INTRODUCTION:

Atenolol is 2-(4-{2-hydroxy-3-[(propan-2-yl)amino]propoxy}phenyl)acetamide (Fig. 1). Atenolol is a beta-adrenergic blocking agent i.e. it blocks the effects of adrenergic drugs and is used in a wide range of cardiovascular conditions, for managing chronic angina and hypertension, and to decrease mortality rate in suspected or known myocardial infarction in hemodynamically stable patients. The key function of beta-adrenergic nerves is to stimulate the heart to beat faster. Atenolol blocks the stimulation of these nerves and reduces the heart rate which helps in treating abnormally rapid heart rhythms. Atenolol can be used either alone in treating hypertension or in combinationwith other anti-hypertensive agents¹.

Figure 1: Chemical structure of Atenolol

Amlodipine besylate is 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate; benzenesulfonic acid (Fig. 2). Amlodipine is used in the treatment of angina and is also a long-acting calcium channel blocker which is used for treating hypertension. It belongs to the group of drugs called dihydropyridine calcium channel blockers. Amlodipine relaxes the smooth muscle in the arterial wall, which decreases total peripheral resistance which help in reducing blood pressure; blood flow to the heart muscle is increased in angina. Amlodipine also exhibits properties of antioxidant and has the ability of enhancing the nitric oxide (NO) production, an essential vasodilator that declines blood pressure. Amlodipine can be used alone or with other antihypertensive and antianginal agents for the treatment of conditions like hypertension, chronic stable angina vasospastic angina, coronary artery disease etc².

Figure 2: Chemical structure of Amlodipine besylate

Aspirin is classified as a non-selective cyclooxygenase (COX) inhibitor, 2-(acetyloxy)benzoic acid (Fig. 3). Aspirin acts as analgesic (treat pain), migraines, inhibits platelet aggregation, prevents blood clotting and reduces inflammation and fever. It is supposed to decreased production of thromboxanes and prostaglandins which is because of irreversible inactivation of the cyclooxygenase (COX) enzyme. Cyclooxygenase is essential for the synthesis prostaglandin and thromboxane. Aspirin acts as an acetylating agent, acetyl group is covalently attached to a serine residue in the active site of the COX enzyme. Various studies demonstrated the results that acetylsalicylic acid used for long-term may decrease the risk of several cancers which includes esophageal, colorectal, lung, breast, liver, prostate and skin cancer³.

Figure 3: Chemical structure of Aspirin

Now a days, HPTLC has become a routine analytical technique because of the advantages of analyzing many sample at a time with small amount of mobile phase which saves the time and cost of analysis, minimum sample preparation and high sample throughput. Literature review reveals that methods have been reported for analysis of Atenolol, Amlodipine besylate and Aspirin by UV spectrophotometry^4-10, method development and validation either alone or in combination with other drugs^11-20 and stability indicating analytical method by HPTLC²¹.

Till date no method is reported for the simultaneous estimation of Atenolol, Amlodipine besylate and Aspirin by HPTLC in bulk drug and in capsule dosage form. For the first time the study reports simultaneous quantitation of Atenolol, Amlodipine besylate and Aspirin by HPTLC in bulk drug and in capsule dosage form which was validated as per ICH Guidelines²².

The properties of all the three drugs are stateted in table-1.

EXPERIMENTAL:

Materials:

Wockhardt Ltd., Aurangabad, Maharashtra, India provided working standards of pharmaceutical grade Atenolol (Batch No.: 174105), Amlodipine besylate (Batch No.: AB/002/7054) and Aspirin (Batch No.: ASI0710032) as generous gifts samples. These samples were analyzed without further purification and were certified to contain 99.91 % Atenolol, 99.02 % Amlodipine besylate and 99.90 % Aspirin on dried basis. Capsules (Brand Name: ATO-GUARD) containing 50 mg of Atenolol, 5 mg of Amlodipine besylate and 100 mg of Aspirin were procured from Perk Pharmaceuticals Ltd., Meerut, Uttar Pradesh, India. Analytical grade reagents and chemicals were procured from Merck Chemicals, Mumbai, India.

