A Review on Development and Validation of RP-HPLC Method for Dapagliflozin and Bisoprolol in Bulk and Pharmaceutical Dosage Form

Abstract

Dapagliflozin is a medication for type 2 diabetes and heart failure, known for lowering blood sugar levels, aiding weight loss, and offering cardiovascular and renal benefits. Its mechanism involves preventing the kidneys from reabsorbing glucose, thus enhancing urinary glucose excretion. In contrast, Bisoprolol is used mainly for high blood pressure and heart conditions; while it provides significant benefits such as blood pressure reduction and improved heart function, some users experience adverse effects like fatigue, dizziness, and shortness of breath. Key future research areas include minimizing environmental impact by developing methods that utilize environmentally friendly or solvent-free techniques, adhering to green chemistry principles. The application of Quality by Design (QbD) principles is anticipated to improve method robustness and control, leading to a better understanding of performance variations. Although conventional techniques like UV spectrophotometry and RP-HPLC are prevalent, there is an urgent need to integrate more sensitive and specific methods, such as LC-MS/MS, particularly useful for ultra-trace analysis and impurity profiling in bioanalytical studies involving biological samples like plasma and urine. Current research emphasizes in vitro validation; future work should pivot towards bioanalytical method development applicable to pharmacokinetic studies, helping to evaluate the real-world performance of dapagliflozin and bisoprolol. Additionally, advancements in automated sample preparation and high-throughput analysis will enhance efficiency in routine quality control by reducing time and costs. More rigorous research is necessary to develop stability-indicating methods that accurately identify, characterize, and quantify potential degradation products, especially for bisoprolol which significantly degrades in harsh conditions. Research on the reproducibility of these analytical methods across various quality control laboratories is essential to ensure compliance with global regulatory standards, particularly those from the FDA and ICH, facilitating the international commercialization of these drugs.

Keywords

Introduction

Dapagliflozin (Figure 1) is a medication utilized for managing type 2 diabetes and heart failure, recognized for its efficacy in lowering blood sugar levels, aiding in weight reduction, and providing several cardiovascular and renal advantages. The drug's mechanism involves inhibiting the reabsorption of glucose in the kidneys, leading to increased urinary glucose excretion. While it is generally well-accepted, there are potential risks associated with its use, notably an increased incidence of genital and urinary tract infections due to its glucose-related effects. Additionally, there's a rarely observed but significant risk of diabetic ketoacidosis (1, 2).

Figure 1: Dapagliflozin

In terms of efficacy, dapagliflozin effectively reduces blood glucose and HbA1c levels along with body weight, benefits that are often maintained over a prolonged duration. For patients suffering from symptomatic heart failure with reduced ejection fraction (HFrEF), dapagliflozin demonstrates a notable reduction in the risk of either cardiovascular death or the worsening of heart failure, which remains effective irrespective of the patient's existing diabetes condition or other heart failure treatments. When considering kidney protection, research indicates that dapagliflozin can enhance renal function and lower serum creatinine levels in individuals with diabetic nephropathy. Moreover, the medication is characterized by a low risk of inducing hypoglycemia, particularly when prescribed alone or alongside Metformin (3, 4). On the downside, the most commonly observed side effects include genital mycotic infections and urinary tract infections, which are directly linked to its pharmacological action of elevating urinary glucose levels. The potential for dehydration and a decrease in blood pressure represents further risks, caused by the drug's tendency to lead to loss of salt and water. These effects can manifest as dizziness or headache, especially when combined with other antihypertensive treatments. Importantly, dapagliflozin is contraindicated for patients with moderate to severe kidney impairment (5, 6). Overall, dapagliflozin stands as a well-tolerated option and can contribute positively to a patients' treatment regimen. Nevertheless, ongoing research is essential to solidify the long-term safety and efficacy profiles for diverse patient populations and comorbid conditions, emphasizing the importance of consulting healthcare professionals to weigh the individualized risks and benefits before initiating therapy.

Bisoprolol (Figure 2), a medication prescribed primarily for high blood pressure and heart conditions, elicits a mixed response among users. While many report substantial benefits, such as effective blood pressure reduction and improved heart function, others experience negative side effects like fatigue, dizziness, and shortness of breath. Clinical evidence underscores bisoprolol's efficacy, especially in chronic heart failure, where it enhances survival rates and decreases hospitalization when combined with ACE inhibitors and diuretics (7).

