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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (8): 2259-2274.doi: 10.3724/SP.J.1006.2023.24202

• TILLAGE & CULTIVATION · PHYSIOLOGY & BIOCHEMISTRY • Previous Articles     Next Articles

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 Online:2023-08-12 Published:2023-02-22
  • Contact: WANG Zhang-Ying E-mail:jiaruixue91@126.com;chenyihang2022@126.com;wangzhangying@gdaas.cn
  • 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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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

Fig. 1

Chromatograms of the different elution programs A: the chromatograms under the first elution condition; B: the chromatograms under the second elution condition; C: the chromatograms under the third elution condition; D-E: the chromatograms under the fourth elution condition, D-E: 10 and 13 carotenoids, respectively. 1: lutein; 2: zeaxanthin; 3: α-cryptoxanthin; 4: ε-carotene; 5: β-cryptoxanthin; 6: α-carotene; 7: β-carotene; 8: δ-carotene; 9: γ-carotene; 10: lycopene; 11: violaxanthin; 12: neoxanthin; 13: antheraxanthin."

Fig. 2

Chromatograms of carotenoid at different column temperatures A-E are chromatograms of carotenoids at 20, 23, 25, 28, and 30℃, respectively. 1: violaxanthin; 2: neoxanthin; 3: antheraxanthin; 4: lutein; 5: zeaxanthin; 6: α-cryptoxanthin; 7: ε-carotene; 8: β-cryptoxanthin; 9: α-carotene; 10: β-carotene; 11: δ-carotene; 12: γ-carotene; 13: lycopene."

Table 1

Resolution of 13 carotenoids under different chromatographic conditions"

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

Fig. 3

Chromatograms of the different flow rates A-C represents chromatograms of carotenoids at flow rates of 0.6, 0.8, and 1.0 mL min-1, respectively. 1: violaxanthin; 2: neoxanthin; 3: antheraxanthin; 4: lutein; 5: zeaxanthin; 6: α-cryptoxanthin; 7: ε-carotene; 8: β-cryptoxanthin; 9: α-carotene; 10: β-carotene; 11: δ-carotene; 12: γ-carotene; 13: lycopene."

Fig. 4

Chromatograms of the different injection volumes A-C represents the chromatograms of carotenoids at injection volumes of 1, 2, and 3 μL, respectively. 1: violaxanthin; 2: neoxanthin; 3: antheraxanthin; 4: lutein; 5: zeaxanthin; 6: α-cryptoxanthin; 7: ε-carotene; 8: β-cryptoxanthin; 9: α-carotene; 10: β-carotene; 11: δ-carotene; 12: γ-carotene; 13: lycopene."

Table 2

Regression equations, correlation coefficients, and detection limits of carotenoids"

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

Table 3

Relative standard deviations (RSD) of precision, repeatability, and stability"

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

Table 4

Recovery rate of 13 carotenoids"

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

Table 5

Carotenoid content of Gangshu 79 in the different extraction and resolution"

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

Table 6

Carotenoid content of different sweetpotato cultivars"

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

Table 7

Comparison of carotenoid detection methods among different species"

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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