HPLC同时测定竹笋中8种酚酸类物质含量的方法研究及其应用

doi:10.12302/j.issn.1000-2006.202205047

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南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (3): 237-244.doi: 10.12302/j.issn.1000-2006.202205047

HPLC同时测定竹笋中8种酚酸类物质含量的方法研究及其应用

黄永健(), 荀航, 张保, 尤俊昊, 姚曦, 汤锋^*()

国际竹藤中心,国家林业和草原局/北京市共建竹藤科学与技术重点实验室,北京 100102

收稿日期:2022-05-30 修回日期:2022-06-20 出版日期:2024-05-30 发布日期:2024-06-14
作者简介:黄永健(huangyongjian@icbr.ac.cn)。
基金资助:
国际竹藤中心基本科研业务费专项资金项目(1632018007);国际竹藤中心基本科研业务费专项资金项目(1632019022)

Simultaneous determination of eight phenolic acids in bamboo shoots by HPLC and its applications

HUANG Yongjian(), XUN Hang, ZHANG Bao, YOU Junhao, YAO Xi, TANG Feng^*()

International Center for Bamboo and Rattan, Key Laboratory of National Forestry and Grassland Administration/Beijing for Bamboo and Rattan Science and Technology, Beijing 100102, China

Received:2022-05-30 Revised:2022-06-20 Online:2024-05-30 Published:2024-06-14

摘要/Abstract

摘要：

【目的】为实现竹笋中3,4-二羟基苯乙酸、对羟基苯甲酸、香草酸、咖啡酸、对羟基苯甲醛、丁香醛、阿魏酸、3-羟基肉桂酸等8种酚酸类化合物的快速检测,建立一种基于高效液相色谱-二极管阵列(HPLC-PDA)检测方法。【方法】采用Symmetry C18(4.6 mm×250 mm,5 μm)色谱柱,乙腈和乙酸-水(体积比0.5∶99.5)为流动相,在流速1.0 mL/min、进样量10 μL和柱温30 ℃的条件下梯度洗脱,检测波长275 nm。【结果】 8种酚酸的分离度均在1.5以上,在1.0~50.0 μg/mL的线性范围内,线性关系良好,相关系数R≥0.999 0,检出限[信号强度(S)与噪声强度(N)之比(S/N)为3]为0.12~0.69 μg/mL,定量限(S/N=10)为0.36~2.08 μg/mL,精密度、重复性、稳定性实验相对标准偏差(RSD)均小于5%,加标回收率为80.99%~129.12%,RSD均小于5%。应用该方法,检测了苦竹(Pleioblastus amarus)和苦篱竹(Arundinaria acerba)竹笋中酚酸类物质的含量,8种酚酸总量为(9.35±0.08)~(38.60±0.12) mg/kg;比较了自然生长和避光处理生长条件下苦竹笋中酚酸含量的变化,发现避光处理的竹笋中6种酚酸含量显著降低,总含量下降达46.2%。【结论】 HPLC-PDA检测方法简单方便,灵敏度高,准确可靠,适用于竹笋中酚酸类物质的测定;避光处理可以有效降低苦竹笋样品中酚酸类物质的含量。

关键词: 竹笋, 酚酸类物质, 高效液相色谱, 苦竹, 苦篱竹, 避光处理

Abstract:

【Objective】 Phenolic acids are present in almost all plant-derived foods, and are associated with the organoleptic and nutritional properties of foods. A high-performance liquid chromatography-photo-diode array (HPLC-PDA) method was established for the simultaneous detection of eight phenolic acids in bamboo shoots: 3,4-dihydroxyphenylacetic acid, 4-hydroxybenzoic acid, vanillic acid, caffeic acid, 4-hydroxybenzaldehyde, syringaldehyde, ferulic acid and 3-hydroxycinnamic acid. 【Method】 Eight phenolic acids were separated on a Symmetry C18 column (4.6 mm×250 mm, 5 μm). Acetonitrile (phase A) and 0.5% acetic acid solution (phase B) were used as the mobile phase, the injection volume was 10 μL, and the detection wavelength was 275 nm. The analysis was performed at 30 ℃ with a flow rate of 1.0 mL/min. 【Result】 Eight phenolic acids were successfully separated (resolution>1.5), and had a good linear relationship (R ≥ 0.999 0) with a linear range of 1.0-50.0 μg/mL. The detection limits (S/N=3) were 0.12-0.69 μg/mL and the quantification limits (S/N=10) were 0.36-2.08 μg/mL. Precision, repeatability, and stability tests yielded relative standard deviations (RSD) of <5%. Additionally, the recoveries of phenolic acids were 80.99%-129.12%, and the RSD values associated with the recoveries were also <5%. The phenolic acid contents in the actual samples of Pleioblastus amarus bamboo shoots and Arundinaria acerba bamboo shoots were determined using an established method. The contents of eight phenolic acids in the actual bamboo shoots samples varied from (9.35±0.08) to (38.60±0.12) mg/kg. A comparative analysis of phenolic acids in P. amarus bamboo shoots grown under a shading treatment and normal conditions was conducted based on this method. Compared to the shoots grown under normal conditions, the contents of six phenolic acids in P. amarus bamboo shoots grown under the shading treatment were lower, and the content of total detected phenolic acids decreased by 46.2%.【Conclusion】 The method developed here is simple, sensitive, accurate and reliable, and is suitable for determining phenolic acids in bamboo shoots. The results showed that a shading treatment downregulated the accumulation of phenolic acids in P. amarus bamboo shoots.

