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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

MilliporeSigma, 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).

Experimental Conditions

Column Used:

Discovery® HS C18 25 cm x 4.6 mm, 5 µm (568523-U)

 

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

 

Gradient:

See Table 2

 

Flow Rate: 

1.0 mL/min

 

Injection Volume:

10 µL

 

Temp:

30 °C

 

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.
Shake well, filter through a 0.45 µm filter membrane.

Pressure drop:

117 bar (1680 psi)

 

Table 1. HPLC conditions

Time
(min)

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

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.

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

Table 3. Specificity data of chlorogenic acid and baicalin

Specificity and repeatability – chlorogenic acid and baicalin

NoCompoundRetention Time (min)Area (%)Tailing FactorRRT
1Chlorogenic acid14.068.41.01.0
2Baicalin37.963.01.02.7
Table 4. Specificity data of chlorogenic acid and baicalin
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

Table 5. Standard repeatability (50 μg/mL) - chlorogenic acid
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

Table 6. Standard repeatability (100 μg/mL) - baicalin

Calibration

Calibration data – chlorogenic acid

Linearity (area mAU*min), Chlorogenic acid
Conc. (µg/mL)Mean Area
0.051520
0.101968
0.205000
0.514644
1.028727
2.058607
5.0137723
10.0295041
20.0603830
50.01547068
60.01837389
R20.9999
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.

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

Table 8. LOD & LOQ of chlorogenic acid

Calibration data – baicalin

Linearity (area mAU*min), Baicalin
Conc. (µg/mL)Mean Area
0.11556
0.22490
0.45549
1.025569
2.051460
4.0111979
10.0272915
20.0595962
40.01227176
R20.9994
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

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

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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