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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硒是人体必需的微量元素，具有多种形态，主要分为无机硒和有机硒，无机硒主要包括硒酸盐[Se(Ⅵ)]、亚硒酸盐[Se(Ⅳ)]等，有机硒主要包括硒代胱氨酸(SeCys2)、硒代蛋氨酸(SeMet)、甲基硒代半胱氨酸(MeSeCys)等硒代氨基酸，研究表明有机硒具有较高的生物活性和生物利用度[1-5]，对健康有益。因此，在硒的营养效应受到日益关注的同时，硒和硒形态的分析研究越来越受到重视。由于硒形态分析与样品基质密切相关，硒形态自身不稳定以及提取试剂的影响[6]，目前尚无统一的食品中硒形态分析检测标准。西兰花复含蛋白质、黄酮、多酚及维生素等[7-8]营养物质，且含硫代葡萄糖苷和较强的聚硒能力，具有抗氧化、抗癌等医用价值[9-10]，对西兰花中硒形态分析研究具有重要的意义。
目前的硒形态分析方法主要有高效液相色谱-原子荧光光谱法(HPLC-AFS)[11-14]、高效液相色谱-电感耦合等离子体质谱法(HPLC-ICP/MS)[15-16]、液相色谱-高分辨率质谱如液相色谱-四极杆/静电场轨道阱高分辨质谱(LC-Q Exactive Orbitrap MS)[4]、气相色谱-串联质谱法(GC-MS/MS)[17]、毛细管电泳-电感耦合等离子体质谱法(CE-ICP/MS)[18]。液相色谱-高分辨率质谱根据化合物的分子离子、碎片离子信息及分子裂解机理等确定分子结构，进行定性定量分析，可对无标准物质的硒形态进行鉴定分析，但因价格昂贵其应用受到较大的限制；GC-MS/MS适用于易挥发硒形态的测定，对于难挥发性硒形态需经过衍生化处理，而多数样品需要衍生，分析步骤繁琐且易发生形态转化；CE-ICP/MS虽然分离度好，但受接口、进样以及化学基体效应等因素的限制，应用受限[19]；HPLC-AFS和HPLC-ICP/MS因接口简单、灵敏度高、方便快捷等优点已成为硒形态检测的主流方法。HPLC-AFS以操作简单、成本低[20]广泛应用于硒形态分析，但HPLC-AFS灵敏度相对较低，对于硒含量低的样品存在一定的局限性；ICP-MS具有高选择性和高灵敏度，与HPLC联用是硒形态最有力的分析技术。目前对于西兰花中硒形态的分析研究多采用HPLC-AFS。陆晓奇等[12]将富硒植物干粉溶于Tris-HCl缓冲液并依次加入纤维素酶、蛋白酶K和蛋白酶XIV，于气浴恒温振荡器中酶解，采用HPLC-UV-AFS检测，样品提取效果好，但提取时间近42.5h；刘为等[13]采用蛋白酶K和蛋白酶E对富硒农产品进行酶解，水浴振荡提取，采用HPLC-HG-AFS分析了富硒西兰花干粉中4种硒形态SeCys2、SeMet、MeSeCys、硒代乙硫氨酸(SeEt)，分析效果较好，但方法检出限为0.86~2.79μg/L，灵敏度有待进一步提高。Pedrero等[21]采用HPLC-ICP/MS对富硒西兰花中Se(Ⅳ)、SeCys2、SeMet、MeSeCys进行检测，10min内实现4种硒形态的分离，分析时间较长。
为了提高检测灵敏度，缩短分析时间，本研究应用HPLC-ICP/MS，以ZORBAX SB-Aq C18反相离子对色谱柱为分析柱，对市售西兰花中Se(Ⅵ)、Se(Ⅳ)、SeCys2、MeSeCys、SeMet共5种硒形态进行研究，为研究西兰花的营养价值提供支持。
1. 实验部分
1.1 仪器
1260型高效液相色谱仪及7700x型电感耦合等离子体质谱仪(美国Agilent公司)；Milliplus 2150超纯水处理系统(美国Millipore公司)；超声波清洗机(宁波新芝生物科技股份有限公司)；冷冻离心机(美国Beckman公司)。
1.2 样品与主要试剂
供试样品：①市售西兰花：从北京市的超市及网上采购的山东、广东、河北、云南等各地西兰花样品；②选一购于浙江临海的市售西兰花样品均质(为a)并进行冷冻干燥成粉末(为b)；③经含Se(Ⅳ)的硒肥强化后的西兰花冷冻干燥粉末(为c, 北京农林科学院农产品加工与食品营养研究所提供)。
