Assessment of Aliskiren Hemifumarate and Amlodipine Besylate in combined Tablet Dosage form by Three Simple UV Spectrophotometric Methods

Background: For a cardiovascular patient's blood pressure to be effectively controlled, more than one drug is required. When coupled with amlodipine besylate (AMLO), a calcium channel blocker, Aliskiren hemifumarate (ALI) is the 1 st nonpeptide, low molecular mass, orally active transition state rennin inhibitor to efficaciously normalize blood pressure and cardiac ailments. Objective: Three innovative, easy, sensitive, exact, and accurate UV spectrophotometric approaches, including simultaneous equation method (SEM), absorbance ratio method (ARM) and 1 derivative (zero-crossing) spectroscopic approach (FDR), were created and authenticated by validation for synchronized estimation of ALI and AMLO in tablet formulation. Materials and Methods: The SEM was used to measure the absorbance of both medicines at 237 and 280 nm. ALI and AMLO were calculated using 237 and 271 nm, respectively, in the ARM. ALI and AMLO, on the other hand, used the FDR technique to transform UV spectra to first derivative spectra, with the first derivative signal captured at 237 and 254 nm, respectively. The wavelength Original Research Article Sen et al.; JPRI, 33(39A): 217-225, 2021; Article no.JPRI.70345 218 interval (Δλ) was kept at 2 and the scaling factor was kept at 1 when transforming zero-order spectra using the first derivative approach. Validation of the proposed processes was done in compliance with the “International Conference on Harmonization” (ICH) recommendations. Results: SEM and ARM both showed a linear outcome in the range of 1-50 μg/ml for ALI and AMLO. FDR, on the other hand, was shown to be more sensitive and linear between 0.5 and 50 μg/ml for both medications. The results of the method validation parameters were within the allowed ranges of the ICH guidelines. Conclusion: The proposed procedures were shown to be relatively quick, sensitive, simple, and cost-effective, and can thus be used for scheduled quality control analysis of ALI and AMLO in the mixed tablet.


Fig. 1. Chemical structures of ALI & AMLO
One of the most prevalent and influential risk factors for cardiovascular ailment is hypertension. Normalizing blood pressure is vital in the handling of cardiovascular illnesses and their consequences. To adequately regulate blood pressure in a cardiovascular patient, multiple drugs are required. Aliskiren hemifumarate (ALI) is the first nonpeptide, low molecular mass, orally active transition state rennin inhibitor to effectively correct blood pressure and cardiac problems when combined with amlodipine besylate (AMLO), a calcium channel blocker [1][2][3]8]. A review of the literature indicates a variety of analytical approaches for estimating ALI and AMLO alone and in combination using UV spectrophotometry [8-10], fluorimetry [11][12], HPLC [13][14][15][16] and highperformance thin-layer chromatography [17]. As a result, the authors of this paper aimed to develop and validate several easier, sensitive, precise, accurate and economical UV spectroscopic approaches for the determination of ALI and AMLO in mixed tablet formulations that might be employed instead of the methods previously published. Projected methods have several advantages, including a straightforward standard and sample preparation approach as well as a broad range of applications.

Materials, Chemicals and Reagents
Glenmark Pharmaceuticals Ltd. (Mumbai, Maharashtra, India) made available the active pharmaceutical ingredient of ALI, which was utilized throughout the study and IPCA Laboratories (Mumbai, Maharashtra, India) provided AMLO as a gift sample. Commercial sources were used to obtain the marketed formulation, Tekamlo® tablet (Novartis Pharmaceuticals Corporation, East Hanover, New Jersey), which contains 300 mg of ALI and 10 mg of AMLO. Loba Chemie Pvt. Ltd. provided the Analytical grade (AR grade) methanol, which was employed as a solvent (Mumbai, India).

Instrument / Apparatus
A UV-1800 with UV Probe (Double beam UV visible spectrophotometer) from Shimadzu Corporation in Kyoto, Japan, and a quartz cell with a 1 cm path length was applied during the study. The weighing was executed with an Adventurer Pro AVG264C (Ohaus Corporation, Pine Brook, NJ, USA).

Standard Solution Preparation
Solutions comprising of ALI and AMLO were prepared by weighing appropriately 11.052 mg of ALI (11.052 mg of aliskiren hemifumarate is similar to 10 mg of aliskiren) and 10 mg of AMLO reference standard drug, which was then transferred to separate 10 ml standard flasks and diluted to 1000 µg/ml with AR grade methanol. To lower the concentration in the working range, necessary dilutions with methanol were made.

