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

Determination of Chlorogenic acid and Baicalin in compounded Lonucerae Japonicae Flos acc. Chinese Pharmacopeia using a Purospher® STAR RP-18e Column

Dean Duan

Merck, Application Scientist, China application lab, Shanghai, China

Introduction

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 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 used to measure analytes in compounded Lonucerae Japonicae Flos with Purospher® STAR RP-18e

Figure 1a.Chemical structures of chlorogenic acid.

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

Figure 1b.Chemical structures of baicalin.

Experimental 

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 (Table 1).

Results

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.

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.

Specificity and Repeatability

Calibration

Calibration data – 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.

Calibration data – 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.

Conclusion

The Chinese pharmacopeia 2020 monograph method was reproduced using a using a Purospher® STAR RP-18 endcapped HPLC column. The limit of detection (LOD) and the limit of quantitation (LOQ) were found to be 0.06 μg/mL and 0.17 μg/mL, 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/mL, respectively, for baicalin. The method can be used for determination of chlorogenic acid and baicalin in compounded Lonucerae Japonicae Flos according to the Chinese pharmacopoeia 2020.

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