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

Section Overview

• Introduction
• Experimental
• Results
• Conclusion
• Materials

Figure 1a. Chemical structures of chlorogenic acid.

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).

Table 1.HPLC conditions

Experimental Conditions
Column Used:	Purospher® STAR RP-18 endcapped 25 cm x 4.6 mm,5 µm (1.51456)
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 25 mL 50% methanol aqueous solution, weigh. Sonicate the sample at 200 W, 40 Hz for 30 minutes. Cool down to room temperature, weigh, make up the weight with 50% methanol aqueous solution. Shake well, filter with 0.45 µm filter membrane
Pressure drop:	133 bar (1920 psi)

Table 2.Mobile phase gradient table

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

Results

Chromatograms at 278 nm for chlorogenic acid

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.

Table 3.Chromatographic data of chlorogenic acid and baicalin

No	Compound	Retention Time (min)	Resolution	Theoretical Plates	Tailing Factor
1	Chlorogenic acid	16.9	--	8152	1.1
2	Baicalin	38.7	41.1	244019	1.0

Specificity and Repeatability

Table 4.Specificity data of chlorogenic acid and baicalin

No	Compound	Retention Time (min)	Area (%)	Tailing Factor	RRT
1	Chlorogenic acid	16.9	68.79	1.1	1.0
2	Baicalin	38.7	63.77	1.0	2.3

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

Standard repeatability (50 μg/mL) Chlorogenic acid - Area (mAU*min)
STD 1	1541148
STD 2	1541252
STD 3	1538214
STD 4	1533013
STD 5	1537813
Mean	1538288
Standard Deviation	3354.9
RSD (%)	0.2

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

Standard Repeatability (100 μg/mL) Baicalin - Area (mAU*min)
STD 1	1305402
STD 2	1304874
STD 3	1302815
STD 4	1297701
STD 5	1299822
Mean	1302123
Standard Deviation	3303.3
RSD (%)	0.3

Calibration

Calibration data – chlorogenic acid

Table 7.Linear range of chlorogenic acid

Conc. (µg/mL)	Mean area (mAU*min)
0.05	678
0.10	1181
0.20	4810
0.5	14051
1.0	27949
2.0	57633
5.0	134684
10.0	293549
20.0	600680
50.0	1541148
60.0	1833226
R2	0.9999

Figure 8. Calibration curve of chlorogenic acid.

Table 8.LOD and LOQ of chlorogenic acid

Conc. (µg/mL)	Mean area (mAU*min)
0.05	678
0.10	1181
0.20	4810
0.50	14051
STEYEX	517.8
Slope	30658.5
LOD (ppm)	0.06
LOQ (ppm)	0.17

Calibration data – baicalin

Table 9.Linear range of baicalin

Conc. (µg/mL)	Mean area (mAU*min)
0.1	1366
0.2	2032
0.4	4905
1.0	24111
2.0	49989
4.0	110286
10.0	267231
20.0	591397
40.0	1218414
R2	0.9992

Figure 9. Calibration curve of baicalin.

Table 10.LOD and LOQ of baicalin

Conc. (µg/mL)	Mean area (mAU*min)
0.10	1366
0.20	2032
0.40	4905
1.00	24111
STEYEX	2295.5
Slope	26338.6
LOD (ppm)	0.29
LOQ (ppm)	0.87

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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