Qbd Based RP-HPLC Method Development and Validation for Simultaneous Estimation of Amlodipine Besylate and Lisinopril Dihydrate in Bulk and Pharmaceutical Dosage Form

The objective of this experiment was to develop and validate a simple, robust, and accurate QbD based Reverse-Phase High-Performance Liquid Chromatography method for Simultaneous estimation of Amlodipine besylate and Lisinopril dihydrate in bulk and Pharmaceutical Dosage form. A box-Behnken design was employed for optimizing the mobile phase, flow rate and pH of buffer, the optimized chromatographic conditions were Phosphate buffer: Methanol (25: 75 v/v), pH of buffer: 6.5 and flow rate: 1mL/min. Furthermore formulation injected and observed that the additives do not interfere with the peak of Amlodipine besylate and Lisinopril dehydrate. Both drugs are well resolved and Retention times were found to be 2.332 min and 3.584 min respectively. Linearity was observed in the concentration range of 10 μg to 50 μg/mL (r2=0.999). The accuracy range was 99.75 to 100.04%. Intra-day and Inter-day precision was found to be less than 2% RSD. The proposed method was useful for the best analysis of Amlodipine besylate and Lisinopril dihydrate in Bulk, pharmaceutical dosage forms and was successfully applied to routine analysis. Original Research Article Babar et al.; JPRI, 33(43A): 143-164, 2021; Article no.JPRI.73764 144

The objective of this experiment was to develop and validate a simple, robust, and accurate QbD based Reverse-Phase High-Performance Liquid Chromatography method for Simultaneous estimation of Amlodipine besylate and Lisinopril dihydrate in bulk and Pharmaceutical Dosage form.

Chemicals and Reagents
Cipla Pharmaceutical Ltd, India, sent a gift sample of lisinopril (LSN) and amlodipine (AMD) reference standards. AMLOPRESS-L (Cipla), a pharmaceutical preparation containing 5 mg of lisinopril and 5 mg of amlodipine comparable to amlodipine besylate, was acquired from a local pharmacy store. Merck Lie Sciences Pvt. Ltd., Mumbai, supplied HPLC grade methanol.

Instrumentation
The proposed work was carried out on Isocratic HPLC (Shimadzu) with LC20AD, PU2080 pump, UV 2075 plus detector, and Phenomenex C18 column (5 µm particle size) was used. The software used was Borwin.

Preliminary analysis of drug
The colour and texture of Amlodipine besylate (AMB) and Lisinopril Dihydrates (LSD) were matched to known drug bank features. Amlodipine is slightly soluble in water and only slightly soluble in ethanol, whereas lisinopril dehydrates is little soluble in water, only slightly soluble in methanol, and almost completely insoluble in ethanol. The solutions were subjected to UV examination by scanning them at 200-400 nm.

Chromatographic condition
Shimadzu series LC 2010 A chromatographic system was used for the analysis (pump Quaternary system). Separation was performed on a Kromasil C8 (4.6mm x 250mm, 5 particle size) column at 30°C, with a flow rate of 1.00mL per min. and an isocratic mobile phase composed of Buffer & Acetonitrile in a 60:40 ratio. Orthophosphoric acid was used to raise the pH to 3.6. The concentrations of lisinopril and amlodipine were determined using a UV detection method at 215nm, with an injection volume of 20L and a run period of 7 minutes.

Selection of detection wavelength
Further dilutions of the standard stock solution were made with water and scanned over the range of 200-400 nm, with the spectra being overlain. Amlodipine and lisinopril were found to have significant absorbance at 215 nm.

Preparation mobile phase
90 ml of HPLC grade Methanol was mixed with 10 ml of water in a 90:10 v/v ratio. Trimethylamine and orthophosphoric acid were used to modify the pH to 4.5, 5.5, and 6.5. The solution was filtered through a 0.45 membrane filter and then sonicated for 10 minutes in a sonicator bath.

