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Article(id=1153429497132081680, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241111004, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1731254400000, receivedDateStr=2024-11-11, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752928621798, onlineDateStr=2025-07-19, pubDate=1741968000000, pubDateStr=2025-03-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752928621798, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752928621798, creator=13701087609, updateTime=1752928621798, updator=13701087609, issue=Issue{id=1153429493357203682, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='5', pageStart='1', pageEnd='326', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752928620900, creator=13701087609, updateTime=1758690311058, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1177595773500932351, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1177595773500932352, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=38, endPage=45, ext={EN=ArticleExt(id=1153429497706701331, articleId=1153429497132081680, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Determination of biotin in formula food for special medical purposes by high performance liquid chromatography-post column derivatization, columnId=1153429494888129034, journalTitle=Journal of Food Safety & Quality, columnName=Highlight: Guangzhou Inspection Testing and Certification Group Co., Ltd., runingTitle=null, highlight=null, articleAbstract=

Objective To establish a method for the determination of biotin in formula food for special medical purposes by high performance liquid chromatography-post column derivatization. Methods The biotin in the sample was dissolved in water and extracted by enzymatic hydrolysis with amylase and papain at 60 ℃ for 1 hour in a water bath. Using Zorbax SB-AQ chromatographic column separation and entering the post column reaction device, the biotin was derived from fluorescein isothiocyanate labeled avidin. Derivatives were detected using a fluorescence detector with an excitation wavelength of 495 nm and an emission wavelength of 525 nm. The results were quantified by the external standard method. Results Under the optimized conditions, the biotin showed good (r2>0.999) linear relationships within the concentration range of 5.00‒75.00 ng/mL. The average recoveries were 97.5%‒100.1%, and the relative standard deviations were 0.86%‒2.9%. The limit of detection was 12 μg/kg, and the limit of quantification was 41 μg/kg. The relative standard deviations of the biotin standard solution and the sample solution to be tested within 24 h were 0.68% and 1.08%, respectively. There was no significant difference between the results of this method and GB 5009.256—2016 National food safety standard-Determination of biotin in food. Conclusion This method has simple pretreatment, high recovery, good sensitivity and precision, and can be used for the determination of biotin content in formula food for special medical purposes.

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目的 建立高效液相色谱-柱后衍生法测定特殊医学用途配方食品中生物素含量的检测方法。方法 样品加水溶解并经淀粉酶和木瓜蛋白酶在60 ℃水浴酶解1 h进行提取, 采用Zorbax SB-AQ色谱柱分离并进入柱后反应装置, 与异硫氰酸荧光素标记亲和素衍生; 衍生物采用荧光检测器检测, 激发波长为495 nm, 发射波长为525 nm, 外标法定量。结果 在优化条件下, 生物素在5.00~75.00 ng/mL线性范围内线性关系良好(r2>0.999), 平均回收率为97.5%~100.1%, 相对标准偏差为0.86%~2.96%, 检出限为12 µg/kg, 定量限为41 µg/kg。生物素标准溶液和样品待测液在24 h内的相对标准偏差分别为0.68%和1.08%, 实际样品测定中, 本方法与GB 5009.256—2016《食品安全国家标准 食品中生物素的测定》测定结果无显著性差异。结论 本方法前处理简单, 回收率高, 灵敏度和精密度好, 可用于特殊医学用途配方食品中生物素含量的测定。

