The ion source temperature was set at 300°C. Nitrogen was used as the drying gas at a flow rate of 10 L min^-1 and as the nebulizer gas at a pressure of 60 psi.Zorbax SB C18 column, 50 x 4.6mm i.d., 3.5mm particle size (Agilent technologies, Santa Clara, CA 95051, USA) with a mobile phase consisting of 0.1% HCOOH (Aq) and acetonitrile in the ratio of 65: 35 (v/v) with UV detection at 250 nm at a flow rate of 1.0 mL min^-1. The column temperature was maintained at 40°C. The sample was prepared in diluent at 1.0 mg mL^-1 concentration and 5 μL of sample solution was injected in LC/MS system. Data acquisition and processing were done using Chemstation software.

 

NMR spectroscopy:

¹H, ¹³C and two dimensional (2D) NMR experiments were performed on 500 MHz Varian Unity Inova FT-NMR instrument in DMSO-d₆. The ¹H chemical shift values were reported on the δ scale in ppm, relative to TMS (δ= 0.00 ppm) and in the ¹³C chemical shift values were reported on the δ scale in ppm, relative to DMSO-d₆ (δ= 39.5 ppm).

 

RESULTS AND DISCUSSION:

Detection of impurity:

A typical HPLC chromatogram of a laboratory batch of key starting material (3) was recorded as per described HPLC method conditions. One major unknown peak was identified in the chromatogram at a relative retention time of about 0.91 with respect to the main peak is shown in figure 2. The LC/MS compatible method was developed and used to detect the impurity. Unknown impurity isolated by preparative HPLC and characterized by complete spectral data. Unknown impurity chemical structure shown in figure 3.

 

 

Figure 2: Typical HPLC chromatogram of olanzapine key starting material (3).

 

 

Figure 3: Structure of unknown impurity, atom numbering used for NMR assignments.

 

Structural elucidation of impurity:

The ⁺ve MS spectrum (Figure 4) of the impurity exhibits protonated molecular ion peak at m/z 381.0, the odd m/z number of [M+H]⁺ ions suggest that impurity contains even number of nitrogen atoms (nitrogen rule). From these results the molecular ion of impurity was found to be at m/z 380. The ¹H NMR spectrum (Figure 5) showed signals at 2.3 ppm (3H), 6.8 ppm (1H), 7.4 ppm (2H), 7.6 ppm (2H), 7.8 ppm (2H) and 8.1 ppm (2H) corresponding to twelve protons. The singlet at 2.3 ppm was considered as methyl and referenced for three protons. The ¹³C NMR spectrum (Figure 6) showed twelve signals corresponding to eighteen carbon atoms signals. The quaternary carbon signals observed at 156.0 ppm (C8), 143.9 ppm (C1&C20), 137.8 ppm (C6&C15), 132.7 ppm (C10), 112.8 ppm (C14) and 98.0 ppm (C12), for these quaternary carbon signals correlations were not observed in HSQC spectrum. The COSY spectrum (Figure-7) showed correlations between the signals at 7.4, 7.6, 7.8 and 8.1 ppm. The HSQC correlations (Figure 8) showed the presence of one methyl and five methine signals. Reviewed HMBC spectrum (Figure 9), the methyl signal at 2.3 ppm showed correlations with carbon signal at 124.0 ppm for C11 and signal at 156.0 ppm for C8 (6.8 ppm). This confirms the presence of thiophene moiety. The splitting and the intensities of the remaining signals in aromatic region confirm the presence of two nitrophenyl moieties. The detailed information for the ¹H NMR and ¹³C NMR spectra can be seen in table 1. Based on this data the structure of unknown impurity was confirmed as 2-(bis(2-nitrophenyl)amino)-5-methylthiophene-3-carbonitrile.

 

 

Figure 4: +ve Mass spectrum of unknown impurity.

 

Figure 5: ¹H NMR spectrum of unknown impurity.

 

 

Figure 6: ¹³C NMR spectrum of unknown impurity.

 

 

Figure 7: COSY NMR spectrum of unknown impurity.

 

Figure 8: HSQC NMR spectrum of unknown impurity.

 

 

Figure 9: HMBC NMR spectrum of unknown impurity and key linkages.

 

Table 1: ^aThe position numbering has given according to impurity (Figure 3).

Positiona	δH (ppm)	δC (ppm)
1	_	143.9
2, 19	8.1	126.2
3, 18	7.5	127.7
4, 17	7.8	135.0
5, 16	7.4	128.7
6	_	137.8
8	_	156.0
10	_	132.7
11	6.8	124.2
12	_	98.0
13	2.3	14.8
14	_	112.8
15	_	137.8
20	_	143.9

 

CONCLUSIONS:

A new process related impurity observed during the impurity profile study of Olanzapine key starting material (3) by HPLC and LCMS methods, isolated by preparative HPLC and structure unambiguously characterized by LC-MS and NMR techniques as 2-(bis(2-nitrophenyl)amino)-5-methylthiophene-3-carbonitrile.

 

ACKNOWLEDGEMENTS:

The authors wish to thank the management of Dr.Reddy’s Laboratories Ltd. For permitting to carry out the present work. The authors also wish to thank the colleagues of Analytical Research & Development and Process Research & Development, Department for supporting this work. Dr. Reddy’s communication number for this research article: IPDO-IPM-00572.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 19.04.2018         Modified on 25.04.2018

Accepted on 30.04.2018         © AJRC All right reserved