Post Market In-vitro Bioequivalence Study on Representative Brands of Ciprofloxacin Tablets (500 mg) Prescribed in Typhoid Disease

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2.1. Weight Variation Test

Twenty individual tablets of each brand were weighed on analytical weighing balance. The mean, average and percentage deviation was assessed. For tablets weighing more than 500mg, the individual tablet must be in range of ±5% according to USP standards.

2.2. Hardness Test

The resistance of a tablet to physical abrasion or crushing depends upon its hardness. Ten tablets of each brand were taken and their crushing strength was determined with a tablet hardness tester (Monsanto hardness tester). Average hardness was assessed.

2.3. Disintegration test

Six tablets from each brand were randomly selected and were employed for disintegration test using freshly prepared 0.1N HCl solution at 37ºC±1. The tablets should comply with in the specified time limit that is ten minutes. The disintegration time is the time taken where no particles of the tablet remains on the basket assembly of the apparatus ^[3].

2.4. Dissolution Test

Dissolution test is performed to assess the amount or percentage of drug released from a formulation under simulated conditions of GIT provided during in-vitro analysis. Six tablets of each brand were taken and dissolved in 900mL of 0.01N HCL solution using apparatus II paddle method. After thirty minutes the sample was taken, filtered and assayed using UV-spectroscope at 277nm wavelength to determine the amount of ciprofloxacin released from the tablets.

2.5. Drug Content Assay

A solution of 1% w/v of ferric chloride was freshly prepared. Another solution of 100 mcg/mL of pure ciprofloxacin was prepared. Five tablets from each brand were crushed and 100mg of the powdered samples were weighed, dissolved in 100mL of 0.1N hydrochloric acid and further dilution was made upto 100mcg/mL or each brand. To 5mL of each brand and pure sample, 1mL of ferric chloride was added and made up to 50mL with 0.1N HCl. The absorbance of each sample was taken at 438nm against blank reagent with an ultraviolet spectrophotometer. The percentage content was calculated for each brand ^[1].

2.6. Anti-bacterial Activity
2.6.1. MIC Determination

Minimum inhibitory concentration is the lowest concentration, amount of the drug at which it shows the highest activity against microorganisms ^{[8, 9]}. MIC was determined by broth dilution method. All seven herbal and seven allopathic formulations along with the standard ciprofloxacin was assessed and their MIC was determined.

2.6.2. Preparation of Inoculum

Standard stock of bacterial isolate was prepared by suspending a loop full of microbial growth in 4mL of distilled water. It was incubated at 37°C for 12 hours and then its turbidity was compared with 0.5 McFarland’s standard giving a bacterial load of about 3.6 × 102 cfu /mL ^[9].

2.6.3. Preparation of Antimicrobial Solutions

One tablet of 500mg was dissolved in 10mL of sterile water. This gives concentration of 50mg/mL. It was further diluted through two-fold dilution method and a series of serial dilutions were made from 1 to 12µg/mL. Similarly herbal formulations were diluted using sterile water.

2.6.4. Preparation of Media

Muller Hinton Broth media was prepared by using manufacturer’s guidelines. It was then autoclaved at 121°C at 15psi pressure for 15 minutes. It was then allowed to cool. This media was poured in sterile test tubes under aseptic conditions using laminar flow Hood.

2.6.5. Inoculation and Incubation

One microliter of the bacterial isolate suspension was taken through micropipette and was inoculated in the test tube containing MH broth media. Then one milliliter of each dilution of the antimicrobial agents of all allopathic was poured in the test tube. These test tubes were incubated at 37°C for 18 – 24 hours.

2.7. Determination of Zone of Inhibition
2.7.1. Preparation of Media

Muller Hinton agar was dissolved is distilled water and simultaneously heated until it gets boiled. Then the media was poured in a conical flask and along with glass petri plates and autoclaved at 121°C and 15psi for 15 minutes. Then under aseptic conditions these petri plates were filled with MHA and allowed to solidify.

