INTRODUCTION:

Metoprolol is chemically (RS)-1-(isopropylamino)-3-[4-(2-methoxyethyl) phenoxylpropan-2-ol. It is a beta-adrenergic blocking agent used in the treatment of hypertension. Telmisartan is an angiotensin II receptor antagonist (angiotensin receptor blocker, ARB) used in the management of hypertension, chemically it is known as 2-(4{[4-Methyl-6-(1-methyl-1H-1,3-benzodiazol2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl}phenyl) benzoicacid. Chlorthalidone is chemically 2-Chloro-5(1-hydroxy-3-oxo-2,3-dihydro-1Hisoindol-1yl) benzene-1-sulphonamide.

It is used in the treatment of High blood pressure, congestive heart failure, symptomatic edema, renal tubularacidosis and prevention of kidney Stones. A new combination of metoprolol, telmisartan and chlorthalidone is available in market as MetXL3D, This combination of drugs are found to be beneficial in thetreatment of hypertension.

Figure 1: Chemical structure of Metoprolol.

Figure2: Chemical structure of telmisartan.

Figure3: Chemical structure of chlorthalidone.

A Thorough computer assisted Literature survey revealed the availability of few analytical methods like HPLC, ^[5-12]UV^[4]and HPTLC^[3]for the determination of metoprolol, telmisartan and chlorthalidone either individually or in combination with other drugs. There is no HPLC method available for above combination of the afore mentioned drugs. The present research work, is aimed to carry out the reverse phase HPLC method for simultaneous estimation of Metoprolol, Telmisartan and Chlorthalidone to meet the ICH and various regulatory authority guidelines. The newly developed method was validated in accordance with the analytical parameter mentioned in the ICH guidelines.^{[1, 2]}

MATERIALS AND METHODS:

Instrumentation:

Chromatography Seperation was performed with Shimadzu LC 10 AT HPLC with Class VP integrated software, Shimadzu Electronic Balance LAB-India pH Meter and Sonicator from Remi, India were also used for the present study

Chemicals and Reagents:

Metoprolol, Telmisartan and Chlorthalidone were generously given as a gift sample by spectrum pharma research solutions, Hyderabad. The commercial formulation (MET XL 3D) tablets contains (Metaprolol=25mg, Chlorthalidone=6.25 mg and Telmisartan=40 mg) were procured from local pharmacy. HPLC grade acetonitrile, sodium dihydrogen ortho phosphate, potassium dihydrogen ortho phosphate and ortho phosphoric acid were purchased from Merk chemicals, India. HPLC grade water used in operation were obtained from Millipore, Bangalore.

Chromatographic condition:

HPLC separation was achieved by using Phenomenex C₁₈column (250×4.5mm, 5µ) with phosphate buffer (pH adjusted to 3.0): acetonitrile as mobile phase in gradient mode at a flow rate of 1ml/min and UV detection at 310 nm.

Preparation of 1M ortho phosphoric acid solution:

Dilute 6.5 ml ortho phosphoric acid solution in to 100ml of water.

Mobile phase preparation:

Solution A:

Dissolve 15.6gm of sodium dihydrogen ortho phosphate in 100ml of water and add 16 ml of 1M ortho phosphoric acid solution and dilute to 2000 ml with water. If necessary adjust the pH 3.0 with 1M potassium dihydrogen ortho phosphate solution or 1M ortho phosphoric acid solution. Filter the solution through 0.45 µm nylon membrane filter and degas it.

Solution B: Acetonitrile:

Diluent preparation:

Mixwell 75 volume of solution of solution A and 25 volume of solution B then degas it.

Preparation of standard stock solution:

Accurately weighed and transferred 10 mg of Metoprolol, 10 mg Chlorthalidone and 10 mg of Telmisartan working standards in to a separate 10 ml clean, dry volumetric flasks separately, added 3/4^thvolume of diluent, sonicated for 30 minutes and made up to the final volume to get the stock solution with concentration of 1000 µg/ml, respectively. Further dilution was made to get the final concentration of 100 µg/ml.

