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: 8 July 2016
Accepted: 24 November 2016
Published: 2 December 2016
Teneligliptin is a new FDA approved drug for treating Diabetes Mellitus. There are no reported evidences for its degradation products during stability studies 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 × 4.6 mm, 5 μm) 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. Their structural elucidation was depicted.
Degradation was observed in base, peroxide and thermal stressed samples, but not in acid and photolytic stressed samples.
Materials and Methods
HPLC grade acetonitrile (Lichrosolv®, Merck life sciences, Pvt. Ltd, Mumbai, India), HPLC water (Lichrosolv®, Merck life sciences, Pvt. Ltd. Mumbai, India), formic acid, 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 and filtered through 0.45-μm-pore-size membrane filters (Millipore, Mumbai, India). 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® 100-5C18 (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 LCMS studies, UPLC system consisting of gradient mode pump with column Acquity UPLC@ BEH C18 (1.7 μm, 2.1 × 50 mm) detected using photodiode array (PDA) detector range 200–400 nm was used. The mass spectrum with ESI mode of ionization was used for the study (LC/MS/MS (Waters, XEVO-TQD).
Chromatographic separation was achieved on Kromasil® 100-5C18 using a mobile phase consisting 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 range of 200–400 nm. The products were ionized by ESI mode for their mass data.
One thousand microgram per milliliter solution of teneligliptin was prepared by dissolving required amount of 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.
Teneligliptin was subjected to forced degradation by acid hydrolysis using 0.1N 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 0.45-μm membrane before its analysis.
Teneligliptin was subjected to forced degradation by base hydrolysis using 0.1N 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 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 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 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 refluxing apparatus. The stressed sample was diluted with methanol and filtered through 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 100–500 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). Both intraday and interday precision were determined.
The recovery of the method was determined by adding known amount of drug to the standard concentration. The recovery was performed at three levels 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.
Robustness and ruggedness
The robustness of developed method is identified by taking the drug sample and analyzing at lower and upper wavelengths and flow rates than actual by changing the conditions in method and calculated the % RSD.
The ruggedness of the method is identified by changing the analyst and performing the analysis with the developed method and calculated the % RSD.
Method development and optimization of chromatographic conditions
System suitability of teneligliptin
Peak height (mAv)
Retention time (minutes)
Linearity of teneligliptin
Peak height (mAv)
Retention time (minutes)
Residual sum of squares
Precision results of teneligliptin
Inter day (day2)
Accuracy results of teneligliptin
Recovery level (%)
Test concentration (μg/mL)
Standard concentration (μg/mL)
Total concentration (μg/mL)
LOD and LOQ results of teneligliptin
SD of lowest concentration in linearity
Y = 4.105x + 22.81
Robustness and ruggedness data of teneligliptin
Change in λmax (246 ± 2 nm)
Change in flow rate (1.0 ± 0.2 mL/min)
HPLC data of Degradation studies
Stress parameters used
Degraded product peaks
0.1 N HCl
0.1 N NaOH
Percentage degradation data of teneligliptin
Duration of stressed condition (hours)
Chromatographic isolation is acquired using Acquity column UPLC @ BEH C18 1.7 μm, 2.1 × 50 mm. The mobile phase in gradient fashion consisting of A (10% acetonitrile in water with 0.1% formic acid) and B (90% acetonitrile with 0.1% formic acid) was used for the liquid chromatography. The temperature of the system was kept constant at 25 °C. Uniform flow rate of 0.3 mL/min is used. The eluted components were detected using photodiode array at range of 200–400 nm. The products were ionized by electrospray ionization mode for their mass data.
Characterization of degradation products
The molecular ion peak for teneligliptin was observed at 427.22 in ESI mode. In base-stressed sample, the major intense fragments with M+ 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 peroxide-stressed sample, the fragments with M+ of 138.08 and 136.18 were observed at a retention time of 1.666 and 1.467 min, respectively. In thermal-stressed sample, the fragments with M+ of 375.72 at a retention time of 0.455 min and fragments 214.20, 310.31, and 155.69 at a retention time of 1.325 min were observed. There is no degradation peaks for teneligliptin in acid- and UV-stressed conditions. The scan mode is PDA with both positive (E+) and negative (E−) scans at total ion current (TIC) chromatogram. Both the scans were compared with diode array spectra.
Teneligliptin is an antidiabetic drug recently approved by 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 RPHPLC. A new RPHPLC method was developed and validated for teneligliptin as per the ICH guidelines and used as a stability indicating method. The linearity range for teneligliptin drug was found to be 100–500 μg/mL. The intraday and interday precision for the developed method was found to be 0.807 and 0.810 respectively and are within the limits. The LOD and LOQ for teneligliptin were found to be 4.04 and 12.25 μg/mL respectively for the developed method. The robustness and ruggedness were found to be 1.000 ± 0.200 and 0.9 ± 0.04 respectively and are within the limits. Teneligliptin pure drug was used for the study and stressed under acid, base, neutral (hydrogen peroxide), UV photolysis, and thermal conditions. The base-stressed sample has shown an extra peak other than drug peak at retention time of 2.966 with an intensity of 312.12 mAV. The oxidative-stress sample has shown two extra degraded peaks at retention time of 2.519 and 2.979 with an intensity of 846.61 and 87.45 mAV, respectively. The thermal-stressed samples have shown a very small degraded peak with a retention time of 3.033 having intensity of 46.65 mAV. The HPLC analysis of stressed samples have shown that no degradation has occurred under the influence of acid and UV light. But the stressed samples under base, peroxide, and thermal have presence of degraded products, which observed as separate peaks in HPLC other than teneligliptin. From the AUC, it is observed that the highest percentage of degradation was observed in oxidative stress followed by base stress and thermal. Although no prominent peaks were observed in the acid and photolytic stress samples, the percentage degradation was limited to 9.18% and 2.61%, respectively. The obtained degraded samples were further analyzed by UPLCMS/MS, to identify the products formed. It is observed that a change in retention times and mass analysis in LCMS is due to the time lag between the sample elution from LC and undergoing ionization in MS. The major molecular ion fragments formed for all the three stress conditions are different except 310.30 ((4-(4-(1-aminovinyl) piperazin-1yl) 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 of 354.30 (4-(4-(1ethyl-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 peaks 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-aminoN-(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 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 degraded products, which is under process.
The authors are thankful to 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 contributed to the main idea and implementation of the work by selecting the drug, performing the wet lab study, analyzing the degradation products, and interpreting the results from LCMS/MS data. SV helped in analyzing the results of RPHPLC method development and validation. NAN performed the LCMS study of the samples and helped in the UPLC method development. YSS performed the wet lab study of hydrogen peroxide stress and thermal stress work for the drug sample and helped in adjusting the pH of mobile phase during the HPLC study. MRL performed the wet lab study of collecting the dilutions of the sample after stress conditions for the drug sample and 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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