Figure 2: Bisoprolol

Efficacy and benefits are particularly noted in patients with heart failure, as clinical trials indicate that bisoprolol significantly bolsters survival rates and minimizes the necessity for hospitalization. Additionally, users often note its ability to lower blood pressure effectively, with favorable results typically manifesting within days. Bisoprolol is also known for its capacity to reduce heart rates. However, potential side effects should not be overlooked. Commonly reported adverse effects include fatigue, dizziness, and shortness of breath. Some individuals have noted less frequent side effects such as dry mouth, weight gain, and diminished libido. In rare instances, severe side effects could arise, warranting immediate medical attention if symptoms such as swelling, breathing difficulties, confusion, or fainting occur (8, 9). Important considerations are essential when using bisoprolol. Individual experiences with side effects can vary drastically in severity and nature. Moreover, users may find a difference in their recovery time following exercise and should be cautious with activities like driving, particularly when initiating treatment or adjusting dosages due to the risk of dizziness (10). Additionally, bisoprolol may interact with other medications, potentially heightening the chances of dizziness and lightheadedness (11). Consultation with a healthcare provider is crucial for anyone experiencing side effects while on bisoprolol, as adjustments in dosage or a change of medication might be necessary to better manage symptoms (12).

2. Review on Development and Validation of Rp-Hplc Method for Dapagliflozin and Bisoprolol in Bulk and Pharmaceutical Dosage Form

Ninama H et al., 2025 developed and validated of a precise and reliable HPTLC method for the concurrent analysis of Dapagliflozin propanediol monohydrate and bisoprolol fumarate in pharmaceutical formulations. adhering to the Q2(R2) guidelines established by the International Council for Harmonization. Chromatographic separation was achieved on HPTLC silica gel 60 F??? plates (10.0 cm × 10.0 cm, 0.20 mm) with a mobile phase of Chloroform: Toluene: Methanol: Ammonia (1:2:6:0.1 v/v/v). Detection was performed at 224 nm. Retention factor (Rf) values for Dapagliflozin propanediol monohydrate and bisoprolol fumarate were 0.22 ± 0.003 and 0.63 ± 0.006, respectively. The method showed excellent linearity with correlation coefficients of 0.9995 and 0.9991 for Dapagliflozin (200–1200 ng/band) and bisoprolol fumarate (100–600 ng/band), respectively. The reported method demonstrated precision in both intraday and interday analyses, with the percentage relative standard deviation of the peak area remaining below 2%. and recovery studies confirmed high accuracy with values ranging from 98.21 to 100.08% for dapagliflozin and 99.19–100.15% for bisoprolol fumarate. Forced degradation studies revealed that dapagliflozin was more susceptible to acidic and oxidative hydrolysis compared to bisoprolol fumarate, with baseline-resolved degradation products observed in all stress conditions (13).

Valiya G et al., 2025 developed and validated a simple reverse-phase LC method was for simultaneous quantification of dapagliflozin propanediol monohydrate and bisoprolol fumarate in bulk and marketed formulation. The optimized chromatographic condition includes Inertsil ODS 3 V (250 mm × 4.6 mm, 5 µm) as the stationary phase, gradient elution of mobile phase A containing 26 mM KH2PO4 containing 0.4 mL/L triethylamine (pH 4.2 adjusted with dilute orthophosphoric acid) and acetonitrile in ratio of 90:10 v/v and mobile phase B containing 26 mM KH2PO4 containing 0.4 mL/L triethylamine (pH 4.2 adjusted with dilute orthophosphoric acid) and acetonitrile in ratio of 25:75 v/v with flow rate of 1 mL/min. The analytical wavelength for detection of both drugs was 223 nm. The forced degradation study was also carried out to estimate the stability of drugs in bulk and formulation. The stress condition applied were acid, alkali, oxidative, thermal, and light. Dapagliflozin propanediol monohydrate was found to be stable and bisoprolol fumarate was degrading under oxidative and light conditions and remaining stable in other conditions applied. The proposed method was found to be linear in the concentration range of 2–24 µg/mL and 1–12 µg/mL with R2 value of 0.9998 and 0.9984 for dapagliflozin propanediol monohydrate and bisoprolol fumarate, respectively. The optimized method was validated as per ICH Q2 (R2) guidelines and was found to be selective, specific, precise, accurate, and robust for quantification of both drugs (14).

Siddique W eta l., 2025 focused on developing and validating Metformin and Dapagliflozin in combination by using high-pressure liquid Chromatography (RP-HPLC). The validation of the method was followed as per the guidelines provided by the International Conference on Harmonization (ICH) and United States Pharmacopeia (USP). Separation of both drugs takes place in less than 4 min. This separation takes place using Phosphate buffer (pH 6.8) and acetonitrile in a 45:55 (v/v) ratio at a 1.0 mL min−1 flow rate. Furthermore, studies on both drugs were conducted by using the bulk and pharmaceutical dosage forms (Tablets). The developed method was accurate as drug recoveries in both cases of Metformin, and Dapagliflozin ranged between (100.8, 99.6, 98.8%) to (100.8, 99.3, and 101.5%) respectively having a concentration range of solutions between 70, 100 and 130 μg mL−1 dilution. The recommended method for simultaneous quantification of Metformin and Dapagliflozin was established and validated and no excipient interactions were found (15).

