Biochemistry & Analytical Biochemistry

Analytical Method Development and Validation for Determination of Assay of Antibacterial Drugs Besifloxacin Hydrochloride and Phenoxyethanol in Gel Formulation

¹Department of Pharmaceutical Sciences, University of Chaudhari Bansi Lal, Haryana, India
²School of Medical & Allied Sciences, K.R. Mangalam University, Haryana, India
³Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science & Technology,Haryana, India
⁴Department of Life Sciences, University of Chaudhari Bansi Lal, Haryana, India
^*Correspondence: Neelam Pawar, Department of Pharmaceutical Sciences, University of Chaudhari Bansi Lal, Haryana, India, Tel: +91-8529002677, Email:

Received: 26-Feb-2022, Manuscript No. BABCR-22-15709; Editor assigned: 28-Feb-2022, Pre QC No. BABCR-22-15709(PQ); Reviewed: 14-Mar-2022, QC No. BABCR-22-15709; Revised: 21-Mar-2022, Manuscript No. BABCR-22-15709(R); Published: 28-Mar-2022, DOI: 10.35248/2161-1009.22.11.426

Introduction

Analytical Method Validation is a process involving confirmation studies that procedure method/system/analyst provides precise and reproducible outcome recognized by research laboratory studies that the performance features of the technique follows the necessities required for the analytical applications. The composition of validated standards in terms of its analyte concentration and matrix used should be much relatable to the sample. High Performance Liquid chromatography is a best method to separate, analyze, and purify virtually any sample; and is used for chemical analysis. It is liquid chromatography in which the mobile phase and sample are pumped (up to 10000 psi) to affect quicker and superior resolution of drug in mixture [1]. Gels are defined as semi rigid systems in which the movement of the dispersing medium is restricted by an interlacing three-dimensional network of particles or solvated macromolecules of the dispersed phase. The word ‘gel’ is derived from ‘gelatin’ [2]. The interlinking of particles gelling agent forms a rigid network. Antibacterial agents are a group of materials that fight against pathogenic bacteria. They work either by killing microorganisms or stopping them from replicating, this allows the body’s natural protection system to eradicate the pathogens. Acne vulgaris is multifactorial, a commensal skin bacteria (acnes) plays a chief role in the formation of acne lesion. It is a contamination of pilosebacious glands, oil glands in the skin [3]. The anti-bacterial drugs used in this research process were Besifloxacin hydrochloride and Phenoxyethanol and their properties (Table 1).

Table 1: Properties of besifloxacin hydrochloride and phenoxyethanol.

Materials and Methods

Present research work was done in the Analytical Research and Development Department of Vyome Therapeutics Ltd., Delhi. Potassium dihydrogen phosphate and Ammonium dihydrogen phosphate were of grade AR/GR and bought from Merck. Acetonitrile, Triethylamine, Orthophosphoric acid, Methanol, Isopropyl Alcohol, Dichloromethane were of HPLC grade and procured from Merck. Dimethyl form amide (HPLC grade) was procured from Merck. Purified water was procured from Millipore and of Milli-Q grade. Vehicle gel and drug X gel 1% procured from of Vyome Therapeutics Ltd.

Method HPLC was then validated to specify that the analytical process used is appropriate for projected use by using several parameters like specificity, linearity, LOD, LOQ, accuracy, stability, precision, robustness and range in analytical solution and system suitability.

The RP-HPLC method was used for determination of assay and validation of anti-bacterial drug in gel formulation. Diluent was selected depending upon on the solubility of impurities, degradation products, starting materials, intermediates and the analyte. Selected diluent should be compatible with the mobile phase to get a better peak shape of analyte [4,5].

Experimental work

Diluent trial for preparation of sample and standard: Five trials were run by using viz water: acetonitrile (30:70), water: dimethyl formamide (50:50), methanol and dichloromethane (75:25), water: methanol: dichloromethane (25:50:25), water: methanol (50:50). Different standard stock solutions were prepared:

Standard solution A (1 mg/ml of Besifloxacin): 27.0 mg of Besifloxacin hydrochloride working standard (equivalent 25.0 mg Besifloxacin) was weighed and transferred in a 25.0 ml of volumetric flask. 10 ml of diluent was added and dissolved by vortexing. Mixture is diluted by adding more diluent with proper mixing.

Standard solution B (0.35 mg of Phenoxyethanol): 35.0 mg of Phenoxyethanol working standard was transfered in a 100 ml of volumetric flask. Drug was dissolved by adding 50 ml of diluent using vortexing. Mixture is diluted by adding more diluent with proper mixing.

