固相萃取-超高效液相色谱法测定人参中5种原人参二醇型人参皂苷的含量

doi:10.3724/SP.J.1123.2019.09003

摘要/Abstract

摘要：

建立了固相萃取-超高效液相色谱测定人参中5种原人参二醇型人参皂苷的方法。人参药材经粉碎后通过水饱和正丁醇溶液进行超声提取，经过亲水作用固相萃取柱净化后，在ACQUITY UPLC BEH Shield RP18色谱柱（100 mm×2.1 mm，1.7 μm）上分离，利用乙腈/水作为流动相进行梯度洗脱，采用光电二极管阵列检测器检测。结果表明，5种原人参二醇型人参皂苷在5~500 μg/mL范围内具有很好的线性关系，相关系数均大于0.999。方法精密度的RSD值在0.95%~2.62%（n=6）之间，22 h内样品稳定性的RSD值在0.90%~2.15%（n=8）之间，日内和日间重复性的RSD值分别为5.35%~6.47%（n=6）和5.56%~6.34%（n=8）。方法的加标回收率在87.16%~101.92%之间，相对标准偏差在1.54%~4.01%（n=6）之间。所建立的方法采用亲水作用色谱模式的固相萃取材料，药材的提取液可直接作为固相萃取的上样溶液进行人参皂苷的富集和净化，并且超高效液相色谱大大缩短了分析时间。该方法简单快速、通量高、重现性好，适用于人参中5种原人参二醇型人参皂苷的定量分析。

关键词: 亲水作用色谱, 固相萃取, 超高效液相色谱, 人参皂苷, 人参

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

宋春颖, 张华蓉, 郭志谋, 闫竞宇, 金高娃, 梁鑫淼. 固相萃取-超高效液相色谱法测定人参中5种原人参二醇型人参皂苷的含量[J]. 色谱, 2020, 38(5): 547-553.

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.

图/表 9

图1 5种原人参二醇型人参皂苷的化学结构

Fig. 1 Chemical structures of the five protopanaxadiol ginsenosides

图2 不同SPE洗脱液条件下的人参提取物的色谱图

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.

图3 不同SPE洗脱体积条件下的人参提取物的色谱图

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.

图4 人参提取物经SPE处理的效果

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.

图5 （a）人参皂苷对照品与（b）人参样品提取物的色谱图

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

表1 5种对照品的线性方程、线性范围、r2、检出限和定量限

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

表2 方法的精密度、稳定性和重复性、

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

表3 5种化合物在实际样品中的加标回收率(n=6)

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

表4 不同人参样品中5种化合物的含量

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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