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Article(id=1153986713459352034, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986709126635984, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241015002, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1728921600000, receivedDateStr=2024-10-15, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1753061472524, onlineDateStr=2025-07-21, pubDate=1737734400000, pubDateStr=2025-01-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1753061472524, onlineIssueDateStr=2025-07-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1753061472524, creator=13701087609, updateTime=1753061472524, updator=13701087609, issue=Issue{id=1153986709126635984, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='2', pageStart='1', pageEnd='324', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1753061471492, creator=13701087609, updateTime=1760345674980, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1184538872999457117, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986709126635984, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1184538872999457118, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986709126635984, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=91, endPage=98, ext={EN=ArticleExt(id=1153986713929114086, articleId=1153986713459352034, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Rapid determination of nucleotides content in infant formula and dairy product by mixed-mode hydrophilic interaction/ion exchange chromatography, columnId=1153986712901505118, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Dairy Processing and Quality Control, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a rapid method for the determination of 5 kinds of nucleotides (cytidine-5’-monophosphate, adenosine-5’-monophosphate, uridine-5’-monophosphate, guanosine-5’-monophosphate and inosine-5’-monophosphate) in infant formulas and dairy products is developed based on mixed-mode hydrophilic interaction/ion exchange chromatography-ultraviolet detection (HILIC/IEX-UV). Methods A zwitterionic hydrophilic interaction chromatography column (Alphasil S-Xion) was used, with mobile phase consisting of 5 mmol/L KH2PO4 (pH adjusted to 3.0 with H3PO4)-acetonitrile (70:30, V:V) to detect nucleotides in infant foods and dairy products. Results The five nucleotides were baseline separated within 10 min, demonstrating efficient chromatographic performance. They exhibited good linearity within the range of 2-50 μg/mL, with limits of detection (LODs) between 0.19 and 0.66 mg/kg, and limits of quantification (LOQs) ranging from 0.62 to 2.19 mg/kg. At spiked levels of 50, 100 and 200 mg/kg, the intraday recoveries ranged from 96.6% to 108.9%, with relative standard deviations (RSDs) of 1.5%-6.3%, while the interday recoveries were between 101.0% and 108.0%, with RSDs of 2.4%-7.3%. Conclusions Compared with the traditional reversed-phase ion-pair chromatography, this method has significant advantages: Effectively reducing the matrix interference, shortening the analysis time, improving the detection efficiency, and especially improving the quantitative accuracy of cytidine-5’-monophosphate. The proposed method is rapid, accurate, and anti-interference, which provides technical support for the efficient analysis of nucleotides in infant formulas and dairy products.

, correspAuthors=Bo-Hui WANG, 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=Yan-Lun XUE, Bo-Hui WANG, Dong-Ming LIANG, Ying-Jie LI, Xing ZHANG, Ke QIAO, Ni-Han SU), CN=ArticleExt(id=1153986750654440031, articleId=1153986713459352034, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=亲水/离子交换混合模式色谱法快速检测婴幼儿食品和乳品中核苷酸含量, columnId=1153986713060888672, journalTitle=食品安全质量检测学报, columnName=本期专题：乳制品加工与质量控制, runingTitle=null, highlight=null, articleAbstract=

目的 建立亲水/离子交换混合模式色谱-紫外检测法(hydrophilic interaction/ion exchange chromatography-ultraviolet detection, HILIC/IEC-UV)快速测定婴幼儿食品和乳制品中5种核苷酸(胞嘧啶核苷酸、腺嘌呤核苷酸、尿嘧啶核苷酸、鸟嘌呤核苷酸和次黄嘌呤核苷酸)含量的分析方法。方法 采用两性离子亲水作用色谱柱(Alphasil S-Xion), 以5 mmol/L磷酸二氢钾水溶液(磷酸调pH 3.0)-乙腈(70:30, V:V)为流动相, 对婴幼儿食品和乳品中的核苷酸进行检测。结果 该方法在10 min内实现了5种核苷酸的高效分离, 5种核苷酸在2~50 μg/mL线性范围内线性关系良好, 检出限(limits of detection, LODs)为0.19~0.66 mg/kg, 定量限(limits of quantification, LOQs)为0.62~2.19 mg/kg。在50、100、200 mg/kg的加标水平, 5种核苷酸的日内回收率为96.6%~108.9%, 相对标准偏差(relative standard deviations, RSDs)为1.5%~6.3%, 日间回收率为101.0%~108.0%, RSDs为2.4%~7.3%。结论 与传统的反相离子对色谱法相比, 本方法具有显著优势: 有效降低了样品基质干扰, 缩短了分析时间, 提高了检测效率, 特别是改善了胞嘧啶核苷酸的定量准确性。该方法快速、准确、抗干扰, 为婴幼儿食品和乳制品中核苷酸的高效分析提供了技术支持。

, correspAuthors=王柏辉, authorNote=null, correspAuthorsNote=
* 王柏辉(1990—), 男, 博士, 高级工程师, 主要研究方向为肉品科学和食品检测。E-mail:
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薛彦伦(1985—), 男, 工程师, 主要研究方向为食品安全。E-mail:

