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Article(id=1153433635551174701, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433633999282214, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250106006, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1736092800000, receivedDateStr=2025-01-06, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752929608475, onlineDateStr=2025-07-19, pubDate=1742832000000, pubDateStr=2025-03-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752929608475, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752929608475, creator=13701087609, updateTime=1752929608475, updator=13701087609, issue=Issue{id=1153433633999282214, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='6', pageStart='1', pageEnd='322', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752929608105, creator=13701087609, updateTime=1758086445549, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1175062977960096080, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433633999282214, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1175062977960096081, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433633999282214, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=208, endPage=215, ext={EN=ArticleExt(id=1153433635983188015, articleId=1153433635551174701, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Identification of vitamin A and unsaturated fatty acid in natural cod liver oil based on a new method, columnId=1153429494506447365, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Development and Detection of Health Foods, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a new method for simple identification of vitamin A (VA) and unsaturated fatty acids in natural cod liver oil. Methods The cod liver oil sample was saponified and injected into a high-performance liquid chromatography system to detect the content of VA. After saponification of cod liver oil and its excipients, injected them into a gas chromatography system to analyze the localization and percentage of linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), and distinguish fish oil from vegetable oil. Results Under the new method, the linear relationship of VA concentration was good within the range of 30.00-1500.00 IU/mL (r2=0.9999); the average recovery rate was 96.1%. The relative standard deviation of instrument precision was 0.18%, the relative standard deviation of method precision was 1.13%, and the relative standard deviation of intermediate precision was 0.98%. There was a significant difference in the content of fatty acids between vegetable oils, fish oil and cod liver oil. Cod liver oil and fish oil were rich in EPA and DHA, while linoleic acid was low. Corn oil, rapeseed oil and olive oil did not contain EPA and DHA, but they were rich in linoleic acid. Conclusion The detection method establish in this article is simple and accurate, and can be used for the determination of VA and unsaturated fatty acid content in cod liver oil, laying the foundation for the development of ideal quality standards.

, correspAuthors=Li-Ping GUAN, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Li-Wei HU, Chao-Hua FANG, Qiu-Shi WANG, Ding-Xin PENG, Li-Ping GUAN), CN=ArticleExt(id=1153433661149012759, articleId=1153433635551174701, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=基于新方法检测天然鱼肝油中维生素A和不饱和脂肪酸, columnId=1153429494632276488, journalTitle=食品安全质量检测学报, columnName=本期专题：保健食品的研发与检测, runingTitle=null, highlight=null, articleAbstract=

目的 建立新方法简便鉴别天然鱼肝油中维生素A (vitamin A, VA)和不饱和脂肪酸的含量。方法 将鱼肝油样品经皂化后注入高效液相色谱系统, 检测VA的含量。将鱼肝油及其辅料皂化后注入气相色谱系统, 分析其中亚油酸、二十碳五烯酸(eicosapentaenoic acid, EPA)和二十二碳六烯酸(docosahexaenoic acid, DHA)定位以及所占百分比, 将鱼油与植物油进行鉴别与区分。结果 在新方法下, VA浓度在30.00~1500.00 IU/mL范围内线性关系良好(r2=0.9999); 平均回收率为96.1%; 仪器精密度的相对标准偏差为0.18%, 方法精密度的相对标准偏差为1.13%, 中间精密度的相对标准偏差为0.98%。各类植物油与鱼油及鱼肝油中脂肪酸含量差异显著, 鱼肝油、鱼油中EPA、DHA含量丰富, 亚油酸含量少; 玉米油、菜籽油、橄榄油中均不含EPA和DHA, 但亚油酸含量丰富。结论 本研究建立的检测方法简便准确, 可用于鱼肝油中VA和不饱和脂肪酸含量测定, 为制定理想的质量标准奠定基础。

, correspAuthors=关丽萍, authorNote=null, correspAuthorsNote=
* 关丽萍(1970—), 女, 博士, 教授, 主要研究方向为食品安全与药物合成。E-mail:
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胡立威(1995—), 男, 硕士, 主要研究方向为食品安全。E-mail:

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胡立威(1995—), 男, 硕士, 主要研究方向为食品安全。E-mail:

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注: a. 视黄醇。

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注: a. 亚油酸; b. EPA; c. DHA。

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注: a. 亚油酸; b. EPA; c. DHA。

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Gradient conditions of mobile phase

