A Review of Analytical Methods for the Estimation of Ivabradine and Metoprolol in Pharmaceutical Formulations and Biological Matrices

M. M. Eswarudu*, B. Vinay Kumar, P. Srinivasa Babu

Department of Pharmaceutical Analysis, Vignan Pharmacy College, Vadlamudi, 522213, Andhra Pradesh, India.

*Corresponding Author E-mail: eswarmunnangi@gmail.com

ABSTRACT:

The combination of Ivabradine (IVA) and Metoprolol (MET) was approved by US-FDA for symptomatic treatment of chronic stable angina pectoris. This Review focuses on recent development in analytical methods for IVA and MET. This review article represents the collection and discussion of various analytical methods available in the literature for the determination of IVA and MET individually and combination available in pharmaceutical and biological samples consisting of HPLC, HPTLC, GC, Thermal methods like TGA, DTA, DSC, and hyphenated techniques such as LC-MS, LC-MS/MS, and UPLC-MS/MS. All reported methods found to be simple, accurate, economic, precise and reproducible in nature. The present review article can be effectively explored to conduct future analytical investigation for the estimation of Ivabradine and Metoprolol.

KEYWORDS: Angina pectoris, Ivabradine, Metoprolol, Analytical Methods, RP-HPLC, LC-MS/MS.

INTRODUCTION:

Ivabradine is a Hyperpolarization-activated, cyclic nucleotide-gated (HCN) Channel Blocker used to reduce the risk of hospitalization for worsening heart failure in adult patients and for treatment of stable symptomatic heart failure as a result of dilated cardiomyopathy in pediatric patients. Chemically Ivabradine is 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. The molecular formula and molecular weight of Ivabradine is C₂₇H₃₆N₂O₅ and 468.594g/mol. It is soluble in ethanol, Dimethyl sulfoxide, and dimethyl formamide¹. Ivabradine lowers heart rate by selectively inhibiting If channels ("funny channels") in the heart in a concentration-dependent manner without affecting any other cardiac ionic channels (including calcium or potassium). IVA binds by entering and attaching to a site on the channel pore from the intracellular side and disrupts If ion current flow, which prolongs diastolic depolarization, lowering heart rate.

The If currents are located in the sinoatrial node and are the home of all cardiac pacemaker activity. Ivabradine therefore lowers the pacemaker firing rate, consequently lowering heart rate and reducing myocardial oxygen demand. This allows for an improved oxygen supply and therefore mitigation of ischemia, allowing for a higher exercise capacity and reduction in angina episodes¹. The chemical structure of Ivabradine is shown in Figure 1. Available some marketed formulations of Ivabradine are listed in Table 1³.

Figure-1: Chemical Structure of Ivabradine

Metoprolol is a beta-blocker used in the treatment of hypertension and angina, and used to reduce mortality due to myocardial infarction. Chemically Metoprolol is 1-[4-(2-methoxyethyl) phenoxy]-3-(propan-2-ylamino) propan-2-ol. The Molecular formula and molecular weight of Metoprolol is C₁₅H₂₅NO₃ and 267.3639 g/mol. It is soluble in water, methanol and sparingly soluble in ethanol. Metoprolol is a beta-1-adrenergic receptor inhibitor specific to cardiac cells with negligible effect on beta-2 receptors. This inhibition decreases cardiac output by producing negative chronotropic and inotropic effects without presenting activity towards membrane stabilization nor intrinsic sympathomimetics². The structure of Metoprolol is shown in Figure 2. Available some marketed formulations of Metoprolol are listed in Table 2⁴.

Figure-2: Chemical Structure of Metoprolol

Both combination of Ivabradine and Metoprolol drugs are used for the treatment of Angina (heart-related chest pain). Literature survey revealed that, reported methods like HPLC, GC HPTLC, LC-MS, LC-MS/MS, UPLC-MS/MS, TGA, DTA and DSC for simultaneous estimation of Ivabradine and Metoprolol in bulk, pharmaceutical dosage form, biological matrices and Impurity profiling in fixed dose combination. The aim of the present review depicts the information about the various methods reported for the determination of Ivabradine and Metoprolol including official pharmacopeial methods. List of trade names of Ivabradine and Metoprolol Combination are shown in table 3⁵.

