Development And Validation Of Stability Indicating Method For The Simultaneous Estimation Of Metformin And Fabuxostat In Bulk And Tablet Dosageform Using RP-HPLC

Pharmaceutical analysis has become an indispensable branch of pharmaceutical sciences and represents an interdisciplinary field that draws its fundamental concepts from chemistry, physics, microbiology, nuclear science, and electronics. Its central role is to evaluate, monitor, and verify the standards established for drugs and pharmaceutical formulations by regulatory bodies so that the purity and quality of medicinal products are assured. Because the safety and therapeutic efficacy of medications depend on adherence to these standards, pharmaceutical analysis is critical throughout drug discovery, formulation development, and production¹.

Analytical testing of pharmaceuticals involves identifying compounds, determining their purity levels, and ensuring their safety by employing various physical, chemical, and instrumental techniques. Spectroscopy, chromatography, and electroanalytical methods are some of the most widely applied approaches for qualitative and quantitative evaluation. Failure to meet the prescribed quality specifications can result in major adverse health consequences, including life-threatening outcomes for patients. Therefore, comprehensive knowledge of analytical principles, experimental procedures, instrumentation, and applications is mandatory for pharmaceutical research and development².

Analytical techniques in pharmaceutical sciences broadly fall into qualitative and quantitative categories. Since drugs may originate from organic, inorganic, or biological sources, the analytical process is based on evaluating a measurable property of the medicinal substance regardless of its origin. Pharmaceutical analytical procedures can be classified into several groups:

(a) Spectroscopic techniques

(b) Chromatographic techniques

(c) Electroanalytical techniques

(d) Biological and microbiological assays

(e) Radioactive methods

(f) Physical methods

(g) Conventional titrimetric assays

Among these, spectrophotometry and chromatographic techniques are most frequently used for identifying and quantifying active pharmaceutical ingredients. Significant advancements in analytical chemistry have led to the development of hyphenated systems such as HPLC, UPLC, LC–MS, and GC–MS. These techniques allow more accurate separation, identification, and analysis of drug substances along with their degradation products. Chromatography, in particular, has evolved as a powerful platform for evaluating the quality and quantity of pharmaceutical compounds³.

Although several analytical protocols exist for single-component and multicomponent dosage forms, the increasing complexity of pharmaceuticals demands rapid, selective, and highly sensitive analytical tools. Hyphenated instruments—especially HPLC, UPLC, and LC–MS—are therefore essential for researchers to develop precise and fast separation methods during quality assessment of pharmaceutical formulations. The fundamental objective of modern analytical research is to design, optimize, and validate reliable methods for both single-drug formulations and combination products⁴.

Combination drug products, which consist of two or more active pharmaceutical ingredients in a single dosage form, are developed to address therapeutic needs not achieved by individual drugs. However, such products pose analytical challenges due to overlapping physicochemical properties and interference from excipients. Consequently, robust method development and validation protocols using HPLC, UPLC, and LC–MS/MS are crucial for multicomponent products.

MATERIAL AND METHODS

FABUXOSTAT-

Gout and inflammatory arthritis are highly painful disorders, and their clinical management is an important responsibility of physicians. Among the different types of arthritis, gout is considered one of the most severe because it results from excessive accumulation of uric acid in the body. When uric acid concentrations rise, needle-like urate crystals form within the joints—especially the big toe—as well as in the kidneys, leading to marked pain, inflammation, swelling, and restricted movement.

To address these complications, an oral combination therapy containing Febuxostat and Metformin has been introduced for the management of gout and diabetes. Febuxostat is a selective xanthine oxidase inhibitor that lowers uric acid synthesis and is widely prescribed in patients suffering from chronic gout and hyperuricemia. Since patients with chronic hyperuricemia continue to experience joint pain, swelling, and stiffness, the addition of an anti-inflammatory agent enhances therapeutic outcomes. Metformin Hydrochloride, a non-steroidal anti-inflammatory drug (NSAID), provides strong anti-inflammatory and analgesic activity in conditions such as gout, inflammatory arthritis, and ankylosing spondylitis.

