Antibiotic Potency Testing is a biological assay used to determine the potency (concentration) of antibiotic active pharmaceutical ingredients (API’s) and the products which contain them. FDA requires that the potency of antibiotic drug products conform to the potency appearing on their labeling in order to be distributed within the United States. USP General Chapter <81> entitled “Antibiotic Potency Assays” provides validated methods for determining the potency of multiple antibiotic active ingredients based upon their actions against specific microorganisms.
How is an Antibiotic Potency Assay Conducted?
USP General Chapter <81> entitled “Antibiotic Potency Assays” provides two (2) method types for determining antibiotic potency: Cylinder-Plate Assay and Turbidimetric Assay.
Cylinder-Plate Assay: This assay extracts the antibiotic active ingredient(s) from the product being tested using solvents and phosphate buffers as outlined in USP <81>. After extraction, the sample preparations are diluted to the median dose of the active ingredient that is found on a Standard curve which is created for the analysis. The sample and corresponding reference Standard preparations are pipetted into penicylinders that are placed into a petri dish already containing a base layer of antibiotic growth medium and a seed layer of antibiotic growth medium which also contains a USP-specified microorganism that is sensitive to the antibiotic being tested. The plates containing the cylinders are then incubated as specified within the method. The Cylinder-Plate Assay is based upon the ability of the antibiotic being assayed and its corresponding Reference Standard to diffuse through the agar and form a ‘zone of clearing’ (‘zone of inhibition’) thereby killing the microorganism which was present within the seed media. The ‘zone’ sizes are measured (in millimeters) using either an automated zone reader or manually using either calibrated calipers or a manual antibiotic zone reader. The size of each ‘zone’ is proportional to the quantity of antibiotic present. Results are calculated using a formula comparing the size of the ‘zone’ obtained from known quantities of antibiotic contained in the Reference Standardto the size of the ‘zone’ obtained from the sample. The Cylinder-Plate Assay is performed by conducting three independent assays on three different days.
Turbidimetric Assay: This assayis based upon the ability of the antibiotic being tested to kill a USP-specified microorganism that is present in solution, thereby reducing the turbidity of that solution. The reduction in turbidity of the test solution is proportional to the quantity of antibiotic that is present. The Turbidimetric Assaydepends upon the inhibition of growth of a USP-specified microorganism in a uniform solution of the antibiotic, using a series of concentrations in a fluid medium that is favorable to the growth of the microorganism in the absence of the antibiotic. The mixture is incubated at the appropriate temperatures and then the turbidity is measured spectrophotometrically. Results are calculated as indicated in USP <81>.
The use of the Cylinder-Plate Assay or Turbidimetric Assay is determined for each antibiotic by the USP.
Although both Antibiotic Potency Assay methods appearing in USP <81> are validated, they still require verification when employed for the first time by a laboratory for a specific sample type (formulation). Method Verification includes a determination of method precision, accuracy, linearity and specificity. This ensures that accurate results are obtained by a laboratory for each sample type under actual conditions of use. Method Verification is performed only once for each sample type (formulation) unless the USP method or sample formulation undergoes change.
At CPT℠, we have the expert capabilities to not only perform these assays but to also provide expert consultation services every step of the way. We employ the use of a Trinity V3 Automated Antibiotic Zone reader and we routinely test all of the following antibiotic products:
Neomycin, Polymyxin B Sulfate, Bacitracin Zinc, Erythromycin, Gentamicin, Nystatin and more.
Please contact CPT℠ regarding your antibiotic testing needs for these active ingredients (singly or in combination) and others which may not appear in the listing above.
Based on the individual needs of our clients, we offer a wide range of support across various departments. Since initial product testing can be fairly complex in terms of a high-quality, safe and trusted product, our clients typically explore the benefits of numerous departments and areas of expertise, including:
Clinical — Efficacy trials can be performed for safety, consumer in-use, and to support claims, such as ‘dermatologist-tested’ and ‘ophthalmologist-tested.
In-Vitro Safety — Our in-vitro safety department assists clients when they are looking to perform non-animal or animal-free toxicology testing.
Microbiology — Antibiotic testing for the pharmaceutical industry is routinely performed at CPT℠. We also perform other compendial assays for Microbial Content, Preservative Efficacy, Sterility, Bioburden, Water Activity and Specialty Microbiology Projects.
Photobiology — From SPF and Water Resistant claims to Photo Allergy and Phototoxicity studies, CPT℠ is the world leader in sunscreen testing.
Analytical Chemistry – From raw materials, batch release, and stability shelf-life testing to “free-of” and low-level detection claims, our Analytical Chemistry Department ensures that all requirements are met based upon regulatory requirements and industry standards.