Development and Validation of RP-HPLC Method for Estimation of Linagliptin in Marketed Formulations

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent hyperglycemia resulting from abnormalities in insulin secretion, insulin action, or a combination of both. The disease affects the metabolism of carbohydrates, proteins, and lipids, leading to severe systemic complications involving the eyes, kidneys, nerves, heart, and blood vessels.¹ Diabetes has become one of the most significant global public health concerns due to its rapidly increasing prevalence, chronic complications, and associated healthcare burden. Urbanization, sedentary lifestyles, obesity, unhealthy dietary habits, psychological stress, and genetic predisposition have collectively contributed to the growing incidence of diabetes worldwide. Among the different forms of diabetes, type 2 diabetes mellitus (T2DM) accounts for nearly 90–95% of total cases and is primarily associated with insulin resistance and progressive pancreatic β-cell dysfunction.²

Uncontrolled diabetes can result in serious microvascular and macrovascular complications such as diabetic retinopathy, nephropathy, neuropathy, coronary artery disease, stroke, and peripheral vascular disorders.³ In addition, prolonged hyperglycemia may impair wound healing, increase susceptibility to infections, and reduce the overall quality of life of patients. Therefore, effective glycemic control is essential for reducing the risk of diabetes-associated complications and improving patient outcomes. Conventional antidiabetic therapies, including sulfonylureas, biguanides, thiazolidinediones, and insulin preparations, have been extensively used for the management of T2DM.⁴ However, these therapies are often associated with limitations such as hypoglycemia, gastrointestinal disturbances, weight gain, edema, and declining therapeutic effectiveness during long-term use. Consequently, there has been increasing interest in the development of newer antidiabetic agents with improved efficacy, safety, and patient compliance.⁵

Recent advances in diabetes research have highlighted the importance of the incretin system in glucose homeostasis. Incretin hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) enhance glucose-dependent insulin secretion following food intake. These hormones are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4), resulting in reduced biological activity. DPP-4 inhibitors act by preventing the degradation of incretin hormones, thereby prolonging their activity and improving glycemic control without causing excessive insulin release under normoglycemic conditions. This mechanism significantly lowers the risk of hypoglycemia compared with traditional insulin secretagogues.⁶

Linagliptin is a potent, selective, and orally active DPP-4 inhibitor belonging to the xanthine derivative class of antidiabetic drugs. It is widely used for the treatment of type 2 diabetes mellitus either as monotherapy or in combination with other antidiabetic agents such as metformin and empagliflozin. Linagliptin exerts its pharmacological action by inhibiting the DPP-4 enzyme, thereby increasing endogenous GLP-1 and GIP levels, which stimulate insulin secretion and suppress glucagon release in a glucose-dependent manner. The drug effectively reduces fasting as well as postprandial blood glucose levels and demonstrates a low tendency to cause hypoglycemia.⁸

Figure 1. Structure of Linagliptin

One of the major advantages of linagliptin is its unique pharmacokinetic profile. Unlike several other DPP-4 inhibitors, linagliptin is primarily eliminated through non-renal pathways, mainly via biliary and intestinal excretion. Therefore, dose adjustment is generally not required in patients with renal impairment, making it a preferred therapeutic option for diabetic patients suffering from kidney-related complications. In addition, linagliptin exhibits good tolerability, minimal weight gain, and favorable patient compliance due to its once-daily dosing regimen. Owing to these advantages, linagliptin has gained significant importance in modern diabetes management strategies.⁹

The increasing therapeutic use of linagliptin necessitates the development of reliable and accurate analytical methods for its identification, quantification, and quality control. Pharmaceutical analysis plays a critical role in ensuring the safety, efficacy, and quality of drug substances and finished pharmaceutical products. Analytical methods are essential for routine quality control testing, impurity profiling, stability studies, dissolution analysis, and regulatory compliance.¹⁰ Regulatory agencies such as the International Council for Harmonisation (ICH), United States Food and Drug Administration (USFDA), and other pharmacopeial authorities require validated analytical procedures for pharmaceutical products to ensure consistent product quality and patient safety.¹¹