Clinical evidence suggests that the combined administration of Febuxostat and Metformin Hydrochloride is safe and highly effective, offering faster and more comprehensive control of uric acid–related complications than monotherapy. The combination provides dual benefits by lowering uric acid levels and simultaneously reducing pain and inflammation. Owing to these advantages, this formulation is clinically approved for the treatment of Diabetes chronic gout, hyperuricemia, and inflammatory arthritis.

Considering the therapeutic importance of this combination, the present work focuses on the development and validation of a novel RP-HPLC method for the simultaneous estimation of Febuxostat and Metformin Hydrochloride in a combined tablet dosage form, including forced degradation studies.

MECHANISM OF ACTION

Febuxostat:

Febuxostat (FEB) is a selective, non-purine inhibitor of the enzyme xanthine oxidase, which is responsible for converting hypoxanthine and xanthine into uric acid. It blocks the molybdenum pterin active site of the enzyme in a non-competitive manner and inhibits both the oxidized and reduced forms of xanthine oxidase. Due to its strong binding affinity, Febuxostat cannot be readily displaced from the enzyme, leading to a significant decrease in overall uric acid production

METFORMIN

Diabetes mellitus is a major global health problem, and its effective management remains an important responsibility for healthcare professionals. It is an endocrine disorder currently affecting more than 100 million people worldwide, accounting for approximately 6% of the population. The condition results from insufficient or ineffective insulin production by the pancreas, leading to abnormal blood glucose levels. Persistent hyperglycemia can progressively damage multiple organ systems, including blood vessels, eyes, kidneys, heart, and nerves.

To provide improved glycemic control and reduce these risks, an oral combination therapy of Metformin Hydrochloride has been developed for the treatment of diabetes mellitus. Metformin Hydrochloride, an orally administered biguanide, is widely prescribed for Type II diabetes and reduces elevated blood glucose levels by enhancing insulin sensitivity and decreasing insulin resistance. It suppresses hepatic gluconeogenesis through inhibition of mitochondrial respiratory chain complex-I and activation of AMP-activated protein kinase (AMPK) signaling pathways.Considering the pharmaceutical significance of this combination, the present study focuses on the development and validation of a novel RP-HPLC method for the simultaneous quantification of Metformin Hydrochloride and fabuxostat in a combined tablet formulation, including forced degradation analysis

Mechanism of Action

MetforminHydrochloride:

Metformin Hydrochloride (MET) is a biguanide agent that exerts its antidiabetic effects primarily by suppressing hepatic gluconeogenesis, thereby reducing glucose output from the liver. It enhances peripheral glucose utilization by increasing glucose uptake in skeletal muscle and activates AMP-activated protein kinase (AMPK), which contributes to improved insulin sensitivity. Metformin also interferes with mitochondrial respiration, further reducing gluconeogenesis and supports glycemic control by minimizing degradation

Table 3.2 Pharmaceutical formulations of Febuxostat and Metformin

Table 3.3 Physico-chemical properties and therapeutic category of Febuxostat and Metformin Hydrochloride

Experimental Investigations

An isocratic RP-HPLC method was developed using a Waters Alliance e2695 HPLC system equipped with a 515 pump and a 2998 Photodiode Array (PDA) detector. Data acquisition and processing were carried out using Empower 2 software. The separation was achieved on an Inertsil C18 column (100 mm × 4.6 mm i.d., 5 µm). An ultrasonic bath sonicator (Frontline FS 4, Mumbai, India), a semi-micro analytical balance, and Whatman No. 41 filter paper were used during the experimental work.

REAGENTS

Febuxostat and Metformin Hydrochloride reference standards were obtained from Abott Pharmaceuticals Pvt. Ltd., Hyderabad, India. Acetonitrile (HPLC grade) was sourced from Merck Chemicals Ltd., Mumbai, India, and HPLC grade water from Rankem, India. Ammonium dihydrogen phosphate and orthophosphoric acid of AR grade were purchased from Finar Reagents, Ahmedabad. Fabustat tablets were obtained from Cipla parmaceuticals pvt  Limited, India.