Final standard solution (0.1 mg of Besifloxacin and 0.035 mg of Phenoxyethanol): 5.0 ml of Solution A and 5.0 ml of Solution B were pipette out in a 50 ml of volumetric flask; to this 25 ml of diluent was added and diluted up to the mark with diluent with proper mixing.

Preparation of test solution: 1000 mg of VTL001 gel sample equivalent to 10 mg of standard was transferred into 100 ml volumetric flask and about 70 ml of diluent was added, vortexes for 5 minutes with recurrent trembling and sonication. Final volume was make-up with diluent. Further dilution of 5 ml test solution in to 10 ml volumetric flask was done. Solution was filtered through 0.45 μm PVDF (Poly Vinylidene Fluoride) syringe filter after discarding 1 ml of filtrate (Table 2) [6,7].

Table 2: Description of trials for mobile phase.

RP-HPLC method trials for mobile phase

In each trial, different solutions were injected into the system following the sequence of blank, standard solution, sample solution and bracketing standard with number of injections 1, 5, 2 and 1 respectively. Chromatogram was recorded for every trial.

For isocratic operation solvent used was mobile phase A: mobile phase B (600:400 ml) [7-9].

Assay determination method for validation of RP-HPLC

The following parameters were considered for the Analytical method validation of title ingredients [10].

• System suitability

• Initial assay

• Accuracy

• Precision

• Specificity

• Linearity and range

• Limit and quantification and detection

• Robustness and ruggedness.

(A) System suitability: To check that the analytical system is running appropriately and can provide accurate and precise results the system suitability parameters are to be set. Chromatographic conditions, mobile phase, diluent and standard preparation should be done as per test methodology as selected by above trials. For verify the system suitability standard was injected and results of 5 injections were checked respectively [11].

(B) Initial assay: Initial assay had been carried out 3 times by the same analyst with the same instrument and same column. The resolution between the standard drug was more than 2.0. The difference between individual assays was not more than 1.0% (RSD). The average of three-assay values was taken for further validation process. Repeatability was demonstrated by taking the RSD of retention time and AUC of chromatogram of 6 replicate injections of standard, which were computed and was within 1%. The average of three assay values was taken for further validation process [12-14].

• Preparation of working standard stock solution: 27 mg of standard drug was transmitted to 100 ml volumetric flask, 60 ml of diluent was poured and sonicated (maintain sonicator water temperature to 25 +2°C) for 5 minutes to thaw it completely and make up the volume with diluent and mix well [15].

• Preparation of final working standard solution: 5 ml of working standard stock solution was poured in to 50 ml volumetric flask and diluted with diluent.

• Preparation of Test solution: 1000 mg of VTL001 gel formulation sample equivalent was transferred to 10 mg of Besifloxacin into 100 ml volumetric flask and 70 ml of diluent was added, vortexed for 5 minutes with intermittent shaking and sonicated for 20 minutes. Final volume make up was done with diluent. Further dilution of 5 ml test solution in to 10 ml volumetric flask was done. The solution was filtered through 0.45 μm PVDF syringe filter after discarding 1 ml of filtrate [16].

(C) Precision: The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple sampling of homogeneous sample. The precision of analytical method is usually expressed as the standard deviation or relative standard deviation coefficient of variation of series of measurement. This had been validated by three types: System precision, Method precision and Intermediate precision. The system precision is checked by using standard Besifloxacin ensure that the analytical system is precise. The retention time and area of six determinations was measured and RSD was calculated. In method precision, a homogenous sample of a single batch should be analyzed six times. This indicates whether a method is giving consistent results for a single batch. The intermediate precision should be carried out to ensure that the analytical results will remain unaffected with change in instrument, analyst, and column and on different days. The method and intermediate precision was performed on Besifloxacin injection [17-19].

(D) Specificity: Photo Diode Array detector may be useful to show that analyte chromatographic peak is not attributable to more than one component. For the determination of specificity up slope similarity, down slope similarity, and 3-point peak purity should be estimated from the sample injection [20-23].

(E) Linearity and range: The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range. Individually samples equivalent to 70%, 80%, 90%, 100%, 110%, 120% of the stated amount of sample were weighed individually and the assay was carried out. A graph of weight taken versus chromatographic area was plotted for both drug peaks. The regression line obtained was linear. From the data obtained, co-relation coefficient, slope and yintercept were calculated. Ideally co-relation coefficient should be around 1. Weight taken of the sample for 70%, 80%, 90%, 100%, 110% were 52.5 mg, 60 mg, 67.5 mg, 75 mg and 82.5 mg respectively.

For the preparation of standard stock solution: Weighed individual quantities of the sample were dissolved in diluents sonicate and volume made up to 50 ml with diluent.

