INTRODUCTION:

At present scenario blood pressure and diabetics are common problems among the individuals. The diabetes and hypertension are two disorders that are found to coexist frequently. The researchers conclude that up to 75% of the patients with diabetes also have hypertension and vice-versa^1-12.

Nowadays multiple components are taking up in the market because of their synergistic effect, patient acceptance, tolerability and multiple actions.

For the study the drugs were selected are metformin, ivabradine, metoprolol and ertugliflozin. hence this investigation was undertaken to develop a new RPHPLC method for the estimation of drugs comes under the category of diabetics and cardio vascular disorders.

Metformin is first choice of antihyperglycemic drug used in the treatment of type II diabetes. The IUPAC name of the drug is 1-carbamimidamido-N,N dimethyl methanimidamide with chemical formula C₄H₁₁N_5. This drug decreases the blood glucose by gluconeogenesis¹³.

Fig.1: Structure of Metformin

Ivabradine is a novel drug used in lowering of heart rate for treating cardiovascular diseases. This drug shows its activity by inhibiting pace maker current in sinoatrial node which thereby improves the flow of blood to the myocardial muscle, resulting in the lowering of heart rate. The chemical formula of the drug is C₂₇H₃₆N₂O₅ with IUPAC name 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

Fig.2: Structure of Ivabradine¹⁴

Metaprolol is the drug used for treating cardiovascular diseases. It is a selective β1 blocker with chemical formula C₁₅H₂₅NO₃ and IUPAC name 1-[4-(2-methoxyethyl) phenoxy] -3-[(propan-2-yl)amino]propan-2-ol. This has maximum bioavailability. This drug is administered in the form of tartrate and succinate derivatives based on the formulation.

Fig.3: Structure of Metoprolol¹⁵.

Ertugliflozin is an inhibitor of type 2 sodium-dependent glucose cotransporter, which helps in the excretion of glucose. Thus, this drug is used in treating hyperglycemic condition. The chemical formula of the drug is C₂₂H₂₅ClO₇with the IUPAC name (1S,2S,3S,4R,5S)-5-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-1-(hydroxymethyl)-6,8-dioxabicyclo [3.2.1] octane-2,3,4-triol. This drug is highly bound to plasma proteins and is well absorbed¹⁶.

Fig.4: Structure of Ertugliflozin

MATERIALS AND METHODS:

Chemicals and Reagents:

Metformin, Ivabradine, Metoprolol, Ertugliflozin API is received as gift samples from Sprectrum Labs. HPLC grade Acetonitrile, Methanol, Millipore MilliQ water and Glacial acetic acid Potassium dihydrogen ortho phosphate, Acetonitrile, HPLC water, triethylamine, dilute orthophosphoric acid were purchased from Merck, Germany, Regis Technologies Inc, USA.

Instrumentation and Chromatographic Conditions:

The HPLC system is used for method development and method validation. Detection was carried by Waters with a diode array detector (model: 2996 detector 2487 separation module). The output signal was supervised and processed using Waters Empower 2 Software. LC GC Ragward Dual Range balance was used to perform weighing.

Kromasil C18 250x4.6, 5m used as stationary phase and phosphate buffer: Acetonitrile taken in the ratio 50:50 as mobile phase. Study was conducted at flow rate of 1 ml/min, at detector wave length of 245nm and run time of 10 min.

Standard Solution Preparation:

Accurately Weighed and transferred 1.25mg of Ertugliflozin, 250mg of Metformin, 2.5mg of Ivabradine and 25mg of Metoprolol working Standards into a 50ml clean dry volumetric flask, add 3/4 th volume of diluent, sonicated for 5 minutes and make up to the final volume with diluents (Water: Acetonitrile (50:50)). 1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml.

Sample Solution Preparation of Ertugliflozin and Metformin:

5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100mL. volumetric flask, 50mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution. 1ml was pipeted out into a 10ml volumetric flask and made up to 10ml with diluent. (2.5ppm of Ertugliflozin and 250ppm of Metformin)

Sample Preparation of Ivabradine and Metoprolol:

Method Validation:

The optimized HPLC method was validated with respect to the following parameters. The validation was performed as per the ICH guidelines.