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薛彦伦(1985—), 男, 工程师, 主要研究方向为食品安全。E-mail:

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薛彦伦(1985—), 男, 工程师, 主要研究方向为食品安全。E-mail:

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Journal of Agricultural and Food Chemistry, 2010, 58(18): 9918-9924., articleTitle=Development and application of an HILIC-MS/MS method for the quantitation of nucleotides in infant formula, refAbstract=null), Reference(id=1184567001256178070, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, doi=null, pmid=null, pmcid=null, year=2015, volume=184, issue=null, pageStart=188, pageEnd=195, url=null, language=null, rfNumber=[31], rfOrder=39, authorNames=SFAKIANAKI AG, STALIKAS CD, journalName=Food Chemistry, refType=null, unstructuredReference=SFAKIANAKI AG, STALIKAS CD. Selective microextraction of mononucleotides from milk using alumina and stannia hollow fibers prior to their determination by hydrophilic interaction liquid chromatography- diode array detection[J]. Food Chemistry, 2015, 184: 188-195., articleTitle=Selective microextraction of mononucleotides from milk using alumina and stannia hollow fibers prior to their determination by hydrophilic interaction liquid chromatography- diode array detection, refAbstract=null), Reference(id=1184567001302315415, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, doi=null, pmid=null, pmcid=null, year=2017, volume=35, issue=8, pageStart=832, pageEnd=836, url=null, language=null, rfNumber=[32], rfOrder=40, authorNames=励炯, 王姣斐, 邱红钰, journalName=色谱, refType=null, unstructuredReference=励炯, 王姣斐, 邱红钰, 等. 改进的高效液相色谱法测定婴幼儿配方奶粉中5种核苷酸[J]. 色谱, 2017, 35(8): 832-836., articleTitle=改进的高效液相色谱法测定婴幼儿配方奶粉中5种核苷酸, refAbstract=null), Reference(id=1184567001361035672, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, doi=null, pmid=null, pmcid=null, year=2017, volume=35, issue=8, pageStart=832, pageEnd=836, url=null, language=null, rfNumber=[32], rfOrder=41, authorNames=LI J, WANG JF, QIU HY, journalName=Chinese Journal of Chromatography, refType=null, unstructuredReference=LI J, WANG JF, QIU HY, et al. Determination of five nucleotides in infant formula milk powder by modified high performance liquid chromatography[J]. Chinese Journal of Chromatography, 2017, 35(8): 832-836., articleTitle=Determination of five nucleotides in infant formula milk powder by modified high performance liquid chromatography, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1184566992154538285, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, xref=null, ext=[AuthorCompanyExt(id=1184566992162926894, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, companyId=1184566992154538285, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Ordos City Inspection and Testing Center, Erdos 017010, China), AuthorCompanyExt(id=1184566992171315503, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, companyId=1184566992154538285, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=鄂尔多斯市检验检测中心, 鄂尔多斯 017010)])], figs=[ArticleFig(id=1184566997024125275, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Fig.1, caption=Chemical structures of the nucleotides, figureFileSmall=W9ygWWU5CB6JnGtdFuzpNw==, figureFileBig=4AJwckLFEwryBXKBukRxag==, tableContent=null), ArticleFig(id=1184566997099622748, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=图1, caption=核苷酸结构式, figureFileSmall=W9ygWWU5CB6JnGtdFuzpNw==, figureFileBig=4AJwckLFEwryBXKBukRxag==, tableContent=null), ArticleFig(id=1184566997158343005, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Fig.2, caption=Chromatograms of the nucleotides separated on an Alphasil VC-C18 column, figureFileSmall=DzbTM5pP/bdLeELxGix//w==, figureFileBig=OLue3XGcwvixFksdjtH+6A==, tableContent=null), ArticleFig(id=1184566997212868958, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=图2, caption=采用Alphasil VC-C18色谱柱分离核苷酸的色谱图

注: a. 核苷酸标准溶液; b. 实际样品; c. 在实际样品b中继续加标CMP; 溶质: 1. CMP; 2. AMP; 3. UMP; 4. GMP; 5. IMP。色谱条件: 色谱柱: Alphasil VC-C18 (250 mm×4.6 mm, 5 μm); 流动相A: 10 mmol/L磷酸二氢钾+1.4 mmol/L四丁基硫酸氢铵, 用100 mmol/L磷酸氢二钾调节pH至3.2; 流动相B: 甲醇; 等度洗脱, A:B=96:4 (V:V); 流速: 1.0 mL/min; 紫外检测波长: 254 nm; 柱温: 25 ℃。

, figureFileSmall=DzbTM5pP/bdLeELxGix//w==, figureFileBig=OLue3XGcwvixFksdjtH+6A==, tableContent=null), ArticleFig(id=1184566997271589215, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Fig.3, caption=Retention and separation of nucleotides under different buffer salt types, figureFileSmall=guF5KfMpU1a63jh6fkXsiA==, figureFileBig=UqenSjkaww2WHQU4Gm2xpA==, tableContent=null), ArticleFig(id=1184566997368058208, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=图3, caption=不同缓冲盐类型条件下核苷酸的保留与分离情况

