Determination of Chlorogenic acid and Baicalin in Compounded Lonucerae Japonicae Flos acc. Chinese Pharmacopeia using a Discovery® HS C18 Column
Dean Duan
MilliporeSigma, Application Scientist, China application lab, Shanghai, China
Section Overview
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.
Figure 1. Chemical structures of chlorogenic acid and baicalin.
Experimental Conditions | ||||
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Column Used: | Discovery® HS C18 25 cm x 4.6 mm, 5 µm (568523-U) |
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Detection: | Waters e2695, 278 nm for chlorogenic acid, 326 nm for baicalin | |||
Mobile phase A: | 0.3% H3PO4 aqueous solution | |||
Mobile phase B: | Acetonitrile gradient grade |
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Gradient: | See Table 2 |
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Flow Rate: | 1.0 mL/min |
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Injection Volume: | 10 µL |
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Temp: | 30 °C |
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Standard | Dissolve appropriate amount of chlorogenic acid and baicalin in 50% methanol aqueous solution to obtain a solution containing 50 µg/mL of chlorogenic acid and 100 µg/mL of baicalin. | |||
sample: | Take appropriate amount of medicine to grind to powder, weigh 2 g of powder into an Erlenmeyer flask with stopper, add into it 25 mL 50% methanol aqueous solution and weigh. Sonicate the sample at 200 W, 40 Hz for 30 minutes. Cool it down to room temperature, weigh, make up the weight with 50% methanol aqueous solution. | |||
Pressure drop: | 117 bar (1680 psi) |
|
Time | A | B |
---|---|---|
0.01 | 90 | 10 |
13 | 90 | 10 |
35 | 71 | 29 |
37 | 71 | 29 |
42 | 75 | 25 |
42.1 | 90 | 10 |
52 | 90 | 10 |
Figure 2. Chromatogram of chlorogenic acid and baicalin standard solution.
Figure 3. Chromatogram of compounded Lonucerae Japonicae Flos.
Figure 4. Chromatogram of solvent blank.
Chromatograms at 326 nm for baicalin
Figure 5. Chromatogram of chlorogenic acid and baicalin standard solution.
Figure 6. Chromatogram of compounded Lonucerae Japonicae Flos.
Figure 7. Chromatogram of solvent blank.
No | Compound | Retention Time (min) | Resolution | Theoretical Plates | Tailing Factor |
---|---|---|---|---|---|
1 | Chlorogenic acid | 14.0 |
| 17933
| 1.0 |
2 | Baicalin | 37.9 | 76.2 | 504124 | 1.0 |
Specificity and repeatability – chlorogenic acid and baicalin
No | Compound | Retention Time (min) | Area (%) | Tailing Factor | RRT |
1 | Chlorogenic acid | 14.0 | 68.4 | 1.0 | 1.0 |
2 | Baicalin | 37.9 | 63.0 | 1.0 | 2.7 |
Standard Repeatability (50 µg/mL), Chlorogenic acid - Area (mAU*min) | |
---|---|
STD 1 | 1547068 |
STD 2 | 1545833 |
STD 3 | 1545193 |
STD 4 | 1551227 |
STD 5 | 1549974 |
Mean | 1547859 |
Standard Deviation | 2629.4 |
RSD (%) | 0.2 |
Standard Repeatability (100 µg/mL), Baicalin - Area (mAU*min) | |
---|---|
STD 1 | 1328157 |
STD 2 | 1325789 |
STD 3 | 1325269 |
STD 4 | 1329275 |
STD 5 | 1327018 |
Mean | 1327102 |
Standard Deviation | 1652.7 |
RSD (%) | 0.1 |
Calibration
Calibration data – chlorogenic acid
Linearity (area mAU*min), Chlorogenic acid | |
---|---|
Conc. (µg/mL) | Mean Area |
0.05 | 1520 |
0.10 | 1968 |
0.20 | 5000 |
0.5 | 14644 |
1.0 | 28727 |
2.0 | 58607 |
5.0 | 137723 |
10.0 | 295041 |
20.0 | 603830 |
50.0 | 1547068 |
60.0 | 1837389 |
R2 | 0.9999 |
Figure 8. Calibration curve of chlorogenic acid.
LOD & LOQ, Chlorogenic acid | |
---|---|
Conc. (µg/mL) | Mean Area (mAU*min) |
0.05 | 1520 |
0.10 | 1968 |
0.20 | 5000 |
0.50 | 14644 |
STEYEX | 627.1 |
Slope | 30189.3 |
LOD (ppm) | 0.07 |
LOQ (ppm) | 0.21 |
Calibration data – baicalin
Linearity (area mAU*min), Baicalin | |
---|---|
Conc. (µg/mL) | Mean Area |
0.1 | 1556 |
0.2 | 2490 |
0.4 | 5549 |
1.0 | 25569 |
2.0 | 51460 |
4.0 | 111979 |
10.0 | 272915 |
20.0 | 595962 |
40.0 | 1227176 |
R2 | 0.9994 |
Figure 9. Calibration curve of baicalin
LOD & LOQ, Baicalin | |
---|---|
Conc. (µg/mL) | Mean Area (mAU*min) |
0.10 | 1556 |
0.20 | 2490 |
0.40 | 5549 |
1.00 | 25569 |
STEYEX | 2275.3 |
Slope | 27686.7 |
LOD (ppm) | 0.27 |
LOQ (ppm) | 0.82 |
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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