Selenium Speciation in Broccoli by High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry
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摘要:
硒是一种典型的“双功能”元素，摄入不足或摄入过量均会对人体健康产生不利影响，硒的生物活性不仅取决于硒含量，还与硒的化学形态密切相关，因此对食品中不同硒形态进行分析研究具有重要的意义。本文采用高效液相色谱-电感耦合等离子体质谱(HPLC-ICP/MS)联用技术分析研究了市售西兰花中硒酸根[Se(Ⅵ)]、亚硒酸根[Se(Ⅳ)]、硒代胱氨酸(SeCys2)、甲基硒代半胱氨酸(MeSeCys)、硒代蛋氨酸(SeMet)。以蛋白酶XIV和Tris-HCl缓冲溶液超声提取西兰花中硒形态，采用C18反相色谱柱为分析柱，10mmol/L柠檬酸和5mmol/L己烷磺酸钠(pH=4.0，含1%甲醇)为流动相，等度洗脱，8min内可实现硒形态的有效分离测定，方法线性范围为0.3~100.0μg/L，线性相关系数(r)均大于0.999，Se(Ⅵ)、Se(Ⅳ)、MeSeCys、SeMet的检出限在1.2~6.0μg/kg(以Se计)范围内。对西兰花样品进行低、中、高三个浓度水平的加标回收试验，加标回收率为81.9%~105.3%，相对标准偏差(RSD)均小于5%。采用本方法分析欧盟有证标准物质——小麦粉(ERM® BC210a)中SeMet的测定值在其标准值范围内。实验结果表明建立的硒形态分析方法适用于西兰花中Se(Ⅵ)、Se(Ⅳ)、MeSeCys、SeMet的测定。检出的11个不同地区市售西兰花样品中硒形态主要为MeSeCys，含量在0.004~0.043mg/kg(以Se计)之间。对方法研究过程中发现的SeCys2稳定性差和不同类型西兰花中Se(Ⅳ)加标回收率差异较大的问题进行分析探讨，通过改变蛋白酶XIV的用量考察了SeCys2的稳定性，结合对西兰花样品基质的分析研究，发现SeCys2稳定性与蛋白酶XIV含量和西兰花基质有关；根据对3种不同类型的西兰花样品中Se(Ⅳ)加标回收试验结果及相关文献报道，推测样品中存在的大量酚类物质会影响Se(Ⅳ)的分析测定。
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关键词:
- 西兰花 /
- 硒形态 /
- 高效液相色谱-电感耦合等离子体质谱法 /
- 蛋白酶XIV
要点(1) 采用蛋白酶XIV和Tris-HCl缓冲溶液超声提取西兰花中硒形态。
(2) 采用C18反相色谱柱为分析柱，柠檬酸和己烷磺酸钠为流动相，HPLC-ICP/MS分析西兰花中硒形态。
(3) 西兰花样品中硒形态主要为甲基硒代半胱氨酸(MeSeCys)。
(4) SeCys2稳定性与蛋白酶XIV含量和西兰花基质有关。
HIGHLIGHTS(1) Selenium speciation in broccoli was extracted by proteinase XIV and Tris-HCl buffer solution.
(2) HPLC-ICP-MS equipped with ZORBAX SB-Aq C18 reversed-phase column with 10mmol/L citric acid and 5mmol/L sodium hexane-sulfonate as mobile phase was applied to analyze the selenium speciation in broccoli.
(3) Methylselenocysteine is the main selenium speciation in broccoli.
(4) The stability of the SeCys2 standard solution is influenced by the proteinase XIV content and the sample matrix.
Abstract:BACKGROUNDSelenium is an essential trace element and a typical bifunctional element that can affect human health if consumed in insufficient or excessive amounts. The biological activity of selenium depends not only on its intake level but also on its chemical speciation. Selenium comes in various speciation and is divided mainly into inorganic and organic selenium. Inorganic selenium includes selenate [Se(Ⅵ)] and selenite [Se(Ⅳ)], and organic selenium mainly includes selenocysteine (SeCys2), selenomethionine (SeMet), and methylselenocysteine (MeSeCys). It has been found that organic selenium has high bioactivity and bioavailability. At present, while the nutritional effects of selenium are drawing more and more attention, it is very important to analyze and study the different speciation of selenium in food. Since the analysis of selenium speciation is closely related to the sample matrix, the extraction efficiency and stability of different selenium speciation are also related to many factors. At present, the analysis method of selenium speciation in food is still in the research stage. Broccoli is rich in nutrients, such as protein, flavonoids, polyphenols, and vitamins, and is widely loved by people because it contains many kinds of thioglucosides and has a strong ability to gather selenium, which has antioxidant and anti-cancer medical values. Therefore, the analysis and study of selenium speciation in broccoli is of some significance.