注: 色谱条件: 色谱柱: Alphasil S-Xion (150 mm×4.6 mm, 5 μm); 流动相A: 10 mmol/L甲酸铵(甲酸调pH 3.0)或10 mmol/L乙酸铵(乙酸调pH 3.0)或10 mmol/L磷酸二氢钾(磷酸调pH 3.0); 流动相B: 乙腈; 等度洗脱, A:B=30:70 (V:V); 流速: 1.0 mL/min; 紫外检测波长: 254 nm; 柱温: 40 ℃。

, figureFileSmall=guF5KfMpU1a63jh6fkXsiA==, figureFileBig=UqenSjkaww2WHQU4Gm2xpA==, tableContent=null), ArticleFig(id=1184566997443555681, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Fig.4, caption=Retention and separation of nucleotides under different acetonitrile ratio conditions, figureFileSmall=S4yf779eQF3uB1dVwW1NkQ==, figureFileBig=2rfKxMn7lBDXde01uWw0rQ==, tableContent=null), ArticleFig(id=1184566997498081634, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=图4, caption=不同乙腈比例条件下核苷酸的保留与分离情况

注: 色谱条件: 色谱柱: Alphasil S-Xion (150 mm×4.6 mm, 5 μm); 流动相A: 10 mmol/L磷酸二氢钾(磷酸调pH 3.0); 流动相B: 乙腈; 流动相C: 水; 等度洗脱, 保持30% A恒定, B相的比例如图4所示, 并由C相将总比例补齐至100%; 流速: 1.0 mL/min; 紫外检测波长: 254 nm; 柱温: 40 ℃。

, figureFileSmall=S4yf779eQF3uB1dVwW1NkQ==, figureFileBig=2rfKxMn7lBDXde01uWw0rQ==, tableContent=null), ArticleFig(id=1184566997569384803, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Fig.5, caption=Retention and separation of nucleotides under different buffer pH conditions, figureFileSmall=QxZkM/GUj/owKMgMUNLbcg==, figureFileBig=c8sx2YSx/lnECsWKfhuYxg==, tableContent=null), ArticleFig(id=1184566997640687972, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=图5, caption=不同缓冲盐pH条件下核苷酸的保留与分离情况

注: 色谱条件: 色谱柱: Alphasil S-Xion (150 mm×4.6 mm, 5 μm); 流动相A: 10 mmol/L磷酸二氢钾(磷酸调pH 2.0、3.0、4.0、5.0); 流动相B: 乙腈; 等度洗脱, A:B=70:30 (V:V); 流速: 1.0 mL/min; 紫外检测波长: 254 nm; 柱温: 40 ℃。

, figureFileSmall=QxZkM/GUj/owKMgMUNLbcg==, figureFileBig=c8sx2YSx/lnECsWKfhuYxg==, tableContent=null), ArticleFig(id=1184566997720379749, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Fig.6, caption=Retention and separation of nucleotides under different buffer concentrations, figureFileSmall=yZEg/O1i5emIAW7T1hSdXw==, figureFileBig=8lD5RpjvPnD+99t689orfQ==, tableContent=null), ArticleFig(id=1184566997791682918, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=图6, caption=不同缓冲盐浓度条件下核苷酸的保留与分离情况

注: 色谱条件: 色谱柱: Alphasil S-Xion (150 mm×4.6 mm, 5 μm); 流动相A: 5、10或20 mmol/L磷酸二氢钾(磷酸调pH 3.0); 流动相B: 乙腈; 等度洗脱, A:B=70:30 (V:V); 流速: 1.0 mL/min; 紫外检测波长: 254 nm; 柱温: 40 ℃。

, figureFileSmall=yZEg/O1i5emIAW7T1hSdXw==, figureFileBig=8lD5RpjvPnD+99t689orfQ==, tableContent=null), ArticleFig(id=1184566997858791783, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Fig.7, caption=Analysis chromatograms of nucleotide standards and actual sample, figureFileSmall=q45n0cGrMYGYFfInoqJp1A==, figureFileBig=wYyx6asdX3CZR1G/yGhdKg==, tableContent=null), ArticleFig(id=1184566997921706344, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=图7, caption=核苷酸标准品及实际样品分析色谱图

注: a. 核苷酸标准溶液; b. 实际样品; 溶质: 1. CMP; 2. AMP; 3. UMP; 4. IMP; 5. GMP。

, figureFileSmall=q45n0cGrMYGYFfInoqJp1A==, figureFileBig=wYyx6asdX3CZR1G/yGhdKg==, tableContent=null), ArticleFig(id=1184566997980426601, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Table 1, caption=

Linear equations, correlation coefficients, LODs, and LOQs for 5 kinds of nucleotides

, figureFileSmall=null, figureFileBig=null, tableContent=
分析物 线性方程 相关系数(r2) LODs/(mg/kg) LOQs/(mg/kg)
CMP Y=8057X-1149 0.9999 0.40 1.33
AMP Y=27356X-3208 0.9999 0.19 0.62
UMP Y=17789X-2214 0.9999 0.41 1.37
IMP Y=11886X-1501 0.9999 0.66 2.19
GMP Y=15586X-2013 0.9999 0.60 1.99
), ArticleFig(id=1184566998039146858, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=表1, caption=