, figureFileSmall=null, figureFileBig=null, tableContent=
时间/min 甲醇/% 水/%
0.00 81 19
5.00 85 15
8.00 95 5
12.00 95 5
13.00 81 19
18.00 81 19
), ArticleFig(id=1175086898201637593, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=CN, label=表1, caption=

流动相梯度条件

, figureFileSmall=null, figureFileBig=null, tableContent=
时间/min 甲醇/% 水/%
0.00 81 19
5.00 85 15
8.00 95 5
12.00 95 5
13.00 81 19
18.00 81 19
), ArticleFig(id=1175086898277135066, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 2, caption=

Temperature program

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速率/(℃/min) 温度/℃ 保持时间/min 运行时间/min
0 160 5 5
5 190 5 16
2 230 20 56
), ArticleFig(id=1175086898331661019, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=CN, label=表2, caption=

升温程序

, figureFileSmall=null, figureFileBig=null, tableContent=
速率/(℃/min) 温度/℃ 保持时间/min 运行时间/min
0 160 5 5
5 190 5 16
2 230 20 56
), ArticleFig(id=1175086898390381276, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 3, caption=

Peak time and VA content

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峰时间/min 含量/(IU/g)
VA醋酸酯 31.51 107
AD滴剂 30.54 4712.79
调配鱼肝油 30.60 21083.56
), ArticleFig(id=1175086898457490141, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=CN, label=表3, caption=

峰时间与VA含量

, figureFileSmall=null, figureFileBig=null, tableContent=
峰时间/min 含量/(IU/g)
VA醋酸酯 31.51 107
AD滴剂 30.54 4712.79
调配鱼肝油 30.60 21083.56
), ArticleFig(id=1175086898532987614, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 4, caption=

Test results of recovery

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加样比例% 加样量/(μg/mL) 实测量/(μg/mL) 回收率/% 平均回收率/%
50 25.00 23.45 93.80 93.37
25.00 22.87 91.48
25.00 23.72 94.88
100 50.00 51.53 103.06 98.34
50.00 48.76 97.52
50.00 47.22 94.44
150 75.00 72.19 96.25 96.58
75.00 73.55 97.93
75.00 71.67 95.56
), ArticleFig(id=1175086898629456607, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=CN, label=表4, caption=

回收率试验结果

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加样比例% 加样量/(μg/mL) 实测量/(μg/mL) 回收率/% 平均回收率/%
50 25.00 23.45 93.80 93.37
25.00 22.87 91.48
25.00 23.72 94.88
100 50.00 51.53 103.06 98.34
50.00 48.76 97.52
50.00 47.22 94.44
150 75.00 72.19 96.25 96.58
75.00 73.55 97.93
75.00 71.67 95.56
), ArticleFig(id=1175086898696565472, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 5, caption=

Instrumental precision test results

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序号 1 2 3 4 5 6
峰面积/A 1410.3 1404.0 1403.4 1406.2 1403.8 1405.3
平均峰
面积/A		 1405.5
RSD/% 0.18
), ArticleFig(id=1175086898776257249, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=CN, label=表5, caption=

仪器精密度试验结果

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序号 1 2 3 4 5 6
峰面积/A 1410.3 1404.0 1403.4 1406.2 1403.8 1405.3
平均峰
面积/A		 1405.5
RSD/% 0.18
), ArticleFig(id=1175086898830783202, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 6, caption=

Method precision test results

, figureFileSmall=null, figureFileBig=null, tableContent=
序号 1 2 3 4 5 6
含量/(IU/g) 4763.05 4679.36 4765.42 4840.19 4805.43 4778.87
平均含量/(IU/g) 4772.05
RSD/% 1.13
), ArticleFig(id=1175086898897892067, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=CN, label=表6, caption=

方法精密度试验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
序号 1 2 3 4 5 6
含量/(IU/g) 4763.05 4679.36 4765.42 4840.19 4805.43 4778.87
平均含量/(IU/g) 4772.05
RSD/% 1.13
), ArticleFig(id=1175086898960806628, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 7, caption=

Test results of intermediate precision

, figureFileSmall=null, figureFileBig=null, tableContent=
仪器1 仪器2
第1d 第2d 第1d 第2d
含量(IU/g) 4762.43 4795.26 4812.66 4705.98
平均含量(IU/g) 4769.08
RSD/% 0.98
), ArticleFig(id=1175086899011138277, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=CN, label=表7, caption=