Preparation of working standard solution:

Aliquots of 0.5, 1, 1.5,2 and 2.5 ml of Metoprolol,0.2, 0.4, 0.6, 0.8 and 1 ml of Chlorthalidone and 1, 2, 3, 4 and 5ml of Telmisartan were pipetted out from the above stock solution and transferred in to a 10 ml volumetric flask and volume was made up to 10 ml with diluents. This gives solution of 5, 10, 15, 20 and 25 µg/ml of Metoprolol, 2, 4, 6, 8 and 10 µg/ml of Chlorthalidone and 10, 20, 30, 40, and 50 µg/ml of Telmisartan respectively.

Sample preparation:

Twenty tablets were weighed, powdered and its average weight equivalent to 1 tablet was transferred in to a 100 ml volumetric flask, 70 ml of diluent added and sonicated for 30 min, further the volume made up with the diluent and filtered. Further dilution were made to get the concentration of 100 µg/ml. From the above filtered solution, pipette out the drug solution to get the dilution of 15 µg/ml of Metoprolol, 6 µg/ml of Chlorthalidone and 30 µg/ml of Telmisartan.

Method validation:

After method development, the validation of the current method has been performed in accordance with the ICH guidelines for assay determination which include Accuracy, Precision, Specificity, Linearity and Range, Robustness and Ruggedness.

System suitability testing:

Data from six injection of 10 µl of the working standard solution of Metoprolol (15 µg/ml), Chlorthalidone (6 µg/ml) and Telmisartan (30 µg/ml) were used for the evaluation of system suitability parameters like tailing factor, number of theoretical plates, retention time and resolution factor.

Specificity:

The specificity study was carried out to verify the interference from the excipient used in the formulation by comparing the chromatogram obtained from the drugs to that of the placebo. Placebo solution was prepared by mixing all the excipients used in the formulation. It was then injected in to HPLC system. Blank solution also injected in to the HPLC system.

Accuracy:

Accuracy of a measurement is defined as the closeness of the measured value to the true value. Accuracy may after be expressed as percent recovery by the assay of the known added amount of analyte.

Previously analyzed sample of Metoprolol, Chlorthalidone and Telmisartan in to which known amount of standard Metoprolol (15 µg/ml), Chlorthalidone (6 µg/ml) and Telmisartan (30 µg/ml) corresponding to 50%, 100% and 150% of target concentration were added. The accuracy was expressed as the percentage of analyte recovered by the proposed method.

Precision:

Precision of analytical method is usually expressed as the standard deviation and relative standard deviation. The repeatability and intermediate precision were determined by analyzing the sample of Metoprolol (15 µg/ml), Chlorthalidone (6 µg/ml) and Telmisartan (30 µg/ml).

RSD = S/x, as percentage (S/xX100)

S = Standard Deviation

X = Mean

Linearity and Range:

Linearity determines how well the graph of the analytical response verse amount of analyte follows a straight line. By taking appropriate aliquots of the standard metoprolol, Chlorthalidone and telmisartan solution with the diluent, six working solution ranging between 5-30 µg/ml Metoprolol, 2-10 µg/ml Chlorthalidone and 10-50 µg/ml of Telmisartan were prepared. Each experiment linearity point was performed in triplicate according to optimized chromatographic conditions. The peak area of the chromatogram was plotted against the concentration of Metoprolol, Chlorthalidone and Telmisartan to obtain the calibration curve.

Robustness:

For demonstrating the robustness of the developed method experimental conditions were purposely altered and evaluated. The method must be robust enough to withstand such slight changes in chromatographic conditions and allow routine analysis of the sample.

Limit of detection (LOD):

The lowest amount of analyte in the sample that can be detected, but not necessary quantified, with acceptable precision and accuracy was determined by comparison of signal to noise ratio value of standard solution with that of the blank. The general signal to noise ratio value for the lowest amount of the detectable analyte found to be 3.

Limit of quantification (LOQ):

The lowest amount of analyte in the sample that can be quantified, with acceptable precision and accuracy was determined by comparing the signal to noise ratio of standard solution with that of the blank. The general signal to noise ratio value for the lowest amount of the detectable analyte found to be 10.

