To develop and validate the stability indicating method for repaglinide and voglibose using HPLC method

Mohd Bilal Sufi; Shantanu Burange*; Rajlaxmi Deolekar

Department of Pharmaceutical Quality Assurance, New Montfort Institute of Pharmacy, Ashti,

Dist – Wardha, (M.S) – 442202

*Correspondence: shantanuburange1799@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5711          

INTRODUCTION

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycaemia caused by defects in insulin secretion, insulin action, or both. It has become a major global public health challenge over the past three decades [1]. According to the International Diabetes Federation (IDF), about one in eleven adults aged 20–79 years had diabetes in 2021, with cases expected to increase substantially by 2045 [2-3]. India accounted for nearly 77 million diabetic patients in 2019, with projections exceeding 130 million by 2045 due to urbanization, dietary changes, and sedentary lifestyles [4]. This growing burden highlights the need for effective pharmacotherapy and reliable analytical methods to ensure the quality, safety, and efficacy of antidiabetic drugs. Diabetes mellitus is classified into type 1 diabetes, type 2 diabetes (T2DM), gestational diabetes, and other specific types. Type 1 diabetes results from autoimmune destruction of pancreatic β-cells, causing absolute insulin deficiency, whereas T2DM is characterized by insulin resistance with progressive β-cell dysfunction [5-6]. Gestational diabetes develops during pregnancy and increases the future risk of T2DM, while other specific forms arise from genetic defects, pancreatic disorders, or drug therapy [7]. T2DM accounts for the majority of diabetes cases worldwide and in India [2,4]. It is characterized by insulin resistance in skeletal muscle, liver, and adipose tissue, along with inadequate insulin secretion [8-9]. Persistent metabolic stress eventually impairs β-cell function, resulting in postprandial hyperglycaemia [6,9]. Poor glycaemic control can lead to microvascular complications such as retinopathy, nephropathy, and neuropathy, as well as macrovascular complications including coronary artery disease, stroke, and peripheral arterial disease [10].

Management of T2DM mainly relies on oral antidiabetic drugs (OADs), particularly in resource-limited settings [11]. Because T2DM has a multifactorial etiology, monotherapy often fails to maintain long-term glycaemic control, making combination therapy necessary to target complementary mechanisms [9]. Combining insulin secretagogues, insulin sensitizers, and carbohydrate absorption inhibitors improves fasting and postprandial glucose control while minimizing adverse effects [12-13]. Drugs with different mechanisms of action provide additive or synergistic effects, enable individualized treatment, and may reduce dose-related adverse effects [13-14]. Combining a rapid insulin secretagogue with an α-glucosidase inhibitor effectively reduces postprandial glucose excursions, particularly in populations consuming high-carbohydrate diets [14-15]. Such benefits have encouraged the development of fixed-dose combinations (FDCs) to improve adherence and simplify therapy [15-16]. Repaglinide is a short-acting oral antidiabetic drug belonging to the meglitinide class [18-19]. It is a substituted benzoic acid derivative (C₂₇H₃₆N₂O₄; molecular weight 450.6 g/mol) with high lipophilicity and extensive plasma protein binding [20]. It lowers blood glucose by binding to SUR1 receptors on ATP-sensitive potassium channels of pancreatic β-cells, stimulating insulin secretion [18-19]. Because of its rapid onset and short duration of action, repaglinide is particularly effective in controlling postprandial hyperglycaemia [18,21].

Repaglinide is rapidly absorbed, reaching peak plasma concentrations within one hour, and has an elimination half-life of 1–2 hours [20,21]. It undergoes extensive hepatic metabolism mainly through CYP3A4 and CYP2C8 and is primarily excreted in bile [22,24]. It is used alone or with other antidiabetic agents, especially when rapid postprandial glucose control is required or metformin is unsuitable [12,19]. Available in 0.5, 1, and 2 mg tablets, it is administered before meals. Its major adverse effects include hypoglycaemia and weight gain, while CYP3A4/CYP2C8-mediated drug interactions may increase hypoglycaemia risk, particularly in elderly patients or those with renal impairment [23-25].

