Simultaneous Estimation of Ciprofloxacin hydrochloride and Dexamethasone sodium phosphate in Ophthalmic Formulations

Abstract

The simultaneous estimation of multiple active pharmaceutical ingredients (APIs) in combined dosage forms has become increasingly significant in the pharmaceutical industry, especially for formulations intended for topical applications such as ophthalmic preparations. This review article focuses on the analytical methodologies developed for the simultaneous estimation of Ciprofloxacin hydrochloride (CIP) and Dexamethasone sodium phosphate (DEX) in ophthalmic formulations. CIP, a broad-spectrum fluoroquinolone antibiotic, and DEX, a potent corticosteroid, are often co-formulated to provide both antibacterial and anti-inflammatory effects in the treatment of ocular infections. However, the co-existence of these chemically distinct compounds poses analytical challenges due to differences in their physicochemical properties. Various analytical techniques have been explored for the simultaneous estimation of CIP and DEX, including UV-Visible spectrophotometry, high-performance liquid chromatography (HPLC), and derivative spectroscopic methods. Among these, HPLC has been widely preferred due to its high sensitivity, selectivity, and accuracy. Validation of these methods, according to US FDA guidelines, ensures reliability in terms of parameters like linearity, precision, accuracy, robustness, and specificity. Spectrophotometric techniques, while simpler and cost-effective, often suffer from overlapping absorption spectra, requiring mathematical correction methods or derivative approaches.

Keywords

Introduction

                   

A)                                                            B)

Figure 1: (A) Chemical structure of Ciprofloxacin hydrochloride and (B) Dexamethasone sodium phosphate

DEX is a corticosteroid that suppresses the immune system and lowers inflammation. Other names for the synthetic glucocorticoid DEX include 9,α-uoro-11β and 17,21-trihydroxy-16α methylpregna-1,4-diene-3,20-dione. It has been widely used to treat conditions related to in inflammation, allergies, and adrenal cortical insuffciency. ^[1–7]

DEX is a potent synthetic glucocorticoid that mimics the effects of cortisol, a hormone naturally found in the adrenal cortex. It works by binding to cytoplasmic glucocorticoid receptors and initiating transcriptional processes that have anti-inflammatory and immunosuppressive effects. Several HPLC techniques for CIP and DEX individual estimation have been described. In pharmaceutical applications, stability-indicating methods for CIP and DEX have been developed and validated here. In one published investigation, the analyte was detected using a photo diode array detector (PDA). A C18 column containing sodium buffer (pH 3.05) and acetonitrile (60:40, v/v) as a mobile

 phase in isocratic mode was used to build the CIP and DEX HPLC technique. Furthermore, an LC-MS/MS method has been developed to evaluate the levels of DEX  and CIP in the tear fluid of the eyes. Similarly, LC-MS/MS is used to evaluate DEX and ofloxacin together. The discovered method was used in drug distribution and ocular pharmacokinetics. To conduct more research on antibacterial and anti inflammatory combinations, a more efficient, sensitive, and practical method for these drugs needs to be developed. Therefore, in this study, we aim to develop a simultaneous estimation analytical approach for CIP and DEX for fast and effective analysis. ^[8–11]

2. Pharmaceutical Significance

CIP: Broad-spectrum bactericidal agent inhibiting bacterial DNA gyrase.

DEX: Corticosteroid reducing in inflammation by inhibiting multiple inflammatory cytokines. Combined formulations offer synergistic benefits but also demand stringent analytical control to ensure the correct dosage of both actives.

3. Analytical Challenges

• Overlapping UV Absorption Peaks: CIP and DEX show absorption in the UV region, often overlapping.
• Polarity and Solubility Differences: Affect chromatographic behavior and separation.
• Stability: DEX is prone to hydrolysis and light degradation, requiring careful sample handling.
• Matrix Interference: Presence of preservatives and excipients in ophthalmic solutions may interfere with analysis.

