A fluoroquinolone antibiotic with broad-spectrum action against the majority of aerobic gram-positive and gram-negative bacteria is ciprofloxacin. It is a quinolone that is quinoline-4(1H)- bearing cyclopropyl, carboxylic acid, fluoro and piperazin-1-yl substituents at positions 1, 3, 6 and 7, respectively, a molecular weight of 331.34 g/mol and its chemical structures is shown in Figure 1.

Among the fluoroquinolone category, ciprofloxacin is typically thought to have the strongest overall antibacterial activity. It is bactericidal and specifically targets DNA gyrase and DNA topoisomerase IV, two significant bacterial topoisomerase enzymes involved in DNA synthesis. In December 1990, the US Food and Drug Administration approved ciprofloxacin as a topical therapy for bacterial corneal ulcers. As of right now, it's the only fluoroquinolone option available on the market in the US for treating corneal ulcers topically. ⁵ Also, for the first-line treatment of common eye conditions including blepharitis, bacterial keratitis, conjunctivitis, endophthalmitis, dacryocystitis, and orbital cellulitis, topical ciprofloxacin has been hailed as an excellent option and previous researches have demonstrated the effectiveness of a topical antibiotic solution containing 0.3% ciprofloxacin in the treatment of such ocular diseases. ^{2, 6} Over 70% of ocular infections are linked to the following pathogens: Staphylococcus aureus, Moraxella species, Pseudomonas aeruginosa, Aspergillus species, Fusarium species, Moraxella aureus, and Streptococcus pneumoniae. Even though topical antibiotics can produce larger drug concentrations in ocular tissues, there are growing reports of clinical failure and suboptimal results when fluoroquinolones are used empirically. The overuse of fluoroquinolone antibiotics and the quickly developing antibiotic resistance may be factors in this. ⁷

In India, the ciprofloxacin ophthalmic solution 0.3% used as a topical antibiotic for ocular diseases, is commonly obtained by patients by "over the counter" medication and is usually stored at room temperature. The lack of knowledge on the significance of pharmaceutical storage in India causes disruptions to the stability and loss of its potency, ultimately contributing to the rise in antibiotic drug resistance. The manufacture recommends to store the topical antibiotic between 20-25°C. Given that summertime room temperatures in the southern part of India are usually higher than the permitted storage temperature, it is likely that the antibiotic concentration (Ciprofloxacin ophthalmic solution 0.3%) has suffered some degree of degradation. Hence, in this article, the potency of the drug needs to be evaluated by analysing its difference in concentration at different storage temperatures using a UV-Spectrophotometer.

To evaluate the potency of 0.3% Ciprofloxacin eye drops at different storage temperatures for a period of 90 days using UV Spectrophotometer.

A Lab India (T-60) UV- Visible spectrophotometer was used for all absorbance measurements.

The Institution Review Board and Ethics Committee of Christian Medical College, Vellore, India, granted permission to conduct the study in accordance with protocol (IRB Min No. 14904 Dated 12-10-2022). Dropper bottles containing ciprofloxacin 0.3% ophthalmic solution (10 ml) were purchased from the pharmacy at CMC Hospital, belonging to the same brand and batch, to prevent inter-batch variability. They were stored at the pharmacy between 20 and 25 degrees Celsius, when they are claimed to have maximum potency. To prepare the stock and working standards, 99% (TLC grade) pure powder of ciprofloxacin chemical was purchased from Sigma Aldrich Inc. India. The dropper bottles were divided into 3 groups (A, B, C) and stored at three different temperatures, away from sunlight and moisture.

Department of Pharmacology and Clinical Pharmacology, Christian Medical College, Bagayam Campus, Vellore.

In a previous study, “Stability and Sterility of Extemporaneously Prepared 0.01% Atropine Ophthalmic Solution in Artificial Tears and Balanced Salt Solution” stability of phenytoin was reported as mean ± SD at different concentrations 8. The study has reported that at room temperature and in refrigerator, the mean and SD value was 99.44±4.34 and 104.86±2.47. With this value, 5% alpha, 80% power and two-sided test; the current study required 7 ciprofloxacin 0.3% eye drops for each temperature.

