INTRODUCTION:

Type 2 diabetes mellitus (T2DM) is the predominant form of diabetes and is currently a major cause of morbidity and mortality worldwide¹.T2DM characterized by insulin insensitivity as a consequence of insulin resistance, declining insulin production, and accompanied by pancreatic beta-cell dysfunction causing insulin deficiency^{2, 3}. T2DM is due primarily to lifestyle factors and genetics⁴. Several lifestyle factors like physical inactivity, sedentary lifestyle, cigarette smoking and generous consumption of alcohol⁵.

Obesity has been found to contribute to about 55% of cases of T2DM⁶. There is a strong inheritable genetic link in T2DM, having relatives with T2DM increases the risks of rising T2DM substantially.

Though Metformin remains the preferred initial monotherapy for T2DM, except it is contraindicated or not tolerated, the latest treatment algorithm from the American Diabetes Association recommends initial combination therapy. Metformin is generally prescribed as a first-line treatment therapy in T2DM and sodium-glucose cotransporter-2 (SGLT2) inhibitors as a suggested option for dual combination treatment⁷. Combination therapy of Empagliflozin and Metformin has the potential to offer better glucose control compared with that achieved with the individual agents. An oral, single-pill, fixed-dose combination of Empagliflozin and Metformin is also present for patients with T2DM, in a choice of dose combinations to assist individualization of therapy⁸.

Empagliflozin (EMP), (2S, 3R, 4R, 5S, 6R) -2- [4-chloro-3-[[4-[(3S)-oxolan-3-yl] oxyphenyl] methyl] phenyl] -6- (hydroxymethyl) oxane-3, 4, 5-triol is an inhibitor of the sodium-glucose co-transporter-2 (SGLT-2), which accounts for about 90 percent of glucose reabsorption into the kidney⁹. Metformin hydrochloride (MET), N, N-dimethylimidodicarbonimidic diamide is a biguanide hypoglycemic drug which shows its effect mainly by increasing peripheral utilization of glucose¹⁰.The Chemical structures of MET and EMP depicted in figure 1.

Literature survey revealed that few published methods are available for determination of MET and EMP either alone or in combinations in pharmaceutical dosage forms and biological matrices including spectrophotometry and chromatographic methods^11-23. HPLC is a rugged analytical tool for the development of stability indicating methods in fast mode^{24- 27}. Based on the reference, we have chosen RP-HPLC for the estimation of MET and EMP in a pharmaceutical dosage form. Also, the established HPLC method has many advantages over the routine HPLC methods reported for MET and EMP as less retention times, higher resolution, improved sensitivity and simple mobile phase.

The present work is aimed to report simple, accurate and rapid RP-HPLC method for the simultaneous determination of MET and EMP in a pharmaceutical formulation as per the ICH guidelines.

Figure 1. Chemical Structure of (a) Metformin (MET) and (b) Empagliflozin (EMP)

MATERIALS AND METHODS:

Chemicals and Reagents

Pure active pharmaceutical ingredients, MET and EMP were kindly supplied by Spectrum Pharma Labs. (Telangana, India). HPLC grade acetonitrile and methanol were procured from Merck Specialties Pvt. Ltd. (Mumbai, India). AR grade Potassium dihydrogen phosphate and ortho-Phosphoric acid were purchased from Spectrochem Pvt. Ltd. HPLC grade water was obtained by using Millipore Milli-Q water purification system (Millipore, Milford, USA). Jardiance Met tablet containing 500mg of MET and 5mg of EMP per tablet supplied from Boehringer Ingelheim pharmaceutical Company (India).

Instrumentation:

The chromatographic separation was achieved on the Waters HPLC Alliance 2695 separating module using a photodiode array detector (waters 2998) with autosampler and column oven. The instrument was controlled by Empower 2 software installed with equipment for data collection and acquisition. UV-VIS spectrophotometer PG Instruments T60 with UV win 6 Software was used for measuring the absorbance of MET and EMP solutions. Ascentis C18 HPLC column (4.6 x 150 mm, 5 μm) was used.

Chromatographic Conditions:

The mobile phase consisted of 0.1% orthophosphoric acid and acetonitrile (60:40, v/v). The mobile phase filtered through 0.45 µm nylon filter and degassed in an ultrasonic bath before use. An injection volume10 μL was used for measurements and UV detection at 260 nm. All analyses were performed at ambient temperature.

