Establishment of HPLC Method for Simultaneous Determination of Ten Carotenoids

doi:10.13560/j.cnki.biotech.bull.1985.2022-0122

Abstract

Abstract:

This study developed a method for simultaneously determining 10 carotenoids of lutein, zeaxanthin, α-cryptoxanthin, β-cryptoxanthin, ε-carotene, α-carotene, β-carotene, （6R）-δ-carotene, γ-carotene, and lycopene by reverse-phase C₃₀ high performance liquid chromatography. Carotenoid extracts for each sample were separated by a reverse-phase C₃₀column（YMC carotenoid C₃₀, 250 mm×4.5 mm, 5 μm）with its column temperature of（25±1）℃, and eluted using the mobile phase A（A1∶B1=9∶1, V/V）and B（A1∶B1=1∶9, V/V）with the flow rate of 1 mL/min. The mobile phase A1 consisted of methanol：water（97∶3, V/V）with 0.05 mol/L ammonium acetate and 0.1%（W/V）2, 6-di-tert-butyl-4-methylphenol（BHT）, and the mobile phase B1 consisted of 100% methyl tert-butyl ether with 0.1%（W/V）BHT. The effluents were then monitored at 450 nm using a photo diode array detector. The calibration curves of these ten carotenoids showed a good linearity with the concentration range of 0.5-20 μg/mL and linear relative coefficients（R²）were > 0.995；limit of detection（S/N=3）was 0.01-1.6 μg/mL, limit of quantitation（S/N=10）was 0.2-4.0 μg/mL, average recovery was 96.29%-104.47%, relative standard deviation was of 0.03%-1.24%, with 40 min of whole process. The method has been applied for measuring carotenoids content in banana fruit and maize kernel samples, i.e., verifying the stability, accuracy and reliability of this method in real agricultural biological samples.

Key words: reverse-phase C₃₀ column, high performance liquid chromatography, carotenoids, method establishment

KONG Qian, HUANG Wen-jie, WU Shao-wen, LI Kun, ZHANG Ming-wei, YAN Shi-juan. Establishment of HPLC Method for Simultaneous Determination of Ten Carotenoids[J]. Biotechnology Bulletin, 2022, 38(11): 80-89.

Figures/Tables 7

References 36

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组分 Compound	保留时间Ret- ention time/min	回归方程Calibration curve	相关系数Correlation coefficients R²	线性范围Linear range/（μg·mL^-1）	检出限Limit of detection（LOD）/（μg·mL^-1）	定量限Limit of quantification（LOG）/（μg·mL^-1）
叶黄素Lutein	8.25	y=0.5502x+0.0013	0.9999	0.5-20	0.06	0.3
玉米黄素Zeaxanthin	9.103	y=0.3099x-0.0144	0.9951	0.5-20	0.4	0.6
α-隐黄素α-Cryptoxanthin	11.41	y=0.7188x+0.0139	0.9999	0.5-20	0.01	0.2
β-隐黄素β-Cryptoxanthin	12.66	y=0.3981x-0.0215	1	0.5-20	0.2	0.4
ε-胡萝卜素ε-Carotene	13.987	y=0.6449x-0.0102	0.9999	0.5-20	0.02	0.2
α-胡萝卜素α-Carotene	15.41	y=0.4497x-0.0447	0.9998	0.5-20	1	2
β-胡萝卜素β-Carotene	17.067	y=0.6936x+0.0179	0.9998	0.5-20	0.2	0.6
（6R）-δ-胡萝卜素（6R）-δ-Carotene	23.767	y=0.3149x-0.0695	0.9999	0.5-20	0.25	1.4
γ-胡萝卜素γ-Carotene	25.653	y=0.0561x+0.0006	0.9998	0.5-20	1.6	4.0
番茄红素Lycopene	29.223	y=0.1429x+0.0011	0.9999	0.5-20	0.5	1.4

组分 Compound	保留时间Ret- ention time/min	回归方程Calibration curve	相关系数Correlation coefficients R²	线性范围Linear range/（μg·mL^-1）	检出限Limit of detection（LOD）/（μg·mL^-1）	定量限Limit of quantification（LOG）/（μg·mL^-1）
叶黄素Lutein	8.25	y=0.5502x+0.0013	0.9999	0.5-20	0.06	0.3
玉米黄素Zeaxanthin	9.103	y=0.3099x-0.0144	0.9951	0.5-20	0.4	0.6
α-隐黄素α-Cryptoxanthin	11.41	y=0.7188x+0.0139	0.9999	0.5-20	0.01	0.2
β-隐黄素β-Cryptoxanthin	12.66	y=0.3981x-0.0215	1	0.5-20	0.2	0.4
ε-胡萝卜素ε-Carotene	13.987	y=0.6449x-0.0102	0.9999	0.5-20	0.02	0.2
α-胡萝卜素α-Carotene	15.41	y=0.4497x-0.0447	0.9998	0.5-20	1	2
β-胡萝卜素β-Carotene	17.067	y=0.6936x+0.0179	0.9998	0.5-20	0.2	0.6
（6R）-δ-胡萝卜素（6R）-δ-Carotene	23.767	y=0.3149x-0.0695	0.9999	0.5-20	0.25	1.4
γ-胡萝卜素γ-Carotene	25.653	y=0.0561x+0.0006	0.9998	0.5-20	1.6	4.0
番茄红素Lycopene	29.223	y=0.1429x+0.0011	0.9999	0.5-20	0.5	1.4