Code numbers were used to blind the principal investigators, co-investigators and statistician from the sources of the samples; the codes were decoded following analysis. Once the eye dropper bottles containing 0.3% Ciprofloxacin were randomly assigned to groups A (Air-Conditioned room, 25°C), B (Room temperature, 30-32°C), and C (Oven temperature, 40°C), they were numbered 1 – 7. On the day of the experiment, quality control was freshly prepared without disclosing its concentration.

28 dropper bottles of Ciprofloxacin 0.3% eye drops (10 ml) were obtained from CMC Hospital pharmacy. They were then randomised into 4 groups with 7 dropper bottles each, in the ratio of 1:1:1:1. 7 samples from one group were used to estimate their baseline concentrations ("concentration at day 0") using a UV Spectrophotometer. Then, the remaining 21 bottles were grouped and named, A, B, C with seven bottles in each group. All bottles were opened to simulate real-world situations and then stored at different temperature conditions (25°C, 30-32°C, 40°C), protected from direct light. A digital data logger was used to track the storage temperatures.

a. Group A (7 bottles) were stored at 25°C in an air-conditioned room.

b. Group B (7 bottles) were stored at 30-32°C at room temperature (A Digital data logger was used to track the temperatures regularly, and its mean was calculated).

c. Group C (7 bottles) were stored at 40°C in the oven.

An assay of Ciprofloxacin 0.3% eye drops by UV Spectrophotometer was done based on the method developed by Safila Naveed and Nimra Waheed. ⁹ After 7 days of storage, 7 samples (1 ml from each dropper bottle) from each group were used to measure the concentration of ciprofloxacin 0.3% eye drops at that particular temperature [AC (25°C), room temperature (30-32°C), oven (40°C)]. The concentration of ciprofloxacin eye drops at 3 different temperatures was also estimated at 14, 30, 45 60, and 90 days. The difference in the initial concentration of ciprofloxacin (Baseline) and concentration of ciprofloxacin at 7, 14, 30, 45, 60 and 90 days was analyzed by a UV Spectrophotometer, at the 3 different storage temperatures. Multiple comparisons were done at each time interval for different temperatures. Follow-up values were expressed in terms of the percentage of initial values, and Paired t-test was used to analyse the difference between the values obtained. The flowchart of the methodology is given in Figure 2.

The strength of the drug was considered using the given formula:

Strength of the drug = [Observed Quantity/Label Quantity] x 100

According to the 2011 WHO Report, the permitted range of strength is between 90% and 110% of label values; samples were deemed to have degraded/ unsatisfactory if the active pharmaceutical ingredient (API) concentration was outside of this range. 10 Descriptive analysis was done to measure the mean concentrations of 7 samples at baseline (day 0) and at different storage conditions (Air-conditioned room temperature, room temperature, Oven temperature) at different time intervals (7, 14, 30, 45, 60, 90 days).

The minimum and maximum mean concentrations at different time intervals were compared to the baseline mean concentration value and expressed as a percentage of the baseline value, which represented the strength of the drug. This was then analysed to determine if the strength of the concentrations lay within the permitted range of 90%- 110%, according to the 2011 WHO Report, to eliminate the degradation possibilities.

A Reliable, efficient, and cost-effective method for quantitative analysis in stability testing research is the UV spectrophotometric method. However, the most accurate, sensitive, and validated approach for quantifying pharmaceuticals and substances is high-performance liquid chromatography (HPLC). Thus, the drug formulation used in our study, if found to have undergone degradation (Strength of the drug concentrations <90% or >110%), would be confirmed with a more accurate and sensitive analytical technique (HPLC).

Department of Pharmacology and Clinical Pharmacology, CMC Vellore, is gratefully acknowledged. The authors would like to thank the Institutional Review Board (IRB) for their approval.

This study was funded by a fluid research grant of the Christian Medical College, Vellore, India through Institutional Review Board (IRB Min No. 14904 Dated 12-10-2022). The funders had no role in study design, data collection, and analysis, decision to publish or preparation of the manuscript.

All the authors declare that there is no competing interest.

The idea was conceived, and study was designed by MS. NJJ and MH did the main laboratory work under MS’s and AK’s guidance. AK, GSD, NC, MH assisted in overall implementation of the study. NJJ prepared the first draft. All the authors gave critical inputs and revised the draft. The final draft was approved by all authors. Each author agrees to be accountable for all aspects of the research work.