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Article(id=1321169934718189568, tenantId=1146029695717560320, journalId=1319373219989389464, issueId=1321169839390053177, articleNumber=null, orderNo=null, doi=10.11847/zgggws1146199, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1729180800000, receivedDateStr=2024-10-18, revisedDate=1736697600000, revisedDateStr=2025-01-13, acceptedDate=1744041600000, acceptedDateStr=2025-04-08, onlineDate=1792921058587, onlineDateStr=2026-10-25, pubDate=1749484800000, pubDateStr=2025-06-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792921058587, onlineIssueDateStr=2026-10-25, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792921058586, creator=13701087609, updateTime=1792921058586, updator=13701087609, issue=Issue{id=1321169839390053177, tenantId=1146029695717560320, journalId=1319373219989389464, year='2025', volume='41', issue='6', pageStart='641', pageEnd='768', issueExtLink='null', onlineDate='null', pubDate='1749484800000', pubDateStr='2025-06-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1792921035859, creator='13701087609', updateTime=1792921035859, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=758, endPage=763, ext={EN=ArticleExt(id=1321169936567877633, articleId=1321169934718189568, tenantId=1146029695717560320, journalId=1319373219989389464, language=EN, title=Isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry for determination of five alternariol mycotoxins in chestnut, columnId=1320438104528376815, journalTitle=Chinese Journal of Public Health, columnName=Inspection technology, runingTitle=null, highlight=null, articleAbstract=
Objective

Establishment of an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) method for the simultaneous determination of five alternariol mycotoxins in chestnuts.

Methods

The optimal mass spectrometry conditions for the five target compounds were determined through parameter optimization. The chromatographic peak elution of the target compounds was analyzed, and the optimal chromatographic column and mobile phase were selected. The extraction efficiency of different extraction reagents was compared by recovery experiments to determine the optimal extraction reagent. Finally, the established method was validated.

Results

The method ultimately used acetonitrile-water-formic acid (70 + 29 + 1) as the extraction reagent for sample pretreatment. A BEH C18 column was used with a mobile phase of acetonitrile -0.15 mmol/L ammonium bicarbonate. The matrix effect of the method could not be ignored, and the internal standard method should be used for quantitative analysis. The limits of detection (LODs) for the five alternariol mycotoxins ranged from 0.10 to 0.50 μg/kg, and the limits of quantitation (LOQs) ranged from 0.30 to 1.50 μg/kg. The recoveries ranged from 80.1% to 111.7%, with relative standard deviations (RSDs) ranging from 2.3% to 8.2%. The correlation coefficients were all ≥ 0.999 0.

Conclusions

This method is simple, rapid, and accurate. The linearity, precision, and recovery meet the methodological requirements, and it is suitable for the simultaneous determination of five alternariol mycotoxins in chestnuts.

, authors=Quanshuai WANG, Weiming SUN, authorsList=Quanshuai WANG, Weiming SUN, authorCompany=null, correspAuthors=Quanshuai WANG, authorNote=null, correspAuthorsNote=
WANG Quanshuai, E-mail:
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目的

建立同时测定板栗中5种交链孢霉菌素含量的同位素稀释–超高效液相色谱–串联质谱法。

方法

通过参数优化确定5种目标物的最优质谱条件，分析目标化合物色谱峰的出峰情况，选择最佳的色谱柱和流动相，通过回收率比较不同提取试剂的提取效果以确定最佳的提取试剂，最后对建立的方法进行方法学验证。

结果

该方法最终使用乙腈–水–甲酸（70+29+1）作为提取试剂对样品前处理。采用BEH C18为色谱柱，流动相为乙腈碳酸氢铵–0.15 mmol/L，方法的基质效应不能忽略，应采用内标法定量分析，5种交链孢霉菌素的检出限为0.10～0.50 μg/kg，定量限为0.30～1.50 μg/kg，回收率80.1%～111.7%，相对标准偏差2.3%～8.2%，相关系数均≥0.999 0 。

