PRESERVATION OF PHARMACEUTICAL PRODUCTS
To minimize the risk of spoilage and to kill any contaminant in a non-sterile medicine after manufacturing, the antimicrobial preservative may be included in a formulation. Ideally, preservatives should be able to kill all microbial contaminants rapidly not be an irritant or toxic to the patient, stable and effective throughout the life of the medicine. They should be selective in reacting with the contaminants and not the ingredients of the medicine. There are two important fundamental
Principles in preservation. The first of these is that the addition of a preservative to a product must not be done to mask any deficiencies in the manufacturing procedures and the second is that the preservative should be an integral part of the formulation, chosen to afford protection in that particular environment.
To protect a pharmaceutical preparation use of a single preservative may seem unrealistic. Increasing attention has focused on the use of mixtures of preservatives and the addition of various potentiators to achieve better results. Preservatives are widely employed in pharmaceutical dosage forms such as emulsions, suspensions, semisolids, parenteral preparations, etc.
The key function of any antimicrobial preservatives is to prevent the growth of unwanted microorganisms in pharmaceutical preparations. We use different physical methods for the protection of pharmaceutical formulations from a microorganism. These methods include sterilization, pasteurization, unfavorable pH, low temperature, minimum nutrient, etc.
Antimicrobial preservatives can be classified into four major groups-
- Quaternary ammonium compounds.
The concentration of preservatives required in an emulsion depends largely on its ability to interact with microorganisms. Microorganisms can reside in the water or the lipid phase or both, the preservatives should be available at an effective level in both phases. Chemical preservatives for semisolids must therefore decrease the quantity available for inhibiting or destroying the microorganisms responsible for spoilage. Multiple-dose eye drops contain an effective antimicrobial preservatives system that ensures maintain sterility during use. Chloroform is the most widely used preservative in oral formulations. Syrups are preserved by maintaining a high concentration of sucrose as part of the formulation.
DEVELOPMENT OF PRESERVATIVE SYSTEM
A single preservative is not suitable for the preservation of all pharmaceutical formulations. The selection of a preservative system depends on an individual, using published information and microbiological studies. A combination of two or more preservatives is used to extend the range and spectrum of preservatives. Germ all 115 has antifungal activity but combined with parabens, shows antibacterial as well as antifungal activity. A combination of antimicrobial preservatives may exhibit synergy. Synergy is exhibited when a combination of two compounds exerts a greater inhibitory effect than the simple additive effect of the two compounds against a single microorganism.
Eye drops and contact lens solutions include phenyl ethyl alcohol and phenoxetol in conjunction with benzalkonium chloride to widen the antimicrobial spectrum. Frequently, a combination of two or more esters of para hydroxy-benzoic acid is used to achieve the desired antimicrobial effect. Methyl para hydroxybenzoic acid and propyl para hydroxybenzoic acid are often used together in a ratio of 10 to 1, respectively. An effective preservative system must retain its antimicrobial activity for the shelf-life of the product.
FACTORS AFFECTING PRESERVATIVE EFFICACY
A wide range of antimicrobial preservatives is available for the preservation of pharmaceutical formulations. The preservative must prevent accidental contamination strange serious or even dangerous decomposition of the product throughout its accepted strange life. The major reasons for a preservative not attaining an effective concentration in the aqueous phase are its interaction with emulgents, solubility in oil, suspended solids, interaction with the container, or pH of the formulation.
- 1. Interaction with formulation components
- 2. Properties of the preservatives
- 3. Effect of containers
- 4. Type of microorganisms
- 5. Influence of pH
1. Interaction with formulation components
Hydrocolloids such as methylcellulose, polyvinylpyrrolidone, alginates, and tragacanth can interact with preservatives and diminish their activity. In pharmaceutical preparations, we use emulgents to produce elegant applications. Preservatives and the emulsified oil phase may interact with emulgent molecules or micelles. Nature of oil, oil-water ratio, type of concentration of emulgent, influence the concentration of preservative needed to protect the system. Many tablet additives cause problems with tablets’ preservatives due to their interaction with added preservatives. Sulphadimidine, Kaolin, Magnesium Trisilicate (therapeutically active ingredient) in the form of suspended solids may also reduce preservative concentration by absorption.
2. Properties of the preservatives
The distribution of the preservatives must be homogeneous and more solubility in the bulk phase is preferable in a multiphase system. Some chemicals such as chlorobutanol may hydrolyze in storage if the pH is unfavorable. Preservatives may react with substances leached from the container and lose their antimicrobial activity.