Standard and Sample Solutions Preparation:

Standard Stock Solution:

The standard stock solution was prepared by dissolving the drugs in the acetonitrile and diluting to the required concentration. The diluent acetonitrile: water in a ratio 50:50 v/v was used as a solvent system. Accurately weighed 250mg MET, 2.5mg of EMP and transferred to 50 mL volumetric flask, to this 5 mL of acetonitrile was added and degassed in an ultrasonic bath. Volume was made up to the mark with diluent and labelled as a standard stock solution (5000µg/ml of MET and 50µg/ml EMP).

Preparation of Sample Solutions:

Weighed twenty tablets were powdered and mixed thoroughly in a mortar. An accurately weighed quantity of powder equivalent to 500 mg of MET and 5mg of EMP was taken into 100 mL volumetric flask. 50 mL of diluent was added, the solution was filtered through 0.45μm nylon filter and sonicated for 15 min, further the volume was made up with diluent to get the required concentration (5000 µg/mL of MET and 50 µg/mL of EMP). Further, concentrations were prepared by dilution with the diluent.

Method Validation:

The proposed method was validated by following the ICH guidelines of Validation of Analytical Procedure: Q2 (R1) by evaluating the linearity, range, specificity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), robustness and system suitability parameters. To assess the linearity and range of the developed method, the standard stock solutions of MET and EMP were suitably diluted with diluent to obtain a series of solutions containing 125, 250, 375, 500, 625 and 750 μg/mL of MET and 1.25, 2.50, 3.75, 5.00, 6.25 and 7.50 μg/mL of EMP respectively. The accuracy and precision of the method were calculated by performing the assay of samples (spiked placebos) prepared at 3 different concentration levels of 50%, 100% and 150%, with three replicates for each concentration. The % recovery and %RSD were calculated for each of the replicate samples. The limit of detection (LOD) and limit of quantification (LOQ) of MET and EMP were determined by the standard deviation of the response (σ) and slope approach as specified in ICH guidelines. The LOD was calculated using the formula 3.3*σ/slope, and the LOQ was calculated using the formula 10*σ/slope. The robustness of the method was established by the slight modification of method parameters such as flow rate, column oven temperature and the percentage of a buffer in the mobile phase. The ruggedness of the method was established by studying the effect of elapsed assay times and by an analyst on the method performance^28,
29.

Forced Degradation Study:

To assess the stability-indicating properties and specificity of the method, forced degradation studies were performed on MET and EMP. Forced degradation was carried out by exposing the drug substance and drug product to different stress conditions. Stressed samples were analysed and the presence of related peaks, retention time and peak purity for the active ingredients were checked^30-32.

Acid Degradation:

To 1 mL of stock solution MET and EMP, 1mL of 2N Hydrochloric acid (HCl) was added and refluxed for 30 min at 60 ⁰C. The solution was cooled to room temperature and neutralized with 2N Sodium Hydroxide (NaOH) and the final solution was made up to target concentration. The chromatograms were recorded by injecting 10 µL solution into the system to assess the stability of the sample.

Alkali Degradation:

To 1 mL of stock solution MET and EMP, 1mL of 2N Sodium Hydroxide (NaOH) was added and refluxed for 30 min at 60 ⁰C. The solution was cooled to room temperature and neutralized with 2N Hydrochloric acid (HCl) and the final solution was made up to target concentration. The chromatograms were recorded by injecting 10 µL solution into the system to assess the stability of the sample.

Oxidative Stress:

To 1 mL of stock solution MET and EMP, 1mL of 20% Hydrogen peroxide (H₂O₂) was added and refluxed for 30 min at 60 ⁰C. Finally, the solution was made up to target concentration. The chromatograms were recorded by injecting 10 µL solution into the system to assess the stability of the sample.

Thermal Degradation:

The standard drug solution was placed in an oven at 60 ⁰C. for 6 hrs to study dry heat degradation. The solution was cooled to room temperature and 10 µL solution was injected into the system to assess the stability of the sample.

Photo Degradation:

The photostability of the drug was also studied by exposing the stock solution to UV Light by keeping the beaker in photostability chamber at 200 Wh/m²at 25 ⁰C^. for 7 days. The chromatograms were recorded by injecting 10 µL solution into the system to assess the stability of the sample.

RESULTS AND DISCUSSION:

In present work stability indicating analytical method for the estimation of MET and EMP in bulk and tablet formulation was developed and validated as per ICH guidelines for analytical method validation, Q2 (R1).