PREPARATION OF MOBILE PHASE

A buffer solution was prepared by dissolving 1.15 g of ammonium dihydrogen phosphate in HPLC water and making up the volume to 1000 mL. The solution was degassed using an ultrasonic bath and filtered through a 0.45 µm nylon membrane, providing a 0.01 M concentration. The pH was adjusted to 5 using orthophosphoric acid. The buffer and acetonitrile were mixed in a 60:40 (v/v) ratio, filtered through a 0.45 µm membrane, and degassed before use.

Preparation of Mixed Standard Stock Solution

A mixed standard stock solution was prepared by accurately weighing 40 mg of Febuxostat and 100 mg of Metformin Hydrochloride into a 100 mL volumetric flask. The drugs were dissolved and sonicated with the mobile phase to obtain final concentrations of 400 µg/mL (Febuxostat) and 1000 µg/mL (Metformin Hydrochloride).

Preparation of Linearity Solutions

Aliquots of 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mL of the mixed standard stock solution were transferred into a series of 10 mL volumetric flasks and diluted with the mobile phase to obtain concentrations of:

• Febuxostat: 10, 20, 30, 40, 50 and 60 µg/mL
• Metformin Hydrochloride: 25, 50, 75, 100, 125 and 150 µg/mL

All solutions were filtered through a 0.45 µm nylon membrane prior to injection.

Preparation of Sample Solution

Twenty fabustat tablets were weighed and the average tablet weight was calculated. The tablets (each containing 40 mg of Febuxostat and 100 mg of Metformin Hydrochloride) were powdered using a mortar and pestle. A sample quantity equivalent to 40 mg of Febuxostat and 100 mg of Metformin Hydrochloride was transferred to a 100 mL volumetric flask, dissolved with the mobile phase, sonicated, and filtered. The volume was made up to obtain 400 µg/mL of Febuxostat and 1000 µg/mL of Metformin Hydrochloride. A 1 mL aliquot of this solution was further diluted to 10 mL with the mobile phase to obtain 40 µg/mL of Febuxostat and 100 µg/mL of Metformin Hydrochloride.

Standard and Sample Assay Solutions

A 20 µL injection of both standard and sample solutions (containing 40 µg/mL of Febuxostat and 100 µg/mL of Metformin Hydrochloride) was injected in six replicates. Peak areas were recorded and the assay percentage was calculated by comparing the sample response with that of the standard. The results are presented in Table 3.5.

Chromatographic Conditions

Recommended Procedure

After systematic evaluation of experimental variables, the developed chromatographic method was optimized for simultaneous quantification of Febuxostat and Metformin Hydrochloride in bulk and tablet formulations

RESULTS AND DISCUSSION

1. Method Optimization

Different mobile phase compositions and chromatographic parameters were tested. Optimal resolution and peak symmetry for both drugs were obtained with the Inertsil C18 column and a mobile phase of 0.01 M ammonium dihydrogen phosphate buffer (pH 5) and acetonitrile (60:40, v/v) at a flow rate of 1 mL/min. UV scans in the 200–400 nm range confirmed 287 nm as the most suitable detection wavelength based on maximal absorbance. The retention times were approximately 2.303 min for Febuxostat and 4.105 min for Metformin Hydrochloride. Representative chromatograms for blank, standard, and sample solutions are illustrated in Figure 3.1

Figure 1.5 a simulation HPLC Chromatogram -two combination

2.1 Performance Calculations

System suitability studies were conducted to evaluate the performance of the proposed RP–HPLC method for the simultaneous estimation of FEB and MET in bulk and tablet formulations. All evaluated system suitability parameters complied with the recommended acceptance criteria, indicating excellent chromatographic performance and confirming the method suitability. The results are summarized in Table 3.6

Figure 1.5 b simulated HPLC Chromatogram of two drugs

2.2 Specificity

To assess the potential interference from common excipients and formulation additives present in combined dosage forms, a placebo solution was injected under the optimized chromatographic conditions. No interfering peaks were observed at the retention times of FEB or MET, confirming the specificity of the method. The representative placebo chromatogram is shown in Figure 3.2.

