Characterization of impurities in Teneligliptin hydrobromide hydrate by using LCMS/MS and NMR

Maruthi R^*, Chandan R. S¹, Anand Kumar Tengli²

Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru-570015, Karnataka.

*Corresponding Author E-mail: maruthireddy666@gmail.com

ABSTRACT:

An accurate and lively LC-MS/MS and NMR analytical methods have been developed for recognition and characterization of key impurities in Teneligliptin. Teneligliptin is an anti-diabetic drug used to treat Diabetes mellitus Type II and three unknown impurities were detected in Teneligliptin bulk drug substance using RP-UFLC method at RT 4.02 min, 5.09 min and 6.2 min. These impurities were isolated by preparative HPLC and further characterized by using LC-MS/MS and NMR techniques. LC-MS/MS analysis was performed using C₁₈reverse phase column and with Methanol and Ammonium Formate (80:20) as a mobile phase. The flow rate was maintained at 0.5mL/min with injection volume as 10 µL. The separations were achieved with binary gradient program and the column is maintained with ambient temperature. The MS conditions adopted for this analysis with scan range of m/z = 50 to 500 with dwell time 3 seconds. NMR was carried out using DMSO as solvent. Based on spectral data, the impurities have been characterized as IMP 3, IMP 5 and IMP E.

KEYWORDS: Teneligliptin, LC-MS/MS, Diabetes mellitus, DMSO and Preparative HPLC, NMR.

INTRODUCTION:

Teneligliptin Hydrobromide Hydrate is a very strong, focused, and durable DPP-4 inhibitor (1, 2, 3, and 4). Glucagon like peptidase (GLP-1) a peptidase discharged from the GIT because of sustenance consumption upgrades insulin emission and stifles glucagon emission from the pancreas, consequently assuming a critical part in controlling postprandial blood glucose level. The peptide is quickly inactivated by debasement by DPP-4 inhibitor, a chemical generally conveyed in the body. DPP-4 inhibitor debasement, expanding the centralization of dynamic GLP-1 in the blood, which fortifies glucose subordinate insulin discharge and in the meantime, smothers glucagon emission, subsequently displaying a glucose bringing down impact. It is successfully used to treat type 2 diabetes mellitus.

The most typically detailed unfavorable responses incorporate hypoglycemia, clogging, and feeling of developed stomach area, stomach distress, sickness, stomach torment, meteorism, stomatitis, skin irritation, rash, pruritus, dermatitis and disquietude. [1-4]

Fig 1: Structure of Teneligliptin

EXPERIMENTAL:

Chemicals and Reagents:

Teneligliptin (99%pure) samples provide by Micro Labs Pvt. Ltd, Bangalore. HPLC grade Acetonitrile was procured from Merck. Analytical reagent grade Ammonium Acetate was purchased from Loba Chemie. DMSO was procured from Merck.

Instrumentation:

Chromatographic separations were obtained using Shimadzu UFLC LC 20 AD with Photo diode array detector. For data analysis LC solutions software is used. For weighing samples Shimadzu electronic weighing balance was used and for sonication of samples and mobile phase Mark ultra sonicator was used.

LC-MS/MS was performed by using Shimadzu LC-MS/MS 8030 system with triple Quadra pole mass spectrometry. NMR was performed on FT NMR spectrometer system – 400MHz (Make: Agilent USA model: 400 MRDD2).

HPLC Experiment:

Chromatography was carried out on a Phenomenex C₁₈column (5µ, 230*4.6mm) with ambient temperature using Shimadzu LC-20AD. Separations were observed at 246nm. Injection volume was 10µL and the flow rate was 1.0mL/min with binary gradient programming. Mobile phase consists of Methanol, Acetonitrile and Potassium dihydrogen orthophosphate (40:20:40 v/v/v/). The pH is changed in accordance with 4.6 using orthophosphoric acid.[5-10]

LCMS and LCMS/MS Experiment:

LC-MS/MS was performed by using Shimadzu LC-MS/MS 8030 system with triple Quadra pole mass spectrometry.[11, 12]

NMR Experiment:

NMR was performed on FT NMR spectrometer system – 400MHz (Make: Agilent USA model: 400 MRDD2).

RESULTS AND DISCUSSION:

The LCMS/MS spectra (Fig 2) of Teneligliptin key impurities showed peaks at RT 3.3 min displayed the protonated molecular ion (M⁺+) at m/z = 426.58, this is corresponding to the molecular formula C₂₂H₃₁N₆OS⁺.

