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Article(id=1319998791030255827, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1319998767835754596, articleNumber=null, orderNo=null, doi=10.11983/CBB25102, pmid=null, cstr=32102.14.CBB25102, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1748966400000, receivedDateStr=2025-06-04, revisedDate=null, revisedDateStr=null, acceptedDate=1751904000000, acceptedDateStr=2025-07-08, onlineDate=1792641836172, onlineDateStr=2026-10-22, pubDate=1757433600000, pubDateStr=2025-09-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792641836172, onlineIssueDateStr=2026-10-22, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792641836172, creator=13701087609, updateTime=1792641836172, updator=13701087609, issue=Issue{id=1319998767835754596, tenantId=1146029695717560320, journalId=1316781310003478668, year='2025', volume='60', issue='5', pageStart='669', pageEnd='853', issueExtLink='null', onlineDate='null', pubDate='1757433600000', pubDateStr='2025-09-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1792641830643, creator='13701087609', updateTime=1792641943258, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1319999240240222831, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1319998767835754596, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1319999240240222832, tenantId=1146029695717560320, journalId=1316781310003478668, issueId=1319998767835754596, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=846, endPage=853, ext={EN=ArticleExt(id=1319998791244165332, articleId=1319998791030255827, tenantId=1146029695717560320, journalId=1316781310003478668, language=EN, title=Optimization of an High-performance Liquid Chromatography Method for the Determination of Salicylic Acid, columnId=1319334540443767589, journalTitle=Chinese Bulletin of Botany, columnName=TECHNIQUE AND METHOD, runingTitle=null, highlight=null, articleAbstract=

INTRODUCTION: The phytohormone salicylic acid (SA) plays multiple important roles in plants, such as disease resistance, seed germination, and leaf senescence. Among these, the role of SA in plant disease resistance is the most studied. Since SA promotes disease resistance at the cost of plant growth, plants need to dynamically regulate the content of SA to balance disease resistance and growth. Therefore, fast and accurate measurement of SA content is a critical basis for plant immunity research.

RATIONALE: High-performance liquid chromatography (HPLC)-fluorescence detector is the most popular method for the quantitative measurement of SA. In order to improve the efficiency and sensitivity of current methods, this study optimized the composition, ion concentration, and pH of the mobile phase, as well as the detection wavelength and detection procedure.

RESULTS: The baseline of the chromatogram was more stable when using acetonitrile instead of methanol in the mobile phase. When the pH of the mobile phase was 5.2, the retention time of SA was the shortest, without interference peak near the SA peak, which was preferred for minimizing the detection time. The higher concentration of sodium acetate (100 mmol∙L-1) in the mobile phase was better than that of lower concentration (20-50 mmol∙L-1). Wavelength scanning revealed that the optimal excitation wavelength was 300 nm and the optimal emission wavelength was 405 nm, under which the highest sensitivity for SA detection was obtained. At a flow rate of 2 mL∙min-1, it took 3.5 min for elution, 3.5 min for column wash, and 3 min for column balance, shortening the measurement time per sample from 50 min to 10 min.

CONCLUSION: These optimizations greatly improved the sensitivity, stability, and efficiency of the SA measurement using HPLC, which will contribute to the plant immunity research.

Optimization of salicylic acid (SA) measurement. (A) SA detection procedure; (B) Chromatogram using the optimized condition. The samples are total SA in Arabidopsis without Psm ES4326 infection. EU: Emission units. The peak labeled in red indicates SA.

