I have treated some chickens with a fixed dose formulation of amoxicillin (100mg/ml) and colistin (85mg/ml) in drinking water (Group A), and by intramuscular route (Group B). My objective is to evaluate the serum profile of the two active ingredients and compare to know the best route of treatment. Because of paucity of assay facilities (HPLC), the only available option is microbiological assay using E.coli as a test organism. I intend reconstituting reference standards of amoxicillin and colistin in the ratio 100/85 and use it to develop my calibration plot (diameter of inhibition zone versus ratio of amox/col) and regression equation. Can this be used and accepted?
Dear Aondover Fidelis Gberindyer
Unfortunately its impossible because you cant distinguish both drugs based on microbiological assay with one bacterial strain. To do it with microbiological assay you should have two bacterial strains, first susceptible only for amoxicillin and second susceptible only for colisitin. Then if cross reactivity will be excluded such metod could be validated. But its impossible to do with one bacterial strain.
In general HPLC methods are recommended for such works and from my knowledge microbiological method should be compared with HPLC to be sure its works correctly. My advice is work with HPLC because it could be finaly less complicated in case two drugs.
Ratio 100/85 could works only in one case if your drugs always in all time points in plasma samples have the same ratio of concentrations 100/85 but it not works like this and pharmacokinetics profiles will be different in different points (ratio will be different and dynamic) and will differ between animals (between animals variability). So finally if you are using one strain with "rigid" calibration curve based on dosing ration you cant see real data. Good example of such work is desrcibed here: https://www.ncbi.nlm.nih.gov/pubmed/21219343
Some nice documents related to validation which could be useful for you:
https://www.epa.gov/sites/production/files/2015-01/documents/final_microbiology_method_guidance_110409.pdf
https://www.fda.gov/downloads/drugs/guidances/ucm070107.pdf
https://www.ema.europa.eu/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf
https://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/UCM207942.pdf
Best regards
Tomasz
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