Analysis of leukotriene B₄ as free acid and its δ-lactone by high-performance liquid chromatography: a study on the stability and formation of leukotriene B₄ δ-lactone

Summary

Reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection (270 nm) was applied to study the stability of LTB₄ δ-lactone in aqueous solutions and its chemical formation from LTB₄. LTB₄ δ-lactone was found to be unstable in aqueous buffered solutions (pH range 4 to 8) and was converted in a pH and time depending manner mainly to LTB₄ and most probably to an isomer of LTB₄ δ-lactone. This compound was spontaneously converted to LTB₄ δ-lactone when incubated in the buffers and exists in equilibrium with LTB₄ δ-lactone at a molar ratio of about 1:1. Incubation of LTB₄ in buffers (pH 4, 6 and 8) and human plasma did not result in formation of detectable amounts of LTB₄ δ-lactone. Conversion of LTB₄ into its δ-lactone form in a yield of 62±6% (mean ± SD, n=3) was achieved by treatment of LTB₄ with a mixture of acetic acid, acetic acid anhydride and granular calcium chloride (1/5/3, v/v/w %) in ethylacetate. Evidence for LTB₄δ-lactone formation from LTB₄ under these conditions was demonstrated by gas chromatography-mass spectrometry.