Voglibose is an oral α-glucosidase inhibitor used to manage postprandial hyperglycaemia [15,26]. It is a nitrogen-containing polyhydroxy compound (C₁₀H₂₁NO₇; molecular weight 267.3 g/mol) with minimal systemic absorption, producing its effect locally in the intestinal lumen. By competitively inhibiting intestinal α-glucosidase enzymes, it delays carbohydrate digestion and glucose absorption, thereby reducing postprandial glucose peaks without stimulating insulin secretion [15,26]. Pharmacokinetic studies show that voglibose has very low systemic bioavailability and is largely excreted unchanged in faeces, contributing to its favourable safety profile but also causing dose-dependent gastrointestinal effects such as flatulence and diarrhoea [26,28-29]. It is used with diet and exercise, particularly in patients with predominant postprandial hyperglycaemia or high-carbohydrate diets [26,28]. Available as 0.2 and 0.3 mg tablets, it is taken immediately before meals. Although generally well tolerated, gastrointestinal adverse effects and caution in patients with intestinal disorders remain important limitations [15,26].

The combination of repaglinide and voglibose provides a rational approach for improving postprandial glycaemic control by simultaneously enhancing insulin secretion and delaying carbohydrate absorption [15]. Repaglinide stimulates meal-related insulin release, while voglibose slows glucose entry into the bloodstream, producing complementary effects that reduce postprandial glucose excursions more effectively than monotherapy [14,15]. Similar combinations with short-acting insulin secretagogues have demonstrated improved glycaemic control and reduced glycaemic variability in T2DM patients. Voglibose-based FDCs have also shown good efficacy and tolerability in Indian patients, particularly those consuming high-carbohydrate diets [14,16]. Consequently, the repaglinide–voglibose fixed-dose combination (FDC) may improve HbA1c, reduce long-term diabetic complications, and enhance patient adherence. These advantages necessitate reliable stability-indicating analytical methods for simultaneous estimation of both drugs in bulk and pharmaceutical formulations.

High-performance liquid chromatography (HPLC), particularly reversed-phase HPLC (RP-HPLC), is widely used in pharmaceutical quality control because of its sensitivity, selectivity, and versatility [32-33]. RP-HPLC separates analytes based on hydrophobic interactions using a C18 stationary phase and a polar mobile phase under high pressure [32-33]. A typical system consists of solvent reservoirs, high-pressure pumps, an injector, analytical column, detector (UV/Visible or PDA), and data acquisition system [32,36]. Chromatographic performance depends on factors such as mobile phase composition, pH, flow rate, and column characteristics, which must be optimized for efficient separation [34,36]. RP-HPLC offers rapid analysis, simultaneous multi-component estimation, high sensitivity, and cost-effectiveness, making it suitable for routine assay, impurity profiling, and stability testing [32-33,36].

Stability-indicating analytical methods are designed to accurately distinguish drugs from degradation products, impurities, and excipients [37]. According to ICH Q1A guidelines, forced degradation studies expose drug substances to acidic, alkaline, oxidative, thermal, and photolytic stress to understand degradation pathways and develop suitable analytical methods [37-40]. For fixed-dose combinations such as repaglinide and voglibose, analytical methods must effectively separate degradation products of both drugs from the parent compounds within a single chromatographic run [36-37]. Method validation also includes evaluation of range, limits of detection (LOD) and quantitation (LOQ), robustness, ruggedness, and system suitability parameters such as theoretical plates, tailing factor, resolution, and repeatability [36-37,41]. Comprehensive validation in accordance with ICH Q2(R2) ensures the reliability of RP-HPLC methods for routine quality control and stability studies [41]. Although several stability-indicating RP-HPLC methods have been reported for repaglinide and voglibose, many involve single-drug analysis, gradient elution, longer run times, or expensive instrumentation, limiting their suitability for routine industrial quality control laboratories [32-36]. Therefore, there remains a need to develop a simple, rapid, economical, and reliable stability-indicating RP-HPLC method for the simultaneous estimation of repaglinide and voglibose in bulk drug and pharmaceutical dosage forms.

