with some
modifications. One gram of freeze dried sample
was mixed with 9 ml diluted sulfuric acid (pH 2.2)
(Merck, Germany), stirred for 30 min and then
centrifuged at 2,500 rpm for 10 min. The supernatant
was filtered through a 0.20-μm nylon membrane
(Vertical, Thailand) and the filtrate used for HPLC
analysis. The HPLC system (Shimadzu LC-10AD;
Shimadzu, Japan) consisted of a low-pressure
pump and a photodiode array detector (SPDM20A;
Shimadzu) adjusted to a lmax 250 nm.
Chromatographic separation was performed with a
C18 column (YMC-Pack ODS-AM, 5 μm, 4.6 mm
ID x 250 mm; YMC, Japan). The isocratic eluent was
0.1 M acetic acid (Merck) in deionized water (RCI
Lab-Scan, Thailand) with a flow rate of 1.5 ml/min.
The temperature of the column was adjusted to 30°C
and the injection volume was a 20-µl filtrate. The peak
area was determined and converted to concentration
of ascorbic acid.