中间精密度试验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
仪器1 仪器2
第1d 第2d 第1d 第2d
含量(IU/g) 4762.43 4795.26 4812.66 4705.98
平均含量(IU/g) 4769.08
RSD/% 0.98
), ArticleFig(id=1175086899095024358, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 8, caption=

Test results of stability

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进样时间/h 峰面积 峰面积变化率/%
0 1410.3 100.00
2 1406.2 99.71
4 1405.2 99.64
24 1407.2 99.78
48 1492.2 105.81
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稳定性试验结果

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进样时间/h 峰面积 峰面积变化率/%
0 1410.3 100.00
2 1406.2 99.71
4 1405.2 99.64
24 1407.2 99.78
48 1492.2 105.81
), ArticleFig(id=1175086899229242088, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 9, caption=

Temperature program 1

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速率/(℃/min) 温度/℃ 保持时间/min 运行时间/min
0 50 1 1.0
25 220 0 7.8
3 230 18 35.0
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升温程序1

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速率/(℃/min) 温度/℃ 保持时间/min 运行时间/min
0 50 1 1.0
25 220 0 7.8
3 230 18 35.0
), ArticleFig(id=1175086899350876906, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433635551174701, language=EN, label=Table 10, caption=

Optimal temperature program

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速率/(℃/min) 温度/℃ 保持时间/min 运行时间/min
0 160 5 5
5 190 5 16
2 230 20 56
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最优升温程序

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速率/(℃/min) 温度/℃ 保持时间/min 运行时间/min
0 160 5 5
5 190 5 16
2 230 20 56
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基于新方法检测天然鱼肝油中维生素A和不饱和脂肪酸
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胡立威 , 方超华 , 王秋实 , 彭鼎新 , 关丽萍 *
食品安全质量检测学报 | 本期专题：保健食品的研发与检测 2025,16(6): 208-215
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食品安全质量检测学报 | 本期专题：保健食品的研发与检测 2025, 16(6): 208-215
基于新方法检测天然鱼肝油中维生素A和不饱和脂肪酸
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胡立威 , 方超华, 王秋实, 彭鼎新, 关丽萍*
作者信息
  • 浙江海洋大学食品与药学学院, 舟山 316022

    • 胡立威(1995—), 男, 硕士, 主要研究方向为食品安全。E-mail:

通讯作者:

* 关丽萍(1970—), 女, 博士, 教授, 主要研究方向为食品安全与药物合成。E-mail:
Identification of vitamin A and unsaturated fatty acid in natural cod liver oil based on a new method
Li-Wei HU , Chao-Hua FANG, Qiu-Shi WANG, Ding-Xin PENG, Li-Ping GUAN*
Affiliations
  • Food and Pharmacy College, Zhejiang Ocean University, Zhoushan 316022, China
出版时间: 2025-03-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250106006
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摘要
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目的 建立新方法简便鉴别天然鱼肝油中维生素A (vitamin A, VA)和不饱和脂肪酸的含量。方法 将鱼肝油样品经皂化后注入高效液相色谱系统, 检测VA的含量。将鱼肝油及其辅料皂化后注入气相色谱系统, 分析其中亚油酸、二十碳五烯酸(eicosapentaenoic acid, EPA)和二十二碳六烯酸(docosahexaenoic acid, DHA)定位以及所占百分比, 将鱼油与植物油进行鉴别与区分。结果 在新方法下, VA浓度在30.00~1500.00 IU/mL范围内线性关系良好(r2=0.9999); 平均回收率为96.1%; 仪器精密度的相对标准偏差为0.18%, 方法精密度的相对标准偏差为1.13%, 中间精密度的相对标准偏差为0.98%。各类植物油与鱼油及鱼肝油中脂肪酸含量差异显著, 鱼肝油、鱼油中EPA、DHA含量丰富, 亚油酸含量少; 玉米油、菜籽油、橄榄油中均不含EPA和DHA, 但亚油酸含量丰富。结论 本研究建立的检测方法简便准确, 可用于鱼肝油中VA和不饱和脂肪酸含量测定, 为制定理想的质量标准奠定基础。