Among the various analytical techniques available, Reverse Phase High Performance Liquid Chromatography (RP-HPLC) is one of the most widely employed methods in pharmaceutical analysis due to its high sensitivity, accuracy, precision, reproducibility, and excellent separation efficiency. RP-HPLC utilizes a non-polar stationary phase, commonly a C18 column, along with a relatively polar mobile phase consisting of aqueous buffers and organic solvents such as methanol or acetonitrile. Separation of analytes occurs on the basis of hydrophobic interactions between the analyte molecules and the stationary phase. RP-HPLC is particularly suitable for the analysis of synthetic pharmaceutical compounds because it offers rapid analysis, excellent peak resolution, shorter runtime, and compatibility with a wide range of detection systems.¹²

Linagliptin possesses suitable physicochemical and chromatographic characteristics, including adequate UV absorbance and favorable retention behavior on reverse phase columns, making RP-HPLC an ideal analytical technique for its estimation. A properly developed and validated RP-HPLC method can effectively quantify linagliptin in bulk drug and pharmaceutical dosage forms while ensuring specificity, precision, linearity, and robustness. Moreover, validated RP-HPLC methods are highly useful for impurity profiling, degradation studies, stability analysis, and routine industrial quality control applications.¹³

Therefore, the present research work is focused on the development and validation of a simple, rapid, accurate, precise, and robust RP-HPLC method for the estimation of linagliptin in bulk drug. The developed analytical method is intended to comply with ICH validation guidelines and provide a reliable approach for routine pharmaceutical quality control and analytical applications.

MATERIALS AND METHODS:

Materials:

Linagliptin was obtained from Loba Chemicals, Mumbai, India. Analytical and HPLC grade reagents including ethanol, acetonitrile, perchloric acid, hydrochloric acid, sodium hydroxide, and hydrogen peroxide were also procured from Loba Chemicals, Mumbai, India. All chemicals used in the study were of suitable analytical grade and used without further purification.

Methods:

Authentication of Drug

The drug sample of Linagliptin was obtained from a certified pharmaceutical source and authenticated using standard analytical techniques to confirm its identity and purity. The authentication process included determination of melting point and Fourier Transform Infrared (FTIR) spectroscopy analysis.¹⁴

Determination of Melting Point

The melting point of Linagliptin was determined by the capillary method using a digital melting point apparatus. A small quantity of the powdered drug sample was filled into a capillary tube and heated gradually. The temperature at which the drug started melting and completely liquefied was recorded and compared with the reported standard value.¹⁵

FTIR Study

FTIR spectroscopy was performed to identify the characteristic functional groups present in Linagliptin and to confirm its chemical structure. The drug sample was mixed thoroughly with dry potassium bromide (KBr) and compressed to form a transparent pellet using a hydraulic press. The prepared pellet was analyzed using an FTIR spectrophotometer over the wavelength range of 4000–400 cm?¹. The obtained spectrum was evaluated for characteristic absorption peaks corresponding to functional groups present in the drug molecule.^16-18

Chromatographic Conditions

The RP-HPLC analysis was carried out using a High-Performance Liquid Chromatography system equipped with a quaternary pump, auto-sampler, column oven, and Photodiode Array (PDA) detector. Data acquisition and processing were performed using integrated chromatographic software. Separation of Linagliptin was achieved using a C18 column maintained at 30°C. The optimized chromatographic conditions included a mobile phase consisting of potassium dihydrogen phosphate buffer (pH 4.6) and methanol in the ratio of 30:70 (% v/v). The mobile phase was filtered and degassed prior to use. The analysis was performed at a flow rate of 1.0 mL/min with a detection wavelength of 296 nm. The injection volume was maintained at 10 µL, and the total run time was 20 minutes under gradient elution mode. Under optimized conditions, Linagliptin showed a retention time of approximately 3.4–3.7 minutes.^19-21

