High performance liquid chromatographic determination of thromboxane B2 in human serum as a methoxime-panacyl ester derivative

Abstract

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 C18/silica mode sequencing approach. The internal standard, 6-ketoprostaglandin F_1α 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$\text{>}$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 > 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 $\text{ex vivo}$ TxB₂ formation.