Development And Validation of HPTLC Method For Simultaneous Estimation of Azelnidipine and Metoprolol in Combined Dosage Form

Hypertension, commonly referred to as high blood pressure, is a medical condition characterized by elevated pressure in the arteries. In stage 2 hypertension, the systolic pressure reaches 140 mm Hg or higher, or the diastolic pressure is 90 mm Hg or more. A blood pressure reading exceeding 180/120 mm Hg is classified as a hypertensive emergency and necessitates immediate medical intervention.

Several factors contribute to the development of stage 2 hypertension: -

• Obesity
• Poor Diet
• Sedentary Lifestyle
• Excessive Alcohol Consumption
• Smoking
• Genetic Predisposition
• Sleep Apnea
• Diabetes
• Kidney Disorders
• Hormonal Imbalances
• Pregnancy-Related Complications.

Drug Profiles: -

Azelnidipine

Azelnidipine is a calcium channel blocker belonging to the dihydropyridine class. It has a slow onset of action and provides a sustained reduction in blood pressure with minimal impact on heart rate. It is also being investigated for use in managing conditions post-ischemic stroke.

Dosage Forms: Tablets (e.g., 8 mg, 16 mg)

Molecular Formula: C33H34N4O

Class: Calcium Channel Blocker

Oral Bioavailability: Approximately 15–30%

Mechanism of Action: Azelnidipine blocks voltage-gated calcium channels, particularly the L-type, preventing calcium influx into vascular smooth muscle cells, which promotes vasodilation.

Half-life: Approximately 16–24 hours, supporting once-daily dosing.

Metoprolol

Metoprolol is a selective beta-1 adrenergic receptor antagonist primarily used to treat cardiovascular diseases. It reduces the heart rate, cardiac output, and blood pressure by inhibiting beta-1 receptors in the heart.

Brand Names: Lopressor, Toprol-XL
Dosage Forms: Immediate and extended-release tablets, and intravenous injection

Molecular Formula: C15H25NO3

Oral Bioavailability: Around 50%

Half-life: 3 to 7 hours

Mechanism of Action: By blocking beta-1 adrenergic receptors, Metoprolol decreases myocardial oxygen demand and improves hemodynamic stability.

Structure of Azelnidipine

MATERIAL AND METHOD

Table:1 Chemical and Reagents Used

Table:2 Instrument specification for HPTLC

Table:3 Instrument Specifications for Weighing Balance

Table:4 Instrument specification for melting point apparatus

Table:5 Instrument Specification for UV double beam Spectrophotometer

Table:6 Procurement of pure APIs

Identification Of Drugs:

By Melting point method: -

Melting point of Azelnidipine & Metoprolol hydrochloride has been determined. The melting points of the compounds were taken by open capillary method.

Table:7 Melting Point of Drugs

By FTIR Spectra Method: -

Table:7 IR Interpretation Spectra for Azelnidipine

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Fig 2: IR Spectra of Metoprolol

Table 8: IR Interpretation Spectra for Metoprolol

By solubility method: -

The solubility of Azelnidipine & Metoprolol practically determined separately by taking 100 mg of both the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes. Solubility data for each study was observed and recorded in Table 9.

Table 9: Solubility Table

Table 10: Solubility Data for Azelnidipine & Metoprolol

Development and Optimization of HPTLC Method

Selection of Wavelength

Drug concentrations were prepared using Water and scanned in the UV-region of 200 – 400 nm and the spectra were recorded to get maximum of analytes in mobile phase. AZE and MET were scanned in UV in which drugs show reasonably good response at 235nm. But during selection, observed that TLC plate was readable in UV scanning So that 235 nm was considered.

Chromatographic Conditions

Mobile phase was selected on the basis of best separation. So, number of trials were tried for the selection of MP to effectively separate the drugs on silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness). And with the best separation of drug bands Chloroform: Ethyl acetate: Methanol in the ratio of 6.5: 3.5: 0.1 was selected as a Mobile phase.

Selection of Stationary Phase

For HPTLC Method, various columns are available but based on literature survey silica gel aluminum plate 60F254 (20 X 10 cm with 250 µm thickness) was selected over the other columns.

Preparation of Solution: -

Preparation of Mobile Phase

HPTLC method was followed by isocratic elution technique. Mobile phase comprised of Chloroform: Ethyl acetate: Methanol in the ratio of 6.5: 3.5: 0.1 ratio because it elutes both drugs peak efficiently in short time with satisfactory resolution, tailing factor and theoretical plates.

Preparation of Standard Stock Solution A: (Azelnidipine Stock Solution)

Accurately weighed quantity of Azelnidipine 100 mg was transferred into 100 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 1000 μg/mL. Withdraw 16 ml from Stock Solution and make up to 10 ml with to get 1600 μg/ml.

