INTRODUCTION:

Benidipine Hydrochloride is a long-acting dihydropyridine calcium channel blocker (CCB) extensively used to manage hypertension and angina pectoris. Unlike conventional CCBs, Benidipine uniquely blocks L-, T-, and N-type calcium channels, contributing to its strong antihypertensive and cardioprotective effects. Initially developed in Japan, it has gained widespread approval due to its prolonged duration of action, superior vascular benefits, and favorable safety profile. Its lipophilic nature allows for sustained calcium channel inhibition, making it an effective once-daily therapy for patients with cardiovascular disorders¹. Chemically, Benidipine Hydrochloride is classified as a lipophilic dihydropyridine derivative with a prolonged duration of action, allowing once-daily dosing. Its high lipophilicity enables prolonged interaction with calcium channels, contributing to sustained blood pressure reduction. These properties make it particularly suitable for patients with cardiovascular and renal comorbidities, offering comprehensive protection against hypertensive complications².

1. Mechanism of Action:

Benidipine Hydrochloride's therapeutic effects stem from its unique ability to simultaneously inhibit L-, T-, and N-type calcium channels, leading to a multifaceted approach to controlling hypertension and providing vascular protection. This triple-channel blockade reduces calcium influx into vascular smooth muscle cells, leading to sustained vasodilation, decreased arterial stiffness, and overall cardiovascular stability. Additionally, the inhibition of T- and N-type channels offers benefits in modulating autonomic nervous system activity, further reducing sympathetic overactivity and enhancing its cardioprotective effects³.

These properties make Benidipine a potent antihypertensive agent that reduces blood pressure through sustained vasodilation and reduces vascular resistance. Its nephroprotective effects are particularly significant. Benidipine helps maintain kidney function by lowering glomerular pressure, decreasing proteinuria, and promoting natriuresis, making it beneficial for hypertensive patients with chronic kidney disease or diabetes-related nephropathy⁴.

2. Pharmacokinetics and Dynamics:

Benidipine is well absorbed following oral administration, with peak plasma concentrations occurring within 2-4hours. Its bioavailability is relatively low due to extensive first-pass metabolism in the liver, but its high lipophilicity aids in prolonged retention within cellular membranes, allowing for sustained drug action⁵.

Benidipine exhibits high plasma protein binding (>95%), primarily to albumin, contributing to its prolonged action duration. This high binding affinity reduces its free active drug concentration in plasma but ensures a slow and sustained release into target tissues, enhancing its antihypertensive efficacy⁶. Enhances endothelial function by increasing nitric oxide availability, which promotes vasodilation and reduces oxidative stress. It also improves arterial compliance, decreasing vascular stiffness and minimizing the progression of atherosclerosis. Furthermore, Benidipine exhibits antioxidative properties that protect blood vessels from oxidative damage, thereby lowering the risk of cardiovascular events such as myocardial infarction and stroke⁷.

4. Analytical Techniques for Benidipine Hydrochloride

Analytical methods are crucial in the quality control, pharmacokinetic assessment, and stability evaluation of Benidipine Hydrochloride. These methods ensure the drug's purity, potency, and compliance with regulatory standards. Various chromatographic and spectroscopic techniques are employed to quantify the drug in bulk and pharmaceutical formulations, detect impurities, and study degradation pathways⁸.

4.1 Chromatographic Methods:

4.1.1 High-Performance Liquid Chromatography (HPLC):

High-Performance Liquid Chromatography (HPLC) is a widely used analytical technique for quantifying and quality control of Benidipine Hydrochloride in bulk drugs and pharmaceutical formulations. HPLC employs a high-pressure liquid mobile phase to pass samples through a stationary phase (typically a silica-based column), enabling efficient separation of the drug from its impurities and degradation products⁹.Commonly used detection methods include UV-visible spectrophotometry and mass spectrometry, which allow precise measurement of Benidipine concentrations in various biological and pharmaceutical matrices¹⁰. HPLC is widely used to quantify Benidipine in bulk and pharmaceutical formulations. It ensures high sensitivity, accuracy, and reproducibility¹¹.

4.1.2 Ultra-Performance Liquid Chromatography (UPLC):

Ultra-Performance Liquid Chromatography (UPLC) is an advanced chromatographic technique offering significantly higher resolution, speed, and sensitivity than conventional HPLC. UPLC operates at much higher pressures (up to 15,000 psi) and uses smaller particle-sized columns (typically <2μm), which improves separation efficiency and reduces analysis time¹². UPLC offers higher resolution and faster analysis times than conventional HPLC, making it useful for pharmacokinetic studies.