Table 1: List of some marketed formulations of Ivabradine

S. No.	Brand Name	Name of the drug and Strength	Manufactured Company
1	Bradia	Ivabradine – 5 mg	Biocon biologics India limited, India.
2	Cora brad	Ivabradine – 5 mg	Mankind Pharma Ltd, India
3	Coralan	Ivabradine – 5 mg,7.5 mg	Ono Pharmaceutical Co., Ltd., Japan
4	Inapure	Ivabradine – 5 mg,10 mg	Sun Pharmaceuticals Ltd, India
5	Ischevia	Ivabradine – 5 mg	Dr. Reddy’s Laboratories Ltd., India
6	Ivabeat	Ivabradine – 5 mg,7.5 mg	Cipla Ltd, India
7	Ivabid	Ivabradine – 5 mg	Torrent Pharmaceuticals Ltd., India
8	Ivables	Ivabradine – 5 mg,10 mg	Lloyd Healthcare Pvt Ltd., India
9	Ivabrad	Ivabradine – 5 mg	Lupin Pharma Ltd., India
10	Ivabratco	Ivabradine – 5 mg	Natco Pharma Ltd., India
11	Ivamac	Ivabradine – 5 mg	Macleods Pharmaceuticals, UK
12	Ivamax	Ivabradine – 5 mg,7.5 mg	Johnlee Pharmaceuticals Pvt Ltd., India
13	Ivangin	Ivabradine – 5 mg	Zydus Cadila, India
14	Ivanode	Ivabradine – 5 mg	Torrent Pharmaceuticals Ltd
15	Ivarest	Ivabradine – 5 mg	Ergos Life Sciences, India

Table 2: List of some marketed formulations of Metoprolol

S. No.	Brand Name	Name of the drug and Strength	Manufactured Company
1.	Toprol XL	Metoprolol succinate, 100 mg	Astra Zeneca pharmaceutical industry company, Sweden.
2.	Lopressor	Metoprolol tartrate, 100 mg	Novartis Pharmaceutical Company, India.

Table 3: List of trade names of Ivabradine and Metoprolol Combination

S. No.	Brand Name	Name of the drug and Strength	Manufactured Company
1.	Ivabrad M TAB	Ivabradine 5 mg Metoprolol 50mg	Lupin Pharma Ltd., India
2.	Ivamet XL TAB	Ivabradine 5 mg Metoprolol 50mg	Ajanta Pharma Ltd., India

