1. INTRODUCTION:

Ocular formulations are intended to be applied on the anterior surface of the eye, delivered intraocularly, periocularly, or in combination with ocular devices. Liquid dosages include drops, suspensions, and emulsions where eye drops represent more than 95% of the marketed ocular products.

Dexamethasone sodium phosphate and chloramphenicol eye drops, commercially marketed as Dexachlor‎are used in this work.

Dexamethasone is a potent corticosteroid, it binds to cytoplasmic glucocorticoid receptors and inhibits function of mediators of inflammatory response, it can be intramuscular, intravenous and sub mucosal. Dexamethasone can be preoperative, perioperative or post-operative appears to be effective in the prevention of post-operative edema¹.

Many works are focused on the determination and assay of dexamethasone by using HPLC method^2,3,4or by using other methods ashydrotropic solubilization technique for quantitative studies of dexamethasone in pharmaceutical preparations⁵.

Chloramphenicol is broad-spectrum antibiotic widely used in ophthalmic dosage forms for management and treatment of superficial eye infections such as bacterial conjunctivitis.⁶

As known, stability is a core quality attribute for any viable drug product that’s whymany works are focused on the study of drug stability^7,8,9,10,11.

Stability refers to the capacity of theproduct or a given drug substance to remain within established specifications of identity, potency, and purity during a specified time period¹². According to world health organization, stability is the ability of a pharmaceutical product to retain its chemical, physical, microbiological and biopharmaceutical properties within specified limits throughout its shelf-life.¹³

The purpose of stability testing is to provide evidence of how the quality of an active pharmaceutical ingredient or finished pharmaceutical product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light. Any aspect related to the change of the physical, chemical, and biopharmaceutical properties of ‎a drug product could be classified as instability.‎ Stability of the drug explains the safety and efficacy of the product during its entire shelf life^14,15.

Lack of stability in pharmaceutical products can lead to ‎many potential adverse effects‎:

a) Loss of drug resulting in a reduction of potency¹⁶.

b) Formation of toxic degradation products, as the formation of anhydro-4-epitetracycline which is a degradant of tetracycline known to cause Fanconi syndrome¹⁷, another example is pralidoxime degradation resulting in cyanide formation^18,19 leading to cytotoxic hypoxia via inhibition of cytochrome-c-oxidase²⁰.

c) Increase in the concentration of the active ingredient as in the case of products in perfusion bags because the loss of vehicle perfusion bags allowing solvent to escape and evaporate so the product within the bag shows an increase in concentration²¹.

d) Changes in rate and extent of absorption on storage leading to alteration in bioavailability²².

e) Increase in number of viable microorganisms during storage²³.

f) Drug degradation may make the product esthetically unacceptable, especially for children and older adults which they differ in many aspects from the other age subsets of population and require particular considerations in medication acceptability²⁴.

Many factors can impact the stability of a pharmaceutical product:

· Moisture: source of moisture in pharmaceutical dosage forms is diverse, comes from manufacturing processes, bulk drug, environmental conditions, excipients. It has a significant impact on product stability, powder flow properties, microbial growth and it impacts on a wide range of chemical, physical, and microbial properties of the finished pharmaceutical product^25,26.

· Excipients: choice of excipients constitutes a major part of preformulation and formulation studies during the preparation of pharmaceutical dosage forms. Drug excipient interactions in solid dosage forms can affect drug product stability in chemical aspects by causing drug degradation or physically such as dissolution slowdown or organoleptic changes^27,28.

· Temperature: eﬀect of temperature on stability was well described by Arrhenius equation^21,29,30. Temperature can affect medication stability^31,32, high temperature leads to drug degradation^33,34 by accelerating hydrolysis, oxidation and reduction reactions.

· PH: influence of pH on drug stability was studied by many searchers^35,36, because pH has great eﬀect on the rate of decomposition of drugs especially in solution³⁷.

· Light: some drugs are photosensitive and their rate of decomposition accelerates when exposed to light so photostability studies of drugs and drug products are an integral part of the product development process in the pharmaceutical industry and light effect and photoprotection methods were clarified by many studies^38,39.

· Oxygen: exposure of some products to oxygen can result in changes in dissolution rate, drugs discoloration and even toxicity and other pharmacological properties associated with negative side effect^40,41.

