超高效液相色谱-串联质谱法同时分析紫薯中15种酚酸类组分

刘文静; 黄彪; 林香信; 方灵; 吴建鸿; 李巍; 吴妙鸿

doi:10.19303/j.issn.1008-0384.2019.10.014

超高效液相色谱-串联质谱法同时分析紫薯中15种酚酸类组分

doi: 10.19303/j.issn.1008-0384.2019.10.014

福建省农业科学院农业质量标准与检测技术研究所/福建省农产品质量安全重点实验室，福建　福州　350003

基金项目: 福建省科技计划公益类专项(2017R1018-2)；福建省农业科学院青年人才创新项目(YC2018-11)

详细信息

作者简介:
刘文静（1982−），女，硕士，助理研究员，主要从事农产品质量安全研究(E-mail：411935637@qq.com)

通讯作者:
林香信（1972−），男，副研究员，主要从事农产品质量安全研究（E-mail：1353645173@qq.com）

中图分类号: TS 215
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出版历程
- 收稿日期: 2019-08-16
- 修回日期: 2019-10-08
- 刊出日期: 2019-10-01

Simultaneous Determination of 15 Phenolic Compounds in Purple Potato by High Performance Liquid Chromatography-tandem Mass Spectrometry

Institute of Agricultural Quality Standards and Testing Technology Research, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory of Agro-products Quality & Safety, Fuzhou, Fujian　350003, China

摘要

摘要: 目的运用超高效液相色谱-串联质谱（UPLC-MS/MS）分析不同品种紫薯中酚酸类组分及含量，为紫薯中酚酸类组分的提取、分析提供理论依据。方法建立超高效液相色谱-串联质谱法同时检测紫薯中15种酚酸类组分的方法，分析8种不同品种紫薯中酚酸类组分的含量。样品经甲醇-水溶液超声提取后，使用Waters T3 C₁₈色谱柱(250 mm × 4.6 mm，5 μm)，以0.1%（v/v）甲酸-5 mmol·L⁻¹乙酸铵水溶液和乙腈为流动相进行梯度洗脱，负离子扫描方式下多反应监测(MRM)模式测定。结果在优化的试验条件下，15种酚酸类组分在相应的浓度范围内具有良好的线性关系，相关系数（r）为0.995 6～0.999 7，方法检出限和定量限分别为0.3～5.8 μg·L⁻¹(S/N ≥3)和1.0～15.2 μg·L⁻¹(S/N ≥10)；加标试验平均回收率73.3%～97.1%，相对标准偏差1.8%～8.9%。结论在所分析的紫薯样品中共检出5种酚酸类组分，含量0.102～1 698.490 mg·kg⁻¹，其中绿原酸含量最高，对香豆酸含量最低。该法灵敏、快速、准确，适用于紫薯中酚酸类组分的定性定量分析。
- 紫薯 /
- 酚酸类组分 /
- 超高效液相色谱-串联质谱
Abstract: Objective A method coupling the ultra-high performance liquid chromatography and the triple-quadrupole tandem mass spectrometry (UPLC-MS/MS) was established to simultaneously determine 15 phenolic compounds in purple potatoes. Method Purple potato specimens were extracted with methanol-water under ultrasound to be separated on a UPLC and analyzed by MS/MS for the phenolic determination. On a Waters T3 C₁₈ column of the UPLC, the extracts was separated by gradient elution with 0.1% (V/V) formic acid-5 mmol·L⁻¹ammonium acetate aqueous solution and acetonitrile as the mobile phase. The elutes were analyzed using the positive electrospray ionization tandem MS under the multiple reaction monitoring mode. Result With the optimized conditions and within the concentration range, linearities on the measurements for 15 phenolics with correlation coefficients ranging from 0.995 6 to 0.999 7 were obtained. The methodology had a limit of detection (LOD) of 0.3–5.8 µg·L⁻¹ (S/N≥3) and that of quantitation 1.0–15.2 µg·L⁻¹ (S/N≥10). The average recoveries on the compounds ranged from 73.3% to 97.1% with a relative standard deviation (RSDs) of 1.8%–8.9%. Conclusion Five categories of phenolic compounds in the concentrations ranging from 0.102 mg·kg⁻¹ to 1 698 490 mg·kg⁻¹ were detected in the purple potato specimens by the method. Among them, chlorogenic acid was the highest in quantity, and coumaric acid the lowest. The newly established methodology was deemed sensitive, rapid, accurate, and suitable for phenolic determination on purple potatoes.
- purple potato /
- phenolic compounds /
- ultra-high liquid chromatography-tandem mass spectrometry