Preparation of standard solution
Weigh correctly 50 mg of Lisinopril and 50 mg of Amlodipine besylate, transfer to a 100 mL volumetric flask, dissolve in 70 mL of mobile phase, and build volume up to the mark with mobile phase to obtain a stock solution containing 500g/ml of Lisinopril and 500g/ml of Amlodipine besylate. The final solution was prepared by pouring 5 mL of this solution into a 100 mL volumetric flask and filling it with mobile phase to obtain 50g/mL of Lisinopril and 50g/mL of Amlodipine besylate, respectively. Figure 1 depicts a typical chromatogram of conventional Lisinopril and Amlodipine.

Preparation of sample solution
For the assay, 20 tablets of Lisinopril labelled as having 5mg and 5 mg of Amlodipine besylate, together with excipients, were precisely weighed and ground into a fine powder. Take an accurate weight of powder equivalent to 5 mg of lisinopril and 5 mg of amlodipine and transfer to a 100 ml volumetric flask, then add 50 ml of mobile phase and sonicate for 10 minutes. Cool it down and increase the volume with mobile phase. Filter a portion of this solution using a 0.45m membrane syringe filter. The final solution was made by putting 5 ml of this filtered solution into a 100 ml volumetric flask and increasing the volume by adding mobile phase to obtain 50g/ml of Lisinopril and 50g/ml of Amlodipine besylate, respectively. Figure 2.0 depicts a typical chromatogram of the samples Lisinopril and Amlodipine.
For Content uniformity, one tablet was placed in to each of ten 100 ml volumetric flask. Approximately 70 ml of mobile phase was added to each volumetric flask &sonicate till tablets were dispersed in the solution. Cool the resultant solutions and make volume up to the mark with the mobile phase. Shake the solution well for uniform distribution. Filtered a portion of solution by using 0.45µm membrane syringe filter & then filtrate was injected for analysis.
A figure 1 & 2 represents the typical sample chromatogram of Lisinopril and Amlodipine respectively.

Design of Experiment
Box-Behnken designs are response surface designs that are specifically designed to require only three levels, denoted as -1, 0 and +1. Box-Behnken designs are offered for three to twentyone factors. They are created by merging twolevel factorial and incomplete block designs. This approach generates designs with desirable statistical features while also requiring a fraction of the experiments required for a three-level factorial. The quadratic model is adequate because there are just three layers. For the majority of these designs, blocking choices are also available. This design may also include categorical factors. The number of runs generated will be doubled by the number of categorical factor level combinations.
Dependent factors were selected as mobile phase, pH of aqueous phase and flow rate and Independent factors were selected as retention time, peak area, theoretical plates and peak asymmetry. The C18 column is used for proposed method.

Following mobile phases selected
 Phosphate buffer: Methanol  Water: Methanol  Water : Acetonitrile The Box-Behnken design produced 12 runs (Table 1) with varying pH, mobile phase percentage, and flow rate. For each mobile phase, the same method was followed. The total number of runs during the three mobile stages was 36. By maximising desired parameters and decreasing undesired ones, optimization involves finding an alternative with the most cost effective or greatest feasible performance under the given restrictions. Maximization, on the other hand, involves attempting to achieve the highest or maximum result or outcome without regard for cost or expense. Water: Acetonitrile Only one peak appeared (Amlodipine) another peak is very small (Lisinopril) Dissatisfactory

Fig. 3. 3D Diagram of Desirability Value
This process begins by creating a desirability function for each individual response. The scale of the individual desirability function spans from i=0 (totally unwanted reaction) to I =1 (entirely desired answer). The experiment was chosen based on the highest attractiveness value. As a result, the first experiment with desirability one (i=1) was chosen for method optimization.

Effect of independent variables on retention time (X):
After applying experimental design, suggested Response Surface Linear Model was found to be significant with model Values of "Prob > F" (p-value) less than 0.0500 indicate model terms are significant. In this case A and B are significant model terms.   Model terms are important when the "Prob > F" (p-value) is less than 0.0500. In this scenario, B denotes important model terms.

3.1.6Effect of independent variables on theoretical plates (Z)
Following     Model terms are important when the "Prob > F" (p-value) is less than 0.0500. In this scenario, A value is significant in terms of model terms.  Calibration curves: Pipette out suitable aliquots from each standard stock solution into a series of 10 ml volumetric flasks for each medication. The capacity was filled to the mark with mobile phase to produce a set of solutions with concentrations ranging from 10, 20, 30, 40, and 50 g/ml for each medication. Separate triplicate dilutions of each medication concentration were made. From these duplicate solutions, 10 l injections of each drug concentration were injected separately into the RP-HPLC apparatus and chromatographed under the conditions stated above. Both medications were evaluated using a UV detector set to 215nm. Peak areas were measured for each peak and plotted against concentrations to create the standard calibration curves.

