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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 1  |  Page : 1-10

Quantitative bioanalytical and analytical methods for estimation of ivabradine hydrochloride in pure and pharmaceutical dosage form


Department of Pharmaceutical Analysis, G. Pulla Reddy College of Pharmacy, Osmania University, Hyderabad, Telangana, India

Date of Submission06-Oct-2019
Date of Acceptance17-Mar-2020
Date of Web Publication26-Jun-2020

Correspondence Address:
Miss. Anjali Polker
Research scholar, Research Fellow (M.Pharmacy) Department of Pharmaceutical Analysis, G. Pulla Reddy College of Pharmacy, Osmania University, Hyderabad 500028, Telangana.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrptps.JRPTPS_110_19

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  Abstract 

Introduction: New analytical and bioanalytical methods were developed for the estimation of Ivabradine hydrochloride in bulk and pharmaceutical dosage form by UV spectrophotometry and high-performance liquid chromatography (HPLC) technique. Objective: The primary objective of the study is to develop a new RP-HPLC method for estimation of Ivabradine Hydrochloride in pure and formulation and to develop a bioanalytical method for analysis of Ivabradine Hydrochloride in biological samples. The methods were validated as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use and USFDA guidelines respectively. Methods: In Reversed-phase (RP)-HPLC method developed telmisartan is the internal standard used. After liquid–liquid extraction, the analyte and the internal standard were chromatographed on Waters 125A (10 µ, 300 × 3.9 mm) C18 column using 20-µL injection volume with a run time of 15 min. An isocratic mobile phase consisting of acetonitrile and ammonium acetate buffer pH 7.8 (60:40% vol/vol) is used to separate drug and internal standard. In Spectrophotometric bioanalytical method developed for the estimation of ivabradine hydrochloride in pure and pharmaceutical dosage form. The solvent system used is absolute methanol and detected at the wavelength of 287 nm. The solvent system used is absolute methanol and detected at the wavelength of 287 nm. Results: The Analytical method is validated according to ICH guidelines over the range of 2–16 μg/ mL, showing accuracy, precision, selectivity, and robustness. For Bioanalytical method the linearity is established in the range of 500–3500 ng/mL with the regression coefficient of r2 = 0.9994. The validated spectrophotometric method is used successfully to study ivabradine hydrochloride in rat plasma and also quantitative determination in marketed tablets. Conclusion: The proposed methods were successfully applied for the quantitation of ivabradine hydrochloride in pharmaceutical dosage form with good recovery and reproducibility.

Keywords: Bioanalytical method, ivabradine hydrochloride, reversed-phase high-performance liquid chromatography, UV-spectrophotometric method


How to cite this article:
Polker A, Padmavathi Y, Kumar R, Babu NR. Quantitative bioanalytical and analytical methods for estimation of ivabradine hydrochloride in pure and pharmaceutical dosage form. J Rep Pharma Sci 2020;9:1-10

How to cite this URL:
Polker A, Padmavathi Y, Kumar R, Babu NR. Quantitative bioanalytical and analytical methods for estimation of ivabradine hydrochloride in pure and pharmaceutical dosage form. J Rep Pharma Sci [serial online] 2020 [cited 2020 Dec 2];9:1-10. Available from: https://www.jrpsjournal.com/text.asp?2020/9/1/1/287578


  Introduction Top


Introduction to drug

Ivabradine hydrochloride[1] is chemically 3-[3-({[(7S)-3,4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl]methyl}(methyl)amino)propyl]7,8-dimethoxy-2, 3, 4, 5-tetrahydro-1H-3-benzazepin-2-one hydrochloride [Figure 1]. Its empirical formula is C27H37ClN2O5. Ivabradine hydrochloride is a first-in-class drug that is selective bradycardic agent with direct effect on the pacemaker If current[2] of the sinoatrial node. It is the only representative of the group of drugs that has been introduced for the treatment of chronic coronary artery disease and chronic heart failure.[3],[4] It is white to off-white solid, soluble in organic solvents such as ethanol (<1 mg/mL at 25°C), DMSO (<20 mg/mL at 25°C), and dimethyl formamide (<25 mg/mL at 25°C), and its melting point is >190°C.[5]
Figure 1: Structure of Ivabradine Hydrochloride

