Introduction
Pharmaceutical preparations, especially multi-dose products, are exposed to contamination during manufacturing, storage and repeated use. To protect these products from microbial growth, preservatives are added. These agents prevent spoilage, maintain product stability and ensure safety for patients. However, not all preservatives are automatically effective in every formulation. Therefore, their antimicrobial strength must be evaluated through standard tests.
Why Evaluation of Preservatives Is Important?
- To ensure the preservative protects the product throughout its shelf life.
- To confirm that microorganisms cannot grow after accidental contamination.
- To verify that the preservative remains active in the specific formulation.
- To meet pharmacopeial standards for safety and effectiveness.
Preservative Effectiveness Test (PET)
This test is also called the Antimicrobial Preservation Effectiveness Test or Challenge Test. The purpose is to check whether the added preservative can destroy or inhibit introduced microorganisms over a certain period.
Different pharmacopeias such as IP, BP and USP provide detailed guidelines for antimicrobial preservative testing. The principles remain the same across systems.
Microorganisms Used in Evaluation
The test product is challenged with standard microorganisms that commonly contaminate pharmaceuticals. These include:
- Bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli
- Yeast: Candida albicans
- Mould: Aspergillus brasiliensis
These organisms represent both Gram-positive and Gram-negative bacteria, as well as fungal contaminants typically encountered during storage and handling.
Procedure for Preservative Effectiveness Test
The test involves adding known counts of microorganisms to the product and observing how the preservative acts over time.
Step 1: Preparation
- Prepare fresh cultures of the test microorganisms.
- Adjust microbial suspension to the required concentration (usually 105 to 106 CFU/ml).
- Inoculate the pharmaceutical product with the microbial load.
Step 2: Incubation
After inoculation, the product is stored under controlled temperature conditions. No additional preservatives or neutralizers should be added unless needed to prevent interference.
Step 3: Sampling at Intervals
Samples are removed and tested for surviving microorganisms at the following time intervals:
- 7 days
- 14 days
- 28 days
The number of surviving organisms at each interval is compared with the initial count.
Acceptance Criteria
The pharmacopeias specify reduction standards based on the type of product:
A) For Parenterals and Ophthalmic Preparations
- Significant reduction of bacteria within 14 days.
- No increase in fungal counts during the test period.
- No recovery of organisms at 28 days.
B) For Oral Liquid Preparations
- Gradual decrease in microbial load over 14–28 days.
- Yeast and mould counts must not rise during the test.
Each product category has slightly different allowable limits because of differences in formulation, pH and route of administration.
Factors Affecting Preservative Action
- pH of the formulation: Many preservatives work only within specific pH ranges.
- Temperature: Higher temperatures may increase or decrease preservative activity.
- Ingredients of the product: Proteins, surfactants and oils can neutralize preservatives.
- Type of container: Plastic materials may absorb the preservative.
- Microbial species: Some organisms, such as Pseudomonas species, are more resistant.
Common Preservatives Used in Pharmaceuticals
- Parabens: Methylparaben, propylparaben – used in syrups, creams, lotions.
- Benzalkonium chloride: Used in ophthalmic solutions.
- Phenoxyethanol: Used in vaccines and cosmetics.
- Benzoic acid and sodium benzoate: Used in acidic liquid preparations.
- Chlorobutanol: Used in sterile solutions and eye drops.
- Thiomersal: Used in vaccines (limited use today).
Need for Revalidation of Preservatives
Preservative systems need to be re-evaluated if:
- Formulation composition changes
- Container or closure system changes
- Manufacturing process is modified
- Storage conditions change
Even a small change can affect preservative stability and effectiveness.
Interpretation of Test Results
- If microbial counts decrease as required → Preservative is effective.
- If microbial counts remain unchanged or increase → Preservative fails the test.
Any failed batch must be investigated for formulation faults, contaminant resistance or incorrect preservative concentration.
Key Points
- Preservatives protect multi-dose products from microbial contamination.
- Evaluation ensures preservatives remain effective throughout shelf life.
- Challenge testing uses bacteria, yeast and moulds to assess stability.
- Acceptance criteria vary for ophthalmic, parenteral and oral preparations.
- Product pH, ingredients and packaging material influence preservative activity.
Detailed Notes:
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