RP-HPLC Method Development and Validation for Simultaneous Estimation of Norfloxacin and Tinidazole in Combined Tablet Dosage Form

1. NORFLOXACIN -

Norfloxacin is a broad-spectrum fluoroquinolone antibiotic with variable activity against gram-positive and gram-negative bacteria. Typically reserved for the treatment of UTIs due to accumulation in the urine. [1]

Struture of Norfloxacine:

Fig. No.1 Structure of Norfloxacin

Mechanism of action:

The bactericidal action of Norfloxacin results from inhibition of the enzymes topoisomerase II (DNA gyrase) and topoisomerase IV, which are required for bacterial DNA replication, transcription, repair, and recombination. Norfloxacin is a broad-spectrum antibiotic agent that is shown to be effective against various Gram-positive and Gram-negative bacterial species. The fluorine atom at the 6 position increases potency against gram-negative organisms, and the piperazine moiety at the 7 position is responsible for anti-pseudomonal activity. [2,3]

Technical Information-

• Physical State: Solid
• Solubility: least soluble at urinary pH of 7.5 with greater solubility occurring at pHs above and below this value
• Storage: Store the medicine in a closed container at room temperature,
• Melting Point: 221°C
• Boiling Point: 760 mmHg
• Indicator: For the treatment of urinary tract infection ^[4]
• Absorption: Rapid
• Volume of Distribution: Not Availability
• Metabolism: Via liver and kidney
• Half Life: 3-4 Hours
• Clearance: Not Available ^[5]

2. TINIDAZOLE

It is a nitroimidazole used to treat trichomoniasis, giardiasis, amebiasis, and bacterial vaginosis ^[6]

Structure of Tinidazole:

Fig. No.2 Structure of Tinidazole

Technical Information

• Physical State:  Solid
• Solubility: solubility of tinidazole in these solvents is approximately 0.2, 10, and 20 mg/ml, & soluble in aqueous buffers.
• Storage: Room temperature and away from excess heat and moisture
• Melting Point     : 127-128° C
• Boiling Point      : 760 mmHg

Mechanism of action-

Tinidazole is a prodrug and antiprotozoal agent. The nitro group of tinidazole is reduced in Trichomonas by a ferredoxin-mediated electron transport system. The free nitro radical generated as a result of this reduction is believed to be responsible for the antiprotozoal activity. It is suggested that the toxic free radicals covalently bind to DNA, causing DNA damage and leading to cell death. The mechanism by which tinidazole exhibits activity against Giardia and Entamoeba species is not known, though it is probably similar. ^[7]

• Absorption- Rapidly and completely absorbed under fasting conditions.
• Volume of Distribution: 50L.
• Metabolism: Hepatic, mainly via CYP3A4
• Half Life- The elimination half-life is 13.2±1.4 hours and the plasma half-life is 12 to 14 hours.
• Clearance: Not Available ^[8]

Introduction To Method Validation-

Validation is a process of establishing document al evidence, which supply a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications. Method validation is process of demonstrating that analytical procedures are suitable for their intended use and that they support the quality, purity, identity and potency of the drug substances and drug products. Main goal of validation process is to challenge the method and determine limits of allowed. Variability for the conditions needed to run the method. ^[9,10]

Types of analytical procedures to be validated: Validation of analytical procedures is directed to the four most common types of analytical procedures.

• Identification test.
• Quantitative test for the impurities content.
• Limit test for the impurities.
• Quantitative test for the active ingredient in samples of drug substance on drug product on other selected components in the drug product. ^[11]

Two steps are required to evaluate an analytical method-

1. First determine the method classification.
2. The second step is to consider the characteristics of the analytical method ^[12]

Reasons for method validation

There are two important reasons for validating assays in the pharmaceutical industry. The first, and by for the most important, is that assay validation is an integral part of the quality control system. These condition that current good manufacturing practice regulation requires assay validation. ^[13]]

Performance parameters examined when carrying out method validation

• Specificity
• Linearity
• Range
• Accuracy
• Precision (Repeatability and Ruggedness)
• Detection and Quantitation limit Robustness ^[15]

MATERIALS AND METHOD-

Materials-

Norfloxacin and Tinidazole, Combination of Norfloxacin and Tinidazole tablet dosage (Noxitef TZ 400mg/600mg) forms, distilled water, Acetonitrile, phosphate buffer, ammonium acetate buffer, glacial acetic acid, methanol, potassium dihydrogen phosphate buffer, tetra hydro furan, tri ethyl amine, ortho-phosphoric acid etc.

Methods-

Diluent: - Based on the solubility of drug the phosphate buffer 4.6 p^H and water used in ratio 60:40 ratio.

Preparation of buffer:

0.01N KH2PO4 Buffer: Accurately weighed 1.36gm of Potassium dihydrogen Ortho phosphate in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water then PH adjusted to 3.5 with dil. Orthophosphoric acid solution.

Buffer: (0.1% OPA)

Accurately 1ml of OPA in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water.

Standard Preparation:

Accurately Weighed and transferred 40mg of Norfloxacin and 60mg of Tinidazole working Standards into a 100ml clean dry volumetric flask, add 3/4th volume of diluent, sonicated for 15 minutes, and make up to the final volume with diluents (600µg/ml of Tinidazole and 400µg/ml Norfloxacin). ^[16]

Working solution of Standard:

1ml from the above stock solution was taken into a 25ml volumetric flask and made up to 10ml. (24µg/ml of Tinidazole and 16µg/ml Norfloxacin)

Sample Preparation Tinidazole and Norfloxacin:

10 to 20 tablets were taken and calculated each tablet weight and equivalent weight of 1 average tablet (400mg/600mg Tablet) was weighed, powdered, and then was transferred into a 500mL volumetric flask, 250mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. (1200µg/ml of Tinidazole and 800µg/ml Norfloxacin)

Working solution of Sample:

1 ml from the above stock solution was taken into a 50ml volumetric flask and made up to 10ml. (24µg/ml of Tinidazole and 16µg/ml Norfloxacin). ^[17]

Validation: Validation of analytical procedures was performed for Glecaprevir and Pibrentasvir using the following parameters.