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表 1 酚酸类组分检测的质谱参数条件

Table 1. Parameters applied for phenolic determination by LC-MS/MS

组分名称 Compound	保留时间 Retention time /min	母离子 Parents ion/ (m·z⁻¹)	子离子 Daughter ion/ (m·z⁻¹)	锥孔电压 Cone voltage /V	碰撞能量 Collision energy/eV
3,4,5-三羟基苯甲酸 3,4,5-Trihydroxybenzoic acid	1.68	168.8	125.0*, 79.0	−36	14, 20
3,5-二羟基苯甲酸 3,5-Dihydroxybenzoic acid	2.62	152.9	108.8*, 80.9	−40	14, 18
绿原酸 Chlorogenic acid	3.25	353.2	191.0*, 92.9	−30	16, 38
对羟基苯甲酸 p−Hydroxybenzoic acid	3.72	136.9	92.9*, 137.4	−36	12, 16
2,5-二羟基苯甲酸 2,5-Dihydroxybenzoic acid	3.82	152.9	108.8*, 80.9	−40	14, 18
香草酸 Vanillic acid	3.85	167.0	151.9*, 108.0	−36	14, 18
咖啡酸 Caffeic acid	3.88	179.1	134.9*, 89.0	−30	16, 32
丁香酸 Syringate	3.97	197.0	181.9*, 123.0	−48	14, 26
对香豆酸 Coumalic acid	4.33	163.0	119.0*, 93.3	−44	16, 26
异绿原酸 A Isochlorogenic acid A	4.39	514.9	352.9*, 190.9	−30	16, 32
芥子酸 Sinapic acid	4.55	223.0	208.0*, 164.0	−44	16, 14
阿魏酸 Ferulic acid	4.59	193.0	133.9*, 177.9	−38	16, 14
3-羟基肉桂酸 3-Hydrocycinnamic acid	4.64	163.0	119.0*, 93.3	−44	16, 26
2-羟基肉桂酸 2-Hydrocycinnamic acid	4.83	163.0	119.0*, 93.3	−44	16, 26
水杨酸 Salicylic acid	5.18	136.9	92.9*, 137.4	−36	16, 12
注：为定量离子。 Note： means quantitative ions.

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表 2 提取溶液对酚类物质提取效果比较

Table 2. Comparison of the effect of extraction solution

提取溶液 Extraction solution	吸光度值 absorbance value	多酚含量 Content/(mg·g⁻¹)
30%甲醇水溶液 Methanol	0.549	3.14
50%甲醇水溶液 Methanol	0.691	3.95
70%甲醇水溶液 Methanol	0.736	4.20

