DEVELOPMENT AND VALIDATION OF RAPID STABILITY-INDICATING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE DETERMINATION OF LINAGLIPTIN AND EMPAGLIFLOZIN IN PURE AND DOSAGE FORMS

Objective: A new, simple, rapid, sensitive, and accurate stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated for the quantitative determination of linagliptin (LNG) and empagliflozin (EMP) in pure and tablet dosage forms. Methods: An isocratic HPLC method, using a C 18 reversed-phase column (150 mm×4.6 mm i.d., particle size 5 μm) with an isocratic binary mobile phase consisting of phosphate buffer and acetonitrile (65:35, v/v), was investigated to separate the drug from its stress degradation products. The flow rate was 1.0 mL/min at ambient temperature and photodiode array detector is used at 226 nm for detection. The developed method was validated for system suitability, linearity, accuracy, precision, limits of detection and quantitation, specificity, stability, and robustness. Results: The retention time of LNG and EMP was found to be 3.276±0.002 and 6.966±0.0006 min, respectively. The calibration curve was found to be linear with the equation y=158926.39X+11.139, with a correlation coefficient of R 2 =0.9991 for LNG and y=22688.45X+4.259, with a correlation coefficient of R 2 =0.9994 for EMP over a concentration range of 2.5–7.5 µg/mL and 5.0–15 µg/mL for LNG and EMP, respectively. The limits of detection were 0.29 and 0.48 µg/mL for LNG and EMP, respectively, and the limits of quantification were 0.89 and 1.5 µg/mL for LNG and EMP, respectively. The recovery values of this method are 101.11% and 101.48% for LNG and EMP, respectively, and the reproducibility is within 0.070 and 0.277 for LNG and EMP, respectively. Conclusion: The proposed method is a rapid stability-indicating HPLC method that can be applied for the determination of LNG and EMP in pure and tablet dosage forms. ABSTRACT Linagliptin (LNG) is a more potent dipeptidyl peptidase (DPP)-4 inhibitor than other drugs that belong to the same class for the treatment of Type II diabetes. LNG is a competitive and reversible DPP-4 enzyme inhibitor that slows the breakdown of insulinotropic hormone glucagon-like peptide-1 for better glycemic control in diabetes patients. Empagliflozin (EMP) is a sodium glucose cotransporter-2 inhibitor indicated as an adjunct to diet and exercise to improve glycemic control in adult patients with Type 2 diabetes. The combination of LNG and EMPis served as an adjuvant to diet and exercise to improve glycemia control in adults with Type-2 diabetes who know to have the cardiovascular disease [1-3].LNGchemically,8-[(3R)-3-aminopiperidin-yl]-7-(but-2yn-1-yl)-3-methyl-1-[(4-methyl

The aim of the present work is to develop and validate simple, fast, and reliable stability-indicating reverse-phase HPLC method with ultraviolet (UV) detection for the simultaneous determination of LNG and EMP in pure and pharmaceutical dosage forms. The proposed method can overcome the problems in all previously reported HPLC methods such as long time of analysis and expensive detectors, as shown in Table 1.

Instrumentation
HPLC apparatus (Agilent 1200, Agilent, USA) equipped with UV detector DAD system. The pH measurements were made on a Hanna pH meter equipped with a combined glass-calomel electrode (Portugal) (HI: 9321).

