Development And Validation of a Stability-Indicating UPLC Method for The Simultaneous Estimation of Clindamycin and Miconazole in Bulk and Combined Dosage Forms

Clindamycin, a lincosamide antibiotic, inhibits bacterial protein synthesis and is widely prescribed for anaerobic infections [1]. Miconazole, an imidazole antifungal agent, acts by disrupting fungal cell membrane integrity and is used to treat dermatophytic and candidal infections [2]. These drugs are often combined in topical dosage forms for managing mixed bacterial-fungal infections, providing broad therapeutic coverage [3]. Conventional methods like UV spectroscopy and RP-HPLC have limitations for simultaneous estimation due to differences in physicochemical properties and overlapping absorption spectra [4]. Ultra-Performance Liquid Chromatography (UPLC) offers advantages such as higher resolution, shorter run times, and lower solvent consumption, making it ideal for complex formulations [5]. Despite the clinical significance of this combination, no stability-indicating UPLC method has been reported for clindamycin and miconazole estimation. Existing RP-HPLC methods lack forced degradation studies and require longer analysis times [6]. According to ICH guidelines Q1A(R2) and Q2(R1), stability-indicating methods are essential to ensure quality and detect degradation products [7]. Therefore, this study aimed to develop and validate a simple, robust UPLC method for simultaneous estimation of clindamycin and miconazole in bulk and combined dosage forms, including forced degradation analysis.

MATERIALS & METHODS

Chemicals and Reagents

Clindamycin and Miconazole reference standards were obtained from Spectrum Laboratories, Hyderabad, India. HPLC-grade solvents such as acetonitrile and methanol were purchased from Merck, Mumbai, India. Analytical-grade potassium dihydrogen orthophosphate and orthophosphoric acid were used for buffer preparation. Double-distilled water was produced using a Milli-Q water purification system. All chemicals and reagents complied with analytical standards [1,2].

Instruments and Software

Chromatographic analysis was performed using a Waters UPLC system (Alliance 2695) equipped with a quaternary pump, autosampler, and 2996 PDA detector, controlled by Empower 2 software. Separation was achieved on an Inertsil ODS C18 column (250 × 4.6 mm, 5 μm). Additional instruments included a pH meter (Eutech, India), ultrasonic bath (PCI Analytics), and analytical balance (Shimadzu AUX220).

Chromatographic Conditions

The optimized chromatographic method employed a C18 reverse-phase column with an isocratic mobile phase consisting of acetonitrile and 0.05 M potassium dihydrogen orthophosphate buffer (adjusted to pH 3.0 with orthophosphoric acid) in a ratio of 70:30 v/v. The flow rate was 1.0 mL/min, and the injection volume was 10 μL. Detection was carried out at λmax 210 nm, which corresponds to the maximum absorbance of both drugs. The column was maintained at ambient temperature (25°C). Diluent used for sample preparation was water:acetonitrile (50:50 v/v) [3,4].

Preparation of Standard Stock Solutions

• Clindamycin Standard: Accurately weigh 100 mg of clindamycin and dissolve in 100 mL of diluent to prepare a 1000 μg/mL stock solution.
• Miconazole Standard: Similarly, weigh 100 mg of miconazole and dissolve in 100 mL of diluent to obtain 1000 μg/mL stock solution.
• Working standards were prepared by serial dilution to cover the linearity range (e.g., 10–100 μg/mL for clindamycin and 5–50 μg/mL for miconazole) [5].

Preparation of Sample Solution

A quantity of the combined dosage form equivalent to 50 mg clindamycin and 25 mg miconazole was accurately weighed and transferred to a 100 mL volumetric flask. Approximately 70 mL of diluent was added, and the mixture was sonicated for 15 minutes to ensure complete dissolution. The volume was made up to the mark with diluent and filtered through a 0.45 μm nylon syringe filter prior to injection [6].

