Validated RP-H-P-L-C Method for the Simultaneous Determination of Betamethasone and Ofloxacin in Bulk and Pharmaceutical Formulations

Introduction

Microbial diseases have a profound effect on public health. Infections can occur in any part of the body, with either the bacteria themselves or the body’s response leading to illness. Humans can acquire bacteria from various sources, including living organisms, atmosphere, water & food. The primary modes of transmission for microbial infections include direct contact, aerosolized droplets, vectors, & contaminated vehicles. Preventive measures significantly decrease morbidity & mortality rates. These measures encompass water disinfection, vaccination of humans & animals, promoting safer sexual practices, & enhancing personal hygiene. The rise of antibiotic resistance in bacteria is an emerging concern that necessitates prudent antibiotic usage.

Bacteria are omnipresent & play a crucial role in maintaining our ecosystem. Only a small fraction of bacteria are responsible for infections & diseases, which have a significant impact on public health. Antimicrobial agents with antimicrobial properties are increasingly accessible, facilitating the treatment of microbial infections compared to viral ones. However, the growing issue of microbial resistance to these antimicrobials poses a serious threat, potentially more severe than diseases caused by viruses & parasites. Ocular microbial infections are among the primary contributors to morbidity & vision impairment. The rise of antibiotic resistance (AMR) is progressively undermining the effectiveness of early antibiotic interventions, which are essential for preserving eyesight.^{1, 2}

In this instance, we evaluated the cause of microbial infections in the eyes observed at Massachusetts Eye & Ear & investigated the molecular epidemiology & antimicrobial resistance profiles of recent isolates.

In primary care, eye infections are a frequent presenting issue. Conjunctivitis, “red eye,” & “corneal ulcer/keratitis” were the top five issues. The practitioner should diagnose the patient as soon as possible & begin the necessary treatment to guarantee a positive visual outcome for the patient. While infections of the cornea or inside the eye pose a major risk to vision, conjunctivitis usually poses no such threat & should be treated immediately by referral to an ophthalmologist.^{3- 5}

In the present state, as antimicrobial & steroid combinations that are frequently used to treat microbial infections, the effectiveness & safety of the combination of Ofloxacin & betamethasone are researched & developed.⁶ According to certain claims, it lessens middle ear mucosal oedema & ocular region redness from infection, which stops microbes from colonizing the allergic site & lessens the sensitivity & allergies of the anti-biotic component inside the liquid drops.⁷

Analyte Background

The combination of two medications is called betamethasone & ofloxacin. A steroid called betamethasone inhibits the synthesis of prostaglandins, which are chemical messengers that induce swelling, redness, & itching. An antibiotic called ofloxacin kills bacteria by stopping their ability to divide & repair. This takes care of your infection.^{8, 9}

Betamethasone

Betamethasone, a corticosteroid which is long-acting, possesses anti-inflammatory & immunosuppressive properties. It can be administered via injection to address various medical settings, including autoimmune disorders, & can also be applied topically to manage inflammatory skin issues such as eczema. Betamethasone demonstrates significant glucocorticoid effects while exhibiting minimal mineralocorticoid activity. For the treatment of plaque psoriasis, it can be used topically in conjunction with a vitamin D analog like calcipotriene. Furthermore, betamethasone functions through both genomic & nongenomic pathways. The genomic pathway is characterized by glucocorticoids activating glucocorticoid receptors, leading to flowing effects that promote the transcription of genes  which are anti-inflammatory, including tyrosine amino transferase (T-A-T), phosphoenolpyruvate carboxykinase (P-E-P-C-K), & the IL-1 receptor antagonist.^{10, 11}

Ofloxacin

Treating microbial infections in the kidney, skin, soft tissues, respiratory tract, & urinary tract, among other regions of the body, is done with this antimicrobial drug. The antimicrobial agent known as fluoroquinolones is a synthetic compound that prevents microbial DNA gyrase from supercoiling, hence stopping DNA replication.^{12, 13}

Scheme 1: Structure of Betamethasone (Source: Drawn in Chemdraw).Click here to View Scheme

Scheme 2: Structure of Ofloxacin (Source: Drawn in Chemdraw).Click here to View Scheme

H-P-L-C is a highly effective analytical technique for the separation & quantification of drugs. Currently, there are no reported RP-H-P-L-C methods in the literature for the estimation of Betamethasone & Ofloxacin, either in combination/individually dosage forms.⁸ A review of existing literature indicates the presence of more economical methods; therefore, it is essential to develop & validate a straightforward, cost-effective stability-indicating simultaneous estimation of Betamethasone & Ofloxacin using RP-H-P-L-C in pharmaceutical dosage forms, in accordance with I-C-H rules (Q2 specifications).^{14, 15}

Materials and Reagents

Betamethasone & Ofloxacin were sourced as pure substances from Symbiotec Pharma Lab.