鱼肝油  /  维生素A  /  不饱和脂肪酸  /  定量检测

Objective To establish a new method for simple identification of vitamin A (VA) and unsaturated fatty acids in natural cod liver oil. Methods The cod liver oil sample was saponified and injected into a high-performance liquid chromatography system to detect the content of VA. After saponification of cod liver oil and its excipients, injected them into a gas chromatography system to analyze the localization and percentage of linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), and distinguish fish oil from vegetable oil. Results Under the new method, the linear relationship of VA concentration was good within the range of 30.00-1500.00 IU/mL (r2=0.9999); the average recovery rate was 96.1%. The relative standard deviation of instrument precision was 0.18%, the relative standard deviation of method precision was 1.13%, and the relative standard deviation of intermediate precision was 0.98%. There was a significant difference in the content of fatty acids between vegetable oils, fish oil and cod liver oil. Cod liver oil and fish oil were rich in EPA and DHA, while linoleic acid was low. Corn oil, rapeseed oil and olive oil did not contain EPA and DHA, but they were rich in linoleic acid. Conclusion The detection method establish in this article is simple and accurate, and can be used for the determination of VA and unsaturated fatty acid content in cod liver oil, laying the foundation for the development of ideal quality standards.

cod liver oil  /  vitamin A  /  unsaturated fatty acids  /  quantitative determination
胡立威, 方超华, 王秋实, 彭鼎新, 关丽萍. 基于新方法检测天然鱼肝油中维生素A和不饱和脂肪酸. 食品安全质量检测学报, 2025 , 16 (6) : 208 -215 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250106006
Li-Wei HU, Chao-Hua FANG, Qiu-Shi WANG, Ding-Xin PENG, Li-Ping GUAN. Identification of vitamin A and unsaturated fatty acid in natural cod liver oil based on a new method[J]. Journal of Food Safety & Quality, 2025 , 16 (6) : 208 -215 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250106006
正文
