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Article(id=1153986590046151442, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986579971429187, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241203001, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1733155200000, receivedDateStr=2024-12-03, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1753061443101, onlineDateStr=2025-07-21, pubDate=1740412800000, pubDateStr=2025-02-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1753061443101, onlineIssueDateStr=2025-07-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1753061443101, creator=13701087609, updateTime=1753061443101, updator=13701087609, issue=Issue{id=1153986579971429187, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='4', pageStart='1', pageEnd='320', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1753061440699, creator=13701087609, updateTime=1758783495950, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1177986619249406427, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986579971429187, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1177986619249406428, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986579971429187, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=209, endPage=214, ext={EN=ArticleExt(id=1153986591392523075, articleId=1153986590046151442, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Determination of 4 kinds of carotenoids in foodstuffs by online solid phase extraction-liquid chromatography, columnId=1153986581653349021, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Application of Modern Analysis Instrument in Food Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To develop a method for the simultaneous determination of lutein, zeaxanthin, α-carotene, and β-carotene in infant formula milk powder and natural foods by online solid phase extraction-liquid chromatography (online SPE-LC). Methods Samples were saponified with 50% potassium hydroxide solution at 70 ℃ for 15 min. Saponified solution was diluted with 70% ethanol, and after high-speed centrifugation, the supernatant was directly analyzed. A styrene-divinylbenzene copolymer-based SelectCore PSS-packed chromatographic column was chosen as the online SPE column, using 80% methanol-water as the initial loading solvent and acetonitrile/methyl tert-butyl ether as the washing solvent. A ChromCore C30 column (4.6 mm×250 mm, 3 μm) was used as the analytical column, with acetonitrile-methanol (26:74, V:V, 0.4 g/L of ascorbic acid) and methyl tert-butyl ether as the mobile phase for gradient elution. The flow rate was 1.0 mL/min, and the detection wavelength was 450 nm. Results The 4 target analytes in infant formula milk powder and natural foods were well separated. Linear correlation coefficients for all target analytes were greater than 0.9999. The limit of quantification of method for lutein, zeaxanthin, α-carotene, and β-carotene were 0.77, 0.72, 0.94, and 1.40 μg/100 g, respectively. Average recovery rates of infant formula milk powder were 99.76%, 103.51%, 99.16%, and 97.92%, respectively. The relative standard deviations of method repeatability for all the analytes were less than 2.5%. Conclusion This method is accurate, reliable, and reproducible and can meet the quantitative requirements of lutein, zeaxanthin, and α-carotene, β-carotene in foodstuffs. It can be applied to the determination of real samples.

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目的 建立在线固相萃取-液相色谱法(online solid phase extraction-liquid chromatography, online SPE-LC)同时测定婴幼儿配方乳粉和天然食品中叶黄素、玉米黄质、α-胡萝卜素和β-胡萝卜素含量的方法。方法 样品采用浓度为50%氢氧化钾溶液在70 ℃下皂化15 min, 皂化液以70%乙醇定容, 高速离心后取上清液直接上机分析。选择聚苯乙烯-二乙烯基苯基质的SelectCore PSS色谱柱作为online SPE柱, 采用80%甲醇-水作为初始上样溶剂, 乙腈/甲基叔丁基醚作为清洗溶剂。采用ChromCore C30 (4.6 mm×250 mm, 3 μm)作为分析柱, 乙腈-甲醇(26:74, V:V, 含0.4 g/L抗坏血酸)与甲基叔丁基醚作为流动相, 梯度洗脱, 流速为1.0 mL/min, 检测波长为450 nm。结果 婴幼儿配方乳粉和天然食品中4个目标物分离良好, 所有目标物的线性相关系数r>0.9999, 叶黄素、玉米黄质及α-胡萝卜素、β-胡萝卜素的方法定量限分别为0.77、0.72、0.94、1.40 μg/100 g, 婴幼儿配方乳粉基质加标平均回收率分别为99.76%、103.51%、99.16%和97.92%; 方法重复性的相对标准偏差小于2.5%。结论 该方法快速、可靠及重复性良好, 可满足食品中叶黄素、玉米黄质和α-胡萝卜素、β-胡萝卜素的定量要求, 可被应用于实际样品的检测中。

