Reversed-Phase Liquid Chromatographic Internal Standard Method Using Losartan Potassium for Quantitative Estimation of Ivabradine Hydrochloride in Pharmaceutical Tablet Dosage Form and Plasma

Makhdoom HS; Saeed A; Saleem F; Sultana K; Mazhar M

Research Article

Austin J Anal Pharm Chem. 2021; 8(2): 1137.

Reversed-Phase Liquid Chromatographic Internal Standard Method Using Losartan Potassium for Quantitative Estimation of Ivabradine Hydrochloride in Pharmaceutical Tablet Dosage Form and Plasma

Makhdoom HS¹*, Saeed A¹, Saleem F¹, Sultana K¹ and Mazhar M²

¹Department of Pharmacy, University of Lahore, Pakistan

²Obsons Pharmaceuticals, Quaid-e-Azam Indutrial Estate, Lahore, Pakistan

*Corresponding author: Makhdoom HS, Department of Pharmaceutical Chemistry, University of Lahore, Pakistan

Received: November 16, 2021; Accepted: December 07, 2021; Published: December 14, 2021

Abstract

A reversed-phase liquid chromatographic method for ivabradine hydrochloride using Diode Array Detector (DAD) and internal standard technique was developed and validated according to ICH and SWGTOX guidelines. The prime objective of this study was to develop a precise and accurate method that can be equally applicable to biological (plasma) as well as non-biological (active pharmaceutical ingredient and pharmaceutical tablets) matrices. Losartan potassium was used as an internal standard due to its easy availability. After liquid-liquid extraction using acetonitrile, the ivabradine hydrochloride and internal standard were chromatographed on Agilent 1200 series HPLC system equipped with DAD detector, auto-sampler and chemstation software. Analytical separation was achieved on Agilent C-18 (5μm, 25cm x 4.6mm) reversed-phase column at 30°C column oven temperature, 10μL injection volume and 286nm wavelength. Isocratic mobile phase system comprised of 60:40 v/v ratio of HPLC grade methanol and water adjusted to pH 6.8 using orthophosphoric acid was employed with 1mL/min flow rate. The method linear range was 0.025-3μg/mL (25-3000ng/mL) for pharmaceutical tablets and plasma with the coefficient of linearity ranged 0.997-0.999. Results for precision, accuracy, recovery, stability and matrix effect studies were within acceptable limits for both plasma and tablets. Method was successfully applied to the commercial tablet products and patient plasma samples to estimate the amount of ivabradine hydrochloride.

Keywords: Ivabradine; Losartan; Plasma; Tablets; Matrix factor; Liquid chromatography

Introduction

The chemical name for ivabradine hydrochloride [1] is 3-(3-{[((7S)-3,4-Dimethoxybicyclo[4.2.0]octa-1,3,5-trien-7-yl) methyl] methyl amino} propyl)-1,3,4,5-tetrahydro-7,8-dimethoxy- 2H-3-benzazepin-2-one, hydrochloride (Figure 1). Its empirical formula is C₂₇H₃₇N₂O₅Cl which corresponds to a molecular weight of 504.24g/mol.

Figure 1: Two-dimensional structure of Ivabradine hydrochloride.

    
    
    Figure 1: Two-dimensional structure of Ivabradine hydrochloride.

Ivabradine hydrochloride is the first selective bradycardic agent which directly affects the pacemaker If current [2] of sinoatrial node. The U.S. Food and Drug Administration approved Corlanor (ivabradine) to reduce hospitalization from worsening heart failure on April 15, 2015. It blocks the hyperpolarizationactivated cyclic nucleotide-gated (HCN) channel responsible for the cardiac pacemaker If current, which regulates heart rate. In clinical electrophysiology studies, the cardiac effects were most pronounced in the Sino-Atrial (SA) node and there were no effects on ventricular repolarization and myocardial contractility [3]. It is whitish to pale yellow crystalline solid which is very much soluble in methanol as compared to ethanol, soluble in DMSO and slightly soluble in 0.1M HCL solution. Melting point of Ivabradine is 190-193°C [4].

Losartan potassium is an angiotensin II receptor antagonist which is used for the treatment of hypertension and diabetic nephropathy [5]. Its chemical name is potassium;[2-butyl-5-chloro-3-[[4-[2- (1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl] phenyl] methyl] imidazol-4-yl] methanol (Figure 2) and empirical formula is C₂₂H₂₂ClKN₆O which corresponds to a molecular weight of 460.12g/ mol [6]. It is soluble in methanol.

