Study of the Physicochemical Stability of a Fortified Eye Drop Based on

Vancomycin

Bhirich Nihal^1,2*, Mohammed Yafout^3,4, Ghita Salime Meknassi^1,2, Soumaya El Baraka⁵^,6,

Brahim Mojemmi^1,2, Ibrahim Sbai El Otmani^3,4.

¹Department of Analytical Chemistry. Faculty of Medicine and Pharmacy,

Mohammed V University, Rabat, Morocco.

²Ibn Sina University Hospital Center, Rabat, Morocco.

³Laboratory of Drugs Sciences, Biomedical Research and Biotechnology.

Faculty of Medicine and Pharmacy, Hassan II University of Casablanca, Morocco.

⁴Ibn Rochd University Hospital Center, Casablanca, Morocco.

⁵Department of Analytical Chemistry, Faculty of Medicine and Pharmacy,

Cadi Ayyad University, Marrakech, Morocco.

⁶Mohammed IV University Hospital Center, Marrakech, Morocco.

*Corresponding Author E-mail: bhirich.nihal@gmail.com

ABSTRACT:

Background: Fortified eye drops containing vancomycin are generally not available in a ready-to-use form. As a result, hospital pharmacies are forced to prepare these solutions from injectable formulations, adapted to ophthalmic specificities. This process raises challenges in terms of stability, since injectable formulations are not designed for prolonged topical use or for storage conditions in ophthalmic vials. The stability of these eye drops is thus influenced by various factors such as storage temperature, exposure to light, pH and potential interactions with packaging materials, which can alter their efficacy and safety. The primary objective: In this study, the focus will be on the evaluation of the physicochemical stability of the fortified vancomycin eye drops. This analysis will determine whether the formulation maintains its therapeutic properties over time, taking into account storage factors such as temperature, pH and interaction with packaging material. Methods: The duration of the study was set at 6 weeks (1 month and a half). The samples prepared for the study will be stored at two different temperatures: room temperature and in the refrigerator (2 to 8°C). The parameters controlled are as follows: clarity (by visual inspection), pH and concentration of active ingredient. Samples for analysis are taken on D0, D1, D3, D5, D7, D14, D21, D28, D35 and D42 for each storage temperature. The bottles are then labeled and numbered with even numbers for eye drops to be stored in the refrigerator (2 to 8°C) and odd numbers for eye drops to be stored at room temperature (nearly 25°C). On each sampling date selected, a bottle is taken and analyzed for each storage temperature in accordance with the adopted schedule. Visual inspection, pH measurement and HPLC dosage will be carried out. Results: Our study allowed us to determine the average physicochemical stability of vancomycin-fortified eye drops before opening. This duration is 6 weeks for eye drops stored between 2 and 8°C and 2 weeks for eye drops stored at room temperature. However, with the appearance of crystals in the sixth week of storage in the refrigerator, it would be prudent to limit ourselves to a storage duration of 1 month at 4°C. A more complete study including microbiological control could be considered in order to be able to recommend a more concise storage duration. Conclusion: This stability study could motivate the initiative to upgrade the status of antibiotic-fortified eye drops from magistral preparation to the status of hospital pharmaceutical preparation complying with the rules of good preparation practices, as is now recommended at hospital pharmacy levels throughout the world. Batches of eye drops could therefore be prepared in advance and stored, thus ensuring the permanent availability of the drug and saving time in the preparation phase.

KEYWORDS: Vancomycin, fortified eye drops, physicochemical stability, eye infection, dosage.

INTRODUCTION:

Fortified eye drops have become a necessity in hospital pharmacies, particularly to meet the specific needs of ophthalmology departments^1,2. These magistral preparations are eye drops made from antibiotics marketed in powder, lyophilisate or injectable solution form, complying with good manufacturing practices for sterile drugs that will allow high concentrations of antibiotics to be administered locally^3,4, meeting the therapeutic requirements of serious eye infections such as bacterial keratitis and endophthalmitis. These eye drops are mainly used in serious microbial keratitis in a patient who is usually hospitalized⁵.

They were developed in the late 1970s to provide clinicians with antibiotic eye drops with a higher concentration than commercially available eye drops. In many cases, the pathogens involved, such as methicillin-resistant Staphylococcus aureus (MRSA)^5-9 and other multidrug-resistant strains, can only be effectively treated with potent antibiotics, such as vancomycin, the administration of which in eye drops is then essential to ensure effective treatment^10,11.

