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HomeSmall Molecule HPLCDetermination of Chlorogenic acid and Baicalin in Compounded Lonucerae Japonicae Flos using a Discovery® HS C18 Column

Determination of Chlorogenic acid and Baicalin in Compounded Lonucerae Japonicae Flos acc. Chinese Pharmacopeia using a Discovery® HS C18 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, cooling blood and detumescence. In this application, one Chinese pharmacopeia 2020 monograph method was validated using a Discovery® HS C18 HPLC column.

2-D chemical structures (bond line structure) of chlorogenic acid and baicalin used to measure analytes in compounded Lonucerae Japonicae Flos using a Discovery® HS C18

Figure 1. Chemical structures of chlorogenic acid and 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 MillexPTFE syringe filters prior to HPLC-UV analysis (Table 1).

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

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 using a Discovery® HS C18 HPLC column

Figure 2. Chromatogram of chlorogenic acid and baicalin standard solution.

Analysis of compounded Lonucerae Japonicae Flos using Discovery® HS C18 HPLC column

Figure 3. Chromatogram of compounded Lonucerae Japonicae Flos.

Separation of solvent blank on Discovery® HS C18 HPLC 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 using Discovery® HS C18 HPLC column

Figure 5. Chromatogram of chlorogenic acid and baicalin standard solution.

Chromatogram of compounded Lonucerae Japonicae Flos to analyze chlorogenic acid and baicalin analytes using Discovery® HS C18 HPLC column

Figure 6. Chromatogram of compounded Lonucerae Japonicae Flos.

Separation of solvent blank on Discovery® HS C18 HPLC column

Figure 7. Chromatogram of solvent blank.

Table 3. Specificity data of chlorogenic acid and baicalin

Specificity and repeatability – chlorogenic acid and baicalin

Table 4. Specificity data of chlorogenic acid and baicalin
Table 5. Standard repeatability (50 μg/mL) - chlorogenic acid
Table 6. Standard repeatability (100 μg/mL) - baicalin

Calibration

Calibration data – chlorogenic acid

Table 7. Linear range of chlorogenic acid
Calibration curve of chlorogenic acid used to determine the analyte in compounded Lonucerae Japonicae Flos with Discovery® HS C18 HPLC column

Figure 8. Calibration curve of chlorogenic acid.

Table 8. LOD & LOQ of chlorogenic acid

Calibration data – baicalin

Table 9. Linear range of baicalin
Calibration curve of baicalin used to determine the analyte in compounded Lonucerae Japonicae Flos with Discovery® HS C18 HPLC column

Figure 9. Calibration curve of baicalin

Table 10. LOD & LOQ of baicalin

Conclusion

The Chinese pharmacopeia 2020 monograph method was reproduced using a Discovery® HS C18 HPLC column. For the shown method, the limit of detection (LOD) and the limit of quantitation (LOQ) were found to be 0.07 µg/mL and 0.21 µg/mL, respectively, for chlorogenicacid, while the limit of detection (LOD) and the limit of quantitation (LOQ) were 0.27 µg/mL and 0.82 µg/mL, respectively, for baicalin. The method can be used for determination of chlorogenicacid and baicalin in compounded Lonucerae Japonicae Flos according to the Chinese pharmacopoeia 2020.

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