Method Development and Optimization

Various chromatographic parameters such as mobile phase ratio, pH, organic modifier concentration, and flow rate were optimized to achieve sharp, symmetrical peaks with satisfactory resolution, retention time, and tailing factor. Different buffer systems (phosphate buffer, formic acid buffer) and organic solvents (methanol, acetonitrile) were tested. The final optimized conditions ensured good system suitability parameters including theoretical plates > 2000, tailing factor < 2.0, and %RSD < 2.0 [7].

RESULTS AND DISCUSSION

The developed UPLC method successfully separated clindamycin and miconazole with sharp, well-resolved peaks under optimized conditions. Validation results confirmed that the method is precise, accurate, robust, and stability-indicating as per ICH guidelines.

Table 1: Optimized Conditions

Fig: 1. Optimized Chromatogram

Method Validation

The developed UPLC method was validated as per ICH Q2(R1) guidelines for the following parameters [8,9]:

1. System Suitability

System suitability was assessed by injecting six replicate injections of the standard solution. Parameters such as retention time, theoretical plates, tailing factor, and %RSD of peak areas were evaluated.

Specificity was confirmed by analyzing blank, placebo, standard, and sample solutions to ensure there was no interference at the retention times of clindamycin and miconazole.

Fig:2. Chromatogram of Blank

Fig: 3. Chromatogram of Placebo

Fig: 4. Chromatogram of Standard

3. Linearity

Calibration curves were constructed by plotting peak area versus concentration over the range of 10–100 μg/mL for clindamycin and 5–50 μg/mL for miconazole. The correlation coefficient (r²) was determined to assess linearity. Clindamycin: y = 10191x + 491.44, r² = 0.9995. Miconazole: y = 9151.6x + 496.44, r² = 0.9999

Fig.5. Calibration Plot of Clindamycin

Fig.6. Calibration Plot of Miconazole

4. Accuracy

Accuracy was determined by recovery studies at 50%, 100%, and 150% of the target concentration. Each level was analyzed in triplicate, and % recovery was calculated.

Accuracy

Fig. 7. Chromatogram For Accuracy At 50% Spike Level

Fig. 8. Chromatogram For Accuracy At 100% Spike Level

Fig. 9. Chromatogram For Accuracy At 150% Spike Level

5. Precision

Precision was evaluated at two levels:

• Repeatability (Intra-day): Six replicate injections of the sample solution were analyzed.
• Intermediate Precision (Inter-day): Analysis was performed on different days by different analysts.

Fig. 10.: Chromatogram for Method Precision

Intermediate Precision

6. LOD and LOQ

The Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated based on the standard deviation of the response and slope method.

• Clindamycin: LOD = 0.10 µg/mL, LOQ = 0.32 µg/mL
• Miconazole: LOD = 0.09 µg/mL, LOQ = 0.28 µg/mL

Robustness was assessed by making deliberate changes to flow rate (±0.1 mL/min), mobile phase composition (±5%), and detection wavelength (±2 nm), and evaluating the effect on system suitability.

Robustness

Standard and sample solutions were evaluated for stability at room temperature and 2–8°C for 24 hours.

Stability of Sample Solution

No significant changes were observed in the assay results. The deviation was less than 2%

Table 9: Stability data

* n = 6 for each parameter

Assay of Marketed Formulation

* n = 6 for each parameter

Fig.11. Assay Of Marketed Formulation

Forced Degradation Studies

To establish the stability-indicating nature of the method, forced degradation was performed under the following conditions [10,11]:

• Acidic Hydrolysis: Sample treated with 1 N HCl and kept at room temperature for 1 hour, then neutralized.
• Alkaline Hydrolysis: Sample treated with 1 N NaOH under similar conditions, then neutralized.
• Oxidative Stress: Sample treated with 3% H?O? for 30 minutes at room temperature.
• Thermal Degradation: Sample exposed to 105°C in a hot air oven for 6 hours.
• Photolytic Degradation: Sample exposed to UV light (254 nm) for 24 hours.