, correspAuthors=张艳海, authorNote=null, correspAuthorsNote=
* 张艳海(1980—), 男, 博士, 工程师, 主要研究方向为中药活性成分和质量标准。E-mail:
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周锋杰(1987—), 男, 硕士, 高级农艺师, 主要研究方向为农产品质量安全与营养品质。E-mail:

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周锋杰(1987—), 男, 硕士, 高级农艺师, 主要研究方向为农产品质量安全与营养品质。E-mail:

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周锋杰(1987—), 男, 硕士, 高级农艺师, 主要研究方向为农产品质量安全与营养品质。E-mail:

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注: 1-6. 上样净化过程; 1-2. 目标物转移过程。

, figureFileSmall=BkpugADMGyiY80EtYepemQ==, figureFileBig=sEuzNc8We5XVqdbALMOznA==, tableContent=null), ArticleFig(id=1177985509730173907, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=EN, label=Fig.2, caption=Online SPE elution profile of the target analyte, figureFileSmall=X2A3RnmlpIV8dHJ7fcZy7Q==, figureFileBig=6R1LV7kuH60IfcnfjIovLA==, tableContent=null), ArticleFig(id=1177985509818254293, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=CN, label=图2, caption=目标待测物的online SPE洗脱谱图

注: A. 胡萝卜素标准品溶液; B. 空白溶剂。

, figureFileSmall=X2A3RnmlpIV8dHJ7fcZy7Q==, figureFileBig=6R1LV7kuH60IfcnfjIovLA==, tableContent=null), ArticleFig(id=1177985509868585943, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=EN, label=Fig.3, caption=Chromatogram of infant formula milk powder, figureFileSmall=OqC7b1j36rOrPh6XCBTFUg==, figureFileBig=IsFrEKf1I5VGPa82kfRukg==, tableContent=null), ArticleFig(id=1177985509944083416, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=CN, label=图3, caption=婴幼儿配方乳粉的色谱图

注: A. 混合标准品; B~G. 不同品牌婴幼儿配方乳粉样品。1. 叶黄素; 2. 玉米黄质; 3. α-胡萝卜素; 4. β-胡萝卜素, 下同。

, figureFileSmall=OqC7b1j36rOrPh6XCBTFUg==, figureFileBig=IsFrEKf1I5VGPa82kfRukg==, tableContent=null), ArticleFig(id=1177985509998609369, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=EN, label=Fig.4, caption=Chromatogram of vegetables and fruits, figureFileSmall=LNj7SRQ5P3nt7FFZ2C+ARQ==, figureFileBig=G4+xJ5UUQlJ4IGvZI8BTqg==, tableContent=null), ArticleFig(id=1177985510157992923, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=CN, label=图4, caption=蔬菜和水果的色谱图

注: A. 混合标准品; B~G分别为菠菜、芒果、猕猴桃、玉米、西红柿、胡萝卜和西兰花。

, figureFileSmall=LNj7SRQ5P3nt7FFZ2C+ARQ==, figureFileBig=G4+xJ5UUQlJ4IGvZI8BTqg==, tableContent=null), ArticleFig(id=1177985510346736603, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=EN, label=Table 1, caption=

Gradient program of SPE pump

, figureFileSmall=null, figureFileBig=null, tableContent=
时间/min 流动相A/% 流动相B/% 流动相C/% 流动相
D/%
0 20 0 80 0
4.5 20 0 80 0
5.0 0 100 0 0
15.0 0 100 0 0
16.0 0 80 0 20
21.0 0 80 0 20
25.0 0 100 0 0
30.0 20 0 80 0
40.0 20 0 80 0
), ArticleFig(id=1177985510451594205, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=CN, label=表1, caption=

SPE泵梯度程序

, figureFileSmall=null, figureFileBig=null, tableContent=
时间/min 流动相A/% 流动相B/% 流动相C/% 流动相
D/%
0 20 0 80 0
4.5 20 0 80 0
5.0 0 100 0 0
15.0 0 100 0 0
16.0 0 80 0 20
21.0 0 80 0 20
25.0 0 100 0 0
30.0 20 0 80 0
40.0 20 0 80 0
), ArticleFig(id=1177985510585811935, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=EN, label=Table 2, caption=