, correspAuthors=覃天福, authorNote=null, correspAuthorsNote=
* 覃天福(1995—), 男, 助理工程师, 主要研究方向为食品安全检测、仪器分析。E-mail:
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Modern Food Technology, 2021(3): 267-274., articleTitle=Determination of biotin content in infant formula for special medical purposes, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1177619578868875356, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, xref=null, ext=[AuthorCompanyExt(id=1177619578873069661, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, companyId=1177619578868875356, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Guangzhou Inspection Testing and Certification Group Co., Ltd., Guangzhou 511447, China), AuthorCompanyExt(id=1177619578881458270, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, companyId=1177619578868875356, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=广州检验检测认证集团有限公司, 广州 511447)])], figs=[ArticleFig(id=1177619581536452768, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Fig.1, caption=Influences of different mass concentrations and flow rates of derivatives on biotin determination, figureFileSmall=QfpPc0WzMs5lSHohz9/U8Q==, figureFileBig=mNURz+O9/KUxfcYrXk1a1A==, tableContent=null), ArticleFig(id=1177619581603561633, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=图1, caption=不同衍生剂质量浓度和流速对生物素测定的影响, figureFileSmall=QfpPc0WzMs5lSHohz9/U8Q==, figureFileBig=mNURz+O9/KUxfcYrXk1a1A==, tableContent=null), ArticleFig(id=1177619581674864802, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Fig.2, caption=Standard chromatogram of biotin, figureFileSmall=z82qwLeNDv7NYfvcs8VdTw==, figureFileBig=7U2meygKBK9ZxxTENRhPng==, tableContent=null), ArticleFig(id=1177619581762945187, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=图2, caption=生物素标准色谱图, figureFileSmall=z82qwLeNDv7NYfvcs8VdTw==, figureFileBig=7U2meygKBK9ZxxTENRhPng==, tableContent=null), ArticleFig(id=1177619581821665444, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Fig.3, caption=Chromatogram of sample, figureFileSmall=pck8oVr1P69kQZGmCbL+PQ==, figureFileBig=cAHNSWQxCUY96lkxqgXx+g==, tableContent=null), ArticleFig(id=1177619581871997093, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=图3, caption=样品色谱图, figureFileSmall=pck8oVr1P69kQZGmCbL+PQ==, figureFileBig=cAHNSWQxCUY96lkxqgXx+g==, tableContent=null), ArticleFig(id=1177619581930717350, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Fig.4, caption=Chromatogram of blank sample, figureFileSmall=uj+Hzt68vLwwVPHIagRCDw==, figureFileBig=0ifnSeDkzQYl1TqGJaQBmw==, tableContent=null), ArticleFig(id=1177619582006214823, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=图4, caption=空白色谱图, figureFileSmall=uj+Hzt68vLwwVPHIagRCDw==, figureFileBig=0ifnSeDkzQYl1TqGJaQBmw==, tableContent=null), ArticleFig(id=1177619582073323688, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Fig.5, caption=Chromatogram of the spiked sample D, figureFileSmall=OsFID8IRLCKuJyK9z5NvxA==, figureFileBig=IMZHKAZfXRW7quFSz8g/4A==, tableContent=null), ArticleFig(id=1177619582161404073, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=图5, caption=加标样品D色谱图, figureFileSmall=OsFID8IRLCKuJyK9z5NvxA==, figureFileBig=IMZHKAZfXRW7quFSz8g/4A==, tableContent=null), ArticleFig(id=1177619582220124330, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Table 1, caption=

Gradient elution program (pump A)

, figureFileSmall=null, figureFileBig=null, tableContent=
时间/min 流速/(mL/min) 流动相A/% 流动相B/%
0.0 1.0 100 0
9.0 1.0 100 0
9.1 1.0 90 10
10.0 1.0 90 10
10.1 1.0 100 0
15.0 1.0 100 0
), ArticleFig(id=1177619582278844587, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=表1, caption=

梯度洗脱程序(泵A)

, figureFileSmall=null, figureFileBig=null, tableContent=
时间/min 流速/(mL/min) 流动相A/% 流动相B/%
0.0 1.0 100 0
9.0 1.0 100 0
9.1 1.0 90 10
10.0 1.0 90 10
10.1 1.0 100 0
15.0 1.0 100 0
), ArticleFig(id=1177619582341759148, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Table 2, caption=

Gradient postcolumn derivatization program (pump B)

, figureFileSmall=null, figureFileBig=null, tableContent=
时间/min 流速/(mL/min) 流动相A/% 流动相B/% 衍生剂/%
0.0 2.0 0 0 100
9.0 2.0 0 0 100
9.1 2.0 90 10 0
10.0 2.0 90 10 0
10.1 2.0 0 0 100
15.0 2.0 0 0 100
), ArticleFig(id=1177619582446616749, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=表2, caption=

柱后衍生程序(泵B)

, figureFileSmall=null, figureFileBig=null, tableContent=
时间/min 流速/(mL/min) 流动相A/% 流动相B/% 衍生剂/%
0.0 2.0 0 0 100
9.0 2.0 0 0 100
9.1 2.0 90 10 0
10.0 2.0 90 10 0
10.1 2.0 0 0 100
15.0 2.0 0 0 100
), ArticleFig(id=1177619582534697134, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Table 3, caption=

Comparison of biotin content determination results under different enzymatic hydrolysis conditions (µg/100 g)

, figureFileSmall=null, figureFileBig=null, tableContent=
样品类别 酶解条件
40 ℃酶解16 h 60 ℃酶解2.5 h 60 ℃酶解1 h
A 24.6±1.2 24.2±0.5 23.8±1.6
B 16.2±0.6 17.3±0.2 16.8±1.2
C 26.8±0.7 27.1±1.5 27.5±0.9
D 25.2±0.8 26.1±1.1 25.5±1.7
E 7.7±1.0 7.6±0.3 7.6±0.5
F 33.2±0.4 32.6±0.7 34.5±1.3
), ArticleFig(id=1177619582601805999, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=表3, caption=

不同酶解条件下测定生物素含量结果比对(µg/100 g)