Instrumentation:

The spotting of samples on HPTLC plate was in the form of 6 mm (width) bands with a Camag syringe 100 microlitre sample capacity (Hamilton, Bonded, Switzerland) on precoated aluminum plate coated with silica gel 60 F₂₅₄, 250 µm thickness, dimension of 20 cm × 10 cm, Merck, Germany by using Camag Linomat V (Switzerland) sample applicator. The prewashing of the plates was done with methanol and activation of plates was done for 5 min at 110 ^oC prior to analysis. The rate of application was kept constant i.e. 0.1 µL/s and distance between the two bands was kept 6 mm. The scanning speed was kept 10 mm/s and the slit dimension was 5 mm × 0.45 mm. Twenty nm was set as monochromator bandwidth and scanning of each track was done thrice. The composition of mobile phase used was n-butanol: water: acetic acid (8: 2: 0.2 v/v/v) and was used per chromatography run. The developed of plate was carried out linearly in a twin trough glass chamber (Camag, Muttenz, Switzerland) of dimension 20 cm × 10 cm and the chamber was saturated with mobile phase for 30 min at room temperature. The mobile phase was run on plates upto a distance of 8 cm. The development HPTLC plates were dried using an air dryer. Scanning of plate were performed using a Camag TLC scanner III at 235 nm and was operated using CATS software (V 3.15, Camag). The radiation source used was deuterium lamp which emitted UV spectrum continuously between 190 and 400 nm.

Preparation of Standard Stock Solutions:

Standard stock solutions of 1000 µg/mL concentration were prepared for Atenolol, Amlodipine besylate and Aspirin in methanol. Further dilutions were carried out from the standard stock solutions and mixed solution was prepared obtaining final concentration of 100 µg/mL of Atenolol, 10 µg/mL of Amlodipine besylate and 200 µg/mL of Aspirin. The stock solution was found to be stable at 2-8 °C in refrigerator.

Optimization of the HPTLC method:

The HPTLC method was developed for simultaneous estimation of assay method for Atenolol, Amlodipine besylate and Aspirin, respectively. From the mixed standard solution (100 µg/mL of Atenolol, 10 µg/mL of Amlodipine besylate and 200 µg/mL of Aspirin) 10 µL samples were spotted on the HPTLC plates in the form of bands and was run in different mobile phases. Optimization of HPTLC method was complex in this case as all the three drugs did not move simultaneously in one mobile phase. After multiple trials it was found that polar mobile phase was required for the movement of the three drugs. Hence, n-butanol: water: acetic acid in the ratio of 8: 2: 0.2 was found optimal. For avoiding the neck less effect, twin trough glass chamber was allowed to saturate for 30 min. The mobile phase was run on plates upto a distance of 8 cm; it took approximately 45 mins of time for development of HPTLC plate. Refer Fig. 4 for densitogram.

Figure 4: Densitogram of Atenolol R_F (0.23), Amlodipine besylate R_F (0.47) and Aspirin R_F(0.70)

Mobile phase: n-butanol: water: acetic acid (8: 2: 0.2 v/v/v).

Concentration of drugs: 100 µg/mL of Atenolol, 10 µg/mL of Amlodipine besylate and 200 µg/mL of Aspirin

Application volume: 10 µL

Validation of the method:

Validation of the optimized HPTLC method was carried out as per the following parameters.

Linearity and range:

From the mixed standard solution, Atenolol, Amlodipine besylate and Aspirin of 100 µg/mL concentrations was prepared and 1 to 6 µL solutions were spotted in the form of bands on HPTLC plate to achieve concentration of 100-600 ng/spot for Atenolol, Amlodipine besylate and Aspirin. Six concentration levels were taken for performing linearity study and three replicates per concentration were applied on the HPTLC plate. The development of plate was carried out in the optimized mobile phase and the calibration graph was obtained by plotting peak areas against the concentrations.