Key words: bamboo shoots, phenolic acids, HPLC, Pleioblastus amarus, Arundinaria acerba, shading treatment

中图分类号:

S718

黄永健,荀航,张保,等. HPLC同时测定竹笋中8种酚酸类物质含量的方法研究及其应用[J]. 南京林业大学学报（自然科学版）, 2024, 48(3): 237-244.

HUANG Yongjian, XUN Hang, ZHANG Bao, YOU Junhao, YAO Xi, TANG Feng. Simultaneous determination of eight phenolic acids in bamboo shoots by HPLC and its applications[J].Journal of Nanjing Forestry University (Natural Science Edition), 2024, 48(3): 237-244.DOI: 10.12302/j.issn.1000-2006.202205047.

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化合物 compound	分离度 resolution
化合物 compound	甲醇-水 methanol-water	甲醇-0.5%乙酸水 methanol-0.5% acetic acid	乙腈-水 acetonitrile-water	乙腈-0.5%乙酸水 acetonitrile-0.5% acetic acid
3,4-二羟基苯乙酸 3,4-dihydroxyphenylacetic acid	—	—	—	—
对羟基苯甲酸 4-hydroxybenzoic acid	5.19	4.71	4.19	3.81
香草酸 vanillic acid	2.11	2.33	3.87	4.28
咖啡酸 caffeic acid	0.60	0.75	1.45	1.56
对羟基苯甲醛 4-hydroxybenzaldehyde	1.12	1.10	1.79	1.82
丁香醛 syringaldehyde	2.05	2.06	9.84	9.85
阿魏酸 ferulic acid	3.22	3.00	3.44	3.69
3-羟基肉桂酸 3-hydroxycinnamic acid	2.81	2.68	5.60	5.87

化合物 compound	线性方程 linear equation	R	线性范围/(μg·mL^-1) linear range	检出限/(μg·mL^-1) LOD	定量限/(μg·mL^-1) LOQ
3,4-二羟基苯乙酸 3,4-dihydroxyphenylacetic acid	y=8 047.8x+1 350.8	0.999 5	1.0~50.0	0.56	1.69
对羟基苯甲酸 4-hydroxybenzoic acid	y=27 830x-9 841.5	0.999 3	1.0~50.0	0.12	0.36
香草酸 vanillic acid	y=12 325x+12 528	0.999 7	1.0~50.0	0.69	2.08
咖啡酸 caffeic acid	y=22 750x-14 252	0.999 1	1.0~50.0	0.66	1.98
对羟基苯甲醛 4-hydroxybenzaldehyde	y=62 943x+14 970	0.999 4	1.0~50.0	0.13	0.40
丁香醛 syringaldehyde	y=30 447x+31 087	0.999 4	1.0~50.0	0.27	0.82
阿魏酸 ferulic acid	y=9 291.6x+3 866.3	0.999 6	1.0~50.0	0.31	0.94
3-羟基肉桂酸 3-hydroxycinnamic acid	y=63 965x+50 228	0.999 0	1.0~50.0	0.12	0.36

化合物 compound	相对标准偏差/%RSD			化合物 compound	相对标准偏差/%RSD
化合物 compound	精密度 precision	重复性 repeatability	稳定性 stability	化合物 compound	精密度 precision	重复性 repeatability	稳定性 stability
3,4-二羟基苯乙酸 3,4-dihydroxyphenylacetic acid	2.14	—	1.51	对羟基苯甲醛 4-hydroxybenzaldehyde	2.51	1.18	2.95
对羟基苯甲酸 4-hydroxybenzoic acid	0.44	4.18	2.93	丁香醛 syringaldehyde	1.10	4.15	3.15
香草酸 vanillic acid	1.88	4.13	2.45	阿魏酸 ferulic acid	1.15	4.38	4.57
咖啡酸 caffeic acid	1.86	2.75	4.32	3-羟基肉桂酸 3-hydroxycinnamic acid	0.46	4.09	4.90

