Design, Optimization, and Justification of RP-HPLC process for the Resolvation of Azelnidipine in bulk plus in Pharmaceutical Components

 

M. Latha1, Sri Charitha Annam2, M. Durga Bhavani3, G. Ravi Kumar4, M. Subba Rao5

1Dept. of Basic Science and Humanities, Dhanekula Institute of Engineering and Technology,

Ganguru, Vijayawada - 521139, AP, India.

2Department of Humanities and Sciences, VNR Vignana Jyothi Institute of Engineering and Technology, Hyderabad. 500090, Telangana, India.

3Dept. of Chemistry, V.R. Siddhartha Engineering College, Kanuru, Vijayawada - 520 007, AP, India.

4Department of Chemistry, Govt. Degree College, Siddipet - 502103, Telangana (Dt), India.

5Dept. of Chemistry, University College of Sciences, Acharya Nagarjuna University,

Guntur - 522510, AP, India.

*Corresponding Author E-mail: mannamsrao@gmail.com

 

ABSTRACT:

As part of this study, a new sensitive, convenient, accurate and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of azelidipine in pharmaceutical and tablet formulations was developed and validated. The three variables chosen were methanol concentration, flow rate, and pH in the mobile phase. Separation was performed using an HPLC method with a UV detector and a Chromosil C18 (250mm x 450mm). Methanol was pumped at a flow rate of 1.0mL/min and adjusted to pH 4.0 with dilute OPA (60:40% v/v) and pH 4.0 with potassium dihydrogen phosphate buffer (pH 4.0), 255nm.

 

KEYWORDS: RP-HPLC method, Validation, Stability-indicating method.

 

 


INTRODUCTION: 

Azelnidipine1-3 [(3-[1-(diphenylmethyl) azetidin-3-yl] 5-propan-2-yl 2-amino-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate)] is a new dihydropyridine derivative used for the treatment of hypertension4-5.

 

Only four RP-HPLC techniques6-10 have been described for the assay of azelnidipine in dosage forms as far as we are aware. As a result, the author attempted to create an appropriate HPLC method in this agreement to identify azelnidipine in pure and pharmaceutical samples. This study describes the development and validation of a novel high-performance liquid chromatography (RP-HPLC) assay11-17 demonstrating the reversed-phase stability of azelidipine in pharmaceutical formulations. The developed RP-HPLC method was developed and validated with high linearity in a relatively short period of time16-19

 

MATERIAL AND METHODS:

Chemicals and Solvents:

Azelnidipine that was analytically pure (100%) and supplied with the proof of analysis came from Torrent Pharmaceutical Ltd. in Ahmedabad, India. Azelnidipine 16 mg market formulation under the trade name Azusa, produced by Ajantha pharma Ltd. in India, was purchased from a neighbourhood pharmacy. The water and methanol were acquired from Merck Specialties Private Limited in Mumbai, India, and were of HPLC grade.

 

Instrumental:

Azelnidipine's HPLC analysis was performed on a Waters LC system that was purchased from Waters Corporation in Ireland and included a 2695 pump, 2996 photodiode array detector, and Cosmosil 18(250mm 4.6 mm, 5µm) column. Using the waters Empower 2 software, the output signal was supervised and integrated. The sample injection volume was 20µL. Using a Loba ultrasonic bath sonicator, the mobile phase was degassed. The materials were weighed using a Denwar Analytical balance.

 

Mobile phase preparation:

A blend of buffer solution of potassium dihydrogen phosphate (pH 4.0) and methanol at a ratio of 40:60% v/v  was prepared and each was used as the mobile phase.

 

Diluent Preparation:

In the present study mobile phase is used as diluent for standard and sample solutions respectively.

 

Prepare a standard solution:

Azelnidipine was weighed precisely at 100.0mg and then put into a 100ml volumetric flask with 60mL of diluent before being dissolved using a sonicator. The solution was marked with methanol and brought to room temperature to achieve a concentration of 1.0mg/mL (stock solution). 20mL of the aforesaid solution (stock) was injected in triplicate into the HPLC system, and the peak areas were recorded. Transfer aliquots of the above solution (stock) into a series of separate 10ml volumetric flasks and dilute to volume with the diluent, accordingly, to yield concentrations ranging from 2.5 to 15µg/m               L

 

Marketed Sample (Dose Forms) Analysis:

Weighed and crushed into a fine powder of ten Azusa pills produced by Ajantha Pharma Ltd. in India and branded as containing 16mg of azelnidipine each. This powder of azelnidipine be deposited to a 100mL volumetric flask with 70mL of methanol after a quantity of the powder that was accurately weighed and accounted for roughly 50mg of the drug was added. The flask's contents were then correctly mixed, diluted with methanol to volume, and subjected to a 30-minute sonication at a controlled temperature. Filter the solution by passing it through a 0.45m membrane filter. It is possible to construct various working sample solutions with concentration ranges of 2.5-15µg/mL by diluting different aliquots of the aforementioned solution (stock) into a series of 10mL volumetric flasks using the same diluent. The protocol was followed, and 20µL of these solutions were added to the HPLC system in triplicate.

