Instrumentation

Shimadzu double-beam spectrophotometer (UV, Japan), Sonicator (Model 3510, UK), Analytical Balance, Italy, Digital Stirring Hot Plate, China, Pure laboratory flex to obtain distilled water

Silica gel F254 (20 × 20 cm) pre-coated TLC plates (Sigma-Aldrich, Germany). The degradation tracing and plate visualization were carried out using a UV lamp with a wavelength of 254.0 nm.

Reagents and chemicals

Pure samples of CAR and IVA were kindly obtained from Global Napi, 6th of October, Egypt. The pureness was certified to be 99.88 ± 0.95 and 99.60 ± 1.06, respectively, depending on their official [1] and reported [8] techniques.

The market-available Carivalan® tablet dosage form, made in France by Les Laboratories Servier Industrie, was purchased. According to the product label, Carivalan ® batch number (29,044) contains 12.5 mg of CAR and 5.0 mg of IVA. 30% H2O2 (ADWIC, Egypt).

Standard solutions

To prepare stock solutions of carvedilol and ivabradine (100.0 µg/ml), in volumetric flasks, 100 ml, 10 mg of each of CAR and IVA was dissolved separately in distilled water.

In two rounded flasks, 10 mg of each of CAR and IVA was refluxed with 10 ml of 30% H2O2 for 8 h at 80 °C. After refluxing, hydrogen peroxide was evaporated using hot plate at 50 °C, transfer oxidative degraded product to volumetric flask, 100 ml, which is completed with distilled water to prepare standard solutions of 100.0 µg/ml for each.

Laboratory mixtures

Exact amounts of the stated drugs were conveyed into different volumetric flasks, 10 ml, with varied ratios and topped off with distilled water to produce variable sets as follows:

Set A: Binary mixture containing CAR and IVA.

Set B: Quaternary mixture containing CAR and IVA along with their oxidative degradation products.

ProceduresStress stability studies

Studied drugs’ stress studies were conducted as per ICH guidelines [30] by applying different stress acidic, basic, photolytic, thermal and oxidative surroundings. Different molar concentrations of HCl and NaOH were tried out where, in each trial, 10 ml of each of the investigated medications was combined individually with either HCl or NaOH. Reflux was applied at 100 °C for 3 h.

To conduct oxidative trials, 10 mg of each medication was refluxed individually in 10 ml of 10, 20 and 30% H2O2 for 8 h at 80 °C.

Drugs’ photostability was tested on their solid powdered form by the exposing them to UV light, 254.0 nm for 10 h.

Finally, drugs’ thermal stress testing was performed in glass ampoules by heating them in a thermostatic oven at 10 °C boosts (50–100 °C) for 10 h.

Spectral characteristics

D0 spectra of each examined drugs were measured at 200.0–400.0 nm.

Construction of calibration graphs

To prepare calibration standards across concentrations of 3.0–30.0 µg/ml for CAR and IVA, from each standard stock solution (100.0 µg/ml), an accurately transferred amounts were conveyed to separate series of volumetric flasks, 10 ml, and finalized with distilled water. The D0 spectra acquired after scanning solutions between 200.0 and 400.0 nm were recorded. The calibration graph for each suggested technique was performed via the mean of three experiments, as shown below:

Factorized dual wavelength coupled with spectrum subtraction technique (FDW-SS)

The regression equations were created by plotting the stored D0 maximum absorbance of CAR and IVA at 285.6 and 287.0 nm, respectively, against the associated concentrations. A factorized ∆A spectrum for IVA was constructed using spectrophotometric software by division of D0 of a certain concentration of IVA by (∆A) at 285.2 nm and 255.0 nm.

Ratio difference technique

The ratio spectra were produced via division of the scanning spectra of the CAR and IVA solutions by the absorption spectra of the IVA (15.0 µg/ml) and CAR (15.0 µg/ml) standard solutions, respectively. CAR's and IVA's concentrations were plotted against their respective amplitude differences at 252.6 and 275.0 nm and 294.0 and 320.0 nm, respectively.

Constant center coupled with spectrum subtraction technique (CC-SS)

The regression equations have been calculated by setting two calibration curves. The first one is between the maximum absorbance of D0 of CAR and IVA at 285.6 nm and 287.0 nm and their related concentrations, respectively. The second one is between the ∆A at 294.0 nm and 320.0 nm versus amplitudes at 294.0 nm for IVA after generating a ratio spectrum by dividing of the stored absorption spectra of IVA by the absorption spectrum of CAR; 15.0 µg/ml.