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0 引言
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在过去的几十年里, 营养保健品引起了全球关注, 并且处于不断增长的趋势[1]。它虽然不能替代药品, 但是在对抗和缓解与生活方式相关的疾病和障碍方面发挥着重要作用, 如降低患癌症、心脏病、高血压、动脉粥样硬化、高胆固醇、糖尿病和许多其他生活方式相关疾病的风险[2‒4]。鱼肝油是从鱼类肝脏中提取出的一类富含维生素A (vitamin A, VA)、维生素D (vitamin D, VD)以及多不饱和脂肪酸的脂肪油[5], 在1979年作为药品被收录在《中国药典》中。鱼肝油中含有丰富的生物活性物质, 可有效调节人体健康状态[6], 其中, VA可预防夜盲症, 还有助于保护皮肤, 调节代谢平衡[7]; VD有助于人体钙、磷的吸收, 预防佝偻病, 还可降低罹患癌症的风险[8‒10]; ω-3不饱和脂肪酸可提高自身免疫力、提高记忆力以及预防和治疗心血管疾病, 同时也具有促进大脑视网膜发育, 改善视力, 促进婴幼儿智力发育和改善非酒精性脂肪肝病的功能[11‒15]。多年来, 鱼肝油是婴幼儿食用保健品之一, 孕妇、儿童食用鱼肝油有高度的安全性且有益健康。因此对鱼肝油中成分的含量检测非常重要。
VA是一种天然微量营养素, 是第一个被发现的脂溶性维生素, VA的生物活性分子形式主要为视黄醇(维生素A醇)、维甲酸、视黄素等, 其中视黄醇是VA对视觉系统起作用至关重要的因素[16], VA缺乏症仍然是造成可预防失明的主要原因。除了治疗视力问题外, VA还有抗炎、抑制细胞凋亡、抗肿瘤和增强免疫力等功能[17‒22]。VA的缺乏和过量摄入都会导致严重的健康后果。VA是孕妇及其胎儿的重要微量营养素。除了对形态和功能发育以及眼部完整性至关重要外, VA还对胎儿器官和胎儿骨骼产生全身性影响[23]。因此, 怀孕期间孕妇对VA的需求量增大。然而在怀孕期间摄入过多的VA也会造成不良影响, 如果摄入过量, 可能会引起胎儿中枢神经和心血管系统的先天性畸形以及自然流产[24-25]。因此孕妇在怀孕期间食用鱼肝油补充VA需要注意其中VA含量以免对自身及胎儿造成影响。药典中VA测定法主要有紫外-可见分光光度法和高效液相色谱法, 紫外分光光度法在测定时需使用校正公式校正非VA物质的无关吸收产生的误差。高效液相色谱法中直接将样品溶解离心及可进样, 无复杂前处理过程, 但是测定的是VA醋酸酯的含量。由于无法直接直接检测出鱼肝油中VA含量, 本研究在药典方法的基础上加以改进, 在前处理过程中加入皂化过程, 并将皂化过的样品注入反相色谱液相系统, 经过优化色谱条件, 成功建立了一个适合油脂样品中VA含量的检测方法。
ω-3多不饱和脂肪酸(polyunsaturated fatty acids, PUFA)在治疗心血管疾病和降低神经精神疾病风险方面发挥着重要作用[26‒28]。海洋来源的长链ω-3 PUFA是细胞膜的关键成分, 可作为功能性食品和膳食补充剂。鱼油中最常见的ω-3 PUFA为二十碳五烯酸(eicosapentaenoic acid, EPA)和二十二碳六烯酸(docosahexaenoic acid, DHA), 定期补充ω-3 PUFA可降低血压并且降低胰岛素、胆固醇和总脂质水平[29-30]。ω-3 PUFA在机体代谢过程中起着重要的介质作用, 它们通过不同的机制调节代谢参数, 包括与其他营养物质的相互作用以及对参与脂质和葡萄糖代谢的基因表达的影响[31]。在饮食中增加ω-3 PUFA的摄入可能是降低代谢综合征患病风险的有效措施[32]。市场中鱼肝油产品多数以鱼油作为辅料调配而成, 鱼油中富含EPA和DHA, 可适当调节鱼肝油产品中EPA和DHA含量占比。然而植物油与鱼油外观并无明显差异, 但植物油中不含EPA和DHA, 且价格低廉, 部分商家会使用植物油代替鱼油作为辅料调配鱼肝油, 大大降低了鱼肝油产品的质量。
市场中, 鱼肝油作为可补充VA、VD、EPA和DHA等营养物质的产品被人们所认可。然而, 目前对鱼肝油的质量没有完善的评价标准。在保健品领域, 许多商家将“人工添加型”鱼肝油(添加人工合成维生素)定义为天然鱼肝油, 在药品领域, 许多人将天然鱼肝油与维生素AD滴剂混淆。天然鱼肝油为天然提取物, 富含天然VA以及天然VD, 不同于人工调配出的鱼肝油, 也不同于富含人工合成维生素的维生素AD滴剂。本研究通过建立含量测定新方法对鱼肝油中VA进行分析研究, 使用气相色谱法分析鱼肝油中各类脂肪酸占比, 达到区分天然鱼肝油和人工合成鱼肝油以及维生素AD滴剂的目的, 有效评价鱼肝油的质量。
1 材料与方法
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1.1 材料与试剂