For the preparation of stock solution B: 10 ml of the above solution was diluted to 50 ml with diluents [24-27].

(F) Detection limit: The Limit Of Detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected. The detection limit is determined by the analysis of sample with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.

For standard dilution, 50 mg of Besifloxacin was added to 50 ml volumetric flask. The contents were dissolved using diluents, sonicate, later the volume was made up to 50 ml with diluent.

For sample dilution, 50 mg of the sample (B.NO: VTL001 vial) was weighed into a 50 ml volumetric flask. The contents were dissolved using diluents, sonicate, later the volume was made up to 50 ml with diluent.

For sample stock solution II: 10 ml filtrate of the above solution was diluted to 50 ml with diluents [28-31].

(G) Quantitation limit: Carried out in order to determine that lowest concentration of analyte which it can be estimated with acceptable precision, accuracy under the stated experimental conditions. For this the sample solution can be further diluted and the minimum concentration at which the sample can be reliably quantified should be found.

Standard dilution: 50 mg of Besifloxacin was weighed and transferred to 50 ml volumetric flask. The contents were dissolved using diluents and sonicate for 5 minutes at room temperature. Later the volume was made up to 50 ml using diluent.

Sample dilution: 50 mg of the sample (B.NO: VTL001 vial) was weighed into a 50 ml volumetric flask. The contents were dissolved using diluents, sonicate, later the volume was made up to 50 ml with distilled water.

Sample stock solution II, III, IV and V were prepared by dilution [32-34].

(H) Robustness and ruggedness: Robustness is the capability of method to remain unchanged by little intentional variations in method parameters. The variations may include change of organic solvent in mobile phase, pH of buffer in mobile phase, column oven temperature and flow rate. According to ICH, the robustness should be initiated in the early phase of method development. In addition, if the results are susceptible to variation in method parameters, the parameters should be controlled and the precautionary statement should include in the documentation of the method.

Determination of ruggedness: Carry out the assay as described above using different analyst and different instrument.

Different instrument: Test for ruggedness was performed on HPLC system VTL-LC-002

Different column: Determination of robustness was performed by use new column of same parameter as selected above [35-37].

(I) Accuracy: The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method should be established across its range. Performed accuracy in three different levels for Drug Besifloxacin and spiked known quantity of Besifloxacin Standard at 50%, 100% and 150% level [38-41].

Results and Discussion

Diluents trial for preparation of sample and standard

In trial 1, standard dissolved in the Water and Acetonitrile (50:50) but the gel formulation was found to be un dissolved. In trial 2, Standard and gel formulation both was found to be very Hazy in Water: Dimethylformide (50:50). In trial 3, Standard dissolved in the Methanol and Dichloromethane (75:25) and almost the gel formulation was dissolved but some un dissolved little lumps of gel components were observed to be suspended in sample. In trial 4, standard and gel formulation both were un dissolved in water, methanol and dichloromethane (25:50:25). In trial 5, Standard and gel formulation both were dissolved in the water and methanol in 50:50.

RP-HPLC method trials

In trial 1, Equilibrate the HPLC system with the mobile phase until a steady baseline is obtained. Inject all solutions as per injection sequence into the chromatograph and record the chromatograms. Chromatogram of Besifloxacin and its symmetry were not good. And the chromatogram was found to be merging with the chromatograms of impurities. To increase the resolution, next trail was taken. In trial 2, chromatogram of besifloxacin and its symmetry were not good. The tailing factor found in this buffer is more than 2. To decrease the tailing, next trail was taken. In trial 3, chromatogram of Besifloxacin and its symmetry and peak shape were good. The chromatogram was found to be clearly separated with the chromatograms of impurities. The system suitability is passed in this trial. The method used in this trial is appropriate for Besifloxacin (Tables 3 and 4).

Table 3: Results of diluents trial for preparation of sample and standard.

Table 4: Results of RP-HPLC method trials.

Chromatogram of blank, placebo, standard and sample are given as respectively (Figures 1-4). Assay of Besifloxacin was found to be 98% and assay of phenoxyethanol was found to be 97.4%. Information about the area and retention time of standards and sample (Table 5).

Figure 1: Chromatogram of blank sample.

Figure 2: Chromatogram of placebo sample.

Figure 3: Chromatogram of standard sample.

Figure 4: Chromatogram of sample.

Table 5: Area and retention time of standards and sample.

Validation of RP-HPLC

System suitability: Standard solutions of Besifloxacin and Phenoxyethanol were injected and standard area and retention time were noted for 5 replicated injections of samples; of which average standard area and retention time were calculated. Data generated with the sample runs are tabulated, which showed that system suitability parameter meets the requirement of method validation (Tables 6 and 7) (Figure 5).