Precision:

Precision of the optimized method was confirmed by repeatability and intermediate precision studies. Repeatability studies were executed by analyzing three different concentrations i.e. 200 ng/spot, 400 ng/spot and 600 ng/spot for Atenolol, Amlodipine besylate and Aspirin six times on the same day. Intermediate precision studies were executed by analyzing the drugs on three different days.

Limit of detection and limit of quantitation:

Limits of detection (LOD) correspond to the concentration of analyte that yields signal-to-noise ratios of 3 and limits of quantification (LOQ) correspond to the concentration of analyte that yields signal-to-noise ratios of 10. For calculating LOD and LOQ, the magnitude of analytical background is measured by spotting a blank and drug in series of concentrations and calculating the signal-to-noise ratio to obtain S/N ratio 3 and 10 for LOD and LOQ, respectively. In the current study, mixed standard solution of Atenolol, Amlodipine besylate and Aspirin was taken to undergo serial dilutions to obtain concentration in the range of 10–500 ng/spot. The spotting of samples was done on HPTLC plates and was run in mobile phase. Signal obtained for the samples was compared with that of blank.

Robustness of the method:

Small changes in the mobile phase composition were introduction by ± 0.1 mL for each component and effect of these small changes on results was studied. Different compositions of mobile phases were tried and robustness study was performed, e.g. n-butanol: water: acetic acid in the ratio of 8.1: 2: 1 v/v/v, 7.9: 2: 1 v/v/v, 8: 2.1: 1 v/v/v, 8: 1.9: 1 v/v/v. Also the mobile phase composition was varied by ± 5 %. The washing of plates prior to analysis was done with methanol and was activated at a temperature of 110 °C for 3, 6, and 8 min. From the time plates were spotted to chromatography and from chromatography to scanning were varied by 10 min. The robustness study was performed at three different concentration levels for 200 ng/spot, 400 ng/spot and 600 ng/spot for Atenolol, Amlodipine besylate and Aspirin.

Specificity:

Method specificity was performed by analyzing standard and test samples. The R_F value and spectrum obtained for Atenolol, Amlodipine besylate and Aspirin in the test samples was confirmed by comparing the R_F and spectrum with standard. The purity of peak for Atenolol, Amlodipine besylate and Aspirin was determined at three regions of the band i.e. at the start, apex and end and the spectrum were compared.

Accuracy:

Accuracy was performed on Atenolol, Amlodipine besylate and Aspirin combination capsules. To this sample known amount of Atenolol, Amlodipine besylate and Aspirin standard powder equivalent to 80, 100 and 120 % of label claim was added, sample and standard were mixed, drug was extracted using suitable solvent and were analyzed.

Analysis of a marketed formulation:

The content of Atenolol, Amlodipine besylate and Aspirin was determined in conventional capsule (Brand name: ATO-GUARD, Label claim: 50 mg of Atenolol, 5 mg of Amlodipine besylate and 100 mg of Aspirin per capsule), twenty capsules were taken, the capsules were opened and the content was removed. The weight equivalent to 50 mg of Atenolol, 5 mg of Amlodipine besylate and 100 mg of Aspirin was transferred into a 100 mL volumetric flask. About 60-70 mL methanol was added as diluent, sonicated in water bath for 30 min with intermittent shaking and diluted upto 100 mL with methanol. Resulting solution was then centrifuged at 2500 rpm for 3-5 min and concentration of the supernatant was found to be 500 µg/mL for Atenolol, 50 µg/mL for Amlodipine besylate and 1000 µg/mL for Aspirin. From the above solution 2 µL of band was applied which gave final concentration of 1000 ng/spot for Atenolol, 100 ng/spot for Amlodipine besylate and 2000 ng/spot for Aspirin. For all the three drugs the spotting was done in triplicate. Development of the plate was carried out in optimized mobile phase. The likelihood of interference of excipients with all the three standards was examined.