2.7.2. Inoculation and Incubation

After solidification of the medium, sterile cotton swab was dipped in the bacterial inoculum and was spread over the medium on plate. Then using a sterile cork borer of 8mm diameter, wells were made and approximately 100uL of the stock solutions of the standard drug and other formulations of allopathic drugs were incorporated. These plates were divided into four portions and were labeled. After allowing the drug to diffuse these plates were incubated at 37ºC for 24 hours ^[8]. Zone of inhibition was measured for all allopathic formulations.

3. Results

3.1. Weight Variation Test

Results for weight variations showed that all the brands were within the specified limit that is ±5%. Not a single tablet deviates from this limit. The average weight of cipro-2 was greater than all other brands. The minimum average weight was that of cipro-5. The results in decreasing order are as, cipro-2 > cipro-4 > cipro-6 > cipro-1 > cipro-3 > cipro-7 > cipro-5 (Table 2).

Table 2. Results of Weight Variation Test

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3.2. Hardness Test

Results showed that cipro-3 has highest value for hardness that is 12 kg/cm², while cipro-5 has the least value that is 7.61 kg/cm². Values for hardness in decreasing order are cipro-3 > cipro-1 > cipro-6 > cipro-2 > cipro-4 > cipro-7 > cipro-5 (Table 3).

Table 3. Results for Hardness test

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3.3. Disintegration Test

Six tablets of each brand were taken and their disintegration tests were performed, temperature was maintained at 37°C±1. The maximum time for disintegration was taken by cipro-3 that was 55 minutes. The minimum time was taken by cipro-7 that was 1 minute. Results for disintegration time in decreasing order are as cipro-3 > cipro-2 > cipro-6 > cipro-4 > cipro-1 > cipro-5 > cipro-7 (Table 4).

Table 4. Results for Disintegration Test

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3.4. Dissolution Test

Dissolution test was performed to check the percentage of drug released from the formulation after thirty minutes. The amount of drug released was analyzed by Uv-spectroscopy. The maximum % of drug released was 101.03 % from cipro-4. The minimum amount of drug was released by cipro-3 that was 39.8%. Cipro-7 released 76.7% of the drug after thirty minutes. Cipro-1, cipro-2, cipro-5 and cipro-6 showed 97.3%, 93.7%. 86.82% and 93.4% respectively (Table 5).

Table 5. Results of Dissolution test

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3.5. Drug Content Assay

The United States Pharmacopoeia (2005) states that the content of ciprofloxacin should not differ from the stated dose by more than 10% ^[6] The results showed that all the brands had amount of active drug >90% except to that of cipro-3 which had 63.45%, cipro-2 had 88.75%,cipro-6 had 88.9% of the total amount of active drug. Cipro-4 had the maximum amount of drug content that was 106.06%. While cipro-1 had 91.28%, cipro-2 had 88.75%, cipro-5 had 91.5%, and cipro-7 had 97.2% of the active drug content respectively (Table 6).

Table 6. Results of Drug Content Assay

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3.6. Determination of MIC and Zone of Inhibition
3.6.1. Minimum Inhibitory Concentrations

Minimum inhibitory concentration was determined for all the brands including seven allopathic brands of ciprofloxacin formulations along with that of the standard ciprofloxacin. Results showed that all the allopathic brands showed antibacterial activity. The least MIC value was shown by cipro-4 (1µg/mL) and the maximum value for MIC was shown by cipro-3 (3.2 µg/mL) (Table 7).

Table 7. Results for MIC of Samples

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3.6.2. Zone of Inhibitions

Zone of Inhibitions was determined after knowing the minimum inhibitory concentrations of all allopathic formulations. As the average MIC value of all the allopathic ciprofloxacin was near to 2µg/mL, zone of inhibitions were determined at 2µg/mL, 4µg/mL, 6µg/mL, 8µg/mL, 10µg/mL and 12µg/mL. Results showed that local brands of ciprofloxacin were more effective and gave bigger zone of inhibition than the multinational brand cipro-1 (Table 8).

Table 8. Results for Zone of Inhibition

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