Glycerol, 3HP, 1,3PDO and AA concentrations were determined by HPLC (JASCO, Tokyo, Japan) equipped with RI detector (ERC, Kawaguchi, Japan) and JASCO intelligent autosampler. Separation of the compounds was done using Aminex HPX-87H chromatographic column connected to a guard column (Biorad, Richmond, CA, USA). The column temperature was kept at 65 °C using chromatographic oven (Shimadzu, Tokyo, Japan). Samples were diluted with Millipore quality water and mixed with 10 % v/v sulfuric acid (20 µL/mL sample) and then filtered. Forty microliter sample was injected in 0.5 mM sulfuric acid mobile phase flowing at a rate of 0.4 mL/min.
For the determination of 3HPA concentration, the modified colorimetric method of Circle et al. 1945 [40 ] as described by Ulmer and Zeng (2007) [41 ] with acrolein as standard was used. Briefly, 200 µL of properly diluted sample was mixed with 150 µL of 10 mM DL-tryptophan solution in 50 mM HCl and 600 µL of concentrated HCl (fuming 37 %). The reaction mixture was incubated for 20 min at 37 °C. The produced purple color was then measured using spectrophotometer at 560 nm.
For the determination of 3HPA concentration, the modified colorimetric method of Circle et al. 1945 [40 ] as described by Ulmer and Zeng (2007) [41 ] with acrolein as standard was used. Briefly, 200 µL of properly diluted sample was mixed with 150 µL of 10 mM DL-tryptophan solution in 50 mM HCl and 600 µL of concentrated HCl (fuming 37 %). The reaction mixture was incubated for 20 min at 37 °C. The produced purple color was then measured using spectrophotometer at 560 nm.