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Determination of Chlorogenic acid and Baicalin in compounded Lonucerae Japonicae Flos with Purospher® STAR RP-18e

Chinese Pharmacopeia method
Dean Duan
MilliporeSigma
China application lab
Shanghai, China

Compounded Lonucerae Japonicae Flos is a kind of Traditional Chinese Medicine (TCM). It has the effects of heat-clearing and detoxicating, also of cooling blood and detumescence.

In this application, one Chinese pharmacopeia 2020 monograph method was validated using a Purospher® STAR RP-18 endcapped HPLC column. The samples were first ground, and the active ingredients chlorogenic acid and baicalin were extracted with an aqueous methanol solution. All samples were filtered through Millex® PTFE syringe filters prior to HPLC-UV analysis.

The limit of detection (LOD) and the limit of quantitation (LOQ) were found to be 0.06 µg/mL and 0.017 µg/kg, respectively, for chlorogenic acid, while the limit of detection (LOD) and the limit of quantitation (LOQ) were 0.29 µg/mL and 0.87 µg/kg, respectively, for baicalin. The method can be used for determination of chlorogenic acid and baicalin in compounded Lonucerae Japonicae Flos.

2-D chemical structures (bond line structure) of chlorogenic acid and baicalin used to measure analytes in compounded Lonucerae Japonicae Flos with Purospher® STAR RP-18e

Figure 1.Chemical structures of chlorogenic acid and baicalin

Determination of chlorogenic acid and baicalin in compounded Lonucerae Japonicae Flos

Table 1.HPLC conditions
Table 2.Mobile phase gradient table

Chromatograms at 278 nm for chlorogenic acid

Analysis of chlorogenic acid and baicalin standards used to determine the analytes in compounded Lonucerae Japonicae Flos with Purospher® STAR RP-18e column

Figure 2.Chromatogram of chlorogenic acid and baicalin standard solution

Analysis of compounded Lonucerae Japonicae Flos using Purospher® STAR RP-18e column

Figure 3.Chromatogram of compounded Lonucerae Japonicae Flos

Separation of solvent blank on Purospher® STAR RP-18e column

Figure 4.Chromatogram of solvent blank

Table 3.Specificity data of chlorogenic acid and baicalin at 326 nm

Chromatograms at 326 nm for baicalin

Chromatographic separation of chlorogenic acid and baicalin standards in compounded Lonucerae Japonicae Flos with Purospher® STAR RP-18e column

Figure 5.Chromatogram of chlorogenic acid and baicalin standard solution

Separation of solvent blank on Purospher® STAR RP-18e column

Figure 6.Chromatogram of compounded Lonucerae Japonicae Flos

Separation of solvent blank on Purospher® STAR RP-18e column

Figure 7.Chromatogram of solvent blank

Table 4.Specificity data of chlorogenic acid and baicalin at 326 nm

Specificity and repeatability – chlorogenic acid and baicalin

Table 5. Specificity: Inject reference solution and determine the retention time of desired analyte in presence of other component like impurities.
Specificity data of chlorogenic acid and baicalin
Table 6.Standard Repeatability (50 µg/mL) - chlorogenic acid
Table 7.Standard repeatability (100 µg/mL) - baicalin

Calibration data – chlorogenic acid

Table 8.Linear range of chlorogenic acid
Calibration curve of chlorogenic acid used to determine the analyte in compounded Lonucerae Japonicae Flos with Purospher® STAR RP-18e column

Figure 8.Calibration curve of chlorogenic acid

Table 9.LOD and LOQ of chlorogenic acid

Calibration data – baicalin

Table 10.Linear range of baicalin
Calibration curve of baicalin used to determine the analyte in compounded Lonucerae Japonicae Flos with Purospher® STAR RP-18e column

Figure 9.Calibration curve of baicalin

Table 11.LOD and LOQ of baicalin

Conclusion

The Chinese pharmacopeia 2020 monograph method was validated using a using a Purospher® STAR RP-18 endcapped HPLC column. The method can be used for determination of chlorogenic acid and baicalin in compounded Lonucerae Japonicae Flos.

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