C. Quan et al. / Agricultural Sciences 4 (2013) 89-96
Copyright © 2013 SciRes. O PEN A CCESS
Table 1. The chromatography conditions of the studied flavonoids CRMs.
CRMs Mobile phase
(Acetonitrile:0.1% phosphoric acid) Wavelength (nm) Injection volume (μL) Flow (mL/min) Concentration (mg/mL)
Baicalein 35:65 275 5 1 0.5
5,7-dihydoxy fl avones 43:57 268 5 1 0.3
Naringenine 35:65 288 2 1 1
homogeneity between bottles, and 7 portions from each
bottle were assayed for homogeneity within bottle. The
results were examined via F-test and t-test. In F-test,
standard deviations were used to examine whether the
deviation between bottles was significantly greater than
the deviation within bottle. In t-test, means were used to
examine whether the mean between bottles was signifi-
cantly different from that of within bottle [10].
A stability study monitored a significant change in
value of the flavonoids CRMs was performed over one
year.
A study of long-term stability for storage at 4˚C was
carried out by HPLC-VWD method [11 ] in which ma-
terial was determined at 1, 3, 6 and 12 months. For each
condition, two solutions were prepared and analyzed in
duplicate. A comparison was made between the purity
determined by HPLC-VWD for different storage time. A
freshly prepared control sample was analyzed at each
time. All the samples were randomized for analysis.
2.6. Uncertainty Evaluation
The uncertainty evaluation for the flavonoids CRMs
derived from the certified value, homogeneity and stabil-
ity study. The expanded uncertainty was calculated as
follows:
( 3)
where, k is the coverage factor, Uc is the combined un-
certainty of the sample, uv, uh, us are the uncertainty
components of the assigned value, homogeneity and sta-
bility study.
3. RESULTS AND DISCUSSION
3.1. Qualitative Analysis Methods
3.1.1. LC-MS Method
The experiment obtained the molecular weight of the
flavonoids CRMs by full scan mass spectra. The results
indicted the molecular ion peak (M + H)+ of mass charge
ratio m/z of baicalein and 5,7-dihydoxy flavones was the
molecular weight plus the atomic weight of hydrogen
(1.0), the molecular ion peak (M − H)− of mass charge
ratio m/z of naringenine was the molecular weight sub-
tract the atomic weight of hydrogen (1.0). Ion fragments
by product ion scan are detailed in Figu re 2. The results
showed the flavonoids mainly fractured in 1,2 C-Ochem-
ical bond and 3,4 C-C chemical bond.
3.1.2. IR Method
The Infrared spectrum of the flavonoids CRMs were
shown in Figu re 3. The results revealed the peaks at
3090, 3097, and 3114 cm−1 belong to hydroxide peaks,
1660, 1653, and 1630 cm−1 belong to associat ed car bon yl
peaks, 1449 ~ 1618 cm−1 belong to benzene ring skeleto n
of C = C peaks of the flavonoids CRMs. The Infrared
spectrums of the flavonoids CRMs were consistent with
those spectrums from literature [13-15] and can be used
for quantification.
3.2. Quantitative Analysis Methods
3.2.1. Mass Balance A pproach
The HPLC chromatogram of the flavonoids CRMs
were shown in Figure 4. The results of the moisture
content and the inorganic elements contents were listed
in Table 2, as indicated, The water content for Baicalein,
5,7-dihydoxy flavones, and naringenine were 0.538%,
0.150%, 0.098% and 0.115%, respectively, all were less
than 0.1%, while the inorganic content all warless than
0.00002% at the studied conditions.
3.2.2. QNMR Method
QNMR was also used to determinate purity values
flavonoids CRMs. The NMR spectrums were shown in
Figu re 5. The C3-H peak areas of baicalein, C8-H peak
areas of 5,7-dihydoxy flavones, C4,-OH peak areas of
naringenine, C2,2-2H peak areas of the benzoic acid in-
ternal standard were selected for integra tion .
3.2.3. Purity Values of the Flavonoids CRMs
The purity values of flavono ids CRMs were calculated
with the average of mass balance approach and QNMR
method. The results are listed in Table 2.
3.3. Homogeneity Study and Stability Study
The F-Test and t-test results of all the studied were
summarized in Table 3. No significant differences were
found for all the studied compounds in homogeneity test
as the calculated F-values were less than the critical
F-values (Table 3). Although a slight difference was ob-
served, this was attributed to the variation of the analyti-
cal method.