Investigation results of linear relationship and limit of quantification

, figureFileSmall=null, figureFileBig=null, tableContent=
待测物 保留时间/min 相关系数(r2) 线性方程 定量限/ng 范围/ng
叶黄素 15.7540 0.99997 Y=0.4959X-0.0054 0.0153 0.4136~103.4000
玉米黄质 16.5780 0.99996 Y=0.3249X-0.0052 0.0144 0.2720~68.0000
α-胡萝卜素 32.7610 0.99995 Y=1.3340X-0.0420 0.0189 1.0944~273.6000
β-胡萝卜素 34.3790 0.99993 Y=0.5350X-0.0389 0.0281 0.5785~144.6300
), ArticleFig(id=1177985510732612579, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=CN, label=表2, caption=

线性关系及定量限考察结果

, figureFileSmall=null, figureFileBig=null, tableContent=
待测物 保留时间/min 相关系数(r2) 线性方程 定量限/ng 范围/ng
叶黄素 15.7540 0.99997 Y=0.4959X-0.0054 0.0153 0.4136~103.4000
玉米黄质 16.5780 0.99996 Y=0.3249X-0.0052 0.0144 0.2720~68.0000
α-胡萝卜素 32.7610 0.99995 Y=1.3340X-0.0420 0.0189 1.0944~273.6000
β-胡萝卜素 34.3790 0.99993 Y=0.5350X-0.0389 0.0281 0.5785~144.6300
), ArticleFig(id=1177985510799721445, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=EN, label=Table 3, caption=

Determination results of recovery rate with added standard (n=5)

, figureFileSmall=null, figureFileBig=null, tableContent=
待测物 加标浓度/(μg/100 g) 平均回收率/% RSDs/%
叶黄素 163.30 99.76 1.64
244.95
326.60
玉米黄质 82.70 103.51 1.53
124.05
165.40
α-胡萝卜素 172.70 99.16 1.75
259.05
345.40
β-胡萝卜素 176.70 97.92 1.64
265.05
353.40
), ArticleFig(id=1177985510866830311, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986590046151442, language=CN, label=表3, caption=

加标回收率测定结果(n=5)

, figureFileSmall=null, figureFileBig=null, tableContent=
待测物 加标浓度/(μg/100 g) 平均回收率/% RSDs/%
叶黄素 163.30 99.76 1.64
244.95
326.60
玉米黄质 82.70 103.51 1.53
124.05
165.40
α-胡萝卜素 172.70 99.16 1.75
259.05
345.40
β-胡萝卜素 176.70 97.92 1.64
265.05
353.40
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在线固相萃取-液相色谱法测定食品中4种类胡萝卜素含量
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周锋杰 1 , 夏钰 2 , 张艳海 2, *
食品安全质量检测学报 | 本期专题：现代分析仪器在食品检测中的应用 2025,16(4): 209-214
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食品安全质量检测学报 | 本期专题：现代分析仪器在食品检测中的应用 2025, 16(4): 209-214
在线固相萃取-液相色谱法测定食品中4种类胡萝卜素含量
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周锋杰1 , 夏钰2, 张艳海2, *
作者信息
  • 1.苏州市农产品质量安全监测中心, 苏州 215104
  • 2.纳谱分析技术(苏州)有限公司, 苏州 215000

    • 周锋杰(1987—), 男, 硕士, 高级农艺师, 主要研究方向为农产品质量安全与营养品质。E-mail:

通讯作者:

* 张艳海(1980—), 男, 博士, 工程师, 主要研究方向为中药活性成分和质量标准。E-mail:
Determination of 4 kinds of carotenoids in foodstuffs by online solid phase extraction-liquid chromatography
Feng-Jie ZHOU1 , XIA-Yu2, Yan-Hai ZHANG2, *
Affiliations
  • 1. Suzhou Agricultural Products Safety and Quality Inspection Center, Suzhou 215104, China
  • 2. NanoChrom Analytical Technology (Suzhou) Co., Ltd., Suzhou 215000, China
出版时间: 2025-02-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241203001
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摘要
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目的 建立在线固相萃取-液相色谱法(online solid phase extraction-liquid chromatography, online SPE-LC)同时测定婴幼儿配方乳粉和天然食品中叶黄素、玉米黄质、α-胡萝卜素和β-胡萝卜素含量的方法。方法 样品采用浓度为50%氢氧化钾溶液在70 ℃下皂化15 min, 皂化液以70%乙醇定容, 高速离心后取上清液直接上机分析。选择聚苯乙烯-二乙烯基苯基质的SelectCore PSS色谱柱作为online SPE柱, 采用80%甲醇-水作为初始上样溶剂, 乙腈/甲基叔丁基醚作为清洗溶剂。采用ChromCore C30 (4.6 mm×250 mm, 3 μm)作为分析柱, 乙腈-甲醇(26:74, V:V, 含0.4 g/L抗坏血酸)与甲基叔丁基醚作为流动相, 梯度洗脱, 流速为1.0 mL/min, 检测波长为450 nm。结果 婴幼儿配方乳粉和天然食品中4个目标物分离良好, 所有目标物的线性相关系数r>0.9999, 叶黄素、玉米黄质及α-胡萝卜素、β-胡萝卜素的方法定量限分别为0.77、0.72、0.94、1.40 μg/100 g, 婴幼儿配方乳粉基质加标平均回收率分别为99.76%、103.51%、99.16%和97.92%; 方法重复性的相对标准偏差小于2.5%。结论 该方法快速、可靠及重复性良好, 可满足食品中叶黄素、玉米黄质和α-胡萝卜素、β-胡萝卜素的定量要求, 可被应用于实际样品的检测中。

胡萝卜素  /  叶黄素  /  玉米黄素  /  婴幼儿配方乳粉  /  天然食品  /  在线固相萃取-液相色谱法

Objective To develop a method for the simultaneous determination of lutein, zeaxanthin, α-carotene, and β-carotene in infant formula milk powder and natural foods by online solid phase extraction-liquid chromatography (online SPE-LC). Methods Samples were saponified with 50% potassium hydroxide solution at 70 ℃ for 15 min. Saponified solution was diluted with 70% ethanol, and after high-speed centrifugation, the supernatant was directly analyzed. A styrene-divinylbenzene copolymer-based SelectCore PSS-packed chromatographic column was chosen as the online SPE column, using 80% methanol-water as the initial loading solvent and acetonitrile/methyl tert-butyl ether as the washing solvent. A ChromCore C30 column (4.6 mm×250 mm, 3 μm) was used as the analytical column, with acetonitrile-methanol (26:74, V:V, 0.4 g/L of ascorbic acid) and methyl tert-butyl ether as the mobile phase for gradient elution. The flow rate was 1.0 mL/min, and the detection wavelength was 450 nm. Results The 4 target analytes in infant formula milk powder and natural foods were well separated. Linear correlation coefficients for all target analytes were greater than 0.9999. The limit of quantification of method for lutein, zeaxanthin, α-carotene, and β-carotene were 0.77, 0.72, 0.94, and 1.40 μg/100 g, respectively. Average recovery rates of infant formula milk powder were 99.76%, 103.51%, 99.16%, and 97.92%, respectively. The relative standard deviations of method repeatability for all the analytes were less than 2.5%. Conclusion This method is accurate, reliable, and reproducible and can meet the quantitative requirements of lutein, zeaxanthin, and α-carotene, β-carotene in foodstuffs. It can be applied to the determination of real samples.

