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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (8): 2259-2274.doi: 10.3724/SP.J.1006.2023.24202

• 耕作栽培·生理生化 • 上一篇    下一篇

超高效液相色谱法同时测定甘薯中13种类胡萝卜素的含量

贾瑞雪1,2(), 陈伊航1,3(), 张荣1, 唐朝臣1, 王章英1,*()   

  1. 1 广东省农业科学院作物研究所 / 广东省农作物遗传改良重点实验室, 广东广州 510640
    2 河北科技师范学院农学与生物科技学院, 河北秦皇岛 066600
    3 西北农林科技大学草业与草原学院, 陕西杨凌 712100
  • 收稿日期:2022-08-31 接受日期:2023-02-10 出版日期:2023-08-12 网络出版日期:2023-02-22
  • 通讯作者: 王章英
  • 作者简介:贾瑞雪, E-mail: jiaruixue91@126.com;
    陈伊航, E-mail: chenyihang2022@126.com第一联系人：**同等贡献
  • 基金资助:
    财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-10);广东省甘薯马铃薯产业技术体系创新团队(2022KJ111);广东省农业科学院食品营养与健康研究中心建设运行经费项目(XTXM202205)

Simultaneous determination of 13 carotenoids in sweetpotato by Ultra- Performance Liquid Chromatography

JIA Rui-Xue1,2(), CHEN Yi-Hang1,3(), ZHANG Rong1, TANG Chao-Chen1, WANG Zhang-Ying1,*()   

  1. 1 Crops Research Institute, Guangdong Academy of Agricultural Science / Key Laboratory of Crop Genetic Improvement of Guangdong Province, Guangzhou 510640, Guangdong, China
    2 College of Agronomy and Biotechnology, Hebei Normal University of Science and Technology, Qinhuangdao 066600, Hebei, China
    3 College of Grassland Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China
  • Received:2022-08-31 Accepted:2023-02-10 Published:2023-08-12 Published online:2023-02-22
  • Contact: WANG Zhang-Ying
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-10);Guangdong Modern Agro-industry Technology Research System(2022KJ111);Construction and Operation of the Food Nutrition and Health Research Center of Guangdong Academy of Agricultural Sciences(XTXM202205)

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摘要：

类胡萝卜素作为评价甘薯食用和加工品质的重要指标, 对于推进甘薯育种至关重要, 但由于缺乏甘薯类胡萝卜素组分及含量的快速检测方法, 限制了甘薯食用品质分析和育种进展。本研究通过优化色谱条件和样品前处理过程, 建立了同时测定甘薯中13种类胡萝卜素含量的超高效液相色谱分析方法。结果表明, 待测样品前处理过程中乙醇∶丙酮∶正己烷(1∶1∶1, v/v/v)作为提取液, 甲基叔丁基醚为复溶液类胡萝卜素得率最高。超高效液相色谱分析条件: 甲醇∶乙腈(1∶3, v/v)含0.01% 2,6-二叔丁基对甲酚(Butylated hydroxytoluene, BHT)和0.1%甲酸作为流动相A相, 甲基叔丁基醚含0.01% BHT作为流动相B相梯度洗脱, 色谱柱YMC Carotenoid C30 (2 mm×100 mm, 3 μm), 流速为0.8 mL min-1, 柱温23℃, 进样量1 μL, 检测波长450 nm, 分析时间8 min。该方法中13种类胡萝卜素线性关系良好, 相关系数大于0.997; 精密度、重复性、稳定性及回收率结果均符合要求。本研究建立的超高效液相色谱方法能快速、准确的同时检测甘薯中13种类胡萝卜素的含量, 可为大批量甘薯种质资源和育种材料的品质分析奠定基础。

关键词: UPLC, 甘薯品种, β-胡萝卜素, 含量测定

Abstract:

Carotenoids as important indexes to evaluate the edible and process quality of sweetpotato, are crucial to promoting sweetpotato breeding. However, the lack of rapid detection methods for the compositions and contents of carotenoids in sweetpotato has limited the progress of edible quality analysis and breeding of sweetpotato. In this study, an Ultra- Performance Liquid Chromatography (UPLC) method was established for simultaneous determination of 13 carotenoids in sweetpotato by optimizing the chromatographic conditions and sample pretreatment process. The results showed that ethanol: acetone: hexane (1:1:1, v/v/v) was used as the extraction solution, and methyl tert-butyl ether was used as the dissolution solution to obtain the highest yield of carotenoids. Analysis conditions of UPLC: methanol: acetonitrile (1:3, v/v) containing 0.01% 2,6-butylated hydroxytoluene (BHT) and 0.1% formic acid as mobile phase A, methyl tert-butyl ether containing 0.01% BHT as mobile phase B gradient elution, YMC Carotenoid C30 (2 mm×100 mm, 3 μm), the flow rate was 0.8 mL min-1, injection volume was 1 μL, the detection wavelength was 450 nm, and the analysis time was 8 min. The linear relationships of 13 carotenoids in this method were good, the correlation coefficients were all greater than 0.997. The test results of precision, repeatability, stability and recovery were all in line with the requirements. The UPLC method in this study can quickly, accurately and simultaneously detect the contents of 13 carotenoids in sweetpotato, which can lay a foundation for quality analysis of germplasm resources and breeding materials in large quantities of sweetpotato.

Key words: UPLC, sweetpotato cultivar, β-carotene, content determination

图1

不同洗脱程序色谱图 A图为第1种洗脱条件下色谱图; B图为第2种洗脱条件下色谱图; C图第3种洗脱条件下色谱图; D~E均为第4种洗脱条件下色谱图, D为10种胡萝卜素, E图为13种胡萝卜素。1为叶黄素, 2为玉米黄质, 3为α-隐黄质, 4为ε-胡萝卜素, 5为β-隐黄质, 6为α-胡萝卜素, 7为β-胡萝卜素, 8为δ-胡萝卜素, 9为γ-胡萝卜素, 10为番茄红素, 11为紫黄质, 12为新黄质, 13为环氧玉米黄质。"

图2

不同柱温类胡萝卜素色谱图 A~E分别为20、23、25、28、30℃条件下类胡萝卜素色谱图。1为紫黄质, 2为新黄质, 3为环氧玉米黄质, 4为叶黄素, 5为玉米黄质, 6为α-隐黄质, 7为ε-胡萝卜素, 8为β-隐黄质, 9为α-胡萝卜素, 10为β-胡萝卜素, 11为δ-胡萝卜素, 12为γ-胡萝卜素, 13为番茄红素。"

表1

不同色谱条件下13种类胡萝卜素分离度"

色谱条件
Chromatographic
conditions		 分离度 Resolution
1 2 3 4 5 6 7 8 9 10 11 12 13 14
色谱柱温度
Chromatographic column
temperature
(℃)		 20 3.09 1.01 1.67 2.28 2.61 1.49 1.87 1.93 1.67 3.94 3.61 3.69 10.11 2.49
23 2.88 0.88 1.62 2.18 2.45 1.57 2.00 1.68 1.80 3.87 3.71 3.70 9.89 2.97
25 2.46 0.67 1.51 2.06 2.26 1.71 2.13 1.33 1.98 3.79 3.79 3.73 9.75 3.37
28 2.38 0.57 1.58 1.91 2.04 1.86 2.29 0.92 2.14 3.62 3.87 3.76 9.48 3.98
30 1.58 — 1.35 1.77 1.89 1.97 2.36 0.59 2.35 3.48 3.89 3.78 9.21 4.30
流速
Flow rate
(mL min-1)		 0.6 4.02 1.19 1.89 2.59 2.99 1.53 1.93 2.29 1.71 4.22 3.60 3.60 10.43 1.25
0.8 2.88 0.88 1.62 2.18 2.45 1.57 2.00 1.68 1.80 3.87 3.71 3.70 9.89 2.97
1 2.64 0.66 1.48 2.00 2.18 2.03 2.44 0.89 2.36 3.70 3.69 3.55 9.75 3.29
进样量
Injection volume (μL)		 1 4.23 1.12 2.01 2.56 2.74 1.80 2.23 1.68 1.99 3.98 3.78 3.74 9.88 2.72
2 2.88 0.88 1.62 2.18 2.45 1.57 2.00 1.68 1.80 3.87 3.71 3.70 9.89 2.97
3 2.80 0.78 1.30 1.88 2.29 1.20 1.58 2.03 1.43 3.79 3.49 3.58 9.94 2.20

图3

不同流速类胡萝卜素的色谱图A~C分别代表流速为0.6、0.8和1.0 mL min-1条件下类胡萝卜素色谱图。1为紫黄质, 2为新黄质, 3为环氧玉米黄质, 4为叶黄素, 5为玉米黄质, 6为α-隐黄质, 7为ε-胡萝卜素, 8为β-隐黄质, 9为α-胡萝卜素, 10为β-胡萝卜素, 11为δ-胡萝卜素, 12为γ-胡萝卜素, 13为番茄红素。"