, authors=Shi Shixi, Yan Shunping*, authorsList=Shi Shixi, Yan Shunping, authorCompany=null, correspAuthors=Yan Shunping, authorNote=null, correspAuthorsNote=
*E-mail:
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植物激素水杨酸(SA)促进植物的抗病性, 但抑制植物生长。植物通过动态调控SA的含量以平衡抗病与生长。高效液相色谱-荧光检测器技术是检测SA含量最常用的方法。该研究优化了流动相的成分、离子浓度、pH值以及检测波长和检测程序。优化后的流动相为10%乙腈、100 mmol∙L-1乙酸钠、pH5.2。优化后的激发光波长为300 nm, 发射光波长为405 nm。优化后的检测程序为: 进样后3.5分钟开始清洗色谱柱, 清洗时间为3.5分钟, 平衡时间为3分钟, 总时长为10分钟。优化后的检测方法显著提高了检测灵敏性、稳定性和高效性。

, authors=史世肸, 严顺平*, authorsList=史世肸, 严顺平, authorCompany=null, correspAuthors=严顺平, authorNote=null, correspAuthorsNote=
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Salicylic acid inhibits gibberellin signaling through receptor interactions. Mol Plant 15, 1759-1771., articleTitle=Salicylic acid inhibits gibberellin signaling through receptor interactions, refAbstract=null), Reference(id=1319998803013382432, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, doi=null, pmid=null, pmcid=null, year=2021, volume=63, issue=null, pageStart=823, pageEnd=827, url=null, language=null, rfNumber=[23], rfOrder=23, authorNames=Yu XD, Xu YR, Yan SP, journalName=J Integr Plant Biol, refType=null, unstructuredReference=Yu XD, Xu YR, Yan SP (2021). Salicylic acid and ethylene coordinately promote leaf senescence. J Integr Plant Biol 63, 823-827., articleTitle=Salicylic acid and ethylene coordinately promote leaf senescence, refAbstract=null), Reference(id=1319998803084685601, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, doi=null, pmid=null, pmcid=null, year=2017, volume=175, issue=null, pageStart=1082, pageEnd=1093, url=null, language=null, rfNumber=[24], rfOrder=24, authorNames=Zhang YJ, Zhao L, Zhao JZ, Li YJ, Wang JB, Guo R, Gan SS, Liu CJ, Zhang KW, journalName=Plant Physiol, refType=null, unstructuredReference=Zhang YJ, Zhao L, Zhao JZ, Li YJ, Wang JB, Guo R, Gan SS, Liu CJ, Zhang KW (2017). S5H/DMR6 encodes a salicylic acid 5-hydroxylase that fine-tunes salicylic acid homeostasis. Plant Physiol 175, 1082-1093., articleTitle=S5H/DMR6 encodes a salicylic acid 5-hydroxylase that fine-tunes salicylic acid homeostasis, refAbstract=null)], funds=[Fund(id=1319998799511138567, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, awardId=2023YFF1001301, language=CN, fundingSource=国家重点研发计划(2023YFF1001301), fundOrder=null, country=null), Fund(id=1319998799603413256, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, awardId=32270306, language=CN, fundingSource=国家自然科学基金(32270306), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1319998794696077540, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, xref=null, ext=[AuthorCompanyExt(id=1319998794704466149, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, companyId=1319998794696077540, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China), AuthorCompanyExt(id=1319998794712854758, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, companyId=1319998794696077540, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=华中农业大学生命科学技术学院, 武汉 430070)])], figs=[ArticleFig(id=1319998798110241019, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=EN, label=Figure 1, caption=Detection of salicylic acid (SA) using methods reported in Zhang et al., 2017

(A) 0.1 μg·mL-1 SA standard sample; (B) The sample is free SA in Arabidopsis without Psm ES4326 infection. EU: Emission units. The peaks labeled in red indicate SA.

, figureFileSmall=I0PaOqxf2oabU95TyvFHtA==, figureFileBig=u41cWq4VnsMEf9SeENcOkA==, tableContent=null), ArticleFig(id=1319998798194127100, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=CN, label=图1, caption=使用Zhang等(2017)的方法检测水杨酸(SA)

(A) 0.1 μg·mL-1 SA标准样品; (B) 样品为未被Psm ES4326侵染的拟南芥游离态SA。EU: 发射单位。红色的峰指示SA。

, figureFileSmall=I0PaOqxf2oabU95TyvFHtA==, figureFileBig=u41cWq4VnsMEf9SeENcOkA==, tableContent=null), ArticleFig(id=1319998798554837245, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=EN, label=Figure 2, caption=Effects of pH of the mobile phase on salicylic acid (SA) separation

(A)-(C) The samples are total SA extracted from Arabidopsis treated with Psm ES4326 for 24 h; (D)-(F) The samples are total SA in Arabidopsis without Psm ES4326 infection. EU is the same as shown in Figure 1. The peaks labeled in red indicate SA.

