Introduction
Preservative are included in pharmaceutical formulations to reduce the susceptibility of the formulated products to microbial spoilage. Such preservatives are benzoic acid, sorbic acid, benzalkonium chloride, phenylmercuric acetate etc.
A preservative must not be expected to completely sterilize formulations which are heavily contaminated as a result of poor raw materials or poor manufacturing procedures. Therefore, there is need to maintain a standard good manufacturing practice (GMP). This entails personal hygiene for the manufacturers, clean and decent environment of manufacture and reduction of the regulative contami+nant load of the ingredients for the manufacture of the pharmaceutical products.
Features of Ideal Preservatives
An ideal preservative must process the following features
- Be able to effectively inhibit the growth of all microorganisms likely to be in the product.
- Freedom from toxicity or irritancy towards the patient
- Sufficient stability to withstand formulation procedures and the intended life span on shelf-life of the product.
- Freedom from inhibition of the preservation by the ingredients of the formulation.
However, such ideality does not exist and the practice is to resort to the least unsuitable preservative for the specific formation
BP and BPC Specifications for a Potential preservatives
The BP and BPC specifications for a potential preservation are:
- The BP specifies that the preservative must be capable of sterilizing the product within 3 hours of the inoculation of 106 vegetative bacterial cells per ml of the product.
- The BPC requires that the bactericidal activity of a potential preservative must be equivalent to the antimicrobial activity of 0.5% w/v phenol
Problems Encountered with Potential Preservatives
- In emulsified products, many potential preservative can react with the varied ingredients that the preservative would be exhausted and not sufficiently available for effective interaction with the microorganisms, and therefore would not adequately protect the product or preparation.
- In eye drop preparations, antimicrobial agents could adequately kill vegetative contaminants that may be introduced into multidose eyedrop formulations before the next dose is withdrawn (between 1 to 5 hours), but the concentrations required to inhibit contaminant growth could cause marked irritary to the cornea.
Effect of preservative concentration, temperature and size of inoculum
There is an exponential relationship between microbial activity and preservative concentration. The value of the exponent (n) the dilution factor varies appreciably for different antimicrobial agents.
Halving the concentration of Phenol (n=6) results in a 64-fold dilution (26) in killing rate, while a similar dilution of chlorhexidine (n=2) reduces killing rate only by 4-fold (22). This property must be considered when selecting a preservative agent.
The concentration exponent or dilution coefficient (n) is the slope of the line obtained by plotting the log of the line required to kill a standard inoculum (log death time) against the log of the concentration. Compounds with low (n) values are not readily inactivated by dilution e.g. mercuric chloride with dilution coefficient (n) of 1.
For formaldehyde and ethylene oxide halving the concentration merely double the time required for disinfection, also a three-fold dilution increase the disinfection time 3-fold.
Phenol has a dilution coefficient of 6, halving the concentration prolongs the disinfection time by 26 (64 fold), whilst a 3-fold dilution brings about a decrease in activity of 36 (729-fold).
Increase in temperature: this always increase antimicrobial activity. Temperature coefficient (10, rate of change of activity per 10oC) vary with preservative, organism and temperature range. A drop from 300C to 200C can result in a 5 fold reduction in the killing rate of E.coli by phenol (10=5). Interaction of concentration exponent and temperature coefficient is complex. If a 0.1% w/v solution of chlorocresol completely killed an inoculum of E.coli at 30oc in 10 minutes (n = 6, 10 = 5) in a laboratory, it would require about 90minutes if used at 20oC and 10% of its chlorocresol by dissolution into the container material. The influence of temperature and concentration changes on microbistatic activities show some similarity with effects on microbicidal activities.
Preservative are removed from solution and used up during the inactivation of microorganisms.
Therefore the preservative capacity is a measure of the total quantity of microorganisms a formulation can inactivate before significant deterioration of efficiency become evident, and varies considerably with the type of preservative.
Factors Affecting the Availability of Preservatives.
In a preserved formulation, only a small proportion of the total preservative is often available for preservative purposes or antimicrobial activities. Most preservatives interact with many of the likely ingredients of a formulation as well as with any microorganisms present in the formulation via various binding interactions. The unavailable preservative may still contribute to the general irritancy of the product.
Factors that affect the availability of preservatives are:
- Effect of pH.
- Effect of container.
- Effect of multiphase systems.
1. Effect of pH:
This refers to the formulation pH. The activity resides generally in either in the neutral molecule or in the conised species. The weekly acidic preservatives are only effective at low pH where neutral molecules will predominate, for example, benzoic acid (Pka = 4.2) has practically no preservative activity above pH5.0, while the p-hydrobenzate esters with their unionisable esterified carboxyl groups have modest activity at neutral pH. Quaternary ammonium preservatives such as (benzalkonium chloride, cetyltrimethyl ammonium bromide (cetrimide) and chlorhexidine have their activity in their cations. They are most effective in neutral or slightly alkaline solutions.
2. Effect of container:
Closures for multidose injection containers most not be of the type such a rubber that can allow permeation of phenolic and other preservative through them with consequent marked decline in antimicrobial activity of the injections within. Volatile preservatives such as chloroform are easily lost by the normal removal of container closure that there use should be limited only to preservation within a sealed container or for extemporaneous products such as eye drop.
Appreciable interactions have been observed between certain plastics and preservatives by adsorption, and solution within the plastic, as is encountered with nylon and certain rubbers with phenol. It is also known that normal surface adsorption onto glass and plastics has significantly removed quaternary ammonium preservatives from solution.
3. Effect of multiphase system
Preservatives within micelles or in the oil phase is not immediately “available” to microorganisms. The extent of oil-water distribution of the preservative is determined by the preservatives’ partition coefficient for that system and the oil-water ratio.
In an emulsified product available preservative is reduced considerably by:
- Distribution between the oil and water phase
- Solubilisation into surfactant micelles within the aqueous phase. Micellar solubitiration is controlled by the relative concentration of preservative and surfactant and a reaction constant unique for each preservative – surfactant pair. The loss of neutral molecules into the oil or micellar phases is favoured over the ionized species. Further loss of “available” preservatives may occur by weak binding with non-micellar surfactants and other hydrocarbon chains, particularly those of polymeric ingredients. Adsorption of the preservative onto the particule surface of particulate suspensions eg magnesium trisilicate, kaolin can both reduce preservative “availability