结论

本方法操作简便、快速准确，线性关系、精密度和回收率等均满足方法学要求，适用于板栗中5种交链孢霉菌素含量的同时测定。

, authors=王权帅*, *, 孙卫明, authorsList=王权帅, 孙卫明, authorCompany=null, correspAuthors=王权帅, authorNote=

王权帅（1985—），主管检验师，硕士，研究方向：食品卫生检验

, correspAuthorsNote=
王权帅，E-mail：
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王权帅（1985—），主管检验师，硕士，研究方向：食品卫生检验

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王权帅（1985—），主管检验师，硕士，研究方向：食品卫生检验

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Journal of Chromatography A, 2021, 1659: 462486., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1321169939289980935, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, xref=null, ext=[AuthorCompanyExt(id=1321169939294175240, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, companyId=1321169939289980935, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Beijing Huairou District Center for Diseases Control and Prevention, Beijing, 101400, China), AuthorCompanyExt(id=1321169939302563849, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, companyId=1321169939289980935, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京市怀柔区疾病预防控制中心，北京 101400)])], figs=[ArticleFig(id=1321169942037250076, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=EN, label=Fig.1, caption=Chromatograms of TeA under different mobile phases, figureFileSmall=/Ci6dgcgweROz+WkkozPLA==, figureFileBig=XxsTQiMvzd/fK5xTDUl+4A==, tableContent=null), ArticleFig(id=1321169942087581725, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=CN, label=图1, caption=不同流动相下TeA的色谱图

注：A：0.15 mmol/L氟化铵；B：0.1%甲酸+0.15 mmol/L碳酸氢铵；C：1.5 mmol/L碳酸氢铵；D：0.15 mmol/L碳酸氢铵。

, figureFileSmall=/Ci6dgcgweROz+WkkozPLA==, figureFileBig=XxsTQiMvzd/fK5xTDUl+4A==, tableContent=null), ArticleFig(id=1321169942255353886, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=EN, label=Fig.2, caption=Chromatograms of five alternariol mycotoxins, figureFileSmall=5ED/EtfrxfRoooLRCIuMWA==, figureFileBig=Doz/0pCjkYCo53k86flA/g==, tableContent=null), ArticleFig(id=1321169942318268447, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=CN, label=图2, caption=5种交链孢霉菌素色谱图, figureFileSmall=5ED/EtfrxfRoooLRCIuMWA==, figureFileBig=Doz/0pCjkYCo53k86flA/g==, tableContent=null), ArticleFig(id=1321169942393765920, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=EN, label=Tab.1, caption=

Mass spectrometry instrument conditions and retention times of each target toxin

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素名称保留时间/min前体离子/（m/z）产物离子/（m/z）Q1电压/VCE/eVQ3电压/V
TeA9.928196.00139.0510.018.022.0
196.00112.0510.028.017.0
AOH10.277257.00146.9513.033.024.0
257.00212.0510.028.020.0
AME10.836271.10256.1010.022.025.0
271.10228.0014.031.022.0
TEN10.453413.20140.9012.017.012.0
413.20271.1512.019.016.0
ALT10.288291.00186.2014.025.017.0
291.00214.1014.020.020.0
13C-TeA9.929206.15117.1010.025.019.0
13C-AOH10.278271.00156.1013.032.026.0
13C-AME10.829274.15254.9514.032.025.0
13C-TEN10.456416.30217.2015.024.021.0
), ArticleFig(id=1321169942469263393, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=CN, label=表1, caption=

质谱仪器条件及各目标毒素的保留时间

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素名称保留时间/min前体离子/（m/z）产物离子/（m/z）Q1电压/VCE/eVQ3电压/V
TeA9.928196.00139.0510.018.022.0
196.00112.0510.028.017.0
AOH10.277257.00146.9513.033.024.0
257.00212.0510.028.020.0
AME10.836271.10256.1010.022.025.0
271.10228.0014.031.022.0
TEN10.453413.20140.9012.017.012.0
413.20271.1512.019.016.0
ALT10.288291.00186.2014.025.017.0
291.00214.1014.020.020.0
13C-TeA9.929206.15117.1010.025.019.0
13C-AOH10.278271.00156.1013.032.026.0
13C-AME10.829274.15254.9514.032.025.0
13C-TEN10.456416.30217.2015.024.021.0
), ArticleFig(id=1321169942565732386, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=EN, label=Tab.2, caption=