RESULTS AND DISCUSSION:

The results obtained after performing validation studies on developed HPTLC method for Atenolol, Amlodipine besylate and Aspirin using n-butanol: water: acetic acid (8: 2: 0.2 v/v/v) as the mobile phase are as follows-

Linearity:

The response of the drug was found linear over the concentration range between 100-600 ng/spotfor Atenolol,Amlodipine besylate and Aspirin, respectively. The regression coefficient values (R²) for Atenolol was 0.9985, for Amlodipine besylate was 0.9972 and for Aspirin was 0.9988.

Precision:

Table 2 depicts the results of repeatability and intermediate precision studies which was performed as recommended by ICH guidelines. The optimized method was found to be precise as the relative standard deviation (RSD) values for repeatability and intermediate precision studies were < 2%.

Table 2: Precision Studies

Concentration (ng/spot)	Repeatability (n=6)			Intermediate precision (n=6)
Concentration (ng/spot)	Measured conc. ± SD	(%) RSD	Recovery (%)	Measured conc. ± SD	(%) RSD	Recovery (%)
Atenolol
200	200.13 ± 3.45	0.28	100.06	199.61 ± 4.21	0.59	99.80
400	399.63 ± 5.71	0.88	99.90	394.56 ± 6.04	0.97	98.64
600	598.74 ± 7.24	0.57	99.79	600.75 ± 8.05	1.21	100.12
Amlodipine besylate
200	200.43 ± 2.76	0.72	100.21	199.00 ± 3.93	0.83	99.50
400	400.92 ± 4.08	1.11	100.23	401.93 ± 5.67	0.57	100.48
600	597.90 ± 6.93	0.86	99.65	596.14 ± 7.22	0.78	99.35
Aspirin
200	198.62 ± 3.02	1.04	99.31	199.56 ± 4.13	0.93	99.78
400	402.69 ± 5.78	0.68	100.67	398.13 ± 6.49	1.24	99.53
600	598.42 ± 8.16	0.98	99.73	600.71 ± 7.84	0.78	100.11

Table 3: Robustness testing

Parameter	SD of Peak Area for Atenolol	% RSD	SD of Peak Area for Amlodipine besylate	% RSD	SD of Peak Area for Aspirin	% RSD
Mobile phase composition (± 0.1 ml)	5.34	0.82	7.02	0.69	9.63	0.92
Amount of mobile phase (± 5%)	9.57	1.21	11.23	0.82	7.38	1.23
Time from spotting to chromatography (10 min)	10.81	0.73	6.82	1.24	12.45	0.89
Time from chromatography to scanning (10 min)	8.03	1.45	8.92	0.95	10.78	0.95

Table 4: Recovery studies

Label claim (mg per capsule)	Amount added (mg)	Total amount (mg)	Amount recovered (mg) ± % RSD	% Recovery
Atenolol
50	40 (80 %)	90	88.72 ± 1.11	98.57
50	50 (100 %)	100	99.31 ± 0.76	99.31
50	60 (120 %)	110	109.89 ± 0.82	99.90
Amlodipine besylate
5	4 (80 %)	9	9.03 ± 0.57	100.33
5	5 (100 %)	10	9.92 ± 1.10	99.20
5	6 (120 %)	11	11.01 ± 0.72	100.09
Aspirin
100	80 (80 %)	180	178.66 ± 0.95	99.25
100	100 (100 %)	200	201.92 ± 0.73	100.96
100	120 (120 %)	220	217.18 ± 1.12	98.71

LOD and LOQ:

Signal-to-noise ratios for LOD and LOQ were 3:1 and 10:1, respectively. The calculated LOD was found to be 80 ng/spotand LOQ was found to be 100 ng/spot for Atenolol, Amlodipine besylate and Aspirin, respectively.