, figureFileSmall=null, figureFileBig=null, tableContent=
样品类别 酶解条件
40 ℃酶解16 h 60 ℃酶解2.5 h 60 ℃酶解1 h
A 24.6±1.2 24.2±0.5 23.8±1.6
B 16.2±0.6 17.3±0.2 16.8±1.2
C 26.8±0.7 27.1±1.5 27.5±0.9
D 25.2±0.8 26.1±1.1 25.5±1.7
E 7.7±1.0 7.6±0.3 7.6±0.5
F 33.2±0.4 32.6±0.7 34.5±1.3
), ArticleFig(id=1177619582664720560, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Table 4, caption=

Test results of recoveries and precision

, figureFileSmall=null, figureFileBig=null, tableContent=
样品 本底值/(µg/100 g) 加标标准品浓度/(µg/mL) 序号 添加体积/µL 添加水平/(µg/100 g) 平均回收率/% 相对标准偏差/%
B 16.8 10.00 1 128 8.4 99.1 1.84
2 256 16.8 98.4 0.86
3 384 25.2 100.1 1.88
D 25.5 1 84 12.8 98.6 1.21
2 168 25.6 98.2 2.44
3 252 38.4 97.5 1.26
F 7.61 1 114 3.8 98.2 2.09
2 228 7.6 99.6 2.96
3 342 11.4 97.7 0.96
), ArticleFig(id=1177619582736023729, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=表4, caption=

回收率和精密度测试结果

, figureFileSmall=null, figureFileBig=null, tableContent=
样品 本底值/(µg/100 g) 加标标准品浓度/(µg/mL) 序号 添加体积/µL 添加水平/(µg/100 g) 平均回收率/% 相对标准偏差/%
B 16.8 10.00 1 128 8.4 99.1 1.84
2 256 16.8 98.4 0.86
3 384 25.2 100.1 1.88
D 25.5 1 84 12.8 98.6 1.21
2 168 25.6 98.2 2.44
3 252 38.4 97.5 1.26
F 7.61 1 114 3.8 98.2 2.09
2 228 7.6 99.6 2.96
3 342 11.4 97.7 0.96
), ArticleFig(id=1177619582798938290, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=EN, label=Table 5, caption=

Comparison of analysis results between this method and national standard method

, figureFileSmall=null, figureFileBig=null, tableContent=
样品 检测方法 生物素含量/(µg/100 g) 均值/(µg/100 g) t值 t0.05,10
1 2 3 4 5 6
A 方法1 24.3 22.9 23.6 24.3 23.2 24.6 23.8 1.410 2.228
方法2 22.8 23.4 22.6 23.8 24.1 23.1 23.3
B 方法1 16.6 17.1 16.2 17.5 16.5 16.7 16.8 0.821
方法2 16.4 15.8 16.6 16.9 16.5 17.1 16.6
C 方法1 28.1 26.5 27.4 26.8 27.8 28.3 27.5 1.016
方法2 27.5 26.4 25.9 27.2 28.2 27.0 27.0
D 方法1 25.3 24.9 25.8 25.7 25.1 26.2 25.5 0.962
方法2 25.8 24.9 26.1 25.1 24.8 24.4 25.2
E 方法1 34.1 33.8 34.7 34.3 35.2 34.9 34.5 0.718
方法2 33.6 33.2 34.1 35.1 34.5 34.9 34.2
F 方法1 7.6 7.5 7.6 7.6 7.7 7.6 7.6 1.544
方法2 7.6 7.6 7.5 7.4 7.7 7.5 7.5
), ArticleFig(id=1177619582861852851, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497132081680, language=CN, label=表5, caption=

本方法与国标方法分析结果的比较

, figureFileSmall=null, figureFileBig=null, tableContent=
样品 检测方法 生物素含量/(µg/100 g) 均值/(µg/100 g) t值 t0.05,10
1 2 3 4 5 6
A 方法1 24.3 22.9 23.6 24.3 23.2 24.6 23.8 1.410 2.228
方法2 22.8 23.4 22.6 23.8 24.1 23.1 23.3
B 方法1 16.6 17.1 16.2 17.5 16.5 16.7 16.8 0.821
方法2 16.4 15.8 16.6 16.9 16.5 17.1 16.6
C 方法1 28.1 26.5 27.4 26.8 27.8 28.3 27.5 1.016
方法2 27.5 26.4 25.9 27.2 28.2 27.0 27.0
D 方法1 25.3 24.9 25.8 25.7 25.1 26.2 25.5 0.962
方法2 25.8 24.9 26.1 25.1 24.8 24.4 25.2
E 方法1 34.1 33.8 34.7 34.3 35.2 34.9 34.5 0.718
方法2 33.6 33.2 34.1 35.1 34.5 34.9 34.2
F 方法1 7.6 7.5 7.6 7.6 7.7 7.6 7.6 1.544
方法2 7.6 7.6 7.5 7.4 7.7 7.5 7.5
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高效液相色谱-柱后衍生法测定特殊医学用途配方食品中生物素含量
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覃天福 * , 黄伟乾 , 吴俊发 , 梁贤玲 , 黄嘉瑜 , 邹昕明
食品安全质量检测学报 | 本期重点：广州检验检测认证集团有限公司 2025,16(5): 38-45
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食品安全质量检测学报 | 本期重点：广州检验检测认证集团有限公司 2025, 16(5): 38-45
高效液相色谱-柱后衍生法测定特殊医学用途配方食品中生物素含量
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覃天福* , 黄伟乾, 吴俊发, 梁贤玲, 黄嘉瑜, 邹昕明
作者信息
  • 广州检验检测认证集团有限公司, 广州 511447

