INTRODUCTION

Ischemia is the most prevalent disease that occurs due to an imbalance of oxygen demand and supply to the heart. Angina is a manifestation of ischemic heart disease, which predominantly contributes to death and impairment of a patient’s quality of life (QoL) [1].

Multiple pathogenesis is involved in ischemia; hence, a single drug may or may not be effective. Effective treatment mainly includes a multifaceted and tailored approach depending on the patient’s disease profile, lifestyle management, pharmacological treatment, and myocardial revascularization.  It is observed that in many angina cases, comorbidities like diabetes and chronic obstructive pulmonary illness accelerate possible intolerance to beta-blockers, in such conditions, newer drug therapy is a good alternative [2]. Ivabradine hydrochloride is considered to be the first choice for normalizing heart rate which acts specifically and particularly on the present cardiac pacemaker current of the sinoatrial node and reduces the heart rate with no alteration in myocardial contractility and present (if any) vascular tone, which makes Ivabradine unique from beta-blockers and calcium channel blockers [1, 3].

Ivabradine hydrochloride’s chemistry nomenclature is 3-(3-{[((7S)-3,4-Dimethoxybcyclo [4,2,0] oct-1,2,3-trien-7-yl) methyl] methyl amino} propyl)- 1,3,4,5-tetrhydro-7,8-dimethoxy-2H-3-benzazpin-2-one, hydrochloride with molecular formula C₇H₆NO₅. HCL and molecular mass 505.05 g/mol. US Food and Drug Administration (USFDA) approved Ivabradine hydrochloride in 2015 as a heart rate-lowering agent [4].

Trimetazidine dihydrochloride is a chemically 1-[(2,3,4-trimethoxyphnyl) methyl] piperazine dihydrochloride with a molecular formula of C₁₄ H₂₂N₂O₃.2HCl and a molecular mass 339.3 g/mol. it is a clinically effective agent that shows anti-ischemic effects without inducing hemodynamic changes, unlike other beta-blockers, calcium channel blockers, or long-acting nitrates [5-9].

The text summary indicated that basic UV, HPLC, HPTLC, and LC-MS methods were considered when evaluating these compounds separately or in combination with other compounds, but simultaneous estimates (SE) were not shown about this new hybrid. The simultaneous estimation (SE), is usually used to evaluate chemical mixtures containing two or more chemicals in the same measurement system. In comparison with other analytical methods, this method has a few unique drawbacks. Consequently, efforts have been made to develop a novel and simple SE method to support the estimation of these active substances in health and pharmaceutical products. The developed method has been validated and successfully used in the formulation development of TMZ and simultaneous estimation of IBH in the unmodified compounds [10-14].

The background survey has confirmed that, at present, no UV spectroscopic analysis is possible for simultaneous estimation of the IBH and TMZ combination in a single unit dosage form. Therefore, the objective of this research work is the development and validation of a new but simple UV spectroscopic analysis tool for simultaneous estimation of IBH and TMZ.

MATERIALS AND METHODS

Apparatus (Jasco V-630 UV-visible spectrophotometer)

For experimental work, a UV-visible spectrophotometer (model Jasco-V 630) with a wide wavelength range of 190 to 1100 nm was employed for measurement. Its facilities include a spectral bandwidth of 1.5 nm, automatic wavelength corrections, a pair of 10 mm quartz cells, etc.

A Sonicator of Biomedica of 2.5 liters was used for sonication purposes. Glassware such as volumetric flasks, pipettes, and beakers were used.

Chemicals and reagents

Ivabradine hydrochloride and Trimetazidine dihydrochloride were both received as gift samples from Lupin Limited, Sikkim, India, and Sharon Bio-Medicine Ltd, Maharashtra, India, respectively. The marketed tablets of IBH Coralan 5 mg and TMZ, Flavedon MR 35 mg were manufactured by Servier India Pvt. Ltd. and Les Laboratoires Servier Industry, respectively, and also purchased from an approved vendor of the institute.

Standard (stock) solutions of IBH

The stock solutions of Ivabradine hydrochloride (100 𝜇g/mL) were prepared in methanol and sonicated for a few minutes which was adjusted further to 100 mL using methanol.

Standard (stock) solutions of TMZ

Trimetazidine dihydrochloride (100 𝜇g/mL) was prepared by dissolving 10 mg of Trimetazidine dihydrochloride in 50 mL of methanol and was sonicated for a few minutes, and the volume was adjusted with methanol.

Preparation of working standards of IBH

From the standard stock solution of IBH, 0.5 mL to 2.5 mL were withdrawn using a pipette in a separate volumetric flask and mixed with methanol to prepare working standards of 5-25𝜇g/mL.

Preparation of working standards of TMZ

From TMZ standard stock solution, 1 mL to 5 mL were drawn using a pipette and mixed with methanol as a solvent to prepare working standards of 10-50𝜇g/mL.