A simple, rapid, inexpensive and precise analytical method, which separates and quantifies repaglinide and voglibose along with their degradation product(s) within a single run and satisfies ICH Q1A and Q2(R2) requirement would be valuable for the pharmaceutical industries that manufacture the repaglinide–voglibose combination for routine assay and content uniformity analysis and studies of their product stability [36-37]. The present aim to fill this gap, a stability indicating RP‑HPLC method was developed and validated for simultaneous estimation of repaglinide and voglibose, which further enhances the quality assurance and compliance in the production of these significant drugs used for the treatment of diabetes.

MATERIALS AND METHODOLOGY

Materials

Repaglinide and Voglibose active pharmaceutical ingredients (APIs) were procured from Swapnroop Drugs and Pharmaceuticals, Aurangabad, India. The marketed formulation, EUREPA V 0.5/0.2 tablets, was obtained from Torrent Pharmaceuticals Ltd. HPLC-grade methanol and 0.1% acetic acid were purchased from Merck Ltd., India. All chemicals and reagents used in the study were of analytical or HPLC grade and were used as received without further purification.

HPLC Instrumentation and Chromatographic Conditions

Chromatographic analysis was carried out using an Agilent 1100 Gradient HPLC system equipped with an Agilent 1100 Gradient Pump, a Diode Array Detector (DAD), and ChemStation software for instrument control, data acquisition, and analysis. Separation was achieved on an Agilent RP-C18 column (4.6 × 250 mm, 5.0 µm particle size) using a mobile phase comprising methanol and 0.1% acetic acid (41:59, v/v). The mobile phase was delivered at a flow rate of 0.7 mL/min, and detection was performed at a wavelength of 239 nm. The column temperature was maintained at 25°C, and the injection volume was 20 µL. The system employed a reciprocating gradient pump for precise mobile phase delivery, which was fully compatible with the DAD detector and ChemStation software, ensuring accurate and reproducible chromatographic analysis.

Determination of Absorption Spectra

Both Repaglinide and Voglibose were accurately weighed and prepared as standard stock solutions on 10 mL each with a concentration of 1000 µg/mL in methanol. Suitable dilutions were made for the working solutions of 10 µg/mL for each drug. The absorption spectra were recorded individually in the range of wavelength 200-400nm with blank of methanol. The λmax was determined for Repaglinide and Voglibose and their overlain spectrums were evaluated for choosing the common detection wavelength for estimation of the two compounds simultaneously in RP-HPLC.

Method Development and Optimization

Different chromatography conditions were tested to achieve good resolution of Repaglinide and Voglibose. Different methanol: OPA mixtures, in proportions ranging from 100:0 to 75:25, and varying flow rates (0.7 mL/min to 1.0 mL/min) were tested in various combinations. All of these above-mentioned chromatographic characteristics have been used to evaluate the chromatographic performance with retention time, peak asymmetry, theoretical plate count, peak width, peak resolution and system suitability.

Table 01 Trials for Mobile Phase Optimization

Optimized Chromatographic Conditions

A C18 analytical column (250 mm × 4.6 mm, 5 µm particle size) was used under isocratic conditions. Methane and 0.1% Orthophosphoric Acid (40:60, v/v) were used as mobile phase after filtering through a membrane filter (0.45 µm) and degassing by sonication. Mobile phase was isocratically pump at a flow rate of 1.0 mL/min and the chromatograms were recorded at the optimized direction wavelength. The volume of injection was kept as 20 μL and the analysis was carried out at the laboratory conditions.