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表 3 15种酚酸类组分检测的方法学验证

Table 3. Methodology validation of UPLC-MS/MS

组分名称 Compound	线性范围 Line arrange/ （µg·L⁻¹）	线性方程 Linear equation	相关系数r	检出限 Limits of detection/ (µg·L⁻¹)	定量限 Limits of quantitation/ (µg·L⁻¹)
3,4,5-三羟基苯甲酸 3,4,5-Trihydroxybenzoic acid	20～8 000	y = 89.9x +3 841.9	0.998 4	3.2	10.2
3,5-二羟基苯甲酸 3,5-Dihydroxybenzoic acid	20～8 000	y = 456.2x + 22 407.5	0.999 2	1.8	5.0
绿原酸 Chlorogenic acid	20～8 000	y = 311.5x + 99.8	0.999 5	2.1	5.0
对羟基苯甲酸 p-Hydroxybenzoic acid	10～4 000	y = 819.2x + 1 232.2	0.997 1	1.2	3.2
2,5-二羟基苯甲酸 2,5-Dihydroxybenzoic acid	10～4 000	y = 857.4x + 805.4	0.997 6	1.2	3.5
香草酸 Vanillic acid	10～4 000	y = 1 875.0x + 9 070.7	0.995 6	0.5	2.0
咖啡酸 Caffeic acid	50～20 000	y = 80.7x + 18.2	0.999 0	3.6	10.0
丁香酸 Syringate	50～20 000	y = 35.2x + 1 100.0	0.998 4	5.8	15.2
对香豆酸 Coumalic acid	10～4 000	y = 1 452.9x + 734.0	0.998 7	0.4	1.5
异绿原酸 A Isochlorogenic acid A	20～8 000	y= 368.1x – 90.0	0.999 0	2.2	6.8
芥子酸 Sinapic acid	20～8 000	y = 340.5x + 214.8	0.999 4	2.0	6.0
阿魏酸 Ferulic acid	20～8 000	y = 607.7x + 28.4	0.999 7	1.5	4.5
3-羟基肉桂酸 3-Hydrocycinnamic acid	10～4 000	y = 1 180.1x + 523.9	0.998 9	0.6	2.0
2-羟基肉桂酸 2-Hydrocycinnamic acid	10～4 000	y = 1 211.5x + 684.7	0.999 1	0.6	2.0
水杨酸 Salicylic acid	10～4 000	y = 2 493.6x + 1 857.8	0.997 6	0.3	1.0

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表 4 紫薯中15种酚类化合物的加标回收试验(n=6)

Table 4. Recovery rates of 15 phenols in actual samples

组分名称 Compound	本底值 Background/ (mg·kg⁻¹)	加标量 Addition /(mg·kg⁻¹)	测量值 Measured value /(mg·kg⁻¹)	回收率 Recovery /%	标准偏差 Standard deviation /%
3,4,5-三羟基苯甲酸 3,4,5-Trihydroxybenzoic acid	ND	0.400	0.317	79.2	4.7
		0.800	0.586	73.3	1.7
		2.000	1.942	97.1	3.1
3,5-二羟基苯甲酸 3,5-Dihydroxybenzoic acid	ND	0.400	0.348	87.0	6.6
		0.800	0.706	88.2	3.1
		2.000	1.854	92.7	5.5
绿原酸 Chlorogenic acid	498.161	10.00	506.931	87.7	5.4
		20.00	513.121	74.8	4.9
		50.00	536.761	77.2	6.3
对羟基苯甲酸 p-Hydroxybenzoic acid	ND	0.200	0.175	87.7	7.0
		0.400	0.343	85.7	3.2
		1.000	0.868	86.8	2.1
2,5-二羟基苯甲酸 2,5-Dihydroxybenzoic acid	ND	0.200	0.154	77.2	4.2
		0.400	0.324	81.1	4.3
		1.000	0.904	90.4	2.7
香草酸 Vanillic acid	9.152	1.000	1.7702	85.5	3.6
		2.000	2.6052	84.5	7.9
		5.000	5.3602	88.9	3.2
咖啡酸 Caffeic acid	ND	1.000	0.783	78.3	5.5
		2.000	1.588	79.4	4.3
		5.000	4.055	81.1	5.9
丁香酸 Syringate	1.384	1.000	0.9774	83.9	6.9
		2.000	2.0304	94.6	8.7
		5.000	4.6534	90.3	8.9
对香豆酸 Coumalic acid	1.017	0.200	1.162	72.3	7.7
		0.400	1.322	76.3	1.9
		1.000	1.791	77.4	8.5
异绿原酸A Isochlorogenic acid A	118.074	10.00	126.644	85.7	5.1
		20.00	135.334	86.3	6.2
		50.00	163.174	90.2	3.8
芥子酸 Sinapic acid	ND	0.400	0.304	76.1	5.6
		0.800	0.603	75.4	3.7
		2.000	1.772	88.6	4.8
阿魏酸 Ferulic acid	ND	0.200	0.150	74.9	5.0
		0.400	0.324	80.9	7.1
		1.000	0.913	91.3	6.2
3-羟基肉桂酸 3-Hydrocycinnamic acid	ND	0.200	0.149	74.5	6.0
		0.400	0.325	81.3	2.0
		1.000	0.894	89.4	7.6
2-羟基肉桂酸 2-Hydrocycinnamic acid	ND	0.200	0.158	79.2	4.2
		0.400	0.351	87.8	4.3
		1.000	0.839	83.9	2.7
水杨酸 Salicylic acid	ND	0.200	0.175	87.3	4.6
		0.400	0.323	80.7	3.9
		1.000	0.968	96.8	2.2
注：ND：未检出。 Note: ND means not detected.