Method Development:

The main objective of present work was to develop a stability indicating RP-HPLC method for estimation of MET and EMP with a short run time. The mobile phases composition and stationary phase play a key role in theoretical plates, peak shape, symmetry and resolution. To achieve symmetrical peak shape with better resolution and peak purity, a variety of chromatographic conditions were studied and optimized for the determination of MET and EMP; such as mobile phases with different compositions, pH and stationary phases with different packing material etc. The UV spectrum displayed MET and EMP have maximum absorption at 260 nm. The choice of stationary phase based on sufficient retention of MET and EMP compounds. AscentisC18 HPLC column contains a silica-based packing. Ascentis C18 HPLC column (4.6 x 150 mm, 5 μm) has the best retention times, theoretical plate count, good peak shapes and resolution for MET and EMP. The orthophosphoric acid buffer with different concentration (0.1, 0.2 and 0.5%) was used to enhance the polarity of the mobile phase, which resulted in a narrowed peak shape. Acetonitrile was used in place of methanol to facilitate faster separation of MET and EMP with good peak shape.

Finally, the mobile phase containing orthophosphoric acid (0.1%) and acetonitrile in 60:40v/v ratio was selected and found to be optimal with more theoretical plates, narrow peak, high peak symmetry and short retention time. Based on the above optimization symmetrical and sharp peak of MET and EMP was obtained on Ascentis C18 HPLC column with 1.0 mL/min flow rate. A typical HPLC chromatogram obtained during simultaneous estimation of MET and EMP has given in Figure 2.

Figure 2: HPLC chromatogram obtained during simultaneous separation of MET and EMP. Chromatographic conditions: Ascentis C18 column (150 mm × 4.6 mm, particle size 5 μm); mobile phase phosphate buffer 0.1% orthophosphoric acid and acetonitrile 60:40 v/v, at a flow rate of 1.0mL/min; and UV detection at 260 nm.

Method Validation:

An optimized method must be validated before actual use. As per ICH guidelines for analytical method validation, Q2 (R1) the system suitability testing was performed. The validation studies were performed as given in the following sections.

Specificity:

The specificity of the method was evaluated by assessing interference from extraneous components in a pharmaceutical dosage form as a placebo solution. The chromatogram indicates the ability of the method to measure the analyte in presence of other excipients. The specificity studies proved the no interference, since no blank and placebo peak detected at the retention time (2.27 and 3.10 min) corresponding to the MET and EMP.

Range and Linearity:

Six different concentrations (125, 250, 375, 500, 625 and 750 µg/mL) of MET, (1.25, 2.50, 3.75, 5.00, 6.25 and 7.50 µg/mL) of EMP were prepared for linearity studies. The calibration curves obtained by plotting peak area against concentration showed a linear relationship. Calibration curves with corresponding residual plots of MET and EMP were shown in Figure 3. The linear regression equations for MET and EMP were found to be y = 20507x + 205140, and y = 57493x + 4331, respectively. The regression coefficient (R²) values for MET and EMP were noted 0.999 and 0.999, respectively. The results obtained showed that there was an excellent correlation existed between concentration and peak area of the drug within the elected concentration range. The results established the linearity and the reproducibility of the assay method.

Figure 3: Linearity plots for (a) MET and (b) EMP

Precision:

The intra-day precision of the developed method was calculated by preparing the samples of the same batch with three concentrations and three replicate each. The inter-day precision was also calculated by assaying the dosage form in triplicate every day for three consecutive days. The Intra-day RSD (%) was found 0.98% for MET and 1.21% for EMP. The Inter-day RSD (%) was at 0.54% for MET and 1.07% for EMP. The results showed that the developed method is precise and within the acceptance limit of 2.0%.

Accuracy:

The recovery parameter was performed by adding a known amount of the drugs in the placebo at three levels: 50%, 100% and 150% of the label claim of the marketed formulation. Three samples at each recovery level were prepared. The solutions were then analyzed and the percent recoveries were calculated from the calibration curve. The mean recovery values were found to be 99.95% and 99.88%. The results obtained revealed that there was no interference of excipients. The results of accuracy are shown in Table 1.

Table 1: Percent recovery data of MET and EMP

Drug	% Simulated dosage nominal	% Mean (n=3)	± SD	RSD (%)
MET	50	100.05	1.02	1.02
EMP	50	99.94	0.58	0.58
MET	100	99.11	0.83	0.84
EMP	100	100.28	1.35	1.35
MET	150	100.70	1.04	1.03
EMP	150	99.42	0.26	0.26

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The sensitivity of the method predicted by LOD and LOQ. The LOD and LOQ for MET and EMP were determined based on a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. The LOD for MET and EMP were 4.59 and 0.08 µg/mL, respectively, whereas LOQ were 13.91 and 0.23 µg/mL, respectively. The values represented that the method is sensitive.