LOD and LOQ were calculated by using following equations

LOD = (3.3× Syx) /b

LOQ = (10.0× Syx) /b

Where sayx is residual variance due to regression; b is slope.

Stability:

The sample solution was injected at 0 hour (comparison sample) and after 24 hour (stability sample) by keeping at ambient room temperature. Stability was determined by using % RSD for sample and standard solutions.

RESULTS AND DISCUSSION:

Method development:

Initially an attempt was made by using various ratio of methanol and acetonitrile in isocratic condition to achieve a reverse phase chromatographic separation, which results in unsuccessful separation of the 3 components. Hence the mobile phase composition was further investigated to get optimum separation of the 3 drugs. The pH of the mobile phase was adjusted to improve the tailing factor. Therefore phosphate buffer: acetonitrile in the ratio of 75:25 v/v were chosen as mobile phase at a flow rate of 1ml/min, in which all the 3 components are eluted but the peaks are very close to each other. To get better separation of the components, system were changed to gradient mode as shown in table 1. Hence phosphate buffer (pH adjusted to 3.0) and acetonitrile were selected as mobile phase in gradient mode at a flow rate of 1ml/min to get separation of all the 3 drugs with high resolution good peak symmetry.

During the development trial several analytical column with different types were tried and finalized in Phenomenex C₁₈ column (250×4.5mm, 5µ) to get improved resolution of the peaks. To detect the drugs various wavelength were tried. Maximum absorption of metaprolol, telmisartan and chlorthalidone were found to be 310nm, hence it is selected as a detection wavelength. The retention time of the Metoprolol, Telmisartan and Chlorthalidone were found to be 4.5, 7.2 and 11.01 respectively. Optimized chromatographic condition and chromatogram are given in table 2and fig 4 respectively.

Table 1: Gradient elution

Time (minute)	Solution A (Phosphate buffer)	Solution B (Acetonitrile)
0.00	75.0	25.0
15.00	75.0	25.0
16.00	50.0	50.0
20.00	0.0	100.0
22.00	50.0	50.0
23.00	75.0	25.0
30.00	75.0	25.0

Table 2: Optimized chromatographic conditions

Parameter	Condition
HPLC column	Reverse phase C₁₈ Phenomenex
Column temperature	30°C
Column dimension	250x4.5 mm× 5µ
Mobile phase	Phosphate buffer (pH adjusted to 3.0) :acetonitrile
Injection volume	10 microliter
Flow rate	1ml/min
Wavelength	310 nm
Run time	30 minute
Retention time (metoprolol)	4.5
Retention time (telmisartan)	7.2
Retention time (chlorthalidone)	11.01

Figure 4: Optimized chromatogram of standard drugs

Table-3: Retention time and peak area of the standard drugs

Peak Number	Retention Time in minute	Peak Area
1	Metoprolol (4.5)	654
2	Telmisartan (7.2)	650
3	Chlorthalidone (11.01)	710

Method validation:

After method development, the validation of the current method has been performed in accordance with the ICH guidelines for assay determination, Accuracy, Precision, Specificity, Linearity, Range and robustness.

System suitability parameter:

System suitability parameters for Telmisartan, Metoprolol and Chlorthalidone were determined. The results are given in the table 4. The method takes only 30 minutes of run time for the separation all drugs. The plate count were found to be ˃ 2000, tailing was ˂ 2 and the % RSD for telmisartan, metoprolol and chlorthalidone were found to be 0.55, 0.23 and 0.34 respectively.

Table 4: System suitability parameters of Telmisartan, Metoprolol andChlorthalidone

System suitability parameters	Metoprolol	Telmisartan	Chlorthalidone
Tailing factor (T)	1.471	1.032	1.333
Theoretical plates (N)	5778	4311	6778
%RSD	0.55	0.23	0.34

Specificity:

Specificity was determined by injecting blank, placebo, and standard solution separately and resultant chromatograms were compared with the chromatogram to check the interference. This method is specific, since there no interfering peak was observed.