, figureFileSmall=Fry7c5ErPeDeohPAfG3wRA==, figureFileBig=yGw/gaOaGf9r0rYdkRf1Fg==, tableContent=null), ArticleFig(id=1319998798609363198, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=CN, label=图2, caption=流动相pH值对水杨酸(SA)分离的影响

(A)-(C) 样品为被Psm ES4326侵染24小时后的拟南芥总SA; (D)-(F) 样品为未被Psm ES4326侵染的拟南芥总SA。EU同图1。红色的峰指示SA。

, figureFileSmall=Fry7c5ErPeDeohPAfG3wRA==, figureFileBig=yGw/gaOaGf9r0rYdkRf1Fg==, tableContent=null), ArticleFig(id=1319998798689054975, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=EN, label=Figure 3, caption=Effects of the concentration of sodium acetate in the mobile phase on salicylic acid (SA) separation

(A) 20 mmol∙L-1 NaAc; (B) 100 mmol∙L-1 NaAc. The samples are total SA in Arabidopsis without Psm ES4326 infection. EU is the same as shown in Figure 1. The peaks labeled in red indicate SA.

, figureFileSmall=I/RAE3zSOXnInjlwslCwXQ==, figureFileBig=YcWvLRi/DucNVGz7EZbufA==, tableContent=null), ArticleFig(id=1319998798777135360, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=CN, label=图3, caption=流动相中乙酸钠浓度对水杨酸(SA)分离的影响

(A) 20 mmol∙L-1乙酸钠; (B) 100 mmol∙L-1乙酸钠。样品为未被Psm ES4326侵染的拟南芥总SA。EU同图1。红色的峰指示SA。

, figureFileSmall=I/RAE3zSOXnInjlwslCwXQ==, figureFileBig=YcWvLRi/DucNVGz7EZbufA==, tableContent=null), ArticleFig(id=1319998798848438529, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=EN, label=Figure 4, caption=Optimization of detection wavelength for salicylic acid (SA)

(A), (B) The excitation wavelength is 296 nm and the emission wavelength is 350-450 nm; (C), (D) The excitation wavelength is 280-320 nm and the emission wavelength is 395 nm; (E), (F) The excitation wavelength is 300 nm and the emission wavelength is 375-415 nm. (A), (C), (E) Two-dimensional signal plots; (B), (D), (F) Fluorescence wavelength-fluorescence energy integration plots. All samples are 1 µg∙mL-1 SA standards. EU is the same as shown in Figure 1.

, figureFileSmall=/KmvPXHY4CilAz7Lt1TD+g==, figureFileBig=mFlQTj73b+LjyH++b/ygTw==, tableContent=null), ArticleFig(id=1319998798915547394, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=CN, label=图4, caption=水杨酸(SA)检测波长的优化

(A), (B) 激发光波长为296 nm, 发射光波长为350-450 nm; (C), (D) 激发光波长为280-320 nm, 发射光波长为395 nm; (E), (F) 激发光波长为300 nm, 发射光波长为375-415 nm。(A), (C), (E) 二维信号图; (B), (D), (F) 荧光波长-荧光能量积分图。样品均为1 µg∙mL-1 SA标准样品。EU同图1。

, figureFileSmall=/KmvPXHY4CilAz7Lt1TD+g==, figureFileBig=mFlQTj73b+LjyH++b/ygTw==, tableContent=null), ArticleFig(id=1319998798999433475, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=EN, label=Figure 5, caption=Optimization of salicylic acid (SA) detection procedure

(A) Graph of detection procedure; (B) Detection results. The samples are total SA in Arabidopsis without Psm ES4326 infection. EU is the same as shown in Figure 1. The peak labeled in red indicates SA.

