A quantitative method was developed and validated to analyze iodoform and its potential metabolite, diiodomethane, in biological fluids from dairy cows, including rumen fluid, duodenal fluid, blood serum, milk, and urine, using liquid-liquid extraction (LLE) and GC-MS/MS. The method showed no matrix effects across different samples, recoveries of spiked samples between 70 and 120%, and relative standard deviations (RSD%) ranging from 0.7 to 14%. Inter- and intravariations in quality control samples were within 3.1-12.3%, with the highest variation noted in iodoform spiking. Normalized internal standard (IS) and matrix factor (MF) calculations confirmed the suitability of diiodomethane-d2 as an internal standard for quantifying both compounds across different matrices. These results validate the method's application for ADME (absorption, distribution, metabolism, and excretion) studies, enabling the investigation of iodoform digestion, metabolism, and excretion in dairy cattle.
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