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表 5 不同紫薯酚酸类组分含量

Table 5. Contents of phenols in purple potato samples （单位：mg·kg⁻¹，n=3）

样品 Sample	香草酸 Vanillic acid	咖啡酸 Caffeic acid	对香豆酸Coumalic acid	绿原酸 Chlorogenic acid	异绿原酸 A Isochlorogenic acid A
福薯9号 Ganshu 9	1.241	3.293	0.137	291.439	34.099
莆紫18号 Puzi 18	1.700	9.819	0.112	1 463.459	112.602
福薯24号 Fushu 24	1.613	3.386	1.173	541.270	58.864
龙紫6号 Longzi 6	1.474	3.806	0.381	1 158.459	135.749
莆紫3号 Puzi 3	1.896	5.409	0.142	644.501	46.524
龙紫4号 Longzi 4	1.835	4.071	0.102	1 250.894	112.611
福宁紫4号 Funingzi 4	1.553	7.869	0.113	469.991	96.853
福宁紫3号 Funingzi 3	2.237	10.722	0.122	1 698.490	202.682

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参考文献(24)

[1]	单珊, 周惠明, 朱科学. 紫薯淀粉理化性质的研究 [J]. 粮食与饲料工业, 2011(4):27−30. doi: 10.3969/j.issn.1003-6202.2011.11.009 SHAN S, ZHOU H M, ZHU K X. Study on the physicochemical properties of purple sweet potato starch [J]. Cereal & Feed Industry, 2011(4): 27−30.(in Chinese) doi: 10.3969/j.issn.1003-6202.2011.11.009
[2]	高秋萍, 阮红, 毛童俊, 等. 紫心甘薯多糖的抗氧化活性研究 [J]. 营养学报, 2011, 33(1):56−60. GAO Q P, RUAN H, MAO T J, et al. Study on the antioxidant activity of polysaccharide from purple sweet potato [J]. Acta Nutrimenta Sinica, 2011, 33(1): 56−60.(in Chinese)
[3]	郝艳玲, 刘增, 牟婷婷, 等. 乌洋芋与紫薯营养成分比较 [J]. 浙江农业学报, 2014, 26(5):1336−1340. doi: 10.3969/j.issn.1004-1524.2014.05.35 HAO Y L, LIU Z, MU T T, et al. Comparison of nutrient composition between purple potato and purple sweet potato. [J]. Acta Agriculturae Zhejiangensis, 2014, 26(5): 1336−1340.(in Chinese) doi: 10.3969/j.issn.1004-1524.2014.05.35
[4]	黄洁, 甘学德, 许瑞丽, 等. 21份紫肉甘薯种质资源的营养品质及其产量评价 [J]. 福建农业学报, 2011, 26(2):215−222. doi: 10.3969/j.issn.1008-0384.2011.02.015 HUANG J, GAN X D, XU R L, et al. Nutrition and Yield of Purple Sweetpotato from Twenty-one Germplasm Source [J]. Fujian Journal of Agricultural Sciences, 2011, 26(2): 215−222.(in Chinese) doi: 10.3969/j.issn.1008-0384.2011.02.015
[5]	蔡湛, 兰余, 赵淑娟, 等. 紫薯的抗氧化及活性成分研究 [J]. 粮食与油脂, 2015, 28(1):43−48. doi: 10.3969/j.issn.1008-9578.2015.01.011 CAI Z, LAN Y, ZHAO S J, et al. Study on composition analysis and antioxidant activity of purple sweet potato [J]. Cereals & Oils, 2015, 28(1): 43−48.(in Chinese) doi: 10.3969/j.issn.1008-9578.2015.01.011
[6]	郭孝萱, 柳嘉, 陆雪娇, 等. 紫薯发酵前后总酚、总黄酮、花色苷、抗氧化性和抗癌作用比较 [J]. 中国食品学报, 2017, 17(3):289−295. GUO X X, LIU J, LU X J, et al. Comparing of Ethanol Extracts from Three Fungi Fermented Purple-fleshed Potatoes on Total Phenolic Content, Flavonoids, Anthocyanins, Antioxidant Activities and Cytotoxicity on HepG2 Cells [J]. Journal of Chinese Institute of Food Science and Technology, 2017, 17(3): 289−295.(in Chinese)