, figureFileSmall=a0+k6izkv0BZgWSKa0VLdg==, figureFileBig=Tp/X0AYJQLaQxCmOui4V0Q==, tableContent=null), ArticleFig(id=1319998799100096772, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=CN, label=图5, caption=水杨酸(SA)检测程序的优化

(A) 检测程序图; (B) 检测结果。样品为未被Psm ES4326侵染的拟南芥总SA。EU同图1。红色的峰指示SA。

, figureFileSmall=a0+k6izkv0BZgWSKa0VLdg==, figureFileBig=Tp/X0AYJQLaQxCmOui4V0Q==, tableContent=null), ArticleFig(id=1319998799293034757, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=EN, label=Figure 6, caption=Salicylic acid (SA) standard curve

(A) Peak areas corresponding to different concentrations of SA standards; (B) The standard curve (with an intercept of 0, R2 indicates the coefficient of determination)

, figureFileSmall=feWPFIaas0eIjb+iUc9Mig==, figureFileBig=5jmnjfFmsZV1OiNeRacRvw==, tableContent=null), ArticleFig(id=1319998799376920838, tenantId=1146029695717560320, journalId=1316781310003478668, articleId=1319998791030255827, language=CN, label=图6, caption=水杨酸(SA)标准曲线

(A) 不同浓度SA标准样品对应的峰面积; (B) 标准曲线(截距为0, R2表示决定系数)

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高效液相色谱法检测水杨酸的优化
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史世肸 , 严顺平 *
植物学报 | 技术方法 2025,60(5): 846-853
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植物学报 |技术方法 2025 , 60 (5) : 846 -853
高效液相色谱法检测水杨酸的优化
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史世肸, 严顺平*
作者信息
  • 华中农业大学生命科学技术学院, 武汉 430070
通讯作者:
Optimization of an High-performance Liquid Chromatography Method for the Determination of Salicylic Acid
Shi Shixi, Yan Shunping*
Affiliations
  • College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
    • Corresponding Author:
    *E-mail:
出版时间: 2025-09-10 doi: 10.11983/CBB25102
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摘要
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植物激素水杨酸(SA)促进植物的抗病性, 但抑制植物生长。植物通过动态调控SA的含量以平衡抗病与生长。高效液相色谱-荧光检测器技术是检测SA含量最常用的方法。该研究优化了流动相的成分、离子浓度、pH值以及检测波长和检测程序。优化后的流动相为10%乙腈、100 mmol∙L-1乙酸钠、pH5.2。优化后的激发光波长为300 nm, 发射光波长为405 nm。优化后的检测程序为: 进样后3.5分钟开始清洗色谱柱, 清洗时间为3.5分钟, 平衡时间为3分钟, 总时长为10分钟。优化后的检测方法显著提高了检测灵敏性、稳定性和高效性。

水杨酸  /  抗病  /  检测方法  /  高效液相色谱  /  优化

INTRODUCTION: The phytohormone salicylic acid (SA) plays multiple important roles in plants, such as disease resistance, seed germination, and leaf senescence. Among these, the role of SA in plant disease resistance is the most studied. Since SA promotes disease resistance at the cost of plant growth, plants need to dynamically regulate the content of SA to balance disease resistance and growth. Therefore, fast and accurate measurement of SA content is a critical basis for plant immunity research.

RATIONALE: High-performance liquid chromatography (HPLC)-fluorescence detector is the most popular method for the quantitative measurement of SA. In order to improve the efficiency and sensitivity of current methods, this study optimized the composition, ion concentration, and pH of the mobile phase, as well as the detection wavelength and detection procedure.

RESULTS: The baseline of the chromatogram was more stable when using acetonitrile instead of methanol in the mobile phase. When the pH of the mobile phase was 5.2, the retention time of SA was the shortest, without interference peak near the SA peak, which was preferred for minimizing the detection time. The higher concentration of sodium acetate (100 mmol∙L-1) in the mobile phase was better than that of lower concentration (20-50 mmol∙L-1). Wavelength scanning revealed that the optimal excitation wavelength was 300 nm and the optimal emission wavelength was 405 nm, under which the highest sensitivity for SA detection was obtained. At a flow rate of 2 mL∙min-1, it took 3.5 min for elution, 3.5 min for column wash, and 3 min for column balance, shortening the measurement time per sample from 50 min to 10 min.