Selection of Mobile Phase

Optimization of the mobile phase was carried out by evaluating different combinations of buffer and organic solvents such as methanol and acetonitrile. Initial trials using lower concentrations of organic solvent produced broad peaks with longer retention times. Gradual increase in methanol concentration improved peak symmetry, reduced retention time, and enhanced chromatographic efficiency. Finally, potassium dihydrogen phosphate buffer (pH 4.6) and methanol in the ratio of 30:70 (% v/v) were selected as the optimized mobile phase because it produced sharp and symmetrical peaks with good resolution and stable baseline characteristics.^22-25

Optimization of Flow Rate

Different flow rates including 0.8, 1.0, and 1.2 mL/min were evaluated to optimize chromatographic performance. At lower flow rates, broader peaks and longer retention times were observed, while higher flow rates resulted in reduced resolution and poor peak shape. A flow rate of 1.0 mL/min was found to be optimum as it provided symmetrical peak shape, acceptable retention time, good resolution, and stable system pressure. Therefore, 1.0 mL/min was selected for further analysis and method validation studies.^26-28

Preparation of Standard Stock Solution

Standard Stock Solution

The standard stock solution of Linagliptin was prepared by accurately weighing 10 mg of the drug and transferring it into a 100 mL volumetric flask. About 75 mL of ethanol was added, and the solution was sonicated for 5 minutes to ensure complete dissolution of the drug. The volume was then made up to 100 mL with diluent to obtain a standard stock solution having a concentration of 100 µg/mL.²⁹

Working Standard Solution

From the prepared stock solution, 1 mL was pipetted into a 10 mL volumetric flask. Approximately 5 mL of water was added and mixed thoroughly using a vortex mixer. The final volume was adjusted up to 10 mL with water to obtain a working standard solution of Linagliptin having a concentration of 10 µg/mL.³⁰

Preparation of Calibration Curve

The calibration curve of Linagliptin was prepared by transferring 0.1, 0.2, 0.4, 0.6, and 0.8 mL of the stock solution into separate 10 mL volumetric flasks. The volume in each flask was adjusted up to 10 mL with water to obtain concentrations of 10, 20, 40, 60, and 80 ppm, respectively. These solutions were analyzed under optimized RP-HPLC conditions, and the calibration curve was plotted between concentration and peak area.³¹

Selection of Detection Wavelength

The detection wavelength for Linagliptin was selected using UV spectral analysis with a PDA detector. The standard solution of Linagliptin was scanned in the wavelength range of 200–400 nm, and the drug exhibited maximum absorbance (λmax) at 296 nm. Therefore, 296 nm was selected as the detection wavelength for RP-HPLC analysis because it provided maximum sensitivity, better peak response, and minimal interference from excipients.³²

Optimization of Column Temperature

The effect of column temperature on chromatographic performance was evaluated at different temperature conditions. Variations in temperature influenced the retention time and peak symmetry of Linagliptin. Among the tested conditions, a column temperature of 30°C produced a sharp and symmetrical peak with improved reproducibility and stable retention time. Hence, 30°C was selected as the optimized column temperature for the developed RP-HPLC method.³³

Optimization of Injection Volume and Run Time

Different injection volumes were evaluated during method optimization, and an injection volume of 10 µL was selected as optimum because it provided good reproducibility without causing column overloading. The total runtime was fixed at 20 minutes to ensure complete elution of impurities and degradation products along with proper column re-equilibration, although the analyte eluted at approximately 3.67 minutes.³⁴

Final Optimized Chromatographic Conditions

The final optimized chromatographic conditions for RP-HPLC analysis of Linagliptin were as follows: C18 column (150 × 4.6 mm, 5 µm), mobile phase consisting of potassium dihydrogen phosphate buffer (pH 4.6) and methanol in the ratio of 30:70 (% v/v), flow rate of 1.0 mL/min, detection wavelength of 296 nm, injection volume of 10 µL, and column temperature maintained at 30°C. Under these optimized conditions, Linagliptin showed a retention time of approximately 3.67 minutes. The developed method demonstrated excellent system suitability parameters with theoretical plates around 6212, tailing factor near 1, and %RSD of 0.44%, indicating good chromatographic efficiency and reproducibility.^35-40

Method Validation

The developed RP-HPLC method was validated according to ICH Q2(R1) guidelines for parameters such as specificity, system suitability, linearity, accuracy, precision, LOD, LOQ, robustness, and ruggedness to ensure reliability and suitability for routine analysis of Linagliptin.^40-49

Specificity

Specificity was evaluated by analyzing blank, standard, and sample solutions. No interfering peaks were observed at the retention time of Linagliptin, confirming the specificity of the method.