System Suitability:

The system suitability parameters with respect to theoretical plates (N), peak symmetry (T), capacity factor (K), selectivity (a), HETP (H), and resolution (Rs) between of Metformin, Ivabradine, Metoprolol and Ertugliflozin peaks were deﬁned.

Linearity:

Constant volume of 20µL injections were made for each concentration six times and chromatographed under the above-mentioned conditions. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs. Linear calibration curves were generated using least-squares linear-regression analysis. Residual analysis was performed to ascertain linearity. A series of concentrations ranging from 125 to 750µg/ml for metformin, 1.25 to 7.5 for ivabradine, 6.25 to 37.5 for metoprolol, and 0.625 to 3.75µg/ml for ertugliflozin. Calibration curve was constructed by plotting the mean peak area (Y-axis) against the concentration (X-axis).

Precision:

Precision of the method was determined with the standard and the real sample. The intraday and interday variation, for determination of Metformin, Ivabradine, Metoprolol and Ertugliflozin were carried out at standard concentration levels of 500,5, 25, 2.5µgmL respectively. Method repeatability was achieved by repeating the same procedure six times on the same day for intraday precision. The intermediate (interday) precision of the method was checked by performing same procedure on diﬀerent days under the same experimental conditions. The repeatability of sample application and measurement of peak area were expressed in terms of relative standard deviation (% RSD).

Accuracy:

Accuracy of the method was carried outby applying the method to drug sample to which known amounts of Metformin, Ivabradine, Metoprolol and Ertugliflozin standard powder corresponding to 50, 100, and 150% of solutions had been added (standard addition method). At each level of the amount three determinations were performed.

Limit of Detection and Limit of Quantitation:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. LOD and LOQ of Metformin, Ivabradine, Metoprolol and Ertugliflozin were determined by calibration curve method. LOD and LOQ were calculated by using following: where S

LOD= 3.3 X SD/Slope

LOQ= 10 X SD/Slope

Robustness:

The robustness was studied by evaluating the effects of small but deliberate variations in the chromatographic conditions. The robustness of the method was studied under deliberate variations in parameters peak resolution, tailing and number of theoretical plates were evaluated. To study the outcome of the flow rate on the developed method, it was changed ± 0.2mL/minute. The effect of column temperature on the developed method was studied at ± 5°C (instead of 30°C). The mobile phase composition was changed ±10% from the initial composition of organic phase. In all the above varied conditions, the aqueous component of the mobile phase was held constant.

Assay:

Assay is a method used to determine the amount of Metformin, Ivabradine, Metoprolol, and Ertugliflozin present in pharmaceutical product. Segluromet and Ivamet XL conventional tablets bearing the label claim of 2.5mg Ertugliflozin and 500mg Metformin and 5mg Ivabradine and 25mg Metoprolol used as sample to conduct assay. Six replicate sample solutions of 28μg/ml concentration were injected and percentage purity was calculated.

RESULTS AND DISCUSSION:

Development of HPLC method and Optimization:

Kromasil C18 250x4.6, 5m used as stationary phase and Buffer: Acetonitrile: taken in the ratio 50:50 as stationary phase. Study was conducted at flow rate of 1 ml/min, at detector wave length of 245nm at run time of 10min.

Fig 5: Optimized chromatogram

System Suitability:

Linearity:

