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Figure 1. HPLC chromatogram of five standard substances (1, Luteoloside; 2, Luteolin; 3, Physalin A; 4, Physalin P; 5,
Physalin O) (A) and the calyces of Physalis Alkekengi L. var. Franchetii (B).
Chromatographic system and conditions
HPLC analysis was carried out with an Agilent 1100 series HPLC
(Palo Alto, CA) incorporating a UV detector. The analytes were
determined at 30°C on an analytical column (Agilent C18, 150×4.6
mm, 5-μm particle size) (Agilent Technologies, Palo Alto, CA). The
mobile phase consisted of the solvent (A) acetonitrile and (B) 0.2%
aqueous phosphoric acid (v/v) using a gradient elution of 20 to 23%
(A) at 0 to 13 min and 23 to 31% (A) at 13 to 37 min and then
returned to initial condition for a 5 min re-equilibration, with total run
time 42 min. The mobile phase was passed under vacuum through
a 0.45-μm membrane filter before use. The analysis was carried out
at a flow rate of 1 ml/min with the detection wavelength set at 350
nm at 0 to 24 min and at 220 nm at 24 to 37 min.
Sample preparation
Crushed dried calyces of P. alkekengi are the 50 mesh, to the
conical flask with lid, 0.5 g of the powder of the calyces and 25 ml
of carbinol were add, then extracted in an ultrasonic bath for 1 h.
The filtrate concentrated to 25 ml volumetric flask. The filtrate were
filtered with 0.45 μm membrane filer to obtain the filtered solution
and an aliquot (20 μl) of filtrate was injected into the HPLC system.
RESULTS AND DISCUSSION
HPLC analysis
Our attempts to use the method with isocratic elution for
the determination of five active ingredients were
unsuccessful. The gradient elutionmethod was, therefore
used for the separation of the ingredients. During method
Xu et al. 2439
development, the mobile phase composition varied using
different combinations of methanol, acetonitrile and
phosphoric acid. It demonstrated a longer retention time
with a decrease in the organic composition. Here, the
mobile phase consisting of (A), acetonitrile and (B) 0.2%
phosphoric acid were chosen to achieve good peak
shape, satisfactory resolution, and relatively short
analysis time. Figure 1 show typical chromatograms of
the standard substances (A), and the calyces of P.
Alkekengi (B). The retention times of Luteoloside,
Luteolin, Physalin A, Physalin P and Physalin O, were
approximately 6.7, 21.9, 27.9, 32.2 and 33.1 min,
respectively, and the total chromatographic run time was
37 min.
Through the pre-test we found the maximum absorption
of the five active ingredients at 220 nm, but the
Luteoloside and Luteolin were not well separated from
adjacent interference peaks and the baseline was not
smooth. Luteoloside and Luteolin also had the maximum
absorption at 350 nm, but Physalin A, Physalin P and
Physalin O were not detected. So the detection
wavelength set at 350 nm at 0 to 24 min and at 220 nm at
24 to 37 min was chosen in the assay and suitable for the
simultaneous determination of the five active ingredients.
A mixture of methanol-water (70:30, v/v) was chosen as
the extraction solvent. A simple ultrasonic method was
used for sample preparation, which was timesaving and
may not lead to losses of the five active ingredients. The
sample clean-up steps to a minimum were a primary
concern; thus, the filtrate, after ultrasonication, was