Chemicals and reagents
Analytical HPLC grade solvents were used in all experiments, including acetonitrile and methanol (LAB-SCAN, Analytical Sciences, Gliwice, UL, Sowinskiego, Poland). Potassium dihydrogen orthophosphate, sodium hydroxide (NaOH), hydrochloric acid (HCl), and hydrogen peroxide (H 2  ABSTRACT Linagliptin (LNG) is a more potent dipeptidyl peptidase (DPP)-4 inhibitor than other drugs that belong to the same class for the treatment of Type II diabetes. LNG is a competitive and reversible DPP-4 enzyme inhibitor that slows the breakdown of insulinotropic hormone glucagon-like peptide-1 for better glycemic control in diabetes patients. Empagliflozin (EMP) is a sodium glucose cotransporter-2 inhibitor indicated as an adjunct to diet and exercise to improve glycemic control in adult patients with Type 2 diabetes. The combination of LNG and EMPis served as an adjuvant to diet and exercise to improve glycemia control in adults with Type-2 diabetes who know to have the cardiovascular disease [1][2][3].LNGchemically

Sheikh et al.
5.0 mg LNG and 10 mg EMP per tablet) and Empacoza plus tablets (Zeta Pharma, Egypt, 5.0 mg LNG and 10 mg EMP per tablet).

Chromatographic conditions
The chromatographic separation was performed using ODS-3 InertsilC18 (150 mm×4.6 mm), 5.0 µm particle size column; the column temperature was maintained at 25±2°C. The autosampler utilized methanol as a rinse solution, the total run time was 6.0 min. The elution quaternary pump ran an isocratic flow using mobile phase consisting of a mixture of phosphate buffer and acetonitrile (65:35, v/v) at a flow rate of 1.0 mL/min. The eluate was monitored at 226 nm using UV diode array detector. The retention time of the drug was found to be LNG and EMP which were found to be3.276±0.002 and 6.966±0.0006 min, respectively. The injection volume was 20 µL. Mobile phase was used as diluent during the standard and test samples preparation.

Preparation of standard solutions
A stock solution of LNG or EMP (10 µg/mL) was prepared by dissolving 10 mg of LNG and EMP in mobile phase in 100 mL volumetric flask, then shaken and sonicated for 10 min till completely dissolved and then complete the volume to 100 mL with mobile phase. The working standard solutions were prepared by diluting aliquots of stock solution with mobile phase to obtain final concentrations ranging from 2.5-7.5 µg/mL to 5.0-15 µg/mL for LNG and EMP, respectively. Working solutions of the drugs were stable for 1 week.

Construction of calibration curves
Aliquots of standard solution, ranging from 2.5-7.5 µg/mL to 5.0-15 µg/mL for LNG and EMP, respectively, were prepared in a series of 10 mL volumetric flasks, 20 µL were injected into the instrument. Detection was performed at the wavelength of 226 nm. The calibration graph was constructed by plotting the peak areas obtained at the wavelength of 226 nm versus the corresponding injected concentrations.

Assay for tablets dosage forms
Twenty tablets of Glyxambi and Empacoza plus were weighed, finely powdered, and an accurately weighed amount of the powdered tablets equivalent to 5.0 mg LNG and 10 mg EMP which were transferred to 100 mL measuring flask and dissolved in 50 mL of mobile phase, sonicated for 10 min, and the solution was filtered through a 0.45 µm membrane filter and then the final solution was completed to volume with mobile phase. The proposed procedure was then completed as mentioned above.

Method optimization
The conditions affecting the chromatographic performance of LNG and EMP were carefully studied to recognize the most suitable chromatographic system. Hence, the optimum chromatographic performances were achieved when using isocratic mobile phase composed of phosphate buffer and acetonitrile (65:35,v/v) with a flow rate of 1.0 mL/min, injection volume 20 μL, column temperature 25°C, and detection wavelength 226 nm. The results of three runs indicate high system suitability ( Table 2). The retention time (t R ) values LNG and EMP were found to be 3.276±0.002 and 6.966±0.0006 min, respectively (Fig. 2).

Method validation
The developed method was validated for system suitability, linearity, sensitivity, precision, accuracy, robustness selectivity, and

Sheikh et al.
specificity and is applied for forced degradation studies as per the ICH guidelines [26].

