High Performance Liquid Chromatographic Determination of Leukotrienes

Authors: Afig HÜSEYİNOV, Canan ÇOKER, H. Hakan AYDIN, Sevgi TÜZÜN, Gülgün KEÇECİOĞLU, Biltan ERSÖZ

Abstract: Leukotrienes are biologically active compounds derived from lipoxygenasecatalyzed metabolism of arachidonic acid in mammalian cells and tissues. The present report describes a high performance liquid chromatographic (HPLC) method for separation and quantitation of both the peptide (LTC_4, LTD_4, LTE_4) and non peptide (LTB_4) leukotrienes along with a solid phase extraction method for their analysis in biological samples. The mobile phase in the HPLC system, consisting of acetonitrile: water: methanol: acetic acid (300:420: 100:0.8) (v:v) with pH value adjusted to 5.1, proved to be suitable for optimal separation. The isocratic elutiOn of mix standards, containing LTB_4, LTC_4, LTD_4, LTE_4 and PGB_2 as the internal standard, with a flow rate of 1 ml/min through a C 18 column, provided high resolution and reproducible retention times (3.7%, 0.5%, 0.3%, and 0.8% for LTC_4, LTE_4, LTD_4,and L TB_4 respectively) . The liquid solid extraction procedure applied using C18 cartridges of 300mg yielded satifactorily high recovery values with the standards dissolved in phosphate buffer saline solution as well as in plasma. It is concluded that this method provides efficient extraction and quantitative determinaton of leukotrienes in biological fluids.

Keywords: Arachidonic Acid, Peptide leukotrienes, LTB_4, High Performance Liquid Chromatography, Solid Phase Extraction