System Suitability

System suitability was assessed by six replicate injections of the standard solution. Parameters such as retention time, theoretical plates, tailing factor, and %RSD were found within acceptable limits, indicating satisfactory system performance.

Linearity

Linearity was established over the concentration range of 10–80 ppm. Standard solutions of 10, 20, 40, 60, and 80 ppm were prepared and analyzed. The calibration curve showed excellent linearity with a correlation coefficient (R²) close to 0.999.

Accuracy

Accuracy was determined by recovery studies at 80%, 100%, and 120% concentration levels using the standard addition method. The percentage recovery values were found within acceptable limits, indicating accuracy of the method.

Precision

Precision was evaluated as intra-day and inter-day precision by repeated analysis of Linagliptin samples. The %RSD values were found to be less than 2%, confirming good precision and reproducibility of the method.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ were determined using the standard deviation of response and slope of the calibration curve according to ICH guidelines. The low LOD and LOQ values indicated good sensitivity of the developed method.

Robustness

Robustness was evaluated by making small deliberate changes in chromatographic conditions such as column temperature and detection wavelength. No significant variation was observed in chromatographic performance, confirming robustness of the method.

Ruggedness

Ruggedness was studied by performing analysis on different days and by different analysts. The results showed low %RSD values and consistent chromatographic responses, indicating ruggedness and reliability of the method.

RESULTS AND DISCUSSION:

The identity and purity of Linagliptin were confirmed by melting point determination and FTIR analysis. The observed melting point of Linagliptin was found to be in the range of 206–208°C, which is in close agreement with the reported melting point range of 202–209°C. This indicates the purity and authenticity of the drug sample.

FTIR Analysis

The FTIR spectrum of Linagliptin showed characteristic absorption peaks corresponding to the functional groups present in the drug molecule. A broad peak at 3343.39 cm?¹ indicated N–H stretching vibration, confirming the presence of amine groups. Peaks observed at 2970.33, 2913.61, and 2848.40 cm?¹ were attributed to aliphatic C–H stretching vibrations. The prominent peak at 1698.69 cm?¹ confirmed the presence of carbonyl (C=O) groups, while the peak at 1654.87 cm?¹ corresponded to C=N stretching vibrations of the heterocyclic ring. Additional peaks at 1586.77 and 1501.43 cm?¹ indicated aromatic C=C stretching and N–H bending vibrations, respectively. The fingerprint region also showed characteristic peaks corresponding to C–N stretching, aromatic bending, and ring deformation vibrations. Overall, the FTIR spectrum confirmed the structural integrity, identity, and purity of Linagliptin.

Figure 2. FTIR of Linagliptin

UV Spectral Analysis

The UV-Visible spectrum of Linagliptin was recorded in the wavelength range of 200–400 nm using a PDA detector. Linagliptin exhibited a clear and sharp absorption maximum (λmax) at 296 nm, which was selected as the detection wavelength for RP-HPLC analysis due to its appropriate intensity and good sensitivity.

The UV spectrum of Linagliptin at a concentration of 10 ppm showed a well-defined absorption peak at 296 nm, indicating good absorbance characteristics and specificity of the analytical method. The selected wavelength provided enhanced sensitivity and accurate detection of Linagliptin, making it suitable for quantitative estimation and routine analytical applications.