SEM and ARM
To obtain drug solutions containing 20 µg/ml of ALI and AMLO, standard solutions containing 1000 µg/ml of ALI and AMLO were properly diluted with AR-grade methanol. UV-Visible spectrophotometer (200-400 nm) was used to scan diluted ALI and AMLO solutions, and matching spectra were acquired and saved on the computer. Based on the spectral pattern, SEM and ARM [3, [18][19][20][21][22] were preferred for the assessment of ALI and AMLO. Basis of the overlain spectra ( Fig. 2A) of both the drugs, 237 nm (λ max of AMLO) and 280 nm (λ max of ALI) were chosen for SEM. However, in ARM, 237 nm (λ max of AMLO) and 271 nm (isosbestic point) were picked, which exhibited an outstanding linear relationship and hence utilized for remaining studies. The resulting solutions were scanned using a double beam UV-Visible spectrophotometer and responses were recorded at 237 and 280 nm for SEM; 237 and 271 nm for ARM. Following that, absorptivity values for both medicines at their appropriate wavelengths were computed using the formula mentioned below: The absorptivity value of each solution individually at its relevant wavelength was recorded and the mean value (Table 1) for that wavelength of each drug was used to calculate ALI and AMLO concentrations.

FDR
The recorded zero-order UV spectra of ALI and AMLO were converted into 1 st and 2 nd derivative spectra. However, based on the zero-crossing point and spectral pattern, FDR [3, [18][19][20][21][22] was preferred for further study. When 2 nm was used as the wavelength interval (Δλ) and 1 as the scaling factor, 1 st derivative spectra exhibited typical zero-crossing spots at 254 nm for ALI and 237 nm for AMLO. Following a thorough examination of overlain spectra, the wavelengths of 254 nm and 237 nm were chosen for further testing (Fig. 2B). For ALI and AMLO, a calibration graph was built in the range of 0.5-50 µg/ml employing the FRD method. The least square approach was applied to acquire the values of the slope, intercept and correlation coefficient post regression analysis.

Sample Solution Preparation
After the tablets were accurately weighed, the mean weight of 20 Tekamlo® tablets comprising 300 mg of ALI and 10 mg of AMLO was computed. The aforementioned formulations were grounded to a fine residue and a quantity equal to 150 mg of ALI and 5 mg of AMLO was weighed and put into a standard flask with a capacity of 50 ml. Standard flasks were shaken and sonicated (10 minutes) after the addition of 30 ml AR grade methanol, and the remaining volume was brought up to the capacity (50 ml) with the same solvent. The subsequent solution was filtered (Whatman filter paper no. 41) and proper dilutions were completed (ALI & AMLO 20 µg/ml).

SEM
Test solutions (formulation) were scanned in the wavelength range of 200 to 400 nm, and observed responses at specific wavelengths were recorded (237 and 280 nm). The following formula was used to assess the unknown concentration of ALI and AMLO existing in the test solution:

ARM
In ARM, the unknown amount of ALI and AMLO that exists in the test solution was calculated by utilizing below stated formula: The absorptivities of ALI at 237 and 271 nm are represented by ax1 and ax2, respectively. Whereas, absorptivities of AMLO at 237 and 271 nm are ay1 and ay2, respectively.
The absorbances observed at 237 and 271 nm for the sample solution are A1 and A2. The detected concentrations of both medicines (ALI and AMLO) in the sample solution are Cx and Cy.

FDR
The spectrum of the test solution was noted between 200 and 400 nm (UV region), processed to 1 st derivative spectra and the amplitude was evaluated at 237 and 254 nm. Utilizing a regression equation, the extent of ALI and AMLO present in the test solution was evaluated in terms of concentration.

Method Validation
The proposed ideas were validated following the strategies of the "International Conference on Harmonization" [7,18-23].

Specificity
A specificity investigation was conducted to determine whether the tablet excipients employed in the formulation interfered with the medicinal substance. Before being filtered (Whatman filter paper no 41), all the formulation excipients (as given in the marketed formulation) were proportioned and diluted with methanol. Both solutions (placebo and standard) were scanned in the UV range and compared to see if excipients and medicines interfered.

Linearity and range
By assessing all of the standard solutions independently, which comprised ALI and AMLO (1, 5, 10, 20, 30, 40, and 50 µg/ml) in methanol and evaluating absorbances at 237 and 280 nm for SEM; 237 and 271 nm for ARM; 237 and 254 nm for FDR, all the three approaches were assessed for linearity and range. The absorbances of reference solution versus concentration were utilized to create calibration graphs in the SEM and ARM, whereas the 1 st derivative response of standard drug solution versus concentration was utilized in the FDR. The slope, intercept and correlation coefficient was computed utilizing the least squares approach of regression analysis.