通讯作者:

* 覃天福(1995—), 男, 助理工程师, 主要研究方向为食品安全检测、仪器分析。E-mail:
Determination of biotin in formula food for special medical purposes by high performance liquid chromatography-post column derivatization
Tian-Fu QIN* , Wei-Qian HUANG, Jun-Fa WU, Xian-Ling LIANG, Jia-Yu HUANG, Xin-Ming ZOU
Affiliations
  • Guangzhou Inspection Testing and Certification Group Co., Ltd., Guangzhou 511447, China
出版时间: 2025-03-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241111004
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摘要
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目的 建立高效液相色谱-柱后衍生法测定特殊医学用途配方食品中生物素含量的检测方法。方法 样品加水溶解并经淀粉酶和木瓜蛋白酶在60 ℃水浴酶解1 h进行提取, 采用Zorbax SB-AQ色谱柱分离并进入柱后反应装置, 与异硫氰酸荧光素标记亲和素衍生; 衍生物采用荧光检测器检测, 激发波长为495 nm, 发射波长为525 nm, 外标法定量。结果 在优化条件下, 生物素在5.00~75.00 ng/mL线性范围内线性关系良好(r2>0.999), 平均回收率为97.5%~100.1%, 相对标准偏差为0.86%~2.96%, 检出限为12 µg/kg, 定量限为41 µg/kg。生物素标准溶液和样品待测液在24 h内的相对标准偏差分别为0.68%和1.08%, 实际样品测定中, 本方法与GB 5009.256—2016《食品安全国家标准 食品中生物素的测定》测定结果无显著性差异。结论 本方法前处理简单, 回收率高, 灵敏度和精密度好, 可用于特殊医学用途配方食品中生物素含量的测定。

柱后衍生  /  高效液相色谱法  /  特殊医学用途配方食品  /  生物素  /  异硫氰酸荧光素标记亲和素

Objective To establish a method for the determination of biotin in formula food for special medical purposes by high performance liquid chromatography-post column derivatization. Methods The biotin in the sample was dissolved in water and extracted by enzymatic hydrolysis with amylase and papain at 60 ℃ for 1 hour in a water bath. Using Zorbax SB-AQ chromatographic column separation and entering the post column reaction device, the biotin was derived from fluorescein isothiocyanate labeled avidin. Derivatives were detected using a fluorescence detector with an excitation wavelength of 495 nm and an emission wavelength of 525 nm. The results were quantified by the external standard method. Results Under the optimized conditions, the biotin showed good (r2>0.999) linear relationships within the concentration range of 5.00‒75.00 ng/mL. The average recoveries were 97.5%‒100.1%, and the relative standard deviations were 0.86%‒2.9%. The limit of detection was 12 μg/kg, and the limit of quantification was 41 μg/kg. The relative standard deviations of the biotin standard solution and the sample solution to be tested within 24 h were 0.68% and 1.08%, respectively. There was no significant difference between the results of this method and GB 5009.256—2016 National food safety standard-Determination of biotin in food. Conclusion This method has simple pretreatment, high recovery, good sensitivity and precision, and can be used for the determination of biotin content in formula food for special medical purposes.