超纯水(电阻率18.2MΩ·cm，由超纯水处理系统制备)；柠檬酸(优级纯，国药集团化学试剂有限公司)；己烷磺酸钠(优级纯，国药集团化学试剂有限公司)；甲醇(HPLC级，美国Sigma公司)；氨水(优级纯，国药集团化学试剂有限公司)；蛋白酶XIV(美国Sigma公司)；三羟基甲基氨基甲烷盐酸盐(99.0%，美国Sigma公司)；亚硒酸根离子溶液(GBW10032)、硒酸根离子溶液(GBW10033)、甲基硒代半胱氨酸(GBW10088)、硒代蛋氨酸(GBW10034)、硒代胱氨酸(GBW10087)购于中国计量科学研究院，欧盟有证标准物质富硒小麦粉(ERM®-BC210a，购于LGC标准品公司)。
本文中的硒含量均是以Se计。
1.3 仪器条件
1.3.1 色谱条件
色谱柱：安捷伦ZORBAX SB-Aq C18(250mm×4.6mm，5μm)；流动相：10mmol/L柠檬酸及5mmol/L己烷磺酸钠(含1%甲醇，pH=4.0)；流速0.8mL/min；进样量20μL。
1.3.2 质谱条件
RF入射功率1550W；载气：高纯氩气；载气流速0.65L/min；补偿气流速0.45L/min；冷却气流速15L/min；采样锥、截取锥：镍锥；射频电压1.80Ⅴ；采样深度8.0mm；泵速0.3r/s；检测同位素：78Se。
1.4 硒形态的样品前处理
取1.0~1.5g西兰花样品于聚丙烯离心管中，若冷冻干燥的粉末则取0.3g，加入12mL 100mmol/L三羟基甲基氨基甲烷盐酸盐缓冲液(Tris-HCl，pH=7.4，含6mg/mL的蛋白酶XIV)，涡旋混匀后于37℃下加热超声3h，在4℃下9000r/min离心10min，取上清液经0.22μm水系滤膜过滤，同时做试剂空白。
2. 结果与讨论
2.1 色谱条件的选择
硒形态分析常用Hamilton PRP-X100阴离子交换色谱柱(4.1mm×250mm，10μm)[22-25]和反相离子对色谱柱[26-27]，其中以Hamilton PRP-X100色谱柱居多，但在分析植物样品时，由于植物样品以酶解法提取时会有较多的酶残留在提取液中，这些大分子物质会对色谱柱造成一定的损害，导致色谱柱的使用寿命缩短[28]。Hamilton PRP-X100与磷酸盐体系分析SeCys2、MeSeCys、Se(Ⅳ)、SeMet和Se(Ⅵ)这5种硒形态在16min内可完成分离，SeMet和Se(Ⅵ)灵敏度明显低于其他三个硒形态，色谱图见图 1a。
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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.张珂等[26]和姚晓慧等[27]使用ZORBAX SB-Aq C18反相离子对色谱柱与柠檬酸体系加入离子对试剂——己烷磺酸钠可以在8min内实现所测硒形态的有效分离，且各硒形态灵敏度较高。其中姚晓慧等[27]采用等度洗脱方式更简便，时间更短。因此本实验采用此色谱条件进行分析研究，色谱图见图 1b。
2.2 样品前处理条件优化
植物中硒的提取方法有超纯水提、酸提[29]、醇提以及酶解法[23]。采用超纯水、酸、醇提取一般只能提取无机硒及水溶性氨基酸，对植物体内与大分子蛋白结合的硒提取效果较差，且酸提取会破坏植物体中硒形态。酶解法可将与蛋白结合的硒分离且方法温和，有利于有机硒的提取[28]，该法时间长，为缩短提取时间和减少硒形态转变，可采用超声辅助萃取和微波辅助萃取。本研究采用超声辅助提取，对提取试剂和蛋白酶的种类、用量以及提取时间进行优化。
2.2.1 提取试剂的选择
本研究选择一个硒含量为0.81mg/kg的西兰花样品，以超纯水、缓冲溶液、蛋白酶XIV和复合蛋白酶作为提取剂，通过超声辅助提取考察对西兰花样品的提取效果。表 1实验数据表明，蛋白酶XIV作为提取试剂时硒的提取效果最佳。因此本研究选用蛋白酶XIV为提取试剂进行后续硒形态的分析研究。
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 2.2.2 蛋白酶XIV用量的优化
选用蛋白酶XIV为提取试剂，为进一步考察蛋白酶XIV含量对提取效果的影响，应用含蛋白酶XIV浓度为(2、4、6、8mg/mL)的Tris-HCl缓冲液(pH=7.4)对西兰花中5种硒形态进行提取，实验结果见图 2。结果表明，5种硒形态含量随蛋白酶XIV浓度增加而增加，当蛋白酶XIV浓度为6mg/mL时提取效果最好；当蛋白酶XIV浓度增加到8mg/mL时SeCys2、MeSeCys、SeMet的提取效果有所下降，Se(Ⅳ)和Se(Ⅵ)提取效果无差别。因此，选择蛋白酶XIV浓度为6mg/mL。