粗制鱼肝油以鮸鱼、鳕鱼、金枪鱼鱼肝油为原料调配出VA与VD的比例为3:1(参考维生素AD滴剂的比例)的鱼肝油, 用于实验研究。
维生素A醋酸酯对照品(100万IU/g, 山东优索化工科技有限公司); 维生素AD滴剂(VA 1800 IU/粒, VD 600 IU/粒, 浙江仟源海力生制药有限公司); 脂肪酸甲酯标准品(美国NU-CHEK-PREP, INC公司); 深海鱼油(华北制药集团维灵保健品有限公司); 鳕鱼(VA 3176.3 IU/g, VD微量, 舟山菜市场采购); 鮸鱼(VA 17373.2 IU/g, VD 803.6 IU/g)、金枪鱼肝油(VA 17472.3 IU/g, VD 28747.4 IU/g)(浙江海力生集团研究院); 玉米油、菜籽油(益海嘉里金龙鱼食品集团股份有限公司); 橄榄油(欧丽薇兰)。
正己烷(色谱纯, 上海阿拉丁生化科技股份有限公司); 乙醇(分析纯)、甲醇(色谱纯)(国药集团化学试剂有限公司); 氢氧化钾(分析纯)、氢氧化钾甲醇溶液(浓度0.5 mol/L)、饱和氯化钠溶液(纯度99.5%, 上海麦克林生化科技股份有限公司); 三氟化硼甲醇溶液(浓度15%, 国药集团化学试剂有限公司)。
1.2 仪器与设备
UV1200型紫外可见分光光度计(上海美谱达仪器有限公司); 1220 Infiniyt II LC高效液相色谱仪、LC210高效液相色谱仪、7890B气相色谱仪、HP-5毛细管色谱柱(0.32 mm×30 m, 0.25 μm)(美国安捷伦科技有限公司); Silversil C18色谱柱(4.6 mm×250 mm, 5 μm)(北京迪马科技公司); 硅胶色谱柱(4.6 mm×150 mm, 5 μm)(大连依利特分析仪器有限公司); HWS26电热恒温水浴锅(上海一恒科学仪器有限公司); TGL-16C高速离心机(上海海安亭科学仪器厂); CH-04BM超声波清洗机(苏州创音唯能超声设备有限公司); 104E/02电子分析天平(精度0.1 mg, 瑞士梅特勒托利多国际有限公司); EYELA OSB-2200旋转蒸发仪(东京理化器械株式会社)。
1.3 实验方法
1.3.1 维生素A含量测定新方法
由于紫外-可见分光光度法与高效液相色谱法都无法准确测定鱼肝油中VA含量, 参考结合药典中几种测定方法, 建立新的VA含量测定方法, 并进行方法学验证。
(1)溶液配制
标准品溶液配制: 称取VA醋酸酯0.025 g于皂化瓶中, 加入乙醇30 mL, 50%氢氧化钾3 mL, 90 ℃水浴锅加热30 min, 冷凝回流。冷却后, 10 mL纯水冲洗冷凝管, 收集皂化液至分液漏斗, 皂化瓶用80 mL水洗涤3次, 洗液并入分液漏斗。用不含过氧化物的乙醚振摇萃取3次, 3次用量分别为60、40、40 mL, 合并乙醚液后用水洗涤3次。洗涤时避免乳化, 酚酞指示液检测是否洗涤干净。分取乙醚提取液, 加入干燥滤纸条除去提取液中残留的水分, 再用乙醚洗涤分液漏斗及滤纸条, 洗液与提取液合并于圆底烧瓶中, 水浴低温蒸发至5 mL左右, 氮气吹干, 迅速加入5 mL甲醇, 密塞, 超声处理助溶, 12000 r/min离心5 min, 作为对照品溶液。
供试品溶液的配制: 称取维生素AD滴剂1.2 g, 参照上述皂化方法, 制得供试品①, 称取鱼肝油0.2 g, 参照上述皂化方法, 制得供试品②。
(2)色谱条件
色谱柱: C18色谱柱(4.6 mm×250 mm, 5 μm); 流动相: A相为甲醇, B相为水, 梯度条件见表1; 进样量10 μL; 流速1.0 mL/min; 检测波长325 nm; 柱温40 ℃。
(3)维生素A含量测定
取对照品溶液、供试品①和供试品②各5 mL, 取进样量注入液相色谱系统, 在1.3.1(2)色谱条件下进行VA的含量分析。
1.3.2 鱼肝油中辅料油的检测
(1)溶液配制
对照品: 精密称取脂肪酸甲酯对照品0.01 g于100 mL容量瓶中, 正己烷定容, 作为对照品溶液。
鱼油样品: 称取鱼油0.10 g置于皂化瓶中, 加入50%氢氧化钾甲醇溶液5 mL, 氮气保护下, 在75 ℃水浴条件下冷凝回流30 min, 待油脂溶解, 冷却后加15%三氟化硼甲醇溶液5 mL, 充氮后75 ℃水浴条件下酯化15 min。静置冷却后加入5 mL正己烷, 完全溶解后加入5 mL饱和氯化钠溶液。
鱼肝油样品: 称取0.10 g鱼肝油置于皂化瓶中, 甲酯化处理过程与鱼油样品相同。
植物油样品: 分别称取玉米油、菜籽油、橄榄油各0.10 g置于皂化瓶, 甲酯化处理过程与鱼油样品相同。
混合油样品: 将适量的鱼油、植物油混合称取0.10 g(鱼油与植物油的体积比为9:1, 植物油种类随机), 甲酯化处理过程与鱼油样品相同。
(2)色谱条件