Preparation of Standard Stock Solution B: (Metoprolol stock solution)

Accurately weighed quantity of Metoprolol 100 mg was transferred into 100 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 1000 μg/mL. Withdraw 25 ml from Stock Solution and make up to 10 ml with to get 2500 μg/mL

Chromatographic Condition

Method Of Validation

Specificity

The analytical method for specificity was evaluated by injecting the following solutions. Diluent was prepared and inject into the HPTLC system in triplicate. Sample solution was prepared with appropriate levels of excipients as a placebo sample and inject into the HPTLC system in triplicate for all the dosage strengths. Placebo was prepared by mixing all excipients without active ingredients. Standard and sample solutions were prepared for assay (100% Conc.) and inject into the HPTLC system in triplicate.

Linearity and Range

Analysis was performed on Precoated silica gel aluminum Plate 60F–254 (20× 10 cm with 250 µm thickness) (E. Merck) pre-washed with methanol and then dried for 30 minutes at 50°C. From the Stock A, 1 µL, 2 µL, 3 µL, 4 µL and 5 µL aliquots were spotted on the TLC plate from 160 µg/mL AZE and 250 µg/mL MET standard stock solution under nitrogen stream using Dosage Applicator, AS30 win to final concentration range 160 – 800 ng/band and 250 – 1250 ng/band for AZE and MET, respectively.

Repeatability

Prepared standard working solution of mixtures having concentration of Azelnidipine (160 μg/ml) and Metoprolol (250 μg/ml) were injected at volume of 20 μL into column by employing optimized chromatographic conditions. Each standard mixture was injected 5 time and peak area was monitored. Each concentration was monitored for repeatability by RSD.

 Intra-day and Inter-day Precision

Method precision was determined by performing intraday and inter day precision. Mixture that represents overall range (Azelnidipine +Metoprolol = 80+125, 160+250 and 240+375 µg/ml) were analyzed on same day at different time interval for intraday precision. Mixture that represents overall range (Azelnidipine +Metoprolol = 80+125, 160+250 and 240+375 µg/ml) were analyzed on different days for inter-day precision.

System Suitability Parameters

Solution of Azelnidipine + Metoprolol (160+250 μg.ml-1) was injected 3 times for determination of System suitability parameters which includes Retention time (Rt), Tailing factor (Tf), Resolution (Rs) and number of theoretical plates. System suitability parameters   for selected concentration were determined by C.V.

Accuracy

Accuracy was determined by performing recovery studies by spiking different concentration of drug to pre-analyzed sample solution of 160 ng/band for AZE and 250 ng/band of MET. To pre-analyzed sample solution, a known amount of working standard solution of AZE and MET were added in 10 mL volumetric flask and made up to mark with different levels i.e., 80%, 100% and 120%. The experiment was performed

Limit of detection and Limit of Quantification

The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation of intercept of calibration curve. The limit of detection (LOD) and the limit of quantification (LOQ):

LOQ = 10 σ/s and LOD = 3.3 σ/s

Where, σ = the standard deviation of the response.

S = the slope of the calibration curve

Robustness

Robustness of proposed method was evaluated by deliberately changing parameters (± 0.2) like Saturation time and Mobile phase composition with the working stock solution (160 ng/band of AZE and 250 ng/band MET).

RESULT AND DISCUSSION

Selection of Wavelength

To determine wavelength for measurement, standard spectra of Azelnidipine & Metoprolol were scanned between 200-400 nm against diluents. Absorbance maxima of Azelnidipine & Metoprolol have detected at 235 nm. Chromatogram was taken at 235 nm, both drugs give good peak height and shape. So,235 nm was selected for Simultaneous estimation of Azelnidipine & Metoprolol in their formulation.

Various Combinations of Solvents were tried for the effective separation of the LEN and EVE on silica gel aluminum plate 60F-254 (20 × 10 cm with 250 µm thickness) (E. Merck). Results of Trials and final selected mobile phase with the discussion shown below.

Trail 1: -

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase: Toluene: Methanol in the ratio of (6.5: 3.5 v/v)

Observations: No Separation Band Observed.

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Fig 4: Trial 1: Azelnidipine & Metoprolol Toluene: Methanol in the ratio of (6.5: 3.5 v/v)

Trail 2: -

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase: Toluene: Methanol in the ratio of (6:4 v/v)

Observations: No Separation Band Observed.

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Fig 5: Trial 2: Azelnidipine & Metoprolol Toluene: Methanol in the ratio of (6:4 v/v)

Trail 3: -

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase: Toluene: Methanol in the ratio of (5:5 v/v)

Observations: No Separation band involved.
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    Fig 6: Trial 3: Azelnidipine & Metoprolol Toluene: Methanol the ratio of (5:5 v/v)

Trail 4: -

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase: Ethyl acetate: Methanol in the ratio of (6.5:3.5 v/v)

Observations: No Separation band involved.

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Fig 7: Trial 3: Azelnidipine & Metoprolol Ethyl acetate: Methanol in the ratio of (6.5:3.5 v/v)

Trail 5: -

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase Ethyl acetate: Methanol in the ratio of (4:6 v/v)

Observations: Spots were not separated.

