Green Analytical Rp-Hplc Method Development And Validation For Simultaneous Estimation Of Anti Biotic Drug In Bulk &Capsule Dosage Form

Chemical structure:

Chemical name          : (2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Molecular formula    : C16H19N3O5S

Molecular weight      : 365.408 g/mol

Appearance   : Off-white solid, Crystals from, Penicillin-type odour, Bitter taste                                             

Melting point :194 °C           

Route of administration : Amoxicillin is administered orally and is available in various forms, including immediate-release or extended-release tablets, chewable tablets, and suspension.

Storage       : Store tablets and capsules at room temperature, away from moisture, heat, and light.

Approval date            : 18 January 1974.

Mechanism of action : Amoxicillin belongs to the class of beta-lactam antimicrobials. Beta-lactams bind to penicillin-binding proteins, inhibiting transpeptidation — a crucial step in cell wall synthesis involving cross-linking. This action activates autolytic enzymes in the bacterial cell wall, resulting in cell wall lysis and bacterial cell destruction.

Drug name : Cloxacillin

Category     :Antibiotic drug

Background   : Cloxacillin is a β-lactam, penicillinase-resistant (anti-staphylococcal) penicillin developed to treat infections caused by penicillinase-producing staphylococci. It belongs to the isoxazolyl penicillin group and is stable against destruction by many bacterial β-lactamase enzymes.

Chemical structure:

Chemical name          : sodium (2S,5R,6R)-6-[[3-(2-chlorophenyl)-5-methyl-1,2-oxazole-4-carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate.

Molecular formula    : C19H17ClN3NaO5S

Molecular weight      : 457.9 g/mol 

Appearance   : white or almost white, odorless crystalline powder.

Melting point :168-172 °C

Route of administration : Cloxacillin is administered by oral, intravenous, intramuscular and is available in various forms, including capsule, oral suspension.

Storage       : Store tablets and capsules at room temperature, away from moisture, heat, and light.

Approval date            : August 1965.

Mechanism of action : Beta-lactam antibiotic. Inhibits bacterial cell wall synthesis by binding to penicillin-binding proteins.

MATERIALS AND METHODS:

Reagent and chemical used:

• Ethanol (HPLC grade)
• Milli-Q-Water (HPLC grade)
• Ammonium acetate (analytical grade)
• Glacial acetic acid (analytical grade)

Instrumentation

• Digital Balance
• SHIMADZU – HPLC-DLC20AD
• Sonica Ultra sonic cleaner – model 2200 MH
• Digital PH Meter

PROCEDURE:

Preparation of buffer solution:

0.77gm of ammonium acetate were dissolved in 800ml of milli-Q-water and adjust the PH for 3.5 by using glacial acetic acid make upto 1000ml

preparation of standard stock solution:

50mg of amoxicillin and 50mg of cloxacillin were dissolved in Milli-Q-water and made upto 100ml volumetric flask with solvent.

1ml stock solution was pipetted out and made upto the 10 ml volumetric flask with solvent. (50µg of amoxicillin & 50µg of cloxacillin\ml)

preparation of sample stock solution:

The average weight of 10 capsules was calculated as 112. 2mg.Then112.2mg of powder were dissolved in Milli-Q-water and made upto 100ml volumetric flask with solvent.

1ml stock solution was pipetted out and made upto the 10 ml volumetric flask with solvent. (50µg\ml)

METHOD DEVELOPMENT:

The developed method was fully validated for the parameters as per ICH guidelines.

Linearity

Weigh accurately and transfer about 50mg of brivaracetam into 100ml of volumetric flask dissolve and make up with solvent. Take 0.5ml, 0.75ml,1ml,1.25ml,1.5ml of stock solution is transfer into 10ml of volumetric flask and make up with solvent upto the volume. To make the linearity concentration of 50%, 75%,100%,125%,150%.

