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Article(id=1153986718123418171, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153986709126635984, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241016004, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1729008000000, receivedDateStr=2024-10-16, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1753061473636, onlineDateStr=2025-07-21, pubDate=1737734400000, pubDateStr=2025-01-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1753061473636, onlineIssueDateStr=2025-07-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1753061473636, creator=13701087609, updateTime=1753061473636, 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=224, endPage=230, ext={EN=ArticleExt(id=1153986719868248679, articleId=1153986718123418171, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Determination of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in health food by high performance 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 establish an analytical mehod for the determination of nicotinamide mononucleotide (NMN) and nicotinamide adenine dinucleotide (NAD+) in health food by high performance liquid chromatography. Methods Determination was performed on Waters BEH-Amide column with mobile phase consisting of 0.1% phosphoric acid solution-acetonitrile (gradient elution). The flow rate was 1.0 mL/min, and the sample size was 2 μL. The column temperature was 35 ℃, and the detection wavelength was set at 263 nm. Health food was extracted with 60% methanol solution in ultrasonic water bath for 10 min. The extract was filtered through micoron filter, and analyzed by high performance liquid chromatography. Results The results showed that NMN and NAD+ exhibited excellent linear relationships, the correlation coefficient (r) was over 0.999 in the range of 10-1000 μg/mL. The method limit of quantitation was 0.50 g/kg. Recoveries were ranged from 95.9% to 98.8%, and relative standard deviations were ranged from 1.19% to 3.05%. Conclusion This method is accurate and suitable to determine NMN and NAD+ in health food.

, correspAuthors=Guo-Jian ZHENG, 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=Nan XIE, Guo-Jian ZHENG), CN=ArticleExt(id=1153986762604011653, articleId=1153986718123418171, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=高效液相色谱法测定保健食品中的烟酰胺单核苷酸和烟酰胺腺嘌呤双核苷酸含量, columnId=1153986581842092705, journalTitle=食品安全质量检测学报, columnName=本期专题：现代分析仪器在食品检测中的应用, runingTitle=null, highlight=null, articleAbstract=

目的 建立高效液相色谱法精确定量保健食品中烟酰胺单核苷酸(nicotinamide mononucleotide, NMN)与烟酰胺腺嘌呤双核苷酸(nicotinamide adenine dinucleotide, NAD⁺)的含量的分析方法。方法 采用Waters BEH-Amide色谱柱, 0.1%磷酸溶液和乙腈为流动相, 进行梯度洗脱, 流速设定为1.0 mL/min, 进样量为2 μL, 柱温箱温度设置为35 ℃, 选择263 nm作为检测波长。保健食品用60%甲醇溶液超声提取10 min, 将提取液通过微孔滤膜过滤后进行高效液相色谱法测定, 外标法定量。结果 在质量浓度10~1000 μg/mL的范围内, NMN和NAD+均展现出优异的线性关系, 其线性相关系数(r)均不低于0.999, 该方法的定量限为0.50 g/kg, NMN和NAD+的回收率在95.9%~98.8%范围内, 相对标准偏差在1.19%~3.05%之间。结论 该方法可以准确测定保健食品中NMN和NAD+含量。

, correspAuthors=郑国建, authorNote=null, correspAuthorsNote=
* 郑国建(1983—), 男, 硕士, 高级工程师, 主要研究方向为食品中营养素的检测。E-mail:
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解楠(1983—), 男, 硕士, 高级工程师, 主要研究方向为食品中营养素的检测。E-mail:

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解楠(1983—), 男, 硕士, 高级工程师, 主要研究方向为食品中营养素的检测。E-mail:

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Optimization of ultrasonic-assisted low eutectic reagents extraction of polysaccharides from artichoke buds by response surface methodology[J]. 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tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986718123418171, language=CN, label=图10, caption=4号样品的色谱图, figureFileSmall=e/a+Fc68OLmnRYTHvSjJyA==, figureFileBig=/ld7nBkbMUZTGkfzTC6Fxg==, tableContent=null), ArticleFig(id=1184567070239900004, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986718123418171, language=EN, label=Table 1, caption=

Recoveries and precisions of the method (n=6)

, figureFileSmall=null, figureFileBig=null, tableContent=
化合物 添加量/(g/kg) 回收率/% RSDs/%
NMN 0.50 95.9 2.10
400.00 96.2 2.07
800.00 98.1 1.41
NAD+ 0.50 96.4 3.05
400.00 97.8 2.78
800.00 98.8 1.19
), ArticleFig(id=1184567070319591781, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986718123418171, language=CN, label=表1, caption=