Table No 1. Linearity of Metformin

S. No	CONC. (µG/ML)	AREA
1	0	0
2	125	1629160
3	250	3211190
4	375	4876809
5	500	6480635
6	625	8014160
7	750	9743404

Fig: 6 Linearity of Metformin

Table No 2. Linearity of Ivabradine

S. NO	CONC.(µG/ML)	AREA
1	0	0
2	1.25	17151
3	2.5	34299
4	3.75	51313
5	5	68665
6	6.25	85851
7	7.5	101642

Fig: 7 Linearity of Ivabradine

Table No 3. Linearity of Metoprolol

S. No	CONC. (µG/ML)	AREA
1	0	0
2	6.25	135020
3	12.5	273742
4	18.75	404523
5	25	536063
6	31.25	680520
7	37.5	807043

Fig: 8 Linearity of Metoprolol

Table No 4. Linearity of Ertugliflozin

S. No	CONC.(µG/ML)	AREA
1	0	0
2	0.625	7055
3	1.25	13901
4	1.875	21025
5	2.5	27574
6	3.125	34555
7	3.75	40903

Fig: 9 Linearity of Ertugliflozin

Table 5: precision results

Injection	Area of Metformin	Area of Ivabradine	Area of Metoprolol	Area of Ertugliflozin
Injection-1	6508907	68803	539561	28188
Injection-2	6485553	70621	548212	27665
Injection-3	6491537	69344	538632	28068
Injection-4	6512843	68894	545171	28098
Injection-5	6518979	69411	546060	27854
Injection-6	6520948	68750	543162	27985
Avg	6505972	69404	544247	27934
SD	14650.3	703.4	3764.8	189.7
%RSD	0.2	1.0	0.7	0.7

Precision:

Precision of the method was determined with the standard and the real sample. The intraday and interday variation, for determination of Metformin, Ivabradine, Metoprolol, and Ertugliflozin

Table 6: Intermediate precision results

Injection	Area of Metformin	Area of Ivabradine	Area of Metoprolol	Area of Ertugliflozin
Injection-1	277873	297175	277873	297175
Injection-2	275427	292447	275427	292447
Injection-3	271531	290662	271531	290662
Injection-4	276641	292422	276641	292422
Injection-5	274090	291307	274090	291307
Injection-6	279951	292914	279951	292914
Avg	275528	291950	275528	291950
SD	2947.5	2289.9	2947.5	2289.9
%RSD	1.1	0.8	1.1	0.8

The repeatability of sample application and measurement of peak area were expressed in terms of relative standard deviation (% RSD). Relative standard deviation (% RSD) was less than hence it is satisfactory to requirements.

Accuracy:

Accuracy of the method was carried outby applying the method to drug sample to which known amounts of Metformin, Ivabradine, Metoprolol, and Ertugliflozin standard powder corresponding to 50, 100, and 150% of solutions had been added (standard addition method). At each level of the amount three determinations were performed. As per ICH guidelines % recovery was between 99-102%W/W.

Table 7: Accuracy results of Metformin

%	TRIAL	AREA(y)	m	C	y-c	Total Conc	Added Conc	Std Conc	Amt Rec	% Rec	AVG %Rec
50%	1	9735144	12934	445	9734699	752.6441163	250	500	252.64	101.06	100.34
	2	9690820	12934	445	9690375	749.2171795	250	500	249.22	99.69
	3	9710136	12934	445	9709691	750.7106077	250	500	250.71	100.28
100%	1	13074312	12934	445	13073867	1010.813901	500	500	510.81	102.16	102.25
	2	13083464	12934	445	13083019	1011.521494	500	500	511.52	102.30
	3	13081981	12934	445	13081536	1011.406835	500	500	511.41	102.28
150%	1	16094234	12934	445	16093789	1244.30099	750	500	744.30	99.24	99.82
	2	16232906	12934	445	16232461	1255.022499	750	500	755.02	100.67
	3	16122931	12934	445	16122486	1246.519715	750	500	746.52	99.54

Table 8: Accuracy results of Ivabradine

%	TRIAL	AREA(y)	m	C	y-c	Total Conc	Added Conc	Std Conc	Amt Rec	% Rec	AVG %Rec
50%	1	101748	13620	200.2	101547.8	7.46	2.5	5	2.46	98.23	99.90
	2	102382	13620	200.2	102181.8	7.50	2.5	5	2.50	100.09
	3	102823	13620	200.2	102622.8	7.53	2.5	5	2.53	101.39
100%	1	136274	13620	200.2	136073.8	9.99	5	5	4.99	99.81	99.61
	2	135873	13620	200.2	135672.8	9.96	5	5	4.96	99.23
	3	136248	13620	200.2	136047.8	9.99	5	5	4.99	99.78
150%	1	170038	13620	200.2	169837.8	12.47	7.5	5	7.47	99.60	100.15
	2	171365	13620	200.2	171164.8	12.57	7.5	5	7.57	100.90
	3	170422	13620	200.2	170221.8	12.50	7.5	5	7.50	99.97