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Fig 8: Trial 5: Azelnidipine & Metoprolol of Ethyl acetate: Methanol in the ratio of (4:6 v/v)

Trail 6: -

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase: Ethyl acetate: Methanol in the ratio of (7:3 v/v)

Observations: Minor Separation observed.
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 Fig 9: Trial 6: Azelnidipine & Metoprolol of Ethyl acetate: Methanol in the ratio of (7:3 v/v)

Trail 7: -

Column:

Mobile Phase Chloroform: Ethyl acetate: Methanol in the ratio of (7:3: 0.1 v/v)

Observations: Adjacent spots observed.

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Fig 10: Trial 7: Azelnidipine & Metoprolol of Chloroform: Ethyl acetate: Methanol in the ratio of (7:3: 0.1 v/v)

Trail 8: -

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase: Chloroform: Ethyl acetate: Methanol in the ratio of (6:4:0.1 v/v)

Observations: Minor Separation observed.

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Fig 11: Trial 8: Azelnidipine & Metoprolol of Chloroform: Ethyl acetate: Methanol in the ratio Trail 9: -

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase Chloroform: Ethyl acetate: Methanol in the ratio of (7:3 0.1 v/v)

Observations: Good separation but broad peaks observed (6:4: 0.1 v/v)

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Fig 12: Trial 9.: Chromatogram of Azelnidipine & Metoprolol Chloroform: Ethyl acetate: Methanol in the    ratio of (7:3: 0.1 v/v)

Chromatographic conditions for optimized mobile phase trial

Column: Silica gel aluminum plate 60F-254 (20 X 10 cm with 250 µm thickness)

Mobile Phase: Chloroform: Ethyl acetate: Methanol in the ratio of (6.5:3.5: 0.1 v/v)

Detection: 292 nm

Saturation Time: 25 minutes

Rf Value: AZE: 0.182. MET: 0.252.

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Fig 13: Optimized mobile phase optimized chromatogram of Std. Azelnidipine: 0.182, Metoprolol: 0.252

Column: -Typically, not used in TLC/HPTLC. If HPLC: C18 column (e.g., 250 mm × 4.6 mm, 5 µm)

Mobile Phase: - Chloroform: Ethyl acetate: Methanol (6.5: 3.5: 0.1 v/v)

Detection: - UV detection at 240 nm (or another wavelength suitable for both drugs)

Rf Value: - Azelnidipine: ~0.6Metoprolol: ~0.30 (values may vary depending on plate and conditions)

Saturation Time: - 20 minutes (for TLC chamber saturation)

Observation   : Well-resolved, sharp spots for Azelnidipine and Metoprolol; no interference from excipients.

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Fig 14: blank Azelnidipine: Metoprolol Chloroform: Ethyl acetate: Methanol in the ratio of (6.5:3.5: 0.1 v/v)

Observation

Introduction Of Method Validation Parameter

Specificity

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Linearity

For the purpose of linearity, accurately weighed amount of Azelnidipine (10 mg), and Metoprolol (10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Azelnidipine, and 100 µg/ml of Metoprolol. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 80+125, 128+200, 160+250, 192+300 and 240+375 µg/ml of Azelnidipine and Metoprolol respectively.

Table 11: Linearity data for Azelnidipine and Metoprolol

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Fig 15: Overlain Linearity Spectra of Azelnidipine and Metoprolol

Table 12: Linearity results for Azelnidipine and Metoprolol

Precision

Repeatability 

The data for repeatability for Azelnidipine and Metoprolol is shown in table 13. The % R.S.D For Repeatability data was found to be 1.10 % for LID and 1.45 % for DIL.

Inter-day precision 

The data for inter day precision for Azelnidipine and Metoprolol is shown in table 14. The % R.S.D for intraday precision was found to be 0.93-1.54 % for Azelnidipine and 0.39-0.85% for Metoprolol.

Table 14: Inter-day precision data for estimation of Azelnidipine and Metoprolol

Intra -day precision 

The data for intra-day precision for Azelnidipine and Metoprolol is shown in table 15. The % R.S.D for intraday precision was found to be 0.10-1.38 % for Azelnidipine and 0.09-1.26 % for Metoprolol.

Table 15: Intra-day precision data for estimation of Azelnidipine and Metoprolol

Accuracy

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Azelnidipine & Metoprolol was found to be 99.48- 99.98% and 99.33-100.59 % respectively. The results are shown in table.9.6-9.9.

Table 17: Recovery data for Metoprolol

LOD and LOQ

The limit of detection (LOD) and Limit of Quantification (LOQ) was found to be as per below:

Selectivity 

There is no interference in the mixture.

Robustness 

The method is found to be robust as the results were not significantly affected by slight variation in Mobile Phase Composition and flow rate of mobile phase. The results are shown in table19. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

Analysis of marketed product 

The proposed method was successfully applied to analysis of the commercially available tablet formulation.  The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Summary of Method Validation