 

RESULTS AND DISCUSSION:

Method development:

By changing one parameter at a time while maintaining all other variables the same, a systematic research of the impact of various parameters was conducted in order to develop the approach. Choosing the right stationary and mobile phases and wave length is the first step in developing a method. The following research projects were carried out to achieve this.

 

Wavelength Detection:

On a UV spectrophotometer, the spectra of a 20µL solution of azelnidipine in methanol was independently recorded. It was possible to see the maximum wavelength of absorption. Azelnidipine's spectra showed that its greatest absorbance occurred at 255nm.

 

Selection of stationary phase:

Octadecyl columns of various sorts, configurations, and manufacturers have been used in preliminary development experiments. On Chromosil C18 (250mm x 450mm), the anticipated separation and peak shapes were finally obtained.

 

Selection of the mobile phase:

On a Cosmosil 18(250mm, 4.6mm, 5µm) column, a variety of solvent mixtures including methanol, water, and acetonitrile with or without various buffers were used as mobile phases in order to obtain a sharp peak, low tailing factor, and ideal separation of azelnidipine under isocratic conditions. The most effective of all the combinations was a buffer solution of potassium dihydrogen phosphate (pH 4.0) and methanol at a ratio of 40:60% v/v because the chromatographic peak was more clearly defined, resolved, and nearly tailing-free.

 

Flow Rate:

The mobilephase was run through a specified column at pour rates varying from 0.5 to 1.5mL/min to determine the effective flow rate for better separation. These investigations revealed that the optimal flow rate for the effective elution of the current analyte was 1.0mL/min. Under these conditions, good results were obtained in terms of shape of the peak, sensitivity and retention time for azelnidipine depicted in chromatogram (Figure 1a).

 

Technique Validation:

Using the following parameters, the devised RP-HPLC technique was thoroughly validated for the assay of azelnidipine.The number of theoretical plates, capacity factor, asymmetry factor, and tailing factor were all estimated. All values were found to be within the parameters (Table1). These results showed that the devised approach might be used for azelnidipine regular pharmaceutical analysis.

 

Selectivity:

The method's selectivity was assessed by injecting a placebo solution and a blank solution into the chromatographic apparatus under the aforementioned chromatographic settings, and then recording each chromatogram. (Figure 1b and c). Indicating that the diluent solution employed in test grounding does not interfere with the measurement of azelnidipine in tablets, the chromatogram of the blank solution (Figure 1b) revealed no peaks at the retention time of the azelnidipine peak. The placebo employed in sample preparation did not interfere with the assay of azelnidipine in formulations, as shown by the fact that the chromatogram of the placebo solution (Figure 1.c) illustrated no peaks during the retention period of the azelnidipine peak.

 

Linearity and Sensitivity:

The Linearity and sensitivity of the currently devised approach were assessed by analysing working standard solutions of azelnidipine at six different concentrations vary from 2.5 to 15.0µg/mL. Regression analysis was used to determine the calibration equation and correlation coefficient from the peak regions measured in relation to their azelnidipine concentration. The calibration plot and the regression data results for azelnidipine (Slope, intercept, and correlation coefficient [r2]) are publicized in Figure 2 and Table.2. These findings demonstrated high linearity and a strong connection between the peak areas and concentrations of azelnidipine. Azelnidipine's LOD was determined to be 0.0848µg/mL. Azelnidipine LOQ was discovered to be 0.3450µg/mL.

 

Precision:

Six replicate injections of the aforementioned standard mixture were used to assess the developed method's precision. When the %RSD value for the peak areas produced for azelnidipine was examined, it was discovered to be within 2.0%, validating the developed method's high level of precision. Table 2 presents an overview of the assay data from this research.

 

Accuracy:

Three replicate injections of each analyte concentration were used to determine the precision of the proposed method for quantifying 50%, 100%, and 150% concentrations of azelidipine. The percentage recovery was then calculated. The results are summarized in Table 3 and show that the proposed RP-HPLC method is very accurate for the determination. The percentage recovery obtained varied from 99.95% and 99.98% for tablet dosage form.