图4

不同进样量类胡萝卜素色谱图 A~C分别代表进样体积为1、2和3 μL条件下类胡萝卜素色谱图。1为紫黄质, 2为新黄质, 3为环氧玉米黄质, 4为叶黄素, 5为玉米黄质, 6为α-隐黄质, 7为ε-胡萝卜素, 8为β-隐黄质, 9为α-胡萝卜素, 10为β-胡萝卜素, 11为δ-胡萝卜素, 12为γ-胡萝卜素, 13为番茄红素。"

表2

13种类胡萝卜素的线性回归方程、相关系数、检出限和定量限"

类胡萝卜素
Carotenoids		 保留时间
Retention time (min)		 线性范围
Linear range
(μg mL-1)		 线性方程
Linear
equation		 相关系数
Correlation
coefficient (r)		 检出限
LOD
(μg mL-1)		 定量限
LOQ
(μg mL-1)
紫黄质 Violaxanthin 0.709 0.1-100 y = 14.2103x-2.2023 0.9997 0.003 0.009
新黄质 Neoxanthin 0.831 0.1-50 y = 11.9700x+5.1485 0.9995 0.004 0.015
环氧玉米黄质 Antheraxanthin 1.057 0.1-100 y = 9.3125x-0.0484 0.9996 0.004 0.015
叶黄素 Lutein 1.370 0.1-100 y = 16.0211x+18.3307 0.9985 0.003 0.012
玉米黄质 Zeaxanthin 1.678 0.1-100 y = 11.7821x+13.2805 0.9971 0.005 0.016
α-隐黄质 α-cryptoxanthin 1.961 0.1-100 y = 20.9719x+10.9579 0.9984 0.003 0.010
ε-胡萝卜素 ε-carotene 2.363 0.1-100 y = 16.7789x+16.6374 0.9975 0.003 0.011
β-隐黄质 β-cryptoxanthin 2.506 0.1-100 y = 17.7172x+5.8782 0.9990 0.004 0.012
α-胡萝卜素 α-carotene 2.874 0.1-100 y = 18.1369x+9.9311 0.9986 0.003 0.010
β-胡萝卜素 β-carotene 3.317 0.1-200 y = 16.3936x-4.5911 0.9993 0.004 0.012
δ-胡萝卜素 δ-carotene 4.326 0.1-100 y = 15.1643x+8.9234 0.9982 0.003 0.011
γ-胡萝卜素 γ-carotene 4.712 0.1-100 y = 9.4632x+3.0909 0.9997 0.005 0.016
番茄红素 Lycopene 5.770 0.1-100 y = 2.9934x+1.1650 0.9990 0.018 0.060

表3

精密度、重复性和稳定性相对标准偏差"

类胡萝卜素
Carotenoids		 精密度Precision RSD (%) 重复性 Repeatability RSD (%) 稳定性Stability RSD (%)
峰面积Area 保留时间
Retention time
(min)		 峰面积
Area		 保留时间
Retention time
(min)		 峰面积
Area		 保留时间
Retention time
(min)
紫黄质 Violaxanthin 0.46 1.31 0.51 1.61 7.03 5.07
新黄质 Neoxanthin 0.89 1.82 1.10 2.23 1.80 6.61
环氧玉米黄质 Antheraxanthin 0.49 1.75 0.79 2.23 6.97 6.95
叶黄素 Lutein 0.55 1.91 0.82 2.43 3.90 7.64
玉米黄质 Zeaxanthin 0.60 2.02 0.99 2.76 6.67 8.16
α-隐黄质 α-cryptoxanthin 0.55 1.55 0.80 2.21 5.36 6.88
ε-胡萝卜素 ε-carotene 0.73 1.14 1.20 1.70 4.75 5.76
β-隐黄质 β-cryptoxanthin 0.74 1.47 1.45 2.25 8.15 6.85
α-胡萝卜素 α-carotene 0.86 1.09 1.05 1.74 6.48 5.75
β-胡萝卜素 β-carotene 8.66 0.92 1.19 1.55 11.08 5.33
δ-胡萝卜素 δ-carotene 0.50 0.61 0.35 1.32 6.69 4.64
γ-胡萝卜素 γ-carotene 0.86 0.48 0.59 1.15 10.51 4.38
番茄红素 Lycopene 5.76 0.45 3.14 1.07 14.05 4.57

表4

13种类胡萝卜素的回收率"