Accuracy

Weigh accurately tablet average weight of sample and transfer into 100ml of volumetric flask dissolve and makeup to the volume with the solvent. Take 0.8ml,1ml,1.2ml of stock solution was transferred into 10ml of volumetric flask and makeup with solvent upto the volume. To make the concentration of 80%, 100%,120%.

Precision

Weigh accurately tablet average weight of sample and transfer into 100ml of  volumetric flask dissolve and makeup to the volume with the solvent.1ml of stock solution was transferred into 10ml of volumetric flask and makeup with the solvent upto the volume. Six-time replicate of injection to produce the similar of the result no maximum deviation.

Robustness

Small deliberate changes in method like flow rate, wavelength, mobile phase and ratio were made but there was no recognized change in the result and were within range as per ICH guidelines. Robustness conditions like flow minus, flow rate, wavelength decreasing, wavelength increasing, mobile phase decreasing, mobile phase increasing, ratio increasing and ratio decreasing was maintained and sample were injected in duplicated manner system suitability parameters were not much effected and all the parameters were passed. % RSD was within the limit.

Ruggedness

Ruggedness is a measure of reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst. The method was fully validated for the parameters as per ICH guidelines.

RESULTS AND DISCUSSION:

The optimized chromatographic method provided satisfactory separation of Amoxicillin and Cloxacillin. The method demonstrated excellent linearity, with a correlation coefficient of 0.9998 for both analytes. The assay results were 98.93% for Amoxicillin and 100.78% for Cloxacillin. Accuracy and precision studies showed satisfactory recovery and low percentage relative standard deviation (%RSD). Robustness studies demonstrated that small deliberate changes in chromatographic conditions did not significantly affect the analytical performance. Ruggedness studies also showed reproducible results between analysts.

Method development:

Optimized chromatographic condition:

Mode of operation      : Gradiant

Stationary phase         : C18 Column (250 mm×5,4.5µ)

Mobile phase  : Buffer: Ethanol (65:35)

Detection wavelength: 276 nm

Flow rate     : 0.8ml/min

Column Temperature : 30℃

Retention time:

Amoxicillin   :7.712minut

 Cloxacillin     :16.970minutes

Run time     :15minutes

Injection volume         : 50 mcg

Figure 1: Optimized chromatogram

Peak table:

Figure 2: Optimized chromatogram of amoxicillin

Figure 3: Optimized chromatogram of cloxacillin

Assay:

Table 1: Assay data for amoxicillin

Table 2: Assay data for cloxacillin

Linearity:

Table 3: linearity data for amoxicillin

Figure 4: calibration curve for amoxicillin

Table 4: linearity data for cloxacillin

Figure 5: calibration curve for cloxacillin

Table 5: Linearity data

Accuracy

Figure 6: Accuracy-80%

Figure 7: Accuracy-100%

Figure 8: Accuracy-120%

Table 6: amoxicillin Data for accuracy

Table 7: cloxacillin Data for accuracy

Precision:

Table 8: Data for precision

Table 9: Data for precision

Robustness

Table 10: amoxicillin Data of robustness (change in flow rate)

Table 11: cloxacillin Data of robustness (change in flow rate)

Ruggedness

Table 12: amoxicillin Data for ruggedness

Table 13: cloxacillin Data for ruggedness

Established method greenness analysis:

Green assessment can be defined as the absence or minimal usage of hazardous chemicals, the elimination of waste, and the decrease of energy consumption the suggested method’s greenness has been investigated by assigning penalty points to all of the specified parameter for the approach by applying a “Greenness evaluation metric of analytical methods (GEMAM)”, after that given a score for every of the twelve green analytical chemistry elements as shown below in “Analytical GREEnness (AGREE)”.

Figure 9: The general result of the greenness evaluation metric of analytical methods (GEMAM) assessment (left) and the corresponding colour scale for reference (right)

Table 14: Different sections & different criteria of GEMAM

Figure 10: The general result of Analytical GREEnness (AGREE)