However, fortified eye drops, such as those containing vancomycin, are generally not available in ready-to-use form. As a result, hospital pharmacies are forced to prepare these solutions from injectable formulations, adapted to ophthalmic specificities¹². This process raises challenges in terms of stability, since injectable formulations are not designed for prolonged topical use or for storage conditions in ophthalmic vials¹³. The stability of these eye drops is thus influenced by various factors such as storage temperature, exposure to light, pH and potential interactions with packaging materials, which can alter their efficacy and safety^14,15.

The assessment of the physicochemical and microbiological stability of these fortified eye drops is therefore essential. This assessment ensures that eye drops retain their therapeutic properties over a defined period.

OBJECTIVE:

Given the mastery of good preparation practices in hospital pharmacies, particularly the preparation of preparations under a laminar flow hood, the microbiological stability of fortified eye drops is generally guaranteed. Indeed, these manufacturing conditions considerably minimize the risks of microbiological contamination, thus ensuring a preparation free of pathogens.Therefore, in the context of this study, the focus will be placed solely on the evaluation of the physicochemical stability of fortified vancomycin-based eye drops. This analysis will determine whether the formulation retains its therapeutic properties over time, considering preservation factors such as temperature, pH and interaction with the packaging material, without worrying about the microbiological aspects, already ensured by the preparation standards.

The results of this research will provide practical recommendations for hospital pharmacies regarding the preparation, storage and use of fortified eye drops, helping to optimize the care of patients with severe eye infections.

In practice, we are faced with the impossibility of producing these eye drops in the evenings and at weekends, and with organizational difficulties. We have therefore sought to set up a system guaranteeing permanent availability of eye drops.

MATERIALS AND METHODS:

Materials:

· Vancomycin

The vancomycin used is in the form of vancomycin hydrochloride, powder for solution to be diluted for infusion, marketed under the specialty Vancomycine Mylan ^®500 mg, batch B1427 with an expiration date of 01/2020.

· Reconstitution solvent

The solvent used for the preparation is sodium chloride 0.9% Soflex ^®, solution for intravenous infusion from Sothema laboratories, batch 182366 with an expiry date of 04/2020.

· Reagents for chromatographic assay

The reagents used for the assay are as follows:

- Distilled water

- Methanol

- Phosphate buffer pH = 3 ; 0.025M

· Visual inspection (colour and clarity) is carried out without specific equipment. The eye drops are brought to the visual assessment of two different manipulators, in ambient and sufficient lighting.

· pH measurement

The pH of the measurements is measured using a HACH senSION + PH31 ^®benchtop pH meter.

· Chromatographic analysis

Infinity II ^®HPLC chain, consisting of an Agilent 1260 Infinity II Isocratic pump Pump^®,a manual injector including a 20µL sample loop, a LiChrosorb ^®5µm RP18 150 x 4.6mm analytical column, an Agilent Technologies variable Wavelength Detector ^®UV Visibledetector. The entire module is controlled by a computer using OpenLAB CDS ^®software.

METHOD:

The preparation of the eye drops was carried out in a clean handling room. 20 vials of VANCOMYCIN MYLAN ® 500mg, powder for solution to be diluted for infusion, were used to prepare the eye drops. To each vial of vancomycin, 10ml of 0.9% NaCl solution was added for the reconstitution of the eye drops at 50mg/ml. The prepared solutions were stored in the original primary packaging material which is type I glass. The vials are recorked with sterile rubber stoppers.

The duration of the study was set at 6 weeks (1 month and a half). The samples prepared for the study will be stored at two different temperatures: room temperature and in the refrigerator (2 to 8°C). The parameters controlled are as follows: clarity (by visual inspection), pH and concentration of active ingredient. Samples for analysis are taken at D0, D1, D3, D5, D7, D14, D21, D28, D35 and D42 for each of the storage temperatures.

The bottles are then labeled and numbered with even numbers for eye drops to be stored in the refrigerator (2 to 8°C) and odd numbers for eye drops to be stored at room temperature (nearly 25°C). On each selected sampling date, a bottle is collected and analyzed for each storage temperature in accordance with the adopted schedule.

· Visual inspection:

Eye drops are stored in colorless, neutral, colorless and transparent glass. The color and clarity of the preparations will be compared to EPPI, in diffuse daylight

· pH measurement:

Part of the contents of the vancomycin eye drops are transferred into a tube suitable for measuring pH.

· Chromatographic analysis:

An analytical method has been developed for the stability study, the method used has already been the subject of appropriate validation in the laboratory and the details of these steps will not be detailed in our work.