[7]	孙海燕. 紫薯多酚类物质的提取及抗氧化研究 [J]. 食品研究与开发, 2015, 36(23):21−24. doi: 10.3969/j.issn.1005-6521.2015.23.006 SUN H Y. Study on Extraction and Antioxidant Activities of Polyphenols from Purple Sweet potato [J]. Food Research and Development, 2015, 36(23): 21−24.(in Chinese) doi: 10.3969/j.issn.1005-6521.2015.23.006
[8]	徐柯, 曾凡坤, 袁美,等. 红薯叶、紫薯块根及不同时期紫薯叶中主要活性成分含量比较 [J]. 食品与机械, 2018, 34(6):30−34. XU K, ZENG F K, YUAN M, et al. Comparison of the content of main active components in sweet potato leaves;purple potato tuber and purple potato leaves of different development stages [J]. Food and Machinery, 2018, 34(6): 30−34.(in Chinese)
[9]	傅玉凡, 陈敏, 叶小利, 等. 紫肉甘薯花色苷含量的变化规律及其与主要经济性状的相关分析 [J]. 中国农业科学, 2007, 40(10):2185−2192. doi: 10.3321/j.issn:0578-1752.2007.10.009 FU Y F, CHEN M, YE X L, et al. Variation Laws of Anthocyanin Content of Roots and Their Relationships with Major Economic Traits in Purple-Fleshed Sweet potato[Ipomoea Batatas (L.) Lam] [J]. Scientia Agricultura Sinica, 2007, 40(10): 2185−2192.(in Chinese) doi: 10.3321/j.issn:0578-1752.2007.10.009
[10]	江连洲, 王晰锐, 张超, 等. HPLC-MS法鉴定不同品种紫甘薯中花色苷组成 [J]. 中国食品学报, 2011, 11(5):176−181. doi: 10.3969/j.issn.1009-7848.2011.05.028 WANG L Z, WANG X R, ZHANG C, et al. Identification of Anthocyanin in Different Varieties of Purple Sweet Potatoes by HPLC-MS [J]. Journal of Chinese Institute of Food Science and Technology, 2011, 11(5): 176−181.(in Chinese) doi: 10.3969/j.issn.1009-7848.2011.05.028
[11]	高玥, 李新生, 韩豪, 等. UPLC-MS/MS定性测定紫薯花青苷方法研究 [J]. 食品工业科技, 2013, 34(3):317−320. GAO Y, LI X S, HAN H, et al. Qualitative approach determination of anthocyanin in purple sweet potatoes by UPLC-MS /MS [J]. Science and Technology of Food Industry, 2013, 34(3): 317−320.(in Chinese)
[12]	OKI T, MASUDA M, FURUTA S, et al. Involvement of anthocyanins and other phenolic compounds in radical-scavenging activity of purple fleshed sweet potato cultivars [J]. Journal of Food Science, 2002, 67(5): 1752−1756. doi: 10.1111/j.1365-2621.2002.tb08718.x
[13]	KANO M, TAKAYANAGI T, HARADA K, et al. Antioxidative activity of anthocyanins from purple sweet potato, Ipomoea batatas cultivar Ayamurasaki [J]. Bioscience, Biotechnology, and Biochemistry, 2005, 69(5): 979−988. doi: 10.1271/bbb.69.979
[14]	LEE Y K, LEE W S, KIM G S, et al. Anthocyanins are novel AMPKαl stimulators that suppress tumor growth by inhibiting mTOR phosphorylation [J]. Oncology Reports, 2010, 24(6): 1471−1477.
[15]	YOSHIMOTO M, YAMAKA O, SUDA I. Physiological function of purple colored flesh sweet potato [J]. Food Processing, 1998, 33(8): 15−17.