方法的回收率和精密度(n=6)

, figureFileSmall=null, figureFileBig=null, tableContent=
化合物 添加量/(g/kg) 回收率/% RSDs/%
NMN 0.50 95.9 2.10
400.00 96.2 2.07
800.00 98.1 1.41
NAD+ 0.50 96.4 3.05
400.00 97.8 2.78
800.00 98.8 1.19
), ArticleFig(id=1184567070407672166, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986718123418171, language=EN, label=Table 2, caption=

Content of NMN and NAD+ in the samples

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样品编号 1 2 3 4 5 6 7 8
NMN/(g/kg) 未检出 636 未检出 522 未检出 889 未检出 未检出
NAD+/(g/kg) 未检出 未检出 未检出 20 未检出 未检出 未检出 未检出
), ArticleFig(id=1184567070478975335, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153986718123418171, language=CN, label=表2, caption=

样品中NMN和NAD+的含量

, figureFileSmall=null, figureFileBig=null, tableContent=
样品编号 1 2 3 4 5 6 7 8
NMN/(g/kg) 未检出 636 未检出 522 未检出 889 未检出 未检出
NAD+/(g/kg) 未检出 未检出 未检出 20 未检出 未检出 未检出 未检出
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高效液相色谱法测定保健食品中的烟酰胺单核苷酸和烟酰胺腺嘌呤双核苷酸含量
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解楠 , 郑国建 *
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(2): 224-230
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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(2): 224-230
高效液相色谱法测定保健食品中的烟酰胺单核苷酸和烟酰胺腺嘌呤双核苷酸含量
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解楠 , 郑国建*
作者信息
  • 上海市质量监督检验技术研究院, 上海 200233

    • 解楠(1983—), 男, 硕士, 高级工程师, 主要研究方向为食品中营养素的检测。E-mail:

通讯作者:

* 郑国建(1983—), 男, 硕士, 高级工程师, 主要研究方向为食品中营养素的检测。E-mail:
Determination of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in health food by high performance liquid chromatography
Nan XIE , Guo-Jian ZHENG*
Affiliations
  • Shanghai Institute of Quality Inspection and Technical Research, Shanghai 200233, China
出版时间: 2025-01-25 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241016004
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摘要
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目的 建立高效液相色谱法精确定量保健食品中烟酰胺单核苷酸(nicotinamide mononucleotide, NMN)与烟酰胺腺嘌呤双核苷酸(nicotinamide adenine dinucleotide, NAD⁺)的含量的分析方法。方法 采用Waters BEH-Amide色谱柱, 0.1%磷酸溶液和乙腈为流动相, 进行梯度洗脱, 流速设定为1.0 mL/min, 进样量为2 μL, 柱温箱温度设置为35 ℃, 选择263 nm作为检测波长。保健食品用60%甲醇溶液超声提取10 min, 将提取液通过微孔滤膜过滤后进行高效液相色谱法测定, 外标法定量。结果 在质量浓度10~1000 μg/mL的范围内, NMN和NAD+均展现出优异的线性关系, 其线性相关系数(r)均不低于0.999, 该方法的定量限为0.50 g/kg, NMN和NAD+的回收率在95.9%~98.8%范围内, 相对标准偏差在1.19%~3.05%之间。结论 该方法可以准确测定保健食品中NMN和NAD+含量。

烟酰胺单核苷酸  /  烟酰胺腺嘌呤双核苷酸  /  保健食品  /  高效液相色谱法

Objective To establish an analytical mehod for the determination of nicotinamide mononucleotide (NMN) and nicotinamide adenine dinucleotide (NAD+) in health food by high performance liquid chromatography. Methods Determination was performed on Waters BEH-Amide column with mobile phase consisting of 0.1% phosphoric acid solution-acetonitrile (gradient elution). The flow rate was 1.0 mL/min, and the sample size was 2 μL. The column temperature was 35 ℃, and the detection wavelength was set at 263 nm. Health food was extracted with 60% methanol solution in ultrasonic water bath for 10 min. The extract was filtered through micoron filter, and analyzed by high performance liquid chromatography. Results The results showed that NMN and NAD+ exhibited excellent linear relationships, the correlation coefficient (r) was over 0.999 in the range of 10-1000 μg/mL. The method limit of quantitation was 0.50 g/kg. Recoveries were ranged from 95.9% to 98.8%, and relative standard deviations were ranged from 1.19% to 3.05%. Conclusion This method is accurate and suitable to determine NMN and NAD+ in health food.