Table 4: Reported Analytical Methods of Ivabradine

S. No.	Method	Method description	Ref. No.
1	HPLC	Stationary phase: Chiral HPLC column Mobile phase: n-hexane and isopropanol: 0.1% triethylamine (60:40) Wavelength: 286 nm	6
2	HPLC	Stationary phase: Nova - Pak C8 (150x4.6 mm, 4 µm) column Mobile phase: Acetonitrile:0.025M Potassium dihydrogen phosphate (22:78 v/v) Plasma Wavelength: 328 nm Linearity: 0.5-100 ng/ml; LOQ: 0.5 ng/ml Urine Wavelength: 283 nm; Linearity: 20-500 ng/ml LOQ: 2.0 ng/ml	7
3	HPLC	Stationary phase: Thermosil C18 (150 × 4.5 mm, 5μm) column Mobile phase: methanol and phosphate buffer pH 6.5 in the ratio of (65:35 v/v) Wavelength: 265nm; Flow Rate: 1 ml/min Retention Time: 4.36 min; Injection Volume: 80 µL Linearity Range: 30-150 µg/ml LOD:  2.97 ng/ml; LOQ:  9.92 ng/ml	8
4	HPLC-UV-DAD	Stationary phase: Knauer C8 (250x4.6 mm, 5 µm ID) Hypersil Gold C8(150x4.6 mm, 5 µm ID) Zorban C8(150x4.6 mm, 5 µm ID) Supelco C18(250x4.6 mm, 5 µm ID) Mobile phase: Acetonitrile:20 mM Ammonium acetate (40:60%v/v) Flow Rate:  1 ml/min; Wavelength (1): 207 nm LOD:0.33µg/ml; LOQ: 1.09 µg/ml Wavelength (2): 286 nm LOD: 1.19 µg/ml; LOQ: 3.97 µg/ml	9
5	RP-HPLC	Stationary phase: SS Wakosil C18 AR, 250 x 4.6 mm,5 µm column) Mobile phase: Methanol:25 mm phosphate buffer (60:40v/v), adjusted to P^H6.5 with orthophosphoric acid Wavelength: 285 nm; Flow Rate: 0.8 ml/min Retention Time: 6.55±0.05 min; Linearity Range: 30-210 µg/ml; Injection Volume: 10 µL Tailing Factor: 1.14; Run Time: 10 mi	10
6	RP-HPLC	Stationary phase: C18 column (VP-ODS, 150x4.6mm, 5µm) Mobile phase: Buffer (P^H7.3): Methanol: Acetonitrile(55:15:30v/v) Wavelength: 285 nm; Flow Rate: 1 ml/min Retention Time: 7.46 min; Linearity: 50%-150% Injection Volume: 20 µL; Run Time: 15 min LLOQ: 80.60 µg/ml; ULOD: 241.80 µg/ml	11
7	RP-HPLC	Stationary phase: Inertsil ODS-3V (250 mmx4.6 mm ,5 µm) column Mobile phase: 0.5% Formic acid (P^H-7.0): Acetonitrile(65:35v/v) Wavelength: 286 nm; Flow Rate: 0.7 ml/min Retention Time: 7 min Linearity Range: 4.2-31.6 µg/mL Injection Volume: 10 µL; Run Time: 12 min LOD: 0.06 µgmL^-1; LOQ: 0.2 µg/mL	12
8	RP-HPLC	Stationary phase: Phenomenex Kinetex C18 (150x4.6 mm, 5 µm) column Mobile phase: 10 mM Ammonium acetate buffer (P^H-6.0): Methanol (50:50 v/v) Wavelength: 285 nm Flow Rate:  1 ml/min Retention Time: 3.1 min Linearity Range: 70.69-131.29 µg/mL Injection Volume: 10 µL	13
9	HPTLC	Stationary phase: Aluminium plate precoated with silica Gel 60 F254 Mobile phase: Chloroform: Methanol (1:1 v/v) Linearity Range: 400-2000 ng/band LOD: 20.73 ng/band; LOQ: 62.83 ng/band	14
10	HPTLC	Stationary phase: High-performance TLC plates (Kieselgel60F254 s, RP-2 F254 s, RP-8 F254 s, RP-18 F254 s) Mobile phase: Aqueous (Methanol-Water and Acetonitrile-Water) and Non-aqueous (Methanol-Acetonitrile and Methanol-dimethyl sulfoxide)	15
11	HPTLC	Stationary phase: pre-coated silica Gel aluminium plate 60 F254, using a Camag Linomat 5 sample applicator Mobile phase: Ethyl acetate:0.389M aluminium acetate in methanol (1:5v/v) Wavelength: 287 nm Linearity Range: 1200-2800 ng/band LOD: 255.86 ng/band; LOQ: 775.33 ng/band	16
12	LC-MS	Stationary phase: Symmetry C18, 20 18 33.9 mm, 5 mm (Waters) guard column in-line with a Kromasil C, 250 18 33 mm, 5µm Mobile phase: Ammonium formate buffer (20 mM) containing 0.1% trifluoroacetic acid–methanol (64:36, v/v) Flow Rate: 0.5 ml/min Injection Volume: 80 µL Linearity: 0.1 - 20 ng/ml LOD:  0.5 ng/ml; LOQ:  0.1 ng/ml	17
13	LC-MS/MS	Stationary phase: Diamonsil C18 column (150 mmx4.6 mm 3.5 µm) Mobile phase: Methanol and aqueous 5 mM ammonium acetate buffer containing 0.2% formic acid (80:20, v/v) Flow Rate: 0.1 ml/min Run Time: 7.0 min Column Temperature: 40°C Injection Volume(plasma): 5.0 µL Injection Volume(urine): 2.0 µL Linearity Range IVA: 0.1013–101.3 ng/mL Linearity Range (N-desmethylivabradine): 0.085–25.5 ng/mL	18
15	LC-HR-MS/MS	Stationary phase: Phenomenex Luna C18 (250x4.6 mm, 5 µm) column Mobile phase: Ammonium format 10 mm (P^H-3.0) and Acetonitrile Wavelength: 286 nm; Flow Rate:  0.7 ml/min Column Temperature: 30±5^0C P^H of Mobile phase: (30±0.2) %Recovery: 99.80-100.75% %RSD: <0.71% - 0.97%	19