5种核苷酸的线性方程、相关系数、LODs和LOQs

, figureFileSmall=null, figureFileBig=null, tableContent=
分析物 线性方程 相关系数(r2) LODs/(mg/kg) LOQs/(mg/kg)
CMP Y=8057X-1149 0.9999 0.40 1.33
AMP Y=27356X-3208 0.9999 0.19 0.62
UMP Y=17789X-2214 0.9999 0.41 1.37
IMP Y=11886X-1501 0.9999 0.66 2.19
GMP Y=15586X-2013 0.9999 0.60 1.99
), ArticleFig(id=1184566998106255723, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Table 2, caption=

Recoveries and precisions of nucleotides in infant formula

, figureFileSmall=null, figureFileBig=null, tableContent=
分析物 浓度/(mg/kg) 日内(n=6) 日间(n=18)
回收率/% RSDs/% 回收率/% RSDs/%
CMP 50 102.8 5.4 103.3 3.3
100 103.1 4.1 101.0 4.1
200 102.6 2.3 103.0 3.9
AMP 50 104.8 6.3 104.9 4.9
100 104.4 3.5 103.1 2.9
200 105.4 1.5 104.6 5.4
UMP 50 107.3 6.0 108.0 3.9
100 97.5 4.4 102.0 4.3
200 96.6 1.7 102.4 7.3
IMP 50 107.6 5.1 107.1 4.3
100 104.0 3.4 103.4 2.4
200 104.9 1.9 104.5 6.1
GMP 50 108.9 5.2 106.8 5.1
100 104.2 3.4 102.1 3.7
200 104.7 1.9 104.5 6.0
), ArticleFig(id=1184566998185947500, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=表2, caption=

婴儿配方食品中核苷酸的回收率和精密度

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分析物 浓度/(mg/kg) 日内(n=6) 日间(n=18)
回收率/% RSDs/% 回收率/% RSDs/%
CMP 50 102.8 5.4 103.3 3.3
100 103.1 4.1 101.0 4.1
200 102.6 2.3 103.0 3.9
AMP 50 104.8 6.3 104.9 4.9
100 104.4 3.5 103.1 2.9
200 105.4 1.5 104.6 5.4
UMP 50 107.3 6.0 108.0 3.9
100 97.5 4.4 102.0 4.3
200 96.6 1.7 102.4 7.3
IMP 50 107.6 5.1 107.1 4.3
100 104.0 3.4 103.4 2.4
200 104.9 1.9 104.5 6.1
GMP 50 108.9 5.2 106.8 5.1
100 104.2 3.4 102.1 3.7
200 104.7 1.9 104.5 6.0
), ArticleFig(id=1184566998265639277, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=EN, label=Table 3, caption=

Actual sample analysis results

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序号 标识量/(mg/kg) 核苷酸含量/(mg/kg)
CMP AMP UMP IMP GMP 总量
1 300 151.4 65.0 73.0 25.2 38.6 353.2
2 350 247.0 39.4 50.8 N.D. 61.6 398.8
3 300 125.3 60.9 67.7 42.6 25.1 321.6
4 N.A. 41.5 N.D. N.D. N.D. N.D. 41.5
5 N.A. 32.9 N.D. N.D. N.D. N.D. 32.9
6 N.A. 21.1 N.D. N.D. N.D. N.D. 21.1
), ArticleFig(id=1184566998378885486, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986713459352034, language=CN, label=表3, caption=

实际样品分析结果

, figureFileSmall=null, figureFileBig=null, tableContent=
序号 标识量/(mg/kg) 核苷酸含量/(mg/kg)
CMP AMP UMP IMP GMP 总量
1 300 151.4 65.0 73.0 25.2 38.6 353.2
2 350 247.0 39.4 50.8 N.D. 61.6 398.8
3 300 125.3 60.9 67.7 42.6 25.1 321.6
4 N.A. 41.5 N.D. N.D. N.D. N.D. 41.5
5 N.A. 32.9 N.D. N.D. N.D. N.D. 32.9
6 N.A. 21.1 N.D. N.D. N.D. N.D. 21.1
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亲水/离子交换混合模式色谱法快速检测婴幼儿食品和乳品中核苷酸含量
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薛彦伦 , 王柏辉 * , 梁栋明 , 李英杰 , 张兴 , 乔轲 , 苏妮汗
食品安全质量检测学报 | 本期专题：乳制品加工与质量控制 2025,16(2): 91-98
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食品安全质量检测学报 | 本期专题：乳制品加工与质量控制 2025, 16(2): 91-98
亲水/离子交换混合模式色谱法快速检测婴幼儿食品和乳品中核苷酸含量
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薛彦伦 , 王柏辉* , 梁栋明, 李英杰, 张兴, 乔轲, 苏妮汗
作者信息
  • 鄂尔多斯市检验检测中心, 鄂尔多斯 017010

    • 薛彦伦(1985—), 男, 工程师, 主要研究方向为食品安全。E-mail:

通讯作者:

