Determination of five protopanaxadiol ginsenosides in ginseng by solid-phase extraction-ultra performance liquid chromatography

doi:10.3724/SP.J.1123.2019.09003

Abstract

Abstract:

A new method based on solid-phase extraction-ultraperformance liquid chromatography (SPE-UPLC) was developed for the determination of five protopanaxadiol ginsenosides in ginseng. The ginsenosides were extracted from ground ginseng samples using water-saturated n-butanol and purified on a hydrophilic solid-phase extraction column. Chromatographic separation was achieved on an ACQUITY UPLC BEH Shield RP18 column (100 mm×2.1 mm, 1.7 μm) by linear gradient elution using an acetonitrile/water mobile phase. Five protopanaxadiol ginsenosides were detected by a photodiode array detector, and they showed a strong positive linear correlation (r²>0.999) in the range of 5-500 μg/mL. In addition, the instrument precision ranged between 0.95% and 2.62% (n=6), with the sample stability between 0.90% and 2.15% (n=8) within 22 h. Intra- and inter-day repeatabilities were 5.35%-6.47% (n=6) and 5.56%-6.34% (n=8), respectively. Sample recoveries and the corresponding relative standard deviations (RSDs) were 87.16%-101.92% and 1.54%-4.01% (n=6), respectively. Hydrophilic chromatography materials were used in SPE, and the extract was directly loaded and purified without pretreatment. Besides, with the use of UPLC, the analysis time was greatly shortened. The developed method is simple and rapid, with high throughput, thus being suitable for the quantitative analysis of the five protopanaxadiol ginsenosides in ginsengs.

Key words: hydrophilic interaction chromatography (HILIC), solid-phase extraction (SPE), ultraperformance liquid chromatography (UPLC), ginsenoside, ginseng

SONG Chunying, ZHANG Huarong, GUO Zhimou, YAN Jingyu, JIN Gaowa, LIANG Xinmiao. Determination of five protopanaxadiol ginsenosides in ginseng by solid-phase extraction-ultra performance liquid chromatography[J]. Chinese Journal of Chromatography, 2020, 38(5): 547-553.

Figures/Tables 9

Fig. 1 Chemical structures of the five protopanaxadiol ginsenosides

Fig. 2 Chromatograms of ginseng extract with different SPE eluents SPE eluent: 2 mL ethanol/water; ethanol volume percentage: (a) 80%, (b) 70%, (c) 60%, (d) 50%.HPLC mobile phase A: ACN; B: H2O; gradient elution time: 0-10 min, 19%A-19%A; 10-16 min, 19%A-29%A; 16-20 min, 29%A-29%A; 20-28 min, 29%A-40%A; 28-34 min, 40%A-90%A; 34-40 min, 90%A-100%A; flow rate: 0.3 mL/min; detection wavelength: 203 nm; column temperature: 30 ℃.Peak identifications: 1. Rb1; 2. Rc; 3. Rb2; 4. Rb3; 5. Rd.

Fig. 3 Chromatograms of ginseng extract with different SPE eluent volumes SPE eluent: 70% (v/v) ethanol/water; volume: (a) 1st 1 mL, (b) 2nd 1 mL, (c) 3rd 1 mL, (d) 4th 1 mL. The chromatographic separation conditions and the peak identifications were the same as those in Fig. 2.

Fig. 4 Effect of the SPE processes on the ginseng extract a. without SPE; b. loading effluent of SPE; c. cleaning effluent of SPE; d. eluent of SPE.The chromatographic separation conditions and the peak identifications were the same as those in Fig. 2.

Fig. 5 Chromatograms of (a) reference standards of ginsenosides and (b) ginseng extract

Table 1 Linear equations, linear ranges, correlation efficients (r2), LODs, and LOQs of the five reference standards

Target	Linear equation	Linear range/ (μg/mL)	r²	LOD/ (μg/g)	LOQ/ (μg/g)
y: peak area; x: mass concentration of the target compound (μg/mL).
Rb1	y=9037.4x-21085	5-500	0.9994	1.51	5.04
Rc	y=9441.5x-25693	5-500	0.9996	1.37	4.57
Rb2	y=90443.5x-20783	5-500	0.9997	1.45	4.84
Rb3	y=9459.8x-39486	5-500	0.9993	1.41	4.70
Rd	y=8315.9x-53136	5-500	0.9997	2.56	8.54

Table 2 Precisions, stabilities, and repeatabilities of the method

Target	Precision (RSD/%, n=6)	Stability (RSD/%, n=8)	Repeatabilities
Target	Precision (RSD/%, n=6)	Stability (RSD/%, n=8)	Intra-day RSD/% (n=6)	Inter-day RSD/% (n=8)
Rb1	0.96	1.00	5.35	5.56
Rc	0.90	0.95	6.47	6.34
Rb2	0.92	0.98	6.23	6.34
Rb3	1.32	2.48	5.69	5.59
Rd	2.15	2.62	5.97	6.06

Table 3 Spiked recoveries of the five compounds in real samples (n=6)

Target	Background/ mg	Added/ mg	Found/ mg	Average recovery/%	RSD/ %
Rb1	0.14	0.19	0.32	95.27	3.19
Rc	0.10	0.12	0.20	91.31	1.65
Rb2	0.075	0.11	0.16	87.16	3.76
Rb3	0.012	0.015	0.025	94.87	4.02
Rd	0.065	0.072	0.14	101.92	1.54

Table 4 Contents of the five compounds in different ginseng samples

Sample	Contents/% (RSD/%, n=3)
Sample	Rb1	Rc	Rb2	Rb3	Rd
A	0.118	0.064	0.074	0.010	0.050
	(1.60)	(1.60)	(1.08)	(4.95)	(2.15)
B	0.100	0.048	0.055	0.008	0.049
	(2.92)	(1.85)	(3.04)	(3.51)	(1.74)
C	0.140	0.058	0.068	0.009	0.052
	(4.62)	(1.11)	(1.48)	(3.25)	(3.62)
D	0.140	0.059	0.070	0.010	0.057
	(1.32)	(0.75)	(0.64)	(0.92)	(0.38)
E	0.160	0.077	0.080	0.010	0.055
	(1.13)	(3.32)	(4.42)	(4.95)	(5.13)
F	0.210	0.096	0.120	0.021	0.056
	(0.98)	(1.12)	(1.26)	(0.96)	(0.80)
G	0.170	0.089	0.110	0.017	0.068
	(1.10)	(3.06)	(2.95)	(6.43)	(2.26)

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