carotene  /  lutein  /  zeaxanthin  /  infant formula milk powder  /  natural food  /  online solid phase extraction-liquid chromatography
周锋杰, 夏钰, 张艳海. 在线固相萃取-液相色谱法测定食品中4种类胡萝卜素含量. 食品安全质量检测学报, 2025 , 16 (4) : 209 -214 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241203001
Feng-Jie ZHOU, XIA-Yu, Yan-Hai ZHANG. Determination of 4 kinds of carotenoids in foodstuffs by online solid phase extraction-liquid chromatography[J]. Journal of Food Safety & Quality, 2025 , 16 (4) : 209 -214 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241203001
正文
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0 引言
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类胡萝卜素是一类具有着色力的脂溶性化合物[1-6], 广泛用于食品工业中, 也作为维生素A前体的食品强化成分, 同时具有增强免疫力、降低患退行性疾病的风险及抗氧化特性等生物功能[7-12]。人体自身不能合成类胡萝卜素, 因此, 叶黄素、玉米黄质和胡萝卜素作为多功能营养素, 被广泛添加到婴幼儿配方食品和成人营养品中[13-17]。
液相色谱法是测定植物组织[18-19]、天然食品[20-21]、配方食品[22-23]和保健食品[24-25]等基质样品中类胡萝卜素的主要方法。食品中叶黄素和胡萝卜素的测定目前主要参考GB 5009.248—2016《食品安全国家标准 食品中叶黄素的测定》和GB 5009.83—2016《食品安全国家标准 食品中胡萝卜素的测定》, 其中样品溶液制备通常需要经过氢氧化钾的皂化和有机溶剂的液-液萃取过程, 碱皂化可以有效的将目标分析物从基质中释放出来, 再采用液-液萃取和净化后, 上机分析。不仅会消耗大量有机试剂, 且由于叶黄素和胡萝卜素对光热敏感, 样品制备步骤越多、流程越长对目标物的准确测定影响越大, 且影响整个方法的分析效率[26]。与传统离线样品前处理相比, 在线样品前处理可以将样品制备与分析过程在密闭系统中自动完成, 即可提升样品分析效率, 又可减少人工操作而导致的目标物损失和误差, 改善方法灵敏度、准确性和重现性[27], 在食品中营养素和兽药残留分析中都有广泛应用[28-33]。
本研究采用在线固相萃取-液相色谱法(online solid phase extraction-liquid chromatography, online SPE-LC)的分析技术, 样品皂化液直接上机分析, 自动完成样品富集、净化和分析的全过程, 同时实现叶黄素、玉米黄质、α-胡萝卜素和β-胡萝卜素的测定, 为开展相关配方食品中目标物的快速高通量检测提供借鉴。
1 材料与方法
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1.1 仪器、试剂与材料
BSA124S万分之一分析天平[赛多利斯科学仪器(北京)有限公司]; Agilent 1260 Infinity II液相色谱系统[包括2台四元泵(G7111A)、自动进样器(G7129B)、柱温箱(G7116B)、阀切换单元(G1170A, 含一个二位六通阀头)、二极管阵列检测器(G7117A)、软件采用Agilent OpenLab CDS软件(版本2.6), 美国安捷伦科技公司]; SelectCore PSS online SPE柱(3.0 mm×30 mm, 40 μm)、ChromCore C30 (4.6 mm×250 mm, 3 μm)[纳谱分析技术(苏州)有限公司]。
乙腈、甲醇(色谱纯, 美国Avantor J.T.Baker公司); 甲基叔丁基醚(methyl tertiary butyl ether, MTBE)[色谱纯, 阿拉丁试剂(上海)有限公司]; 抗坏血酸、2,6-二叔丁基对甲酚(butylated hydroxytoluene, BHT)、氢氧化钾、乙醇(分析纯, 国药集团化学试剂有限公司)。
6种品牌的婴幼儿配方乳粉均购于超市, 其中5种营养成分表中含叶黄素; 玉米、胡萝卜、西红柿、菠菜、西兰花、猕猴桃和芒果等蔬菜、水果随机采购于当地的菜市场。
1.2 实验方法
1.2.1 标准品溶液配制
