The microbiological examination of products can often prove challenging when the product is antimicrobial. This is often by design, but can also be inherent to the excipients, inherent to the active pharmaceutical ingredient(s) (API) or a side-effect of some other selected activity thereof. The former is often the case in products which require preservative systems such as multiple-use dosage forms, personal care products such as shampoo and conditioner, while the latter is the case for many cancer drugs which target life-cycle specific reactions, and in the most challenging products: antibiotics, which are specific and very potent in their activity towards the analyte of interest, microorganisms.
USP<61> Microbiological Examination of Non-sterile Products: Tests for Enumeration, and <1227> Validation of Microbial Recovery From Pharmacopeial Articles, outline three basic and common methods for the neutralization of antimicrobial properties. These include chemical inhibition, dilution, and filtration and washing.
Chemical inhibition is achieved as follows, and as outlined in USP<1227>: Bisulfate can be used as a neutralizer against glutaraldehydes and mercurials, whose action is typically targeting non-sporulating bacteria. Glycine can be used to neutralize aldehydes which target cells which are actively multiplying, replicating, or otherwise in a growth phase. Lecithin is used against Quaternary Ammonium compounds (QACs) as well as Parabens and bis-biguanides, all of which are active against bacteria. Magnesium and calcium ions can be used to neutralize ethylenediaminetetraacetic acid (EDTA). polysorbate is used to counteract biocidal activity of QACs, Iodine, and parabens. Thioglycollate is effective against mercurial antimicrobial agents (which are employed with increasing rarity due to environmental and toxicological concerns) which target Staphylococci and spores. Thiosulfate can be used to neutralize mercurials, halogens, and aldehydes, which have biocidal potential against Staphylococci.
Dilution is covered in the chemical inhibition section in USP<1227>, where it is listed as effective against Phenolics, Alcohols, Aldehydes, and Sorbate. Dilution is effective as a neutralization method for a number of antimicrobial and bactericidal agents, as the concentration of most of these compounds is a major component of their efficacy (and/or potency). Concentration and efficacy are related exponentially, and depend on constants related parameters such as bioavailability and biochemical kinetics. For this reason, some compounds are not particularly susceptible to neutralization by dilution. It is good to work with suppliers and manufacturers to determine any known compounds present, and any information regarding their activity. However, it is often the case that this information is obtained by experimentation which is resource prohibitive, and it is more likely that a neutralization strategy will be determined by trial and error in many laboratories.
Filtration and Washing is the final neutralization method discussed in detail in chapters 61 and 1227 of the United States Pharmacopoeia. This method essentially uses a membrane with a pore-size small enough to retain microorganisms while allowing smaller molecules and materials in solution to pass through. The material of construction for this membrane should be selected carefully, as different antimicrobials may adsorb to the filter rather than passing through, which then retain or even concentrate inhibitory properties on the membrane. Rinse solutions can also aid in the removal of antimicrobial substances. Just passing a rinse solution through can often remove inhibitory compounds. They can also ensure additional neutralization by incorporating chemical neutralizers.
These approaches demonstrate well understood methods of chemical or physical inactivation of common antimicrobials used in industrial and pharmaceutical applications, which fall into a few different categories, including saturation of chemical activity, sequestration of chemicals from solution, dilution beyond relevant activity concentration, and complete removal from solution. In addition, pH can often be an issue, and is sometimes overlooked. A simple pH check and adjustment is sometimes the easiest solution to a problem with inhibition. Another strategy is enzymatic treatment. Penicillinase as mentioned in 1227 as effective against penicillin. Applying these theories to novel or unrepresented antimicrobial substances is often the first step in understanding appropriate neutralization strategies.