Determination of Chlorogenic acid and Baicalin in compounded Lonucerae Japonicae Flos with Purospher^® STAR RP-18e

Chinese Pharmacopeia method
Dean Duan
MilliporeSigma
China application lab
Shanghai, China

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 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.

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.

Figure 1. Chemical structures of chlorogenic acid and baicalin

Determination of chlorogenic acid and baicalin in compounded Lonucerae Japonicae Flos

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

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

Table 3. Specificity data of chlorogenic acid and baicalin at 326 nm

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

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 4. Specificity data of chlorogenic acid and baicalin at 326 nm

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 – chlorogenic acid and baicalin

Table 5. Specificity: Inject reference solution and determine the retention time of desired analyte in presence of other component like impurities.
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 6. Standard Repeatability (50 µg/mL) - chlorogenic acid

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 7. Standard repeatability (100 µg/mL) - baicalin

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 data – chlorogenic acid

Table 8. 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

Figure 8. Calibration curve of chlorogenic acid

Table 9. 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 10. 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

Figure 9. Calibration curve of baicalin

Table 11. 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 validated using a using a Purospher^® STAR RP-18 endcapped HPLC column. The method can be used for determination of chlorogenic acid and baicalin in compounded Lonucerae Japonicae Flos.