精密称取叶黄素和玉米黄质标准品适量, 加0.1% (m:m) BHT的乙醇溶液溶解并定容到100 mL棕色容量瓶中, 制成质量浓度均为0.5 mg/mL的储备溶液; 再精密称取α-胡萝卜素和β-胡萝卜素适量, 加0.1% (m:m) BHT的二氯甲烷溶液溶解并定容到100 mL, 制成质量浓度分别为0.518 mg/mL和0.530 mg/mL的储备溶液。使用前按照标准GB 5009.248—2016与GB 5009.83—2016附录对标准品溶液浓度进行校正。再精密移取各储备液适量, 加0.1% (m:m) BHT的乙醇溶液稀释10倍, 制成中间液; 再移取中间液, 加甲醇稀释制成叶黄素、玉米黄质、α-胡萝卜素和β-胡萝卜素质量浓度分别为2.0680、1.3600、5.4720和2.8926 μg/mL的混合标准品溶液(校正后质量浓度)。
1.2.2 样品前处理
取样品(蔬果需提前粉碎均质)2~5 g, 精密称定, 置50 mL棕色离心管中, 加入5 mL抗坏血酸水溶液(0.2 g/mL), 使样品溶解, 再加入15 mL BHT-乙醇溶液(0.2 g/100 mL), 涡旋混匀30 s, 加入5 mL质量浓度为50%的氢氧化钾溶液, 涡旋混匀后, 置70 ℃水浴中皂化15 min, 迅速冷却后, 加70%乙醇, 定容至50 mL, 以5000 r/min离心5 min, 取上清液, 过0.22 μm尼龙膜后, 上机分析, 进样体积50~100 μL。
1.2.3 色谱条件
全自动online SPE-LC系统见图1所示, 初始位置1-6。其中二位置六通阀的具体切换时间: 0.0~4.5 min, 1-6; 4.5~6.5 min, 1-2; 6.5~40.0 min, 1-6。
online SPE柱为SelectCore PSS (30 mm×3.0 mm, 40 µm), 其中流动相A为水, 流动相B为乙腈, 流动相C为甲醇, 流动相D为MTBE, 梯度过程见表1, 流速为1.0 mL/min。分析色谱柱为ChromCore C30 (4.6 mm×250 mm, 3.0 µm), 流动相A为乙腈, 流动相B为乙腈-甲醇(26:74, V:V, 含0.4 g/L抗坏血酸), 流动相C为MTBE; 梯度洗脱: 0~6.5 min, A:B=25:75 (V:V); 6.5~7.0 min, 100% B; 7.0~32 min, 100%~64% B, 0%~36% C; 32~35 min, 64%~20% B, 36%~80% C; 35~40 min, B:C=20:80 (V:V); 流速, 1.0 mL/min; 柱温为25 ℃, 紫外检测波长为450 nm, 采集频率10 Hz; 进样体积为50~100 μL。
1.3 数据处理
色谱数据的采集和数据处理采用Agilent OpenLab CDS 2.5软件, 标准曲线、重复性等采用Excel 2010进行数据结果的统计分析。
2 结果与讨论
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2.1 在线固相萃取条件考察
样品皂化溶液中通常含有强碱、水溶性蛋白多糖、水溶性的基质小分子、脂肪酸钠盐及包含目标待测物在内的脂溶性成分。因此本研究根据目标物的理化特征及皂化基质成分组成, 选择了聚合物基质的SelectCore PSS填料作为online SPE柱。洗脱转移条件是影响online SPE过程及回收率的主要因素, 因此为确认目标物的SPE转移时间, 采用质量浓度为21.2 μg/mL的β-胡萝卜素标准品溶液, 进样10 μL, 在上样和除杂洗脱过程结束后, 在4.5 min切换至1-2, 采用分析泵初始流动相比例(A:B=75:25, V:V)转移目标物, 结果由图2所示, 目标物在2 min内可基本转移完全, 依此确认了转移时间为2 min。
2.2 皂化条件确认
为确认皂化时间和温度, 实验分别参考GB 5009.248—2016、GB5009.83—2016及文献[27-29], 采用婴幼儿配方乳粉(含叶黄素)作为样品基质, 考察皂化温度和时间对目标物提取过程的影响。皂化温度分别对比室温、53 ℃和70 ℃, 皂化时间分别考察了15、30和45 min。结果表明不同皂化温度对婴幼儿配方乳粉样品中叶黄素的含量影响不大, 在70 ℃下叶黄素的提取率略高; 皂化温度对样品中胡萝卜素的影响较大, 其中53 ℃和70 ℃显著高于室温, 53 ℃较70 ℃略高; 在70 ℃的皂化温度下, 对比不同皂化时间对叶黄素和β-胡萝卜素提取效果的影响, 结果随着皂化时间增加, 叶黄素和胡萝卜素的含量显著下降, 因此皂化15 min较为适宜。从目标物提取率和时间的整体上考虑, 最终选择皂化温度为70 ℃和皂化时间15 min。
2.3 方法学考察
2.3.1 线性关系及定量限考察