Figure 2: Two-dimensional structure of losartan potassium.

    
    
    Figure 2: Two-dimensional structure of losartan potassium.

Literature survey [7-19] reveals that limited analytical methods for determination of ivabradine hydrochloride employing internal standard in pharmaceutical preparations. The reported methods include few UV-Spectrophotometric and liquid chromatographic determinations using Diode array detector or tandem mass spectrometry. Most of these methods are either applicable to pharmaceutical preparations or biological samples. The one that was applicable to both type of samples utilized internal standard which is expensive and not easily available [20]. This study was aimed to develop and validate a simple, inexpensive and reliable method for determination of ivabradine that can be applicable on both biological and non-biological samples (Active pharmaceutical ingredient or pharmaceutical dosage forms). Validated method was applied to the commercially available pharmaceutical dosage forms and patient plasma. The use of internal standard rendered this method least affected by the potential matrix interferences.

Materials and Methods

Chemicals

Certified reference materials of Ivabradine hydrochloride and losartan potassium were provided by Obsons Pharmaceuticals (Lahore, Pakistan). Three commercial brands of Ivabradine marketed under brand names Coralan (5mg and 7.5mg tablets, Servier Laboratories, Pakistan), Ivaset (5mg and 7.5mg tablets, Highnoon Laboratories, Pakistan) and Ivatab (5mg tablets, Nabi Qasim Industries, Pakistan) were purchased from local market of Lahore- Pakistan. High-Performance Liquid Chromatography (HPLC) grade methanol and water were purchased from Honeywell (Seelze, Germany). Blank plasma was purchased from Biorad.

Instrumentation

Reversed Phase Liquid Chromatographic analysis of Ivabradine hydrochloride was carried out using an Agilent 1200 series HPLC system equipped with automatic injector, Chemstation software and diode array detector. Chromatographic separation was achieved on Ocatadecyl Silane (ODS) C-18 column (5μm, 25cm x 4.6mm). Other equipments used include Micro-analytical balance (Shimadzu), pH meter (Innotech), ultrasonic bath (Elmasonic), centrifuge (Eppendorf), microanalytical pipettes (Eppendorf), Nitrogen evaporator (PCi Analytics) and hot air oven (Pol-eko).

Chromatographic conditions

Isocratic mobile phase system comprised of 60:40 v/v ratio of HPLC grade methanol and water adjusted to pH 6.8 using orthophosphoric acid. 1mL/min mobile phase flow rate, 30°C column oven temperature, 10μL injection volume and 286nm wavelength were selected. The chromatographic data obtained thus was processed using Chemstation software.

Methods

Preparation of standard stock solution of Ivabradine hydrochloride (100μg/mL): Stock solution was prepared by dissolving 2.5mg of certified reference material of ivabradine hydrochloride in methanol quantity sufficient to 25mL, filtered it through 0.45μm syringe filter and stored in labeled storage flask. Solution was protected from light. The working standard solutions were prepared by appropriate dilution of this stock solution.

Preparation of internal standard stock solution of Losartan potassium (100μg/mL): Stock solution of certified standard of losartan potassium was prepared by dissolving 10mg in methanol q.s.100mL, filtered it through 0.45μm syringe filter and stored in labeled storage flask.

Preparation of mobile phase and diluent: Simple isocratic Mobile phase consisted of 600mL HPLC grade Methanol and 200mL HPLC grade water. The pH of buffer was adjusted to 6.0 with phosphoric acid. This mixture was filtered through 0.2μm cellulose acetate filter, transferred to solvent reservoir and degassed in ultrasonic bath prior to use. Mobile phase was used as diluent wherever required.

Preparation of blank sample solution of drug-free placebo tablets: 5mg powdered drug-free placebo tablets were weighed and transferred to a 100ml volumetric flask. Contents were dissolved in small amount of diluent using ultrasonic bath. Final volumes were made up to the mark using diluents, filtered through 0.45μm syringe filter and stored in labeled storage flask. This solution was used as blank placebo sample for the preparation of calibration levels, positive and negative quality control samples used in this study to evaluate matrix effect.

Preparation of calibration levels and positive Quality Control (QC) standards in drug-free placebo tablets solution: These samples were prepared by spiking appropriate volumes of stock standard solution of ivabradine hydrochloride (100μg/mL) in blank placebo tablets solution.

