High performance liquid chromatographic determination of leukotrienes

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 (LTC4, LTD4, LTE4) and non peptide (LTB4) 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 LTB4, LTC4, LTD4, LTE4 and PGB2 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 LTC4, LTE4, LTD4,and L TB4 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.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 (LTC4, LTD4, LTE4) and non peptide (LTB4) 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 LTB4, LTC4, LTD4, LTE4 and PGB2 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 LTC4, LTE4, LTD4,and L TB4 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