From Rankem Chemical Division H-P-L-C-grade methanol & acetonitrile were obtained in India.

From Rankem Potassium-hydrogen-phosphate was also acquired, India.

(Rankem, India) Pure milli-Q water was utilized, filtered through 0.45µ Millipore filters.

Chromatographic Settings and Instrumentation

The WATERS H-P-L-C model (System 2695), equipped with a photodiode array detector, utilized for method authenticate & development, featuring an automated sample injector. Kromosil C18 (4.6 mm ID x 250 mm x 5µ) served as the separation medium. The Mobile Phase (MP) consisted of 0.01N KH₂PO₄ as phase A & Acetonitrile as phase B in a 55:45 ratio. The study was performed in isocratic mode at a FR of 1.0 mL/min, with an injection vol 10 µL. The column was maintained at a temp of 30 degree centigrade, & the total run time was 8 mins. Data acquisition occurred at a detection wave vector of 278 nm using Empower 2 software.

Figure 1: Enhanced Chromatogram of Betamethasone & Ofloxacin | Source: attached is the chromatogram file in folder labeled Figure 1Click here to View Figure

Solutions Preparation

Diluent: (50:50v/v) ratio of Water & Acetonitrile.

Buffer Preparation

KH₂PO₄ Buffer

Precisely measure 0.1% of ortho phosphoric acid in a 1000 ml flask which is volumetric, add approximately 900 ml of milli-Q water, degas by sonication, & then adjust the vol with water to achieve a pH of 2.8.

Preparation of Standard solution

Accurately Weighed & transferred 2mg of Betamethasone & 6mg of Ofloxacin working Standards into 25 ml clean dry flask which is volumetrics, add 10ml of diluent, sonicated for 10 mins & make up to the final vol with diluents. (80µg/ml Betamethasone & 240µg/ml Ofloxacin)

Standard Working Solution

1ml of standard concentrated solution was transferred to 10ml flask which is volumetric & made up with diluent. (8µg/ml Betamethasone & 24µg/ml Ofloxacin)

Preparation of Sample Concentrated solution

A total of 0.01 mg w/v of Betamethasone & 0.03% w/v of Ofloxacin was extracted from the sample container & transferred into a clean, dry 25 mL flask which is volumetric. Approximately 7 mL of diluent was added, & the mixture was sonicated until fully dissolved. The vol was then adjusted to the mark with the same solvent & ran through through a 0.45-micron injection filter using a syringe. Subsequently, 1 mL of the Concentrated solution was pipetted into a 10 mL flask which is volumetric & madeup to the mark with diluent, resulting in concentrations of 40 µg/mL for Betamethasone & 120 µg/mL for Ofloxacin.

Sample Working Solution

2ml of standard concentrated solution was transferred to 10mL flask which is volumetric & made up with diluent. (8µg/ml Betamethasone & 24µg/ml Ofloxacin)

Method Authenticate

The authenticate of the H-P-L-C method was conducted for the concurrent estimation of Betamethasone & Ofloxacin drug substances in accordance with I-C-H rules, to establish that the method is suitable for regular study.

System suitability

The assessment of system suitability was conducted for each authenticate parameter by introducing a system suitability solution comprising Betamethasone at a concentration of 8 µg/ml & Ofloxacin at 24 µg/ml. The elution profile illustrating system suitability is presented in Depiction 2, with the corresponding values detailed in Chart 1.

Specificity (Selectivity)

The evaluation of interference in the enhanced method indicates that no interfering Max outs should be present in the blank & placebo samples at the retention times of these drugs. Consequently, this method is considered specific. A representative elution profile is illustrated in Depiction 4, & the experimental data is presented in Chart 1. 