Table 1.6 System Suitability and Performance Parameters for FEB and MET

2.3 Linearity

Aliquots of 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mL of mixed standard stock solutions containing FEB (400 µg/mL) and MET (1000 µg/mL) were transferred into separate 10 mL volumetric flasks, and the volume was adjusted to mark with the mobile phase to obtain final concentrations of 10, 20, 30, 40, 50 and 60 µg/mL for FEB, and 25, 50, 75, 100, 125 and 150 µg/mL for MET. The prepared solutions were filtered through a 0.45 µm nylon membrane, and 20 µL of each concentration was injected in triplicate. A calibration plot of peak area versus concentration (µg/mL) was constructed for both analytes (Figure 3.3). Linear regression analysis was performed to determine the slope, intercept and correlation coefficient, and the results are presented in Table 3.7 and Table 3.8

3. RESULTS AND DISCUSSION

3.1 Method Optimisation

Several chromatographic conditions were explored during the optimisation of the RP-HPLC  method to achieve reliable separation of Febuxostat and Metformin Hydrochloride. Different mobile phase ratios and operational parameters were tested, and the best chromatographic performance—characterised by sharp peaks, good symmetry, and clear separation—was obtained using an Inertsil C18 column (100 mm × 4.6 mm, 5 µm). The selected mobile phase consisted of 0.01 M ammonium dihydrogen phosphate buffer (adjusted to pH 5.0 with orthophosphoric acid) and acetonitrile in a 60:40 (v/v) ratio, delivered at a flow rate of 1 mL/min, ensuring consistency and repeatability of the results.

Both analytes were scanned in the wavelength range of 200–400 nm using a PDA detector to identify an appropriate detection wavelength. Based on maximum absorbance and signal suitability, 287 nm was chosen for quantification, with peak area serving as the basis for analysis. Under the optimised conditions, Febuxostat eluted at 2.303 minutes and Metformin Hydrochloride at 4.105 minutes. Representative chromatograms of the blank, standard, and sample solutions are presented in Figure 3.1.

figure 1.6: Chromatogram of blank, standard, and sample solutions of Febuxostat and Metformin Hydrochloride.

3.2 Method Validation

The optimised RP-HPLC method was validated according to the ICH Q2(R1) guidelines to confirm its suitability for routine quality control analysis.

3.3 Performance Calculations

System suitability testing was performed to evaluate the reliability and efficiency of the analytical method for simultaneous estimation of FEB and MET in bulk and tablet formulations. Parameters such as retention time, theoretical plates, resolution, and tailing factor were within the acceptable ranges, confirming that the chromatographic system was functioning properly. The detailed system suitability results are summarised in Table 3.6.

3.4 Specificity

Specificity was assessed to ensure that excipients and formulation additives did not interfere with the detection of Febuxostat or Metformin Hydrochloride. A placebo solution containing all tablet excipients was injected under the same chromatographic conditions. No additional peaks or overlaps were observed, demonstrating that the method is specific to the active ingredients. The chromatogram for the placebo sample is shown in Figure 3.2

3.5 Linearity

To assess linearity, aliquots of 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mL of the combined standard stock solutions containing 400 µg/mL of FEB and 1000 µg/mL of MET were accurately transferred into a series of 10 mL volumetric flasks. The volumes were adjusted to the mark with the mobile phase to obtain final concentrations of 10, 20, 30, 40, 50, and 60 µg/mL for FEB, and 25, 50, 75, 100, 125, and 150 µg/mL for MET, respective shown in Figure 3.3, and the regression parameters are summarised in Table 3.7 and Table 3.8.

Table 3.7 Linearity of FEB and MET

Figure 3.3: Standard calibration curves of FEB and MET.