4. Analytical Techniques for Simultaneous Estimation

4.1 UV-Visible Spectrophotometry

The quantitative assessment of a material's transmission or reflection characteristics as a function of wavelength is known as spectrophotometry. These techniques have the benefit of requiring little time and effort. These techniques also have outstanding precision. Over the past few years, there has been a sharp rise in the usage of UV-Vis spectrophotometry, particularly when it comes to the examination of medicinal dosage forms.

4.1.1 Simultaneous Equation Method                                                                                                                                             

This approach can be used to estimate medications whose spectra appropriately overlap, meaning that if the sample contains two absorbing pharmaceuticals, each of which absorbs at the others  λmax, both drugs can be identified using the simultaneous equation method. Based on absorbance measurements at two wavelengths (for example, 240 nm for DEX and 272 nm for CIP). Requires both medicines' absorptivity levels at both wavelengths.

4.1.2 Q-Analysis (Absorbance Ratio)

Use one of the components' λmax and isoabsorptive point. Ideal for quick and easy analysis in straightforward solutions. The ratio of absorption at two chosen wavelengths—one representing the iso-absorptive point and the other the λmax of one of the two components—is determined using the absorption ratio method.CIP and DEX absorption maxima are located at 272nm and 242 nm, respectively.

4.1.3 Derivative Spectrophotometry

Separates overlapping spectra to improve selectivity. For qualitative analysis and estimation, derivative spectroscopy employs the first or upper derivatives of absorbance concerning wavelength. In the 1950s, the idea of derivatizing spectral data was originally proposed, and its many benefits were demonstrated. However, the difficulty of producing derivative spectra with early UV-visible spectrophotometer meant that the technique was not given much thought. ^[12]

Advantages: Simple, inexpensive, fast.
Limitations: Less specific, requires clear solutions and little matrix interference.

4.2 High-Performance Liquid Chromatography (HPLC)

Through advancements in column technology and instrumental components (pump, column, injection volume, and detectors), high-performance liquid chromatography was created as an analytical method in the 1960s. Because of the relatively high operating pressure produced by early columns, the HPLC was originally known as high-pressure liquid chromatography. Enhancing the separation changed the meaning of great performance. ^{[13, 14]}

Types of HPLC

HPLC is divided on the basis of separation chemistry. All these techniques can be used the same instrumentation.

1. Reversed - phase chromatography

In this type uses a non-polar (hydrophobic) stationary phase and a polar (usually including some water) mobile phase. This is the most common type of HPLC separation in use today.

II. Normal - phase chromatography

In this type uses a polar (hydrophilic) stationary phase and a non-polar (usually with no water) mobile phase. This was the sort of separation to that the term “Chromatography” was first applied.

III. Ion - exchange chromatography

This is the most common type of HPLC separation in use today. In this type uses a non-polar (hydrophobic) stationary phase and a polar (usually including some water) mobile phase are used.Use a derivatized stationary phase support to uniformly attach charged groups to the surface. Typically, an aqueous buffer serves as the mobile phase. This method is mostly used to separate large molecules like proteins, nucleic acids, or large peptides, or to analyze ions like strong acids or bases.
IV. Size - exclusion chromatography

Size-exclusion chromatography makes use of a stationary phase with a regulated distribution of pore sizes. The ability of analyte molecules to pierce the network of holes is the basis for separation.

4.2.1 Reversed-Phase HPLC (RP-HPLC)

Nowadays, this is the most often used kind of HPLC separation. This kind employs a polar (often containing some water) mobile phase and a non-polar (hydrophobic) stationary phase. Typically employs a C18 column with mobile phases such as phosphate buffer and acetonitrile or methanol. Detection at dual wavelengths, or 254 nm for both drugs.

HPLC method development:

Advantages: High accuracy, reproducibility, and resolution.

Applications

1. Pharmaceutical industry

• Drug quality control: HPLC is use to analyze the analyte in the pharmaceutical formulation to determine their purity, identity, stability and safety.
• Pharmacokinetics: It is used to study what body does to the drug(ADME). HPLC used to study how drug are absorbed, distributed, metabolized, and excreted from the body.

