Amlodipine besylate (standard) was obtained as a gift from Beximco Pharmaceuticals Limited. Hydrochloric Acid (HCl), 37%; Methanol was purchased from Active Fine Chemicals Ltd., Bangladesh.

On the basis of local market share, four national brands of marketed Amlodipine besylate tablets were purchased from retail pharmacy situated inside and outside of city area. These brands here are represented as ALT₁, AMT₂, AMT₃ and AST₄. Here “L”, “M” and “S” stand for “large”, “medium” and “small” market share. This study was done in late 2021. The samples were properly checked for their license number, batch number, manufacturing date and expiry date before purchasing. Amlodipine besylate tablets with 5 mg Amlodipine packaged in blister packing were stored at 25˚C ± 2˚C for four weeks before the quality determination study in order to evaluate any change.

Appearance and identification marking of the tablets were visually inspected to check the presence of any physical flaws and legible identifying markings for ensuring tablet-to-tablet uniformity. The sizes, shape, and color of the tablets were also checked for their uniformity.

For each brand, twenty tablets were randomly selected and weighed individually with the help of scientech electronic balance (USA). The average weights were determined and the percentage deviations from mean values were calculated using the formula [13]:

Individual weight − Averag eweight Average weight × 100 %

Copley Tablet Hardness tester (England) was used to evaluate the tablet hardness of randomly selected 10 tablets. The instrument reads in kilogram units [13].

The crown thickness of the individual tablet was measured with a micrometer. Tablet thickness should be controlled within a ± 5% variation of a standard value [13].

20 tablets were weighed accurately by using an electronic balance. The Copley friabilator (England) was run for 4 minutes at 25 rpm or 100 revolutions to expose the tablets to rolling and repeated shocks resulting from free fall within the apparatus [13]. After run completion, the tablets were collected and weighed again and friability was calculated using the following formula:

Friability = I w − F w I w × 100 %

where I_w is the weight of the tablets before the test and F_w is the weight of the tablets after test. A maximum mean weight should not be more than 1%.

The test was conducted on 1 to 2 g test specimens. In the case of a large crystal form specimen, particle size was reduced to about 2 mm by quickly crushing in a mortar pestle. The test specimen was distributed as evenly as practicable to a depth of about 5 mm on a tray by gentle shaking. The tray was placed in the moisture analyzer and the test was started.

The disintegration test was done by a tablet disintegration tester (Copley, England). One dosage unit was placed in each of the 6 tubes of the basket and a disc was added to each of the tubes. The apparatus was operated using 800 mL distilled water as the immersion fluid, maintained at 37˚C ± 2˚C At the end of the specified time, the basket was lifted from the fluid and the dosage units were observed [14].

3 tablets of Amlodipine besylate were weighted and taken into a 50 mL volumetric flask. 2 mL of distilled water was added into the volumetric flask and swirled to disintegrate the tablets; it was made up to mark with methanol. The solution was then filtered through Whatman no.1 filter paper. 1 mL of this solution was taken into a 25 mL volumetric flask and diluted up to mark with methanol. The absorbance of each solution was measured at 237 nm. Using the standard amlodipine besylate solution of known concentration, the amount of amlodipine in

each brand was determined.

As a model independent approach, here two adjustment factors, namely difference factor (f₁) and similarity factor (f₂), were applied to the dissolution data to compare the dissolution profile of a pair of pharmaceutical products; f₁ values between 0 and 15, and f₂ values between 50 and 100 were used to define the pharmaceutical equivalence of two dissolution profiles [15].

Difference factor, f 1 = { [ ∑ 1 n | R t − T t | ] / ∑ 1 n R t } ⋅ 100

Similarity factor, f 2 = 50 ⋅ log { [ 1 + ( 1 / n ) ∑ 1 n ( R t − T t ) 2 ] − 0.5 ⋅ 100 }

where n is the number of dissolution sample times, R_t is the mean percent dissolved at each time point for the reference and T_t is the mean percent dissolved at each time point for the test dissolution profile.

Statistical analysis:

Statistical analysis of the assay, disintegration and dissolution (30-min time point) data was performed by applying a one-way analysis of variance. The mean, standard deviation (SD) and %RSD were calculated using Microsoft Excel 2010 (Microsoft Corporation). The data were presented as mean ± SD or mean ± %RSD, as applicable.

According to BP, for tablets weighing ≤ 130 mg, the percentage difference allowed is ±10% and for tablets weighing 130 - 324 mg, it is ±7.5%. All brands of amlodipine besylate were within the standard limit and as such, all the brands passed the uniformity of weight test (Table 1). Among all the brands ALT₁ showed minimal deviations from the mean weight (1.22) and brand AST₄ showed maximum deviation (4.91).