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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.2.2.3 缓冲溶液加入量的优化
Tris-HCl缓冲溶液作为核酸和蛋白质溶剂，在适宜的pH下，与蛋白酶XIV配合使用作为硒形态分析的提取剂具有较好的效果[30]。本研究选择Tris-HCl缓冲溶液为提取溶液，并考察了缓冲溶液(pH=7.4，含6mg/mL蛋白酶XIV)加入量(6、10、12、15mL)对5种硒形态的提取效果，结果见图 3。
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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.随缓冲溶液体积增加，各形态的提取效果先逐渐升高后降低，与林樾[31]研究结果一致，可能是适宜的缓冲溶液体积使样品溶解度变高，蛋白质分子更易扩散，酶对样品的水解更加完全，从而达到更好的提取效果。当到达最大溶解度后，随缓冲溶液体积提高，样品浓度变小导致提取率下降。另外提取溶剂的增加会导致硒浓度降低，因此最终Tris-HCl缓冲溶液的加入体积为12mL。
2.2.4 提取时间的优化
为考察提取时间对提取效果的影响，设置了1h、3h、5h、7h四个提取时间对5种硒形态的提取效果进行研究。实验结果显示，提取时间从1h延长至3h，提取出的各硒形态含量均有提高(仅SeCys2有轻微下降)。3h与5h提取效果相当，略低于7h提取效果，增加提取时间提取效果未有显著变化，长时间酶解条件下会引起SeMet以及SeCys2稳定性降低[32-33]，因此最终选择提取时间为3h，既缩短分析时间也减少硒形态间的转换。
2.3 方法线性范围和检出限
分别配制0.0、0.5、1.0、5.0、10.0、25.0、50.0和100.0μg/L的Se(Ⅵ)、Se(Ⅳ)、SeCys2、MeSeCys和SeMet共5种硒形态混合标准系列，在优化好的实验条件下进行线性范围实验。实验结果表明在0.3~100.0μg/L范围内5种硒形态线性关系良好，线性相关系数(r)均大于0.999。
在空白样品中分别加入0.5、0.4、0.3、0.2、0.1、0.05μg/L的标准溶液进行硒形态含量测定，根据各硒形态的测定结果信号值与3倍信噪比(S/N)相对应的浓度为硒形态的检出限，定量限为检出限的3倍。当市售西兰花样品的称样量为1.0g，加入提取剂为12mL时，计算方法检出限，结果见表 2。
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.2.4 方法正确度和重复性
2.4.1 样品加标回收和精密度试验
选取一个市售西兰花样品，同时制备6个样品，分别添加三个不同浓度水平的5种硒形态混合标准溶液进行加标回收和精密度试验，测定结果见表 3。Se(Ⅵ)、Se(Ⅳ)、MeSeCys、SeMet的加标回收率在81.9%~105.3%范围内，SeCys2的加标回收率为7.91%~10.5%。5种硒形态的RSD均小于5%。
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 针对西兰花中SeCys2加标回收率低的现象进行了进一步研究，在空白试剂中加入不同浓度(1、2、4、6mg/mL)的蛋白酶XIV及10μg/L的SeCys2标准溶液，应用所建立的方法进行加标回收试验，考察提取体系对SeCys2稳定性的影响，发现随蛋白酶XIV浓度升高，SeCys2信号值降低，出现的三个未知峰(U1、U2、U3)信号值逐渐升高，说明蛋白酶XIV的浓度会影响SeCys2的稳定性，测定的色谱图见图 4。虽然经优化选择的含蛋白酶XIV提取体系对西兰花中硒形态的提取效果较好，但蛋白酶XIV浓度会影响SeCys2的稳定性，并依据西兰花样品中SeCys2加标回收率低的情况，说明蛋白酶XIV浓度和西兰花基质会影响SeCys2的准确测定。
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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.另外，对西兰花进行Se(Ⅳ)加标回收试验时发现一个现象，即不同的西兰花样品对Se(Ⅳ)的测定有影响。对选择的三个西兰花样品(a、b、c)进行Se(Ⅳ)加标回收试验，称取一定的样品，加入1.2mL浓度为100μg/L的Se(Ⅳ)标准溶液和提取试剂，按所建分析方法进行分析测定，发现三个样品的加标回收率依次为：81.6% > 69.9% > 1.5%，进行Kruskal-Wallis秩和检验，三者加标回收率具有显著性差异(P < 0.05)，具体结果见表 4。