CONCLUSION: These optimizations greatly improved the sensitivity, stability, and efficiency of the SA measurement using HPLC, which will contribute to the plant immunity research.

Optimization of salicylic acid (SA) measurement. (A) SA detection procedure; (B) Chromatogram using the optimized condition. The samples are total SA in Arabidopsis without Psm ES4326 infection. EU: Emission units. The peak labeled in red indicates SA.

salicylic acid  /  disease resistance  /  measurement  /  high-performance liquid chromatography  /  optimization
史世肸, 严顺平. 高效液相色谱法检测水杨酸的优化. 植物学报, 2025 , 60 (5) : 846 -853 . DOI: 10.11983/CBB25102
Shi Shixi, Yan Shunping. Optimization of an High-performance Liquid Chromatography Method for the Determination of Salicylic Acid[J]. Chinese Bulletin of Botany, 2025 , 60 (5) : 846 -853 . DOI: 10.11983/CBB25102
正文
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植物激素水杨酸(salicylic acid, SA)是一种酚类化合物, 在植物体内行使多种功能, 包括抗病反应、种子萌发、气孔开闭及叶片衰老等(Yan and Dong, 2014; Yu et al., 2021)。特别值得一提的是, SA在植物抗病反应中起核心作用(Fu and Dong, 2013; Mishra et al., 2024)。植物体内SA含量降低将显著减弱植物的抗病性(Gaffney et al., 1993; Delaney et al., 1994; Nawrath and Métraux, 1999), 而外源SA处理可显著增强植物的抗病性(Cao et al., 1994)。SA受体NPR1和NPR3/4通过调控大量抗病相关基因的表达来激活抗病反应(Fu et al., 2012; Ding et al., 2018; Kumar et al., 2022)。SA在激活抗病反应的同时会抑制植物的生长(Yu et al., 2022)。因此, 植物需要通过多种机制来动态调控SA的含量。在无病原体侵染时, 植物合成的SA较少; 在病原体侵染后, 植物会增加SA的合成。此外, 植物还可通过葡萄糖基化修饰将游离态SA转换为结合态SA, 即SA葡萄糖苷(SA 2-O-β-D-glucoside, SAG)和SA葡萄糖酯(salicylate glucose ester, SGE), 从而减弱SA对生长的抑制作用(Lim et al., 2002; Song, 2006)。因此, 在研究植物抗病反应时需要同时检测游离态SA和结合态SA。
前人建立了多种检测植物中SA的方法。Raskin等(1989)建立了基于高效液相色谱(high-performance liquid chromatography, HPLC)-荧光检测器检测植物提取物中SA的方法。Zhang等(2017)对该方法进行了改进。Balcke等(2012)建立了超高效液相色谱(ultra- performance liquid chromatography, UPLC)-串联质谱的检测方法, 在检测SA的同时还可检测提取物中茉莉酸和脱落酸等多种激素的含量。这些方法均使用C18反相色谱柱, 而Aboul-Soud等(2004)使用基于离子交换的HPLC法检测SA。Schmelz等(2003)建立了气相色谱-质谱联用(gas chromatography-mass spectrometry, GC-MS)检测方法, 可同时检测包括SA在内的多种植物激素和挥发性有机物。Huang等(2005)建立了基于生物传感器检测SA的方法。DeFraia等(2008)和Marek等(2010)改进了该方法, 使其能够检测不同基因型拟南芥(Arabidopsis thaliana)之间SA水平的差异。叶灿等(2025)进一步改进了该方法, 使其还可检测不同基因型水稻(Oryza sativa)之间SA水平的差异, 并用于高通量筛选水稻SA代谢突变体。