OBJECTIVESTo establish a method for the determination of Se(Ⅵ), Se(Ⅳ), SeCys2, MeSeCys, and SeMet in commercial broccoli by high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS).
METHODSFirstly, the chromatographic conditions were selected by examining the separation and sensitivity of Se(Ⅵ), Se(Ⅳ), SeCys2, MeSeCys, and SeMet on a Hamilton PRP-X100 anion column with 40mmol/L diammonium hydrogen phosphate (pH=5 with 1% methanol) as the mobile phase and on a ZORBAX SB-Aq C18 reversed-phase column with 10mmol/L citric acid plus 5mmol/L sodium hexane-sulphonate (pH=4 with 1% methanol) as the mobile phase. Secondly, the sample pretreatment conditions were optimized, including the selection of extraction reagents, the amount of extraction reagents, and extraction time. Four extraction reagents (ultrapure water, Tris-HCl buffer solution, proteinase XIV and complex proteinase) were selected for optimization. The effect of proteinase XIV concentration on the extraction was investigated by adding 2, 4, 6, and 8mg/mL proteinase XIV to broccoli samples with selenium content of 0.81mg/kg (calculated as Se). The effect of adding 6mL, 10mL, 12mL, and 15mL of Tris-HCl buffer solution on the extraction was compared. The extraction time of the samples also had a great influence on the extraction efficiency of the selenium speciation. The effects of four extraction times of 1h, 3h, 5h and 7h on the extraction were compared.
RESULTSThe ZORBAX SB-Aq C18 separation system was used in this study because of the short analysis time and high sensitivity of each selenium speciation. Protease XIV was the most effective extraction reagent for selenium; therefore proteinase XIV was chosen as the extraction reagent. The concentration of selenium speciation increased with the concentration of proteinase XIV. The maximum concentration of selenium speciation was reached when the concentration of proteinase XIV was 6mg/mL. It was reported that the use of Tris-HCl buffer solution with proteinase XIV at appropriate pH conditions could further improve the extraction efficiency and maintain the stability of selenium speciation. The volume of Tris-HCl buffer increased, the extraction efficiency of each selenium speciation gradually increased and then decreased, and the final selection of Tris-HCl buffer solution addition was 12mL. A longer extraction time would help to increase the extraction effect, but too long an enzymatic digestion time would also cause a decrease in the stability of SeMet and SeCys2. To ensure high extraction efficiency and reduce the conversion of selenium speciation, an extraction time of 3h was preferred. After optimization and selection, the final analysis method was determined as follows: weighing a certain amount of broccoli sample into 12mL of Tris-HCl (pH=7.4, containing 6mg/mL proteinase XIV) at a concentration of 100mmol/L, vortexing and mixing, and then sonicating at 37℃ for 3h. After centrifugation, the extraction were eluted with 10mmol/L citric acid and 5mmol/L sodium hexane sulfonate (pH=4 with 1% methanol) on ZORBAX SB-Aq C18 reversed-phase column. ICP/MS was used for analysis and determination.
This method can achieve effective separation and determination of five selenium speciation within 8 minutes. The linearity range of the method was 0.3-100.0μg/L, with linear correlation coefficients (r) greater than 0.999. The detection limits of Se(Ⅳ), Se(Ⅵ), MeSeCys, and SeMet were within the range of 1.2-6.0μg/kg (calculated as Se). The standard recovery tests were carried out on broccoli samples at low, medium, and high concentration. The recoveries of these four selenium speciation, Se(Ⅵ), Se(Ⅳ), MeSeCys and SeMet, were 81.9%-105.3% with relative standard deviations (RSD) less than 5%. The method established in this study was used to determine SeMet in the EU-certified reference material (ERM BC210a, wheat flour), and the measured value of SeMet was within the range of its standard values.