Fig. 2: HPLC Spectrum of Teneligliptin

POSITIVE IONIZATION:

Teneligliptin (Mol weight: 426.58 g/mol; (M+2))

Fig 3: LCMS/MS spectra of Teneligliptin

Fig 4: LCMS/MS spectra of IMP 3

Fig 5: Structure of IMP 3

Fig 6: LCMS/MS spectra of IMP 5

Fig 7: Structure of IMP 5

Fig 8: LCMS/MS spectra of IMP E

Fig 9: Structure of IMP E

Fig 10: Mass fragmentation of Teneligliptin

NMR Study of Teneligliptin key impurity:

The NMR data (₁H, ₁₃C) of Teneligliptin impurity was recorded in DMSO at 400MHz for H₁and 100MHz for ₁₃C on Agilent FT-NMR 400MHz spectrometer.

Fig 11: ¹H NMR of Teneligliptin

The proton NMR spectrum (Fig 11) of Teneligliptin has showed C=C-CH₃ peaks at 1.9ppm, R₂ N-CH₃ proton peaks from 2 to 3ppm(α to nitrogen C is attached to nitrogen), ArNH₂ peaks from 3 to 5ppm, Ar-H peaks from 6 to 7ppm and R-C=o-NH₂ peaks from 7 to 8ppm.

Fig 12: ¹³C NMR of Teneligliptin

The C- NMR of Teneligliptin (Fig 12) has showed R-CH₃ peak at 13.7ppm, R-CH₂-NH₂ peaks from 30 to 65ppm, C=C peaks from115 to 140ppm, C in aromatic ring shows peaks from 125 to 150ppm and RNC=O gives peak at 169.

Fig: 13 ¹H NMR of IMP E

The proton NMR spectrum (Fig 13) of Teneligliptin has showed C=C-CH₃ peaks at 1.7ppm, R₂ N-CH₃ proton peaks from 2 to 3ppm(α to nitrogen C is attached to nitrogen), ArNH₂ peaks from 3 to 5ppm, Ar-H peaks from 6 to 7ppm, Ar-OH peak at 12.01ppm and R-C=O-NH₂ peaks from 7 to 8ppm.

Fig: 14 ¹³C NMR of IMP E

The C- NMR of Teneligliptin (Fig 14) has showed R-CH₃ peak at 13.138ppm, R-OH peak at 29, R-CH₂-NH₂ peaks from 30 to 65ppm, C=C peaks from115 to 140ppm, C in aromatic ring shows peaks from 120 to 150ppm and RC=O gives peak at 171.

Fig: 15 ¹H NMR of IMP 3

The proton NMR spectrum (Fig 15) of Teneligliptin has showed C=C-CH₃ peaks at 1.7ppm, R₂ N-CH₃ proton peaks from 2 to 3ppm(α to nitrogen C is attached to nitrogen), ArNH₂ peaks from 3 to 5ppm, Ar-H peaks from 6 to 7ppm and R-C=O-NH₂ peaks from 7 to 8ppm.

Fig: 16 ¹³C NMR of IMP 3

The C- NMR of Teneligliptin (Fig 16) has showed R-CH₃ peak at 14.7ppm, R-CH₂-NH₂ peaks from 30 to 65ppm, C=C peaks from115 to 140ppm, C in aromatic ring shows peaks from 120 to 150ppm and RNC=O gives peak at 169.

Fig: 17 ¹H NMR of IMP 5

The proton NMR spectrum (Fig 17) of Teneligliptin has showed C=C-CH₃ peaks at 1.763ppm, R₂ N-CH₃ proton peaks from 2 to 3ppm(α to nitrogen C is attached to nitrogen), ArNH₂ peaks from 3 to 5ppm, Ar-H peaks from 6 to 7ppm and R-C=O-NH₂ peaks from 7 to 8ppm.

Fig: 18 ¹³C NMR of IMP 5

The C- NMR of Teneligliptin (Fig 18) has showed R-CH₃ peak at 12.138ppm, R-CH₂-NH₂ peaks from 30 to 65ppm, Peak at 81ppm is solvent peak, C=C peaks from115 to 140ppm, C in aromatic ring shows peaks from 120 to 150ppm and RNC=O gives peak at 169.

CONCLUSION:

A rapid and precise LC-MS/MS and NMR method was developed for identifying impurities in Teneligliptin Hydrobromide Hydrate. Combination of these methods enabled a complete structural prediction of three major impurities which are present in very low levels before isolation and purification. After isolation these impurities were subjected to NMR and structure was elucidated.

CONFLICT OF INTEREST:

The authors have no conflict of interest.

ACKNOWLEDGEMENT:

We would like to thank the Principal, JSS College of Pharmacy, Mysuru and JSS Academy of Higher Education & Research, Mysuru for providing the facilities in successfully completion of the research work.

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Received on 21.12.2019 Modified on 23.02.2020

Research J. Pharm. and Tech. 2020; 13(8):3569-3576.

DOI: 10.5958/0974-360X.2020.00631.9