Separation of leukotrienes by HPLC

The vast majority of separations described for the leukotrienes have used isocratic reversed phase conditions with an ODS stationary phase, although a single report exists which used ion-pair reversed phase chromatography with gradient conditions. Examples of these separations will not be discussed, together with some of the problems associated with HPLC of the leukotrienes. 

The most popular mobile phases for the separation of the leukotrienes all contain methanol for example, a mobile phase of methanol-water-acetic acid-ammonium hydroxide (67 33 0.08 0.04), pH 6.2 in combination with an ODS column was used to resolve leukotrienes B4, C4, W-trans-C and D4 in the elution order given (Metz et al., 1982) (Fig. 11.5.4). Under these conditions, the major leukotrienes were well resolved from the diHETEs which are often co-synthesised in in vitro test systems. The addition of acetic acid and ammonium hydroxide to the mobile phase is reported to maximise the sharpness of the eluting peaks and enhance the reproducibility of k and a values (Metz et al., 1982). The mechanisms by which the ammonium and acetate ions improve chromatography was postulated to be through ion-pairing of the free carboxyl and amino functions on leukotrienes C4 and D4. 

In addition to providing an adequate separation of the leukotrienes from diHETEs, the use of methanol-based mobile phase systems has also been observed to provide good resolution of leukotriene C4 and D4 from the monoHETEs (Osborne et al., 1983). These authors used a 

An ion-pair separation of the leukotrienes has been reported to facilitate the resolution of leukotriene B4, (5S, 12S)-6-/ra 5-leukotriene B4, (5S, 12i )-6-tran -leukotriene B4, leukotriene C4 and D4 (Ziltener et al., 1983). The initial mobile phase was 70% methanol-30% water-0.1 % acetic acid (v/v) with 2.5 mM 1-pentanesulphonic add as the counter-ion (final pH 3.9). The final mobile phase was 100% methanol-0.1% acetic acid (v/v), and 2.5 mM 1-pentanesulphonic acid, pH 3.9. The stationary phase was an ODS column. The addition of the counter-ion was reported to reduce the long retention times of leukotriene C4 and leukotriene D4 at pH 5.7 which was the optimal pH for chromatographic resolution. 

The chromatography of leukotriene A 4 presents certain problems since it contains an allyhc 5,6-epoxide which breaks down almost instantaneously at neutral or acidic pH. To overcome this chemical instability, alkaline chromatographic conditions are required which use a mobile phase of acetonitrile-0.01 M borate buffer, pH 10 (30 70) in combination with either an octyl or ODS colunm (Wynalda et al., 1982). As silica-based columns are somewhat labile at alkaline 

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