3. Effect of containers
Formulations packed in a glass container retain their preservative content longer if the closer is airtight. Preservatives may penetrate through the plastic container and interact with it. Rubber also reacts with many preservatives but the industry uses it for teats and closures. Contamination of pathogens may occur due to containers and closures. Screw-capped containers and corks are common sources of mold spores.
4. Type of microorganisms
There is a chance of pathogenic contamination from the soil in plant products. e.g. Clostridium species, Bacillus antacids. These soil microorganisms can cause the spoilage of pharmaceutical products. Soil organisms are common in dust, which may gain access to preparation during processing or packing. Many products prepared from animal sources may contain pathogens like Salmonella typhi.
5. Influence of pH
The solution may be chemically unstable by adjustment of PH and may affect the activity of the preservative. Benzoic acid (weak acid preservative) is mainly required to be predominantly in an undissociated form to exert antimicrobial activity. Preservatives are less dependent upon pH, although at high pH values cationic active quaternary ammonium compounds are more active.
PRESERVATIVES EFFICACY TEST: CHALLENGE TEST
This test is applied to the formulated medicine in its final container to determine whether it is adequately protected against microbial spoilage. The test demonstrates multiple-dose parenteral, otic, nasal, oral, topical, and ophthalmic products made with aqueous bases or vehicles. The effectiveness of any added antimicrobial preservatives and their presence is declared on the label of the product concerned. The tests and standards apply only to the product in the original, unopened container in which the manufacturer supplies it.
For the initial cultivation of the test microorganisms, use a Soybean Casein Digest Medium or any other medium. It should not be less nutritive than the said medium.
Choice of test microorganisms and inoculums preparation
The intention is to use microorganisms that are likely to arise in the raw material used in the product and which occur in the manufacturing environments and represent a particular health hazard if they grew in the product. A preservative should be active against as wide a range of microorganisms as possible hence the choice should be of both Gram-positive and Gram-negative bacteria yeasts and molds in the IP test. The test microorganisms used in preservative efficacy tests are Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, Candida albicans ATCC 10231, and Aspergillus brosilliensis ATCC 16404. The microorganisms used in the test should not be more than five passages made from the original culture, to prevent any phenotypic changes in the strains.
Fresh stock culture of test microorganism is subcultured on the surface of soybean casein digest agar medium. Incubate the bacteria culture at 30 to 35°C for 18 to 24 hours and incubate the culture of Candida albicans and Aspergillus Brasiliense to 20 to 25°C for 48 hours and 7 days respectively. Using the sterile saline solution, harvest the bacteria and Candida Albicans and dilute suitably with sterile saline solution to bring the count to about 1×10 CFU per ml. Similarly, harvest Aspergilliensis Brasiliense culture with a sterile saline solution containing 0.05% w/v of polysorbate 80 and adjust the spore count to about 1x 10° CFU per mi with sterile saline solution. Alternatively, the stock culture microorganisms may be grown in a suitable liquid medium and the cells may be harvested by centrifugation, washed, and resuspended in sterile saline solution to give the required microbial or spore count.
Inoculate each original product container or product tube with one of the standardized microbisuspensionsion using a ratio equivalent to 0.1 ml of inoculums suspension to 20 ml of product and mix. The final concentration should be between 1 x10³ and 1×10 microorganisms per ml of product. Determine the number of viable microorganisms by the plate count method in each inoculum’s suspension and from these. Calculate the initial concentration of microorganisms per ml product being examined. Incubate the inoculated tubes at room temperature. Determine the viable count by the plate count method at 7, 14, and 28 days after inoculation. Calculate the percentage of reduction in CFU per ml for each organism at the stated test intervals and express changes in terms of the percentage of initial concentration.
The Interpretation of results
The preservation is effective in the product examined if
i). For parenteral, ophthalmic, sterile nasal, and otic preparation:
a. The concentration of viable bacteria is not more than 10% of the initial concentration at 7 days and not more than 0.1 % of the initial concentration at 14 days and there is a further decrease in the count at 28 days.
b. Yeast and mold count do not increase at 7, 14, and 28 days from the initial count.
ii). For topical preparations made with an aqueous base, non-sterile nasal preparation, and emulsions
a. The concentration of viable bacteria is not more than 1% of the initial concentration at 14 days and there is a further decrease in the count at 28 days.
b. Yeast and mold count do not increase at 14 and 28 days from the initial count.
iii). For oral preparations
a. The concentration of viable bacteria is not more than 10% of the initial concentration at 14 days and there is a further decrease in the count at 28 days.
b. There is no increase in yeast and mold count at 1 and 28 days from the initial count.