nicotinamide mononucleotide  /  nicotinamide adenine dinucleotide  /  health food  /  high performance liquid chromatography
解楠, 郑国建. 高效液相色谱法测定保健食品中的烟酰胺单核苷酸和烟酰胺腺嘌呤双核苷酸含量. 食品安全质量检测学报, 2025 , 16 (2) : 224 -230 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241016004
Nan XIE, Guo-Jian ZHENG. Determination of nicotinamide mononucleotide and nicotinamide adenine dinucleotide in health food by high performance liquid chromatography[J]. Journal of Food Safety & Quality, 2025 , 16 (2) : 224 -230 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241016004
正文
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0 引言
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烟酰胺单核苷酸(nicotinamide mononucleotide, NMN), 是生物体内一个至关重要的前体分子[1], 它直接参与将烟酰胺核糖(nicotinamide riboside, NR)转化为关键的烟酰胺腺嘌呤双核苷酸(nicotinamide adenine dinucleotide, NAD+)的过程[2-4]。NAD+在生物体内的作用广泛而深远, 它不仅是抗衰老策略中的核心分子[5-6], 也在疾病预防中发挥着举足轻重的作用[7-8]。此外, NAD+还深度嵌入到细胞分裂、蛋白质合成、以及能量代谢等多个基本生命活动之中, 是维持机体正常生理功能不可或缺的一环[9]。多项研究表明, 通过外源性补充NMN, 可以有效提升体内NAD+的水平[2], 从而促进DNA修复[10], 对抗因衰老引起的多种代谢失衡和老年相关疾病[11], 包括但不限于阿尔兹海默病[12]、糖尿病[13]、心血管疾病[14]以及慢性炎症[15]等, 它们在很大程度上影响着老年人的生活质量和寿命。2016年MILLS等[9]的研究为NMN的抗衰老潜力提供了有力的实验证据, 研究发现长期对小鼠进行NMN灌胃处理, 能够显著减轻小鼠因年龄增长而出现的生理机能衰退, 展现出NMN在延缓衰老过程、促进健康长寿方面的巨大潜力。
鉴于NMN和NAD+在促进健康方面的广泛益处, 市场上迅速涌现出众多以它们为核心成分的创新食品、营养膳食补充剂及保健品。为了保障这些产品的品质与安全性, 精确测定其中NMN和NAD+的含量成为了至关重要的一环。目前, 针对这两种成分的检测, 主要技术包括液相色谱法(liquid chromatography, LC)[11,16-18]和液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry, LC-MS/MS)[19-24]。如冯雪萍等[16]通过运用反相-LC成功实现了保健食品中的NMN含量的准确测定; 刘晓谦等[21]采用LC-MS/MS技术对铁皮石斛提取物中的NMN和NAD+进行了分析。除此之外, UMMARINO等[25]还开发了基于酶联-荧光法的新型检测技术。
尽管LC-MS/MS灵敏度高、特异性好[26-27], 但是在实际应用中该方法可能存在基质效应干扰目标物的准确定性和定量[28]; 另一方面该设备成本高昂, 对实验室的硬件要求较高[29]; 同时, 保健食品中NMN和NAD+的添加量往往较高, 而LC-MS/MS在处理高浓度样品时可能需要额外的样品稀释步骤, 这也增加了操作的复杂性。文献中的LC通常选用C18色谱柱, 会遇到NMN和NAD+这类强极性化合物保留能力弱的问题[17]。它们在C18色谱柱上的快速洗脱使得检测信号易受基质中复杂杂质的影响, 从而导致检测结果的准确性降低。这种保留行为的不足, 是LC在检测NMN和NAD+时需要考虑并克服的重要问题。
本研究致力于开发一种高效液相色谱技术, 实现对NMN和NAD+的快速、精确测定。本研究的成果将为保健食品的研发、生产以及质量控制等领域提供更加科学、可靠的技术支持, 促进该行业的健康发展。