4.1.3 Reverse-Phase Liquid Chromatography (RP-HPLC):

Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) is a widely utilized analytical technique for separating and quantifying Benidipine and its related impurities. It employs a non-polar stationary phase (typically a C18 or C8 column) and a polar mobile phase composed of aqueous buffers and organic solvents (such as acetonitrile or methanol). Its ability to provide sharp peak resolution and efficient separation makes it an indispensable technique in the pharmaceutical analysis of Benidipine¹³.

4.1.4 High-Performance Thin Layer Chromatography (HPTLC):

High-Performance Thin Layer Chromatography (HPTLC) is an advanced chromatographic technique that enhances the traditional thin-layer chromatography (TLC) method by offering improved separation efficiency, reproducibility, and automation capabilities. It is widely used for the identification, quantification, and purity assessment of Benidipine in bulk drugs and pharmaceutical formulations¹⁴. To optimize HPTLC performance, key factors such as mobile phase composition, plate preconditioning, sample application techniques, and detection wavelengths must be carefully controlled. Coupling HPTLC with densitometry or mass spectrometry further enhances its analytical capabilities, allowing for highly sensitive and specific quantification of Benidipine and its metabolites¹⁵.

4.1.5 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS):

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is an advanced analytical technique widely used for the highly sensitive and specific detection of Benidipine in complex biological and pharmaceutical matrices. This method combines the high separation efficiency of liquid chromatography (LC) with the superior identification and quantification capabilities of tandem mass spectrometry (MS/MS), making it an indispensable tool in pharmacokinetic, bioavailability, and drug metabolism studies¹⁶.

4.2 Spectroscopic Methods:

4.2.1 UV-Visible Spectroscopy:

UV-visible spectroscopy is a widely utilized analytical technique for quantitatively estimating Benidipine based on its strong absorption in the ultraviolet (UV) region. This method relies on the interaction of UV-visible light with the electronic transitions in the molecular structure of Benidipine, allowing for the precise determination of its concentration in pharmaceutical formulations and biological samples¹⁷. UV-visible spectroscopy is commonly employed with other analytical techniques, such as HPLC and LC-MS, to provide complementary information about the drug’s stability, degradation products, and formulation characteristics. Advanced UV-visible spectrophotometers equipped with diode array detection (DAD) further enhance the specificity and sensitivity of the analysis, enabling real-time monitoring of Benidipine in various matrices¹⁸.

4.2.2 Fourier Transform Infrared Spectroscopy (FTIR):

Fourier Transform Infrared Spectroscopy (FTIR) is a powerful analytical technique used to identify functional groups, detect molecular interactions, and confirm the structural integrity of Benidipine. This method is based on the absorption of infrared radiation by molecular bonds, producing a unique spectral fingerprint that can be used for compound identification and purity assessment¹⁹.

4.2.3 Nuclear Magnetic Resonance Spectroscopy (NMR):

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful analytical technique used for detailed structural elucidation, impurity profiling, and interaction studies of Benidipine. It operates by detecting the magnetic properties of atomic nuclei, primarily hydrogen (¹H-NMR) and carbon (¹³C-NMR), within the molecular framework of the compound (20). Advanced NMR techniques, such as 2D-NMR (COSY, HSQC, and HMBC), further enhance its ability to determine molecular interactions, including drug-excipient compatibility in formulation development. Additionally, NMR plays a crucial role in stability studies, helping to monitor chemical changes under stress conditions²¹.

5. Analytical Method Development:

Analytical method development for Benidipine is critical in ensuring accurate, precise, and reproducible analysis of the drug in bulk and pharmaceutical formulations. This process involves selecting the most suitable analytical techniques to separate, identify, and quantify Benidipine and its impurities. Effective method development improves drug formulation consistency, stability assessment, and regulatory compliance¹⁷. These strategies ensure method robustness, reproducibility, and compliance with pharmaceutical regulations, enabling reliable quality control and pharmacokinetic analysis of Benidipine²².

6. Validation of Analytical Methods:

Analytical method validation is a critical process that ensures the accuracy, precision, specificity, and robustness of analytical techniques used for Benidipine Hydrochloride. Regulatory agencies such as the International Council for Harmonisation (ICH) and the U.S. Food and Drug Administration (FDA) mandate stringent validation protocols to establish the reliability of analytical methods in pharmaceutical quality control and regulatory submissions²³. Validation ensures the reliability and accuracy of the analytical method. Accuracy is evaluated through recovery studies, while precision includes intra-day (repeatability) and inter-day (intermediate) consistency. Specificity confirms no interference from excipients or impurities. Linearity and range establish a direct relationship between concentration and response. LOD and LOQ define the lowest detectable and quantifiable concentrations. Robustness checks method stability under slight variations in conditions. System suitability testing (SST) ensures the system's performance through checks like retention time and peak symmetry²⁴.

