To ensure consistency, each antibiotic must be weighed and diluted according to the specifications listed in the following sections. Only high-grade, pure substances should be used to avoid variability in results.
Stock Solution Preparation
Category | Penicillin-G potassium (K) | Oxytetracycline (dihydrate) | Sulfadiazine |
First solvent** | Water-methanol (50 + 50, v/v) | Methanol | Methanol |
Molecular weight* | 372.48g/mol | 496.46g/mol | 250.28g/mol |
Salt content* | K = Mw 39.1 = 11.17% | No | No |
Crystalline water | No | . 2 H2O = Mw 36.0 10.78% | No |
Loss of drying* water content | Typical 1% | Unknown* | Typical 0.5% |
Impurity* | Typical 1-2% | Typical 1-2% | Typical 0.5% |
Corrected weighing for 50mg | Typical 57.6mg | Typical 55.0mg | Typical 50.5mg |
* ‘Typical’ or ‘Unknown’ values must be checked and confirmed by the supplier’s product specifications and batch’s Certificate of Analysis (CoA).
** First dissolve solvent in ‘corrected weight’, then solvent water, and finally dissolve into raw milk.
Dilutions
Category | Penicillin-G 50 mg | Oxytetracycline 50 mg | Sulfadiazine 50 mg |
First solvent | Water-methanol (50 + 50, v/v) | 50 ml Methanol | 50 ml Methanol |
Concentration First Solvent† | 50 mg/50 ml ⇒ 1000 µg/ml | ||
Second solvent (water) | 1.0 ml into 100 ml ⇒ 10,000 ng/ml (ppb)††† | ||
First dilution (in milk) | 1.0 ml into 100 ml of milk ⇒ 100 ng/ml (ppb) | ||
Concentration | 100 ppb | 100 ppb†† | 100 ppb†† |
Second dilution | 2.0 ml into 50 ml of milk | Not applicable | Not applicable |
Second dilution's concentration | 4 ppb†† | Not applicable | Not applicable |
† Portion into small vials (e.g., 5 ml). Shelf-life maximum 200 days at −18°C.
†† Test concentrations to be used on the day of final dilution, kept on ice or at max 4°C.
††† ppb (i.e., ‘parts per billion’) = ng/ml (i.e., ‘nanogram/millilitre’).
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