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Telmisartan (internal standard)

Chemically, telmisartan[6] is 2-(4-{[4-methyl-6-(1-methyl-1H-1,3-benzodiazol-2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl}phenyl)benzoic acid [Figure 2]. The empirical formula is C33H30N4O2, which corresponds to a molecular weight of 514.617 g/mol. Telmisartan is white to off-white, odorless powder, slightly soluble in methanol, sparingly soluble in methylene chloride, dissolves in one molar NaOH, but practically insoluble in water. It is used for the treatment of hypertension.
Figure 2: Structure of Telmisartan

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Literature review[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19] reveals that few UV spectrophotometric, high-performance liquid chromatography (HPLC), and liquid chromatography (LC)-mass spectrometry (MS)/MS-ESI methods have been reported so far for the determination of ivabradine hydrochloride alone and its combination with other drugs. There are few methods available for the estimation of ivabradine hydrochloride in biological samples. This study was planned for the development of bioanalytical and analytical methods for the analysis of ivabradine hydrochloride in pure and pharmaceutical dosage form. RP-HPLC method is extremely quick and efficient. Quantitation of drug with the use of internal standard makes the work unique.


  Materials and Methods Top


Chemicals

Ivabradine hydrochloride was kindly provided by MSN Laboratories (Hyderabad, India), telmisartan (internal standard) by Sanofi (Banglore, India), Ivabrad 5 tablets (Lupin, Uttar Pradesh, India). The tested chemicals were supplied from the following sources: acetonitrile (HPLC Grade) from Merck (Mumbai, India), methanol (HPLC grade) from SDFCL (Thane India), water (HPLC grade) from Merck, ammonium acetate buffer (HPLC grade) from SDFCL, and disodium EDTA (LR grade) from SDFCL.

Instrumentation

HPLC analysis of ivabradine hydrochloride is carried out using an Agilent, 1100 series, RP-HPLC system equipped with automatic injector, Chemstation software, and variable wavelength detector. Chromatographic separation is achieved on C18 column (300 × 3.9mm, 10 µm). Bioanalytical estimation is carried out using UV-Visible spectrophotometer (Shimadzu, 1800 series), a semi microbalance (Shimadzu, BL220H, Japan), pH meter (Elico, LI 127, India), ultrasonic bath sonicator (PCI Analytics, India 6.5L 200 H), refrigerated centrifuge (Eltek, India), and hot air oven (Tempo Equipment, India).[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31]

Methods

Analytical method

Preparation of standard stock solution: A stock solution of ivabradine hydrochloride 1000 µg/mL is prepared in acetonitrile, and the working standard solutions were prepared daily by appropriate dilution of the stock solution with diluent.

Mobile phases (MPs) of varying compositions of solvents such as acetonitrile, water, and ammonium acetate buffer (pH, 7.8) were used for the method development.

Preparation of internal standard stock solution: A stock solution of standard telmisartan 1000 µg/mL is prepared in methanol, and the working standard solutions were prepared daily by appropriate dilution of the stock solution with diluent.

Preparation of mobile phase: MP consists of acetonitrile and 0.01 M ammonium acetate buffer (60:40). The pH of ammonium acetate buffer is maintained to 7.8. The MP is filtered through 0.2-μm cellulose acetate filters and degassed in sonicator before use.

Preparation of 0.01M Ammonium acetate buffer pH 7.8: A total of 0.28 g of ammonium acetate is dissolved in 300 mL of HPLC water to produce 0.01 M solution. The pH of this solution is adjusted to 7.8 with sodium hydrochloride solution.

Bioanalytical method

Preparation of stock solutions: A stock solution of ivabradine hydrochloride 1000 µg/mL is prepared in methanol, and the working standard solutions were prepared daily by appropriate dilution of the stock solution with methanol.

The absorbance of resulting solution is measured against respective blank solution in visible region, that is, 200–400 nm, which shows a maximum absorbance at 287 nm.

Plasma sample preparation: The blood samples were collected from retro-orbital puncture into disodium ethylenediaminetetraacetic acid (EDTA) vials (20 mg disodium EDTA in 1 mL water, 1 mL of blood requires 50 µL of disodium EDTA). Plasma is separated from blood samples by centrifugation at 10,000revolutions per minute for 10 min. After centrifugation, plasma layer gets separated, and it is collected and stored at -20°C for further use.[32],[33]

Extraction procedure:

  • A pool of blank rat plasma is obtained.


