The adequacy with which a formulated medicine is protected from spoilage by the use of a preservative cannot easily be predicted from a study of the activity of the preservative in simple aqueous solutions.
A person with limited knowledge of pharmaceutical microbiology might, for example, expect that it would be possible to confirm that a medicine was adequately preserved simply by conducting an assay for the preservative and showing it to be present at a concentration higher than that required to inhibit the growth of common contaminants (i.e. higher than minimum inhibitory concentrations quoted in reference books.
Not infrequently, however, this logic does not apply, because the preservative activity is reduced as a result of it interacting with other components of the formulation or the container, or by a change in the environmental conditions within the product.
As a consequence, it is necessary to measure preservative efficacy not by a chemical assay but by a pharmacopoeial preservative efficacy test where the manufactured medicine is inoculated with a range of test organisms whose death rate is measured over a 28-day
Contaminating microorganisms do not invariably grow uniformly throughout a medicine packed in its final market container; they may be concentrated
near the surface as a result of the higher oxygen availability there or, in the case of an emulsion, they grow in the water phase rather than the oil phase.
The concentration of preservative available at the site where the organisms are growing is therefore the principal determinant of how effectively the
medicine is protected from spoilage, and the ‘free’ preservative concentration (that which is actually available to kill microorganisms) may be significantly
lower than the calculated value. Fig. 1 below shows the major factors that influence preservative activity.
Several groups of common preservatives are affected by pH. This may be a consequence of:
- a change in ionization of the preservative molecule which alters the relative proportions of its undissociated and dissociated forms, which possess different intrinsic antimicrobial potencies;
- an effect on cell surface charge influencing adsorption of preservative molecules onto the
microbial cell; or
- a change in the solubility or stability of the preservative molecule.
The weak organic acids (e.g. benzoic and sorbic acids) are the most commonly cited examples of preservatives whose ionization and activity are pH dependent, but there are several others.
These acids are effective in formulations that are naturally acidic or can be
buffered to a low pH. This is because in these conditions they exist as the undissociated molecules, which are more lipid soluble and more effective than the
ionized forms that predominate when the ambient pH exceeds the molecule’s pKa.
Phenolic preservatives exhibit similar but less marked pH dependence. Parabens are also slightly affected in the same way.
This situation contrasts with that seen with quaternary ammonium compounds, which are most effective in neutral or slightly alkaline conditions.
Bacterial cells are usually negatively charged, and a rise in pH increases the number of such charges and so promotes the binding of positively charged molecules such as quaternary ammonium compounds.
Even though many liquid medicines contain a buffer to restrict pH change, it is not uncommon for the product specification to quote a permissible pH range that is sufficiently large to have a significant impact on preservative activity and for the product pH to
drift within that range during the shelf life, which may be 2 years or more.
Slow precipitation duringbstorage (e.g. parabens precipitating with falling pH)
is a further problem that is not necessarily detected by chemical assays because the assay procedure may redissolve the precipitate.
The oil–water partition coefficient is another molecular property that can have a marked influencebon preservatives when they are used in emulsions.
As microorganisms grow in the aqueous phase or at the oil–water interface, a preservative that accumulates in the oil phase is essentially inactive, although
again this will not necessarily be apparent because a chemical assay is likely to show that the correct amount of preservative is present in a given weight
or volume of a sample.
This is a factor that makes parabens less useful choices as cream preservatives because they are usually a lot more soluble in vegetable oil than they are in water, although their partitioning might be reduced by substitution of mineral oil for vegetable oil.
Again phenolics and some other preservatives are similarly affected, so when one is selecting preservatives for multiphase formulations, it is useful to consult publications such as the Handbook of Pharmaceutical Excipients (Rowe et al., 2016), where lists of solubilities and partition coefficients
may indicate the likely extent of the problem.
Entrapment of preservatives within micelles of surfactants or emulsifying agents is a related phenomenon, where again the preservative is present but
unavailable to inhibit microbial spoilage.
Surfactants such as lecithin, polysorbate 80 and Lubrol W are so effective in removing preservative in this way that they are commonly used as inactivators (neutralizers) to prevent preservative carry-over and erroneous results
in bioburden and preservative efficacy determinations.
Dissolution and dialysis techniques
together with mathematical models may provide some indication of the extent of preservative loss in such complex formulations. Complexation between anionic surfactants (e.g. sodium lauryl sulfate) and cationic preservatives (e.g. chlorhexidine and quaternary
ammonium compounds) is also a potential problem in emulsion formulation.
Preservatives may be removed from solution by adsorption onto suspended solids, such as bentonite, kaolin, magnesium trisilicate and talc. In the case of bentonite, the potential to adsorb cationic drugs has been investigated as a means of retarding drug release
to achieve a long-acting formulation.
Antacid products, in particular, may prove difficult to protect because
of preservative adsorption, and there have been several reports of products based on aluminium hydroxide, magnesium trisilicate or kaolin being vulnerable to preservative inactivation.
This is reflected in the United States Pharmacopeia (United States Pharmacopeial Convention, 2016), which specifies less stringent preservative performance criteria for antacids
than for other oral products. The problems posed by adsorption are compounded by the fact that the
phenomenon may also be pH dependent, so the most favourable pH for preservative activity itself may be
one that promotes adsorption. Other hydrocolloids used as viscosity-raising agents in oral and topical products (e.g. alginates, tragacanth, cellulose derivatives and polyvinylpyrrolidone) may also reduce preservative activity. In some cases, this is simply a charge effect where an anionic polymer (e.g. alginate)
complexes a cation (e.g. a quaternary ammonium compound).
Another major mechanism by which preservative activity may be compromised is interaction with the
container or, in the case of volatile agents such as chlorbutanol, permeation through the container and loss by evaporation. Preservatives may adsorb onto the internal surface or penetrate into the material of the container itself.
This problem has become
more significant as plastic has tended to replace glass as a packaging material. Rubber stoppers in vials may also cause preservative loss.
Most plastics, but particularly those commonly used for container manufacture such as polypropylene and polyethylene, have the potential to remove significant amounts of parabens
and other common preservatives. The surface-tovolume ratio of the product in its container is likely to have a bearing on the magnitude of the problem;
small containers have a relatively larger surface and may exhibit proportionately greater loss.