Table 1: System suitability chart

Figure 2: System suitability Elution profile of Betamethasone & Ofloxacin | Source: attached is the chromatogram file in folder labeled Figure 2.Click here to View Figure

Table 2: Specificity data

Figure 3: Specificity & Overlay representation of H-P-L-C Elution profile of Betamethasone & Ofloxacin | Source: attached is the chromatogram file in folder labeled Figure 3Click here to View Figure

The elution profile presented above indicates that there was no interference detected in the blank & placebo solutions at the retention times of Betamethasone & Ofloxacin. All compounds are well separated with satisfactory resolution.

In order to assess the stability-indicating characteristics of the H-P-L-C method, samples of Betamethasone & Ofloxacin were subjected to stress settings including acid, base, oxidation, thermal exposure, light, & water. The resulting degraded samples were analyzed using a photodiode-array detector, & the Max out purity for both Betamethasone & Ofloxacin was confirmed. The settings for forced degradation are detailed in Chart 3, while the corresponding outcomes are presented in Chart 4. 

Table 3: Forced degradation settings for Betamethasone and Ofloxacin.

The outcomes indicated that degradation Max outs were noted when the samples were subjected to acid exposure. The stress study revealed that none of the degradants co-eluted with the Max outs of the active drug that were formed.

Table 4: Degradation profile outcomes

Figure 4: Purity plots | Source: attached is the chromatogram file in folder labeled Figure 4.Click here to View Figure

L-O-D and L-O-Q

The detection boundary indicates the lowest concentration of an analyte in a sample that can be recognized, even if it cannot be quantified. In contrast, the L-O-Q is the minimum concentration of an analyte that can be accurately & precisely measured using the specified method. The L-O-D values for Betamethasone & Ofloxacin are shown in Chart 5, accompanied by the relevant elution profile depicted in Depiction 4.

Table 5: Summary of L-O-D

Figure 5: Typical representation of H-P-L-C Elution profile of L-O-D Solution. Based on above outcomes for L-O-D, S/N ratio of each component was within the boundary | Source: attached is the chromatogram file in folder labeled Figure 8Click here to View Figure

The L-O-Q values obtained for Betamethasone & Ofloxacin are listed in Chart 5 & 6 corresponding representative elution profile is shown in Depiction 4.

Table 6: Summary of L-O-Q

Figure 6: Typical representation of H-P-L-C Elution profile of L-O-Q Solution. Based on above outcomes for L-O-D, S/N ratio of each component was within the boundary | Source: attached is the chromatogram file in folder labeled Figure 9Click here to View Figure

Linearity

The method’s linearity was established for Betamethasone & Ofloxacin by examining solutions that varied from 25% to 150% of the specified boundary (refer to Chart 7). The correlation coefficient obtained for both Betamethasone & Ofloxacin was 0.999, which signifies a strong linear relationship (see Figure 7).

Figure 7: Linearity plot of BetamethasoneClick here to View Figure

Figure 8: Linearity plot of OfloxacinClick here to View Figure

Table 7: Linearity data

Assay data

Festive-D Eye/Ear Drops bearing the label claims Betamethasone 0.01% W/v, Ofloxacin 0.03% w/v. Assay was performed with the above formulation. Average % Assay for Betamethasone & Ofloxacin obtained was 99.10 & 100.33% respectively. Assay data shown in chart no 8.

Preparation of Sample Concentrated solution

A sample containing 0.01 mg w/v of Betamethasone & 0.03% w/v of Ofloxacin was pipetted from the sample container into a clean, dry 25 mL flask which is volumetric. Approximately 7 mL of diluent was added, & the mixture was sonicated until fully dissolved. The vol was then adjusted to the mark with the same solvent & filtered through a 0.45-micron injection filter using a syringe. Subsequently, 1 mL of the Concentrated solution was pipetted into a 10 mL flask which is volumetric & diluted to the mark with diluent. Additionally, 2 mL of the standard Concentrated solution was transferred to a 10 mL flask which is volumetric & brought to vol with diluent, resulting in concentrations of 8 µg/mL for Betamethasone & 24 µg/mL for Ofloxacin. 

Table 8: Assay data of Betamethasone & Ofloxacin

AT – Average Peak area of in test solution

AS – Mean peak ares of in standard solution

WS – Weight of working standard taken in mg

P – Assay of working standard in % on dried basic

L.C – Label Claim

FV – Filled volume(1ml of vail).

Accuracy

The accuracy of the method was assessed by utilizing solutions that included spiked samples of Betamethasone & Ofloxacin at concentrations of 50%, 100%, & 150% of the working strength. All solutions were prepared in triplicate & subsequently analyzed. The % recovery outcomes for each impurity are presented in Chart 9.