化合物 compounds	样品含量/ (mg·kg^-1) sample content	添加含量/ (mg·kg^-1) additive content	检测含量/ (mg·kg^-1) detection content	添加回收率/% spiked recovery	相对标准偏差/% RSD	化合物 compounds	样品含量/ (mg·kg^-1) sample content	添加含量/ (mg·kg^-1) additive content	检测含量/ (mg·kg^-1) detection content	添加回收率/% spiked recovery	相对标准偏差/% RSD
3,4-二羟基苯乙酸 3,4-dihydroxyphenylacetic acid		1.00	1.25	124.87	4.36	对羟基苯甲醛 4-hydroxybenzaldehyde		1.00	1.21	108.30	4.28
	—	2.00	2.07	103.70	2.63		0.12	2.00	2.30	108.65	4.59
		4.00	4.36	109.05	3.15			4.00	3.76	90.83	4.76
对羟基苯甲酸 4-hydroxybenzoic acid		1.00	1.85	109.98	2.32	丁香醛syringaldehyde		1.00	4.48	100.12	4.22
	0.75	2.00	2.88	106.49	3.78		3.48	2.00	5.29	90.16	2.15
		4.00	5.82	126.69	2.49			4.00	6.95	86.58	4.63
香草酸 vanillic acid		1.00	2.98	80.99	3.44	阿魏酸 ferulic acid		1.00	2.09	110.36	2.44
	2.16	2.00	3.99	90.80	4.41		0.99	2.00	3.10	105.58	4.16
		4.00	5.54	84.30	3.41			4.00	5.72	118.14	2.05
咖啡酸 caffeic acid		1.00	2.14	82.56	4.73	3-羟基肉桂酸 3-hydroxycinnamic acid		1.00	1.94	129.12	1.31
	1.31	2.00	3.15	91.64	3.92		0.64	2.00	2.50	92.86	4.03
		4.00	5.02	92.67	1.20			4.00	4.83	104.63	4.29

化合物 compound	竹笋中酚酸含量/(mg·kg^-1) content of phenolic acids
化合物 compound	苦竹笋(宜宾) Pleioblastus amarus bamboo shoots (Yinbin)	苦竹笋(乐山) Pleioblastus amarus bamboo shoots (Leshan)	苦竹笋(南平) Pleioblastus amarus bamboo shoots (Nanping)	苦篱竹笋(肇庆) Arundinaria acerba bamboo shoots (Zhaoqing)
3,4-二羟基苯乙酸 3,4-dihydroxyphenylacetic acid	—	0.55±0.02	—	—
对羟基苯甲酸 4-hydroxybenzoic acid	0.91±0.03	2.06±0.10	4.09±0.18	2.32±0.07
香草酸 vanillic acid	1.25±0.04	7.13±0.17	—	—
咖啡酸 caffeic acid	1.36±0.01	—	6.11±0.21	3.34±0.17
对羟基苯甲醛 4-hydroxybenzaldehyde	0.85±0.02	0.27±0.01	0.09±0.00	—
丁香醛 syringaldehyde	10.43±0.51	0.47±0.02	0.45±0.01	2.30±0.06
阿魏酸 ferulic acid	1.24±0.04	27.83±0.79	10.52±0.52	9.68±0.40
3-羟基肉桂酸 3-hydroxycinnamic acid	0.96±0.03	0.28±0.00	0.12±0.01	0.23±0.01
总含量 total content	16.94±0.64	38.60±0.12	21.38±0.93	17.89±0.69

HPLC同时测定竹笋中8种酚酸类物质含量的方法研究及其应用

Simultaneous determination of eight phenolic acids in bamboo shoots by HPLC and its applications

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流动相 mobile phase	流动相占比/% mobile phase preportion
流动相 mobile phase	0 min	5 min	10 min	25 min	33 min	39 min	45 min	45.01 min	50 min
A	5	5	10	10	16	24	24	5	5
B	95	95	90	90	84	76	76	95	95

化合物 compound	物质含量/(mg·kg^-1) material content		变化率/% change of content	化合物 compound	物质含量/(mg·kg^-1) material content		变化率/% change of content
化合物 compound	避光处理 shading treatment	CK	变化率/% change of content	化合物 compound	避光处理 shading treatment	CK	变化率/% change of content
3,4-二羟基苯乙酸 3,4-dihydroxyphenylacetic acid	—	—	—	丁香醛 syringaldehyde	3.43±0.05	10.43±0.51	-68.1
对羟基苯甲酸 4-hydroxybenzoic acid	0.74±0.01	0.91±0.03	-19.8	阿魏酸 ferulic acid	0.96±0.03	1.24±0.04	-22.7
香草酸 vanillic acid	2.16±0.04	1.25±0.04	68.7	3-羟基肉桂酸 3-hydroxycinnamic acid	0.64±0.03	0.96±0.03	-30.5
咖啡酸 caffeic acid	1.33±0.04	1.36±0.01	-2.5	总含量 total content	9.35±0.08	16.94±0.64	-46.2
对羟基苯甲醛 4-hydroxybenzaldehyde	0.12±0.00	0.85±0.02	-85.9

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