Preparation of Standard Stock Solution

Preparation of Repaglinide and Voglibose accounted by transferring their quantities into 10 mL volumetric flasks and the previously processed quantities was used. The quantities of Repaglinide and Voglibose were transferred respectively into 10 mL volumetric flasks. Approximately 7 mL of methanol was added to each solution, and the solutions were sonicated around 15 minutes to completely dissolve them. The volume was then adjusted to the mark with methanol to make standard stock solutions containing 1000 µg/mL of each drug.

Preparation of Working Standard Solutions

Working standard solutions were prepared by appropriate dilution of the stock solutions with the optimized mobile phase. Repaglinide calibration standards were prepared in the concentration range of 5–25 µg/mL, whereas Voglibose standards were prepared in the concentration range of 1.5–7.5 µg/mL. All prepared solutions were filtered through a 0.45 µm membrane filter before chromatographic analysis.

Specificity

Specificity of the developed RP-HPLC method was evaluated by injecting blank solution, placebo solution, standard solution, and sample solution under the optimized chromatographic conditions. The chromatograms were examined for any interference from excipients, impurities, degradation products, or solvents at the retention times corresponding to Repaglinide and Voglibose. The absence of interfering peaks confirmed the specificity of the method.

Linearity

To assess the linearity of the developed method, five levels were used for Repaglinide (5, 10, 15, 20 and 25 µg/mL) and Voglibose (1.5, 3.0, 4.5, 6.0 and 7.5 µg/mL). The concentrations were injected in triplicate, under optimized chromatographic conditions. Calibration curves were drawn with the mean peak area as function of the mean concentration and linear regression analysis was carried out for the calculation of the slope, intercept and correlation coefficient [42-43].

Accuracy

The standard addition technique was used to assess the accuracy of the method at 50%, 100% and 150% levels of the method concentration. A set amount of reference standard solutions of Repaglinide and Voglibose were spiked to the pre-analysed tablet sample solutions and were subjected to analysis under optimum chromatographic conditions. The percentage recovery was obtained to check the accuracy of the method developed [44].

Repeatability

To assess the repeatability of the developed RP-HPLC method, six replicate injections were performed with RP-HPLC of the standard solutions of Repaglinide and Voglibose having 15 µg/mL and 4.5 µg/mL, respectively, with identical chromatographic conditions. The peak areas of successive injections were measured and the %RSD was calculated to represent the system precision [45].

Intraday Precision

Intraday precision was assessed by analysing samples at three different concentrations of Repaglinide released (5, 15 and 25 µg/mL) and Voglibose released (1.5, 4.5 and 7.5 µg/mL) three times a day in the same chromatographic conditions. The peak areas were measured and the percentage relative standard deviation (%RSD) was calculated to assess the repeatability of the analytical method within-day [45].

Interday Precision

To assess the interday precision, freshly prepared standard solutions of Repaglinide (5 µg/mL, 15 µg/mL and 25 µg/mL) and Voglibose (1.5 µg/mL, 4.5 µg/mL and 7.5 µg/mL) were analysed on three consecutive days with the same chromatographic conditions. Comparisons were done for the analytical results that were obtained on different days and the percentage relative standard deviation (%RSD) was computed to compare intermediate precision [45].

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

The developed RP-HPLC method was sensitive enough to estimate LOD and LOQ as per ICH Q2(R1) guidelines by using Calibration Curve method.  [46] Values were calculated using the standard deviation of the response (σ) and the slope (S) of the calibration curve by the following equations:

Here σ is the standard deviation of the response while S is the slope of the calibration curve.

Robustness

The robustness of the developed RP-HPLC method has been checked by introducing deliberate small variations in those chromatographic parameters; the flow rate (0.8 and 1.2 mL/min), mobile phase composition (40:60 and 42:58, v/v) and the detection wavelength (238 and 240 nm), while keeping all other conditions unchanged. Appropriate parameters for retention time, peak area, peak symmetry and system suitability were checked for the effect of these variations [48].