Extraction efficiency (%) of two different extraction solvents

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素乙腈–水–甲酸（70+29+1）甲醇–水–甲酸（70+29+1）
TeA80.580.3
AOH97.870.2
AME80.355.9
TEN86.051.6
ALT89.572.5
), ArticleFig(id=1321169942628646947, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=CN, label=表2, caption=

2种不同提取溶剂提取效率/%

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素乙腈–水–甲酸（70+29+1）甲醇–水–甲酸（70+29+1）
TeA80.580.3
AOH97.870.2
AME80.355.9
TEN86.051.6
ALT89.572.5
), ArticleFig(id=1321169942687367204, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=EN, label=Tab.3, caption=

Matrix effects (%) of five alternariol mycotoxins

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素板栗品种平均基质效应
3113怀黄怀九燕红
TeA–51.9–59.6–57.5–49.0–54.5
AOH–24.6–28.2–25.8–19.2–24.5
TEN–40.5–47.0–46.5–40.9–43.7
AME22.115.912.124.018.5
ALT–21.6–25.6–22.8–20.9–22.7
), ArticleFig(id=1321169942750281765, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=CN, label=表3, caption=

5种交链孢霉菌素的基质效应/%

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素板栗品种平均基质效应
3113怀黄怀九燕红
TeA–51.9–59.6–57.5–49.0–54.5
AOH–24.6–28.2–25.8–19.2–24.5
TEN–40.5–47.0–46.5–40.9–43.7
AME22.115.912.124.018.5
ALT–21.6–25.6–22.8–20.9–22.7
), ArticleFig(id=1321169942813196326, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=EN, label=Tab.4, caption=

Recovery rate and precision of five alternariol mycotoxins (n=6)

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素名称加标量/（μg/kg）回收率范围/%平均回收率/%RSD/%
TeA4.080.1～105.293.17.2
10.083.0～100.792.37.0
20.084.2～99.992.72.9
AOH4.080.3～105.791.77.2
10.082.9～105.192.27.5
20.084.4～98.090.42.3
AME0.480.7～97.489.45.0
1.089.6～107.399.56.8
2.081.6～104.493.07.0
TEN0.488.1～109.099.96.7
1.080.8～101.391.06.0
2.084.0～99.792.13.1
ALT2.089.1～110.792.48.2
5.081.3～108.390.57.2
10.094.7～111.7103.15.3
), ArticleFig(id=1321169942880305191, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=CN, label=表4, caption=

5种交链孢霉菌素加标回收率及精密度（n=6）

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素名称加标量/（μg/kg）回收率范围/%平均回收率/%RSD/%
TeA4.080.1～105.293.17.2
10.083.0～100.792.37.0
20.084.2～99.992.72.9
AOH4.080.3～105.791.77.2
10.082.9～105.192.27.5
20.084.4～98.090.42.3
AME0.480.7～97.489.45.0
1.089.6～107.399.56.8
2.081.6～104.493.07.0
TEN0.488.1～109.099.96.7
1.080.8～101.391.06.0
2.084.0～99.792.13.1
ALT2.089.1～110.792.48.2
5.081.3～108.390.57.2
10.094.7～111.7103.15.3
), ArticleFig(id=1321169942959996968, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=EN, label=Tab.5, caption=

Linear equations, correlation coefficients, limits of detection, and limits of quantitation of the method (n=6)

