高效液相色谱紫外等吸收波长法快速测定秦皮中秦皮甲素和秦皮乙素

doi:10.3724/SP.J.1123.2023.03018

摘要/Abstract

摘要：

研究建立了一种快速、环保和节约对照品的秦皮化学成分含量测定方法。秦皮样品采用25%(v/v)乙醇水溶液超声提取5 min。样品提取溶液采用Poroshell 120 EC-C₁₈色谱柱(50 mm×4.6 mm, 2.7 μm)进行分析,0.1%甲酸水溶液-乙腈(94∶6, v/v)等度洗脱,流速1.5 mL/min。采用紫外分光光度仪对等浓度的秦皮甲素和秦皮乙素进行紫外光谱扫描,初步筛选得到2个对照品的等吸收波长,并用高效液相色谱仪对筛选得到的等吸收波长进行确证,从而获得2个对照品的等吸收检测波长341 nm。新建立的高效液相色谱紫外等吸收波长法的方法验证结果显示,秦皮甲素和秦皮乙素在各自的浓度范围内具有良好的线性关系(r=1.0000),检出限和定量限分别为1.5 μmol/L和3.0 μmol/L,平均加标回收率分别为99.0%(RSD=1.4%)和97.5%(RSD=0.9%)。通过比较高效液相色谱紫外等吸收波长法和传统高效液相色谱外标法对10批秦皮样品中秦皮甲素和秦皮乙素的含量测定结果,显示两种方法测定结果一致。此外还采用t检验对该方法与《中国药典》2020版方法的含量测定结果进行比较,对比结果表明两种测定方法无显著性差异(P>0.05)。该方法耗时10 min,有害溶剂消耗0.5 mL,只使用1个对照品,具有快速、简便、环保和对照品消耗少的特点,适用于秦皮中秦皮甲素和秦皮乙素的含量测定,为秦皮质量评价的技术提升提供了依据。

关键词: 高效液相色谱, 等吸收波长, 核壳型色谱柱, 秦皮甲素, 秦皮乙素

Abstract:

Fraxini Cortex is a traditional Chinese herbal medicine that has been used for thousands of years to treat dampness-heat diarrhea, dysentery, red or white vaginal discharge, painful swelling or redness of the eyes, and nebula. It contains various chemical components, including coumarins, iridoids, phenolic acids, and flavonoids. Coumarins are important active ingredients in Fraxini Cortex and have antibacterial, anti-inflammatory, antioxidant, antitumor, and antiviral activities. Aesculin and aesculetin are two major coumarin components of Fraxini Cortex that are widely used in its quality evaluation. Previous HPLC methods for determination of aesculin and aesculetin present several limitations, such as long analysis times and high solvent and reference compound consumption.

In this study, a rapid, eco-friendly and cost saving HPLC method for the determination of aesculin and aesculetin in Fraxini Cortex was established by using the core-shell column and equal absorption wavelength (EAW). Different factors influencing the extraction process, such as the extraction solvent, temperature, and time, were assessed to obtain the optimal extraction conditions. The results showed that Fraxini Cortex samples could be well extracted by ultrasonic extraction for 5 min with a 25% ethanol aqueous solution. A core-shell column was used, and different mobile phases and flow rates were investigated to obtain the best rapid-HPLC separation conditions. The optimized HPLC conditions were as follows: a Poroshell 120 EC-C₁₈ column (50 mm×4.6 mm, 2.7 μm), acetonitrile-0.1% formic acid aqueous solution (6∶94, v/v) as the eluent, a flow rate of 1.5 mL/min, and a column temperature of 25 ℃.

The EAW of aesculin and aesculetin was a key factor in their determination using a single reference compound. EAW selection was performed in two steps. First, the UV spectra of two equimolar concentrations of the reference compounds (aesculin and aesculetin) were compared to determine the EAW of the two analytes. The EAW results were then verified by the HPLC analysis of the reference compound solutions. The final EAW of aesculin and aesculetin was 341 nm. The determination of aesculin and aesculetin using only one reference compound (i. e., aesculin) was achieved by HPLC-UV at this EAW.

The newly developed HPLC method revealed a good linear relationship between the two target analytes (r=1.0000). The limits of detection (LODs) and limits of quantification (LOQs) were 1.5 μmol/L and 3.0 μmol/L, respectively, and the average recoveries of aesculin and aesculetin were 99.0% and 97.5%. The stabilities of the sample solutions were examined, and the two analytes demonstrated good stability for 24 h. The contents of the target analytes in 10 batches of Fraxini Cortex were determined using the proposed EAW method and the classic external standard method (ESM), and comparable concentrations were obtained. The contents of aesculin and aesculetin in the 10 batches of Fraxini Cortex were 0.26%-2.80% and 0.11%-1.47%, respectively. A t-test was conducted to compare the results of the proposed EAW technique with those obtained via the method reported in the Chinese Pharmacopoeia, and no significant difference between the two assay methods was noted (P>0.05). Comparison of the newly established EAW method with those reported in the literature revealed that our method required only 10 min to complete and used as little as 0.5 mL of the solvent and only one standard. Therefore, the developed EAW method is a rapid, simple, eco-friendly, and cost-effective analytical method that is suitable for the determination of aesculin and aesculetin in Fraxini Cortex and its related products. The proposed technique is an improved method for determining aesculin and aesculetin and contributes to the enhancement of the quality evaluation of Fraxini Cortex.