 

Robustness and Ruggedness:

The experimental circumstances were purposefully changed for the evaluation of robustness studies of the current proposed method, and the impact on retention time, peak asymmetry, and% assay was assessed by varying flow rate and column temperature. The impact of a different column, analyst, and system was also examined concurrently as part of the method's robustness.

 

The resolution of azelnidipine in the aforementioned two investigations was within the acceptable range, demonstrating the good resilience and roughness of the established approach. Tables 3 and 4 provide a summary of these research' findings.

 

Analysis of Pharmaceutic Formulations:

The estimation of azelnidipine in tablet dosage form was successfully accomplished using the newly devised approach. The assay results showed that the proposed RP-HPLC technique is specific for the analysis of azelnidipine with no interference from the excipients used to construct these tablets (99.98% for azelnidipine). (Table 5).

 


 


Figure 1: Typical HPLC chromatogram(s) showing (a) the peak of Azelnidipine (b) No Interference of Diluent for Azelnidipine and (c) No Interference of Placebo for Azelnidipine



Figure 2: linearity curve for Azelnidipine

 

Table:1 System suitability parameters for azelnidipine by the planned RP-HPLC method


Compound

Retention time

Theoretical Plates

Tailing factor

Peak area


Azelnidipine

1.543

2445

1.17

3753078


 

 


Table: 2: Linearity and Precision studies of Azelnidipine

Linearity of response for azelnidipine

% level (Approx.)

Concentration (µg/mL)

Average abs.

Results of Precision

S.No

Name

Area

25

2.5

1775485

1

Solution-1

3700666

50

5

2377432

2

Solution-2

3614019

75

7.5

2973219

3

Solution-3

3757254

100

10

3753078

4

Solution-4

3679361

125

12.5

4270740

5

Solution-5

3768674

150

15

4922169

6

Solution-6

3780279

 

Slope

25744

AVG *

3716709

 

Intercept

106312.13

STD DEV* 

64160.6

 

RSQ

0.9998

% RSD *

1.72

 

LOD (µg/mL)

848

 

 

 

 

 

LOQ (µg/mL)

0.345

 

 

 

 

*Average of six determinations

 

Table: 3: Results of Recovery Reading (Accuracy) and Robustness

Recovery reading (accuracy) Results

 

50% area

100% area

150% area

Robustness

Robust conditions

Azelnidipine

Injection-1

2364282

3636078

4922169

Theoretical Plates

RT

Peak Area

Injection-2

2343217

3598456

4923456

Flow Rate

0.9 ml/min

2698

1.543

2318990

Injection-3

2432652

3612056

4900876

1.2 ml/min

2540

1.403

1924718

Avg.*

2380050

3615530

4915500

Temp

at 33oC

2486

1.55

2140965

Amount recuperated*

1.96

9.97

14.95

at 37oC

2562

1.52

2103232

%Recovery*

99.96

99.98

99.95

 

 

*Average of six determinations

 


Table: 4: Results of ruggedness studies of Azelnidipine

S. No.

Name

Analyst-1

Analyst -2

Area

Area

1

Inoculation-1

3700666

3637802

2

Inoculation-2

3614019

3614213

3

Inoculation-3

3757254

3745754

4

Inoculation-4

3679361

3679361

5

Inoculation-5

3768674

3735684

6

Inoculation-6

3780279

3711679

Avg.* 

3716709

3687416

Std Dev* 

64160.56

53304.5

% RSD* 

1.72

1.44

*Average of six determinations

 

Table. 5: Analysis of marketed tablets of azelnidipine

Drug

Label claim

Quantity found*

%Assay

AZUSA

16mg

15.98 mg

99.98

*Average of three determinations

 

CONCLUSION:

In conclusion, a reversed-phase HPLC technique that is precise, quick, easy, sensitive, reproducible, and uses UV detection was described for the assessment of azelnidipine in both bulk and tablet dosage form. When the created method was validated against the previously described methods listed below, it showed good levels of sensitivity, selectivity, repeatability, and recovery17-20. The established method presented a great deal of usefulness for future research capabilities in terms of shorter runtime, with less precision and high accuracy, based on the citations mentioned aboveof azelnidipine respectively.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Pharma Train, Hyderabad and Ajantha Pharma Ltd., India for providing gift sample of Azelnidipine for the present research and the Department of Chemistry, University Science College, Acharya Nagarjuna University, Guntur for providing all necessary lab facilities to carry out the present research.

 

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Received on 22.08.2023            Modified on 18.11.2023

Accepted on 30.01.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(5):2175-2179.

DOI: 10.52711/0974-360X.2024.00342