postcolumn derivatization  /  high performance liquid chromatography  /  formula food for special medical purposes  /  biotin  /  fluorescein isothiocyanate labeled avidin
覃天福, 黄伟乾, 吴俊发, 梁贤玲, 黄嘉瑜, 邹昕明. 高效液相色谱-柱后衍生法测定特殊医学用途配方食品中生物素含量. 食品安全质量检测学报, 2025 , 16 (5) : 38 -45 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241111004
Tian-Fu QIN, Wei-Qian HUANG, Jun-Fa WU, Xian-Ling LIANG, Jia-Yu HUANG, Xin-Ming ZOU. Determination of biotin in formula food for special medical purposes by high performance liquid chromatography-post column derivatization[J]. Journal of Food Safety & Quality, 2025 , 16 (5) : 38 -45 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241111004
正文
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0 引言
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特殊医学用途配方食品[1]是以满足特定人群的特殊医学或营养需求为目的的食品, 广泛应用于临床治疗和预防疾病的过程中[2], 以及婴幼儿和儿童的生长发育阶段[3], 可以作为母乳的替代品, 以满足婴儿的营养需求, 也可以根据相关的医学指导提供相应的营养支持, 满足慢性疾病患者日常中重要的膳食补充[4-6]。根据相关法规, 特殊医学用途配方食品必须符合特定的营养需求, 而且必须标明使用范围和剂量。
生物素是一种重要的水溶性维生素[7], 对人体的正常生长、代谢和发育起着重要的作用, 在人体的生理过程中参与多种代谢酶的催化反应, 如羧化酶和戊二酰辅酶A羧化酶等, 此外, 生物素还参与胆固醇和脂肪酸的合成以及葡萄糖的代谢过程, 以及蛋白质的代谢过程[8-9]。然而, 生物素在人体内不能自主合成, 需要通过摄入食物来获得。人体缺乏生物素, 会出现食欲减退、抑郁、嗜睡、皮炎等一系列症状, 引起免疫力下降、婴幼儿发育延迟的问题[10-11]。生物素是特殊医学用途配方食品中重要的营养成分, 根据GB 25596—2010《食品国家标准 特殊医学用途婴儿配方食品通则》、GB 29922—2013《食品国家标准 特殊医学用途配方食品通则》和GB 14880—2012《食品国家标准 食品营养强化剂使用标准》, 明确规定了其添加形式和添加量, 是该类食品中重要的检测项目。
目前, 广泛应用的生物素检测方法是微生物法[12-13], 除此之外还有酶联免疫法[14]、荧光层析柱法[15]、荧光分光光度法[16]、生物传感器法[17]、液相色谱法[18-20]、酶联免疫吸附法[21]、液相色谱-串联质谱法[22-23]等。其中, 荧光分光光度法可靠性存在缺陷, 要求待测液无色、无浑浊; 液相色谱-串联质谱法对样品基质敏感, 不适用于基质较为复杂的特殊学医用途配方食品; 滴定分析法适用于常量生物素的测定; 液相色谱法紫外检测灵敏度较低; 微生物法是目前国内检测生物素比较成熟的一种方法, 灵敏度高, 但菌株专一性较差, 结果准确度低, 检验周期较长, 操作复杂, 环境要求较高, 菌种难保存等问题; 而酶联免疫吸附法利用生物素与亲和素之间的抗原-抗体反应, 具有较强特异性和专一性, 操作简单、选择廉价, 但容易出现假阳性。
鉴于此, 本研究采用高效液相色谱-柱后衍生法[24-26], 通过优化提取条件、色谱分离条件等, 分离出生物素, 利用生物素与异硫氰酸荧光素标记的亲和素发生抗原-抗体反应, 荧光检测器检测, 建立测定生物素含量的检测方法, 以此为特殊医学用途配方食品中生物素含量的准确检测提供参考。
1 材料与方法
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1.1 试剂与材料
维生素B7 (d-生物素)标准品(纯度≥99%, 上海安谱璀世标准技术服务有限公司); 异硫氰酸荧光素标记亲和素(来源鸡蛋白, 美国Sigma公司); 淀粉酶(酶活力10万U/g)、木瓜蛋白酶(酶活力800 U/mg)(上海源叶生物科技有限公司); 甲醇、异丙醇(色谱纯, 美国赛默飞世尔科技有限公司); 无水乙醇、盐酸、磷酸二氢钾、氢氧化钠(分析纯, 广州化学试剂厂)。
市售已注册的特殊医学用途配方食品, 分别为: 无乳糖配方食品(A)、乳蛋白部分水解配方食品(B)、氨基酸配方食品(C)、早产/低出生体重婴儿配方食品(D)、全营养配方液(E)、全营养配方粉(F)。