Table 9: Accuracy results of Metoprolol

%	TRIAL	AREA(y)	m	C	y-c	Total Conc	Added Conc	Std Conc	Amt Rec	% Rec	AVG %Rec
50%	1	808363	21568	867.4	807495.6	37.43952	12.5	25	12.44	99.52	99.31
	2	806634	21568	867.4	805766.6	37.35936	12.5	25	12.36	98.87
	3	808385	21568	867.4	807517.6	37.44054	12.5	25	12.44	99.52
100%	1	1086649	21568	867.4	1085781.6	50.34225	25	25	25.34	101.37	100.04
	2	1072938	21568	867.4	1072070.6	49.70654	25	25	24.71	98.83
	3	1078927	21568	867.4	1078059.6	49.98422	25	25	24.98	99.94
150%	1	1352522	21568	867.4	1351654.6	62.66945	37.5	25	37.67	100.45	100.18
	2	1345215	21568	867.4	1344347.6	62.33066	37.5	25	37.33	99.55
	3	1353343	21568	867.4	1352475.6	62.70751	37.5	25	37.71	100.55

Table 10: Accuracy results of Ertugliflozin

%	TRIAL	AREA(y)	m	C	y-c	Total Conc	Added Conc	Std Conc	Amt Rec	% Rec	AVG %Rec
50%	1	41149	10936	210.8	40938.2	3.74	1.25	2.5	1.24	99.47	100.65
	2	41297	10936	210.8	41086.2	3.76	1.25	2.5	1.26	100.56
	3	41485	10936	210.8	41274.2	3.77	1.25	2.5	1.27	101.93
100%	1	55145	10936	210.8	54934.2	5.02	2.5	2.5	2.52	100.93	100.64
	2	54978	10936	210.8	54767.2	5.01	2.5	2.5	2.51	100.32
	3	55073	10936	210.8	54862.2	5.02	2.5	2.5	2.52	100.67
150%	1	68238	10936	210.8	68027.2	6.22	3.75	2.5	3.72	99.21	99.42
	2	68415	10936	210.8	68204.2	6.24	3.75	2.5	3.74	99.64
	3	68312	10936	210.8	68101.2	6.23	3.75	2.5	3.73	99.39

Limit of Detection and Limit of Quantitation:

The detection limit and quantitation limit of Metformin, Ivabradine, Metoprolol and Ertugliflozin were within the limits as per the ICH guideline. Results were shown in table no 11.

Table 11: Accuracy results of LOD and LOQ

parameter	Metformin	Ivabradine	Metoprolol	Ertugliflozin
LOD	0.06	0.03	0.09	0.02
LOQ	0.18	0.10	0.28	0.05

Robustness:

Study conducted at the flow rate on the developed method, at ± 0.2mL/minute, column temperature of ± 5°C and the mobile phase composition of ±10% from the initial composition of organic phase. System suitability parameters are reaching satisfactory results of ICH guideline. Thus, the method was found to be robust with respect to variability in applied conditions.

Table 12: Accuracy results of Metformin

S. No	Standard Area	Sample Area
1	6480619	6508907
2	6480619	6485553
3	6480619	6491537
4	6480619	6512843
5	6480619	6518979
6	6480619	6520948
AVG	6480619	6506461
% of Drug	100.20

Table 13: Accuracy results of Ivabradine

S. No	Standard Area	Sample Area
1	69452	68803
2	69452	70621
3	69452	69344
4	69452	68894
5	69452	69411
6	69452	68750
AVG	69452	69303.83
% of Drug	99.59%

Table 14: Accuracy results of Metoprolol

S. No	Sample Area	Standard Area
1	539561	536825
2	548212	536825
3	538632	536825
4	545171	536825
5	546060	536825
6	543162	536825
AVG	543466.3	536825
% of Drug	101.03

Assay:

Assay is a method used to determine the amount of of Metformin, Ivabradine, Metoprolol and Ertugliflozin present in pharmaceutical product. Segluromet and Ivamet XL conventional tablets bearing the label claim of 2.5 mg Ertugliflozin and 500mg Metformin and 5 mg Ivabradine and 25mg Metoprolol used as sample to conduct assay. Six replicate sample solutions of 28μg/ml concentration were injected and percentage purity was calculated.