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.西兰花粉末(c)，即经硒强化后的西兰花冷冻干燥粉末Se(Ⅳ)加标回收率明显降低，进一步增加蛋白酶XIV的用量到8mg/mL，增加提取试剂体积至12mL，提取效果并无明显改变，说明不存在因酶含量以及提取液体积不足引起竞争提取的问题。推测可能是样品本身存在的某些物质会影响Se(Ⅳ)加标回收率。初步推测源于西兰花中存在的酚类化合物影响了Se(Ⅳ)的测定，Cuderman等[34]报道了类似的现象，曾将酚类化合物(单宁和芦丁)按照1:100(w/w)的比例加入到硒标准溶液中时，37℃采用蛋白酶酶解24h后发现Se(Ⅳ)响应值下降20%，说明酚类化合物会影响Se(Ⅳ)测定。Tian等[35]报道在西兰花的幼苗阶段施加硒酸钠发现酚酸(酚类化合物的一种)含量增加；同样，Gui等[36]研究发现，在西兰花结球期，施加硒酵母和亚硒酸钠发现酚酸含量明显上升，因此，推测c样品中Se(Ⅳ)加标回收率显著下降是由于西兰花样品中酚类物质含量较高造成的，具体原因有待研究。
2.4.2 方法正确度验证
由于目前无西兰花基质的有证标准物质，于是选用欧盟有证标准物质小麦粉(ERM® BC210a, 标准值为11.03±1.05mg/kg，以Se计)进行试验，采用本研究建立的方法对ERM® BC210a中SeMet进行测定，SeMet的测定值为10.11±0.05mg/kg(n=3)，测定值在其标准值范围内。
2.5 样品分析
应用所建立的分析方法对从山东、北京、广东、河北、云南等地采购的20多个市售西兰花样品进行硒形态分析，发现在11个检出硒形态的样品中主要形态为MeSeCys，含量在0.004~0.043mg/kg之间，部分样品中还存在少量的Se(Ⅵ)、Se(Ⅳ)和SeMet，但含量均低于定量限，样品色谱图见图 5。样品色谱图显示，在保留时间为340s和550s左右，还存在两个未知的含硒化合物(Ua、Ub)，具体物质有待进一步研究。已有文献[37-38]报道，西兰花中存在较高含量的MeSeCys，源于西兰花作为十字花科中的次级聚硒植物，可通过甲基化的方式转变硒的储存方式[13]。
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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.同时采用此方法对经含Se(Ⅳ)的硒肥强化后的西兰花冷冻干粉进行分析。结果显示，经含Se(Ⅳ)硒肥强化后的西兰花冷冻干粉中硒形态含量依次为SeMet、MeSeCys和少量的SeCys2，这与陆晓奇等[12]对富硒植物硒形态研究中得出富硒西兰花中硒主要存在形态为MeSeCys(37.1%)、SeMet(27.8%)和SeCys2(25.9%)以及刘为等[13]实验结果显示富硒西兰花蛋白中SeCys2、SeMet和MeSeCys占比高，结果近似。
市售西兰花中主要检出MeSeCys，推测经硒肥强化后，硒在植物体中富集，转化为不同形态的有机硒。虽然本研究中SeCys2加标回收率不理想，但如果样品中存在SeCys2且含量较高时仍可以分析检出。
3. 结论
本研究通过对样品提取、分析条件的选择和优化，选择了含蛋白酶XIV的Tris-HCl缓冲溶液进行样品提取，建立了HPLC-ICP/MS测定市售西兰花中Se(Ⅳ)、Se(Ⅵ)、MeSeCys、SeMet的方法，对采集的全国不同地区市售的20多份西兰花样品进行分析测定，结果表明市售西兰花中硒形态以MeSeCys为主，存在少量Se(Ⅵ)、Se(Ⅳ)和SeMet，也存在少量未知含硒化合物。
在分析研究中发现，SeCys2稳定性受蛋白酶XIV浓度及西兰花样品基质影响，同时推测若样品中存在大量酚类物质将会影响Se(Ⅳ)的测定，具体原因有待进一步分析探究。
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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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