Figure 1.5 c simulated HPLC Chromatogram -two drug combination

1.8 Optical and regression parameters of FEB and MET

Linearity

Linearity was evaluated by preparing a series of dilutions from the mixed standard stock solutions of FEB (400 µg/mL) and MET (1000 µg/mL). Aliquots of 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mL were transferred into separate 10 mL volumetric flasks and diluted to volume with the mobile phase. These dilutions produced FEB concentrations of 10, 20, 30, 40, 50, and 60 µg/mL, along

Table 1.9 Results of accuracy studies of FEB

Table 1.10 Results of accuracy studies of MET

4. PRECISION

Repeatability was examined through both method precision and system precision.
For method precision, six replicate injections of a homogeneous sample solution containing 40 µg/mL of Febuxostat and 100 µg/mL of Metformin Hydrochloride, prepared from a single batch of Fabustat 40tablet powder, were introduced into the HPLC system. This test confirmed the consistency of the analytical procedure. The results are summarised in Table 3.11 and Table 3.12.

For system precision, six consecutive injections of a standard solution containing the same concentrations (40 µg/mL FEB and 100 µg/mL MET) were analysed to verify the performance and stability of the chromatographic system. The findings of system precision are presented in Table 3.13 and Table 3.14.

Intermediate precision (ruggedness) was assessed by analysing variations across different working conditions, including separate days, different analysts, and alternative instruments. Six injections of four sets of homogeneous sample solutions (40 µg/mL FEB and 100 µg/mL MET) from the same tablet batch were evaluated under these varying conditions. This ensured that the method remained reliable despite normal laboratory variations. The results of intermediate precision are provided in Table 3.15 and Table 3.16.

Reproducibility, which reflects precision between laboratories, was determined by injecting eight sets of six replicate preparations of the same concentration levels from the same batch of Fabustat 40 tablet powder. These results demonstrate the method’s robustness across different laboratory settings and are also listed in Table 3.15 and Table3.16

Table 2.1 Method precision of Febuxostat

Table 2.2 Method precision of Metformin Hydrochloride

2.3 System precision of Febuxostat

Table 2.4 System precision of Metformin Hydrochloride

Table 2.6 Ruggedness and reproducibility of Metformin Hydrochloride

6.5 Limit of Detection and Limit of Quantitation

The limit of detection (LOD) represents the lowest concentration of an analyte that can be detected, but not necessarily quantified, under the experimental conditions. The limit of quantitation (LOQ) refers to the lowest concentration that can be measured with acceptable accuracy and precision.

LOD and LOQ were calculated using the following standard equations, and the values obtained for Febuxostat and Metformin Hydrochloride are presented in

The robustness of the method was assessed by intentionally varying chromatographic conditions, including modifying the organic phase of the mobile phase by ±10% and adjusting the flow rate by ±0.1 mL/min. These deliberate changes did not produce significant alterations in the system suitability parameters.

The results, summarized in Table 2.7 and Table 2.8, confirm that the developed method maintains its performance despite minor variations in chromatographic conditions, demonstrating its reliability for routine analysis.

Table 2.7 Robustness data of Febuxostat

* mean of six determinations

Table 2.8 Robustness data of Metformin

6.8 Solution Stability Study

A solution stability assessment was performed to confirm that the sample solutions containing 40 µg/mL of Febuxostat and 100 µg/mL of Metformin Hydrochloride remained stable for up to 48 hours at room temperature. The study involved repeated injections (seven replicates) of a uniformly prepared sample solution derived from Fabustat 40tablet powder. Injections were carried out at predetermined intervals: 0, 8, 16, 24, 32, 40, and 48 hours.

Throughout the study period, the %RSD values for both Febuxostat and Metformin Hydrochloride were below 2%, acceptable consistency and confirming that no significant degradation occurred. These findings demonstrate that the prepared sample solutions maintain stability for up to 48 hours under ambient conditions.

The detailed results of the solution stability evaluation for both drugs are provided in Table 2.9 and Table 2.10.