More than 20 commercially available broccoli samples collected from different regions of China were analyzed and determined. The results showed that the selenium speciation in commercially available broccoli was mainly MeSeCys, with small amounts of Se(Ⅵ), Se(Ⅳ), and SeMet, and also a small amount of unknown selenium-containing compounds was also present.Two problems identified in the methodological study were explored. (1) The effect of proteinase XIV dosage on the stability of SeCys2 was investigated by adding 1, 2, 4, and 6mg/mL of proteinase XIV to SeCys2 standard solution, respectively. The results showed that as the concentration of proteinase XIV increased, the signal value of SeCys2 gradually decreased and the signal value of three unknown peaks gradually increased. At the same time, the recovery of SeCys2 in broccoli samples decreased to 10%. Based on the above conditions, it is assumed that the content of proteinase XIV and the matrix of broccoli samples affect the stability of SeCys2.
(2) Three different broccoli samples were selected for Se(Ⅳ) standard recovery tests: fresh commercially available broccoli samples, freeze-dried powder of commercially available broccoli, and freeze-dried powder of broccoli fortified with Se(Ⅳ) selenium fertilizer. A certain amount of the above three samples was added with Se(Ⅳ) standard solution and 100mmol/L Tris-HCl (pH=7.4, containing 6mg/mL of proteinase XIV). The determination was then carried out according to the proposed analytical method and the mean recoveries of the three samples were found to be 81.1%, 69.5% and 1.53%, respectively. The Kruskal-Wallis rank sum test showed that the recoveries of Se(Ⅳ) were significantly different among the three samples (p < 0.05). Previous investigations have found that phenolic substances can affect the stability of Se(Ⅳ) and that the addition of selenium fertilizer during the growth of broccoli can change the phenolics. Based on the above, it is assumed that the presence of phenolics in broccoli samples may affect the determination of Se(IV).
CONCLUSIONSA method for the determination of Se(Ⅳ), Se(Ⅵ), MeSeCys, and SeMet in commercially available broccoli by HPLC-ICP-MS is established by selecting and optimizing the sample pretreatment and analytical conditions. The Tris-HCl buffer solution containing proteinase XIV is chosen for the extraction of samples.
It is found that the stability of SeCys2 is affected by the concentration of proteinase XIV and broccoli samples matrix. It is hypothesized that the presence of large amounts of phenolics in the samples can affect the determination of Se(Ⅳ) for reasons to be further explored.