  • A total of 100 μL of plasma sample is spiked with appropriate volume of the stock solution, and 400 μL of acetonitrile is added.


  • The mixture is vortexed for 1 min.


  • Centrifuged at 10,000rpm for 10 min at 4°C.


  • The organic layer is separated and filtered through a 0.2-μm cellulose acetate filter.


  • The organic layer is evaporated on a Savant vacuum evaporator at about 60°C. The residue is reconstituted in 10 mL of solvent and is used for the measurement of the absorbance at the absorption maxima of ivabradine hydrochloride.


  • Blank plasma and standard preparation: The separated organic layer is taken from processed blank plasma, and after filtration, its absorbance is measured directly without spiking it with either sample or solvent. Standard solution of ivabradine hydrochloride 500 ng/mL spiked in plasma is prepared, and absorbance is measured. Blank plasma absorbance is also recorded.

    Preparation of quality control (QC) standards in plasma: Calibration samples are prepared by spiking with appropriate amounts of sample into 90 μL of control plasma. From 1 mg/mL stock solution of drug, 5, 10, 15, 20, 25, 30, and 35 μL samples are taken and volume is made up to 10 mL with methanol. From each concentration, 10 μL of sample is taken and spiked into 90 μL control plasma to get 500, 1000, 1500, 2000, 2500, 3000, and 3500 ng/mL, and the absorbance of these concentrations is measured spectrophotometrically at 287 nm against blank plasma.

    Matrix effect: Calibration sample is prepared by spiking with appropriate amounts of sample. From 1 mg/mL stock solution of drug, 5, 10, 15, 20, 25, 30, and 35 μL samples are taken and volume is made up to 10 mL with methanol. From each concentration, 10 μL of sample is taken and the final volume is made with methanol to get 500, 1000, 1500, 2000, 2500, 3000, and 3500 ng/mL, and the absorbance of these concentrations is measured spectrophotometrically at 287 nm against methanol as blank, and the matrix effect is calculated and tabulated in [Table 1].
    Table 1: Calibration curve data of reversed-phase high-performance liquid chromatography method

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      Results and Discussion Top


    For the validation of the developed methods in the determination of ivabradine hydrochloride under experimental conditions, the analytical and bioanalytical characteristics were calculated.

    Validation of analytical method

    The optimized RP-HPLC method is validated according to ICH Q2 (R1) guidelines.[34]

    Linearity

    Linear regression analysis is performed for checking the linearity of the data obtained. The response of the drug is found to be linear in the concentration range 2–16 μg/mL. The linear regression equation for ivabradine hydrochloride is y = 0.1954x + 0.0828 with r2 = 0.9997.

    The calibration curve data are shown in [Table 1], and the calibration plot is shown in [Figure 3] and chromatogram in [Figure 4].
    Figure 3: Calibration curve of Ivabradine Hydrochloride

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    ,
    Figure 4: Overlay chromatogram of Ivabradine Hydrochloride (2–16 μg/mL)

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    Acceptance value for coefficient of determination should not be less than 0.999 and the found value is 0.999 which falls in the acceptance criteria.

    System suitability tests

    System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such. Following system suitability test parameters were established as shown in [Table 2].
    Table 2: Results of system suitability parameter

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    Specificity

    The specificity study is performed by injecting the blank. The chromatograms of blank and standard solutions were compared, and no interference due to solvent from MP at retention time of ivabradine hydrochloride peak was found. No interfering peaks were noticed in the chromatogram, suggesting that solvents do not interfere in the estimation of drug.

    Analyte solution stability

    Sample solution of ivabradine hydrochloride is prepared and analyzed initially and at different time interval by keeping at room temperature. The results are given in [Table 3].
    Table 3: Analyte solution stability data of reversed-phase high-performance liquid chromatography method

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    Stability for analytical solution showed the %CV- % coefficient of variation in the acceptance range, proving the stability of the analytical solution.