Table 2.9 Solution stability of Febuxostat upto 48 hrs at room temperature

Table 2.10 Solution stability of Metformin Hydrochloride upto 48hrs at room temperature

6.9 Forced Degradation Studies of Febuxostat and Metformin Hydrochloride

Forced degradation studies were carried out to understand the degradation pathways of Febuxostat (FEB) and Metformin Hydrochloride (MET) under various stress conditions—including acidic and alkaline hydrolysis, oxidative stress, thermal degradation, and photolysis. These experiments also served to verify the stability-indicating capability of the developed RP-HPLC method. Each stress condition was applied using appropriate reagents: 1 N HCl for acid hydrolysis, 1 N NaOH for alkaline degradation, 3% hydrogen peroxide for oxidative stress, dry heat at 60°C for thermal degradation, and controlled light exposure providing a minimum of 1.2 million lux hours and not less than 200 watt-hours/m² UV energy (320–400 nm) for photolytic degradation. The results of these studies are presented in Table 3.21 and Table 3.22.

Thought for 5s

6.9.1 Acid Hydrolysis Degradation Study

Accurately weighed quantities of 40 mg FEB and 100 mg MET were transferred into a 100 mL volumetric flask. To initiate degradation, 1 mL of 2 N HCl was added, and the mixture was refluxed at 60°C for 30 minutes. After cooling, the solution was diluted to volume with the mobile phase to obtain concentrations of 400 µg/mL FEB and 1000 µg/mL MET.

A 1 mL aliquot of this degraded solution was further diluted to 10 mL with the mobile phase to yield final test concentrations of 40 µg/mL FEB and 100µg/mL MET. A 20 µL portion of this solution was injected into the HPLC system, and the resulting chromatogram (Figure 3.4) was used to evaluate the extent of degradation.

6.9.2 Preparation of Samples for LC–MS Analysis

For LC–MS characterization, the acid-degraded samples at concentrations of 40 µg/mL FEB and 100 µg/mL MET were injected directly into the system without neutralization. All samples were filtered through a 0.45 µm nylon membrane prior to analysis to remove any particulates.

6.9.3 LC–MS Conditions and Characterization of Degradation Products

The degradation samples were analyzed individually by LC–MS. Chromatographic separation was performed using an isocratic mobile phase containing 0.01 M ammonium dihydrogen phosphate buffer (pH adjusted to 5 with orthophosphoric acid) and acetonitrile (60:40, v/v) at a flow rate of 1 mL/min. Mass spectrometric analysis was conducted using atmospheric-pressure electrospray ionization (ESI) in positive ion mode.

This LC–MS method enabled identification of both the parent compounds and their major degradation products formed under aciMET conditions. FEB exhibited a retention time of 2.325 min, whereas MET eluted at 4.080 min. Additional degradation peaks appeared at 1.804 min and 2.995 min, corresponding to MET degradation products (Figure 3.5).

After heating in 2 N HCl at 60°C for 30 minutes, MET predominantly degraded into:

Figure 1.13  Mass spectrum of        2-(2-chlorophenyl) acetic acid (degradation product-2)

In acid degradation studies of Febuxostat and Metformin Hydrochloride, there is no degradation product was found from Febuxostat.

Figure 1.15 Mass spectrum of Febuxostat

6.10 Alkali Hydrolysis Degradation Studies

For alkaline degradation, accurately weighed quantities of 40 mg Febuxostat and 100 mg Metformin Hydrochloride were transferred into a 100 mL volumetric flask. To initiate degradation, 1 mL of 2 N NaOH was added, and the mixture was refluxed at 60°C for 30 minutes. After cooling, the solution was diluted to volume with the mobile phase to obtain concentrations of 400 µg/mL FEB and 1000 µg/mL MET.

A 1 mL aliquot of this solution was further diluted to 10 mL with the mobile phase to give final test concentrations of 40 µg/mL FEB and 100 µg/mL MET. A 20 µL portion of this diluted solution was injected into the HPLC system. The resulting chromatogram used to assess the degradation behavior of FEB and MET is shown in Figure 3.14.

6.10.1 Oxidative Degradation Studies

For oxidative stress testing, 40 mg FEB and 100 mg MET were weighed and placed into a 100 mL volumetric flask. To induce oxidation, 1 mL of 3% hydrogen peroxide (H₂O₂) was added, and the mixture was kept at 60°C for 30 minutes. The solution was then diluted to the mark with the mobile phase to achieve concentrations of 400 µg/mL FEB and 1000 µg/mL MET.