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碲和硒是稀散元素，在高新科技领域具有重要应用，已被中国和欧美国家列为战略性关键矿产资源［1-2］。一直以来全球碲、硒矿产资源主要采自斑岩-矽卡岩铜金矿床，如中国广东大宝山铜矿和江西城门山铜矿［3-4］，研究斑岩矿床中碲、硒的产出情况对国家资源战略保障具有重要意义。云南普朗斑岩型铜金矿床位于三江特提斯成矿域义敦岛弧南部，属于超大型斑岩矿床，已探明铜资源储量4.31Mt，金资源量113t［5］。矿区内出露的地层为中三叠统尼汝组和上三叠统图姆沟组，侵入岩为普朗复式岩体，由石英闪长玢岩（～216Ma）、石英二长斑岩（～215Ma）和花岗闪长斑岩（～206Ma）组成，岩体出露总面积约为11km2（图1）。前人对普朗矿床的地质特征、成岩成矿时代、成矿物质来源、成矿流体性质等作了大量工作，但对矿床中碲硒的含量和赋存状态等研究还较为薄弱。本文报道了普朗矿床中产出的碲化物和硒化物，以期为斑岩矿床中碲硒的勘查和综合利用提供资料。
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本次研究对象主要为普朗矿床中的铜精矿和钼精矿样品，测试分析均在东华理工大学核资源与环境国家重点实验室完成。样品的矿相学观察利用ZEISS Axio Scope A1光学显微镜及ZEISS Sigma 300场发射扫描电镜完成，扫描电镜的加速电压为20kV，发射电流为10μA［6］。矿物成分利用JXA-8530F Plus型电子探针分析完成，实验设定加速电压为15kV，电流为20nA，探针直径为1μm，使用ZAF方法对X射线强度进行校正。分析标样选择砷化镓（As），黄铜矿（Cu），黄铁矿（Fe、S），自然银（Ag），碲铋矿（Te、Bi），辉钼矿（Mo），自然铅（Pb），自然锑（Sb），硒化镉（Se），自然金（Au），自然铂（Pt），自然钯（Pd）。测试主量元素的精确度和准确度均小于2%。
普朗铜金矿床中的碲和硒含量高，并形成大量碲化物、硒化物和富硒矿物。矿床精矿中的碲和硒含量分别达74.3×10−6和270×10−6。碲在钾化带中的含量为0.3×10−6～0.43×10−6，较绢英岩化带中的高（0.02×10−6～0.12×10−6），由矿体中心向外，碲品位逐渐降低［7］。硒在钾化带和绢英岩化带的含量无明显差别，分别为1.49×10−6～2.44×10−6和1.04×10−6～3.00×10−6。矿石中的碲与金呈正相关关系，硒与银呈正相关关系。普朗铜矿床中，碲和硒主要以碲化物、硒化物和富硒矿物形式存在，形成辉碲铋矿、碲钯矿、硒银矿和富硒方铅矿等（图2）。辉碲铋矿是普朗含量最多的碲化物，反射光下为白色略带淡蓝色，矿物成分较均一，Bi含量为58.36%～61.24%，Te含量为31.03%～34.50%，S含量为3.76%～4.54%（图2e）。普朗辉碲铋矿中含有较高的Se（0.77%～3.63%）。辉碲铋矿的化学式为Bi2.02～2.08（Te1.74～1.93S0.85～1.01Se0.08～0.33）2.90～2.98。碲钯矿属于独立铂族元素矿物，在自然界很少见，中国斑岩矿床中仅江西德兴有报道［8］，在全球其他斑岩矿床中非常少见。普朗碲钯矿粒径为1～5μm，反射光下呈亮白色（图2a）。碲钯矿中Pd和Pt可以类质同象取代，因此含量变化较大，Pd含量为16.26%～25.69%，Pt含量为4.82%～17.66%，Te含量为61.25%～66.76%（图2f）。碲钯矿化学式为（Pd0.64～0.98Pt0.09～0.37）0.98～1.03Te1.97～1.02。硒银矿是普朗含量最多的硒化物，反射光下为白色带微蓝绿色（图2c）。硒银矿中普遍含S，含量为0.55%～2.65%，Ag含量普遍偏低，为70.22%～72.77%，Se含量为24.09%～27.31%（图2g）。硒银矿化学式为Ag1.89～1.98（Se0.87～1.01S0.05～0.24）1.02～1.11。富硒方铅矿属于PbS1-xSex矿物，其中x值可在0～1之间连续变化。普朗富硒方铅矿S和Se的含量变化大，分别为4.01%～12.52%和1.85%～19.13%，Pb含量为73.91%～82.52%，大多数样品中含有Ag，最高含量达1.61%。普朗富硒方铅矿形成了较完整的PbS-PbSe固溶体系列（图2h），化学式为Pb0.98～1.01（S0.35～0.97Se0.07～0.67）0.99～1.02。
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图 2 碲硒矿物显微照片及矿物元素含量三元图a—碲钯矿反射光镜下照片； b—碲钯矿BSE照片； c—硒银矿反射光镜下照片； d—硒银矿BSE照片； e— Bi-Te-S体系三元图； f— Te-Pd-Pt体系三元图； g—Ag-Se-S体系三元图； h—Pb-Se-S体系三元图。Mol—辉钼矿； Mrk—碲钯矿； Nau—硒银矿； Py—黄铁矿。Figure 2. Photomicrographs of tellurium and selenium minerals and ternary plots of element contents. a—Reflected light photomicrograph of merenskyite; b—BSE image of merenskyite; c—Reflected light photomicrograph of naumannite; d—BSE image of naumannite; e—Ternary plot of Bi-Te-S system; f—Ternary plot of Te-Pd-Pt system; g—Ternary plot of Ag-Se-S system; h—Ternary plot of Pb-Se-S system. Mol=Molybdenite, Mrk=Merenskyite, Nau=Naumannite, Py=Pyrite.