Table 5: Reported Analytical Methods of Metoprolol

S. No.	Method	Method description	Ref. No.
1	HPLC With Fluorescence Detector	Stationary phase: Ace C18 column (5 µm, 250 mmx4.6 mm id) Mobile phase: Methanol: Water (50:50, v/v) Flow Rate:  1 ml/min Wavelength Excitation: 276 nm Wavelength Emission:   296 nm Plasma Linearity Range: 3-200 ng/ml Extraction recovery: 95.6±1.53% LOD: 1 µg/ml; LOQ: 3 µg/ml Urine Linearity Range: 5-300 ng/ml Extraction recovery: 96.4±1.75% LOD: 1.5 µg/ml; LOQ: 5 µg/ml	20
2	Preparative HPLC	Stationary phase: Symmetry C18 column (250 mm, id 30 mm, 5 µm) Mobile phase: Mixture of water (pH-3; adjusted with TFA): Acetonitrile (80:20) Injection Volume: 5 mL Flow Rate: 15 ml/min; Wavelength: 280 nm	21
3	HPLC-MS	Stationary phase: C18 2.7 µm column (Cortecs2, 1x100 mm, waters) Mobile phase: a) Water: 0.1%(v/v) of Formic Acid b) Acetonitrile: 0.1%(v/v) of Formic Acid Flow Rate: 0.25 ml/min	22
4	LC-MS	Stationary phase: Thermo scientific Hypersil Gold PFP 3 µm,150 x 2.1 mm Mobile phase: a) 20 mM Ammonium formate and 0.1%(v/v) formic acid in water b) 0.1%v/v formic acid in methanol Column Temperature: 30°C Flow Rate: 0.4 ml/min	23
5	LC-MS/MS	Stationary phase: Ultimate XB-C18 column (150 x 2.1mm ID,5 µm) Mobile phase: Methanol-Water containing 0.2% formic acid(65:35v/v) Flow Rate: 0.2 ml/min Linearity Range: 3.03-4.16.35 ng/ml; LOQ: 3.03 ng/ml	24
6	GC	Stationary phase: A fused HP5 silica capillary column (25 mm x 0.32 mm) Film Thickness: 1.05 µm of 5% phenyl methyl silicone Carrier Gas: pure helium Flow Rate: 4 ml/min Makeup Gas: Argon: methane (95:5) Flow Rate: 30 ml/min Oven Temperature: 105°C Injection Port Temperature: 200°C Detector Temperature: 325°C Purge off time: 0.5 min	25
7	GC	Stationary phase: 195 cm glass column, 2 mm Id Packed Column: a)3% JXR on Gas Chrom Q100-120 mesh b)3% OV-17 on Gas Chrom Q100-120 mesh Carrier Gas: Argon: 5% methane Flow Rate: 50 ml/min Column Temperature: 160°C Detector Temperature: 300°C Urine Conc. of Metoprolol -RSD 192.5 nanogram/ml       2.5% 85.5 nanogram/ml         4.6% Plasma Conc. of Metoprolol - RSD 88.1 nanogram/ml          1.9% 8.8 nanogram/ml             8.1% 4.8 nanogram/ml             14.5%	26
8	GC	Stationary phase: Fused-silica tubing (25 m x 0.32 mm id), persilylated at 400°C and coated with phenyl poly siloxane (10% phenyl) Carrier gas: Helium with an inlet pressure 1.4 bar Linear Velocity: 35 cm/s Makeup Gas: Argon: Methane (95:5) Flow Rate: 20 ml/min Column Temperature: 170°C Concentration Range (LR): 0-800 nmol/L	27
9	TG/DTA	SDT – Q600 modulus (TA, Instruments) Controlled by Thermal Advantage software (V.5.5.24. TA Instruments) Sample masses of C.A = 4±0.1 mg at 10°C/min Flow Rate: 50 ml/min	28
10	DSC	Q10Differential Calorimetric Modulus (TA instruments) Controlled by Thermal Advantage Software (V.5.5.24, TA instruments) Sample masses of c.a = 3.0 ± 0.1 mg at 10°C/min Flow Rate: 50 ml/min	29