, figureFileSmall=null, figureFileBig=null, tableContent=
毒素名称线性方程相关系数/rLOD/（μg/kg）LOQ/（μg/kg）
TeAy=0.009 81x+0.002 880.999 90.501.50
AOHy=0.073 79x+0.056 960.999 40.200.60
TENy=0.097 14x+0.003 360.999 60.150.45
AMEy=0.054 47x+0.006 170.999 90.100.30
ALTy=0.067 17x+0.011 240.999 60.300.90
), ArticleFig(id=1321169943031300137, tenantId=1146029695717560320, journalId=1319373219989389464, articleId=1321169934718189568, language=CN, label=表5, caption=

方法的线性方程、相关系数、检出限和定量限 （n=6）

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毒素名称线性方程相关系数/rLOD/（μg/kg）LOQ/（μg/kg）
TeAy=0.009 81x+0.002 880.999 90.501.50
AOHy=0.073 79x+0.056 960.999 40.200.60
TENy=0.097 14x+0.003 360.999 60.150.45
AMEy=0.054 47x+0.006 170.999 90.100.30
ALTy=0.067 17x+0.011 240.999 60.300.90
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同位素稀释–超高效液相色谱–串联质谱法测定板栗中5种交链孢霉菌素
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王权帅 *, * , 孙卫明
中国公共卫生 | 检验技术 2025,41(6): 758-763
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中国公共卫生 |检验技术 2025 , 41 (6) : 758 -763
同位素稀释–超高效液相色谱–串联质谱法测定板栗中5种交链孢霉菌素
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王权帅*, * , 孙卫明
作者信息
  • 北京市怀柔区疾病预防控制中心，北京 101400
通讯作者:
王权帅，E-mail：
作者简介:

王权帅（1985—），主管检验师，硕士，研究方向：食品卫生检验

Isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry for determination of five alternariol mycotoxins in chestnut
Quanshuai WANG , Weiming SUN
Affiliations
  • Beijing Huairou District Center for Diseases Control and Prevention, Beijing, 101400, China
    • Corresponding Author:
    WANG Quanshuai, E-mail:
出版时间: 2025-06-10 doi: 10.11847/zgggws1146199
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摘要
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目的

建立同时测定板栗中5种交链孢霉菌素含量的同位素稀释–超高效液相色谱–串联质谱法。

方法

通过参数优化确定5种目标物的最优质谱条件，分析目标化合物色谱峰的出峰情况，选择最佳的色谱柱和流动相，通过回收率比较不同提取试剂的提取效果以确定最佳的提取试剂，最后对建立的方法进行方法学验证。

结果

该方法最终使用乙腈–水–甲酸（70+29+1）作为提取试剂对样品前处理。采用BEH C18为色谱柱，流动相为乙腈碳酸氢铵–0.15 mmol/L，方法的基质效应不能忽略，应采用内标法定量分析，5种交链孢霉菌素的检出限为0.10～0.50 μg/kg，定量限为0.30～1.50 μg/kg，回收率80.1%～111.7%，相对标准偏差2.3%～8.2%，相关系数均≥0.999 0 。

结论

本方法操作简便、快速准确，线性关系、精密度和回收率等均满足方法学要求，适用于板栗中5种交链孢霉菌素含量的同时测定。

交链孢霉菌素  /  超高效液相色谱–串联质谱法  /  同位素稀释  /  板栗
Objective

Establishment of an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) method for the simultaneous determination of five alternariol mycotoxins in chestnuts.

Methods

The optimal mass spectrometry conditions for the five target compounds were determined through parameter optimization. The chromatographic peak elution of the target compounds was analyzed, and the optimal chromatographic column and mobile phase were selected. The extraction efficiency of different extraction reagents was compared by recovery experiments to determine the optimal extraction reagent. Finally, the established method was validated.

Results

The method ultimately used acetonitrile-water-formic acid (70 + 29 + 1) as the extraction reagent for sample pretreatment. A BEH C18 column was used with a mobile phase of acetonitrile -0.15 mmol/L ammonium bicarbonate. The matrix effect of the method could not be ignored, and the internal standard method should be used for quantitative analysis. The limits of detection (LODs) for the five alternariol mycotoxins ranged from 0.10 to 0.50 μg/kg, and the limits of quantitation (LOQs) ranged from 0.30 to 1.50 μg/kg. The recoveries ranged from 80.1% to 111.7%, with relative standard deviations (RSDs) ranging from 2.3% to 8.2%. The correlation coefficients were all ≥ 0.999 0.