In this research the work was on eye drop pharmaceutical product, as known Liquid dosage forms are less stable than solid dosage forms, so they require a little extra care.

The aim of this work is to predict the chemical and physical stability of Dexachlor eye drops stored at room ‎temperature while the recommended condition storage is between (2-8°C). ‎

2. MATERIALS AND METHODS:

Experimental work was done in medico labs, Homs, Syria.

2.1 Materials:

Drug trade name: Dexachlor. pharmaceutical form: eye drops packaged in bottle of 10ml. composition: dexamethasone Na phosphate (1mg) and chloramphenicol (4mg/1ml). therapeutic category: anti-inflammatory agents, anti-infective agents, ophthalmological drugs. company: Medico Labs. manufacturing date: 12.2020 expiration date: 12.2022 batch number:197

Properties of Dexachlor:

Dexamethasone is a potent corticosteroid with anti-allergic, anti-exudative and anti-proliferative properties. Chloramphenicol is a broad spectrum bacteriostatic antibiotic which acts by inhibiting thebacterial protein synthesis. The corticosteroid – antibiotic association reduces inflammation while concurrently attacking the existing infection or preventing the outbreak of infection.

Recommended storage conditions:

In refrigerator between (2-8°C).

Samples:

30 samples of Dexachlor eye drops were stored between (2-8°C) according to the recommended storage instructions, then 3 samples of these 30 samples were used as reference samples (keep stored in the refrigerator) and the other 27 samples were stored at room temperature away from light and humidity according to a specific schedule time (three samples were used for each period): 24h, 48h, 72h, 6days, 8 days, 15days, 25days, 35days, 70days. After the end of each period, physical and chemical tests were carried out.

2.2 Instruments:

· HPLC device (Type LC -2030c shimadzu, Kyoto, Japan)‎

· HPLC column (Macherey-Nagel, EC 250\4.6 Nucleosil 100-5 C-18, Düren Germany)

· PH meter (Mesalabs, America)

· Electronic analytical and precision Balance (Sartorius, Germany)

· Heating and stirrer device (Selecta, Spain)

· Ultrasonic bath device (Wise Clean, Korea)

· Filters nylon: 0.45µm (Sartorius)

· Other instruments: glass ware (beakers, funnels, pipettes and erlenmeyer flasks)

2.3 Reagents and solvents:

water grade HPLC, acetonitrile grade HPLC, glacial acetic acid, acetate buffer

2.4 Methods:

2.4.1 Physical tests:

2.4.1.1 Visual test:

This test was carried out according to USP standard. The eye drops should not contain anyforeign particles and must be free from turbid.

Procedure:

Clarity was determined ‎optically by putting eye drops in test tubes behind white background and compared with standard solutions (water).

2.4.1.2 pH test:

PH is an important parameter in aqueous drug product formulation, especially since it involves drug solubility, activity, absorption, stability, sorption and patient comfort. The pH is also related to certain physical characteristics such as the viscosity. The rate of hydrolysis of product may vary depending on the pH of the solution. Determination of the pH will give information about formulation stability.

Procedure:

5ml of each sample was taken into a 10ml beaker and pH was measured by using the pH meter. All tests were carried out at room temperature.

2.4.2 Chemical test (Content assay):

2.4.2.1 Standard solution preparation:

50mg of dexamethasone standard material was dissolved in 50ml of purified water, and then it was put in ultrasonic bath for 5 minutes for dissolving. The solution was then filtered, then a 5ml of solution was transferred to a 50ml flask and 20mg chloramphenicol was added. The volume was completed to 50ml with purified water.

2.4.2.2 Sample solution preparation:

5ml of Dexachloreye drop solution was transferred to a 50ml volumetric flask. The volume was completed to 50 ml with purified water.

2.4.2.3 HPLC calibration:

Three standard solutions of chloramphenicol and dexamethasone sodium phosphate were prepared, and for each standard solution, three successive injections into HPLC system have been carried out before each sample assay, and then the retention time (RT), area under curve (AUC), and relative standard deviation (RSD) were calculated.

2.4.2.4 Samples assay:

This test was carried out according to USP by using HPLC.