Table 6: Reported Analytical Methods for Combination of Ivabradine and Metoprolol

S. No.	Method	Method Description	Ref. No.
1	RP-HPLC	Stationary phase: Agilent C18(150 mm x 4.6 mm; 5µm) Mobile phase: Buffer 0.01N KH2PO4 (pH=3.75): Acetonitrile (50:50) Flow Rate: 0.8 ml/min; Wavelength: 260 nm Retention Time: IVA:  3.309 min; MET: 2.461 min %RSD: IVA:  0.7; MET: 0.7 LOD: IVA: 0.28 µg/ml; MET: 0.41 µg/ml LOQ IVA: 0.85 µg/ml; MET: 1.23 µg/ml Injection Volume: 10 µL Run Time: 6 min Column Temperature: 30°C Linearity:  IVA: 5-30 µg/ml; MET: 25-150 µg/ml	30
2	RP-HPLC	Stationary phase: Denali C18 (150 mm x 4.6 mm,5 µm) Mobile phase: Ortho Phosphoric acid (0.1%) buffer: Acetonitrile (60:40 v/v) Flow Rate: 0.8 ml/min Wavelength: 260 nm Retention Time MET: 3.520 min; IVA:   2.290 min Linearity Range IVA:  5-30 µg/ml Linearity Range MET: 25-150 µg/ml	31
3	RP-UPLC	Stationary phase: SB C8 (100x3.0 mm, 1.8 mm) column Mobile phase: 0.01N Potassium Dihydrogen Ortho Phosphate: Acetonitrile (50:50 v/v) Flow Rate:  0.3 ml/min Wavelength: 260 nm Retention Time IVA: 1.156 min Retention Time MET: 0.810 min Linearity Range IVA: 5-30 µg/ml Linearity Range MET: 25-150 µg/ml LOD IVA:  0.03 µg/ml; MET: 0.12 µg /ml LOQ IVA:  0.08 µg /ml; LOQ MET: 0.35 µg /ml	32
4	UPLC-MS/MS	Stationary phase: Acquity BEH C18 (2.1 mm x 50 mm,1.7 µm) Mobile phase: 0.1% Formic Acid in Water: Acetonitrile Flow Rate:  0.40 ml/min; Injection Volume: 2.0 µL LLOQ: IVA: 0.2 ng/ml; (N-demethyl IVA): 0.05 ng/ml MET:  5.0 ng/ml; (α-hydroxy MET): 1.0 ng/ml	33

CONCLUSION:

The present review provides a summary of various analytical methods reported for the determination of IVA and MET in bulk, pharmaceutical formulations and also in various biological matrices like blood plasma and urine. Analytical methods consisting of chromatography, hyphenated techniques, and Thermal methods were employed for determination of IVA and MET in bulk, pharmaceutical dosage forms and biological matrix. From this survey, it is revealed that a handful of analytical methods are obtainable on HPLC and HPTLC and very few articles are available based on hyphenated methods (LC-MS/MS) and thermal methods. The reported data for analysis of IVA and MET revealed that HPLC with UV detection is the most frequent technique employed for the determination of IVA and MET in pharmaceutical dosage forms. For analysis of IVA and MET in biological matrices like blood plasma, urine LC-MS with MS detection is

appropriate since this strategy gives precise outcomes and minimal effort. Furthermore, employing MS techniques in LC offered unique selectivity and sensitivity as well as a choice of method for analysis of IVA, MET and its metabolites in biological samples. This review will be useful in further development of the analytical methods for this combination and also gives a glimpse of the drug Profile.