1.2 仪器与设备
U3000双三元液相色谱仪(自带双泵系统和配荧光检测器, 美国赛默飞世尔科技有限公司); Milli-Q去离子水发生器(美国Millipore公司); SW-22恒温振荡水浴锅(美国优莱博公司); KQ3200V超声波清洗器(昆山市超声仪器有限公司); PE28 pH计、MX105/A分析天平(精度0.01 mg)[梅特勒托利多仪器(上海)有限公司]; ZorbaxSB-AQ色谱柱(4.6 mm×150 mm, 5 μm)、ZorbaxSB-AQ保护柱(4.6 mm× 12.5 mm, 5 μm)(美国安捷伦公司); IKA-RO15磁力搅拌器(德国IKA公司)。
1.3 试验方法
1.3.1 样品前处理
精确称取固体试样10 g或液体试样30 g(精确至0.001 g)至250 mL锥形瓶中, 加约60 ℃水至60 mL左右, 加入100 mg淀粉酶和木瓜蛋白酶, 在60 ℃恒温水浴锅下酶解1 h, 取出, 冷却至室温, 用1.0 mol/L盐酸溶液调pH至4.5, 转移至100 mL棕色容量瓶中, 加水至刻度, 摇匀, 静止10 min, 用滤纸过滤, 弃去初滤液约5 mL, 收集续滤液, 取续滤液过0.45 µm水相滤膜, 用棕色样品瓶收集, 供液相色谱仪分析。
1.3.2 标准溶液及试剂的配制
生物素标准储备液的配制: 称量25 mg(精确至0.01 mg)生物素至250 mL棕色玻璃容量瓶中。加入乙醇水(50:50, V:V)超声溶解并定容, 在4 ℃下保存, 可放置1个月。
生物素标准中间液的配制: 移取2.00 mL生物素标准储备液于100 mL棕色容量瓶中并用水定容, 再移取5.00 mL稀释液于100 mL棕色容量瓶中并用水定容, 现配现用。
生物素标准系列工作液的配制: 分别准确量取生物素标准中间液7.50、5.00、2.50、1.00、0.50 mL于10 mL棕色容量瓶中并用水定容, 现配现用。
盐酸溶液的配制(1.0 mol/L): 移取85.0 mL盐酸, 用水稀释并定容至1000 mL, 摇匀。
氢氧化钠溶液的配制(2.0 mol/L): 称取40 g氢氧化钠于500 mL容量瓶中, 加入400 mL水搅拌完全溶解, 待冷却后用水稀释至刻度。
磷酸二氢钾溶液的配制(0.1 mol/L, pH=6.0): 称取13.6 g磷酸二氢钾于1000 mL烧杯中, 加入900 mL水溶解, 用2.0 mol/L氢氧化钠溶液调pH为6.00±0.05, 转移到1 L容量瓶中, 加水定容。
磷酸二氢钾溶液的配制(0.1 mol/L, pH=7.0): 称取13.6 g磷酸二氢钾于1000 mL烧杯中, 用2.0 mol/L氢氧化钠溶液调pH为7.0±0.05, 转移至1 L容量瓶中, 加水定容。
异硫氰酸荧光素标记亲和素溶液的配制(2.0 µg/mL): 称取2.5 mg异硫氰酸荧光素标记亲和素于50 mL棕色容量瓶中, 用pH 7.0的0.1 mol/L磷酸二氢钾溶液溶解并稀释至刻度, 4 ℃下保存。再移取20.0 mL该溶液于500 mL棕色容量瓶中, 用pH 7.0 0.1 mol/L磷酸二氢钾溶液稀释至刻度, 现配现用。
1.3.3 生物素含量的计算
生物素含量计算公式(1)如下:
$\text { 生物素 } /(\mu \mathrm{g} / 100 \mathrm{~g})=\frac{c \times V}{m} \times \frac{100}{1000}$
式中: c为待测液的质量浓度, ng/mL; V为定容体积, mL; m为称样质量, g。
1.3.4 色谱分析条件
色谱柱: ZorbaxSB-AQ (4.6 mm×150 mm, 5 μm); 保护柱: ZorbaxSB-AQ (4.6 mm×12.5 mm, 5 μm); 柱温: 35 ℃; 检测器: 荧光检测器; 激发波长: 495 nm; 发射波长: 525 nm; 进样量: 100 µL; 流动相A: 0.1 mol/L磷酸二氢钾溶液(pH=6.0):甲醇=94:6 (V:V); 流动相B: 异丙醇; 衍生剂溶液: 荧光素标记亲和素溶液; 衍生温度: 35 ℃; 梯度洗脱程序见表1、柱后衍生程序表见表2。
1.4 数据处理
实验重复3次测定。采用Thermo液相色谱仪自带Chromeleon 7色谱工作站系统进行自动采集数据和定性、定量分析; 仪器配套的电脑Windows 7专业版系统自带Office 2007软件进行数据计算, 以及自带图型处理工具绘制图表。
2 结果与分析
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2.1 样品前处理的确定
2.1.1 提取方式的选择
生物素常作为营养强化剂与油脂、维生素、矿物质等原料添加于食品基质中, 几乎都是以游离的形式存在, 能够很好的溶于水中, 因此提取样品中生物素常采用的方式有: 直接提取、酶解、酸水解。酸水解方式通常需要高温高压下进行, 设备要求耐腐蚀、高温和高压, 而且副反应较多, 反应产物复杂; 由于本次分析样品为特殊医学用途配方食品, 其配方复杂、工艺类型较多和品种多样, 当样品采用直接提取方式时, 试样液存在难以过滤的问题, 且用滤纸过滤得到的滤液出现浑浊, 此时滤液过0.22 µm水相滤膜依然得不到澄清的待测液, 然而当样品溶解并经木瓜蛋白酶和淀粉酶酶解后得到酶解液, 酶解液用滤纸过滤得到的滤液过0.45 µm水相滤膜即可得到澄清待测液, 为了避免用不澄清的待测液直接上机分析使色谱柱堵塞而破坏色谱柱, 所以本研究选择酶解方式进行提取。