Table 9: % Recovery data

System accuracy

The system accuracy was performed by analyzing six replicate injections of working solution at 100% of the specified boundary with respect to the working strength of Betamethasone & Ofloxacin. Outcomes of Max out area are summarized in Chart 10.

Table 10: System accuracy data

The % R-S-D for the Max out areas of Betamethasone & Ofloxacin obtained from six replicate injections of working solution was within the boundary.

Method Accuracy

A frequently used statistical term is the standard error of the mean of a population of observations. The standard error of the mean is calculated as the square root of the total of the squared deviations of each individual result from the mean, divided by the total number of outcomes minus one. The standard error of the mean, denoted as S, is expressed as follows

The standard error of the mean shares the same units as the measured property. The square of the standard error of the mean is referred to as variance (S²). This is defined as the standard error of the mean divided by the mean, represented as S/x. This value is occasionally multiplied by 100 to express it as a percentage, known as the percent relative standard error of the mean, which provides a more dependable indication of accuracy.

The accuracy of the method was assessed through the study of a sample consisting of Betamethasone & Ofloxacin, involving six separate sample preparations. The outcomes are compiled in Table 11.

Table 11: Method accuracy data

From the above outcomes, the % R-S-D of method accuracy study was within the boundary for Betamethasone & Ofloxacin.

Intermediate accuracy differs from repeatability in that it reflects the accuracy achieved within a single laboratory over an extended duration, typically several months, & takes into account a wider range of variables than repeatability. Specifically, it includes variations such as different analysts, calibration Standards, reagent batches, columns, & spray needles. While these factors remain consistent within a single day, they are not s-chart over longer periods, thus exhibiting random behavior in the context of intermediate accuracy. As a result, since intermediate accuracy considers more influencing factors, its value, represented as standard error of the mean (as detailed in the following section), is greater than that of the repeatability standard error of the mean. The data collected is presented in Chart 12.

Table 12: Intermediate accuracy data

From the above outcomes, the % R-S-D of method accuracy study was within the boundary for Betamethasone & Ofloxacin.

Robustness

The chromatographic parameters were intentionally modified to assess the robustness of the current method. To evaluate this robustness, a system suitability solution was prepared according to the established methodology & injected into the H-P-L-C under various altered settings. These settings included adjustments to the FR (± 10%), column oven temp (± 5°C), & MP composition (± 10%) from the original method specifications. No significant changes were noted in the method’s performance with respect to flow, temp, or MP, & the system suitability criteria were met as per the methodology. The outcomes regarding robustness are presented in Table 13.

Table 13: Robustness outcomes

Conclusion

A straightforward, precise, & reliable method has been established for the concurrent assessment of analytes in chart formulations. The system suitability was evaluated by performing 6 injections of the standard, with outcomes consistently meeting the acceptance criteria (Boundary of <2). A linearity assessment was conducted across levels ranging from 25% to 150%, yielding an R2 value of 0.999. Various authenticate parameters, including accuracy, accuracy, boundary of detection L-O-D, L-O-Q, & robustness, were confirmed to be within accept chart boundary. The percentage recovery was recorded at 100.09% for Betamethasone & 100.62% for Ofloxacin. This method is characterized by its simplicity, accuracy, sensitivity, rapidity, & cost-effectiveness, with a total runtime of fewer than 8 mins. It is practically applicable for the determination of assay in chart formulations.

Acknowledgement

We would like to express my sincere gratitude to Spectrum Pharma Research Solutions for the opportunity to conduct part of the experiment.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

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Abbrevations List

RP-H-P-L-C: Reverse Phase High Performance Liquid Chromatography

H-P-L-C: High Performance Liquid Chromatography

R-S-D: Relative Standard Deviation

L-O-D: Limit of Detection  

L-O-Q: Limit of Quantification

Temp: Temperature

Vol: Volume

KH₂PO₄: potassium dihydrogen phosphate

μm: micrometer

nm: nanometer

⁰C: Degree Centigrade

mins: Minutes

T-A-T: tyrosine amino transferase

AMR: antibiotic resistance

P-E-P-C-K: phosphoenolpyruvate carboxykinase

I-C-H: International Council for Harmonisation

mL: milliliter

mm: millimeter

v/v: volume per volume

µg: microgram

Std: Standard

dev: Deviation

FR: Flow Rate

MP: Mobile Phase