Method development, validation, and stability studies of teneligliptin by RP-HPLC and identification of degradation products by UPLC tandem mass spectroscopy
© The Author(s). 2016
Received: 9 July 2016
Accepted: 27 July 2016
Published: 9 August 2016
Teneligliptin is a new FDA approved drug for treating Diabetes Mellitus. There are no reported evidences for their identified degradation products and their effects on humans.
A simple and new stability indicating RP-HPLC method was developed and validated for identification of Teneligliptin and its degradants on Kromasil 100- 5C18 (250×4.6mm, 5μm) column using pH 6.0 phosphate buffer and acetonitrile (60:40 v/v) as a mobile phase in isocratic mode of elution at a flow rate of 1.0 mL/min. The column effluents were monitored by a variable wavelength UV detector at 246 nm. The method was validated as per ICH guidelines. Forced degradation studies of Teneligliptin were carried out under acidic, basic, neutral (peroxide), photo and thermal conditions for 48 hours at room temperature. The degradation products were identified by HPLC and characterized by UPLC with tandem mass spectroscopy (LC/MS/MS).
UPLC MS/MS data shown major peaks, observed at 375.72, 354.30, 310.30, 214.19, 155.65, 138.08 and 136.18 m/z.
Degradation was observed in base, peroxide and thermal stressed samples, but not in acid and photolytic stressed samples.
Materials and reagents
HPLC grade acetonitrile (Lichrosolv®, Merck Life Science, Pvt. Ltd., Mumbai, India), HPLC water (Lichrosolv®, Merck Life Science, Pvt. Ltd., Mumbai, India), formic acid and potassium dihydrogen o-phosphate (Thermo Fisher Scientific Pvt. Ltd., Mumbai, India), and sodium hydroxide (S D Fine-Chem. Ltd., Mumbai, India) were used for the study. Teneligliptin pure drug and its tablet formulation were obtained from Ajanta Pharma Limited, Mumbai, India.
The HPLC system (Agilent Technologies, Compact LC-G4286A made in Germany) with variable wavelength UV detector was used. Reversed-phase Kromasil® 100-5-C18 (250 × 4.6 mm, 5-μm particle size) column was used for chromatographic separation. The chromatographic and integrated data were recorded using EZChrom Elite Compact Software in computed system (Version 3.30B, Sr. no. 08051601100, Scientific Software, Inc.). For the LC/MS studies, UPLC system consisting of gradient mode pump with column Acquity UPLC@ BEH C18 (1.7 μm, 2.1 × 50 mm) detected using photo diode array (PDA) detector range 200–400 nm was used. The mass spectrum with electrospray ionization (ESI) mode of ionization was used for the study (LC/MS/MS (Waters, XEVO-TQD).
Chromatographic separation was achieved on Kromasil® 100-5-C18 using a mobile phase consisting of a mixture of pH 6.0 phosphate buffer and acetonitrile (60:40 v/v) under isocratic mode of elution. The mobile phase was prepared and filtered through membrane filters (0.45 μm) and sonicated for 30 min prior to use. Separation was performed using 1 mL/min flow rate at room temperature, and the run time was 25 min. The injection volume was 20 μL and the detection wavelength set at 246 nm.
Chromatographic separation was achieved on Acquity UPLC@ BEH C18 1.7 μm, 2.1 × 50 mm using the gradient mobile phase consisting of A (10 % acetonitrile in water with 0.1 % formic acid) and B (90 % acetonitrile with 0.1 % formic acid). A flow rate of 0.3 mL/min is maintained for the study. The eluted components were detected using PDA at a range of 200–400 nm. The products were ionized by ESI mode for their mass data.
1000 μg/mL solution of teneligliptin was prepared by dissolving the required amount of the drug in methanol. The solution was adequately diluted with methanol for accuracy, precision, linearity, limit of detection, and quantification studies.
Stability sample preparation
The collected samples of acid and base hydrolysis were neutralized with sodium hydroxide and hydrochloric acid, respectively. Further dilution was carried out with methanol and the remaining stressed samples also diluted with methanol. All the samples were filtered before analysis.
The teneligliptin was subjected to forced degradation by acid hydrolysis using 0.1 N HCl maintained at 35 °C for 48 h. The sample after the stress was neutralized with sodium hydroxide and diluted with methanol and filtered through a 0.45-μm membrane before its analysis.
The teneligliptin was subjected to forced degradation by base hydrolysis using 0.1 N NaOH maintained at 35 °C for 48 h. The sample after the stress was neutralized with hydrochloric acid and diluted with methanol and filtered through a 0.45-μm membrane before its analysis.
Hydrogen peroxide (neutral) degradation
Forced degradation of teneligliptin was studied under the influence of (3 %) hydrogen peroxide maintained at 35 °C for 48 h. The stressed sample was diluted with methanol and filtered through a 0.45-μm membrane before its analysis.
The influence of UV light on the stability of teneligliptin was studied by exposing the sample in UV light at 365 nm for 48 h. The stressed sample was diluted with methanol and filtered through a 0.45-μm membrane before its analysis.
The effect of increased temperature on teneligliptin was studied by heating the sample at 69 °C for 48 h in a refluxing apparatus. The stressed sample was diluted with methanol and filtered through a 0.45-μm membrane before its analysis.
The system suitability was determined by six injections of teneligliptin (300 μg/mL). The developed method was found to be suitable for use as the tailing factor and peak resolution for teneligliptin were within the limits.