Linearity
The linearity of LNG and EMP was established by eight-point calibration curve, concentration ranging from 2.5 to 7.5 µg/mL and 5.0 to 15 µg/mL for LNG and EMP, respectively. The graph of the peak area against concentration proved linear graph with regression equations; y=158,926.39x+11.139 and y=22,688.45x+4.259, with a correlation coefficients (R 2 =0.9991 and 0.9994) for LNG and EMP, respectively.

Sensitivity
The limit of detection is defined as the injected quantity giving S=N of 3 (in terms of peak area) and were found to be 0.29 and 0.48 µg/mL for LNG and EMP, respectively. The limit of quantification is defined as the injected quantity giving S=N of 10 (in terms of peak area) and was found to be 0.89 and 1.50 µg/mL for LNG and EMP, respectively (Table 2).

Precision
The intraday repeatability (precision) of the developed method was assessed by analyzing six replicate injections of the standard solution at three different concentrations on the same day. The same was done for interday precision test except that the injection of the samples was every day for 5 days. The precision of the method was determined by calculating relative standard deviation (RSD %). The results in Table 3 show that the method is reproducible and there were high intra-and inter-day precisions (RSD ≤0.435%).

Accuracy
Accuracy of the method was established by performing recovery studies according to the ICH guidelines. Spiked samples were prepared by spiking pre-analyzed sample solutions with standard drug at three different concentration levels (50%, 100%, and 150% level). Mean percentage recovery values at three different concentrations of the two drugs were calculated. The % mean recovery was ranged from 100.13 to 101.20% and 100.40 to 101.70% for LNG and EMP, respectively (Table 3).

Robustness
The robustness of the present method was evaluated in terms of temperature, flow rate, column to column, wavelength of detection, and injection volume ( Table 4). The slight variations in the examined factors had no significant effect on the shape of the peak. The results of coefficient of variation % indicate that the method is more sensitive to changes in the wavelength and the flow rate greater than to changes in the other factors. Compared with retention times (t R -values), peak areas were more affected with the slight changes in the chromatographic conditions.

Selectivity and specificity of the method
The resulted peak after tablet analysis is found to be homogeneous and there are no coeluting peaks indicating specificity of the method. Comparison between the chromatogram of the raw LNG and EMP and that of extracted LNG and EMP from tablets indicate that the excipients in the formulation did not interfere with the determination of LNG and EMP.

Accuracy and application
Analysis of LNG and EMP in Empacoza plus and Glyxambi tablets by the proposed method showed high accuracy with a mean recovery range of 100.71±0.541% and 100.81±0.589% and 101.48±0.254% and 101.64±0.289% for LNG and EMP, respectively ( Table 5). The results were compared with a reported method [20]. The values of t and f indicate that there is no significant difference between both methods.

Stability tests
The results (Fig. 3) of stress degradation indicate that LNG and EMP are strongly affected with reflux with HCl or NaOH. Reflux with H 2 O 2 and exposure to UV radiation leads to degradation of EMP and LIN, but the effect here is weaker than that in the case of HCl and NaOH. There is no interference with the peak of the intact drug, indicating that the method is stability indicating (Fig. 3). Hence, the proposed analytical method is also useful for the determination of LNG and EMP stability in sample of pharmaceutical dosage form.

CONCLUSION
Precise, simple, accurate, robust, and cost-effective stability-indicating HPLC method was developed for the routine and quantification analysis of LNG and EMP in pure forms and tablets. The method was successfully validated in terms of linearity, precision, and accuracy as per the ICH guidelines. Compared with the published chromatographic methods, this method represents a strong reduction of the analysis time and it is considered as a stability-indicating method. A high recovery of LNG and EMP in tablets was achieved. The proposed method ensured a precise and accurate determination of LNG and EMP in tablet formulations and is a stabilityindicating method. No interference from the excipients was noticed. Hence, it can be concluded that the proposed method was a good approach for obtaining reliable results and found to be suitable for the routine analysis and quality control and percentage degradation of pharmaceutical preparations containing both drugs either individually or in combination.