组分Compound	本底值Background value /（μg·mL^-1）	加标量Standard addition /（μg·mL^-1）	检测值Detection value（μg·mL^-1，n=3）	回收率Recovery（%，n=3）	相对标准偏差Relative standard deviation（%，n=3）
叶黄素Lutein	2	0.5	2.5198	100.79	0.03
	2	3	4.9340	98.68	0.41
	2	8	10.1317	101.32	0.51
玉米黄素Zeaxanthin	2	0.5	2.4072	96.29	0.10
	2	3	5.0634	101.27	0.40
	2	8	10.5531	101.75	1.24
α-隐黄素α-Cryptoxanthin	2	0.5	2.5367	101.47	0.07
	2	3	4.9766	99.53	0.44
	2	8	10.1745	101.75	0.49
β-隐黄素β-Cryptoxanthin	2	0.5	2.5228	100.91	0.09
	2	3	4.9333	98.67	0.51
	2	8	10.002	100.02	0.40
ε-胡萝卜素ε-Carotene	2	0.5	2.6117	104.47	0.05
	2	3	4.9320	98.64	0.41
	2	8	10.0587	100.59	0.45
α-胡萝卜素α-Carotene	2	0.5	2.5573	102.29	0.10
	2	3	4.8304	96.61	0.36
	2	8	10.0834	100.83	0.51
β-胡萝卜素β-Carotene	2	0.5	2.5474	101.90	0.07
	2	3	4.9520	99.04	0.44
	2	8	10.2073	102.07	0.49
（6R）-δ-胡萝卜素（6R）-δ-Carotene	2	0.5	2.4594	98.37	0.24
	2	3	4.9525	99.05	0.49
	2	8	10.2073	101.46	0.61
γ-胡萝卜素γ-Carotene	2	0.5	2.5811	103.24	0.22
	2	3	5	100.00	0.57
	2	8	10.1171	101.17	0.88
番茄红素Lycopene	2	0.5	2.4726	98.90	0.10
	2	3	4.9596	99.19	0.47
	2	8	10.1059	101.06	0.46

组分Compound	本底值Background value /（μg·mL^-1）	加标量Standard addition /（μg·mL^-1）	检测值Detection value（μg·mL^-1，n=3）	回收率Recovery（%，n=3）	相对标准偏差Relative standard deviation（%，n=3）
叶黄素Lutein	2	0.5	2.5198	100.79	0.03
	2	3	4.9340	98.68	0.41
	2	8	10.1317	101.32	0.51
玉米黄素Zeaxanthin	2	0.5	2.4072	96.29	0.10
	2	3	5.0634	101.27	0.40
	2	8	10.5531	101.75	1.24
α-隐黄素α-Cryptoxanthin	2	0.5	2.5367	101.47	0.07
	2	3	4.9766	99.53	0.44
	2	8	10.1745	101.75	0.49
β-隐黄素β-Cryptoxanthin	2	0.5	2.5228	100.91	0.09
	2	3	4.9333	98.67	0.51
	2	8	10.002	100.02	0.40
ε-胡萝卜素ε-Carotene	2	0.5	2.6117	104.47	0.05
	2	3	4.9320	98.64	0.41
	2	8	10.0587	100.59	0.45
α-胡萝卜素α-Carotene	2	0.5	2.5573	102.29	0.10
	2	3	4.8304	96.61	0.36
	2	8	10.0834	100.83	0.51
β-胡萝卜素β-Carotene	2	0.5	2.5474	101.90	0.07
	2	3	4.9520	99.04	0.44
	2	8	10.2073	102.07	0.49
（6R）-δ-胡萝卜素（6R）-δ-Carotene	2	0.5	2.4594	98.37	0.24
	2	3	4.9525	99.05	0.49
	2	8	10.2073	101.46	0.61
γ-胡萝卜素γ-Carotene	2	0.5	2.5811	103.24	0.22
	2	3	5	100.00	0.57
	2	8	10.1171	101.17	0.88
番茄红素Lycopene	2	0.5	2.4726	98.90	0.10
	2	3	4.9596	99.19	0.47
	2	8	10.1059	101.06	0.46