    Sensitivity (limit of quantification)

    LOQ is the least concentration in the calibration curve, which is 2.2 μg/mL [Table 4].
    Table 4: Sensitivity data of reversed-phase high-performance liquid chromatography method

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    The analyte response at the LOQ is more than five times the blank response. Analyte peak (response) is identifiable, discrete, and reproducible. It agreed the acceptance criteria.

    Precision

    Repeatability: The repeatability data are shown in [Table 5].
    Table 5: Repeatability data of reversed-phase high-performance liquid chromatography method

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    Interday precision: The interday precision data are shown in [Table 6].
    Table 6: Interday precision data of reversed-phase high-performance liquid chromatography method

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    Intraday precision: The intraday precision data are shown in [Table 7].
    Table 7: Intraday precision data of reversed-phase high-performance liquid chromatography method

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    The calculated % relative standard deviation values are very low, indicating the precision of the method. The %RSD values less than or equal to 2 is the acceptance criteria.

    Accuracy (drug substance)

    Accuracy of the method is determined at three different concentration levels. Mean and %RSD values were calculated and are shown in [Table 8].
    Table 8: Accuracy data of reversed-phase high-performance liquid chromatography method

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    The calculated %RSD values indicate that the method is accurate.

    Recovery studies (drug product)

    The recovery values indicate that extraction efficiency for the analyte is consistent and reproducible. The results are shown in [Table 9].
    Table 9: Recovery studies data of ivabradine formulation

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    The % recovery should be between 90% and 110%, and the found results were between the acceptance range.

    Assay (drug product)

    The assay of Ivabrad 5 tablets containing ivabradine hydrochloride is found to be 96.04%. The assay values are shown in [Table 10].
    Table 10: Assay of ivabradine hydrochloride tablets

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    Validation of bioanalytical method

    After completion of several trials with different solvents and varying solubility-enhancing techniques, spectrum with good absorbance and stability, that is, trial 6 was selected [Table 11]. The absorption spectra are recorded in wavelength region of 200–400 nm (UV region). The λmax of the drug was found to be at 287 nm.
    Table 11: Trials for method development and method optimization

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    Matrix effect

    It is necessary to develop an extraction method, which gives consistent and reproducible recovery of the analyte from plasma. Drug is extracted from plasma by solvent extraction technique using acetonitrile. It is shown in [Table 12], and the spectra are shown in [Figure 5].
    Table 12: Matrix effect data

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    ,
    Figure 5: Spectra of treated plasma

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    The quantitative measure of matrix effects[35] is the matrix factor (MF). The value of MF less than one signifies suppression, MF value greater than one signifies enhancement, and MF value equal to one implies that the analytical method is free from matrix effect. The obtained value of MF is 0.9994. It can be considered as free from matrix effect. The values are noted in [Table 12].

    As such in untreated plasma, disturbance was noted; therefore, it made the plasma treatment necessary step. The plasma is treated with different volumes of acetonitrile to enhance the results. 400 µL of acetonitrile showed good results. The treated plasma is then filtered and this process is implemented for the preparation of samples throughout the study. The spectrum is shown in [Figure 5].

    Method validation

    The optimized UV-spectrophotometric method is validated according to the United States Food and Drug Administration (USFDA) guidelines[36] “Bioanalytical Method Validation.”

    Linearity

    Linear regression analysis is performed for checking the linearity of the data obtained. The response of the drug is found to be linear in the concentration range 500–3500 ng.

    The linear regression equation for ivabradine hydrochloride is y = 0.0000215x – 0.00628571 with r2 = 0.9994.

    The calibration curve data are shown in [Table 13], and calibration plot is shown in [Figure 6], and calibration spectra in [Figure 7].
    Table 13: Linearity data of ivabradine hydrochloride in plasma

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    ,
    Figure 6: Calibration graph of ivabradine hydrochloride spiked in plasma

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    ,
    Figure 7: Overlay spectrum of ivabradine hydrochloride spiked in plasma (500–3500 ng/mL)

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    Selectivity

    There is no interference observed in blank plasma.

    Sensitivity (lower limit of quantification)

    LLOQ is the least concentration in the calibration curve, which is 500 ng/mL.

    The analyte response at the LLOQ is more than five times the blank response. It can be accepted.