色谱柱: HP-5毛细管色谱柱(0.32 mm×30 m, 0.25 μm); 进样口温度: 250 ℃; 检测器温度: 270 ℃; 流动相: 氮气, 1 mL/min; 柱温: 升温程序(表2)。
气体流速: 氮气(载气): 25 mL/min, 氢气: 30 mL/min, 空气: 400 mL/min。进样量: 1 μL, 分流比40:1。
(3)鱼油、鱼肝油、各类植物油以及混合油脂肪酸检测
取上述各样品溶液, 按照1.3.2(2)色谱条件进样, 分析其中亚油酸、EPA和DHA定位以及所占百分比, 将鱼油与植物油进行鉴别与区分。
1.4 数据处理
本研究中引用的图谱和表格采用Excel 2021和SPSS 24.0进行数据处理, 每组实验均3次平行, 以平均值作为最后结果。
2 结果与分析
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2.1 维生素A含量测定新方法
紫外分光光度计法所测得吸光度不是VA的独有吸收, 在规定条件下, 非VA物质的吸收所引起的误差可用校正公式校正[33], 但鱼肝油中成分相对复杂, 经皂化后的鱼肝油仍无法使用紫外分光光度计法测得VA含量, 需再经过净化制备后才能使用紫外分光光度法有效测得其VA含量。高效液相色谱法适用于VA醋酸酯及其制剂中VA含量测定, 鱼肝油中所含有的VA为天然VA, 不以VA醋酸酯的形式存在。
药典方法中, 样品皂化后注入液相系统进行制备, 将制备后的VA样品再次进行紫外分光光度法检测, 步骤相对烦琐且易产生误差。而VA醋酸酯的高效液相法没有皂化过程, 不进行制备, 直接将样品溶解后注入正相色谱系统进行检测, 无法测得天然鱼肝油的VA含量。本研究将皂化过的样品注入反相色谱液相系统, 经过优化色谱条件, 成功建立了一个适合油脂样品中VA含量的检测方法, 其保留时间在21 min左右, 分离度较好(图1)。
2.1.1 鱼肝油中维生素A含量测定
该方法可以有效分析测定鱼肝油中VA含量, 由于使用色谱柱不同, 出峰时间在31 min左右(表3), 计算得维生素AD滴剂VA含量为4712.79 IU/g, 自制调配鱼肝油中VA含量为21083.56 IU/g。药典方法中高效液相法测定的VA是VA醋酸酯, 而鱼肝油中天然VA的存在形式不是VA醋酸酯。天然VA经皂化过程转化为视黄醇, 可以通过本研究建立的VA检测方法进行分析。
2.2 方法学验证
2.2.1 专属性
称取适量VA醋酸酯对照品, 经皂化操作后, 在检测VA色谱条件下进样, 考察视黄醇出峰状态。称取适量辅料(药用大豆油), 经皂化操作后, 在检测VA色谱条件下进样, 检测辅料在维生素A醇出峰保留时间内是否有干扰。视黄醇出峰时间在21 min左右, 峰型和分离度较好, 空白辅料在视黄醇出峰处无干扰(图2)。
2.2.2 线性关系
取VA对照品溶液分别稀释2、5、10、20、50倍, 皂化后在检测VA色谱条件下分别进样, 记录色谱图。以VA浓度为横坐标(X, IU/mL), 峰面积(Y)为纵坐标, 建立标准曲线。线性回归方程为Y=14.007X+65.252, r2=0.9999。VA含量测定新方法在30.00 IU/mL至1500.00 IU/mL的浓度范围内线性关系良好。
2.2.3 准确性
由于样品的特殊性, 选择50%、100%、150%的浓度进行加样, 每个浓度分别制备3份, 将实测值与理论值比较, 计算回收率, 结果见表4。由表4可知平均回收率为96.10%, 该方法回收率高。
2.2.4 精密度
取皂化后的VA对照品溶液进样, 进样量10 μL, 重复测定6次, 记录色谱图峰面积, 并计算相对标准偏差(relative standard deviation, RSD)值为0.18%(表5)。
精密度试验: 使用皂化方法制备6份VA供试品, 在VA含量检测色谱条件下进样, 重复测定6次, 计算样品中VA含量, 并计算RSD值为1.13%(表6)。
中间精密度试验: 取同一批VA待测样品, 在不同的时间使用不同的仪器对其VA含量进行测定, 记录VA含量测定结果, 计算RSD值为0.98%(表7)。各精密度试验RSD值均小于2%, 该方法精密度良好。
2.2.5 稳定性
取皂化后的VA供试品, 分别在0、2、4、24、48 h进样, 记录色谱图, 计算峰面积变化率, 结果见表8, 皂化后的样品在24 h内稳定性良好。48 h峰面积的增加可能与溶剂挥发有一定关系。
2.2.6 方法学验证结果
根据该VA含量测定新方法的方法学试验结果, 综合上述数据, 视黄醇浓度在30.00~1500.00 IU/mL范围内线性关系良好(r2=0.9999); 平均回收率为96.10%; 仪器精密度的相对标准偏差为0.18%, 方法精密度的相对标准偏差为1.13%, 中间精密度的相对标准偏差为0.98%; 皂化后的供试品溶液在24 h内具有良好的稳定性。得出该方法具有专属性高、精密度高、回收率高、简便易行的特点, 能够准确可靠地测定VA的含量。