ACKNOWLEDGEMENTS:

The authors are thankful to Vignan Pharmacy College, Vadlamudi, for providing all necessary facilities for carrying out this review work.

CONFLICTS OF INTEREST STATEMENT:

All the authors declare that they do not have any conflicts of interest.

REFERENCES:

1. Drug Profile “Ivabradine” September, 2015 https://go.drugbank.com/drugs/DB09083.

2. Drug Profile “Metoprolol” June, 2005 https://go.drugbank.com/drugs/DB00264.

3. Brand names “Ivabradine” https://www.mims.com/india/drug/search?q=ivabradine.

4. Brand names “Metoprolol” https://www.mayoclinic.org/drugs-supplements/metoprolol-oral-route/description/drg-20071141.

5. Brand names “Combination of Ivabradine and Metoprolol” https://www.medplusmart.com/compositionProducts/Ivabradine-5-MG-and-Metoprolol-50-MG/31278.

6. Tabassum I et al. A review on analytical methods for Ivabradine determination in Pharmaceutical Dosage Forms, Pharma Tutor. 2018;6(10):26-30.

7. Pierre Klippert et. Determination of ivabradine and its N-demethylated metabolite in human plasma and urine, and in rat and dog plasma by a validated high-performance liquid chromatographic method with fluorescence detection. Journal of Chromatography B.1998; 719:125–133.

8. Md. Muzaffar-ur- Rehman, G. Nagamallika, Validated RP-HPLC Method for the Determination of Ivabradine Hydrochloride in Pharmaceutical Formulation, International Journal of Pharmaceutical Sciences and Drug Research 2017;9(5):228-233.

9. Joanna Nowakowska, Piotr Pikul, Marcin MarszaBB, and Krzesimir Ciura, Application and Validation of Simple Isocratic HPLC-UV-DAD Method with Dual Wavelength Detection for Ivabradine Determination and Its Application in the Study of Stress Degradation, Journal of Chemistry Volume 2017; 2069571.

10. Sunitha Seerapu and B. P. Srinivasan, Development and Validation of RP-HPLC Method for the Estimation of Ivabradine Hydrochloride in Tablets, Indian Journal of Pharmaceutical Sciences, 2010:72 (5): 667-671.

11. Md. Rezowanur Rahman, Md. Asaduzzaman and S. M. Ashraful Islam, Development and validation of RP-HPLC method for analysis of Ivabradine Hydrochloride in tablet dosage forms. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2012;(3):1032-1043.

12. Shweta Maheshwaria, Amit P. Khandharb, Anurekha Jaina, Quantitative Determination and Validation of Ivabradine HCL by Stability Indicating RP-HPLC Method and Spectrophotometric Method in Solid Dosage Form, Eurosean Journal of Analytical Chemistry.2010;5(1):53-62.

13. Selva Kumar P, Pandiyan K, Rajagopal K, Development and Validation of Rapid RP-HPLC Method For Dissolution Release of Ivabradine Hydrochloride in Solid Oral Dosage Form, World Journal of Pharmacy and Pharmaceutical Sciences. 2022; 3(7):1877-1888.

14. M. C. Damle, R. A. Bagwe, Development and Validation of Stability-Indicating HPTLC Method for Ivabradine HCl, An International Journal of Pharmaceutical Sciences, Pharma Science Monitor. Jan-Mar 2015: 6(1): 141-152.

15. Piotr Pikul, Joanna Nowakowska, Krzesimir Ciura, Chromatographic analysis of ivabradine on polar, nonpolar and chemically modified adsorbents by HPTLC. Journal of food and drug analysis.2013;21:165 – 168.

16. Mitesh H. Motisariya, Kalpana Govindbhai Patel, Purvi A. Shah, Validated stability-indicating high performance thin layer chromatographic method for determination of Ivabradine hydrochloride in bulk and marketed formulation: An application to kinetic study, Bulletin of Faculty of Pharmacy, Cairo University .2013; 51: 233–241.

17. Maryse Franc¸ ois-Bouchard, Gilles Simonin, Marie-Jeanne Bossant, Claire Boursier - Neyret, Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry, Journal of Chromatography B, 2000;745:261–269.