Key words: high performance liquid chromatography (HPLC), equal absorption wavelength (EAW), core-shell chromatographic column, aesculin, aesculetin

中图分类号:

O658

钱正明, 吴梦奇, 谭国英, 金李玲, 李宁, 谢菊英. 高效液相色谱紫外等吸收波长法快速测定秦皮中秦皮甲素和秦皮乙素[J]. 色谱, 2023, 41(8): 690-697.

QIAN Zhengming, WU Mengqi, TAN Guoying, JIN Liling, LI Ning, XIE Juying. Rapid determination of aesculin and aesculetin in Fraxini Cortex by high performance liquid chromatography-ultraviolet at equal absorption wavelength[J]. Chinese Journal of Chromatography, 2023, 41(8): 690-697.

图/表 8

图1 不同提取条件对秦皮甲素、秦皮乙素含量的影响

Fig. 1 Effects of different extraction conditions on the contents of aesculin and aesculetin a. volume percentage of ethanol in aqueous solution; b. ratio of sample weight (g) to extraction solvent volume (mL); c. extraction time; d. extraction temperature.

图2 秦皮甲素和秦皮乙素的紫外光谱

Fig. 2 UV spectra of aesculin and aesculetin a. equivalent mass concentration; b. equivalent concentration.

图3 等浓度的秦皮甲素、秦皮乙素对照品溶液在不同检测波长下的峰面积

Fig. 3 Peak areas of aesculin and aesculetin with equivalent concentration at different wavelengths

表1 秦皮甲素和秦皮乙素的线性范围、线性方程、相关系数、检出限和定量限

Table 1 Linear ranges, linear equations, correlation coefficients (r), limits of detection (LODs) and limits of quantification (LOQs) of aesculin and aesculetin

Compound	Linear range/(μmol/L)	Linear equation	r	LOD/(μmol/L)	LOQ/(μmol/L)
Aesculin	14.8-443.8	Y=2.7820X+1.1814	1.0000	1.5	3.0
Aesculetin	14.8-444.6	Y=2.7499X+1.7877	1.0000	1.5	3.0

图4 (a)空白溶液、(b)对照品溶液和(c)样品溶液的色谱图

Fig. 4 Chromatograms of (a) blank, (b) reference and (c) sample solutions

表2 秦皮样品中秦皮甲素和秦皮乙素的含量

Table 2 Contents of aesculin and aesculetin in Fraxini Cortex samples %

No.	Aesculin (ESM)	Aesculetin
No.	Aesculin (ESM)	ESM	EAW
S1	1.22	0.11	0.11
S2	2.11	0.19	0.19
S3	1.86	0.22	0.22
S4	2.14	0.37	0.37
S5	0.77	1.48	1.47
S6	0.83	0.23	0.22
S7	2.80	0.12	0.11
S8	0.68	0.33	0.33
S9	0.41	0.40	0.40
S10	0.26	0.11	0.11

图5 采用(a)本方法和(b)《中国药典》方法时秦皮样品的色谱图

Fig. 5 Chromatograms of Fraxini Cortex sample by (a) this method and (b) the method in the Chinese Pharmacopoeia

表3 本方法与文献报道的比较

Table 3 Comparison of this method with methods in literature reports

No.	Sample extraction			HPLC separation		Harmful solvent	Number of reference compound	Ref.
No.	Method	Solvent	Time/min	Mobile phase	Time/min	Harmful solvent	Number of reference compound	Ref.
1	RE	50 mL methanol	60	20 mL acetonitrile-0.1% (v/v) phosphoric acid aqueous solution	20	50.0 mL methanol and 1.6 mL acetonitrile	2	[1]
2	UE	24 mL 94% (v/v) methanol aqueous solution	8.5	50.4 mL methanol-0.1% (v/v) formic acid aqueous solution	63	42.6 mL methanol	2	[10]
3	RE	50 mL methanol	60	60 mL acetonitrile-0.1% (v/v) phosphoric acid aqueous solution	60	50.0 mL methanol and 7.2 mL acetonitrile	2	[11]
4	UE	10 mL synthesized DES containing 20% (v/v) water	30	12.5 mL methanol-0.1% (v/v) formic acid aqueous solution	25	3.8 mL methanol	2	[12]
5	UE	10 mL 25% (v/v) ethanol aqueous solution	5	8.25 mL acetonitrile-0.1% (v/v) formic acid aqueous solution	5.5	0.5 mL acetonitrile	1	this method

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