    Optical conditions and statistical data of regression equation

    The optimum conditions such as Beer’s Law limits, sensitivity, and other regression characteristics such as slope (m), intercept (C), and correlation coefficient were calculated and presented in [Table 14].
    Table 14: Optimized UV-spectroscopic conditions

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    Accuracy (DS)

    Accuracy of the method is determined at four different concentration levels. Mean and %CV values were calculated, and are shown in [Table 15]. The results indicate that the method is accurate.
    Table 15: Accuracy data of ivabradine hydrochloride in plasma

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    % CV for analyte concentration in five replicate standard samples should not be more than 15.0 in the acceptance criteria.

    Recovery studies

    They represent the extraction efficiency of a method. They are performed at lower quality control (LQC), middle quality control (MQC), and higher quality control (HQC). Six replicates at LQC, MQC and HQC level were prepared for recording determination. Mean relative recovery was found to be 101.62. The data for relative recovery are given in [Table 16]. The results indicate that extraction efficiency for the analyte is consistent and reproducible.
    Table 16: Recovery data of ivabradine hydrochloride tablets

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    Recovery data of ivabradine hydrochloride tablets is %C: (B/A) × 100.

    % Recovery between 85% and 115% is the acceptance criteria.

    Precision

    Precision of the data is reported in terms of repeatability [Table 17], intraday [Table 18], and interday precision [Table 19].
    Table 17: Repeatability data of ivabradine hydrochloride in plasma

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    ,
    Table 18: Intraday precision data of ivabradine hydrochloride in plasma

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    ,
    Table 19: Interday precision data of ivabradine hydrochloride in plasma

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    The calculated %CV values are very low, indicating that the method is precise. The %CV for acceptance of sample should not be more than 15%.

    Stability of analyte solutions

    After completion of the storage times for different types of stabilities, sample is tested by comparing the instrument response with that of freshly prepared solution.

    Freeze and thaw, short-term temperature, and stock solution stability studies were performed for the plasma samples spiked with the drug. They are noted in [Table 20][Table 21][Table 22].
    Table 20: Freeze and thaw stability of plasma samples

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    ,
    Table 21: Short-term stability of plasma samples

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    ,
    Table 22: Stock solution stability of plasma samples

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    There should not exist much difference between test and freshly prepared solutions in terms of absorbance in the set acceptance criteria.

    Assay of ivabradine hydrochloride tablets

    The data obtained after analyzing five replicates of formulation samples spiked in plasma is given in [Table 23].
    Table 23: Assay data of ivabradine hydrochloride tablets

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    The % recovery found is 98%, which falls in the acceptance range, which is 90%–110%.


      Conclusion Top


    Analytical and bioanalytical methods were developed for the estimation of ivabradine hydrochloride in pure and pharmaceutical dosage form.

    In analytical method, ivabradine hydrochloride is estimated using telmisartan as internal standard on Agilent RP-HPLC. The developed analytical method is validated according to ICH guidelines. MP consisted of acetonitrile and ammonium acetate buffer pH 7.8 (60:40% vol/vol), and Waters 125A (10 µ, 300 × 3.9mm) C18 column is used. The method is linear in the range of 2–16 μg/mL.

    A new sensitive bioanalytical method is developed on Shimadzu, UV-1800 spectrophotometer for the estimation of ivabradine hydrochloride in rats. Analysis is carried out at λmax, 287 nm. The developed method is optimized and validated according to USFDA guidelines. The sensitivity of the method LLOQ is determined at 500 ng/mL. The method is linear in the range of 500–3500 ng/mL. All the validation parameters were found to be within the acceptable limits. As the method is sensitive, it is successfully applied to the analysis of ivabradine hydrochloride in spiked rat plasma.

    Developed RP-HPLC method is sensitive and reproducible, which can be used for routine QC of ivabradine hydrochloride dosage forms. Bioanalytical method developed can be used for pharmacokinetic studies of new formulations developed for ivabradine hydrochloride.

    Financial support and sponsorship

    Nil.

    Conflicts of interest

    There are no conflicts of interest.



     
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        Figures

      [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
     
     
        Tables

      [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12], [Table 13], [Table 14], [Table 15], [Table 16], [Table 17], [Table 18], [Table 19], [Table 20], [Table 21], [Table 22], [Table 23]



     

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