High performance liquid chromatographic determination of thromboxane B2 in human serum as a methoxime-panacylester...

A high performance liquid chromatographic (HPLC) method was developed to measure thromboxane B2 (TxB2) levels in human serum. Serum samples (2 mL) were extracted using solid phase extraction columns in a C 18/silica mode sequencing approach. The internal standard, 6-ketoprostaglandin F1 alpha, was added to the serum extracts. The eicosanoids were doubly derivatized, first with panacyl bromide, then with methoxyamine to form methoxime-panacyl ester derivatives. The eicosanoid derivatives were chromatographed using a reverse phase HPLC system with UV detection (254 nm). Assay linearity was demonstrated with fortified TxB2 standards in 3% bovine serum albumin over a range of 25 to 500 ng/mL (r greater than or equal to 0.994). There was no significant interday difference or bias in assay results for pooled standards at 75, 226 and 376 ng/mL concentrations (p greater than 0.05). Pooled estimates of precision at these levels indicate an assay relative standard deviation 6-9%. The HPLC assay was used to quantitate TxB2 levels in human serum. Results were consistent with previously published values when drug-free serum was analyzed to assess ex vivo TxB2 formation.