Ruggedness

Ruggedness was determined with the assay performed by the different analysts and on different days under identical chromatographic conditions. All the standard solutions were analysed and compared through the calculation of percentage relative standard deviation (%RSD), and the dependence on the reproducibility of the developed method when comparing the two was evaluated.

Assay of Marketed Tablet Formulation

The twenty tablets of the EUREPA V 0.5/0.2 were weighed accurately and the average weight of each tablet has been calculated. The tablets were finely powdered and both drugs were transferred in to a volumetric flask, using 100% methanol, equivalent to the labelled claim. This was sonicated for about 15 min to achieve complete extraction of the drugs followed by dilution to volume with methanol. The solution was filtered through membrane filter of 0.45 µm and suitable dilutions were made in the optimized mobile phase to attain a final concentration of 10 µg/mL of Repaglinide and 3 µg/mL of Voglibose. The sample solution was then prepared and injected into the HPLC system and the concentration of the drug was determined from the peak areas measured from the sample solution with that from the standard solution which correspond to the amount of the prepared drug [46].

Forced Degradation Studies

Acid Degradation Study

The acid degradation was performed by transferring appropriate aliquot of the standard stock solution of Repaglinide and Voglibose in volumetric flask then adding 0.1 N hydrochloric acid (HCl). This solution was then held at room temperature for 2 h for easy acid hydrolysis. Once the degradation period was completed, the solution was neutralized with the same amount of 0.1 N sodium hydroxide (NaOH), diluted to the proper concentration with the optimized mobile phase, and passed through a 0.45 µm membrane filter. The optimized chromatographic conditions were employed for injection of the filtered solution into RP-HPLC system and the chromatogram was subsequently screened for possible degradation products and peak purity [49].

Alkaline Degradation

To obtain alkaline degraded samples, a proper aliquot of the standard stock solution was treated with 0.1 N sodium hydroxide (NaOH) solution. The mixture was left standing for 2 h at temperature so that alkaline hydrolysis will take place. The optimized mobile phase was then used to dilute the solution to an appropriate analytical concentration after the degradation period, the solution was neutralized with an equal amount of 0.1 N hydrochloric acid (HCl). After filtering through a 0.45 µm membrane filter, the solution was injected into the RP-HPLC system. Evaluation of chromatograms, with regard to the formation of degradation products, was made, and the ability of these degradation products and analytes to separate [49].

Oxidative Degradation Study

Oxidative degradation was carried out by treating the standard drug solution with 3% hydrogen peroxide (H₂O₂). Oxidative degradation was induced by allowing mixture to react at room temperature for 2 h. The solution was then diluted to the correct concentration with the optimum mobile phase, filtered through a 0.45 µm membrane filter and the solution used for the test. The filtered sample was analysed with the optimized RP-HPLC method and chromatograms were analysed for both oxidative degradation and the separation of the degradation peaks from the principal peaks of the drug [49].

Neutral Degradation Study

Hydrolytic degradation occurred at neutral pH where an aliquot of the standard solution was stirred in with purified water and the mixture allowed to stand at room temperature for 2 h. The solution after the stress treatment was then filtered through 0.45 µm mobile phase membrane filter to bring its concentration to the optimized value. The injected filtered sample was chromatographed in the RP-HPLC system and the chromatogram was analysed to check the degradation degree and to ascertain the separation of degradation products from the peaks of the intact peaks of the drugs [49].

Photolytic Degradation Study

Standard solutions of Repaglinide and Voglibose were placed under the ultraviolet (UV) light for 24 hours which was called Photolytic degradation. Sample solutions were diluted to the required concentration in providing analytical concentration by optimizing the mobile phase in order to filter the solutions through 0.45 µm membrane filter before chromatographic analysis after being exposed. In order to confirm the stability indicating capability, the chromatograms of the degraded samples were examined after injecting on the RP-HPLC system under the optimized chromatographic conditions to confirmed that the developed RP-HPLC method was ability to identify the degradation product and to completely separate the drug peaks from degradation ones [49].

