High Performance Liquid Chromatography
High performance liquid chromatography was performed as previously described on a Beckman model 344 liquid chromatography system (Beckman Instruments Inc, Fullerton, Cal) using an Ultrasphere C18 column (4.7 x 250 mm) with 5 |un particle size (Beckman) together with an Ultrasphere C18 precolumn (4.7 x 45 mm). The gradient program of Shak was used with mobile phase A—a solution of methanol, acetonitrile, water, and acetic acid in a ratio of 30:25:45:0.02, and mobile phase B—a solution of methanol and acetonitrile in a ratio of75:25; absorbance was measured at 280 nm. Leukotriene B4 standards, prostaglandin Bt, and [H]LTB4 (New England Nuclear, Boston) were used as standards. The LTB4-COOH and LTB4-OH were prepared from LTB4 as described previously and used for identification of LTB4 metabolites. No peaks corresponding to LTB4 metabolites were detected in any specimen analyzed by HPLC. Consequently, radioimmunoassay should accurately reflect the concentration of LTB4.
Initially, BAL samples were analyzed by HPLC; then fractions corresponding to the retention time of LTB4 were collected and analyzed by radioimmunoassay (12 specimens). Analysis of several split specimens with and without HPLC demonstrated nearly identical LTB4 levels (data not shown). Subsequently, radioimmunoassay was done on BAL specimens after eicosanoid extraction, without HPLC (27 specimens).