The C18 LiChrosorb ® 5µm column: 150 x 4.6mm, reference AGA0040150X046, Agilent Technologies® is selected and the analysis will be carried out at a wavelength of 280nm with a mobile phase methanol/water/phosphate buffer pH = 3; 0.01M (25/60/15) with a flow rate of 1mL.mn ^-1.

a. Calibration range:

The calibration range is carried out by preparing solutions of different concentrations of the substance sought, around the target value. These different solutions are prepared from a reference substance whose content is perfectly known and controlled.

A stock solution ( S_m) of concentration 50mg/mL is prepared by adding 10ml of distilled water to exactly 500mg of vancomycin. This is diluted 100 times to reach a concentration of 500mg/L. From this, standard solutions of concentration 5mg/L, 25mg/L, 50mg/L, 75 mg/L and 100mg/L are prepared by dilution.

Each calibration point is analyzed 3 times for the construction of the calibration range. The results expressed in AUC are collected and processed by Excel ^®. A calibration line is plotted and the correlation coefficient R²is calculated.

b. Vancomycin dosage in samples:

Before being analyzed, the samples are diluted so that the theoretical final concentration obtained is of the order of those used for the calibration range. It should also be remembered that since the detector used is a UV detector, it is recommended to work at low concentrations to apply the Beer-Lambert law. The samples are therefore diluted with distilled water by a factor of 1000.

The samples thus diluted are analyzed in duplicate in the same way as for the calibration solutions. The chromatograms obtained are recorded and analyzed. The use of the equation of the calibration curve will allow the vancomycin concentration of the different samples to be calculated.

RESULTS:

1. Calibration range:

The results obtained for the calibration range are interpreted in Fig. 1:

Figure 1: Vancomycin calibration line.

2. Sample dosage

Each sample is assayed 2 times. The vancomycin concentration (C) is determined from the equation of the calibration line determined as follows: C = 5.1878 AUC + 4.5805.

The results obtained are listed in figures 2,3,4:

3. pH measurement:

The results of the pH measurements are listed in Figure 5:

4. Visual inspection (color and clarity)

The results obtained for the color and clarity of the eye drops presented in Table I:

Table I: Results of the control of the clarity of vancomycin eye drops

I	No. eye drops at 2 to 8°C	Color	No. eye drops at 25°C	Clarity
J ₁	2	COLORLESS	1	CLEAR
J ₃	4	COLORLESS	3	CLEAR
J ₅	6	COLORLESS	5	CLEAR
D ₇(1S)	8	COLORLESS	7	CLEAR
D ₁₄(2S)	10	COLORLESS	9	CLEAR
D ₂₁(3S)	12	COLORLESS	11	CLEAR
D ₂₈(4S)	14	COLORLESS	13	CLEAR
J ₃₅(5S)	16	COLORLESS	15	CLEAR
J ₄₂(6S)	18	COLOURLESS (Appearance of white crystals at the bottom of the bottle)	17	CLEAR

DISCUSSION:

The study investigated the stability of fortified vancomycin eye drops, exploring different storage temperatures: room temperature (25°C), refrigeration (2–8°C), and freezing (-20°C). Freezing was found to be the most effective storage method, providing 75–90 days of stability while inhibiting bacterial growth^16,17. However, its use may be limited by the thawing process, which is lengthy and not suitable for emergency situations. Refrigeration has also been shown to be effective in slowing vancomycin degradation and preventing bacterial growth^18,19,20, which motivated its inclusion in this study. Room temperature storage was added for comparative purposes to assess the impact of environmental conditions on the stability of the eye drops.

The duration of the study was set at six weeks to assess the possibility of preparing the eye drops in advance while maintaining their efficacy under optimal conditions²¹. For the measurement of vancomycin, an HPLC method using a C18 column and a mobile phase composed of methanol and water buffered at pH 3 was chosen. The analyses were carried out at a wavelength of 280nm, allowing a precise evaluation of the vancomycin concentration while favoring the selectivity of the method (Figure 6). Although the use of 220 nm would have facilitated the detection of degradation products, the choice of 280 nm aligned with the main objective of study^22,23,24.

Figure 6: Typical chromatogram showing the vancomycin peak

The analysis schedule was designed to follow for the first month the sampling frequencies proposed by the Methodological Guide for stability studies of preparations developed under the aegis of the French Society of Clinical Pharmacy (SFPC) and the Evaluation and Research Group on Protection in Controlled Atmosphere (GERPAC)^25,26.

It was therefore decided to work the first month at sampling times corresponding to approximately 1/24th, 1/12th, ¼, ½ and ¾ of the said duration (D1, D3, D5, D7, D14, D21 and D28). For the remaining two weeks, sampling is carried out at intervals of 7 days.