Preparation of test sample solution of pharmaceutical tablets: Powdered tablets containing 5mg and 7.5mg of ivabradine hydrochloride were separately weighed and transferred to a 100ml volumetric flask. Contents were dissolved in small amount of diluent using ultrasonic bath. Final volumes were made up to the mark using diluents, filtered through 0.45μm syringe filter and stored in labeled storage flasks.

Patient’s plasma sample collection: The patient’s peripheral blood samples were collected into Ethylene Diamine Tetra Acetate (EDTA) purple-top collection vials. Plasma is separated from blood samples by centrifugation at 11,000 revolutions per minute (rpm) for 10 minutes. After centrifugation, plasma layers were separated, collected in labeled glass tubes and stored at -20°C for further use.

Preparation of calibration levels and positive quality control (QC) standards in plasma: These samples were prepared by spiking appropriate volumes of stock standard solution of ivabradine hydrochloride (100μg/mL) in blank plasma to compensate matrix effect.

Extraction procedure

500μL of spiked calibration levels, positive quality control sample, negative quality control sample in matrices and test samples (pharmaceutical test sample or plasma) were taken in labeled centrifuge tubes. 100μL of internal standard solution and 2mL of acetonitrile were added in all tubes. The tubes were vortexed and then centrifuged at 11000rpm for 10 minutes. The clear supernatants were transferred to clean labeled glass tubes and evaporated using nitrogen evaporator at about 50°C. The residues were reconstituted using 200μL diluents, vortexed, filtered using 0.45μm micro-syringe filters and then transferred to labeled HPLC glass vials with inserts.

Results and Discussion

The developed RP-HPLC internal standard method for determination of ivabradine hydrochloride in biological (plasma) and non-biological (pharmaceutical tablets) matrices was validated according to the ICH guidelines [21].

Analytical method validation for Pharmaceutical tablets

Concentration levels used in the validation study were prepared in blank placebo tablets and analyzed after extraction to compensate for matrix effect. The summarized validation study results for estimation of ivabradine hydrochloride in pharmaceutical tablets using proposed RP-HPLC internal standard method is presented in Table 1.

Table 1: Results of Analytical Method Validation for Tablets.




  
    Sr. no.
    Parameter
    Results
  
  
    1
    Limit of Detection (μg/mL)
    0.01
  
  
    2
    Limit of Quantitation (μg/mL)
    0.025
  
  
    3
    Linear range (μg/mL)
    0.025-3.00
  
  
    4
    Coefficient of linearity (r2)
    0.997
  
  
    5
    Inter-day Precision (%CV)
    0.32-0.45
  
  
    6
    Intra-day Precision (%CV)
    0.61-0.87
  
  
    7
    Accuracy (%recovery)
    92.81-98.97
  
  
    8
    Stability (%CV)
    0.178



Table 1: Results of Analytical Method Validation for Tablets.

System suitability tests

It is the criterion to be considered in analytical method developments according to the pharmacopoeias. System suitability testing is an integral part of many analytical procedures. System suitability tests result are presented in Table 2.

Table 2: Results of system suitability parameter.




  
    Sr. no.
    Parameter
    Ivabradine hydrochloride
  
  
    1
    Retention time (min)
    4.56
  
  
    2
    Theoretical plates (N)
    2150
  
  
    3
    Tailing factor
    1.1
  
  
    4
    Precision (%RSD)
    0.3
  
  
    5
    Resolution (R)
    20



Table 2: Results of system suitability parameter.

Linearity study

Concentration level range 0.025-3.00μg/mL for ivabradine hydrochloride was used to conduct linearity study. Each concentration level was run in triplicate per day for five days. Linear response of ratio of ivabradine hydrochloride and losartan potassium (internal standard) was observed in this linearity range with the coefficient of correlation (r2) value ranged 0.997. The overlay chromatograms of five calibration levels and the linearity curve are shown in Figures 3 and 4 respectively. Acceptance value for coefficient of correlation should not be less than 0.985 therefore an acceptance criterion has been met.

Figure 3: Overlay chromatograms of five calibration levels of Ivabradine hydrochloride.

    
    
    Figure 3: Overlay chromatograms of five calibration levels of Ivabradine hydrochloride.

Figure 4: Linearity Curve of Ivabradine hydrochloride in Pharmaceutical Tablet Dosage Form.

    
    
    Figure 4: Linearity Curve of Ivabradine hydrochloride in Pharmaceutical
Tablet Dosage Form.

Specificity study

The mobile phase was injected as reagent blank followed by the injection of standard solution to check interference of mobile phase solvents at retention time of drug. Negative quality control sample was injected followed by the standard solution in placebo tablet matrix to access the matrix interference at retention time of drug. Absence of interfering peaks suggested that solvents and tablet matrix do not interfere in the estimation of ivabradine hydrochloride.