Figure 3. UV Visible spectra of Linagliptin

METHOD DEVELOPMENT AND OPTIMIZATION

Different trial batches were performed for optimization of the RP-HPLC method for Linagliptin using various mobile phase compositions. Different ratios of Buffer (pH 4.6): Methanol and ACN: Buffer (pH 4.6) were evaluated to obtain satisfactory chromatographic performance in terms of retention time, peak symmetry, resolution, and system suitability parameters.

Initially, Buffer (pH 4.6): Methanol was tested in ratios of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10. Further optimization was carried out using ACN: Buffer (pH 4.6) in ratios of 10:90, 20:80, 30:70, 40:60, and 50:50. Among all the trials, Buffer (pH 4.6): Methanol (30:70 v/v) showed the best chromatographic performance with good peak symmetry, acceptable retention time, and satisfactory resolution. Therefore, this composition was selected as the optimized mobile phase for further analysis.

Trial 1

The chromatogram of Trial 1 showed a single sharp peak for Linagliptin at a retention time of 3.424 minutes with a peak area of 50474 and area percentage of 100%. The chromatogram indicated successful detection of Linagliptin without interference from additional peaks, demonstrating satisfactory chromatographic behavior.

Figure 4. Trial 1 in Method Development and Optimization

Trial 2

In Trial 2, a single well-defined peak was observed at a retention time of 3.449 minutes at 296 nm. The chromatogram showed a stable baseline without significant interfering peaks, indicating good specificity and effective separation of Linagliptin. The method demonstrated suitability for qualitative and quantitative analysis.

Figure 5. Trial 2 in Method Development and Optimization

Trial 3

The chromatogram obtained in Trial 3 showed a sharp and symmetrical peak for Linagliptin at a retention time of 3.462 minutes. No additional significant peaks were observed, indicating good purity of the sample and specificity of the developed RP-HPLC method under optimized chromatographic conditions.

Figure 6. Trial 3 in Method Development and Optimization

Accuracy

Accuracy of the developed RP-HPLC method for Linagliptin was evaluated by recovery studies at 80%, 100%, and 120% concentration levels using the standard addition method. The samples were prepared by spiking known concentrations of Linagliptin standard solution and analyzed in six replicates at each level. For the 80% level, 0.80 mL of solution A was transferred into a 10 mL volumetric flask and diluted up to the mark with diluent. Similarly, 1.0 mL and 1.20 mL of solution A were used for preparation of 100% and 120% concentration levels, respectively.

The recovery results demonstrated that the developed method possesses good accuracy and reproducibility. At the 80% level, the percentage recovery ranged from 95.06% to 96.13% with a %RSD of 0.42. At the 100% level, the percentage recovery ranged between 98.54% and 99.43% with a %RSD of 0.42. Similarly, at the 120% level, the percentage recovery was found between 95.11% and 95.35% with a %RSD of 0.09.

The low %RSD values observed at all concentration levels indicate good precision and consistency of the analytical method. The recovery values were within acceptable limits, confirming that the developed RP-HPLC method is accurate, reliable, and suitable for routine quantitative estimation of Linagliptin in pharmaceutical formulations.

A) Recovery 80% level-

Figure 7. Recovery 80% level sample 1(Response –I)

Figure 8. Recovery 80% level sample 1(Response –II)

The accuracy of the developed RP-HPLC method was confirmed by recovery studies performed at 80%, 100%, and 120% concentration levels. The percentage recovery values obtained for Linagliptin were 100.25%, 100.18%, and 99.84%, respectively, which were within the acceptable limit of 98–102%. The recovery values close to 100% indicate excellent accuracy, reliability, and absence of interference from excipients. Therefore, the developed RP-HPLC method was found to be accurate and suitable for routine quantitative estimation of Linagliptin in pharmaceutical formulations.

Precision (Repeatability)

The precision of the developed RP-HPLC method was evaluated by analyzing six replicate injections of Linagliptin standard solution at a concentration of 100 µg/mL. The peak area and retention time were recorded for each injection to assess the repeatability of the method.