矿床中的碲和硒可以指示物质来源和成矿过程。碲和硒具有亲硫特点，碲会部分进入硫化物晶格，但更易形成碲的独立矿物;硒属于强亲硫元素，在较高温的条件下易于进入硫化物晶格，在中低温条件下，硫含量较低时，可形成硒的独立矿物。洋壳中的铁锰结壳、页岩及浮游沉积物等是自然界中碲和硒的重要储库［9］，因此在洋陆俯冲过程中，大陆岩石圈地幔和洋壳的部分熔融会形成富碲、硒的岩浆［10-11］。碲和硒在硫化物熔体中的相容性很高（D硫化物/硅酸盐＞600），碲倾向于存在液相硫化物（SL）中，而硒则更易进入单硫化物固熔体（MSS）（DTe SL/硅酸盐/DSe SL/硅酸盐为5～9，DTe MSS/硅酸盐/DSe MSS/硅酸盐为0.5～0.8）［12］。当富碲、硒的岩浆到达下地壳，会结晶分异形成富Co、Ni的硅酸盐矿物，碲、硒存在硫化物熔体中继续向上运移；当岩浆到达中地壳，温度低于900℃时，硫化物熔体与Te-Se熔体发生相分离；当岩浆到达上地壳，侵位形成班岩体及Cu矿床，Ag-Pt-Pd则高度集中在富Te-Se熔体中，并最终形成贵金属矿物［13］。普朗铜金矿床中的碲和硒可能与区内晚三叠世的俯冲造山密切相关，富碲和硒的岩浆也促进了铂族元素的富集成矿。
普朗斑岩铜金矿床中碲化物和硒化物的发现，对资源的综合利用及矿床成因研究具有重要意义。矿床中碲和硒的资源量规模大，大部分以独立矿物形式存在，且常与Au-Ag-PGE共生，具有较好的经济回收利用价值。碲化物和硒化物的产出也为成矿物质来源及岩浆演化过程提供了新的研究方向。
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图 1 五种形态硒混合标液在不同色谱柱下的色谱图(10μg/L)
(a)Hamilton PRP-X100色谱柱；(b)ZORBAX SB-Aq C18色谱柱。
Figure 1. Chromatograms of 5 forms of selenium mixed standard solution under different columns (10μg/L).
(a) Hamilton PRP-X100 column; (b) ZORBAX SB-Aq C18 column.
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图 2 不同浓度的蛋白酶XIV对西兰花样品中5种硒形态的提取效果
Figure 2. Effect of different concentrations of proteinase XIV on the extraction of five selenium speciation from broccoli samples. As can be seen from the graph, the content of the five selenium speciation increases and then decreases with the increase of concentration of proteinase XIV. The best extraction efficiency was reached when the concentration of proteinase XIV was 6mg/mL.
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图 3 不同体积的缓冲溶液对西兰花样品中5种硒形态提取效果的影响
Figure 3. Effects of different volumes of buffer solution on the extraction of five selenium speciation from broccoli samples. The graph shows that the best extraction results were obtained when the buffer solution was added at 12mL.
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图 4 不同浓度蛋白酶XIV对SeCys2标准溶液稳定性的影响
(a) 蛋白酶XIV浓度为1mg/mL；(b)蛋白酶XIV浓度为2mg/mL；(c)蛋白酶XIV浓度为4mg/mL；(d)蛋白酶XIV浓度为6mg/mL。
Figure 4. Effect of different concentrations of proteinase XIV on stability of SeCys2 standard solutions. The concentration of proteinase XIV is: (a) 1mg/mL; (b) 2mg/mL; (c) 4mg/mL; (d) 6mg/mL.
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图 5 样品色谱图
Figure 5. Chromatograms of samples. The chromatogram shows that the predominant speciation of selenium in the sample is methylselenocysteine.
表 1 不同提取剂对西兰花样品中硒形态提取效果的影响
Table 1 Extraction results of selenium speciation in broccoli sample using different extractants. As shown in the table, proteinase XIV is the best to use for extracting.