取混合标准品溶液分别进样1、2、5、10、20和50 μL,以目标峰的峰面积响应为纵坐标(Y), 以进样量为横坐标(X, ng), 考察线性相关关系, 结果所有目标待测物的相关系数r2>0.9999, 表明各化合物的线性相关关系较好(表2)。按照信噪比(S/N)为10:1计算, 叶黄素、玉米黄质、α-胡萝卜素和β-胡萝卜素的仪器定量限分别为0.0153、0.0144、0.0189和0.0281 ng。按照取样量2 g, 定容体积50 mL, 进样体积50 μL计算定量限, 叶黄素、玉米黄质、α-胡萝卜素和β-胡萝卜素的方法定量限分别为0.77、0.72、0.94和1.40 μg/100 g。
2.3.2 重复性试验
取某品牌的婴幼儿配方奶粉5份, 每份2 g, 精密称定, 按照确定的皂化方法制备上机溶液, 结果叶黄素含量的相对标准偏差(relative standard deviation, RSD)<2%; 玉米黄素和β-胡萝卜素含量的RSD<2.5%, 表明方法重复性较好。
2.3.3 加样回收率试验
取婴幼儿配方乳粉, 每份2 g, 精密称定, 按照低、中、高3水平分别加入混合标准品, 每个加标水平各制备5份平行样品, 按照1.2.2中样品溶液制备方法制备样品溶液, 上机分析。测定加标样品中目标物的总量, 计算加标回收率。由见表3结果可见, 叶黄素、玉米黄质及α-胡萝卜素、β-胡萝卜素的平均回收率分别为99.76%、103.51%、99.16%和97.92%, 表明该方法测定准确度良好, 满足样品中目标物定量要求。
2.4 样品测定
分别取不同品牌的婴幼儿配方乳粉按照1.2.2中样品制备方法, 制备上机溶液, 进行测定。由图3所示, 婴幼儿配方乳粉B、C、D、E和G中均含有叶黄素, 同时含有微量的玉米黄质及β-胡萝卜素, 其中叶黄素、玉米黄质与其他叶黄素顺式异构体分离良好, 叶黄素与玉米黄质及α-胡萝卜素与β-胡萝卜素的分离度分别为4.6和5.9。本研究还选取了西兰花、西红柿、玉米、猕猴桃、芒果和菠菜等蔬菜、水果, 对其中目标的类胡萝卜素成分进行分析, 结果由图4可见, 菠菜、玉米、西兰花和猕猴桃中叶黄素的含量较高, 胡萝卜、西红柿、芒果及西兰花中胡萝卜素的含量相对较高, 总体上, 各目标峰分离良好, 通过该方法可以对各目标待测物进行定量。
2.5 与标准方法比较
现行食品中叶黄素和胡萝卜素测定分别参考GB 5009.248—2016和GB 5009.83—2016方法进行测定, 二者样品前处理过程均需皂化和有机溶剂的液-液萃取过程, 其中皂化时间均为30 min, 后续处理过程约为30~40 min; 分析条件均采用C30色谱柱进行分离分析, 其中叶黄素和α-胡萝卜素、β-胡萝卜素的分析时间分别约为28 min和30 min(加平衡时间)。本研究方法的皂化时间为15 min, 皂化液直接上机分析, 同时测定目标物, 上机的分析时间为40 min, 因此采用本研究方法的总分析时间较标准方法减少近76%, 显著提升了方法检测效率。且该方法无需有机溶剂(环己烷、乙醚、石油醚等), 更绿色和环境友好。
3 结论
收起
本研究采用online SPE-LC建立了婴幼儿配方乳粉和天然食品中叶黄素、玉米黄素和胡萝卜素的同时检测方法。通过对样品皂化条件和online SPE过程关键条件的考察, 确定了优化的样品制备和净化及转移条件。系统的方法学考察结果表明该方法的线性相关性、精密度和平均回收率均能满足目标物的测定要求。采用实际的婴幼儿配方乳粉和蔬菜水果等天然食品进行分析, 表明样品中各目标物分离良好, 可对目标物实现准确定量。
本研究所构建的方法与标准方法比较, 实现了从烦琐手动操作到高效自动化分析的转变, 显著提升了分析效率和准确性, 为科学研究和实际应用提供了强有力的技术支持。该方法也将为实现高通量、高灵敏度和高准确性的样品检测提供新的思路和解决方案。
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241203001
  • 接收时间：2024-12-03
  • 首发时间：2025-07-21
  • 出版时间：2025-02-25
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  • 收稿日期：2024-12-03
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    1.苏州市农产品质量安全监测中心, 苏州 215104
    2.纳谱分析技术(苏州)有限公司, 苏州 215000

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* 张艳海(1980—), 男, 博士, 工程师, 主要研究方向为中药活性成分和质量标准。E-mail:
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2种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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