Kavana DC et al., 2025 validated techniques including UV spectrophotometry, reversed-phase high-performance liquid chromatography (RP-HPLC), and high-performance thin-layer chromatography (HPTLC) have emerged for analyzing dapagliflozin alone and in combination with other antidiabetic agents. UV spectrophotometric methods demonstrate simplicity and cost-effectiveness, utilizing various solvents and detecting wavelengths between 210-280 nm. RP-HPLC methods employ C18 columns with diverse mobile phase compositions, achieving efficient separation with retention times typically under 6 minutes. Method validation parameters encompass linearity, precision, accuracy, specificity, and robustness following International Conference on Harmonisation (ICH) guidelines. Reported linear ranges vary from 5-100 μg/mL for UV methods and 10-250 μg/mL for HPLC methods, with correlation coefficients exceeding 0.99. Recovery studies indicate accuracies between 98-102%, while precision studies show relative standard deviations below 2%. Several stability-indicating assays confirm method selectivity and ability to detect degradation products (16).

Shukla S eta l., 2025 focuses on the green innovations in analytical methodologies for metoprolol determination. It examines alternative techniques that minimize environmental impact, including eco-friendly solvents, reagentless detection methods, and energy-efficient approaches. Key green technologies such as chromatography, spectrometry, and electrochemical techniques are discussed, with an emphasis on their potential to replace or enhance traditional methods. It introduces Analytical GREEnness Metric Approach, Green Analytical Procedure Index, Blue Applicability Grade Index, Carbon Footprint Reduction Index and Click analytical chemistry tools as critical frameworks for assessing the sustainability and efficiency of green analytical approaches. These tools provide a standardized approach to evaluate various green methods based on factors such as waste reduction, solvent use, energy consumption, and overall environmental footprint. Additionally, the review examines how these frameworks can guide the development of new green technologies in metoprolol analysis. The study concludes with recommendations for future research, emphasizing the importance of integrating green chemistry principles into pharmaceutical analysis and quality control while minimizing environmental impacts (17).

Hadole PM et al., 2025  aimed to develop a normal-phase TLC densitometry/ high-performance thin-layer chromatography (HPTLC) protocol, assisted with an Automatic Development Chamber 2 (ADC 2) for the efficient and robust pharmaceutical quantification of a thrombopoietin receptor agonist Eltrombopag Olamine (ELTO) using experimental design. TLC densitometry scans were performed at 399 nm in reflectance mode optimizing the retention of ELTO at an Rf value of 0.47 ± 0.02. The application of ADC 2 during the development minimized the impact of temperature and humidity. The BBD, supported with response surface methodology (RSM), was used to assess the method’s operable design region for increased robustness. The established linearity range was from 250 to 1500 ng per spot, with a regression coefficient (r2) of 0.9998. The developed protocol was validated according to Q2(R2) recommendations of the International Council for Harmonization (ICH). NP-HPTLC is unique, efficient, precise, reproducible, and robust for the rapid quantification of ELTO. The method also uses minimal amount of organic solvents compared to well-known techniques such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). The protocol is timesaving, allowing the simultaneous analysis of multiple samples, making it an effective alternative for determining ELTO in pharmaceutical formulations (18).

Kant P eta l., 2024 emphasized the vital role of process validation in assuring that manufactured products consistently meet predefined specifications and quality attributes throughout the manufacturing process. It highlights that thorough validation studies are crucial, evaluating essential parameters such as accuracy, sensitivity, specificity, and repeatability of the approved and documented test procedures of manufacturers. Validation is underlined as a fundamental aspect of quality assurance, essential for compliance with regulatory requirements while upholding the highest standards of quality. The article stresses the importance of utilizing objective measures, robust statistical tools, and comprehensive analyses within the validation process, which are critical for understanding and managing the inherent variability present in manufacturing processes. By prioritizing the detection and control of variability, process validation assures consistent quality and productivity over the product's life cycle. The review explores various approaches, processes, and critical monitoring steps required during the manufacturing phase, reinforcing the necessity for pharmaceutical validation. The study articulates how these practices contribute to maintaining product integrity, reliability, and compliance within the pharmaceutical industry, ultimately enhancing patient safety and therapeutic efficacy (19).