* 王柏辉(1990—), 男, 博士, 高级工程师, 主要研究方向为肉品科学和食品检测。E-mail:
Rapid determination of nucleotides content in infant formula and dairy product by mixed-mode hydrophilic interaction/ion exchange chromatography
Yan-Lun XUE , Bo-Hui WANG* , Dong-Ming LIANG, Ying-Jie LI, Xing ZHANG, Ke QIAO, Ni-Han SU
Affiliations
  • Ordos City Inspection and Testing Center, Erdos 017010, China
出版时间: 2025-01-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241015002
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摘要
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目的 建立亲水/离子交换混合模式色谱-紫外检测法(hydrophilic interaction/ion exchange chromatography-ultraviolet detection, HILIC/IEC-UV)快速测定婴幼儿食品和乳制品中5种核苷酸(胞嘧啶核苷酸、腺嘌呤核苷酸、尿嘧啶核苷酸、鸟嘌呤核苷酸和次黄嘌呤核苷酸)含量的分析方法。方法 采用两性离子亲水作用色谱柱(Alphasil S-Xion), 以5 mmol/L磷酸二氢钾水溶液(磷酸调pH 3.0)-乙腈(70:30, V:V)为流动相, 对婴幼儿食品和乳品中的核苷酸进行检测。结果 该方法在10 min内实现了5种核苷酸的高效分离, 5种核苷酸在2~50 μg/mL线性范围内线性关系良好, 检出限(limits of detection, LODs)为0.19~0.66 mg/kg, 定量限(limits of quantification, LOQs)为0.62~2.19 mg/kg。在50、100、200 mg/kg的加标水平, 5种核苷酸的日内回收率为96.6%~108.9%, 相对标准偏差(relative standard deviations, RSDs)为1.5%~6.3%, 日间回收率为101.0%~108.0%, RSDs为2.4%~7.3%。结论 与传统的反相离子对色谱法相比, 本方法具有显著优势: 有效降低了样品基质干扰, 缩短了分析时间, 提高了检测效率, 特别是改善了胞嘧啶核苷酸的定量准确性。该方法快速、准确、抗干扰, 为婴幼儿食品和乳制品中核苷酸的高效分析提供了技术支持。

婴幼儿配方食品  /  乳制品  /  亲水/离子交换混合模式色谱  /  核苷酸

Objective To establish a rapid method for the determination of 5 kinds of nucleotides (cytidine-5’-monophosphate, adenosine-5’-monophosphate, uridine-5’-monophosphate, guanosine-5’-monophosphate and inosine-5’-monophosphate) in infant formulas and dairy products is developed based on mixed-mode hydrophilic interaction/ion exchange chromatography-ultraviolet detection (HILIC/IEX-UV). Methods A zwitterionic hydrophilic interaction chromatography column (Alphasil S-Xion) was used, with mobile phase consisting of 5 mmol/L KH2PO4 (pH adjusted to 3.0 with H3PO4)-acetonitrile (70:30, V:V) to detect nucleotides in infant foods and dairy products. Results The five nucleotides were baseline separated within 10 min, demonstrating efficient chromatographic performance. They exhibited good linearity within the range of 2-50 μg/mL, with limits of detection (LODs) between 0.19 and 0.66 mg/kg, and limits of quantification (LOQs) ranging from 0.62 to 2.19 mg/kg. At spiked levels of 50, 100 and 200 mg/kg, the intraday recoveries ranged from 96.6% to 108.9%, with relative standard deviations (RSDs) of 1.5%-6.3%, while the interday recoveries were between 101.0% and 108.0%, with RSDs of 2.4%-7.3%. Conclusions Compared with the traditional reversed-phase ion-pair chromatography, this method has significant advantages: Effectively reducing the matrix interference, shortening the analysis time, improving the detection efficiency, and especially improving the quantitative accuracy of cytidine-5’-monophosphate. The proposed method is rapid, accurate, and anti-interference, which provides technical support for the efficient analysis of nucleotides in infant formulas and dairy products.

