FIG. 2.

HPLC determination of the Tyr and 4-HPP intracellular and supernatant concentrations. (A) HPLC chromatograms, monitored at 274 nm, of culture supernatants from wild-type and ΔhpdA strains submitted to solid-phase extraction onto Octadecyl (C₁₈) SPE columns. Arrows 1 and 2 represent Tyr and 4-HPP, respectively. The separation was achieved by applying the following gradient with 0.02 M acetic acid (solvent A) and different amounts of HPLC-grade methanol (solvent B) as follows: 3% to 35% solvent B from 0 to 5 min, 35% solvent B from 5 to 12 min, 35% solvent B to 3% solvent B from 12 to 13 min, and 3% solvent B from 13 to 15 min. Standard solutions were also submitted to solid-phase extraction onto Octadecyl (C₁₈) SPE columns (Mallinckrodt Baker) and injected into the C₈ column. The intracellular and supernatant concentrations of Tyr (B) and 4-HPP (C) were measured by HPLC determination (A). The concentration of Tyr and 4-hydroxyphenylpyruvate was determined by comparing areas of standards and samples. mAU, milliabsorbance unit.