上述SA检测方法各有优缺点。例如, UPLC-串联质谱法的检测灵敏度高, 但是所用仪器昂贵; 基于生物传感器的方法适合高通量检测, 但灵敏度偏低。在这些方法中, HPLC-荧光检测器联用技术应用最为广泛。本研究进一步优化了该方法, 主要包括流动相成分、离子浓度、pH值以及检测波长和检测程序, 提高了检测的灵敏性、稳定性和高效性。
1 材料与方法
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1.1 实验材料
实验材料和仪器试剂包括拟南芥(Arabidopsis thaliana L.) Col-0、丁香假单胞菌(Pseudomonas syringae pv. maculicola, Psm) ES4326、高效液相色谱仪(Waters, Cat No.2695)、荧光检测器(Waters, Cat No. 2475)、色谱柱(Agilent, ZORBAX StableBond C18, 4.6 mm × 250 mm, 5 μm)和SA (Sigma-Aldrich, Cat No. 247588)。
1.2 试剂配方
90 %甲醇: 90 mL 100%甲醇, 加ddH2O定容至100 mL。5%三氯乙酸(trichloroacetic acid, TCA): 5 g TCA, 加ddH2O溶解后定容至100 mL。β-葡萄糖苷酶溶液: 用100 mmol∙L-1乙酸钠(pH5.2)溶解β-葡萄糖苷酶, 浓度为80 units·mL-1。萃取溶剂: 乙酸乙酯:环戊烷:异丙醇=100:99:1 (v/v/v)。样品溶解液: 90% 100 mmol∙L-1乙酸钠(pH5.2)+10%乙腈。SA标准溶液: 1 mg·mL-1 SA溶解于100%甲醇。
1.3 实验方法
1.3.1 拟南芥种植
拟南芥种植条件: 22°C, 相对湿度为70%, 光照强度为120 µmol∙m-2∙s-1, 光周期为12小时光照/12小时黑暗。
1.3.2 病原菌侵染
将丁香假单胞菌Psm ES4326在28°C摇床以200 r∙min-1旋转培养至OD600=0.3。用10 mmol∙L-1 MgCl2溶液稀释至OD600=0.001。用无针注射器注射拟南芥第3、4和5片真叶, 24小时后取样用于提取SA。
1.3.3 拟南芥叶片中SA的提取
称取约0.2 g拟南芥叶片, 加入液氮研磨成粉末。在粉末中加入1 mL 90%甲醇, 涡旋混匀后用超声波震荡提取20分钟。用离心机5 000 ×g离心10分钟, 将上清液转移至2 mL圆底离心管。在沉淀物中加入0.8 mL 100%甲醇, 涡旋混匀后用超声波震荡提取10分钟。用离心机5 000 ×g离心10分钟, 将上清液与第1次提取的上清液合并, 再平均分为2份, 一份用于检测游离态SA, 另一份用于检测总SA。在通风橱中, 使用恒温金属浴50°C加热至样品完全干燥。向其中一份样品加入0.5 mL 5% TCA, 涡旋混匀后用超声波震荡5分钟, 用于检测游离态SA。向另一份样品加入0.2 mL β-葡萄糖苷酶溶液, 涡旋混匀后用超声波震荡5分钟, 并在37°C孵育90分钟, 然后加入0.5 mL 5% TCA, 用于检测总SA。使用萃取溶剂萃取3次, 每次加入0.5 mL萃取溶剂, 涡旋混匀后静置, 待分层后吸出上层有机相, 转移至2 mL离心管。在通风橱中, 使用恒温金属浴50°C加热至样品完全干燥。加入0.2 mL样品溶解液, 涡旋混匀后用超声波震荡5分钟, 并用离心机10 000 ×g离心5分钟。取上清液用0.22 μm微孔过滤器过滤, 将过滤液装入样品瓶。此时样品制备完成, 可进行HPLC检测, 或于-20°C保存。
1.3.4 前人检测SA的实验方法
流动相由乙酸钠(200 mmol∙L-1, pH5.5)和甲醇组成。初始甲醇浓度为3% (v/v), 持续12分钟, 在12.5分钟线性上升至7% (v/v), 并保持38分钟。1分钟后, 恢复初始条件, 系统平衡7分钟后进行下一次进样。整个过程中流速保持在0.8 mL·min-1。使用荧光检测器检测SA, 激发光波长为296 nm, 发射光波长为410 nm (Zhang et al., 2017)。