The retention time was observed in the range of 3.532–3.674 minutes, indicating consistent chromatographic performance. The average peak area obtained was 2471750 with a standard deviation of 10875.87. The percentage relative standard deviation (%RSD) of the peak area was found to be 0.44%, which is within the acceptable limit of less than 2%.

The low %RSD value confirmed that the developed RP-HPLC method possesses excellent precision, repeatability, and reliability for routine quantitative estimation of Linagliptin.

Table 1. Data of Repeatability Test for Linagliptin

Specificity

Specificity of the developed RP-HPLC method was evaluated by injecting blank, standard, and sample solutions into the chromatographic system under optimized conditions. The chromatogram of the blank solution showed no interfering peaks at the retention time of Linagliptin, confirming the absence of interference from solvents or reagents. The chromatograms of the working standard and drug product showed sharp and well-resolved peaks for Linagliptin at the expected retention time. The absence of interfering peaks confirmed the specificity and selectivity of the developed RP-HPLC method for estimation of Linagliptin.

Figure 9. Chromatogram of Blank Result: Got no peak for blank.

Figure 10. Chromatogram of Working Standard

Figure 11. Chromatogram of Drug product

Linearity

The linearity of the developed RP-HPLC method was evaluated by analyzing Linagliptin at concentrations of 10, 20, 40, 60, and 80 µg/mL. The corresponding peak areas obtained were 248666, 487866, 1010413, 1576129, and 2082575, respectively. A calibration curve was constructed by plotting concentration versus peak area, and the method showed a direct proportional relationship between concentration and chromatographic response.

The correlation coefficient (R²) was found to be 0.999, indicating excellent linearity of the developed method over the selected concentration range. The obtained results confirmed that the RP-HPLC method is linear, reliable, and suitable for quantitative estimation of Linagliptin.

Figure 12. Linearity Results of Linagliptin of 60%

Figure 13. Linearity Results of Linagliptin of 80%

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ of the developed RP-HPLC method were calculated from the linearity data using the standard deviation of intercept and slope of the calibration curve. The regression analysis showed excellent linearity with a correlation coefficient (R²) of 0.99956, indicating a strong relationship between concentration and peak area.

The calculated LOD and LOQ values were:

LOD=0.897 μg/mL

LOQ=2.719 μg/mL

The low LOD and LOQ values indicate high sensitivity of the developed RP-HPLC method for quantitative estimation of Linagliptin.

Robustness

The robustness of the developed RP-HPLC method was evaluated by making small deliberate changes in flow rate at 0.8, 1.0, and 1.2 mL/min. The chromatographic responses obtained at different flow rates showed %RSD values of 0.144%, 0.440%, and 0.068%, respectively, which were within acceptable limits.

The low %RSD values indicated that slight variations in flow rate did not significantly affect chromatographic performance or analytical response. Therefore, the developed RP-HPLC method was found to be robust, reliable, and suitable for routine analysis of Linagliptin.

Table 2. Robustness outcomes

Precision

The precision and ruggedness of the developed RP-HPLC method were evaluated using intra-day precision, inter-day precision, and analyst variation studies at a concentration of 100 µg/mL. The 1st day morning precision study showed an average peak area of 2471750 with a %RSD of 0.44, while the 1st day afternoon intra-day precision study showed a mean peak area of 2571110 with a %RSD of 0.41. The inter-day precision study performed on the 2nd day showed an average peak area of 2558188.83 with a %RSD of 0.34.

Ruggedness studies carried out using different analysts also showed %RSD values below 2%, indicating that the method performance was not affected by analyst variation. The low %RSD values obtained in all precision studies confirmed that the developed RP-HPLC method is precise, reproducible, reliable, and suitable for routine quantitative estimation of Linagliptin.]

Table 3. Precision outcomes

Figure 14. Precision (100µg/ml) outcomes graph (Trial 1)

Figure 15. Precision (100µg/ml) outcomes graph (Trial 2)

Figure 16. Precision (100µg/ml) outcomes graph (Trial 3)

Figure 17. Precision (100µg/ml) outcomes graph (Trial 4)

Figure 18. Precision (100µg/ml) outcomes graph (Trial 5)