Precision
The repeatability, intra and inter-day precision of the procedures were assessed and expressed in terms of percent RSD (Coefficient of variation) of the acquired data. Precision experiments were completed in triplicate on the same day and 3 distinct days for both drugs at 2 dissimilar concentration levels (10 and 20 µg/ml).

Accuracy
To confirm the applicability and consistency of the proposed processes, recovery studies were conducted using the traditional addition approach. Standard medication solutions of ALI and AMLO (10, 15, and 20 µg/ml) were added to pre-analyzed sample solutions containing ALI and AMLO (10, 15, and 20 µg/ml) at 3 dissimilar levels, 50, 100, and 150 %. Finally, the RSD (%) and the proportion of medicines retrieved were calculated. The study's accuracy was assessed by utilizing the formula to evaluate the proportion of reference ALI and AMLO recovered from the formulation: % Recovery= [(Amt. of the drug found post addition of std. drug -Amt. of the drug found before the addition of std. drug) / Amount of std. drug added ×100

Limit of Detection (LOD) and Limit of Quantification (LOQ)
The sensitivity of the projected approaches was assessed using the parameters LOD and LOQ.
The following equations were utilized to compute the LOD and LOQ of ALI and AMLO as specified in ICH guidelines.
Where 'S'=Slope of the calibration curve; ' '= Standard deviation of the response.

Solution Stability
Solutions' stability was verified by supervising any fluctuations in absorbance and comparing the spectral pattern to newly created solutions while holding them at ambient temperature and evaluating them at regular intervals.

RESULTS AND DISCUSSION
For synchronized assessment of ALI and AMLO in marketed tablets, three UV spectrophotometric methods, SEM, ARM, and FDR, were created and tested. They are very accurate, precise, easy and responsive. Both medicines' response was noted in SEM at 237 and 280 nm. For the identification and quantification of ALI and AMLO in ARM, wavelengths of 237 and 271 nm were employed ( Fig. 2A). The FDR approach was based on converting stored zero-order UV spectra to first derivative spectra, then evaluating the first derivative signal at 237 and 254 nm (Fig.  2B) for ALI and AMLO, respectively. In FDR, a wavelength interval (Δλ) of 2 nm and a scaling factor of 1 was exercised. It was testified that there was no interference amongst excipients and typical pharmaceuticals after comparing the overlain spectra of placebo and drug solutions. SEM and ARM revealed a linear association with ALI and AMLO in the range of 1-50 µg/ml. In the concentration range of 0.5-50 µg/ml, the FDR approach, on the other hand, was found to be linear. The overlapping spectra of ALI and AMLO are shown in Fig. 2C and 2D.
For SEM and ARM, calibration graphs were created using the absorbance of a standard drug solution against concentration. The 1 st derivative signal of a standard drug solution vs concentration was utilized to plot the calibration graph for the FDR. To compute values of the slope, intercept, and correlation coefficient for ALI and AMLO at their relevant wavelengths, regression analysis was executed by exploring the least square approach. Precision experimentations were assessed in terms of % RSD, which met the ICH guideline acceptable limits (<2) and demonstrated high repeatability and low intra-and inter-day variability, indicating exceptional precision of the outcomes (Table 2). Recovery rates for both drugs ranged from 98 to 103 percent, implying that the recommended approaches are effective (Table  2). There was no tablet excipient interference, based on the % recovery. Furthermore, the sensitivity of the projected approaches was supported by low LOD and LOQ values ( Table 2).   The solution's stability was tested at room temperature and found to be two days stable. The proposed methods for quantifying ALI and AMLO in tablet formulation (Tekamlo® tablet: 300 mg of ALI and 10 mg of AMLO) were effectively executed. Because sample solutions were checked 6 times and investigational results were determined to be between 96 and 101 % for both medications, the recommended methodologies can be utilized for the synchronized assessment of ALI and AMLO in collective tablet formulations (Table 3).

CONCLUSION
Three novel UV spectrophotometric procedures have been developed that are effortless, responsive, accurate and precise. For synchronized assessment of ALI and AMLO in combination tablet dose form, spectrophotometric methods such as SEM, ARM and FDR were devised. According to ICH criteria, all of the procedures were validated. The proposed approaches were discovered to be extremely responsive, accurate, straightforward, and cost-effective. Furthermore, each of the established UV-spectrophotometric methods requires relatively little sample preparation and has a greater concentration range and sensitivity. As a result, the three described methodologies can be used to investigate the quality of ALI and AMLO in mixed formulations regularly.

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, the research was not funded by the producing company rather it was funded by personal efforts of the authors.