7. Literature Review:

7.1. Hasan I. Shaikhmulani and co-workers published in the year 2022 for the purpose of RP-HPLC method development and validation for the simultaneous estimation of Benidipine Hydrochloride and Chlorthalidone in pharmaceutical dosage form²⁵.

Parameters	Description
Column Name	C-18 Agilent Zorbax Bonus – RP (250 × 4.6 mm, 5 µm)
Mobile Phase	Methanol and 0.1% Orthophosphoric acid (45:55 v/v)
Flow Rate	1.0 mL/min
Detection	Photodiode Array Detector at 238 nm
Retention Time	Benidipine Hydrochloride: 1.09 min, Chlorthalidone: 3.52 min
Precision	<2% RSD
Calibration Range	Benidipine Hydrochloride: 32–48 µg/mL, Chlorthalidone: 100–150 µg/mL
Limit of Detection (LOD)	Benidipine Hydrochloride: 2.05 µg/mL, Chlorthalidone: 7.71 µg/mL
Limit of Quantification (LOQ)	Benidipine Hydrochloride: 6.22 µg/mL, Chlorthalidone: 23.38 µg/mL
Validation	Conducted as per ICH guidelines for specificity, linearity, accuracy, precision, robustness, LOD, and LOQ

7.2. Majan Naim and co-workers published in 2018 for the purpose of Stability-indicating RP-HPLC method development and validation for the simultaneous estimation of Telmisartan and Benidipine Hydrochloride in pharmaceutical dosage form²⁶.

Parameters	Description
Column Name	Thermo Scientific C18 Hypersil BDS (250 × 4.6 mm, 5 µm)
Mobile Phase	Phosphate buffer (pH 4.0) and Methanol (50:50 v/v)
Flow Rate	1.0 mL/min
Detection	Photodiode Array Detector at 210 nm
Retention Time	Telmisartan: 3.273 min, Benidipine Hydrochloride: 4.807 min
Precision	<2% RSD
Calibration Range	Telmisartan: 20–60 µg/mL, Benidipine Hydrochloride: 2–6 µg/mL

7.3 Tabrej Mujawar and co-workers published in the year 2022 for the purpose of RP-HPLC method development and validation for the simultaneous estimation of Metoprolol and Benidipine in bulk and tablet dosage forms²⁷.

Parameters	Description
Column Name	C18 column (250 × 4.6 mm, 5 µm)
Mobile Phase	Acetonitrile and 0.1% Orthophosphoric acid in water (45:55 v/v) (pH 3.0)
Flow Rate	1.0 mL/min
Detection	UV Detector at 230 nm
Retention Time	Metoprolol: 2.983 min, Benidipine: 7.383 min
Precision	<2% RSD
Calibration Range	Metoprolol: 50–250 µg/mL, Benidipine: 4–20 µg/mL

7.4 Manoj M. Kadam and co-workers published in the year 2023 for the purpose of RP-HPLC method development and validation for the estimation of Benidipine in bulk and tablet dosage form²⁸.

Parameters	Description
Column Name	Agilent C18 reverse-phase column (100 × 4.6 mm, 2.5 µm)
Mobile Phase	Methanol: Water (40:60 v/v) (pH 5.0)
Flow Rate	1.0 mL/min
Detection	UV Detector at 237 nm
Retention Time	Benidipine: 3.33 min
Precision	<2% RSD
Calibration Range	10–50 µg/mL (r² = 0.9997)
Limit of Detection (LOD)	0.03935 µg/mL
Limit of Quantification (LOQ)	0.1192 µg/mL
Validation	Conducted as per ICH guidelines for accuracy, precision, robustness, LOD, LOQ, and recovery

7.5 Payal G. Jain and co-workers published in the year 2018 for the purpose of RP-HPLC method development and validation for the simultaneous estimation of Benidipine Hydrochloride and Telmisartan in tablet dosage form²⁹.

Parameters	Description
Column Name	Inertsil ODS C18 column (150 × 4.6mm, 5µm)
Mobile Phase	0.05M Potassium Dihydrogen Phosphate Buffer (pH 4.5, adjusted with 1% OPA) and Acetonitrile (40:60 v/v)
Flow Rate	1.0 mL/min
Detection	PDA Detector at 267 nm
Retention Time	Benidipine Hydrochloride: 2.977 min, Telmisartan: 5.167 min
Precision	<2% RSD
Calibration Range	Benidipine Hydrochloride: 2–6 µg/mL, Telmisartan: 20–60 µg/mL
Validation	Conducted as per ICH guidelines for specificity, linearity, accuracy, precision, robustness, LOD, and LOQ

7.6 Naveenarani Dharuman and co-workers published in the year 2023 for the purpose of Environmentally benign RP-HPLC method for the simultaneous estimation of Benidipine Hydrochloride and Chlorthalidone using Analytical Quality by Design (AQbD)³⁰.