infant formula  /  dairy product  /  hydrophilic interaction/ion exchange chromatography  /  nucleotide
薛彦伦, 王柏辉, 梁栋明, 李英杰, 张兴, 乔轲, 苏妮汗. 亲水/离子交换混合模式色谱法快速检测婴幼儿食品和乳品中核苷酸含量. 食品安全质量检测学报, 2025 , 16 (2) : 91 -98 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241015002
Yan-Lun XUE, Bo-Hui WANG, Dong-Ming LIANG, Ying-Jie LI, Xing ZHANG, Ke QIAO, Ni-Han SU. Rapid determination of nucleotides content in infant formula and dairy product by mixed-mode hydrophilic interaction/ion exchange chromatography[J]. Journal of Food Safety & Quality, 2025 , 16 (2) : 91 -98 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241015002
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0 引言
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核苷酸是人体所需的一类重要的免疫物质, 能帮助激活淋巴细胞活力, 提高免疫球蛋白水平, 参与了人体几乎全部生理代谢过程[1]。经研究, 母乳中含有胞嘧啶核苷酸(胞苷酸, cytidine-5’-monophosphate, CMP)、腺嘌呤核苷酸(腺苷酸, adenosine-5’-monophosphate, AMP)、尿嘧啶核苷酸(尿苷酸, uridine-5’-monophosphate, UMP)、鸟嘌呤核苷酸(鸟苷酸, guanosine-5’-monophosphate, GMP)和次黄嘌呤核苷酸(肌苷酸, inosine-5’-monophosphate, IMP)等多种核苷酸, 为提高婴幼儿的免疫调节功能和记忆力发挥着重要作用[2-3]。由于牛乳中核苷酸含量很低, 我国GB 14880—2012《食品营养强化剂使用标准》对婴幼儿配方食品中上述5种核苷酸的添加量做出了明确规定, 以核苷酸总量计, 其添加量应达到0.12~0.58 g/kg。为保证婴幼儿的健康成长, 有必要进行婴幼儿配方食品和乳品中的核苷酸含量测定。
核苷酸主要由嘌呤碱/嘧啶碱、核糖/脱氧核糖和磷酸组成(图1)。目前, 液相色谱法、液相色谱-质谱法、毛细管电泳法等方法均可用于核苷酸的定量分析[4-7]。由于核苷酸作为营养强化剂的规定添加量较高, 实际应用中多采用高效液相色谱法进行定量分析, 包括离子交换色谱[7-9]、反相离子对色谱[10-11]和极性修饰反相色谱[12-15]等。然而, 这些分析方法各有局限性。例如, 离子交换色谱易受缓冲盐浓度和pH影响, 实际样品分析中需严格控制流动相pH以避免基质干扰[16]; 反相离子对色谱虽可增强核苷酸的保留, 但存在平衡时间长、柱寿命低、保留时间不稳定等问题, 限制了其应用[17]; 尹丽丽等[12]和郑红等[13]采用极性修饰反相C18色谱柱, 在纯水相条件下分析了5种核苷酸, 并系统考察了多种样品前处理方法以降低基质干扰。然而, 无论是离子对色谱法还是极性反相色谱法, 文献报道的检测结果均显示CMP保留弱且易受基质干扰, 导致无法准确定量[18-20]。
自1990年ALPERT提出亲水相互作用色谱法(hydrophilic interaction liquid chromatography, HILIC)以来, 分析科学家开发了大量的HILIC固定相[21-23], 并对HILIC的保留行为进行了深入研究[24-26], 目前该分离模式已广泛应用于糖、糖肽、糖蛋白、极性药物等强极性化合物的分离[27-29]。鉴于核苷酸具有强极性和电离特性, HILIC模式可实现其直接保留, 避免添加离子对试剂带来的不利影响。然而, 目前HILIC模式在核苷酸分离方面的研究报道仍然较少[30-32], 在分离选择性和抗基质干扰方面有一定局限。本研究选用一种表面同时键合氨基和羧基的两性离子亲水作用色谱固定相, 对5种核苷酸的保留机制进行深入研究, 建立了婴幼儿配方食品和乳品中核苷酸的快速分析方法, 以期为婴幼儿食品质量监控提供参考和依据。
1 材料与方法
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1.1 材料、试剂与仪器
各品牌婴幼儿配方乳粉及成人乳制品购买自本地超市, 空白奶粉样品由国内某乳品生产企业研发部门提供, 该样品为企业内部质控使用, 可确保无待测核苷酸存在。
CMP(纯度99%, CAS: 63-37-6)、AMP(纯度98%, CAS: 61-19-8)、UMP(纯度98%, CAS: 58-97-9)、乙腈(纯度99.9%)(上海阿拉丁生化科技有限公司); GMP(纯度97%, CAS:85-32-5)(上海泰坦科技股份有限公司); IMP(纯度98%, CAS: 131-99-7)(北京曼哈格生物科技有限公司); 磷酸氢二钾(纯度99%)、乙酸铵(纯度98%)、甲酸(纯度98%)(北京百灵威科技有限公司); 甲酸铵(纯度99%)、磷酸(纯度98%)(天津市大茂化学试剂厂); 醋酸(纯度99.8%, 西陇科学股份有限公司); 淀粉酶(酶活度1.5 U/mg, 西格玛奥德里奇贸易有限公司); 超纯水由实验室纯水机制得, 电阻率18.2 MΩ·cm。