1 材料与方法
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1.1 材料、试剂与仪器
8批次保健食品采购于淘宝。
NMN标准品、NAD+标准品(纯度不低于95%, 美国Sigma公司); 甲酸、甲醇、乙腈(色谱纯, 美国Thermo Fisher Scientific公司); 盐酸、乙醇、冰醋酸(分析纯, 国药集团化学试剂有限公司)。
Agilent 1260高效液相色谱仪(配有四元泵、自动进样器、柱温箱和二极管阵列检测器, 美国安捷伦公司); MX104/A型电子天平(感量为0.1 mg, 瑞士梅特勒托利多公司); Wiggens Votex 3000涡旋振荡仪(德国WIGGENS公司); SK250LHC型超声波清洗机(上海科导公司); Eppendorf 5804离心机(德国艾本德公司); Millipore Milli-Q超纯水(美国密理博公司); Waters BEH-Amide色谱柱(4.5 mm×250 mm, 5 μm, 美国沃特斯公司); 0.22 μm聚四氟乙烯滤膜(上海安谱实验科技股份有限公司)。
1.2 实验方法
1.2.1 色谱条件
采用Waters BEH-Amide色谱柱(4.5 mm×250 mm, 5 μm)。流动相A为0.1%(体积比)磷酸溶液, 流动相B为乙腈, 梯度洗脱程序: 0~1 min, B相比例保持90%不变; 1~12 min, B相比例从90%线性变化至50%; 12~15 min, B比例保持50%不变; 15.1 min时, B相比例变化至90%, 并维持此比例平衡5 min。柱温为: 35 ℃。流速为: 1.0 mL/min。进样量为: 2 μL。检测波长设定为263 nm。
1.2.2 标准溶液的配制
(1)标准储备溶液
准确称取NMN和NAD+标准品各50 mg(精确至0.1 mg), 用60%甲醇溶液转移至两个洁净的50 mL容量瓶中, 涡旋使其完全溶解。用60%甲醇溶液定容至刻度, 充分摇匀, 配制成1 mg/mL的标准储备溶液。
(2)标准系列溶液
从预先配制好的标准储备溶液中, 依次准确移取适量体积的溶液至多个清洁干燥的10 mL容量瓶中, 并用60%甲醇溶液定容至刻度, 充分摇匀。即可得到一系列质量浓度分别为10、20、50、100、200、500、1000 μg/mL的标准系列溶液。
1.2.3 样品处理
进行3组平行实验。精确称取试样0.5 g(精确至0.1 mg)于50 mL离心管中。向离心管中加入40 mL 60%甲醇溶液, 使用涡旋振荡器使样品与溶剂充分混合。将离心管置于超声设备中超声提取10 min。结束后, 将全部液体用60%甲醇溶液分2次转移至50 mL容量瓶中, 用60%甲醇溶液定容至刻度, 混合均匀。取部分样液经9000 r/min离心5 min, 取离心后的上清液, 通过0.22 μm聚四氟乙烯滤膜过滤后测定。若测定的样品浓度超出线性范围, 根据需要, 使用60%甲醇溶液进行适当稀释后重新测定。
1.2.4 提取溶剂的优化
精确称取0.5 g待测试样, 随后将其置于含有40 mL不同甲醇浓度比例的提取溶剂中。通过涡旋混合确保样品充分分散后, 利用超声波处理10 min。之后, 将所得溶液转移至50 mL容量瓶中, 并用相同的提取溶剂定容至刻度线, 混匀。
从上述溶液中移取约3 mL的样品液, 置于离心管中, 在9000 r/min的转速下离心处理5 min。小心吸取上层清液, 通过孔径为0.22 μm的聚四氟乙烯滤膜进行过滤, 将过滤后的清液用于后续的检测分析。
1.2.5 提取次数的优化
取0.5 g样品置于50 mL离心管中, 分别尝试了1、2、3次不同的提取次数, 每次提取均使用15 mL的提取溶剂, 并设定超声处理时间为10 min以确保充分提取。随后, 以9000 r/min的转速进行离心, 收集上清液, 最终使用60%甲醇溶液将上清液定容至50 mL。
1.3 数据处理
使用Excel 2016进行数据处理, 使用Origin 9.1作图。
2 结果与分析
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2.1 检测波长的确定
对图1中的NMN和NAD+的紫外光谱图进行分析。发现两者在紫外光区域均有特征性的吸收峰, 但最大吸收峰的位置略有不同: NMN在266 nm处有最大吸收峰, NAD+在260 nm处有最大吸收峰。为确保两种化合物具有相近的检测灵敏度, 将检测波长设定为263 nm, 满足实验对于精确度和准确性的要求。