1.3.5 优化后的参数设置
进样针高度为6 mm (样品瓶使用200 μL内插管)。进样体积为10 μL。柱温箱的温度为25°C。激发光波长为300 nm, 发射光波长为405 nm。流动相的流速为2 mL·min-1。流动相A: 乙酸钠(100 mmol∙L-1, pH5.2)。流动相B: 100%乙腈。梯度洗脱, A泵+B泵总流量2 mL·min-1; 0.0-3.5分钟, 90%A+10%B (洗脱); 3.5- 4.0分钟, 线性变化至80%A+20%B; 4.0-6.5分钟, 80%A+20%B (柱清洗); 6.5-7.0分钟, 线性变化至 90%A+10%B; 7.0-10.0分钟, 90%A+10%B (柱平衡)。设置起始样品对应的样品盘、序号和样品总数, 采用自动进样可实现连续检测。
2 结果与讨论
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2.1 使用前人的方法检测SA
我们先依据Zhang等(2017)的实验方法检测SA标准样品, 发现SA的保留时间为22.2分钟, SA峰比较宽, 且基线不平(图1A), 这不利于计算SA峰面积。当我们用该方法检测拟南芥叶片提取物中的SA时, 发现SA峰的左侧还有1个紧密相邻的峰(图1B), 这对SA峰面积的计算造成了干扰。上述结果表明该实验方法还可继续优化。
2.2 流动相的pH值对SA分离的影响
在HPLC实验中, 甲醇和乙腈是流动相中常用的有机溶剂。与甲醇相比, 乙腈具有更低的极性、黏度、紫外吸收及更强的洗脱能力(Snyder et al., 2012)。因此, 我们用乙腈代替甲醇, 取得了良好的分离效果(图2)。
流动相的pH值也影响SA的分离。SA的pKa为3.01, 根据电解质电离公式pH=pKa-log([HA]/[A-]), 当流动相pH值为4.5时, SA的电离率为96.87%; 当流动相pH值为5.0时, SA的电离率为98.99%; 当流动相pH值为5.5时, SA的电离率为99.68%。因此, 我们测试了这3种不同pH值流动相对SA分离的影响。结果表明, 当流动相pH值为4.5时, SA峰值明显低于其它2种流动相(图2A-C); 当流动相pH值为5.5时, SA峰右侧出现干扰峰(图2C); 当流动相pH值为5.0时, SA的峰值与流动相pH值为5.5时相当, 且无干扰峰(图2B)。
我们选取被Psm ES4326侵染24小时后的拟南芥叶片测定总SA (图2A-C)。为进一步优化流动相的pH值, 我们测试了不同pH值流动相在分离未被Psm ES4326侵染拟南芥的总SA的差异。结果显示, 当流动相pH值为4.75时, SA峰和右侧相邻的峰重叠(图2D); 当流动相pH值为5.0时, SA峰右侧的干扰峰消失, 左侧出现互不干扰的峰(图2E); 当流动相pH值为5.2时, SA峰左侧的峰进一步远离SA峰, 保留时间相差1分钟(图2F)。这表明pH5.2的流动相比pH5.0的具有更好的分离效果。
2.3 流动相中乙酸钠浓度对SA分离的影响
流动相中的离子浓度会影响物质的分离。上述实验使用的流动相的乙酸钠浓度为50 mmol∙L-1。为进一步测试乙酸钠浓度对SA分离的影响, 我们分别用含有20和100 mmol∙L-1乙酸钠的流动相进行实验。当流动相中含50 mmol∙L-1乙酸钠时, SA峰的左侧还有1个峰(图2E)。当流动相中含20 mmol∙L-1乙酸钠时, SA峰与相邻峰无法区分(图3A)。当流动相中含100 mmol∙L-1乙酸钠时, SA峰与相邻峰区分更加明显(图3B)。这表明含100 mmol∙L-1乙酸钠的流动相比含50 mmol∙L-1乙酸钠的具有更好的分离效果。
2.4 优化SA的检测波长
为了提高检测的灵敏度, 我们优化了SA的检测波长。前人的研究(Zhang et al., 2017)使用激发光波长为296 nm, 发射光波长为410 nm。首先, 我们在296 nm激发光下扫描了350-450 nm发射光的强度, 发现最优的发射光波长为395 nm (荧光信号强度为9.5) (图4A, B)。其次, 我们扫描了280-320 nm的激发光, 并检测了395 nm发射光的强度, 发现最优激发光波长为300 nm (荧光信号强度为10.5) (图4C, D)。最后, 我们在300 nm激发光下扫描了375-415 nm发射光的强度, 发现最优发射光波长为405 nm (荧光信号强度为11.4) (图4E, F)。上述实验结果表明, 优化检测波长可以大幅提高检测SA的灵敏度。
