Urine, solid phase extraction 3621 Subject

The most popular methods for extracting biological samples (in particular plasma, urine, and tissue) include solvent extraction with protein precipitation (PP), solid-phase extraction (SPE), and liquid-liquid extraction (LLE). While urine or bile can be directly injected for an LC-MS analysis, plasma must be first extracted to remove proteins. In some cases, it is desirable to clean up urine and bile samples and thus such samples are also most commonly subjected to SPE. 

Levels of radioactivity were highest in the liver and kidneys of male and female rats at two hours post final dose and averaged 3.A63 ug equivalents/g and 4.073 ug equivalents/g in male and female rat liver, respectively, and 2.383 and 2.132 yg equivalents/g in male and female rat kidney. Urine samples were analyzed by direct injection onto the HPLC column, whereas feces and liver samples were homogenized and extracted with methanol and subjected to solid phase extraction (if necessary) prior to HPLC analysis of the organic extract. The major radiolabeled component in male and female rat urine co-chromatographed with flunixin. Lesser amounts of the two hydroxylated metabolites, 5-hydroxyflunixin and 2 -methyIhydroxyflunixin, were also detected. Two major radiolabeled residues, identified by retention characteristics as flunixin and 5-hydroxyflunixin, were observed in rat feces. The major radiolabeled component in male and female rat liver co-chromatographed with authentic flunixin standard. The monohydroxymetabolite, 5-hydroxyflunixin was also detected in male rat liver. 

Urine. Urine samples collected from cows 821 and 59 in the 0-12 hour period after the administration of a 30 mg dose (injection 2) were utilized in the isolation and identification of metabolites. The isolation procedure involved ethyl acetate extraction of urine followed by solid phase extraction of the ethyl acetate extract on C-18 Bond Elut cartridges. The extract was derivatized into methyl esters with diazomethane and subjected to silica and alumina chromatography for further cleanup prior to analysis. The detailed procedure is described in flow diagram 1. 

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