Visual checks revealed macroscopic stability of the eye drops, although crystalline deposition was observed at 6 weeks under refrigeration, suggesting possible precipitation beyond this period as in some studies^27,28,29.

Concerning the dosage, the HPLC method showed excellent linearity (R² = 0.9999). For drugs with a non-narrow therapeutic margin (case of vancomycin), the variation in active ingredient concentration accepted is ± 10% of the value at time D0²⁵.

For eye drops stored in the refrigerator, the concentration of active ingredient remains within the acceptable range (Figure 2) over the entire duration of the study (6 weeks) even if a downward trend in said concentration is observed over time. At D28, an increase (98%) in the concentration compared to the trend is observed. Since this is an isolated deviation, it could be explained by the fact that the bottles of the specialty used initially have an acceptable variation in dose when the product is released.

For eye drops stored at room temperature, the vancomycin concentration falls below 90% by the fourteenth day (2 weeks) and continues to decline to reach 78.25% at 6 weeks (Figure 3).

At D5 (sample 5), we observed an isolated deviation (87.55%) also outside the trend. As in the case, it could be explained by the variations tolerated during the industrial release of batches of the specialty used.

Figure 4 allows a comparison of the profiles of the evolution of the vancomycin concentration of the eye drops for the different storage temperatures. The vancomycin-fortified eye drops are stable, under the conditions studied (before opening, protected from light) for 6 weeks between 2 and 8°C and 14 days at room temperature.

For the pH, eye drops stored between 2 and 8°C, no significant variation in pH (mean of 3.34) was observed compared to D0 (pH = 3.33) throughout the duration of the study. The evolution profile (Figure 5) shows pH stability up to 6 weeks. For those stored at room temperature, no significant variation in pH was observed compared to D0 (3.33±0.4). These results are consistent with those found in the literature^16,17,18.

However, although there is no significant change, the pH profile of eye drops stored at room temperature (Figure 5) shows a clear trend towards more basic pH values. It appears that as the pH of the eye drops increases, the vancomycin concentration decreases. This is explained by the fact that vancomycin is easily degradable in a basic environment as reported by studies that have performed forced chemical degradation tests^30,31.

It should be noted that pH is not subject to strict limits defined in the context of stability studies^25,32 since the human eye can tolerate a pH range from 3 to 10^16,33.

From 3 weeks onwards, another peak is observed on the chromatograms at a retention time tr of 2.7min (Figure 7). This should correspond to a degradation product. However, the decrease in the AUC of vancomycin is not compensated by that of the new peak. There could therefore be other degradation products not identified on the chromatogram. These could absorb at a different wavelength than that of vancomycin (280nm). Working with a wavelength of 220nm might have made it possible to detect them.

Figure 7: Chromatogram of an eye drop sample after 6 weeks at room temperature showing the appearance of degradation impurities.

In the literature, three vancomycin degradation products resulting from deamination at the aspartic acid level have been identified: succinimide 11, CDP Im and CDP IM^34,35. The two CDP1s differ by the position of a chlorine atom on an aromatic ring and have no antibiotic activity. In aqueous solution, vancomycin can indeed undergo a conversion of its asparagine derivative to isoaspartate via an imide cycle to form CDP-I^36,37which is structurally very close to vancomycin. It is therefore the latter which can be identified on the chromatogram. The precise identification of impurities requires having the corresponding standards or using an identification technique such as mass spectrometry.

CONCLUSION:

Our study allowed us to determine the average physicochemical stability of vancomycin-fortified eye drops before opening. This duration is 6 weeks for eye drops stored between 2 and 8°C and 2 weeks for eye drops stored at room temperature. However, with the appearance of crystals in the sixth week of storage in the refrigerator, it would be prudent to limit the shelf life to 1 month at 4°C. A more complete study including microbiological control could be considered to be able to recommend a more concise shelf life.

In practice, this stability study could motivate the initiative to upgrade the status of antibiotic-fortified eye drops from magistral preparation to the status of hospital pharmaceutical preparation complying with the rules of good preparation practices, as is now recommended at hospital pharmacy levels throughout the world. Batches of eye drops could therefore be prepared in advance and stored, thus ensuring the permanent availability of the drug and saving time in the preparation phase.

CONFLICT OF INTEREST:

Authors declare that there is no conflict of interest.

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Received on 26.11.2024 Revised on 12.03.2025

Accepted on 14.05.2025 Published on 10.02.2026

Available online from February 16, 2026

Research J. Pharmacy and Technology. 2026;19(2):788-794.

DOI: 10.52711/0974-360X.2026.00113

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