Induced tripartite amplitude difference coupled with ratio subtraction technique (ITAD-RS)

The regression equations were computed by plotting the maximum absorbance of the stored D0 of CAR and IVA at 285.6 nm and 287.0 nm versus their corresponding concentrations, respectively.

The equality factor of IVA ratio spectra was calculated by using amplitude ratios' average at 296.0 nm (P296.0) and 242.7 nm (P242.7) using CAR's oxidative degradate spectrum as a divisor.

Via dividing the ratio spectrum of a certain concentration of CAR using CAR's oxidative degradate's spectrum as a divisor by ∆P at 242.7 nm and 296.0 nm after multiplying the former by F, (FP242.7–P296.0), factorized induced ratio spectrum for CAR was created.

Through the division of the ratio spectrum of IVA using CAR's oxidative degradate's spectrum as a divisor by the value of the ∆P at 242.7 nm and 296.0 nm, factorized ratio spectrum for IVA was created.

Laboratory mixtures' analysis

Different mixtures in different sets, set A (binary mixture of CAR and IVA) and set B (quaternary mixture of CAR and IVA with their oxidative degradates), were analyzed by carrying out the following manipulation actions for each method:

Analysis of set A containing CAR and IVA in their binary mixturesFactorized dual wavelength coupled with spectrum subtraction technique (FDW-SS)

The D0 of IVA was obtained by noting ΔA at 285.2 nm and 255.0 nm and multiplying by IVA's factorized ΔA spectrum in the prepared laboratory mixtures of CAR and IVA. CAR's D0 spectrum was resulted from the subtraction of IVA's D0 spectrum from the spectra of laboratory mixture.

Ratio difference technique

The D0 of binary laboratory mixture in set A was divided separately by the absorption spectrum of standard IVA' and CAR' (15.0 µg/ml, each). The amplitudes of the obtained ratio spectra were noted at 252.6 and 275.0 nm for CAR and 294.0 and 320.0 nm for IVA. The concentrations of CAR and IVA were resolved from their related regression equations.

Constant center coupled with spectrum subtraction technique (CC-SS):

Via employing the formerly manipulated ratio spectra in RD method, the following steps were employed: Firstly, IVA's ratio spectrum in each mixture was recorded at 294.0 nm and 320.0 nm using CAR's D0 as a divisor. Secondly, the postulated amplitude at 294.0 was acquired from the correspondent related regression equation followed by subtracting the postulated amplitude from the recorded one to obtain a constant value; CAR/CAR'. Finally, via multiplying the constant value by the spectra of 15.0 µg/ml standard CAR', the parent D0 of CAR was obtained in which its concentration might be determined using the matching regression equation built at its maxima.

As a result, the D0 of IVA could be calculated via subtraction of the acquired spectra of CAR from the spectra of its related mixtures' spectra. The concentration of IVA was estimated using the relevant regression equation.

Analysis of set B containing CAR and IVA with their oxidative degradates in their quaternary mixturesInduced tripartite amplitude difference coupled with ratio subtraction technique (ITAD-RS)

CAR/CAR's degradate ratio spectrum in mixtures of set B was acquired after multiplication of CAR's factorized induced ratio spectrum by (FP242.7 − P296.0) of each mixture’s ratio spectrum. After multiplication of the former ratio spectrum, CAR/CAR's degradate by the D0 spectrum of the CAR oxidative degradates, CAR's D0 was calculated.

The ratio spectrum comprising, IVA + IVA's degradate + CAR's degradate/CAR's degradate was acquired by subtraction of the obtained ratio spectrum; CAR/CAR's degradate from that of quaternary mixture.

For IVA, its ratio spectrum in the combination can be acquired by multiplication of ΔP at 242.7 nm and 296.0 nm by the formerly set IVA's factorized ratio spectrum. IVA's parent D0 was acquired after multiplication of the acquired ratio spectrum by the spectrum of the CAR oxidative degradate.

CAR and IVA concentrations were figured using their respective regression equations generated at their maxima.

Application to pharmaceutical formulation

Five Carivalan® tablets were crushed and blended together. Amounts equivalent to 25.0 mg CAR and 10.0 mg IVA were precisely moved to beaker, 100 ml, dissolved in 30 ml distilled water, sonicated and filtered into a volumetric flask, 100 ml, then accomplished with distilled water.

Further dilutions were proceeded to produce concentrations of 10.0 µg/ml for CAR and 4.0 µg/ml for IVA. The proposed techniques were used for the analysis of the examined Carivalan® following the described measures under laboratory-prepared mixtures analysis to compute their concentrations utilizing the related regression equation for each proposed drug.