Accuracy:

To pre-analyzed sample solution, a definite concentration of standard drugs (50%, 100% and150% level) was added. The percentage recoveries for Metoprolol, Telmisartan and Chlorthalidone are within acceptable limit of 98-102%. The % RSD for the above drugs are found to be less than 2. Hence the proposed method is accurate and the results are shown in table 5 and 6 respectively.

Table 5: Accuracy data for Metoprolol, Telmisartan and Chlorthalidone

Pre-analyzed Amount (µg/ml)			Spiked amount (µg/ml)			% Recovery
Met	Tel	Chl	Met	Tel	Chl	Met	Tel	Chl
15	30	6	7.5	15	3	99.90	99.01	99.25
15	30	6	7.5	15	3	99.45	99.22	99.46
15	30	6	7.5	15	3	99.20	99.90	98.76
15	30	6	15	30	6	99.45	98.99	98.43
15	30	6	15	30	6	98.55	99.43	99.10
15	30	6	15	30	6	98.67	98.56	98.89
15	30	6	22.5	45	9	99.34	98.78	98.89
15	30	6	22.5	45	9	98.34	99.56	99.65
15	30	6	22.5	45	9	100.05	99.04	98.76

Table 6: Percentage Mean Recovery Studies for Metoprolol, Telmisartan and Chlorthalidone

	Metoprolol	Telmisartan	Chlorthalidone
Mean recovery	99.22%	99.17%	99.02%
SD	0.122	0.122	0.09488
%RSD	0.115	0.115	0.077

Precision:

Precision of analytical method is usually expressed as the standard deviation and relative standard deviation. The precision of the test method was performed by injecting 6 replicates standard preparation and the % RSD was calculated (Table 7)

Table 7: Results of System Precision

Sample No	Area of Telmisartan	Area of Metaprolol	Area of Chlorthalidone.
1	254	567	656
2	254	561	675
3	243	544	644
4	265	564	668
5	254	544	645
Mean	247	545	644
S.D	2655	2677	3331
% R S D	0.20	0.10	0.54

Linearity:

Linearity determine how well the graph of the analytical response verse amount of analyte follow a straight line. Metoprolol showed a linearity of response between 5-25 µg/ml, Telmisartan showed a linearity of response between 10-50 µg/ml and for Chlorthalidone linearity of response was between 2-10 µg/ml, the results are given in table 8, 9and fig 5, 6and 7respectively.

Table 8: Linearity data for Metoprolol, Telmisartan and Chlorthalidone

Metoprolol		Telmisartan		Chlorthalidone
Concentration µg/ml	Peak area	concentration µg/ml	Peak area	Concentration µg/ml	Peak area
5	197	10	278	2	242
10	375	20	548	4	498
15	575	30	845	6	737
20	785	40	1120	8	982
25	971	50	1405	10	1222

Table 9: Analytical performanceparameters for Metoprolol, Telmisartan and Chlorthalidone

Parameter	Metoprolol	Telmisartan	Chlorthalidone
Linearity Range	5-25 µg/ml	10-50 µg/ml	2-10 µg/ml
Correlation Coefficient	0.999	0.999	0.998

Figure 5: Linearity curve of Metoprolol

Figure 6: Linearity curve of Telmisartan

Figure 7: Linearity curve of Chlorthalidone

Robustness:

Few chromatographic conditions are deliberately altered to evaluate the robustness of the developed HPLC method. The resolution was established by changing the flow rate, column temperature and composition of the mobile phase within allowable limits from actual chromatographic conditions. It was obtained that were no marked change in mean retention time and the % RSD was within the limit. The results are shown in table 10, 11, 12 and13 and figure 8, 9, 10and11.