Conclusions

This method is simple, rapid, and accurate. The linearity, precision, and recovery meet the methodological requirements, and it is suitable for the simultaneous determination of five alternariol mycotoxins in chestnuts.

alternariol monomethyl ether  /  ultra high performance liquid chromatography-tandem mass spectrometry  /  isotope dilution  /  chestnut
王权帅, 孙卫明. 同位素稀释–超高效液相色谱–串联质谱法测定板栗中5种交链孢霉菌素. 中国公共卫生, 2025 , 41 (6) : 758 -763 . DOI: 10.11847/zgggws1146199
Quanshuai WANG, Weiming SUN. Isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry for determination of five alternariol mycotoxins in chestnut[J]. Chinese Journal of Public Health, 2025 , 41 (6) : 758 -763 . DOI: 10.11847/zgggws1146199
正文
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交链孢霉菌（Alternaria）是一种在自然界中普遍存在的植物致病菌和腐生菌，由于对环境有极强的适应性，因此极有可能污染多种谷物、水果、蔬菜等农作物[1–2]。交链孢霉毒素（Alternaria toxins）是由交链孢霉菌属（Alternaria species）产生的次级代谢产物。目前全世界已发现70多种不同的交链孢霉菌素，按照化学结构的不同，主要分为5类，分别为四氨基酸类衍生物、二苯–α–吡喃酮衍生物、苝醌类衍生物、混杂多环结构化合物和长链氨基多元醇的丙三羧酸酯类化合物[3]。其中，目前认为污染最为广泛、研究最为深入的主要有细交链孢菌酮酸（TeA）、交链孢酚（AOH）、腾毒素（TEN）、交链孢烯（ALT）和交链孢酚单甲醚（AME）等[4–6]。交链孢霉菌素作为一种当前关注度很高的真菌毒素，不仅给农业上造成经济损失，人在食用被其污染的食物后，会产生急性毒性、诱变性、致癌性和致畸性等毒害作用，尤其是对于婴儿、儿童和素食主义者等易接触的人群产生危害的风险则更大[7–10]。由于板栗味道甜美、营养丰富、脂肪含量低，故而在我国板栗是一种非常令人喜爱的坚果。北京是我国重要的板栗产地之一，尤其怀柔板栗已成为板栗行业的一张名片[11]。目前我国已经开始加强对坚果中交链孢霉菌素污染的研究[12]，但关于板栗中交链孢霉菌素的污染状况研究和检测方法还鲜有报道，因此建立一种同时检测板栗中多种交链孢霉菌素的方法具有十分重要的现实意义。现有文献资料报道的交链孢霉菌素检测技术涉及多种检测仪器，方法多种多样，主要有酶联免疫吸附测定法、薄层色谱法、气相色谱法、气相色谱质谱法、液相色谱法和液相色谱–质谱联用法等[13–19]。随着液相色谱质谱联用仪越来越广泛地使用，液相色谱–质谱联用法逐渐成为检测交链孢霉菌素的最佳方法。前处理方法主要有固相萃取法和QuEChERS法[20–21]，但直接使用同位素稀释法进行前处理的研究鲜有报道。因此，本研究采用同位素稀释法对板栗样本前处理，利用超高效液相色谱–串联质谱法同时测定5种交链孢霉菌素。优化和建立一种快速、准确、灵敏度高的方法，能够满足同时测定板栗中5种交链孢霉菌素含量的要求。
1　材料与方法
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1.1　材料、仪器与试剂