2.2 色谱柱及流动相的确定
在考察NMN与NAD+分子的色谱分离过程时, 鉴于两者均富含羟基基团, 展现出显著的极性特征, 传统的C18色谱柱在分离这两种化合物时存在保留不强、容易受试样中其他杂质干扰的问题。本研究使用Agilent XDB C18色谱柱(4.5 mm×250 mm, 5.0 µm), 并以0.1%磷酸溶液与乙腈的混合液作为流动相进行实验(图2), 结果显示NMN与NAD+在柱上的保留能力不佳, 两者间难以实现有效分离。为了优化分离效果, 考察了更适合极性化合物分析的亲水色谱柱, 采用Waters BEH-Amide亲水色谱柱(4.5 mm×250 mm, 5.0 µm)进行后续实验。
在考察流动相体系时, 首先选用了水与乙腈组成的流动相体系, 相应的色谱图见图3。结果显示, Waters BEH-Amide亲水色谱柱对NMN和NAD+均展现出了良好的保留能力。然而, 在该流动相条件下, NMN的色谱峰出现了较为严重的前伸现象, 这可能归因于NMN较强的亲水性, 使其与色谱柱中的酰胺基团发生了相互作用, 从而影响了峰形的质量。随后考察了加入少量酸的流动相体系, 具体为0.1%磷酸溶液与乙腈的混合液, 并观察其对NMN和NAD+的洗脱效果(图4)。实验结果表明, 向流动相中添加微量的磷酸能够有效抑制NMN的电离, 显著改善了NMN的色谱峰形, 使其更加清晰和尖锐。
基于上述实验结果, 最终确定以0.1%磷酸溶液与乙腈组成的流动相体系, 并辅以梯度洗脱的方法, 以确保NMN和NAD+在Waters BEH-Amide亲水色谱柱上能够实现高效且准确的分离与分析。
2.3 提取溶剂的确定
鉴于NMN和NAD+在极性溶剂如水、甲醇、乙醇及乙腈中具备良好的溶解性, 本研究选择实验室常规使用的水与甲醇作为样品提取溶剂, 以优化提取过程。
为评估不同溶剂组合对目标化合物提取效率的影响, 本研究选取了包含白藜芦醇、花青素、吡咯喹啉醌、虾青素及辅酶Q10等多种成分的保健食品作为代表性基质, 并设定加标浓度为10 mg/g。不同溶剂条件下的提取效果结果如图5所示。
根据图5的结果可知, 在提取试样中的NMN和NAD+时, 甲醇溶液展现出了良好的提取效能。特别地, 当甲醇浓度调整至60%时, 提取效率达到了最高, 此时, 回收率超过了98%。
综上所述, 为了获得最佳的提取效果, 选定60%甲醇溶液作为本研究及后续相关工作中提的最佳溶剂。
2.4 超声时间的确定
通常情况下, 超声波在提取过程中通过其机械效应、空化效应和热效应等机制, 有助于加速溶剂渗透进入样品内部, 促进目标化合物从固相转移到液相, 从而提高提取效率[30]。因此, 考察不同超声时间对回收率的影响是优化提取条件的重要步骤。
根据图6的结果显示, 超声时间对于NMN和NAD+的提取回收率具有一定影响。具体而言, 超声处理5 min后, 已经能够基本完成NMN和NAD+的提取过程, 此时回收率稳定在98%左右, 表明在短时间内超声波即能高效地将目标化合物从样品中提取出来。然而, 为了确保提取过程的充分性和提取效率的最大化, 基于冗余设计原则, 将超声时间延长至10 min。实验证明, 当超声时间设为10 min时, 回收率可保持在98%以上。
综上所述, 为了确保提取效率并优化实验条件, 将超声时间设定为10 min, 这样既能保证目标化合物的高效提取, 又能确保实验结果的稳定性和可靠性。
2.5 提取次数考察
为优化提取流程, 考察提取次数对提取效率的影响, 加标浓度为10 mg/g。
通过比较不同提取次数下的回收率, 并将结果绘制成图7。由图7可知, 仅进行1次提取时, 回收率已高达97%以上, 表明单次提取即能提取样品中大部分的目标化合物。当提取次数增加至2次时, 回收率虽有轻微上升, 达到98%以上, 但与直接使用40 mL提取液进行单次提取的效果相近(如2.3所述)。
在综合考虑提取效率、资源消耗以及工作效率等多方面因素后, 最终采用单次40 mL提取液的方案进行提取。该方案既保证了较高的回收率, 又简化了操作流程, 提高了实验效率。
2.6 线性关系和检出限
对NMN和NAD+的标准工作溶液进行了仪器测定, 其质量浓度范围为10~1000 μg/mL。以化合物的质量浓度为横坐标(X, μg/mL), 相应的峰面积为纵坐标(Y), 绘制了标准曲线。对于NMN, 其线性回归方程为Y=1.72749X-1.94237, 线性相关系数r=0.99995, 表明浓度与峰面积之间具有极高的线性关系。对于NAD+, 其线性回归方程为Y=2.78483X-3.41243, 线性相关系数r=0.99995, 也显示了极好的线性相关性。
