3. Results and discussion
3.1. Optimization of chromatographic conditions
In order to obtain the most useful chemical information and
better separation in the chromatograms, the column, mobile phase,
and the detection wavelength were investigated. Two reversedphase
columns, Agilent Zorbax SB-C18 column (5 lm, 4.6
250 mm) and Agilent HC-C18 column (5 lm, 4.6 250 mm) were
first investigated. The Agilent Zorbax SB-C18 column was found
to be more suitable which gave good peak separation with stable
baselines. The effect of mobile phase composition on chromatographic
separation was then investigated. The results indicated
that no differences were found between methanol–water and
acetonitrile–water. Considering the higher toxicity of acetonitrile,
the binary mixture of methanol–water was chosen. In order to
enhance the resolution, as well as to restrain the ionization of
the polyphenols and to eliminate the peak tailing of target compounds,
1% (v/v) glacial acetic acid was added to the mobile phase.
On the ultraviolet spectra with chromatograms of HPLC-UV of
eight target compounds in PPPs and reference standards, maximum
absorbance values around 254 and 280 nm were observed.
More detectable peaks could be obtained and the baseline was well
improved around 280 nm at which the better characterization of
polyphenols can be attributed. Hence, characteristic chromatographic
patterns were obtained by using 280 nm as the detection
wavelength. Optimal chromatographic condition used in this study
is shown in Section 2.2.
3.2. Method validation of quantitative analysis
3.2.1. Linearity and limits of detection
Linear regression analysis for each compound was performed
by plotting the peak area (y) against the concentrations (x, mg/L)
of the mixed standard solutions which was shown in Table 1. Good
linearity was observed in the investigated ranges for all the