Acchrom S6000型高效液相色谱仪(配有四元梯度泵、自动进样器、柱温箱、UV检测器)、Alphasil S-Xion色谱柱(150 mm×4.6 mm, 5 μm)、Alphasil VC-C18色谱柱(250 mm× 4.6 mm, 5 μm)[华谱科仪(北京)科技有限公司]; Milli-Q EQ 7008型超纯水系统(美国Millipore公司); XM-P101H型超声波清洗仪[小美超声仪器(昆山)有限公司]; Seven Direct SD20型pH计、ML104T 102型电子天平(精度0.0001 g)[梅特勒-托利多仪器(上海)有限公司]。
1.2 实验方法
1.2.1 标准溶液配制
分别准确称取10 mg(精确至0.1 mg)核苷酸标准品CMP、AMP、UMP、IMP和GMP至5个10 mL棕色容量瓶中, 用超纯水溶解并定容, 配制成1 mg/mL的核苷酸单标储备液, 放置于4 ℃冰箱中避光保存(有效期30 d)。
分别准确移取各单标储备液1 mL至1个10 mL棕色容量瓶中, 用水定容, 配制成100 µg/mL的混合标准工作溶液。用水对混合标准工作溶液进行逐级稀释, 配制成2、5、10、20、50 μg/mL的标准溶液(有效期7 d), 作为外标法定量的工作曲线。
1.2.2 样品前处理
样品前处理按GB 5413.40—2016《食品安全国家标准 婴幼儿食品和乳品中核苷酸的测定》中的“5.1试样制备”来进行。
(1)试样预处理
含淀粉的试样: 称取混合均匀的固体试样约5 g(精确至0.1 mg)于100 mL锥形瓶中, 加入约0.2 g淀粉酶, 加入20 mL热水(30~40 ℃)充分溶解试样, 或称取混合均匀的液体试样约20 g(精确至1 mg)于100 mL锥形瓶中, 加入约0.2 g淀粉酶摇匀, 于(37±2) ℃培养箱内酶解30 min。取出冷却至室温。
不含淀粉的试样: 称取混合均匀的固体试样约5 g(精确至0.1 mg)于100 mL锥形瓶中, 加入20 mL热水(50~60 ℃)充分溶解试样, 冷却至室温, 或称取混合均匀的液体试样约20 g(精确至1 mg)于100 mL锥形瓶中。
(2)待测液的制备
用醋酸溶液调节试样溶液pH至4.1, 移入50 mL容量瓶中, 定容, 滤纸过滤, 所得滤液用0.45 µm微孔滤膜过滤, 待上机使用。
1.2.3 色谱条件
色谱柱: Alphasil S-Xion色谱柱(150 mm×4.6 mm, 5 μm); 流动相A: 5 mmol/L磷酸氢二钾(用磷酸调pH为3.0); 流动相B: 乙腈; 等度洗脱: A:B=70:30 (V:V); 流速: 1.0 mL/min; 进样体积: 10 μL; 柱温40 ℃; 紫外检测波长: 254 nm; 以保留时间定性, 外标法定量。
1.3 数据处理
采用华谱科仪Chromloong色谱软件采集液相色谱原始数据, 并进行目标化合物的积分和信噪比计算。使用Microsoft Excel 2019进行线性方程、回收率和相对标准偏差的计算。回收率和精密度测定中, 每个加标浓度设置6个平行样, 日内重复性分析当日完成, 日间重复性连续测定3 d(共18个平行样)。本研究中化学结构式使用ChemBioDraw 14.0绘制, 色谱图和保留行为变化趋势图使用Origin 2019绘制。
2 结果与分析
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2.1 分析方法的建立
5种核苷酸为强极性、可电离化合物。在本研究中采用Alphasil VC-C18色谱柱, 并通过在流动相中添加离子对试剂四丁基硫酸氢铵实现了核苷酸的分离, 结果如图2所示。然而, 在该条件下, CMP的保留能力较弱(图2a), 在实际样品分析中容易受到基质干扰, 并可能导致CMP的保留时间发生漂移(图2b)。进一步实验发现, 在处理后的实际样品中添加CMP会导致两个峰信号同时增强(图2c), 这些干扰显著增加了CMP定性与定量分析的难度和不确定度。
为解决上述问题, 本研究采用两性离子亲水作用色谱柱(Alphasil S-Xion), 在HILIC模式下对核苷酸进行保留, 通过考察缓冲盐类型、有机相比例、缓冲盐pH、缓冲盐浓度等因素对核苷酸保留的影响, 确定核苷酸在该色谱柱上的保留机制, 并最终建立核苷酸的最佳分离方法。
2.1.1 缓冲盐类型的影响
实验首先考察了不同缓冲盐类型对5种核苷酸分离的影响(图3)。从各化合物的log k’值来看, 使用甲酸铵或乙酸铵作为缓冲盐时, 核苷酸峰的分离选择性基本一致, 均无法实现CMP和GMP的有效分离。然而, 采用磷酸二氢钾作为缓冲盐时, 这两个化合物之间的分离效果得到了显著改善。此外, 磷酸二氢钾的使用对UMP和IMP的分离选择性也有所不同。因此, 磷酸二氢钾作为添加剂更有利于实现5种核苷酸的选择性分离。
2.1.2 有机相比例的影响
采用10 mmol/L磷酸二氢钾(pH 3.0)为缓冲盐, 考察不同有机相比例条件下各核苷酸的保留和分离情况(图4)。结果可见, 随着乙腈比例的降低, 核苷酸的保留值整体呈现降低的趋势, 表现出显著的亲水保留行为。此外, 各核苷酸的分离选择性也发生了变化, 如CMP的保留值由最强变为最弱, 而UMP的保留值由最弱变为居中。由于S-Xion色谱柱表面同时键合有氨基与羧基基团, 随着水相比例升高, 核苷酸与两性离子S-Xion固定相均表现出较显著的电离状态, 二者之间的离子交换作用带来了分离选择性的差异。为更清楚地阐明离子交换作用的影响, 下面将重点考察缓冲盐pH和浓度对5种核苷酸保留的影响。