Habeeb MR eta l., 2024 aimed to establish a validated, eco-friendly, and sustainable approach utilizing a fluorescence detector coupled with high-performance liquid chromatography for quantifying the antihyperglycemic agent dapagliflozin (DAPA), in human plasma. This method employed a C18 Microsorb MV (4.5 × 250 mm, 5 μm [particle size]) column at 40°C, with 40:60% v/v isocratic elution of acetonitrile and (0.1%) orthophosphoric acid as the mobile phase at 1 mL/min flow rate. DAPA and the internal standard demonstrated their greatest response by performing excitation at 225 nm (λex) and recording chromatograms at an emission wavelength (λem) equal to 305 nm. The presented approach demonstrated high linearity between 50 and 2000 ng/mL and full adherence to the guidelines of the US Food and Drug Administration regarding the validation of bioanalytical methods. The described technique was effectively used for quantification of DAPA in human plasma samples from a healthy male participant who received a tablet of 10 mg DAPA. Analytical Eco-Scale, Analytical GREEnness metric, and the recently created ChlorTox Scale were utilized for greenness assessment. Additionally, the “Red, Green, and Blue 12” model was used in whiteness evaluation (20).

Kang SJ et al., 2023 derived an optimal drug release formulation with human clinical bioequivalence in developing a sitagliptin phosphate monohydrate-dapagliflozin propanediol hydrate fixed-dose combination (FDC) tablet as a treatment for type 2 diabetes mellitus. However, in the quality evaluation of the double-layer tablets, the hardness was 12–14 kilopond, the friability was 0.2%, and the disintegration was within 3 min. In addition, the stability test revealed that the double-layer tablet was stable for 9 months under room temperature storage conditions and 6 months under accelerated storage conditions. In the drug release test, only the FDC double-layer tablet showed the optimal drug release pattern that satisfied each drug release rate. In addition, the FDC double-layer tablet showed a high dissolution rate of over 80% in the form of immediate-release tablets within 30 min in a pH 6.8 dissolution solution. In the human clinical trial, we co-administered a single dose of a sitagliptin phosphate monohydrate-dapagliflozin propanediol hydrate FDC double-layered tablet and the reference drug (Forxiga®, Januvia®) in healthy adult volunteers. This study showed clinically equivalent results in the stability and pharmacodynamic characteristics between the two groups (21).

3. Future Scope

The future scope for research and review regarding the analytical method development and validation of dapagliflozin and bisoprolol in both bulk and pharmaceutical dosage forms is extensive and multifaceted. Key areas identified for future exploration include the following: Current analytical techniques frequently incorporate organic solvents such as acetonitrile, which pose environmental concerns. Future research should aim to develop and validate methods employing more environmentally friendly solvents or even solvent-free techniques. This shift would align with principles of green chemistry and reduce the ecological impact of analytical processes. The adaptation of QbD principles in analytical method development is anticipated to enhance robustness and ensure systematic control throughout the analytical process. This approach will facilitate a better understanding of variations that may affect method performance. Traditional methods such as UV spectrophotometry and RP-HPLC are commonly used; however, there is a significant opportunity to adopt more sensitive and specific detection techniques, including LC-MS/MS. These advanced methods are particularly beneficial for ultra-trace analysis, impurity profiling, and in-depth bioanalytical studies utilizing biological matrices like plasma and urine. Current research predominantly emphasizes in vitro validation methods. Future investigations should focus on developing and validating bioanalytical methods applicable in preclinical and clinical pharmacokinetic studies. This research is crucial for assessing the in vivo performance and bioavailability of the combined formulation of dapagliflozin and bisoprolol. The exploration of automated sample preparation and high-throughput analysis systems is important. Such advancements could significantly diminish analysis time and costs, enhancing the efficiency of routine quality control measures. While stability-indicating methods exist, there is a need for more extensive research utilizing advanced techniques to identify, characterize, and quantify potential degradation products and related substances. Particular attention should be given to substances that degrade significantly under certain conditions, such as bisoprolol, which demonstrates considerable degradation in acidic and oxidative environments. It will be vital to conduct research on the transferability of developed analytical methods across different quality control laboratories. Ensuring that these methods align with global regulatory standards, such as those set forth by the FDA and ICH, will be critical for the international commercialization of dapagliflozin and bisoprolol.

CONCLUSION

Analytical methods for the simultaneous estimation of dapagliflozin and bisoprolol in bulk and pharmaceutical dosage forms have been developed and validated, demonstrating accuracy, precision, linearity, robustness, and specificity. These methods, including RP-HPLC and HPTLC, are stability-indicating and suitable for routine quality control analysis according to ICH Q2(R1) guidelines.

CONFLICT OF INTEREST

None

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