Determination of Chlorogenic acid and Baicalin in Compounded Lonucerae Japonicae Flos using a Discovery^® HS C18 Column

Dean Duan^{Chinese Pharmacopeia method, 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, cooling blood and detumescence. In this application, one Chinese pharmacopeia 2020 monograph method was validated using a Discovery^® HS C18 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 0.07 µg/mL and 0.021 µg/kg, respectively, for chlorogenic acid, while the limit of detection (LOD) and the limit of quantitation (LOQ) were 0.27 µg/mL and 0.82 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:	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 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 278 nm

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

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	18.7	1	chlorogenic acid	14.0
2	baicalin	20.5	2	baicalin	37.9

Specificity and repeatability – chlorogenic acid and baicalin

Table 5. Specificity data of chlorogenic acid and baicalin

Specificity: Inject reference solution and determine the retention time of desired analyte in presence of other component like impurities.
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

Table 6. Standard repeatability data of chlorogenic acid

Standard repeatability (50 µg/mL), chlorogenic acid
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 7. Standard Repeatability data of baicalin

Standard Repeatability (100 µg/mL), baicalin
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 data – chlorogenic acid

Table 8. Linear range of 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

Table 9. LOD & LOQ 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

Figure 8. Calibration curve of chlorogenic acid

Calibration data – baicalin

Table 10. Linear range of 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

Table 11. LOD & LOQ 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

Figure 9. Calibration curve of baicalin

Conclusion

The Chinese pharmacopeia 2020 monograph method was validated using a Discovery® HS C18 HPLC column. The method can be used for determination of chlorogenic acid and baicalin in compounded Lonucerae Japonicae Flos.