Analytical Method Development and Validation of Amoxicillin and Dicloxacillin in API and its Dosage Form by RP-HPLC

Introduction

A moderate spectrum bacteriolytic antibiotic called amoxicillin is used to treat bacterial infections brought on by susceptible microorganisms. Because it is better absorbed after oral administration, it is typically the medicine of choice within the class. Amoxicillin works by preventing the bacterial cell wall from being produced. It is soluble in water, methanol, and ethanol, but very marginally soluble in toluene.^1-4

Materials and Methods

Chemicals and Reagents

Concept Pharmaceuticals Ltd. (Aurangabad, India), provided the Amoxicillin and Dicloxacillin which were utilized as a working standard. Gift sample was given to carried out the analysis for a quantitative analysis, Flumox® Capsules were employed. Analytical grade potassium dihydrogen orthophosphate was used. The High-performance liquid chromatography (HPLC) grade acetonitrile, methanol, and water were acquired from Merck Chem Ltd., Mumbai. For HPLC, all solutions were made with double-distilled R.O. water.^5-6

Instrumentation

The HPLC system model Waters 2695 Empower Waters

UV double beam UV 3000

Digital weighing machine

pH meter

Ultra Suction pump.⁷

Chromatographic Conditions

The estimation of Amoxicillin and Dicloxacillin was done by RP-HPLC. The Phosphate buffer had a pH of 4.6 and the mobile phase was optimized consisting of Methonol:Phosphate buffer mixed in the ratio of 70:30 %v/v. A Symmetry C18 (4.6 x 150 mm, 5 µm, Make XTerra) column was used as stationary phase. The detection was carried out using UV detector at 273 nm. The solutions were chromatographed at a constant flow rate of 1.0 mL/min.^8-9

Preparation of Mobile Phase

To establish a relevant HPLC method, couple of different mobile phases were attempted which were decided based on the readily available solvents, short run time, results and sensitivity of assay. Aqueous solvent, or MilliQ water, and an organic solvent, methanol, were determined to be the mobile phases for this experiment. Separate solvents were filtered via 0.45 m membrane filters and sonicated to remove any gas.¹⁰

Preparation of Sample Solution

A 10 mL clean, dry volumetric flask was filled with precisely weighed tablet powder containing 8 mg of Amoxicillin and 12.5 mg of Dicloxacillin. About 7 mL of diluent was added, sonicated to completely dissolve, and then volume was adjusted to the required amount using the same solvent (Stock solution). Further, 0.6 mL of the above stock solution was pipetted into a 10 mL volumetric flask and diluted up to the mark with diluent.¹¹

Preparation of Standard Solution

Accurately measured 10 mg of the standard medication Amoxicillin and Dicloxacillin were dissolved in 10 mL of methanol with 1000 g/mL of each antibiotic, individually. It was fully dissolved using sonication. A membrane filter paper was used to further refine it. By using the appropriate dilution, this standard stock solution was utilized to prepare the concentrations required to generate the calibration curve.¹²

Results

The overlay spectra are shown in Figures 1, 2, 3

Trial 1

Mp              :        Water:Methanol (50:50 %v/v)

Column       :       Thermosil C.18 (4.6*150 mm) 5 µm

Fr                :        1.0 mL/min

Wl               :        260 nm

Ct                :        Ambient

St                :        Ambient

Iv                :        10 µL   

Chromatogram for Amoxicillin and Dicloxacillin

The given chromatogram in Figure no 4 shows that peaks are well separated.³

Retention time of Amoxicillin – 2.003 min

Retention time of Dicloxacillin - 5.067 min

Optimized chromatogram was obtained with the following conditions

Column             :            Symmetry C.18 (4.6 x 150 mm, 5 µm, Make: Xterra) or equivalent

Buffer pH          :            4.6

Mp                    :            70% Methonol: 30% pb ph-4.6

Fr                      :            1 mL per min

Wl                     :            273 nm

Temperature     :            ambient

Rt                      :            7 min

Acceptance criteria (Table 2)

• Resolution between two drugs allows a resolution of 1.5 to be achieved with about not more than 2000 theoretical plates and tailing issue should not be but 9.

