HPLC Analysis of Organic Acids

Organic acids were analyzed as described previously (Osanai et al., 2015 (link)). Freeze-dried supernatants were resolved in 100 μL of filtered 3 mM perchloric acid. The resolved samples were analyzed by HPLC using an LC-2000Plus System (JASCO, Tokyo, Japan) with a photodiode array detector and two RSpak KC-811 columns (Showa Denko, Tokyo, Japan). Organic acids were quantified using 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer; peaks were detected at 445 nm. The temperature of the column was 60°C, and flow rates of 3 mM perchloric acid and 0.2 mM bromothymol blue solutions were 0.7 and 1.2 mL/min, respectively.