2.5 优化SA检测程序
上述实验设置柱清洗开始时间为进样后6.0分钟, 清洗3分钟, 然后平衡3分钟, 总时长为12分钟。由于柱体积和输液管路体积的存在, 柱清洗体现在色谱图上的时间为进样后8.0分钟, 比设置的柱清洗开始时间滞后2分钟, 但与SA峰相隔3分钟(图3B)。因此, 为了提高实验效率, 我们重新优化了检测程序, 将柱清洗开始时间提前至进样后3.5分钟, 柱清洗时间增加至3.5分钟, 平衡时间保持不变(3分钟), 使总时长缩短为10分钟(图5A)。结果表明, 该检测程序不仅能很好地分离SA (图5B), 也有助于更好地清洗色谱柱。
2.6 制作SA标准曲线
为了测试优化效果, 我们检测了0.01-10 µg∙mL-1 SA标准样品, 并制作SA标准曲线(图6)。该曲线的R2为0.999 94, 表明优化的检测方法能够准确测定SA的浓度。
2.7 讨论
高效液相色谱-荧光检测器技术是检测SA最常用的方法。本研究在Zhang等(2017)的基础上对检测方法进行优化。在Zhang等(2017)的方法中, 流动相为甲醇、200 mmol∙L-1乙酸钠, pH5.5, SA保留时间为22.2分钟, SA峰与干扰峰相邻, 基线波动大, 总检测时长达50分钟(图1B)。Zhang等(2017)的方法不利于SA的精确检测, 也不利于高通量检测。本研究将流动相优化为10%乙腈、100 mmol∙L-1乙酸钠, pH5.2, 这使得SA保留时间缩短至4.5分钟, 有效避免了干扰峰(图3B)。结合检测程序的优化, 将单个样品检测时间缩短至10分钟(图5B), 显著提高了检测效率。
本研究还优化了荧光检测的激发光波长和发射光波长。在Zhang等(2017)的方法中, 激发光波长为296 nm, 发射光波长为410 nm, 检测到EU值约为9 (图4B)。优化后激发光波长为300 nm, 发射光波长为405 nm, 检测到EU值约为11.4 (图4F), 灵敏度提高了约25%, 有利于精确测定SA浓度。
本研究中确定的流动相组成和检测波长具有通用性, 可应用于其它HPLC系统。需要注意的是, 在使用其它HPLC系统时, 流动相的流速和清洗时间需要继续优化, 以确保柱压稳定, 但可能会影响检测时间。
作者贡献声明
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史世肸: 设计并完成实验, 分析数据, 撰写论文; 严顺平: 构思并设计实验, 修改论文。
基金
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  • 国家重点研发计划(2023YFF1001301)
  • 国家自然科学基金(32270306)
文献
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2025年第60卷第5期
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doi: 10.11983/CBB25102
  • 接收时间：2025-06-04
  • 首发时间：2026-10-22
  • 出版时间：2025-09-10
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  • 收稿日期：2025-06-04
  • 录用日期：2025-07-08
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国家自然科学基金(32270306)
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    华中农业大学生命科学技术学院, 武汉 430070

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species		 占总种数比例
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total species (%)		 属
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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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