2.3 气相色谱条件优化
参照鳕鱼肝油掺假植物油的指纹图谱鉴定实验[34], 将初始色谱条件设置为: HP-5毛细管色谱柱(0.32 mm×30 m, 0.25 μm); 进样口温度220 ℃; 检测器温度280 ℃; 流动相为氮气; 流速1 mL/min; 色谱柱升温程序1(表9)。
气体流速: 氮气(载气): 30 mL/min, 氢气: 40 mL/min, 空气: 450 mL/min。进样量: 1 μL, 分流比50:1。由于使用的色谱柱与文献[34]中不同, 分析结果显示多个峰分离度欠缺, 且出峰不完全, 基线上飘严重(图3), 难以判断10 min后脂肪酸甲酯中各脂肪酸对应的出峰时间。
如图3所示, 在初始色谱条件下10 min后基线出现偏离, 原因可能为该时段升温速率过快, 对部分条件改进后, 色谱条件为: HP-5毛细管色谱柱(0.32 mm×30 m, 0.25 μm); 进样口温度250 ℃; 检测器温度270 ℃; 流动相为氮气; 流速: 1 mL/min; 色谱柱升温程序2(表10)。分析结果显示脂肪酸甲酯对照品在0~60 min内出峰正常, 且能准确定位到亚油酸、EPA和DHA 3种不饱和脂肪酸(图4)。
气体流速: 氮气(载气): 25 mL/min, 氢气: 30 mL/min, 空气: 400 mL/min。进样量: 1 μL, 分流比40:1。
2.4 各类油中脂肪酸含量
实验主要分析各类油中亚油酸、EPA和DHA含量。根据脂肪酸甲酯对照品的测定, 亚油酸出峰时间在22.5 min左右, EPA出峰时间在28.6 min左右, DHA出峰时间在35.8 min左右(图4)。
鱼油中含有的主要不饱和脂肪酸有亚油酸、EPA和DHA, 其中EPA、DHA含量丰富, 占比大于亚油酸, 在总脂肪酸占比为8.64%和26.21%(图5)。鱼肝油中主要脂肪酸与鱼油基本相同, 含有丰富的亚油酸、EPA和DHA, 其亚油酸含量占比略高于鱼油, EPA和DHA占比相对鱼油较低, 在总脂肪酸占比为3.08%和16.38%。各类植物油中亚油酸含量丰富, 其中玉米油亚油酸占比85.22%, 菜籽油亚油酸占比29.78%, 橄榄油亚油酸占比69.47%。3种植物油中均未检测到EPA和DHA含量。混合油样品模拟添加了植物油后鱼肝油状态, 其中亚油酸占比47.55%, EPA和DHA占比分别为4.88%和14.77%。鱼油与植物油混合体积比为9:1, 植物油的添加, 减少了鱼油中原有的EPA和DHA占比。由于植物油中含有大量的亚油酸及其他不饱和脂肪酸, 混合油中虽然只有10%的体积比, 却导致EPA和DHA占比大幅下降。
3 结论
收起
紫外分光光度计法和高效液相色谱法(VA醋酸酯法)不适用于鱼肝油VA含量测定。紫外分光光度计法可能更适用于单一结构的VA样品检测, 药典高效液相法所定量的VA有效成分为VA醋酸酯, 但实验结果显示天然鱼肝油中VA不以VA醋酸酯的形式存在。本研究建立的新方法通过皂化过程, 将鱼肝油中的天然VA转化为视黄醇, 通过反相液相色谱系统测得其VA含量。通过方法学验证, 该方法专属性、精密度高、回收率等符合要求, 是一个通用的VA含量测定方法。
使用气相色谱系统在特定的色谱条件下, 分别定位测定了鱼肝油、鱼油以及各类植物油和混合油中的亚油酸、EPA和DHA。鱼肝油、鱼油中EPA、DHA含量丰富, 亚油酸含量少; 玉米油、菜籽油、橄榄油中均不含EPA和DHA, 但亚油酸含量丰富。通过制定亚油酸、EPA和DHA的比例限度, 能够控制鱼肝油辅料的选择, 且有效控制产品质量。
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250106006
  • 接收时间：2025-01-06
  • 首发时间：2025-07-19
  • 出版时间：2025-03-25
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  • 收稿日期：2025-01-06
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    浙江海洋大学食品与药学学院, 舟山 316022

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* 关丽萍(1970—), 女, 博士, 教授, 主要研究方向为食品安全与药物合成。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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