The linearity of teneligliptin was studied from the standard concentrations ranging from 100 to 500 μg/mL. The calibration curve of peak intensity versus concentration was plotted, and correlation coefficient and regression line equation were determined.
The precision of the method was determined by six (n = 6) injections of teneligliptin (300 μg/mL), and the % RSD of peak areas were calculated. The obtained RSD was within the range (≤2).
The recovery of the method was determined by adding a known amount of the drug to the standard concentration. The recovery was performed at three levels of 80, 100, and 120 % of teneligliptin standard concentration. The three samples were prepared for each recovery level, and % recoveries were calculated.
Limits of detection (LOD) and limit of quantification (LOQ)
The LOD and LOQ are the lowest level and lowest concentration of the analyte, respectively, in a sample that would yield signal-to-noise ratios of 3.3 for LOD and 10 for LOQ. These are determined from the standard deviation of the peak response and the slope of the calibration curve.
Method development and optimization of chromatographic conditions
System suitability of teneligliptin
Peak height (mAv)
Retention time (min)
Linearity of teneligliptin
Peak height (mAv)
Retention time (min)
Precision results of teneligliptin
Accuracy results of teneligliptin
Recovery level (%)
LOD and LOQ results of teneligliptin
SD of the lowest concentration in linearity
Y = 4.105x + 22.81
HPLC data of degradation studies
Stress parameters used
Degraded product peaks
0.1 N HCl
0.1 N NaOH
0.3 % H2O2
Characterization of the degradation products
The molecular ion peak for teneligliptin was observed at 427.22 in ESI mode. In the base-stressed sample, the fragments of 354.30 at a retention time of 1.195 min, 310.30 and 214.19 at a retention time of 1.345 min, and 178.73 and 155.65 at a retention time of 1.205 min were observed. In the peroxide-stressed sample, the fragments of 138.08 and 136.18 were observed at a retention time of 1.666 and 1.467 min, respectively. In the thermally stressed sample, the fragments of 375.72 at a retention time of 0.455 min and 214.20, 310.31, and 155.69 at a retention time of 1.325 min were observed. There are no degradation peaks for teneligliptin in acid- and UV-stressed conditions.
Teneligliptin is an antidiabetic drug recently approved by the FDA. There are no reports available for the stability of the drug and their possible degraded products till date. In the present research work, we aimed to perform stability studies on teneligliptin and develop and validate a method for its estimation and identification by RP-HPLC. A new RP-HPLC method was developed and validated for teneligliptin as per the ICH guidelines and used as a stability-indicating method. The teneligliptin pure drug was used for the study and stressed under acid, base, neutral (hydrogen peroxide), UV photolysis, and thermal conditions. The HPLC analysis of the stressed samples has shown that no degradation occurred under the influence of acid and UV light. But the stressed samples under base, peroxide, and thermal have presence of the degraded products, which was observed as separate peaks in HPLC other than the teneligliptin. The obtained degraded samples were further analyzed by UPLC/MS/MS, to identify the products formed. The major molecular ion fragments formed for all the three stress conditions are different except 310.30 ((4-(4-(1-aminovinyl)piperazin-1-yl)pyrrolidin-2-yl)(thiazolidin-3-yl)methanone), 214.19 (N,N-diethyl-1-phenyl-1H-pyrazol-5-amine), and 155.65 (1-(pyrrolidin-3-yl)piperazine) were observed in both base and thermal stress conditions. A characteristic 354.30 (4-(4-(1-ethyl-3-methyl-1H-pyrazol-5-yl)piperazin-1-yl)-N-(mercaptomethyl)-N-methylpyrrolidine-2-carboxamide) and 375.72 ((4-(4-(3-methyl-1-vinyl-1H-pyrazol-5-yl)piperazin-1-yl)pyrrolidin-2-yl)(thiazolidin-3-yl)methanone) molecular ion peak was observed in base condition and thermal condition, respectively. The products formed with photolytic stress were completely different with molecular ions at 138.08 (N,N-diethyl-1H-pyrazol-5-amine) and 136.18 (2-amino-N-(mercaptomethyl)-N-methylacetamide), which are not observed in other stress conditions. From the data, it is observed that comparatively less degradation occurred for photolysis stress than for base and thermal stress. The fragmentation pattern shows that the degraded products are similar for the base and thermal stress samples. Further study is required for determining the degraded products’ toxicity by quantifying the samples.
The present study helps in identifying the degraded products of teneligliptin in bulk and formulations, during their storage and transport conditions. This research work is the first to report its stability studies with degraded product identification, which is helpful for determining the toxicity of the degraded products and also to caution the storage conditions. The products formed could also be the starting materials during its synthesis, which has to be studied. Further study is required for establishing the toxicity profile of the degraded products, which is under process.
The authors are thankful to the Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Guntur, and CSIR-Indian Institute Integrative Medicine, Mumbai, for providing the facilities and timely support for carrying out this research work.
TNVGK conceived the main idea and implementation of the work by selecting the drug and performed the wet lab study. TNVGK also analyzed the degradation products and interpreted the results from the LC/MS/MS data. SV helped in analyzing the results of the RP-HPLC method development and validation. NAN performed the LC/MS study of the samples and helped in the UPLC method development. YSS performed the wet lab study of the hydrogen peroxide stress and thermal stress work for the drug sample. YSS also helped in adjusting the pH of the mobile phase during the HPLC study. MRL performed the wet lab study of collecting and dilutions of the sample after stress conditions for the drug sample. MRL also helped in the mobile phase preparation during the HPLC study. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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