From this oxidized solution, 1 mL was transferred into a 10 mL flask and diluted to volume with the mobile phase to obtain 40 µg/mL FEB and 100 µg/mL MET. A 20 µL aliquot was injected into the HPLC system, and the chromatographic profile obtained is presented in Figure 3.15.

6.10.2 Thermal Degradation Studies

For thermal degradation, 40 mg FEB and 100 mg MET were accurately weighed, dissolved, and diluted to 100 mL with the mobile phase to produce concentrations of 400 µg/mL FEB and 1000 µg/mL MET. This solution was then placed in a hot-air oven maintained at 105°C for 6 hours to induce heat-related degradation.

After thermal treatment, a 1 mL aliquot was diluted to 10 mL using the mobile phase to obtain test concentrations of 40 µg/mL FEB and 100 µg/mL MET. A 20 µL sample was injected into the HPLC system, and the chromatogram illustrating thermal degradation is shown in Figure 3.16.

6.10.3 Photolytic Degradation Studies

For photostability assessment, 40 mg FEB and 100 mg MET were weighed and dissolved in the mobile phase, followed by dilution to 100 mL to yield 400 µg/mL FEB and 1000 µg/mL MET. The prepared solution was then exposed to intense light providing at least 1.2 million lux hours of visible illumination and a minimum of 200 watt-hours/m² of near-UV radiation (320–400 nm).

Following exposure, 1 mL of the photolyzed solution was diluted to 10 mL with the mobile phase to achieve final concentrations of 40 µg/mL FEB and 100 µg/mL MET. A 20 µL volume of the resulting solution was injected into the HPLC system, and the corresponding chromatogram is shown in Figure 3.17

Figure 2.2 Chromatogram of photolytic degradation studies for FEB and MET

Table 3.2 Results of stability indicating assay of FEB and MET

SUMMARY

A reverse-phase HPLC method was successfully developed and validated for the simultaneous quantification of Febuxostat and Metformin Hydrochloride in bulk drug samples and combined dosage forms, following ICH guidelines. The primary goal of the study was to design a rapid, accurate, and reliable analytical procedure suitable for routine quality control. The chromatographic conditions selected provided clear separation of both analytes without any co-eluting or interfering peaks.

During method development, multiple HPLC columns and mobile phase combinations were evaluated. Optimal chromatographic performance was achieved using an Inertsil C18 column (100 mm × 4.6 mm, 5 μm) on a Waters Alliance e2695 system equipped with a 2998 PDA detector. The mobile phase consisting of 0.01 M ammonium dihydrogen phosphate buffer (pH adjusted to 5 with orthophosphoric acid) and acetonitrile in a 60:40 (v/v) ratio was pumped at 1 mL/min. Detection was carried out at 287 nm, with quantification based on peak area.

Under these optimized conditions, Febuxostat and Metformin Hydrochloride eluted at 2.303 min and 4.105 min, respectively, with a resolution value of 7.52, confirming effective separation. The method demonstrated excellent linearity for Febuxostat (10–60 µg/mL) and Metformin Hydrochloride (25–150 µg/mL), both exhibiting correlation coefficients of 0.999. Recovery values ranged from 99.58–99.75% for Febuxostat and 99.53–99.74% for Metformin, confirming the accuracy of the method. The RSD values for accuracy, method precision, and system precision were all below 2%, indicating high precision and reproducibility. LOD values were 0.52 µg/mL for Febuxostat and 1.27 µg/mL for Metformin, while LOQ values were 1.57 µg/mL and 3.87 µg/mL, respectively.Stability testing revealed that both analytes were stable for up to 48 hours at room temperature. Forced degradation studies involving acid, alkali, oxidative, thermal, and photolytic conditions confirmed that Febuxostat exhibited lower susceptibility to acid and thermal stress, whereas Metformin showed greater sensitivity to acidic and photolytic conditions.

CONCLUSION

Deals with development and validation of stability indicating method for simultaneous estimation of Febuxostat and Metformin in bulk and tablet dosage forms using RP-HPLC as per ICH guidelines. A summary of this chapter are shown in Table 14.2.