2.1.2 酶解条件的选择
样品在加水溶解后, 加入100 mg淀粉酶和木瓜蛋白酶, 分别置于以下条件进行: (1) 40 ℃酶解16 h; (2) 60 ℃酶解2.5 h; (3) 60 ℃酶解1 h。酶解结束后放置至室温, 调节pH至4.5, 定容, 混匀, 过滤, 上机分析测定生物素的含量, 结果见表3。结果表明, 3种酶解条件下特殊医学用途配方食品中生物素的提取效果无明显差异, 为了提高实验效率, 最终选择酶解条件为60 ℃酶解1 h。
2.1.3 沉淀蛋白方式的选择
目前常用沉淀蛋白质的方式[27-29]有: 有机溶剂蛋白变性法、沉淀剂胶体法、调节等电点法等。卢兰香等[30]在特殊医学用途婴儿配方食品中生物素含量的测定中研究发现3种沉淀方式均具有良好的沉淀效果, 但乙腈和亚铁氰化钾-乙酸锌两种沉淀方式会形成胶体吸附杂质致降低生物素的提取效果, 所以本研究选择调节pH 4.5为沉淀蛋白方式。
2.2 波长的确定
荧光检测波长通常是通过多-激发、发射波长扫描, 以此确定最佳激发波长和发射波长。实验利用生物素标液与荧光素标记亲和素衍生剂混合后在35 ℃恒温水浴锅进行模拟柱后衍生反应, 得到生物素-荧光素标记亲和素的产物, 将此产物做荧光光谱扫描, 设定激发波长分别为400 nm和450 nm, 进行多-发射波长扫描, 结果均显示只有一个峰, 且在525 nm处具有较高响应, 可以选择作为发射波长, 然后以测得的波长525 nm设定为发射波长, 进行多-激发波长扫描, 结果在260 nm和495 nm处均具有响应, 考虑到本次测定样品的基质比较复杂, 为了减少杂质的影响, 故选择495 nm为激发波长。因此最终确定检测波长: 激发波长为495 nm, 发射波长为525 nm。
2.3 色谱柱的选择
生物素分子的特殊化学结构, 带有两个五元杂环和一条戊酸的侧链, 在中性溶液中部分以离子形式存在, 具有较强的极性, 在传统的RP-C18柱上保留较弱, 且容易受流动相的pH影响, 而ZorbaxSB-AQ柱对极性化合物有较强的保留能力, 具有亲水性的表面, 能够在耐受100%水溶液流动相和较低的pH时, 分析极性化合物依然能获得良好的峰型和重复性, 可以有效地分离和浓缩样品中的生物素, 所以本方法选择安捷伦的ZorbaxSB-AQ色谱柱(4.6 mm× 150 mm, 5 μm)作为分析色谱柱。由于方法的前处理相对比较简单, 中间无除杂和净化操作, 且分析的样品基质较为复杂, 本方法还选择了ZorbaxSB-AQ色谱柱(4.6 mm×12.5 mm, 5 μm)作为保护柱, 以便更好的保护色谱柱, 延长其使用寿命。
2.4 色谱条件的优化
2.4.1 流动相的选:
本研究采用0.1 mol/L磷酸二氢钾溶液(pH=6.0):甲醇(94:6, V:V)作为洗脱液时, 生物素出峰时间约在7.5 min, 且具有良好的峰型和分离度, 目标峰出峰后, 利用中等极性有机溶剂异丙醇:流动相A (10:90, V:V)作为冲洗液冲洗色谱柱中保留的杂质, 以便避免样品中杂质干扰下一个样品。所以本研究选择0.1 mol/L磷酸二氢钾溶液(pH=6.0):甲醇(94:6, V:V)作为洗脱阶段的流动相, 异丙醇:流动相A (10:90, V:V)作为冲洗阶段的流动相。
2.4.2 衍生条件的选择
实验发现柱后衍生剂的浓度和流速直接影响结果的测定。本研究采用了单一变量法, 考察了衍生剂流速和质量浓度对衍生效果的影响, 比较了不同衍生剂浓度和流速时生物素的单位质量浓度(为标准曲线最大点质量浓度75.0 ng/mL)峰面积变化, 结果见图1。研究表明, 随着衍生剂质量浓度和流速的增加, 生物素的单位质量浓度峰面积逐渐增大; 当衍生剂质量浓度为2.0 µg/mL和流速为2.0 mL/min时, 生物素的单位质量浓度峰面积达到平衡。所以本方法选择衍生剂的质量浓度为2.0 µg/mL, 流速为2.0 mL/min。
2.5 方法学验证
2.5.1 标准曲线的绘制
对生物素标准系列工作液进行测定, 以生物素的质量浓度为横坐标(X, ng/mL), 生物素的色谱峰面积为纵坐标(Y)进行线性回归, 得到生物素的线性方程Y=120.72X+34.841, 在5.00~75.00 ng/mL线性范围内线性关系良好(r2>0.999)。
2.5.2 定性和专属性试验
准备两个锥形瓶, 一个称取含有生物素的特殊医学用途配方食品的锥形瓶标记为样品测试, 一个不含有样品的空锥形瓶标记为空白测试, 按照本研究样品前处理项下操作, 然后配制一个质量浓度为20 ng/mL的生物素标准溶液, 按照本研究色谱条件项下的条件上机分析测定样品溶液、空白溶液和标准溶液。结果表明, 标准溶液和样品溶液约在7.5 min出现色谱峰, 说明该色谱峰为目标峰, 详细见生物素标准色谱图(图2)和样品色谱图(图3)。而空白溶液上没有出现色谱峰, 详细见空白色谱图(图4), 说明相关试剂在相同条件下对目标峰无干扰, 表明该方法具有良好的专属性。