Robustness of the method:

Standard deviation and % RSD of peak areas were calculated for each deviation in parameters. The low values of % RSD i.e. less than 2, shows that the developed method is robustness which is shown in Table 3.

Specificity:

The purity of peak for Atenolol, Amlodipine besylate and Aspirin was evaluated by comparing the spectra at peak start, apex and end positions of the band i.e., r (S, M) = 0.9983 and r (M, E) = 0.9978. A good correlation (r = 0.9987) was also attained between sample and standard spectra of Atenolol, Amlodipine besylate and Aspirin, respectively.

Recovery studies:

Good recoveries of Atenolol, Amlodipine besylate and Aspirin were obtained in the range from 98.57 to 100.96 % which was performed by standard addition method as shown in Table 4.

Analysis of a formulation:

Results obtained after performing studies on the amount of Atenolol, Amlodipine besylate and Aspirin in capsules, showed that the percentage recovery was in the range of 98.97 to 100.40 % which suggested that there was no interference from the excipients that are present in capsules. Two different lots of Atenolol, Amlodipine besylate and Aspirin combination capsules were examined using the projected procedures (Table 5).

Table 5: Analysis of commercial formulation

Atenolol (50 mg)	Atenolol found (mg per capsule)
Atenolol (50 mg)	Mean ± SD (n= 6)	Recovery (%)
1^st Lot	49.85 ± 5.25	99.70
2^nd Lot	50.11 ± 5.12	100.22
Amlodipine besylate (5 mg)	Amlodipine besylate found (mg per capsule)
Amlodipine besylate (5 mg)	Mean ± SD (n= 6)	Recovery (%)
1^st Lot	5.02 ± 1.27	100.40
2^nd Lot	4.98 ± 1.71	99.60
Aspirin (100 mg)	Aspirin found (mg per capsule)
Aspirin (100 mg)	Mean ± SD (n= 6)	Recovery (%)
1^st Lot	99.23 ± 7.83	99.23
2^nd Lot	98.97 ± 8.28	98.97

CONCLUSION:

Introduction of HPTLC into pharmaceutical analysis symbolizes a major step in quality assurance. The developed HPTLC method is specific, accurate, precise, robust. Statistics confirms that the developed method is appropriate for the study and quantitative determination of Atenolol, Amlodipine besylate and Aspirin as bulk drug and in pharmaceutical formulation without any excipients interference. The proposed HPTLC method is simple, less expensive, rapid and flexible than a HPLC.

ACKNOWLEDGEMENT:

The authors are very thankful to Wockhardt Ltd., Aurangabad, Maharashta, India for giving Atenolol, Aspirin and Amlodipine besylate standard as the gift sample. The authors would also like to thank, Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Pune, India for providing needed facilities for carrying out the work.

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Received on 10.07.2020 Modified on 23.12.2020

Research J. Pharm. and Tech 2022; 15(1):213-218.

DOI: 10.52711/0974-360X.2022.00035

Properties	Atenolol	Amlodipine besylate	Aspirin
Molecular formula	C₁₄H₂₂N₂O₃	C₂₀H₂₅ClN₂O₅.C₆H₅SO₃H	C₉H₈O₄
Molecular weight	266.33	567.1	180.15
Category	Anti-hypertensive	Anti-hypertensive	Analgesic, antipyretic
Melting point	152-155 °C	199-201 ^oC	138 - 140 °C
pKa	9.6	9.4	3.5
Appearance	White or almost white powder	White powder	White crystalline powder
Solubility	Sparingly soluble in water, soluble in alcohol and practically insoluble in ether	Slightly soluble in water; sparingly soluble in ethanol and methanol	Slightly soluble in water, soluble in ethanol, DMSO, dimethylsulfoxide
Storage	To be kept at 25 ± 2 ºC and protected from heat, light and moisture	To be kept at 25 ± 2 ºC and protected from heat, light and moisture	to be kept at 25 ± 2 ºC and protected from heat, light and moisture