2. Biotechnology and Biochemistry

• Bio-pharmaceutical analysis: HPLC used in the bio-pharmaceuticals such as, monoclonal antibodies and recombinant proteins.
• Protein expression and purification: HPLC is used to optimize the protein expression and purification conditions.

3. Food and beverages analysis

• Detection of food additives: HPLC is used to detect and quantify food additives, such as preservatives, sweeteners, and colorants.
• Analysis of food contaminants: HPLC used to determine the food contaminants such as  micotoxins, heavy metals, and polycyclic aromatic hydrocarbons, etc.^[15]

Limitations: Expensive instrumentation, more time-consuming.

4.3 High-Performance Thin-Layer Chromatography (HPTLC)

HPTLC is an advanced type of thin-layer chromatography (TLC) that improves quantitative analysis and separation effectiveness. Compared to TLC, HPTLC uses precoated plates with lower particle sizes, which improves sensitivity and resolution. Involves optimizing the mobile phase and silica gel plates. Ideal for semi-quantitative analysis and regular quality control. Densitometry is used for detection at the appropriate λmax.

4.4 Other Advanced Techniques

4.4.1 LC-MS/MS: LC-MS/MS Liquid Chromatograph Mass Spectrometry (LC-MS) is a potent analytical method that identifies and quantifies them by combining with mass spectrometry (MS). While MS determines the mass-to-charge ratio of the ions, which provides information about the molecular weight and structure of the separated chemical, LC separates the compounds according to their varying affinities for the stationary and mobile phases. Mostly employed in pharmacokinetic research. Provides exceptional selectivity and sensitivity.

4.4.2 Chemometric Methods: Chemometric analyzes complex chemical data reduction, pattern recognition, and model generation for a variety of analytical techniques using the mathematical and statistical method. To resolve overlapping data, apply statistical modeling (e.g., PLS, PCR) to UV spectra.

4.4.3 Capillary Electrophoresis: Narrow-bore capillaries are used in capillary electrophoresis (CE), a separation method, to separate molecules according to their size and charge in an electric field. Its adaptable approach has uses in several fields, such as environmental monitoring, biotechnology, and pharmaceuticals. High eco-friendliness and efficiency. Calls for the creation of intricate methods.

5. Sample Preparation and Extraction

Ophthalmic preparations frequently require a straightforward dilution with buffer (pH ~4) or distilled water. Before injection, membrane filtration (0.45 μm) is used. Stability is ensured by adequate pH correction and light protection. To ensure the stability and integrity of both analytes during analysis, meticulous attention to sample preparation and extraction techniques is necessary for the accurate and repeatable simultaneous estimation of Ciprofloxacin Hydrochloride and DEX in ocular formulations.

5.1. Dilution and Solvent Selection: Usually aqueous in base, ophthalmic formulations may include additional excipients, viscosity boosters, and preservatives. Therefore, a basic dilution of the formulation using an acceptable solvent, like distilled water or a suitable buffer solution, is typically required for the initial sample preparation. Because it provides the best conditions for both CIP and DEX, a buffer with a pH of about 4 is frequently used to preserve the medications' solubility and chemical stability. To bring the analytes within the calibration range of the analytical method (such as HPLC or UV-Vis), the dilution factor is selected depending on the expected concentration range of the analytes. To prevent matrix effects and peak distortion in HPLC, it is frequently desirable to employ a mobile phase or diluent with a comparable composition.

5.2. Filtration: The material is put through membrane filtration to get rid of any particles that could obstruct chromatographic systems or interfere with analysis. Standard membrane filters have pore sizes of 0.45 μm because they effectively remove particles without adsorbing substantial amounts of the analytes. A 0.22 μm filter may be employed for extremely sensitive analyses, especially when employing sophisticated detection techniques like mass spectrometry or fluorescence.