Robustness:

The robustness of an analytical method is the ability to remain unchanged by minor changes in parameters. The experimental conditions were deliberately modified and the chromatographic resolution of MET and EMP was assessed. To study the effect of the organic solvent (acetonitrile) on the resolution, the concentration was changed 2 units on either side from 40 to 42 and 38, while other chromatographic conditions were kept constant. To study the effect of flow rate on the resolution, the flow rate was changed ± 0.1 units from 1.0 to 1.1mL/min and 0.9mL/min, while other conditions were kept constant. The resolution between MET and EMP was not less than 1.5 in the study.

Forced degradation study:

Forced degradation study required to demonstrated specificity when developing stability indicating analytical method. All the stress conditions applied were adequate to degrade MET and EMP in the pharmaceutical formulation. The results of stress studies of MET and EMP are shown in Table 2 and Table 3, respectively. MET and EMP were degraded and remained 95.77% and ~95.32% respectively when 2N HCl was used at 60°C for 30 min. The MET and EMP were degraded and remained ~95.83% and ~95.45% respectively when 2N NaOH was used at 60°C for 30 min. The MET and EMP were degraded and remained ~94.32% and ~94.35% respectively under 20% H2O2 at 60°C for 30 min. The MET and EMP were degraded and remained ~98.30% and ~98.17% respectively under 60°C for 6hrs. The MET and EMP were degraded and remained ~98.49% and ~98.23% respectively under overall illumination of 200 Wh/m² at 25°C in photostability chamber for 7 days. From these applied stress studies, it was thus concluded that MET and EMP were not stable in strongly acidic, strong basic and oxidative conditions, but stable in thermal and photolytic conditions and developed method can be considered highly specific for the intended use. The chromatograms of stress studies of MET and EMP are given in Fig. 4 (b), (c), (d), (e) and (f).

Figure 4: (a) A typical HPLC chromatogram of a sample solution containing MET and EMP. HPLC chromatogram of MET and EMP obtained from degradation studies, (b) Acid hydrolysis (2N HCl at 60°C for 30 min); (c) Base hydrolysis (2N NaOH at 60°C for 30 min); (d) Oxidative degradation (20% H₂O₂ at 60°C for 30 min); (e) Thermal degradation (60°C for 6hrs); (f) Photodegradation (overall illumination of 200Wh/m²at 25°C for 7 days).

Table 2: Degradation study of MET

Condition	RT	Purity Angle	Purity Threshold	% Drug degraded
Acid Hydrolysis	2.28	1.04	1.49	4.23
Base Hydrolysis	2.28	0.89	1.30	4.17
Oxidative (Peroxide)	2.25	1.00	1.46	5.68
Thermal	2.28	0.77	1.26	1.70
Photo (UV Light)	2.27	0.40	0.76	1.51

Table 3: Degradation study of EMP

Condition	RT	Purity Angle	Purity Threshold	% Drug degraded
Acid Hydrolysis	2.92	1.89	2.15	4.68
Base Hydrolysis	2.92	1.91	2.38	4.55
Oxidative (Peroxide)	2.89	1.77	2.06	5.65
Thermal	2.94	1.57	1.87	1.83
Photo (UV Light)	2.94	2.38	2.86	1.77

System Suitability Parameters:

System suitability parameters were measured to make sure the system performance. For system suitability parameters, six replicates of mixed standard solution were injected. All critical parameters were within the acceptance criteria on all days. Parameters such as resolution, capacity factor, tailing factor, theoretical plate, retention volume, and asymmetry factor of the peaks were calculated. The injection precision RSD was found 0.11% for MET and 0.08% for EMP.

CONCLUSION:

A simple, accurate, precise and rapid RP-HPLC method was validated for simultaneous estimation of MET and EMP as per the ICH guidelines. The performed validation exercise proved that the HPLC method was linear over the proposed working range as well as accurate, precise and specific. The good recovery percentage of dosage form showed that the excipients have no interference in the determination. The RSD (%) was also lower than 2.0, show a high degree of precision of the proposed method. The validated analytical method was also ascertained to be robust concerning flow rate, temperature and composition of the mobile phase. The validated method was consistent as well as capable to determine and detect any expected change in the drug product assay during stability studies. Thus, the method was qualified to exhibit and detect any probable change in the drug product assay during stability studies. The validated method can be used for routine analysis of MET and EMP in the combined dosage form and the quality control in bulk manufacturing as well.

ACKNOWLEDGEMENT:

I am truly grateful to the Director of the School of Pharmacy, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India for encouraging to carry out this work and providing research support.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 17.12.2020 Modified on 23.01.2021

Research J. Pharm.and Tech 2022; 15(2):830-836.

DOI: 10.52711/0974-360X.2022.00138