[16]	王荞薇, 谢媛媛, 王义明, 等. 银杏叶中银杏酚酸类成分含量测定方法研究 [J]. 中国药学杂志, 2015, 50(2):167−173. WANG Q W, XIE Y Y, WANG Y, et al. Simultaneous Determination of Five ginkgolic Acids in Ginkgo Folium by HPLC [J]. Chinese Pharmaceutical Journal, 2015, 50(2): 167−173.(in Chinese)
[17]	谢越, 俞浩, 汪建飞, 等. 高效液相色谱法同时测定滁菊样品中的9种酚酸 [J]. 分析化学, 2013, 41(3):383−388. XIE Y, YU H, WANG J F, et al. Simultaneous Determination of Nine Phenolic Acids in Dendranthema Morifolium(Ramat) Tzvel.cv. Chuju Samples by High Performance Liquid Chromatography [J]. Chinese Journal of Analytical Chemistry, 2013, 41(3): 383−388.(in Chinese)
[18]	孙崇臻, 王超, 蔡子哲, 等. 高效液相色谱测定蜂蜜中的脱落酸、黄酮和酚酸 [J]. 食品科学, 2013, 34(10):281−285. doi: 10.7506/spkx1002-6630-201310062 SUN C Z, WANG C, CAI Z Z, et al. Determination of Flavonoids, Phenolic Acids and Abscisic Acid in Honeys of Different Floral Origins by HPLC [J]. Food Science, 2013, 34(10): 281−285.(in Chinese) doi: 10.7506/spkx1002-6630-201310062
[19]	洪燕, 韩燕全, 夏伦祝, 等. 超高效液相色谱法同时测定不同产地苍耳子9种酚酸类成分的含量 [J]. 中国药学杂志, 2013, 48(13):1109−1112. HONG Y, HAN Y Q, XIA L Z, et al. Simultaneous Determination of Nine Phenolic Acid Components in Xanthii Fructus [J]. Chinese Pharmaceutical Journal, 2013, 48(13): 1109−1112.(in Chinese)
[20]	张娜, 王国祥, Abacar Jose Daniel, et al. 超高效液相色谱法分析稻米酚酸化合物组分及其含量 [J]. 中国农业科学, 2015, 48(9):1718−1726. doi: 10.3864/j.issn.0578-1752.2015.09.05 ZHANG N, WANG G X, DANIEL A J, et al. , Determination of Phenolic Acids in Rice by Ultra-High Performance Liquid Chromatography [J]. Scientia Agricultura Sinica, 2015, 48(9): 1718−1726.(in Chinese) doi: 10.3864/j.issn.0578-1752.2015.09.05
[21]	REGNAULT-ROGER C, HADIDANE R, BIARD J F, et al. High performance liquid and thin-layer chromatographic determination of phenolic acids in palm (Phoenix dactilifcra) products [J]. Food Chemistry, 1987, 25(1): 61−71.
[22]	LEE J, CHAN B L S, MITCHELL A E. Identification/quantification of free and bound phenolic acids in peel and pulp of apples(Malus domestica) using high resolution mass spectrometry (HRMS) [J]. Food Chemistry, 2017, 215: 301−310. doi: 10.1016/j.foodchem.2016.07.166
[23]	HUANG Y, SUN H Y, QIN X L, et al. A UPLC-MS/MS method for simultaneous determination of free and total forms of a phenolic acid and two flavonoids in rat plasma and its application to comparative pharmacokinctic studies of polygonum capitatum extract in rats [J]. Molecules, 2017, 22(3): 353−365. doi: 10.3390/molecules22030353
[24]	SZCWCZYK K, OLCCH M. Optimization of extraction method for LC-MS based determination of phenolic acid profiles in different impatiens, species [J]. Phytochemistry Letters, 2017, 20(6): 322−330.