RESULT AND DISCUSSION

Determination of Absorption Spectra (λmax)

The UV absorption spectra of Repaglinide and Voglibose were obtained separately in methanol solutions in the absorption wavelength range of 200–400 nm to determine the absorption maximum of each drug and to decide the necessary conditions for their simultaneous estimation. The maximum absorbance (λmax) of Repaglinide was 243 nm and for Voglibose was 232 nm. In addition of both spectra showed suitable spectral features for the simultaneous analysis and helped in choosing a suitable detection wavelength for chromatographic investigations. The chosen wavelength was adequate to detect both analytes with good sensitivity and reproducible detector response for the further RP-HPLC method development and validation.

Fig. 01: UV absorption spectrum of Repaglinide (10 µg/mL) in methanol

Fig. 02: UV absorption spectrum of Voglibose (10 µg/mL) in methanol

Fig. 03: Iso-electric point determination spectrum

Mobile Phase Optimization

Different mobile phase composition (0.1% methanol: orthophosphoric acid (OPA)) were evaluated, changing the ratio of the components, to optimise the chromatographic conditions. Early chromatography experimentation resulted in low resolution, out-of-shape peaks and unacceptably low theoretical plate count. The use of progressively more optimum compositions of solvents produced substantially better results in the chromatography. The optimized chromatographic condition of Methanol : 0.1% OPA (40:60, v/v) at flow rate of 1.0mL/min provided sharp symmetrical and well resolved peak for both drugs with satisfactory retention time, peak symmetry and theoretical plate count. The optimized chromatogram showed complete baseline separation between Repaglinide and Voglibose thereby establishing the applicability of the developed mobile phase for the simultaneous estimation of Repaglinide and Voglibose and further validation study.

Table 02 Chromatographic Performance Data of Mobile Phase Optimization Trials

Fig. 04: Chromatogram of Trial-06 mobile phase composition

Linearity

Developed RP-HPLC method was linear for both Repaglinide and Voglibose over the following concentration ranges: 5–25 µg/mL for Repaglinide and 1.5–7.5 µg/mL for Voglibose. A good linear relationship between concentration and peak area was obtained as shown from the calibration curves within the experimental ranges. Repaglinide showed the regression coefficient R² of 0.9998, while Voglibose, which had R² of 0.9995 is outstanding in the linearity. Standard deviations and percentage relative standard deviation (%RSD) obtained for each concentration level were low and indicated good detector response. The results of these experiments showed that the method developed is suitable for quantitative estimation of both the drugs with good accuracy in the selected concentration ranges as per ICH guidelines.

Table 03 Linearity Data for Voglibose

Table 04 Linearity Data for Repaglinide

Fig. 05: Calibration curve of Voglibose

Fig. 06: Calibration curve of Repaglinide

Accuracy (Recovery Studies)

The developed RP-HPLC method was evaluated by recovery studies from the standard addition technique at 50%, 100% and 150% concentration levels as a measure of accuracy. The mean percentage recoveries obtained for repaglinide were between 99.25% to 100.82% and for voglibose were between 99.36% to 99.99%. The %RSDs were <1% for both cases, which provided good reproducibility to the recovery studies. Near recovery values of 100% indicated that no interference from formulation excipients occurred and also exhibited excellent accuracy while conforming to ICH validation requirements, both for the drugs.