5.3. pH Adjustment: Both CIP and DEX depend on the pH of the sample solution to remain chemically stable and in their ionized state. DEX is more stable in mildly acidic to neutral environments than CIP, which is a weak base. In order to avoid hydrolysis or degradation of either component and to guarantee constant retention lengths and peak shapes in chromatographic analysis, the pH should be adjusted to a slightly acidic range (about 4 to 5).

5.4. Light Protection: Because CIP and DEX are both light-sensitive, photodegradation and impurity formation may result. This is kept to a minimum by preparing samples in low light and storing them in amber vials or covered in aluminum foil for processing and analysis.

5.5. Storage and Stability: Samples should be evaluated as soon as they are prepared. Samples should be stored at low temperatures (such as 2–8°C) and shielded from light if storage is required. To ascertain the longest period that the prepared sample can be stable without experiencing appreciable degradation, stability tests ought to be carried out. ^[16]

6. Method Validation

According to US FDA criteria, method validation guarantees that an analytical technique is appropriate for its intended purpose, offering precision, accuracy, and reliability within the designated range and stability conditions. Selectivity, accuracy, precision, recovery, calibration curves, sensitivity, repeatability, and stability are among the particular metrics that the FDA specifies must be validated. ^{[17, 18]}

Validation is carried out in compliance with US FDA regulations, assessing:

Validation of HPLC method focus mainly on following:

• Identification test
• Quantitative measurements of the content of related substances
• Semi-quantitative and limit test for control of related substances
• Quantitative test for assay of major components (e.g drug substances and preservatives) in sample of drug substances or drug product (assay, content uniformity, dissolution rate etc.)

6.1 Specificity: In chromatography, specificity is the capacity of the technology to reliably quantify the analyte response in the presence of all conceivable sample components. For chromatographic procedures, developing a separation requires proving specificity. To examine the test compound response, execute the study in a test mixture containing all potential components (placebo formulation, excipients, degradation products, etc.) is compare it with the sample containing just the analyte. By subjecting the analyte to enough stress to degrade it to 80–90% purity, additional possible components are produced. It is common for degradation to involve heat at 50–60°C, acid (1N HCl), basic (1 N NaOH), and oxidants (3% H2O2). Following analysis of the resultant mixture, the analyte peaks are assessed for peak purity and resolution from the closest eluting peak.

6.2 Linearity: Linearity is the range of the method to elicit test results that are directly proportional to analyte concentration within a given range. Range is the interval between the upper and lower limit of the analyte (inclusively) that has been demonstrated to be determined with precision, accuracy, and linearity using the method as written. The linear relationship should be evaluated across the range of the analytical procedure. Acceptability of linearity data is judged by examining the regression coefficient and y-intercept of the linear regression line for the response versus concentration plot. The correlation coefficient >0.999 is considered acceptable. The y-intercept should be less than a few percent of the response obtained for the analyte target level. Taking the regression line as a mean, a percentage RSD was calculated for the data.

6.3 Precision: Precision is the measure of the degree of repeatability of analytical conditions under normal operation, and is normally expressed as the percent relative standard deviation (RSD) for a statistically significant number of samples. The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions. Repeatability is the short time interval study under the same conditions (intra assay precision). According to the FDA, instrument or injection repeatability minimum of 10 injections of a sample test the performance of the chromatographic instrument. Intermediate precision results from within-lab variations due to random changes on different days, analysts, temperature, etc. The precision criteria for the assay method are that instrument RSD < 1%, and the intra-assay precision is < 2%. For impurity assay, the instrument precision is < 5%, and intra-assay precision will be < 10%. Reproducibility, which determines whether the test same sample in multiple laboratories. The precision of an analytical procedure is typically expressed as the variance, standard deviation, or coefficient of variation of a series of measurements.

a. Repeatability

Repeatability expresses the precision beneath the constant operational conditions over a brief interval of time. Repeatability is also termed intra-assay precision.

b. Intermediate precision

Intermediate precision expresses among laboratories variations: Variation in days, analysts, and equipment, etc.

c. Reproducibility

Reproducibility expresses the precision between laboratories (collaborative studies typically applied to standardization of methodology).