2.1.3 缓冲盐pH的影响
图5展示了核苷酸在不同缓冲盐pH条件下的保留行为, 从整体趋势来看, 在低pH条件下各核苷酸保留值相对较高, 随着缓冲盐pH的升高, 核苷酸保留值逐渐减弱直至不保留。由于S-Xion固定相表面的氨基和羧基均可电离, 在低pH条件(如pH 2)下, S-Xion表面氨基被激活而羧基被抑制, 与核苷酸中的磷酸基团产生静电吸引作用; 而随着pH升高, S-Xion表面氨基被抑制而羧基被激活, 与核苷酸中的磷酸基产生静电排斥作用而导致其保留的丧失。对于CMP、AMP、GMP表现出的保留值随pH升高略有升高再显著下降的趋势, 则可能是由于其结构中碱基的碱性较强, 在低pH条件下碱基被电离并在一定程度上抵消了固定相表面氨基与核苷酸结构中羧基的静电吸引作用。
2.1.4 缓冲盐浓度的影响
在30%乙腈比例及pH 3.0条件下, 考察不同缓冲盐浓度对核苷酸保留的影响。从图6结果可见, 随着流动相中缓冲盐浓度的升高, 所有核苷酸的保留均呈现下降的趋势, 即呈现出显著的离子交换作用。
通过对缓冲盐类型、乙腈比例、缓冲盐pH、缓冲盐浓度的系统考察, 结果可知, 采用磷酸盐为缓冲盐, 并在较低乙腈比例的条件下, 可以更好地实现5种核苷酸的选择性分离。在该分离条件下, 核苷酸的保留行为呈现典型的亲水/阴离子交换混合模式色谱的原理。
2.1.5 色谱分析方法的建立
综合上述讨论结果, 并充分考虑实际样品分析中可能存在的基质干扰, 优化并确定了最佳色谱分离条件(详见1.2.3)。在该最佳条件下, 各核苷酸与潜在基质干扰实现了良好的分离, 如图7所示。实验结果表明, 该方法有效避免了样品基质对核苷酸检测的干扰。更重要的是, 所有目标核苷酸均能在10 min内实现快速分离和检测, 显著提升了分析效率。
2.2 方法学考察
2.2.1 线性范围和检出限
将1.2小节配制的不同浓度标准溶液重复分析3次, 用所得到的峰面积平均值(Y)为纵坐标, 核苷酸的质量浓度(X, μg/mL)为横坐标建立回归曲线, 结果表明5种核苷酸在2~50 μg/mL质量浓度范围内线性关系良好, 线性相关系数(r2)均可达到0.9999。以信噪比(signal to noise ratio, S/N)大于等于3为检出限(limits of detection, LODs), S/N大于等于10为定量限(limits of quantification, LOQs), 5种核苷酸的LODs在0.19~0.66 mg/kg之间, LOQs在0.62~2.19 mg/kg之间, 均可满足定量检测的要求。相关结果如表1所示。
2.2.2 回收率和精密度
为了评估方法的准确性和精密度, 在空白奶粉基质中添加了3个浓度水平(50、100和200 mg/kg)的核苷酸标准品, 每个浓度平行测定6次, 计算日内回收率和相对标准偏差(relative standard deviation, RSD)。通过连续3 d重复上述实验, 进一步评估了日间回收率和RSDs。结果显示(表2), 该方法的日内回收率在96.6%~108.9%之间, RSDs在1.5%~6.3%之间; 日间回收率范围为101.0%~108.0%, RSDs为2.4%~7.3%。结果表明该方法具有良好的准确性和重复性, 适用于核苷酸的定量分析。
2.3 实际样品分析
按照本研究建立的方法, 对市场上多个不同品牌的婴幼儿配方食品和乳制品中的核苷酸含量进行测定, 结果列于表3中。从结果可见, 对于标识有核苷酸含量的样品, 其检测结果基本与标示量一致, 且添加量以CMP为主。对于未标识添加量的成人乳制品, 仅从中检测到了少量CMP, 而AMP、UMP、IMP、GMP未检出。
3 讨论与结论
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本研究开发了一种高效、准确地测定婴幼儿配方食品和乳制品中核苷酸的分析方法。该方法采用亲水作用两性离子色谱柱, 在亲水/离子交换混合模式条件下实现了5种核苷酸的快速分离分析。相较于传统的反相离子对色谱方法, 本方法具有显著优势: 首先, 它有效降低了样品基质干扰, 提高了分析结果的可靠性; 其次, 分析时间大幅缩短, 显著提升了检测效率; 最后, 本方法特别改善了CMP的定量准确性, 解决了传统方法中CMP易受干扰的问题。综上所述, 本研究建立的核苷酸检测方法具有快速、准确、抗干扰等优点, 为婴幼儿配方食品和乳制品中核苷酸的高效分析提供了有力的技术支持, 在食品安全监管、营养评估等相关领域具有广阔的应用前景。
文献
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241015002
  • 接收时间：2024-10-15
  • 首发时间：2025-07-21
  • 出版时间：2025-01-25
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  • 收稿日期：2024-10-15
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    鄂尔多斯市检验检测中心, 鄂尔多斯 017010

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* 王柏辉(1990—), 男, 博士, 高级工程师, 主要研究方向为肉品科学和食品检测。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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