Parameters	Description
Column Name	Agilent Eclipse Plus C18 (250 × 4.6mm, 5µm)
Mobile Phase	Ethanol and Potassium Dihydrogen Orthophosphate buffer (pH 3.5) (40:60 v/v)
Flow Rate	1.0mL/min
Detection	UV Detector at 230nm
Retention Time	Benidipine: 5.1 min, Chlorthalidone: 3.1 min
Precision	<2% RSD
Calibration Range	Benidipine: 3.2–4.8µg/mL, Chlorthalidone: 5.0–7.5µg/mL
Validation	Conducted as per ICH guidelines with green chemistry principles ensuring eco-friendliness

7.7 Patel Advaita and co-workers published in the year 2019 for the purpose of RP-HPLC method development and validation for the simultaneous estimation of Benidipine Hydrochloride and Metoprolol Succinate in tablet dosage form³¹.

Parameters	Description
Column Name	C18 Hypersil BDS (250 × 4.6 mm, 5 µm)
Mobile Phase	Potassium Dihydrogen Phosphate Buffer (pH 4.0) and Methanol (65:35 v/v)
Flow Rate	1.0mL/min
Detection	UV Detector at 269nm
Retention Time	Metoprolol Succinate: 3.4 min, Benidipine Hydrochloride: 5.9min
Precision	<2% RSD
Calibration Range	Metoprolol Succinate: 25–75µg/mL, Benidipine Hydrochloride: 4–12µg/mL
Validation	Conducted as per ICH guidelines for specificity, linearity, accuracy, precision, robustness, LOD, and LOQ

7.8 Kadali Jagadeesh and co-workers published in the year 2020 for the purpose of Stability-indicating HPLC method development and validation for the simultaneous estimation of Benidipine and Chlorthalidone in bulk and tablet dosage forms³².

Parameters	Description
Column Name	Kromasil C18 (250 × 4.6mm, 5µm)
Mobile Phase	Methanol and 0.1M Dipotassium Hydrogen Phosphate Buffer (pH 4.5) (40:60 v/v)
Flow Rate	1.0 mL/min
Detection	Photodiode Array (PDA) Detector at 260nm
Retention Time	Benidipine: 4.573min, Chlorthalidone: 6.422 min
Precision	Benidipine: 0.106% RSD, Chlorthalidone: 0.031% RSD
Calibration Range	Benidipine: 2–6µg/mL (R² = 0.9997), Chlorthalidone: 6.25–18.75µg/mL (R² = 0.9998)
Validation	Conducted as per ICH guidelines for specificity, linearity, accuracy, precision, robustness, LOD, and LOQ

7.9 Bhoomi D. Patel and co-workers published in the year 2019 for the purpose of RP-HPLC method development and validation for the simultaneous estimation of Benidipine Hydrochloride, Telmisartan, and Chlorthalidone in a combined tablet dosage form³³.

Parameters	Description
Column Name	C18 Hypersil BDS (25cm × 0.46cm)
Mobile Phase	Buffer (pH 3.0) and Methanol (50:50 v/v)
Flow Rate	1.0mL/min
Detection	UV Detector at 230nm
Retention Time	Chlorthalidone: 4.887 min, Benidipine: 6.690 min, Telmisartan: 8.813min
Precision	<2% RSD
Calibration Range	Benidipine: 2–6µg/mL, Telmisartan: 20–60 µg/mL, Chlorthalidone: 6.25–18.75µg/mL
Validation	Conducted as per ICH guidelines for specificity, linearity, accuracy, precision, robustness, LOD, and LOQ

8. CONCLUSION:

The accurate analysis of Benidipine Hydrochloride is crucial for ensuring drug efficacy, safety, and regulatory adherence. Chromatographic techniques such as HPLC, UPLC, and LC-MS/MS, along with spectroscopic methods like UV-visible, FTIR, and NMR spectroscopy, serve as essential tools for quality control, pharmacokinetic profiling, and stability assessment. These analytical methods help detect impurities and degradation products and ensure batch-to-batch consistency in pharmaceutical formulations. Moreover, emerging trends in automation, green chemistry, and nanotechnology-based analytical approaches promise to enhance Benidipine analysis's sensitivity, efficiency, and environmental sustainability, paving the way for more precise and eco-friendly drug monitoring strategies.

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Received on 15.05.2025 Revised on 24.06.2025

Accepted on 26.07.2025 Published on 08.10.2025

Available online from October 15, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(4):328-333.

DOI: 10.52711/2231-5675.2025.00051

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.