类胡萝卜素
Carotenoids		 本底值
Background content		 回收率Recovery rate (%) 平均值回收率
Average recovery rate (%)
添加量1 μg
Addition amount 1 μg		 添加量2 μg
Addition amount 2 μg		 添加量3 μg
Addition amount 3 μg
紫黄质 Violaxanthin 0 138.35 132.15 96.65 122.38
新黄质 Neoxanthin 0 74.64 91.43 91.32 85.80
环氧玉米黄质 Antheraxanthin 0 116.19 110.63 97.25 108.02
叶黄素 Lutein 1 104.71 103.42 117.10 108.41
玉米黄质 Zeaxanthin 1 111.90 112.67 130.56 118.38
α-隐黄质 α-cryptoxanthin 1 96.12 95.93 110.95 101.00
ε-胡萝卜素 ε-carotene 1 97.73 98.90 113.73 103.45
β-隐黄质 β-cryptoxanthin 1 91.99 91.58 105.84 96.47
α-胡萝卜素 α-carotene 1 92.94 93.89 108.49 98.44
β-胡萝卜素 β-carotene 1 80.54 80.11 93.19 84.61
δ-胡萝卜素 δ-carotene 1 83.79 89.19 105.73 92.90
γ-胡萝卜素 γ-carotene 1 92.51 93.31 110.18 98.67
番茄红素 Lycopene 1 84.82 87.86 106.71 93.13

表5

不同提取溶剂和复溶液组合广薯79类胡萝卜素含量"

提取溶剂
Extraction solution		 复溶液Dissolution solution F检验
F-test		 显著性
Significant
甲醇:甲基叔丁基醚(3:1, v/v)
Methanol:Methyl tert-butyl ether (3:1, v/v)		 甲醇:甲基叔丁基醚(1:1，v/v)
Methanol:Methyl tert-butyl ether (1:1, v/v)		 甲醇:甲基叔丁基醚(1:3，v/v)
Methanol:Methyl tert-butyl ether (1:3, v/v)		 甲基叔丁基醚
Methyl tert-butyl ether
乙醇:丙酮:正己烷(1:1:1，v/v/v) Ethanol: acetone: n-hexane (1:1:1, v/v/v) 18.06 ± 1.88 37.33 ± 5.31 172.23 ± 14.88 214.49 ± 19.92
乙醇:丙酮:正己烷(1:1:2，v/v/v) Ethanol: acetone: n-hexane (1:1:2, v/v/v) 19.44 ± 3.38 39.28 ± 3.33 199.29 ± 34.94 193.91 ± 18.14
甲醇:丙酮:正己烷(1:1:1，v/v/v) Methanol: acetone: n-hexane (1:1:1, v/v/v) 17.11 ± 1.58 32.46 ± 0.71 152.99 ± 12.03 205.36 ± 16.49
甲醇:丙酮:正己烷(1:1:2，v/v/v) Methanol: acetone: n-hexane (1:1:2, v/v/v) 17.06 ± 1.72 38.42 ± 3.35 167.99 ± 28.8 195.71 ± 33.25
提取溶剂 Extraction solution 1.074 0.374
复溶液 Dissolution solution 366.885 < 0.001
提取溶剂×复溶液 Extraction solution×dissolution solution 1.293 0.279

表6

不同甘薯品种类胡萝卜素含量"

类胡萝卜素
Carotenoids		 含量 Content (μg g-1)
龙薯9号Longshu 9 广薯87
Guangshu 87		 普薯32
Pushu 32		 广薯79
Guangshu 79		 济薯26
Jishu 26		 烟薯25
Yanshu 25
紫黄质 Violaxanthin 2.22 0.79 1.16 0.41 0.62 0.72
新黄质 Neoxanthin 1.54 1.60 ND ND 0.50 0.39
环氧玉米黄质 Antheraxanthin 4.07 5.79 1.11 3.19 6.30 3.55
叶黄素 Lutein 3.18 ND ND ND ND 0.81
玉米黄质 Zeaxanthin 0.41 0.99 ND 0.10 ND 0.67
α-隐黄质 α-cryptoxanthin 0.88 ND 0.78 2.88 ND 2.70
ε-胡萝卜素 ε-carotene ND 0.41 2.62 2.00 ND 1.28
β-隐黄质 β-cryptoxanthin ND ND ND ND ND ND
α-胡萝卜素 α-carotene ND ND ND ND ND ND
β-胡萝卜素 β-carotene 58.66 27.86 178.08 205.91 3.79 125.34
δ-胡萝卜素 δ-carotene ND ND ND ND ND ND
γ-胡萝卜素 γ-carotene ND ND 0.00 ND ND ND
番茄红素 Lycopene ND ND 0.00 ND ND ND
总含量 Total content 70.97 37.49 183.76 214.49 11.29 135.15