18. Chengtao Lua, Yanyan Jiaa, Jing Yanga, Xin Jina, Ying Songa, Wenxing Liua, Yi Dinga, Xiaoli Sunbn, Aidong Wena, Simultaneous determination of ivabradine and N-desmethylivabradine in human plasma and urine using a LC-MS/MS method: application to a pharmacokinetic study. Acta Pharmaceutica Sinica B, 2012;2(2):205–212.

19. Prinesh N. Patel Roshan M. Borkar, Pradipbhai D. Kalariya, Rahul P. Gangwal, Abhay T. Sangamwar, Gananadhamu Samanthulaa and Srinivas Ragampeta, Characterization of degradation products of Ivabradine by LC-HR-MS/MS: a typical case of exhibition of different degradation behaviour in HCl and H2SO4 acid hydrolysis. Journal of Mass Spectrometry, 2015;50: 344–353.

20. Bilal Yilmaz, Ali Asci, Sakir Arslan, Determination of metoprolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection, Journal of Separation Science, 2010;33:1904–1908.

21. R. Buchi Reddy, Kishor R. More, Leena Gupta, Mukesh S. Jha, Laki Magar, Identification, synthesis, isolation and characterization of new impurity in metoprolol tartrate tablets. Journal of Pharmaceutical and Biomedical Analysis.2016;117:104–108.

22. Mariani A. Ciciliati, E´der T. G. Cavalheiro, Studies of thermal behavior of metoprolol tartrate, Journal of Thermal Analysis and Calorimetry. 2019;138(5):3653-3663.

23. Sjoukje Postma-Kunnen, Jan Peter Yska, Gerard Hommema, Sikke Koopmans, Bob Wilffert, Eric N. van Roon, A validated high-resolution accurate mass LC-MS assay for quantitative determination of metoprolol and α-hydroxymetoprolol in human serum for application in pharmacokinetics. Journal of Applied Bioanalysis. June 2017;3(3): 49-57

24. Sha Li, Xingli Wang, Kelong Peng, Zhiguo Ma, Xiaoqi Zhang, Shaolian Fu, Xiaofei Li, Linlin Li, Aihua Hong and Jie Jiang, Rapid and Sensitive LC-MS/MS Method for the Determination of Metoprolol in Beagle Dog Plasma with a Simple Protein Precipitation Treatment and Its Pharmacokinetic Applications, MDPI, Molecules. 2012; 17:2663-2674.

25. Penny D. Colbourne, Glen B. Baker, and Ronald T. Coutts, A Rapid and Sensitive Electron-capture Gas Chromatographic Procedure for Analysis of Metoprolol in Rat Brain and Heart. Journal of Pharmacological and Toxicological Methods. September 1997:38(1):27-31.

26. Magnar Ervik, Quantitative determination of metoprolol in plasma and urine by gas chromatography, Actu pharmacol. et toxicol. 1975; 36, suppl. V, 136-144.

27. Ervik, Kerstin Kylberg-Hansenn and Lars Johannson, Determination of metoprolol in plasma and urine using high-resolution gas chromatography and electron-capture detection, Journal of Chromatography.1986; 381:168-174.

28. Mariani A. Ciciliati, E´der T. G. Cavalheiro, Studies of thermal behavior of metoprolol tartrate, Journal of Thermal Analysis and Calorimetry. 2019; 138(5):3653-3663.

29. Mariani A. Ciciliati, E´der T. G. Cavalheiro, Studies of thermal behavior of metoprolol tartrate, Journal of Thermal Analysis and Calorimetry.2019;138(5):3653-3663.

30. Bidkar J.S, Vare S.R, Dama G. Y., Shelke M. M. and Dhokare A. M, Development and validation of stability indicating RP-HPLC Method for the Estimation of Metoprolol and Ivabradine in Solid Dosage Form, World Journal of Pharmaceutical Research.2019; 8(7):1742-1768.

31. Sangameshwar B. Kanthale, Sanjay S. Thonte, Debarshi Kar Mahapatra, Stability indicating RP-HPLC method for the simultaneous estimation of ivabradine and metoprolol in bulk and tablet formulation, Journal of Applied Pharmaceutical Science. April 2019; 9(04):137-144.

