一种同时测定十种类胡萝卜素的液相色谱方法的建立

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

摘要/Abstract

摘要：

建立了一种同时定量分析叶黄素、玉米黄素、α-隐黄素、β-隐黄素、ε-胡萝卜素、α-胡萝卜素、β-胡萝卜素、（6R）-δ-胡萝卜素、γ-胡萝卜素、番茄红素10种类胡萝卜素的方法。该方法的色谱条件为：YMC Carotenoid C₃₀（250 mm×4.5 mm，5 μm）色谱柱，柱温（25±1）℃，紫外检测波长450 nm，流速1 mL/min，进样体积10 μL，流动相分别由A1和B1按照不同比例混合制得A相和B相，A相中A1∶B1体积比为9∶1，B相中A1∶B1体积比为1∶9。其中，A1相为97%甲醇-水，含有0.05 mol/L乙酸铵和0.1%（W/V）2，6-二叔丁基-4-甲基苯酚（BHT）；B1相为100%甲基叔丁基醚（MTBE），含有0.1%（W/V）BHT，梯度洗脱。在0.5-20 μg/mL范围内，10种类胡萝卜素的质量浓度与峰面积呈良好的线性关系，相关系数（R²）均大于0.995，检出限（LOD，S/N=3）和定量限（LOQ，S/N=10）分别为0.01-1.6 μg/mL、0.2-4.0 μg/mL，且平均回收率在96.29%-104.47%之间，相对标准偏差（RSD）范围为0.03%-1.24%，整个过程可以在40 min内完成。应用此方法对香蕉和玉米样品中的类胡萝卜素含量进行测定，验证此方法在真实农业生物样品中的稳定性、准确性和可靠性。

关键词: 反相C₃₀柱, 高效液相色谱, 类胡萝卜素, 方法建立

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

孔谦, 黄文洁, 吴绍文, 李坤, 张名位, 晏石娟. 一种同时测定十种类胡萝卜素的液相色谱方法的建立[J]. 生物技术通报, 2022, 38(11): 80-89.

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.

图/表 7

表1 液相色谱分析的洗脱梯度

Table 1 Elution program for liquid chromatography

时间 Time/min	流动相A Mobile phase A/%	流动相B Mobile phase B/%
0	90	10
10	60	40
20	50	50
25	10	90
29	10	90
29.5	90	10
40	90	10

图1 10种类胡萝卜素单标色谱峰的保留时间（A）和光谱特性结果（B）

Fig. 1 Retention time of chromatogram peaks（A）and spectroscopic properties（B）of 10 carotenoids sole standards

图2 10种类胡萝卜素混合标准品的HPLC色谱图 1：8.25 min，叶黄素；2：9.103 min，玉米黄素；3：11.41 min，α-隐黄素；4：12.66 min，β-隐黄素；5：13.987 min，ε-胡萝卜素；6：15.41 min，α-胡萝卜素；7：17.067 min，β-胡萝卜素；8：23.767 min，（6R）-δ-胡萝卜素；9：25.653 min，γ-胡萝卜素；10：29.223 min，番茄红素

Fig. 2 Chromatograms of 10 carotenoids mixed standards by HPLC 1：8.25 min，lutein；2：9.103 min，zeaxanthin；3：11.41 min，α-cryptoxanthin；4：12.66 min，β-cryptoxanthin；5：13.99 min，ε-carotene；6：15.41 min，α-carotene；7：17.07 min，β-carotene；8，23.77 min，（6R）-δ-carotene；9：25.65 min，γ-carotene；10：29.22 min，lycopene

表2 10种类胡萝卜素保留时间、线性回归方程、相关系数、线性范围、检出限和定量限

Table 2 Retention time，calibration curves，correlation coefficients，linear ranges，LODs and LOQs of ten carotenoids

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

表3 10种类胡萝卜素的回收率和精密度

Table 3 Recoveries and accuracies of 10 carotenoids

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

图3 香蕉果肉和玉米籽粒中不同类胡萝卜素的HPLC色谱图样品测定使用的紫外波长450 nm，A/B/C/D香蕉色谱图；E/F/G/H玉米色谱图。A和E样品提取：不使用乙醇沉淀-使用提取溶剂1-使用皂化；B和F样品提取：不使用乙醇沉淀-使用提取溶剂2-使用皂化；C和G样品提取：使用乙醇沉淀-使用提取溶剂1-使用皂化；D和H样品提取：不使用乙醇沉淀-使用提取溶剂1-不使用皂化

Fig. 3 HPLC chromatogram of carotenoids from banana fruit and maize kernel The UV wavelength of 450 nm，A/ B/C/D for banana chromatogram；E/F/G/H for maize kernel chromatogram. A and E extractions：No ethanol precipitation-solvent 1- no saponification；B and F extractions：no ethanol precipitation-solvent 2-no saponification；C and G extractions：no ethanol precipitation-solvent 1-no saponification；D and H extractions：no ethanol precipitation-solvent 1-no saponification

图4 HPLC方法分析香蕉和甜玉米样品中类胡萝卜素含量 A-B：香蕉果肉样品的HPLC色谱图和类胡萝卜素绝对含量；C-D：玉米籽粒样品的HPLC色谱图和类胡萝卜素绝对含量

Fig. 4 Determination of carotenoids in banana fruit and maize kernel samples using HPLC A-B：Chromatograms and absolute contents of carotenoids in banana fruit samples detected by HPLC. C-D：Chromatograms and absolute contents of carotenoids in maize kernel samples detected by HPLC

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