It was found that information obtained of quality parameters for developed methodology was within the limits.

Acceptance criteria(Table 3)

• % Relative Standard Deviation (RSD) of five completely different sample solutions should not two.
• The % RSD obtained was within the limit, thus the strategy was rugged.

Acceptance criteria (Table 4)

• The percentage recovery at every level ought to be between 97-103%.
• The results obtained for recovery at five hundredth, 100%, a hundred and fiftieth area unit were inside the boundaries. Thus, the strategy was correct.

Linearity (Table 5)

The dimensionality variation was found to lie between twenty fifth to one hundred and twenty fifth and the chromatograms square measure is shown below.

Area of different concentrations of Amoxicillin and Dicloxacillin

Analytical performance parameters of Amoxicillin and Dicloxacillin 

Acceptance criteria (Table 6)

• Correlation coefficient (R2) should not be but 0.999.
• The correlation obtained was 0.999 which is within the acceptance limit. The one-dimensionality was established within the variation of twenty-five to 150 µg/mL

Limit of Detection (LOD) of Amoxicillin and Dicloxacillin

With reference to the overall noise, the’ lowest concentration of sample was prepared, and the quantitative signal to noise relationship was assessed.

Limit of Quantitation (LOQ) of Amoxicillin and Dicloxacillin

The lowest concentration of the sample was prepared with respect to the whole noise, and the quantitative signal to noise relationship was assessed.

Acceptance criteria (Table 7)

• Signal to noise magnitude relation ought to be three for LOD resolution.
• The results obtained were inside the limit.

Acceptance criteria (Table 8)

• Signal to noise quantitative relation ought to be ten for LOQ resolution.
• The results obtained were within limits.

Results of Robustness (Table 9)

• The standard sample and samples of Augmentin and antibacterial were injected by continuously changing the conditions of activity. There was no important modification observed within the parameters like resolution, tailing issue, uneven issue, and plate count.
• The degree of test result repeatability was determined under a range of standard testing circumstances, including different labs, analysts, instruments, reagent lots, elapsed assay periods, assay temperatures, different days etc, unaffected by small but deliberate variations in method parameters.
• Adequate method development - separation on column described in method was uniform and no column deterioration was observed.
• Resolution of the critical pair of peaks in the chromatogram.

Acceptance criteria for variation in flow (Table 10)

• Percentage RSD must not be two.
• The %RSD obtained for flow of rate, variation in mobile phase was found to be below two, that is at five intervals the acceptance criteria. Hence the method is accepted .

Discussion

Method Development

Selection of Detection wavelength

Bactericide and Larotid 10 mg were dissolved in the mobile component. The spectra of the solution were obtained and scanned from 200 to 400 nm. The wavelength for Larotid and the bactericide was selected using the overlay spectrum. The detecting wavelength was chosen to be the isobestic purpose.^13-14

Validation Parameters

Precision

Precision of the strategy was administered for each sample and the customary solutions as delineated beneath experimental work. The corresponding chromatograms and results are shown below.

System Suitability

The system suitability of the strategy was checked by injecting five completely different preparations of the Larotid and its antibacterial effect. The parameters of system suitability were checked.

Intermediate Precession (Ruggedness)

At completely different levels of huskiness, such as day-to-day and system-to-system variation, there was no significant change in the assay content and system quality metrics.

Accuracy

Sample solutions at totally different concentrations (50%, 100% & 150%) were ready and also the recovery was calculated.

Conclusion

Based on the assay results and validation parameters, it was determined that the proposed method for the simultaneous estimation of Amoxicillin and Dicloxacillin in combined capsule dosage form was straightforward, quick, accurate, and precise. It can also be used for the routine estimation of Amoxicillin and Dicloxacillin dosage form.

Consent and ethical approval

It is not applicable

Conflict of interest

None

Acknowledgements

We gratefully acknowledge ABIPER and RGUHS University for providing all needed things to prepare this manuscript.