LCMS-8050超高效液相色谱–三重四级杆串联质谱仪（日本岛津公司），Waters BEH （美国Waters公司、C 182.1 mm×150 mm、1.7 μm），BSA623S-CW电子天平（德国sartorius公司），VORTEX-5调速振荡器（海门其林贝尔仪器制造有限公司），Centrifuge 5810R高速低温离心机（德国sigma公司），水平摇床（北京市景山人民公社医疗器械修配厂）。 标准品： TeA（100.1 μg/mL），AOH（100.0 μg/mL） ，TEN（100.0 μg/mL） ，AME（102.3 μg/mL）和ALT（10.48 μg/mL）均购自美国ROMER公司。内标：本实验使用的内标物质D3-TEN（96%）和D3-AME（97%）购自Toronto公司。13C14-AOH（10.02 μg/mL）和13C14-TeA（25.66 μg/mL）购自Pribolab公司。乙腈（色谱纯、美国Fisher公司），甲醇（色谱纯、美国Fisher公司），甲酸（色谱纯、北京迪马公司）。
1.2　实验方法
1.2.1　标准溶液的配制
混合标准使用液（TeA和AOH：1.0 μg/mL；TEN和AME：0.1 μg/mL）：分别准确吸取一定量的标准溶液，用乙腈定容至10 mL，涡旋混匀。–20 ℃保存。D3-TEN（200 μg/mL）配制：准确称取1 mg D3-TEN标准品，用乙腈溶解并稀释至5 mL。D3-AME（100 μg/mL）配制：准确称取0.5 mg D3-AME标准品，用乙腈溶解并稀释至5 mL。混合同位素内标使用液（13C14-AOH：0.5 μg/mL，13C14-TeA：1.0 μg/mL，D3-TEN：1.0 μg/mL，D3-AME：1.0 μg/mL）：分别准确吸取一定量的标准溶液，以乙腈定容至10 mL，涡旋混匀，–20 ℃保存。
1.2.2　样品制备
3113、怀九、怀黄和燕红4种板栗样品先用去皮机脱掉外壳，然后以高速粉碎机粉碎、混合均匀，储存于样品袋中，密封保存。
1.2.3　提取及净化
称取5 g（精确到0.001 g）试样，置于 50 mL离心管中，加入20 mL乙腈–水–甲酸（70+29+1，体积比）溶液，涡旋混合器混匀，置于摇床上振荡提取30 min，取1 mL提取液至1.5 mL离心管中，4 ℃冷冻离心机10 000 r/min离心5 min。准确转移0.5 mL上清液于另一1.5 mL离心管中，加入1.0 mL水，涡旋混匀，4 ℃冷冻离心机10 000 r/min离心5 min，吸取上清液过0.22 μm微孔滤膜。吸取180 μL处理好的试样于300 μL衬管内，加入20 μL内标使用液，涡旋混匀，上机测定。
1.2.4　仪器条件
1.2.4.1　液相色谱条件色谱柱
Waters BEH C18（2.1 mm×150 mm，1.7 μm）；进样量：10 μL；柱温：40 ℃。流动相A：乙腈；流动相B：0.15 mmol/L碳酸氢铵；流速：0.3 mL/min。梯度洗脱程序：0.0～1.0 min，95% B；1.0～5.0 min，60% B；5.0～7.0 min，30% B；7.0～8.0 min, 15% B；8.0～9.0 min，5% B；9.0～12.0 min，5% B；12.0～12.1 min，95% B；12.1～14.0 min，95% B。
1.2.4.2　质谱条件离子化模式
电喷雾电离负离子模式（ESI-）；质谱扫描方式：多反应监测；接口温度：300 ℃；脱溶剂管温度：200 ℃；加热模块温度：500 ℃；干燥气流速：10.00 L/min；加热气流速：10.00 L/min；雾化气流速：3.00 L/min；
1.2.5　标准曲线制备