Table 05 Summary of Accuracy Studies of Voglibose

Table 06 Summary of Accuracy Studies of Repaglinide

Intraday Precision

To assess the precision of intraday analysis, the levels of Repaglinide (5, 15, 25 µg/mL) and Voglibose (1.5, 4.5, 7.5 µg/mL) were chosen and were analysed at the same day, and under the same chromatographic conditions. The assay values obtained were found to be within 0.11-0.68% of the nominal concentrations for Repaglinide and 0.15-1.07% for Voglibose and %RSD values were within good limits. The low variation between replicates from the analyses indicated that the developed RP-HPLC method had good repeatability and precision that were used during one analytical day. Intraday precision was found to be comparable and acceptable as per the ICH acceptance criteria.

Table 07 Intraday Precision for Voglibose

Table 08 Intraday Precision for Repaglinide

Interday Precision

To check the interday precision, freshly prepared standard solutions of Repaglinide (5.0, 15.0, 25.0 µg/mL) and Voglibose (1.5, 4.5 and 7.5 µg/mL) were injected on different days under exactly the same chromatographic conditions. Assay values obtained after every day showed a good reproducibility with %RSD in the range of 0.01% to 0.34% for Repaglinide and 0.09% to 0.92% for Voglibose. The very little variation between the analytical days resulted excellent intermediate precision and good reproduction of the developed RP-HPLC method. The result indicated the reliability of the method for the routine pharmaceutical quality control analysis.

Table 09 Interday Precision for Voglibose

Table 10 Interday Precision for Repaglinide

Repeatability

Repeatability of methods was determined by injecting replicate amounts of standard solutions into the chromatographic system. Repaglinide at the concentration of 15 µg/mL was found to have an assay value of 97.39% and a %RSD of 0.09% and voglibose at the concentration of 4.5 µg/mL was found to have an assay value of 101.42% and %RSD of 0.02%. These low %RSD values for both analytes showed excellent injection repeatability and instrument precision which proved the consistency and reliability of the developed chromatographic method.

Table 11 Repeatability for Voglibose and Repaglinide

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

ICH recommended calibration curve approach was used to determine the sensitivity of developed RP-HPLC method by determining LOD and LOQ. The values obtained revealed that the method had a good detection and quantification limit for low concentrations of Repaglinide and Voglibose with an adequate range. Low detection and quantification limits were obtained, thus demonstrating the usefulness of the developed method for stability studies, as well as for routine QC analysis, that also involved trace-level estimation.

Ruggedness

The developed analytical method was tested its ruggedness by assaying at various analytical conditions, such as different analysts and operation day. Repaglinide had a mean assay of 99.38% with a low %RSD value of 0.352% whereas the voglibose gave a mean assay of 100.90% and low %RSD value of 0.512%. The negligible variation of the assay was confirmed by excellent reproducibility and ruggedness of developed RP-HPLC method.

Table 12 Ruggedness Study for Voglibose and Repaglinide

Robustness

Robustness studies were conducted by intentionally causing small changes in the flow rate, mobile phase composition and detection wavelength. No significant changes in chromatographic performance such as retention time, peak area, peak symmetry, and/or resolution, were observed when applying these intentional modifications. The %RSD values for Repaglinide and Voglibose were always less than 2% which is within the acceptable limit, and this meant that the developed RP-HPLC method was quite robust and it was able to yield reliable analytical results even when there were slight variations in the chromatographic conditions.

Table 13 Robustness Study for Voglibose and Repaglinide

Assay of Tablet Formulation

The developed and validated RP-HPLC method was then applied to the analysis of marketed tablet formulation of Repaglinide and Voglibose as EUREPA V 0.5/0.2. The mean assay value of repaglinide was found to be 99.72% of labelled claim, while the mean assay value of voglibose was 100.58% of labelled claim. The low standard deviation and %RSD were considered to show good precision and reproducibility for the assay procedure. The results obtained agreed with the developed analytical approach indicating the validity of the developed method for routine quality control analysis of combined pharmaceutical dosage forms.