ANALYSIS OF THE MARKETED FORMULATION
Twenty tablets were weighed and finely ground into powder. The tablet powder containing 5 mg of amlodipine and 5 mg of lisinopril was transferred to a 100 ml volumetric flask and dissolved in mobile phase for 30 minutes in an ultra sonicator. Finally, mobile phase was used to bring the volume up to the required level. The solution was passed through a 0.45 m membrane filter paper before being filtered. This solution was diluted further with mobile phase, and a standard stock solution of AMD was added to produce a mixed sample solution comprising 5 mg amlodipine and 5 mg lisinopril.
Under the chromatographic conditions mentioned above, a total of 20 l of sample solution was injected into the sample injector five times. At 215 nm, the area of each peak was measured.
The peak area of AMD and LSN was used to calculate the amount of each drug present in the sample (n = 5). A typical chromatogram of AMD and LSN in tablet formulation (Fig.1).

Method Validation
The proposed RP-HPLC method was validated as per ICH guidelines.

Linearity
Several aliquots of standard AML and LIS solutions were placed in various 10 ml volumetric flasks and the capacity was filled with mobile phase to achieve a final concentration of AML and LIS of 10-50 g/ml, respectively. The UV-Vis detector at 215 nm was used for the evaluation, and the peak area for each peak was recorded.

Specificity
The RP-HPLC method's specificity was determined by comparing the chromatograms of mixed standards and sample solutions. Retention time (t R), resolution (R S), and tailing factor (T f) were all computed. There was a strong association between the results of mixed standards and sample solutions as shown in Table. 16.

Precision
Six repetitions of the sample made from commercial tablets were injected to determine method precision, and the assay was calculated to measure the repeatability of retention periods and peak area of standard and sample. The method's precision was validated by utilising a tablet stock solution. The intraday and interday precision tests were conducted by repeating the assay six times on the same day for intraday precision and on different days for interday precision studies. The findings of this study are as follows (Table. 17):

Recovery
The approach's accuracy was determined through recovery trials at three levels (80%, 100%, and 120%) using the usual addition method. The percentage of analyte recovered was used to calculate the accuracy. The proposed method's accuracy was verified in accordance with ICH norms. For AML, a tablet powder equivalent to 5 mg AML was placed in three separate 100 ml volumetric flasks, and then 8 mg (80%), 10 mg (100%), and 12 mg (120%) of standard AML were added to each volumetric flask. The mobile phase [phosphate buffer solution: methanol (75:25 v/v)] was then poured to each volumetric flask and sonicated for 5 minutes. The solutions were then filtered, and 1 ml of the filtrate from each was placed in separate 10 ml volumetric flasks and diluted with mobile phase to the desired concentration. The solutions were injected into the chromatographic apparatus in triplicate, and the peak area was calculated to produce the percent recovery and standard deviation. The same approach was followed with Lisinopril dehydrate (Table. 18).

Robustness
The proposed method's robustness was tested by altering the solvent ratio in the mobile phase, flow rate, and wavelength range. The sample solutions were introduced into the chromatographic apparatus in 10 l increments. Peak area was analysed, as well as its standard deviation and percent RSD (Table. 19).

Limit of detection and Limit of quantification (LOD, LOQ)
The suggested method's LOD and LOQ were obtained by gradually injecting lower amounts of the standard solutions under the specified chromatographic conditions. L.O.Q. = 10(SD/S) L.O.D. = 3.3(SD/S) Where SD denotes the standard deviation of the answer and S denotes the slope of the calibration curve. The slope S can be calculated using the analyte calibration curve.

CONCLUSION
With a short analytical time, the new approach provides good resolution between Amlodipine besylate and Lisinopril dehydrate. The approach is simple, accurate, fast, and precise, and it can be used for regular drug analysis without requiring any sophisticated sample preparation.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, I was not funded by the producing company rather it was funded by personal efforts of the authors.