在评估方法的灵敏度时, 采用了信噪比的方法来确定检出限(limit of detection, LOD)和定量限(limit of quantification, LOQ)。以3倍信噪比对应的浓度被定义为LOD, 以10倍信噪比对应的浓度被定义为LOQ, 因此方法LOD为0.15 g/kg, LOQ为0.50 g/kg。
2.7 回收率和精密度
在保健食品基质(该基质包含白藜芦醇、花青素、吡咯喹啉醌、虾青素及辅酶Q10等多种成分)中进行了加标回收实验, 以评估方法的准确性和可靠性。实验中, 设定了3个不同的加标浓度水平: 第一点为方法的LOQ (0.50 g/kg), 第二点则对应于保健食品中目标分析物的平均含量, 第三点则是该平均含量的两倍。每个加标浓度均进行了6次重复测定, 以确保数据的稳定性和可靠性。
实验结果显示, 所有目标分析物的总体回收率均落在95.9%至98.8%的范围内, 表明该方法在保健食品基质中的回收效率良好。同时, 相对标准偏差(relative standard deviation, RSD)的计算结果显示为1.19%至3.05%, 这一较低的RSD值反映了测量结果良好的重现性和精密度, 结果见表1, 空白基质和加标回收色谱图见图8、9。
2.8 试样测定
采用已建立的分析方法, 对市场上随机采购的8批次保健食品进行了检测分析, 这些样品均标注含有NMN或同时含有NMN与NAD+成分。通过检测, 数据见表2, 样品4的色谱图见图10。
根据表2中的检测结果, 在所检测的8个保健食品样品中, 第2、4、6号样品被检测出含有NMN, 尤为值得注意的是, 4号样品中不仅检测到了NMN, 还同时检测出了NAD+。其余5个批次的样品中均未检测到NMN和NAD+的存在。这一结果揭示了市场上部分保健食品可能存在成分标识与实际含量不符的问题。
鉴于上述情况, 建议相关监管部门加大对保健食品市场的监督与管理力度, 特别是要针对声称添加了NMN和NAD+等热门保健食品成分的产品进行重点审查, 确保产品标识的真实性与准确性。通过加强监管, 有效防止虚假宣传和不实标注, 保护消费者的合法权益, 维护市场的公平与秩序。
3 结论
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本研究成功开发了一种高效、精准的高效液相色谱法, 用于测定保健食品中NMN与NAD+的含量。该方法具有良好的的线性范围, 在10~1000 μg/mL的质量浓度范围内, 其线性相关系数高达0.999或以上, 确保了测量结果的准确。此外, 该方法的LOQ为0.50 g/kg, 充分满足了保健食品的检测需求。
在保健食品实际基质中, 通过设定3个不同浓度的加标水平进行方法验证, 该方法表现出了优异的回收率和精密度。该方法回收率稳定在95.9%至98.8%之间, RSDs在1.19%至3.05%之间, 这充分证明了该方法在复杂基质中的稳定性和可靠性。
通过应用此方法对市场上实际购买的保健食品样品进行检测, 获得了NMN和NAD+的准确含量数据, 验证了该方法在测定保健食品中这两种重要生物活性成分方面的广泛适用性和高度可靠性。这一研究成果不仅为保健食品的质量控制和市场监管提供了强有力的技术支持, 也为相关产业的发展奠定了坚实基础。
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2025年第16卷第2期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241016004
  • 接收时间：2024-10-16
  • 首发时间：2025-07-21
  • 出版时间：2025-01-25
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  • 收稿日期：2024-10-16
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    上海市质量监督检验技术研究院, 上海 200233

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* 郑国建(1983—), 男, 硕士, 高级工程师, 主要研究方向为食品中营养素的检测。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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