2.5.3 回收率和精密度试验
取3种特殊医学用途配方食品作为代表进行加标回收试验, 每种样品进行3个水平加标, 每个加标水平同时做6个平行试验。准确称取样品, 向每个加标样品中分别一定体积的生物素标准溶液后(生物素标准溶液浓度、加标体积见表4), 静置10 min后, 按照1.3.1项下样品前处理操作, 在本方法的色谱条件下测定, 代表性加标样品B添加水平为16.8 µg/100 g的色谱图见图5, 测试结果见表4, 由表4可知, 平均回收率在97.5%~100.1%之间, 相对标准偏差在0.86%~2.96%之间, 表明该方法具有良好的准确度和精密度, 可用于测定特殊医学用途配方食品中生物素的含量。
2.5.4 稳定性实验
选取质量浓度为20 ng/mL的生物素标准工作液和氨基酸配方食品待测液分别于制备后的0、2、4、8、12、24 h在相同色谱条件下进样分析, 记录每个时间点的色谱峰峰面积, 计算得到在24 h内标准工作液和样品待测液中生物素色谱峰峰面积的相对标准偏差(n=6)分别为0.68%和1.08%, 表明标准工作液和样品待测液在24 h内稳定性良好。
2.5.5 方法检出限和定量限
本研究选择不含有生物素的非全营养配方食品作为阴性样品, 对阴性样品做加标试验(加标水平为: 5 µg/100 g), 以仪器3倍信噪比(S/N=3)和10倍信噪比(S/N=10)计算的检出限。可得生物素的方法检出限为12 µg/kg, 定量限为41 µg/kg, 对比GB 5009.256—2016《食品安全国家标准 食品中生物素的测定》方法中生物素的检出限为2.0 µg/100 g、定量限为4.0 µg/100 g, 具有良好的灵敏度, 可用于特殊医学用途配方食品中生物素的定性定量分析。
2.6 实际样品检测
为了验证方法能否应用于实际样品的测定, 实验用本方法(方法1)和GB 5009.256—2016(方法2)对市售不同牌子的6种特殊医学用途配方食品进行6平行测定结果进行比对评价。方法1样品的前处理是采用木瓜蛋白酶和淀粉酶进行60 ℃水浴酶解1 h后得到酶解液, 酶解液经调pH至4.5沉淀蛋白质, 滤纸过滤, 滤液用0.45 µm水相滤膜得到待测液即可上机分析测定; 方法2样品的前处理是硫酸溶液121 ℃水解30 min得到水解液, 水解液经调pH至4.5沉淀蛋白质, 滤纸过滤, 滤液再次调pH至6.8, 灭菌、接种和培养约20 h后进行测定; 通过计算t值检验评价两种方法的差异性(结果见表5), 结果表明, 6种样品的测定结果均大于80%的明示值, 计算t值均小于2.228(t0.05,10), 表示同样品两组结果间的差异无统计学意义, 说明两种方法测定结果无显著性差异, 方法1的样品前处理操作可靠, 具有检验周期短、简便、准确度高的特点, 因此本研究方法可以应用于特殊医学用途配方食品中生物素的检测。
3 结论
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本研究利用生物素-亲和素系统结合异硫氰酸荧光素标记技术, 建立了高效液相色谱-柱后衍生法测定特殊医学用途配方食品中生物素含量的分析方法。样品经过酶解提取、色谱柱分离后, 进行衍生化反应, 增加了目标物的荧光响应强度; 使用淀粉酶和木瓜蛋白酶优化样品前处理方法, 以及采用在线柱后衍生, 简化了前处理步骤, 缩短检验周期; 通过方法学验证表明该方法线性和精密度良好、回收率高, 灵敏度高, 稳定性和重现性好, 可批量测定特殊医学用途配方食品中生物素含量, 为食品安全保障提供技术支持。
文献
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2025年第16卷第5期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241111004
  • 接收时间：2024-11-11
  • 首发时间：2025-07-19
  • 出版时间：2025-03-15
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  • 收稿日期：2024-11-11
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    广州检验检测认证集团有限公司, 广州 511447

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* 覃天福(1995—), 男, 助理工程师, 主要研究方向为食品安全检测、仪器分析。E-mail:
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2种不同金属材料的力学参数
科
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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