The hardness of the different brands of Amlodipine besylate tablets was found to be between 2.29 kg/cm² - 9.52 kg/cm². Only one brand, AMT₂ exhibited hardness of more than 4 kg (Table 1). The low hardness value may be the result of the improper amount of binder and lubricant. Besides mixing time of lubricants, moisture content of the excipients and non-uniform size distribution of granules may also result in low hardness of tablets.

Thickness of the tablets of all the brands of Amlodipine besylate was in the acceptable range as they lie within ± 5% variation (Table 1). The results revealed that the tablet’s thickness ranged between 2.03 mm to 3.67 mm. These differences between generics in thickness are probably the consequence of differences in the added adjuvants according to each company’s formulation or differences in the coating layer applied in the different products.

One of the very effective tests indicating the compactness of the tablet’s core and coat is the friability test. High friability (i.e., low capacity to withstand friction) means that the drug is more likely to withstand mechanical erosion, which may cause loss of the active drug and thus compromise its efficacy. Results of this test as shown in Table 1 revealed that all tablets understudy met the USP specification, as the maximum mean weight loss from the samples taken was not more than 1.0%. So, the low hardness value can be compensated as all the brands met the standard specifications of friability.

Brands ALT₁, AMT₂, AMT₃ and AST₄ showed 4.45%, 4.75%, 6.17% and 5.27% of loss on drying, respectively. Brand AMT₃ showed maximum moisture content which is consistent with its lowest hardness value result (Table 1).

The physical assay on disintegration is related to the capacity of solid pharmaceutical forms to release their active ingredients, because before their solubilization the tablets must disintegrate into small particles, increasing the contact surface with the dissolution medium and favoring absorption and bioavailability of the drug. All the tested brands disintegrated within 30 minutes and thus conformed with regard to their disintegration time. ALT₁ had the fastest disintegration time, 23 sec. whereas AMT₃ had the slowest disintegration time, 3 min 3 sec. The conformity of the brands of tablets to the standard specification for disintegration time can be explained for the appropriate use of disintegrating agent by the manufacturers. However, variations in disintegration time from brand to brand were observed in the study (Table 2).

Percent of drug release for brands ALT₁, AMT₂, AMT₃ and AST₄ was 95.34%, 92.33%, 84.29% and 92.49%, respectively (Table 1). All the brands of Amlodipine besylate met the official standard (Figure 3).

Dissolution studies give an idea of the amount of drug available for the absorption after oral administration. Drugs with poor dissolution profiles will not be available in the body system or target organ/tissue to elicit therapeutic effect. Moreover for a BCS Class I drug of high solubility and high permeability like amlodipine, a good dissolution rate will ultimately ensure permeability and systemic absorption of the drug. Pharmaceutical equivalence between drugs can be determined from the dissolution result and it must be performed to ensure

bioequivalence of Amlodipine besylate. The four brands that we tested showed more than 75% dissolution rate within 30 minutes and did comply with the specification.

According to USP, amlodipine besylate tablets must contain not less than 90.0% and not more than 110.0% of the labeled amount of amlodipine (C₂₀H₂₅N₂O₅Cl) [14]. The results of dosage assays presented in Table 1 showed that the average content of amlodipine besylate among the analyzed drugs ranged from 103.89% to 105.53% and thus complied with the USP standard of drug content (90% - 110%). Brand AST₄ showed a maximum percent of drug content, 105.53% and brand AMT₃ showed a minimum percent of drug content, 103.89% (Table 1).

Comparison of therapeutic performances of different medicinal products containing the same active substance is a critical mean of assessing the possibility of alternative usage between the innovator and any essentially similar medicinal products. Difference factor (f₁) and similarity factor (f₂) were applied to the release rate and the data obtained showed that the tested brands of amlodipine besylate were pharmaceutically equivalent and can be used as alternatives to any of the brands with insignificant differences in their qualities (Table 2). ALT₁ was considered the benchmark for better average drug release and its highest market share. Pharmaceutical equivalency of AMT₂ is the closest to that of the benchmark brand ALT₁ whereas AST₄ is the furthest to that of ALT₁.

The low hardness value for brand ALT₁ can be omitted as it has the lowest average weight of 70.15 mg and also showed acceptable friability.

All in all, the pharmaceutical quality of the tested generic brands can be regarded as acceptable according to the above quality control tests. The tested generic products met the pharmacopoeial criteria for in vitro drug dissolution and drug content. The tested generic products are pharmaceutically equivalent and

^aPharmaceutically Equivalent.

therefore interchangeable. The efficacy associated with the use of the generic formulations would be comparable to the innovator brand.