Figure 5: Chromatograms of samples for recording system suitability parameter.

Table 6: Data of standard solution of besifloxacin and phenoxyethanol.

Table 7: System suitability parameters.

Initial assay: Initial assay had been carried out 3 times to check RSD of Retention Time and Area of standard drug Besifloxacin and the results are tabulated, which showed that RSD of Retention Time and Area of six replicate is within 2%, which proves repeatability (Table 8).

Table 8: Area and retention time of standard drug besifloxacin.

Chromatograms of 3 runs of initial assay with standard and sample: Initial assay 1, Initial assay 2 and Initial assay 3 are represented in respectively (Figures 6-8).

Figure 6: Chromatograms of initial assay 1.

Figure 7: Chromatograms of initial assay 2.

Figure 8: Chromatograms of initial assay 3.

The results of the three trials of Initial assay are summarized in, which showed that RSD of 3 initial Assays performed in different timings are within 2% and this complies with ICH guidelines and thus, initial assay parameter meets the requirement of method validation (Table 9).

Table 9: Summation of initial results (Repeatability).

Precision: Results obtained from system precision studies are tabulated in, concluding that retention time and area response are consistent as evidenced by the values of relative standard deviation and thus, system precision parameter meets the requirement of method validation (Table 10).

Table 10: Results of injecting besifloxacin.

Results of method precision and intermediate precision are tabulated in Table 11 collectively. Results of Table 10 and concluded that method is precise and rugged. Chromatograms of 6 samples of precision are presented (Table 11) (Figures 9 and 10).

Figure 9: Chromatograms of first three precision assays.

Figure 10: Chromatograms of fourth, fifth, sixth precision assays.

Table 11: Data interpretation of method precision and intermediate precision.

Specificity: Chromatogram of specificity is schematically represented in Figures 10 and the values of peak point are tabulated (Table 12 and Figure 11).

Figure 11: Typical chromatogram of peak purity.

Table 12: Three-point peak purity.

Linearity and range: A Graph of concentration versus chromatographic response (area) is plotted. From the graph it can be seen that the correlation co-efficient is almost equal to 1 and regression line obtained is linear the linearity plot concentration (Figure 12).

Figure 12: Linearity plot concentration of besifloxacinvs area.

Detection limit: Chromatograms of the detection limit. Here Besifloxacin cannot be detected because the Signal vs. Noise Ratio (S/N) should be 3/1 but here the area of signal is similar to area of noise (Figure 13).

Figure 13: Chromatograms of detection limit.

From it can be seen that lowest concentration (0.058 μg/ml) of an analyte in a sample can be detected. But it couldn’t be quantified (Table 13).

Table 13: Area for limit of detection of besifloxacin after 5 times dilution.

Quantitation limit: Chromatograms of quantitation limit test and assay of Besifloxacin is tabulated. From the above data it can be seen that sample recovery of Besifloxacin is 100.74%. Hence sample concentration up to 117 μg/ml can be quantified with the acceptable accuracy (Figure 14 and Table 14).

Figure 14: Chromatograms for quantitation limit.

Table 14: Results of assay of besifloxacin for quantitation test.

Robustness and ruggedness: Chromatogram of robustness, which was carried out by assay using different column and assay of Besifloxacin is tabulated in from the above results, it can be concluded that the recovery is well within the limit collectively showed the data of robustness and ruggedness (Figure 15) (Tables 15 and 16).

Figure 15: Chromatogram of standard and sample for robustness.

Table 15: Assay of besifloxacin by using different column.

Table 16: Results of robustness and ruggedness.

Accuracy: Accuracy testing showed % RSD for all recovery values is 1.31%. There is clear difference between the assay carried out under different conditions and that of Accuracy is within ± 2%. This proves that lack of influence operational and environmental variables does not effect on the test results by using this method (Table 17).

Table 17: Accuracy data.

Conclusion

From the above results, it can be concluded that the recovery is well within the limit. Hence, the method is accurate. The recovery is in between 98% to 101% and the % RSD for all recovery values is 1.41% which is in limits. In HPLC method, the conditions were optimized to obtain an adequate separation of eluted compounds. Initially, various mobile phase compositions were tried, to separate title ingredients. Mobile phase and flow rate selection was based on peak parameters (height, tailing, theoretical plates, capacity or symmetry factor), run time, resolution. The system with Acetonitrile: methanol: buffer with 1.0 ml/min flow rate is quite robust. Since all the acceptance criteria’s of the parameters selected for validation are satisfied, the method stands validated.

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