32. Suresh Gandi, A. Manikandan, S. Venkat Rao, Novel Stability indicating RP-UPLC Method for simultaneous Determination of Ivabradine and Metoprolol drug materials in bulk and their Pharmaceutical Dosage Forms , Research Journal of Pharmacy and Technology, Volume-13(1):2020.

33. Wei Sun, Ruimin Chen, Wanshu Li, Hui Zhang, Jifeng Ye, Xiao Cuia and Chengke Huang, Simultaneous determination of ivabradine, metoprolol and their metabolites in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry and its application in a pharmacokinetic study, The Royal Society of Chemistry 2015;7:8459-8465.

34. Prajakta Gopinath Thete, Ravindranath Bhanudas Saudagar. Analytical Method Development and Validation for the Determination of Ivabradine HCl by RP-HPLC in bulk and Pharmaceutical Dosage form. Asian J. Pharm. Tech. 2019; 9(2):89-92.

35. Noopur K. Gandhi, Sindhu B. Ezhava. Stability indicating Analytical Method Development using Quality by Design (QbD) approach for simultaneous estimation of Ivabradine and Metoprolol. Research Journal of Pharmacy and Technology. 2021; 14(11):5937-4.

36. Suresh Gandi, A. Manikandan, S. Venkat Rao. Novel Stability indicating RP-UPLC Method for simultaneous Determination of Ivabradine and Metoprolol drug materials in bulk and their Pharmaceutical Dosage Forms. Research J. Pharm. and Tech. 2020; 13(1):250-254.

37. Mitesh D Phale, Purnima D Hamrapurkar. A Validated and Simplified RP-HPLC of Metoprolol Succinate from Bulk Drugs. Asian J. Research Chem. 2(2): April. -June, 2009 page 119-122.

38. M. T. Mohite, V. V. Dudhabale, K. V. Chandgude. Validation of Stability Indicating RP-HPLC, Method of Analysis for assay of Ivabradine HCl in SR Tablet. Asian J. Pharm. Ana. 2019; 9(3):133-137.

39. Nirmala Rangu, B. Chaitanya Kumari, Ganesh Akula, A Jaswanth. Formulation and Evaluation of Metoprolol Orodispersible Tablets by Super Disintegration Method. Asian J. Pharm. Res. 2018; 8(3):119-124.

40. T. E. Gopala Krishna Murthy, V. Akash. Development of Metoprolol Tartrate Sustained Release Formulations by using Modified Starches. Asian J. Res. Pharm. Sci. 2018; 8(4): 241-246.

41. Sojitra Rajanit, ViraniParas, Hashumati Raj. Absorption Correction Method for Simultaneous Estimation of Nifedipine and Metoprolol Succinate in Their Synthetic Mixture Using from Spectrophotometry. Asian J. Pharm. Tech. 2015; Vol. 5: Issue 1, Pg 13-16.

42. Sagar B. Wankhede, Nitin R. Dixit, Somnath S. Zambare, Sohan S. Chitlange. Development and Validation of RP-HPLC Method for Quantitative Estimation of Atorvastatin Calcium and Metoprolol Succinate in Combined Dose Capsule Formulation. Asian J. Research Chem. 3(3): July- Sept. 2010; Page 663-665.

43. Shailesh Dadge, Chaitali Dhale, Savita Yadav, Janhavi Rao. Stability Indicating HPTLC Method for Estimation of Metoprolol Tartrate in Bulk and in Pharmaceutical Formulation. Asian J. Research Chem. 2018; 11(4):755-762.

44. Shahana. V. P, Gayathri Ramya M, Rajesh A, Kathirvel. S. A Comprehensive Validation Method and Development of RP-HPLC for Simultaneous Estimation of Metoprolol, Telmisartan and Chlorthalidone in Bulk and its Formulation. Asian J. Research Chem. 2018; 11(6): 827-834.

Received on 15.05.2022                    Modified on 05.07.2022

Accepted on 18.09.2022                   ©AJRC All right reserved

Asian J. Research Chem. 2022; 15(6):506-512.

DOI: 10.52711/0974-4150.2022.00086