Specificity: Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Linearity:

Standard Preparation:

Accurately Weighed and transferred 40mg of Norfloxacin and 60mg of Tinidazole working Standards into a 100ml clean dry volumetric flask, add 3/4th volume of diluent, sonicated for 15 minutes, and make up to the final volume with diluents. (600µg/ml of Tinidazole and 400µg/ml Norfloxacin). ^[18]

Accuracy:

Sample Preparation:

10 to 20 tablets were taken and calculated each tablet weight and equivalent weight of 1 average tablet (400mg/600mg Tablet) was weighed, powdered, and then was transferred into a 500mL volumetric flask, 250mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. (1200µg/ml of Tinidazole and 800µg/ml Norfloxacin)

Working solution of Standard:

1ml from the above stock solution was taken into a 25ml volumetric flask and made up to 10ml. (24µg/ml of Tinidazole and 16µg/ml Norfloxacin) ^[19]

Robustness: Small deliberate changes in method like Flow rate, mobile phase ratio, and temperature are made but there was no recognized change in the result and are within range as per ICH Guide lines.

Robustness conditions like Flow minus (0.7ml/min), Flow plus (0.9ml/min), mobile phase minus, mobile phase plus, temperature minus (21°C) and temperature plus(31°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit. ^[20]

LOD sample Preparation: 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flasks and made up with diluents. From the above solutions 0.3ml each of Norfloxacin & Tinidazole, solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents

LOQ sample Preparation: 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flask and made up with diluent. From the above solutions 0.9ml each of Norfloxacin& Tinidazole, solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluent. ^[21]

Degradation studies:

Oxidation:

To 1 ml of stock solution of Glecaprevir and Pibrentasvir, 1 ml of 20% hydrogen peroxide (H₂O₂) was added separately. The solutions were kept for 30 min at 600c. For HPLC study, the result and solution were diluted to obtain 20µg/ ml & 16µg/ml solution and 10µl were injected in to the system and the chromatograms were recorded to assess the stability of sample.

Acid Degradation Studies:

To 1 ml of stock solution Glecaprevir and Pibrentasvir, 1 ml of 2N Hydrochloric acid was added and refluxed for 30mins at 600c.The result ant solution was diluted to obtain 20µg/ml & 16µg/ml solution and 10µl solutions were injected into the system and the chromatograms wererat corded to assess the stability of sample.

Alkali Degradation Studies:

To 1 ml of stock solution Glecaprevir and Pibrentasvir, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 600c. There solution was diluted to obtain 20µg/ml&16µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Dry Heat Degradation Studies:

The standard drug solution was placed in oven at 105°C for 6h to study dry heat degradation. For HPLC study, the resultant solution was diluted to 20µg/ml&16µg/ml solution and10µl were injected into the system and the chromatograms wererat corded to assess the stability of the sample.

Photo Stability studies:

The photochemical stability of the drug was also studied by exposing the 400µg/ml&&160µg/ml solution to UV Light by keeping the beaker in UV Chamber for 1days or 200-Watt hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain 20µg/ml&16µg/ml solutions and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Neutral Degradation Studies:

Stress testing under neutral conditions was studied by refluxing the drug in water for 1 h r s at a temperature of 60ºC. For HPLC study, the resultant solution was diluted to 20µg/ml&16µg/ml solution and 10µl were injected in to the system and the chroma to grams wererat corded to assess the stability of the sample. ^[22,23]

RESULTS AND DISCUSSION-

1. System suitability:

All the system suitability parameters are within range and satisfactory as per ICH guidelines.

Table No. 1 System suitability studies of Norfloxacin and Tinidazole method

Fig. No. 3 Chromatogram of blank

Fig. No. 4  Typical chromatogram of Norfloxacin and Tinidazole

Linearity: Six Linear concentrations of Norfloxacin (4ppm- 24ppm) and Tinidazole (6ppm- 36ppm) are prepared and injected. Regression equation of the Norfloxacin and Tinidazole are found to be, y = 99753x + 36144and y = 117845x + 57896and the regression co-efficient was 0.999.

Table No. 2 Calibration data of Norfloxacin and Tinidazole method

Fig. No.5  Calibration curve of Norfloxacin

Fig. No.6 Calibration curve of Tinidazole

Precision-

Intraday precision (Repeatability): Intraday Precision was performed and % RSD for Norfloxacin and Tinidazole were found to be 1.3% and 0.5% respectively.

Table No. 3 Repeatability results for Norfloxacin and Tinidazole

Accuracy:

Three concentrations 50%, 100%, 150%, were injected in a triplicate manner and amount Recovered and % Recovery were displayed in Table 6.5.

Table No. 4 Accuracy table of Norfloxacin%

Table No. 5 Accuracy table of Tinidazole

LOD-

Limit of detection was calculated by in Norfloxacin and Tinidazole method and LOD for Norfloxacin was found to be 0.03 and Tinidazole was 0.06 respectively.

LOQ:

Limit of Quantification was calculated by into Norfloxacin and Tinidazole method and LOQ for Norfloxacin and Tinidazole were found to be 0.10 and 0.19 respectively.

Robustness:

Small deliberate changes in method like Flow rate, mobile phase ratio, and temperature are made but there was no recognized change in the result and are within range as per ICH Guide lines.

Table No. 6 Robustness data of Norfloxacin and Tinidazole method

Degradation data-

SUMMARY AND CONCLUSION-