20μl of each sample were injected three times into the HPLC system according to the time schedule (for each test, three different samples were used). Phase mobile were composed of 30% acetonitrile and 70% acetate buffer, and flow rate was 1.2ml/min. Then the RT, AUC and RSD were calculated.

3. RESULTS AND DISCUSSION:

3.1 Visual Tests:

All samples, which were stored in the refrigerator and at room temperature, were in conformity with the specifications (clear solution, colorless, odorless, free of precipitation and ‎foreign particles).

3.2 Buffering Test:

According to USP, pH value of ophthalmic ‎preparations is an important factor for the stability of the active pharmaceutical ingredient, that’s why the PH for eye drops must be between (6-7.5) during the shelf life of product.the resultsdemonstrate that there are no statistically significant differences in term of pH between samples stored at the room temperature and samples stored in the refrigerator. All results of buffering test were in conformity with pre-established acceptance criteria

3.3 Assay test:

3.3.1 Standardsolutions:

RT ‎for dexamethasone sodium phosphate and chloramphenicol standard solutions were 4.475 min (RSD = 0.398%) and ‎‎6.908 min (RSD= 0.23%), respectively.

3.3.2 Reference samples (refrigerated samples):

Table 1 and table 2show the, RT, AUC of reference samples stored in refrigerator for 70 days for dexamethasone sodium phosphate and chloramphenicol respectively.

Table 1: RT and AUC of refrigerated samples after 70 days

Compound: chloramphenicol	Ret. time	area
Ref. sample after 70-d…1.icd	5.992	4749301
Ref. sample after 70-d..2.icd	6.015	4714306
Ref. sample after 70-d..3.icd	6.011	4660548
Average	6.006	4708052
Rsd %	0.204	0.950
Maximum	6.015	4749301
Minimum	5.992	4660548
Standard deviation	0.012	44706

Table 2: RT and AUC of refrigerated samples after 70 days

Compound: dexamethasone sodium phosphate	Ret. time	area
Ref. sample after 70-d…1.icd	4.190	1493872
Ref. sample after 70-d..2.icd	4.198	1487814
Ref. sample after 70-d..3.icd	4.202	1468267
Average	4.197	1483317
Rsd %	0.142	0.902
Maximum	4.202	1493872
Minimum	4.190	1468267
Standard deviation	0.006	13382

3.3.3Samples stored at room temperature:

According to USP, the amount of Dexamethasone sodium phosphate in eye drops solution must be between 90-115% of the labeled amount, where the amount of chloramphenicol in eye drops solution must be between 90-130% of the labeled amount.

According to theresults:

· HPLC quantitative analysis of Dexamethasone sodium phosphate samples showed that the refrigerated samples stored for (1day, 25 days and 70 days ) are conform in term of API concentrations (110.28 %, 110.12 %, 111.48 %, respectively).

· HPLC quantitative analysis of Dexamethasone sodium phosphate samples showed that the samples stored at room temperature for (1day, 2 days, 3 days, 6 days, 8 days, 15 days, 25 days, 35 days, 70 days) are conform in term of API concentrations (113.62% , 113.77%, 114.41%, 114.29%, 113.14%,112.77%, 115.56%, 107.45%, 111.19% , respectively) .

· HPLC quantitative analysis of chloramphenicol samples showed that refrigerated samples stored for (1day, 25 days and 70 days) are conform in term of API concentrations (127.42 %, 123.13 %, 114.48 %, respectively)

· HPLC quantitative analysis of chloramphenicol samples showed that the samples stored at room temperature for (1day, 2 days, 3 days, 6 days, 8 days, 15 days, 25 days, 35 days, 70 days) are conform in term of API concentrations (125.5 %-125.8%, 127.2%, 124.2%, 122.2%, 122.6%, 118.5%, 117.77%, 103.52% respectively).

4. CONCLUSION:

The results for refrigerated samples and room temperature samples passed the tests of clarity, pH and content assay.

Storage of samples at room temperature up to 70 days has no impact on the concentration of chloramphenicol and dexamethasone sodium phosphate and the drug keeps its stability.

5. ACKNOWLEDGEMENTS:

Author would like to thank medico labs for its support in this work.

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Received on 04.11.2023 Modified on 21.12.2023

Research J. Pharm. and Tech 2024; 17(6):2773-2777.

DOI: 10.52711/0974-360X.2024.00435