6.4 Accuracy: Accuracy of an analytical method is the closeness of the agreement between the value that is expected as the reference value and the value found. It is determined by applying the method to samples to which a known amount of analyte is added. These should be analyzed against a blank to ensure the interference exists in the sample. The accuracy is then calculated from the test result as a percentage recovered by assay. The accuracy criteria for assay method (FDA) are that the mean recovery will be 100 + 2% at each concentration over a range of 80-120% of the target concentration. For the impurity method, the mean recovery is within 0.1% absolute of the theoretical concentration. To document accuracy according it should contain nine samples of three different sets of high, medium, and low concentration (for example, three concentrations, three replicates each).

6.5 Solution stability: In the HPLC method, validation is performed to generate reproducible and reliable results. The stability of sample solutions, standards, reagents, and mobile phase must be determined before initiating the method validation studies. It is performed to ensure the sample is stable enough to allow for delays instrument instrument-related problems, or overnight analyses using an autosampler. During validation of stability, samples are prepared under normal conditions, normal storage, and sometimes in the HPLC system to determine different storage conditions the conditions as refrigeration or protection from light.

6.6 Limit of Detection (LOD): In HPLC method validation, LOD is the lowest amount of analyte in the sample that can be detected, not necessarily quantified to the exact value. Several approaches are used to detect LOD are possible. For an analytical procedure that exhibits baseline noise, an alternative is based on signal-to-noise ratio. It is determined by comparing the signal from the blank sample with a known low analyte concentration. According to the USFDA guidelines signal-to-noise ratio between 3:1 and 2:1 is generally acceptable for detection limit.

The Detection limit expressed as :

DL= x3.3σS

        -------------------------------------- 1

Where σ is the standard deviation of response and S is the calibration curve slope, which are used to calculate the limit of detection.

6.7 Limit of quantification (LOQ): The limit of Quantification of an analytical procedure is the lowest amount of analyte in a sample that can be quantitatively determined in the presence of another component present in the sample. US FDA guidelines. Quantitative limit is determined based on the signal-to-noise ratio observed in the sample. It is generally an S/N ratio (10:1) and usually confirmed by injecting a standard, which gives an acceptable S/N ratio. LOQ is determined with suitable precision and accuracy.

The Quantitation limit expressed as:

QL= x10σS

------------------------ 2

 Where σ is the standard deviation of response and S is the calibration curve slope, which are used to calculate the limit of quantification.

6.8 System Suitability: To generate reproducible and reliable results. In HPLC method validation, system suitability refers to a set of predefined criteria used to determine whether the HPLC is producing optimal results and reliable data. It is an integral part of liquid chromatography methods by checking parameters like resolution, peak integrity, tailing factor, and repeatability (retention time) to ensure that system that define system is suitable for the intended analysis. The quality control samples are strongly referred to in the system suitability test.

7. Applications ^[19]

• Routine Quality Control of commercial eye drops.
• Stability Studies under ICH stress conditions (light, acid/base hydrolysis).
• Dissolution Testing in ophthalmic formulations.
• Pharmacokinetics when used with LC-MS/MS.

CONCLUSION

The simultaneous estimation of CIP and DEX in ophthalmic formulations is essential for quality control and therapeutic efficacy in combination drug therapy for ocular infections. Various analytical techniques, particularly UV-Visible spectrophotometry and high-performance liquid chromatography (HPLC), have been successfully employed for their simultaneous quantification. Each method offers specific advantages in terms of sensitivity, selectivity, cost-effectiveness, and suitability for routine analysis. Critical aspects such as appropriate sample preparation, pH adjustment, protection from light, and the use of membrane filtration significantly influence the accuracy and reproducibility of results. The choice of analytical method should be guided by formulation complexity, regulatory requirements, and available resources. Continued research and optimization of analytical methodologies will further enhance the reliability and efficiency of simultaneous drug estimation in ophthalmic dosage forms, supporting better compliance with pharmacopoeial standards and ensuring patient safety.

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