Table 10: Robustness –flow rate (0.9 ml/min)

System Suitability Parameter	Metoprolol	Telmisartan	Chlorthalidone
Tailing Factor	1.333	1.412	1.343
Platte Count	2522	3522	4532

Table 11: Robustness- flow rate (1.1ml/min)

System Suitability Parameter	Metoprolol	Telmisartan	Chlorthalidone
Tailing Factor	1.333	1.212	1.012
Platte Count	2621	3522	4364

Table 12: Robustness-temperature (25°C)

System suitability parameter	Metoprolol	Telmisartan	Chlorthalidone
Tailing Factor	1.022	1.333	1.012
Platte Count	2621	3522	4522

Table 13: Robustness-temperature (35°C)

System suitability parameter	Metoprolol	Telmisartan	Chlorthalidone
Tailing Factor	1.111	1.322	1.022
Platte Count	4563	2621	4522

Figure 8: Chromatogram of Robustness– Flow variation (0.9ml/min) of Metaprolol, Telmisartan and Chlorthalidone

Figure 9: Chromatogram of Robustness – flow variation (1.1 ml/min) for Metoprolol, Telmisartan and Chlorthalidone

Figure 10: Chromatogram of Robustness – Temperature (25°C) of Metoprolol, Telmisartan and Chlorthalidone

Figure 11: Chromatogram of Robustness – Temperature (35°C) of Telmisartan, Metoprolol and Chlorthalidone

Limit of detection (LOD) and Limit of quantification (LOQ):

LOD and LOQ values for drugs are found to be lo lower, hence the developed method is highly sensitive. The results are given in the table-14.

Table-14: Results of LOD and LOQ

SAMPLE	LOD	LOQ
Metoprolol	0.79 µg/ml	2.40 µg/ml
Telmisartan	0.76 µg/ml	2.28 µg/ml
Chlorthalidone	0.13µg/ml	0.42 µg/ml

Stability of sample solution:

The sample solution injected after 24 hours by keeping at ambient room temperature did not show any appropriate change. The deviation in the assay is not more than 2 and the results are shown in table 15.

Table15: Stability data of metoprolol, Telmisartan and Chlorthalidone

Drug	% Assay at 0 hour	% Assay at 24 hour	Deviation
Metoprolol	99.87	98.65	0.87
Telmisartan	99.85	99.12	0.52
Chlorthalidone	100.11	99.47	0.45

Assay:

The percentage labeled claim for Metoprolol, Telmisartan and Chlorthalidone in their formulation (Met XL 3D) were found to be 99.96%, 99.76% and 99.83% individually. The % RSD values for drugs were within the acceptable limit (≤ 2) and the result were given in table-16 and figure 12, respectively.

Table 16: Assay results of Metoprolol, Telmisartan and Chlorthalidone (n=6)

S. No	Drug	Percentage Label Claim
1	Metoprolol	99.96
2	Telmisartan	99.76
3	Chlorthalidone	99. 83

Figure 12: Assay chromatogram of metoprolol, telmisartan and chlorthalidone

CONCLUSION:

A new Gradient RP HPLC method described in the present research work provides a simple, convenient and reproducible approach for the simultaneous estimation of Metoprolol, Telmisartan and Chlorthalidone. This method was thoroughly validated, therefore it can be recommended for routine analysis and for checking quality during the stability studies of the cited drugs. The method employed Phenomenex C₁₈ column (250×4.5mm× 5µ) as stationary phase, phosphate buffer (pH adjusted to 3.0): acetonitrile were used as mobile phase in gradient mode of separation with a flow rate of 1ml/min. In this method, the number of theoretical plate were above 2000. The retention time of Metoprolol, Telmisartan and Chlorthalidone were found to be 4.5, 7.2 and 11.01 min respectively. Tailing factor is less than 2 and %RSD of peak area is less than 2. The regression coefficient r²was nearly0.999 and all the drugs are found to be follow the straight line. The percentage recoveries obtained from accuracy study ranged between 98-102% showed that the method is accurate. The lowest LOD and LOQ values suggest that the method is sensitive. The solution stability studies of the drugs shows that the method is stable up to 24 hrs. A small variation in the robustness studies did not show any variation which showed the reliability of the method. Therefore the developed method can be used for routine quality control analysis of the above mentioned drugs.

ACKNOWLEDGEMENT:

Authors are thankful to their respective management for providing facilities and co-operation to carry out the present research work

CONFLICT OF INTERSET:

The authors have no conflict of interest.

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Received on 09.10.2018 Modified on 01.12.2018

Asian J. Research Chem. 2018; 11(6): 827-834.

DOI: 10.5958/0974-4150.2018.00146.3