分别移取混合标准储备液适量于不同的10 mL容量瓶中，用20%乙腈–水溶液定容至刻度，涡旋混匀。TeA、AOH和ALT的浓度为1.0、2.0、5.0、10.0和20.0 μg/L，TEN和AME的浓度为0.1、0.2、0.5、1.0和2.0 μg/L。分别准确吸取20 μL内标混合液于各内插管中，加入180 μL对应的混合标准曲线浓度点，涡旋混匀，配成混合标准曲线溶液系列。以目标物与对应内标的浓度比为横坐标，以目标物峰面积与对应内标峰面积的比为纵坐标，绘制标准曲线。
1.2.6　基质效应
基质效应（matrix effect, ME）的计算按照Frenich A G的方法计算[22]，其计算公式为：ME（%）=[（A2-A1）/A1]×100%，其中A1为标准物质溶于有机溶剂中的平均峰面积，A2为标准物质溶于基质空白中的平均峰面积。ME在±20%之间为可接受范围。
1.2.7　检出限和定量限
取样量按5 g计算，以3倍信噪比（S/N）下的浓度为方法检出限（limit of detection, LOD），10倍信噪比（S/N）下的浓度为方法定量限（limit of quantitation, LOQ）。
1.3　统计分析
通过LCMS-8050仪器所配置的LabSolution工作站进行数据采集处理，使用Office Excel 2007 进行统计分析。
2　结　果
收起
2.1　流动相选择（图1、2）
分别采用0.15 mmol/L氟化铵、0.15 mmol/L碳酸氢铵、1.5 mmol/L碳酸氢铵和0.1%甲酸+0.15 mmol/L碳酸氢铵作为流动相，分析色谱峰的出峰情况。图1表明，当采用0.15 mmol/L氟化铵和0.1%甲酸+0.15 mmol/L碳酸氢铵作为流动相时，均严重影响了TeA的出峰；而使用碳酸氢铵作为流动相时，TeA的色谱峰可以很好地分离并达到很好的出峰效果。碳酸氢铵浓度对TeA的出峰影响很大，当使用1.5 mmol/L碳酸氢铵作为流动相时，TeA色谱峰有严重的拖尾，而使用0.15 mmol/L碳酸氢铵时TeA的色谱图峰形对称，无拖尾或前延现象，故本方法采用0.15 mmol/L碳酸氢铵作为流动相。碳酸氢铵的起始浓度对TeA的出峰情况有一定的影响，当起始浓度为60%时，TeA峰型不好且响应值很低，当起始浓度提高到95%时，峰型和响应值均大大改善。因此，选择95%作为0.15 mmol/L碳酸氢铵的起始浓度。在该流动相条件下，5种交链孢霉菌素峰形最佳且分离良好（图2）。
2.2　质谱条件优化（表1）
当采用多反应监测模式定量分析时，质谱的仪器条件对目标物的分析灵敏度非常有影响。首先对喷针位置优化，最终确认当喷针位置为–1时，目标物的响应值最佳；其次通过目标物的响应强度，优化接口温度、脱溶剂管温度、加热模块温度、干燥气流速、加热气流速和雾化气流速；最后优化目标物的前体离子、产物离子、Q1电压、Q3电压和碰撞电压，最终确定5种交链孢霉菌素及4种内标化合物的保留时间及特征离子、电压等质谱仪器条件。
2.3　提取试剂的选择（表2）
选择合适的提取试剂对于交链孢霉菌素的准确测定非常重要，本研究对乙腈–水–甲酸（70+29+1）和甲醇–水–甲酸（70+29+1）2种不同提取试剂的提取效率进行分析，用回收率指标比较。当使用乙腈–水–甲酸作为提取试剂时，5种交链孢霉菌素的回收率为80.5%～97.8%；而采用甲醇–水–甲酸提取时，的TEN回收率仅51.6%、AME的回收率也只有55.9%，无法满足实验要求。因此本实验采用乙腈–水–甲酸（70+29+1）作为提取试剂。
2.4　基质效应（ME）分析（表3）
5种真菌毒素中，TeA、AOH、TEN和ALT 4种产生基质抑制效应，只有AME产生基质增强效应。按品种来分析，4种不同品种的板栗基质所导致的基质效应差异不大；TeA、AOH、TEN和ALT的平均基质效应分别为–54.5%、–24.5%、–43.7%和–22.7%，AME的基质效应在12.1%～24.0%。ALT无内标可以使用，由于ALT与AOH的基质效应基本一致，因此本方法对于ALT的定量使用13C14-AOH进行内标校准计算。
2.5　方法的回收率与精密度（表4）