Analyte solution stability study

Pure drug standard solution (1μg/mL) and drug solution (1μg/ mL) in tablet matrix were subjected to stability study. Solutions were analyzed immediately after preparation and then at different time intervals (1-24 hours at room temperature). The results are shown in Table 1. The precision and accuracy/recovery were found within acceptance criteria. Therefore, drug solution as well as drug solution in tablet matrix is stable for the period of 24 hours at room temperature. Results are presented in Table 1.

Limit of detection (LOD) & Limit of quantitation (LOQ)

Three replicate injections per day for five days of different concentrations of ivabradine hydrochloride in lowest concentration ranges were evaluated to determine LOD and LOQ of proposed method. The response of the drug that can be precisely detected using proposed method was 0.01μg/mL. Signal-to-Noise (S/N) ratio at this concentration was 3.1:10 which is acceptable for estimation of LOD. The lowest concentration in the linearity curve that showed precise and accurate result selected as LOQ for the proposed method is the least concentration in the calibration curve, which is 0.025μg/mL. The S/N ratio at this concentration was 10.3:1 which is acceptable for estimation of LOQ.

Precision and accuracy

Precision and Accuracy of this method were determined at three different concentration levels of drug. Results are presented in Table 1. Both accuracy and precision were in acceptable ranges which reflected that method is accurate and precise.

Bio-Analytical method validation in plasma

The simplest method for extraction of ivabradine from plasma using acetonitrile was developed and validated using SWGTOX guidelines [22].

Linearity

Linearity was accessed in the range of 0.025-3.0μg/mL (25-3000ng/ mL) of ivabradine. Matrix effect was compensated by preparing drug concentrations in blank plasma as well as use of internal standard. The response was found linear over the entire concentration range used. The coefficient of linearity was 0.997.

Matrix effect

Matrix factor was used to determine matrix effect. Matrix factor is the ratio of response in extracted plasma to that of standard solution. Matrix factor less than one represents suppression, greater than one signifies enhancement whereas equal to one depicts that there is no matrix effect in the analytical method [23]. Matrix effect was studied in the concentration range of 10-3000ng/mL. The matrix factor determined was 0.95 which shows that there is least matrix effect causing response suppression.

Accuracy and precision

Accuracy and precision studies at three different concentrations of ivabradine (30, 1000 and 2500ng/mL) were evaluated. The precision and accuracy were found within acceptable ranges of %CV≤10 and ±20%, respectively as shown in Table 3.

Sr. no.	Parameter	Results
1	Limit of Detection (ng/mL)	10
2	Limit of Quantitation (ng/mL)	25
3	Linear range (ng/mL)	25-3000
4	Coefficient of linearity (r2)	0.999
5	Inter-day Precision (%CV)	5.21-10.60
6	Intra-day Precision (%CV)	1.14-6.32
7	Accuracy (%recovery)	93.2-99.5
8	Stability at room temp. (%CV)	5.34-11.82
9	Stability at 2-8oC (%CV)	3.61-12.13
10	Freeze and Thaw Stability (%CV)	2.98-10.17
11	Matrix Effect (Matrix Factor)	0.95

Sr. no.	Sample identity	Amount of Ivabradine obtained*
1	Coralan tablets, 5mg	4.82mg
2	Coralan tablets, 7.5mg	7.39mg
3	Ivaset tablets, 5mg	4.96mg
4	Ivaset tablets, 7.5mg	7.43mg
5	Ivatab tablets, 5mg	4.95mg
6	Plasma A	55.6ng/mL
7	Plasma B	28.7ng/mL
8	Plasma C	63.9ng/mL
*Average of three replicates.

Reversed-Phase Liquid Chromatographic Internal Standard Method Using Losartan Potassium for Quantitative Estimation of Ivabradine Hydrochloride in Pharmaceutical Tablet Dosage Form and Plasma

Abstract

Introduction

Materials and Methods

Chemicals

Instrumentation

Chromatographic conditions

Methods

Extraction procedure

Results and Discussion

Analytical method validation for Pharmaceutical tablets

System suitability tests

Linearity study

Specificity study

Analyte solution stability study

Limit of detection (LOD) & Limit of quantitation (LOQ)

Precision and accuracy

Bio-Analytical method validation in plasma

Linearity

Matrix effect

Accuracy and precision

Recovery

Stability study

Conclusion

References