Table 15: Accuracy results of Ertugliflozin

S. No	Sample Area	Standard Area
1	28188	27887
2	27665	27887
3	28068	27887
4	28098	27887
5	27854	27887
6	27985	27887
AVG	27976.33	27887
% of Drug	100.12

CONCLUSION:

A new chromatographic separation was achieved for the simultaneous estimation of Metformin, Ivabradine, Metoprolol and Ertugliflozin classified under anti-hypertensive and anti-diabetic drugs. The developed HPLC technique is precise, speciﬁc, robust, and accurate. Statistical analysis proves that the method is suitable for routine analysis of Metformin, Ivabradine, Metoprolol and Ertugliflozin in pharmaceutical dosage form. Further the same method will be used for analyzing these drugs in biological sample.

ACKNOWLEDGEMENT:

The authors wish to thank the management of Vels University, for supporting this work. The authors wish to thank Spectrum labs, for providing facilities to conduct this work. The authors wish to acknowledge the Hetero labs for providing the samples for their research. They would also like to thank colleagues for their support to complete research work.

CONFLICT OF INTEREST:

The authors have no conflict of interests to disclose other than what has been acknowledged above.

REFERENCES:

1. Available at https://www.who.int/news-room/facts-in-pictures/detail/diabetes,21st February,2018.

2. Diabetes Care. 2004; 27(5): 1047-1053. Available at https://doi.org/10.2337/diacare.27.5.1047

3. Government survey found 11.8% prevalence of diabetes in India, 2 min read. 2019; IST Neetu Chandra Sharma. Available at https://www.livemint.com/science/health/government-survey-found-11-8-prevalence-of-diabetes-in-india-11570702665713.html.

4. Available at https://www.dailyexcelsior.com/dr-rampal-delivers-lecture-on-diabetes-in-up/

5. Available at https://www.who.int/news-room/fact-sheets/detail/hypertension

6. Brenda B. Spriggs. 2019; Availble at https://www.medicalnewstoday.com/articles/150109.php

7. Anchala, Raghupathy, Kannuri, Nanda, Pant, Hira, Khan, Hassan, Franco, Oscar, Angelantonio, Emanuele, Dorairaj and Prabhakaran. Hypertension in India: A systematic review and meta-analysis of prevalence, awareness, and control of hypertension. Journal of Hypertension. 2014; 32(6): 1170–1177.

8. Smita S. Swami, Sanjay C. Swami, Vinayak W. Patil, Anuradha M. Kanhere. hypertension and diabetes in india: a review. International Journal of Clinical Biochemistry and Research. 2015;2(1):54-58.

9. Rajeev Gupta, Krishna K Sharma, Sailesh Lodha, Sunil Gupta, Anand S Meenawat, Anuj Maheshwari, Bhupendra N Mahanta, Dinesh C Sharma, Arthur J Asirvatham, Shreya Gupta and Surendra K Sharma. High-Performance Liquid Chromatography (HPLC) method for the simultaneous estimation of metformin hydrochloride and ertugliflozin in pharmaceutical formulation. Journal of the Association of Physicians of India. 2018; 66.

10. Tong-Yuan Tai, Lee-Ming Chuang, Chien-Jen Chen and Boniface J Lin. Link Between Hypertension and Diabetes Mellitus Epidemiological Study of Chinese Adults in Taiwan. Diabetes Care. 1991; 14 (11) 1013-1020.

11. David M. Maahs, Gregory L. Kinney, Paul Wadwa, Janet K. Snell-Bergeon, Dana Dabelea, John Hokanson, James Ehrlich, Satish Garg, Robert H. Eckel and Marian J. Rewers. Hypertension Prevalence, Awareness, Treatment, and Control in an Adult Type 1 Diabetes Population and a Comparable General Population.

12. Treatment of Hypertension in Adults With Diabetes. Diabetes Care. 2007;26(1):80-82.

13. Available at Drug bank, Metformin, https://www.drugbank.ca/drugs/DB00331.

14. Available at Drug bank, Ivabradine, https://www.drugbank.ca/drugs/DB09083.

15. Available at https://www.drugbank.ca/drugs/DB00264.

16. Available at https://www.drugbank.ca/drugs/DB11827.

Received on 26.02.2020 Modified on 05.04.2020

Research J. Pharm. and Tech. 2021; 14(4):2055-2061.

DOI: 10.52711/0974-360X.2021.00365