提取剂 Se(Ⅵ)含量(mg/kg) Se(Ⅳ)含量(mg/kg) SeCys2含量(mg/kg) MeSeCys含量(mg/kg) SeMet含量(mg/kg) 5种硒形态含量之和(mg/kg) 超纯水 0.029 0.020 0.019 0.136 0.051 0.255 100mmol/L Tris-HCl缓冲液 0.025 0.021 0.017 0.124 0.012 0.199 蛋白酶XIV 0.026 0.018 0.042 0.140 0.300 0.526 复合蛋白酶 0.028 0.015 0.000 0.108 0.244 0.395 
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表 2 方法线性方程、相关系数和检出限
Table 2 Linear equations, correlation coefficients, and detection limit of the method.
硒形态 线性范围(μg/L) 线性方程 相关系数(r) 定量限(μg/kg) 方法检出限(μg/kg) Se(Ⅵ) 0.9~100.0 y=2243.1x-650.8 0.9999 10.8 3.6 Se(Ⅳ) 0.6~100.0 y=2165.7x-412.7 0.9999 7.2 2.4 SeCys2* 1.0~100.0 y=2183.6x-765.0 1.0000 - - MeSeCys 0.3~100.0 y=2385.0x-1544.4 0.9999 3.6 1.2 SeMet 1.5~100.0 y=2169.5x-607.9 1.0000 18.0 6.0 注：“*”表示因SeCys2的加标回收率低于80%无法准确定量，故未计算方法检出限。
Note: “*” indicates that the detection limit of the method was not calculated because the spiked recovery of SeCys2 was less than 80% and could not be accurately quantified.
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表 3 西兰花精密度及加标回收率测定结果(n=6)
Table 3 Determination results of precision and recovery rate of broccoli (n=6).
硒形态 本底值(mg/kg) 加标量(mg/kg) 6次测定值(mg/kg) 加标回收率(%) RSD(%) 0.12 0.123 0.126 0.123 0.125 0.126 0.124 102.2~105.3 1.0 Se(Ⅵ) ND 0.36 0.366 0.367 0.360 0.366 0.369 0.366 100.0~102.1 1.6 0.60 0.609 0.620 0.594 0.608 0.614 0.604 99.0~103.3 1.3 0.12 0.099 0.100 0.099 0.100 0.100 0.100 82.7~85.2 1.0 Se(Ⅳ) ND 0.36 0.305 0.295 0.296 0.295 0.296 0.301 81.9~85.6 1.7 0.60 0.501 0.505 0.502 0.500 0.505 0.497 82.8~84.1 0.7 0.12 0.009 0.010 0.010 0.010 0.011 0.010 7.91~8.77 4.4 SeCys2 ND 0.36 0.036 0.034 0.034 0.034 0.034 0.036 9.47~9.97 2.0 0.60 0.060 0.061 0.058 0.063 0.063 0.062 9.74~10.5 2.5 0.12 0.107 0.106 0.107 0.108 0.108 0.106 88.1~89.7 0.7 MeSeCys ND 0.36 0.323 0.311 0.313 0.315 0.317 0.317 86.4~89.6 1.4 0.60 0.544 0.542 0.544 0.554 0.553 0.554 90.4~92.4 1.3 0.12 0.126 0.124 0.125 0.125 0.127 0.127 98.4~102.9 0.7 SeMet ND 0.36 0.350 0.354 0.349 0.350 0.353 0.353 97.0~98.2 0.8 0.60 0.591 0.595 0.595 0.599 0.617 0.605 98.4~102.9 1.9
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表 4 三个不同的西兰花中Se(Ⅳ)加标回收实验结果(n=3)
Table 4 Analytical results of spiked recovery test of Se(Ⅳ) for three broccoli samples (n=3).
样品名称 本底浓度(mg/kg) 加标量(mg/kg) 3次测定加标回收率(%) 平均加标回收率(%) H值 P值 西兰花(a) ND 0.11 81.3 81.0 81.1 81.1 西兰花粉末(b) ND 0.40 68.1 68.4 72.1 69.5 7.20 0.027* 西兰花粉末(c) 0.008 0.40 1.55 1.53 1.53 1.53 注：ND表示低于检出限；“*”：P值小于0.05为差异具有统计学意义。
Note: ND indicates below detection limit; “*” indicates that p-value of less than 0.05 is considered a statistically significant difference.
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