表7

不同物种间类胡萝卜素检测方法比较"

物种Species 检测方法Detection method 部位Part 类胡萝卜素种类 Carotenoid composition 提取方法Extraction method 提取溶剂
Extraction solution		 复溶液
Dissolution solution		 色谱柱
Chromatographic column		 洗脱程序Elution programs 温度Temperature
(℃)		 流速Flow rate
(mL min-1)		 进样量Injection volume
(μL)		 分析时间Time
(min)		 参考文献References
甘薯
Sweetpotato		 UPLC
UPLC		 薯肉
Flesh		 13 非皂化
Non saponification		 乙醇:丙酮:正己烷(1:1:1)
Ethanol: acetone: n-hexane (1:1:1)		 甲基叔丁基醚
Methyl tert-butyl ether		 YMC Carotenoid C30 (2 mm×100 mm，3 μm) 梯度洗脱
Gradient elution		 23 0.8 1 8 本研究
This study
甘薯
Sweetpotato		 分光光度计法
Spectrophotometry		 薯肉
Flesh
 1 非皂化
Non saponification		 丙酮
Acetone		 — — — — — — — [8]
甘薯
Sweetpotato		 HPLC
HPLC		 薯肉
Flesh		 17 非皂化
Non saponification		 — — Wakopack Navi C30 (30 mm×250 mm，5 μm) 梯度洗脱
Gradient elution		 35 0.85 — 60 [6]
甘薯
Sweetpotato		 HPLC
HPLC		 愈伤组织
Callus		 6 非皂化
Non saponification		 丙酮
Acetone		 二氯甲烷:丙酮(1:1)
Methylene chloride: acetone (1:1)		 YMC Carotenoid C30 (4.6 mm×250 mm，3 μm) 梯度洗脱
Gradient elution		 22 0.7 — 60 [10]
甘薯
Sweetpotato		 HPLC
HPLC		 茎叶
Leaves		 2 非皂化
Non saponification		 甲醇
Methanol
 — Wondasil C18 (4.6 mm×250 mm，5 μm) 梯度洗脱
Gradient elution		 20 1 10 30 [34]
柑橘
Citrus		 HPLC
HPLC		 果皮
Peel		 7 皂化
Saponification		 丙酮
Acetone		 — C18 (3.9 mm×300 mm，10 μm) 等度洗脱
Isocratic elution		 25 1 20 — [22]
菠萝蜜
Jackfruit		 HPLC
HPLC		 果肉
Flesh		 16 皂化
Saponification		 乙醇:丙酮:正己烷(1:1:2)
Ethanol: acetone: n-hexane (1:1:2)		 甲醇和甲基叔丁基醚
Methanol and methyl tert-butyl ether		 YMC Carotenoid C30 (4.6 mm×250 mm，5 μm) 梯度洗脱
Gradient elution		 — 1 — 60 [23]
香蕉
Banana		 HPLC
HPLC		 果肉
Pulp		 9 皂化
Saponification		 乙醇:丙酮:正己烷(1:1:2)
Ethanol: acetone: n-hexane (1:1:2)		 甲醇:甲基叔丁基醚(1:1)
Methanol: methyl tert-butyl ether (1:1)		 YMC Carotenoid C30 (4.5 mm×250 mm，5 μm) 梯度洗脱
Gradient elution		 20 1 10 50 [24]
草莓
Strawberry		 UPLC
UPLC		 果实
Fruit		 4 皂化
Saponification		 丙酮:石油醚(1:2)
Acetone: petroleum ether (1:2)		 丙酮
Acetone		 C18 (2.1 mm×50 mm，1.7 μm) 等度洗脱
Isocratic elution		 27.4 0.5 50 5 [26]
南瓜
Pumpkin		 UPLC
UPLC		 果肉
Pulp		 4 皂化
Saponification		 乙醇
Ethanol		 — C18 (2.1 mm×100 mm，1.7 μm) 等度洗脱
Isocratic elution		 30 0.5 10 8 [17]
小麦
Wheat		 UPLC
UPLC		 麦粒
Kernel		 4 非皂化
Non saponification		 正己烷:丙酮(80:20)
N-hexane: acetone (80:20)		 甲醇:乙酸乙酯(68:32)
Methanol: ethyl acetate (68:32)		 YMC Carotenoid C30 (4.6 mm×100 mm, 3 μm) 梯度洗脱
Gradient elution		 35 0.4 8 25 [27]
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