Table 14 Assay of Voglibose and Repaglinide (% Label Claim)

Fig. 07: Chromatogram of assay of Voglibose and Repaglinide (10 µg/mL + 3 µg/mL) (Injection 02)

Forced Degradation Studies

Acid Degradation

The stability of Repaglinide and Voglibose under acidic condition was investigated to evaluate Acid degradation studies. Repaglinide showed a degradation of 3.80% which is considered to be relatively good in an acidic environment. Voglibose, on the other hand, exhibited 6.17% degradation rate, implying lower acid resistance. The chromatograms showed good separation of degradation peaks from the drug peaks, and hence it can be concluded that the developed RP-HPLC method was specific and could diagnose a degradation pathway of the Drug, because all peaks were obtained under acidic condition.

Basic Degradation

The alkaline stability of the two drugs was tested by using the alkaline hydrolysis. In alkaline environment, repaglinide degraded to 18.67% which showed it was very susceptible to degradation under alkaline line. For Voglibose, complete degradation (100% degradation) was observed under the same stress condition. This is extremely poor stability and this degradation occurred in alkaline media. The developed chromatographic method proved to be selective towards the various degradation products in the extent of chromatogram degradation, without compromising the resolution.

Oxidative Degradation

The oxidative degradation experiments conducted with H2O2 revealed degradation of Repaglinide (4.45%) and Voglibose (17.89%) respectively. The results showed that Voglibose is highly susceptible to oxidative stress as compared to Repaglinide. Both the peaks of the drugs were well separated from each other and their possible oxidative degradation products confirming that the developed RP-HPLC method can be used for the accurate determination of both drug molecules even in the presence of their oxidative degradation products.

Neutral Degradation

Both drugs stability was studied in aqueous environment under neutral hydrolytic degradation conditions. Thus, Voglibose was found to be comparatively very susceptible to neutral hydrolysis with 10.63% degradation and repaglinide 4.16% degradation. These chromatograms showed the good separation of the peaks of the drug and the peaks of degradation products and verified the stability-indicating characteristic of the analytical method developed.

Table 15 Forced Degradation Study – Repaglinide (After 2 h Stress)

Table 16 Forced Degradation Study – Voglibose (After 2 h Stress)

Photolytic Degradation (UV Light, 24 Hours)

The chosen UV light intensity was assessed by photolytic degradation studies and exposed the drug samples to this UV light for 24 hours. Both drugs appear to have excellent photostability with repaglinide exhibiting 2.97% degradation and Voglibose showing only 0.34% degradation. No degradation products were found to interfere in the chromatograms and drug peaks were always well-resolved and sharp in the chromatograms. The results showed that the developed RP-HPLC method could selectively estimate both drugs upon exposure to photolytic stress conditions and it was also proven to be a viable stability indicating analytical method as per ICH guidelines.

Table 17 Photolytic Degradation Study (24 h) – Voglibose and Repaglinide

Fig. 08: Chromatogram of photolytic degradation under UV light exposure (24 h)

CONCLUSION:

A robust method for simultaneous RP-HPLC estimation of Repaglinide and Voglibose in presence of their degradation compounds have been developed and validated in a simple manner and excellently, which is also accurate and precise. Well resolved, symmetrical peaks were obtained with satisfactory retention times and excellent chromatographic performance obtained using optimized chromatographic conditions. All the validation parameters of the developed method were followed with excellent linearity, accuracy, precision, specificity, resolution, robustness, ruggedness and sensitivity, fulfilling the ICH Q2(R2) criteria for the acceptance of the method. The method demonstrated stability-indicating properties under forced degradation conditions such as: acidic degradation, alkaline degradation, oxidation, neutral degradation and photolytic degradation where peak of the degradation products were well separated from the analyte peaks. The method was validated and applied to the assay of marketed tablet formulations, and gave results which were accurate and reproducible. Hence, the developed RP-HPLC method can be applied in the routine quality control analysis, assay determination and stability studies of Repaglinide-Voglibose combinations in pharmaceutical dosage form.

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