RP-HPLC-UV Method for Simultaneous Determination of Nine Isoflavonoids in Huangqi-Gegen Herbal Pair

A new simple and efficient RP-HPLC-UV method was developed for the simultaneous analysis of nine isoflavonoids in Huangqi-Gegen herbal pair. Effective chromatographic separation of these components was achieved on a Kromasil C18 column (4.6×250 mm, i.d.; 5 μm) with gradient elution of methanol and 0.2% formic acid aqueous solution at a flow rate of 1.0 mL/min in 60 min. Detection was performed at 250nm. The method was proved to be linear in the ranges of 81.28-406.4, 670.4-3352, 136.96-684.8, 33.376-166.88, 22.848-114.24, 10.17650.88, 31.04-155.2, 44.544-222.72 and 26.56-132.8 ng for the nine isoflavonoid: 3'-hydroxypuerarin, puerarin, daidzin, calycosin-7-O-β-D-glucoside, genistin, ononin, daidzein, calycosin and formononetin, respectively. The average recoveries were 99.27, 102.38, 98.46, 103.06, 101.29, 99.71, 102.28, 97.89 and 100.78 % respectively; RSD was 1.79, 2.02, 1.44, 1.37, 1.26, 1.81, 1.29, 0.97 and 1.77 % respectively. The developed method was successfully applied for the quantitative analysis of constituents in huangqi gegen herbal pair.


Materials
Test preparation: According to the proportion of Huangqi-Gegen decoction prescription, weighed 20 g of huangqi pieces, 10 g of gegen pieces, add 10 times of water, reflux extraction two times, each time for 1 h, the combined extracts were concentrated under reduced pressure and constant volume at 250 mL, shake and filtrate (0.45 μm).

Chromatographic conditions and system suitability:
Kromasil 100-5 C 18 (4.6 × 250 mm, id., 5 μm) column, pre-column (Security Guard, C 18 , 4.0×3.0mm, Phenomenex); using methanol as mobile phase A, 0.1% citric acid solution as mobile phase B, gradient elution (Table 2); flow rate of 1.0 mL/min, column temperature was 25°C, the detection wavelength was 250 nm, the injection volume was 10 μL.Theoretical plates of the test solution chromatogram with each corresponding absorption peaks were not less than 7000 and peaks separation greater than 2.0.
Methodological studies: Limits of detection and quantification: The Limits of Detection (LOD) and Quantification (LOQ) for each studied component were defined as the lowest concentration of component in the diluted standard solution producing a signal-to-noise ratio (S/N) of 3:1 and 10:1, respectively.

Calibration curves:
Working solutions containing all nine references were prepared by diluting the stock  solution to proper concentration in order to draw calibration curves.Each calibration curve contained six different concentrations and was performed in triplicate: Precision tests: Precision drawing reference solution, into the liquid chromatograph, six replicates.
Repeatability tests: Precision drawing test solution into the liquid chromatograph, repeated six times.

Stability tests:
Stability was tested with a sample solution at room temperature and analyzed at 0, 2, 4, 8, 12 and 24 h.

Recovery tests:
According to the proportion of Huangqi-Gegen decoction prescription, weigh the same batch of Huangqi Pieces 2 g, Pueraria Herbal 1 g, 6 copies.Accurately weighed, add precision to a considerable amount of the reference solution, the solvent evaporated, with the same approach to measuring, calculating recoveries.

Application:
The optimized method was applied to analyse the contents of nine components in Huangqigegen decoction, the samples solutions were prepared as described above.The content HPLC analysis of each compound analysed was calculated from the corresponding calibration curve.

RESULTS AND DISCUSSION
Methodological studies: Limits of detection and quantification, Calibration curves: All calibration curves were constructed from peak areas of the reference standards versus their concentrations (Table 3 and 4).Precision tests: resulting peak area RSD was 0.94, 1.13, 0.77, 1.81, 0.93, 1.31, 0.98, 1.04 and 1.17% (1-9), respectively.The results showed good precision instruments.

Application:
The results are shown in Fig. 1 and Table 5.

Table 1 :
Structure of nine isoflavones in Huangqi-Gegen herbal pair

Table 2 :
The mobile phase gradient elution procedure

Table 3 :
The linear relationship of isoflavones No y = ax+b (y = Peak area, x = Injection Quality, ng)

Table 5 :
The linear relationship of isoflavones