Calibration curve for IBH and TMZ

From these standard (stock) solutions, different working standards were prepared with methanol and examined in the whole UV range to determine that Ivabradine hydrochloride is at 286 nm and Trimetazidine dihydrochloride is at 231 nm λ max. The linearity framework was explored by sequentially diluting the stock solution to fix the range of 5–25 𝜇g/mL for Ivabradine hydrochloride and 10–50 g/mL for Trimetazidine dihydrochloride (Figure 3).

Table 1 represents recovery study  of both drugs based on concentration range.

Table 1. Recovery result of  IBH and TMZ

Development of simultaneous estimation of IBH and TMZ

In this step, the prepared sample solutions of both drugs were scanned in the UV range of 200–400 nm, and overlay spectra were taken. By application of the simultaneous equation, the method with 286 nm (IBH 𝜆max) and 231 nm (TMZ 𝜆max) was selected for overlay spectra for analysis of both drugs together. Concentrations range of 5, 10, 15, 20, and 25 𝜇g/mL for IBH and 10, 20, 30, 40, & 50 𝜇g/mL for TMZ were prepared in methanol. The concentration of drugs x (IBH) and y (TMZ) in sample solutions was determined by the SE method using the following formula:

Where, 𝐶𝑥 and 𝐶𝑦 are the concentration of IBH and TMZ, 𝐴1 and 𝐴2 are the absorbance of sample solution at 286 nm and 231 nm, respectively, 𝑎𝑥1 and 𝑎𝑥2 are absorptivity of IBH at 286 nm and 231 nm, and 𝑎𝑦1 and 𝑎𝑦2 are absorptivity of TMZ at 286 nm and 231 nm, respectively [15-20].

Analysis of marketed tablet formulation and bulk API (Active Pharmaceutical Ingredient) mixture

Marketed tablet formulation, 10 tablets of IBH (Corlanor 5mg) and TMZ (Flavadon MR 35 mg) taken into a mortar and pestle, triturated into powder form. The powder mixture equivalent to 5 mg IBH and 35 mg TMZ was taken into a 100 mL flask. To this, 50 mL of methanol was mixed and sonicated for a few minutes. After sonication, the volume was adjusted with the same solvent and filtered through Whatman’s filter paper (0.45 microns). The resultant filtered sample was further diluted to get a 20 µg/ml concentration. Finally, this was evaluated at 286 nm and 231 nm, for IBH and TMZ estimation.

Similarly, an API mixture of IBH and TMZ was prepared by weighing a 1:7 ratio of IBH and TMZ. From this mixture, 40 mg of powder was weighed, which equates to 5 mg of IBH and 35 mg of TMZ. This mixture was taken to a 100 mL flask, and methanol was mixed and sonicated for a few minutes. The resulting solution was then diluted to for final concentration of 20 µg/mL. The resulting solution was evaluated at 286 nm and 231 nm for IBH and TMZ respectively. The result is shown in Table 2.

Valıdatıon of method

1. Linearity: The calibration curves for IBH and TMZ were separately plotted using absorbance on the y-axis and concentration on the x-axis. For IBH, the working standard linearity range was found to be 5–25 𝜇g/mL, and for TMZ 10–50 𝜇g/mL, which were scanned using 286 nm and 23nm, respectively, to get the absorbance at a specific concentration. After plotting the graph, the correlation coefficient values (𝑅2) of IBH and TMZ were 0.9989 and 0.9993, which indicates a direct relation between concentration and absorbance. The curves of IBH and TMZ are shown in Figures 1 and 2, respectively.
2. LOD and LOQ (Limit of detection and limit of quantification): The calibration curve was used to determine both LOD and LOQ, which were estimated based on the formula for LOD =3.3*(σ/S) and LOQ =10*(σ/S). Using y-intercept, where sigma is the standard deviation (SD) of the reaction (y-intercept) and S is the slope of the plot. The LOD of IBH and TMZ were reported at 2.350 μg/ml and 3.790 μg/ml, respectively. IBH had a limit of quantification (LOQ) at 7.122 µg/mL, while that for TMZ was 11.485 µg/mL. The table of LOD and LOQ of IBH and TMZ are 1 and 2, respectively.
3. Precision: Evaluation of precision or reproducibility of the method performed by determination of inter-day plus intra-day independent assays for 3 different samples selected from the calibration curve range, for IBH 10, 15, and 20 µg/mL, and for TMZ 20, 30, and 40 µg/mL, and percentage RSD was estimated. These precision results are shown in Table 3.
4. Accuracy: The accuracy study was performed at three levels using 50%, 100%, and 150%. This was studied by a recovery study using the standard addition method. A recovery study was performed to check for any interferences due to excipients. Close to 100% recovery results revealed that the developed method is accurate and there was no interference due to other excipients. The accuracy study results is shown in Table 4.

Table 2. Results of Analysis of Marketed Formulation and Bulk API

Table 3. Precision Study

Table 4. Results of Recovery Studies