以板栗为基质，分别测定加标量为低、中、高3个浓度（TeA、AOH加标浓度为4.0、10.0、20.0，AME和TEN加标浓度为0.4、1.0和2.0 μg/kg，ALT加标浓度为2.0、5.0和10.0 μg/kg）的加标回收率，平行测定6次。本实验的回收率为80.1%～111.7%，相对标准偏差（RSD）为2.3%～8.2%。方法的精密度和回收率满足分析检测的要求。
2.6　方法线性方程、相关系数、检出限和定量限（表5）
5种交链孢霉菌素在线性范围内均有良好的线性关系，相关系数均大于0.999 0。5种交链孢霉菌素的LOD为0.10 μg/kg～0.50 μg/kg，LOQ为0.30 μg/kg～1.50 μg/kg。因此本实验的线性关系、检出限与定量限均满足分析检测的要求。
3　讨　论
收起
色谱柱的选择对目标化合物的分离、选择和灵敏度有非常重要的作用。本实验比较了BEH C18和HSS PFP色谱柱，分析显示，PFP色谱柱对TeA的分离不够理想，而C18色谱柱对5种交链孢霉菌素的分离效果均非常理想，故本方法采用BEH C18色谱柱分析目标物。
由于TeA的化学结构与其他4种差异较大，故5种交链孢霉菌素的分析难点在TeA。由于TeA含有羧基，所以呈酸性且具有水溶性，因此在提取溶剂中加入一定量的酸，能有效地提高TeA的回收率[23–24]，故在提取溶剂中加入了1%甲酸。此外，邢家溧等[24]对于婴幼儿奶粉中交链孢霉菌素的提取采用2次提取的方法，2次的提取溶剂分别为乙腈–水（84+16）和乙腈–甲醇–水（45+10+45）。可能由于板栗样本无奶粉基质复杂，本研究在实验时，用乙腈–水–甲酸（70+29+1）1次提取就可以达到很好的提取效果，故未再用含甲醇的提取试剂进行2次提取。
板栗样本经前处理得到的提取液经超高效液相色谱–串联质谱分析时，可能产生基质效应，从而影响检测结果的准确度。本方法中TeA、AOH、TEN和ALT 4种交链孢霉菌素产生的基质抑制效应均大于20%，虽然AME平均基质效应在20%以内，但3113和燕红产生的基质效应在20%以上。因此基质效应均不可忽略，本方法使用内标法定量分析以消除基质效应对结果的影响。
关于交链孢霉菌素的研究很多，但板栗基质中交链孢霉菌素的研究文献相对较少。LOD和LOQ是方法验证中非常重要的2个指标，Wang Y J等[25]对干果的3种交链孢霉菌素测定方法中，AOH、TEN和AME的LOD分别为0.5、0.5和0.1 μg/kg。本实验所得到AME的LOD值一样，其余2种目标物的LOD低于该报道。Zhou J等[26]研究了板栗中交链孢霉菌素的测定方法，ALT和TEN的LOQ分别为5.0 μg/kg和0.4 μg/kg，本方法TEN的LOQ值与其基本一致，ALT的LOQ值低于其实验结果。
本研究采用同位素稀释–超高效液相色谱–串联质谱法同时检测板栗中5种交链孢霉菌素，内标法定量。建立的方法操作简便、快速准确，且线性关系、精密度和回收率等也满足方法学要求，适用于对板栗中5种交链孢霉菌素的同时检测。

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doi: 10.11847/zgggws1146199
  • 接收时间：2024-10-18
  • 首发时间：2026-10-25
  • 出版时间：2025-06-10
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  • 收稿日期：2024-10-18
  • 修回日期：2025-01-13
  • 录用日期：2025-04-08
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    北京市怀柔区疾病预防控制中心，北京 101400

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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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