Article(id=1217529313067451125, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250213007, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1739376000000, receivedDateStr=2025-02-13, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768211208835, onlineDateStr=2026-01-12, pubDate=1752508800000, pubDateStr=2025-07-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768211208835, onlineIssueDateStr=2026-01-12, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768211208835, creator=13701087609, updateTime=1768211208835, updator=13701087609, issue=Issue{id=1217529305693864468, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='13', pageStart='1', pageEnd='320', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768211207077, creator=13701087609, updateTime=1768212057891, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217532874337730593, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217532874337730594, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1217529305693864468, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=144, endPage=154, ext={EN=ArticleExt(id=1217529314103444246, articleId=1217529313067451125, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Determination of the content of 5 kinds of caffeoylquinic acid in Lonicera japonica Thunb. from Sandao real estate area by quantitative analysis of multi-components by single marker, columnId=1217529307778438039, journalTitle=Journal of Food Safety & Quality, columnName=Highlight: Application of Modern Analysis Instrument in Food Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a method to simultaneously determine the content of 5 kinds of components of Lonicera japonica Thunb. herbs from Sandao real estate area (neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C) by quantitative analysis of multi-components by single marker. Methods Phenomenex Synergi Hydro-RP80A (250 mm×4.6 mm, 4 μm) column was used with acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) as mobile phase, the detection wavelength was 327 nm, the column temperature was 20 ℃, and the flow rate was 0.7 mL/min. The injection volume was 2 µL. Results The linear ranges of neochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C were 0.41-16.40, 7.22-289.00, 0.21-8.50, 1.65-66.00 and 0.37-14.80 μg/mL (r≥0.9993), respectively. The average recovery rate was 95.25%-100.42%, and the relative standard deviation (RSD) of precision, repeatability and stability were less than 2.0%. The content of neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in 25 batches of samples was determined by external standard method, and there was no significant difference between the measured values and the calculated values of quantitative analysis of multi-components by single marker method. Conclusion This method is accurate, reliable and simple for the simultaneous determination of the content of 5 kinds of compounds in Lonicera japonica Thunb., which solves the bottleneck problem of lack of reference substances in the quality control of Lonicera japonica Thunb.. The content of caffeoylquinic acid in Lonicera japonica Thunb. from different origins varies greatly, and it is necessary to further strengthen the processing of Lonicera japonica Thunb.. This study provides an important basis for the identification, quality evaluation and formulation of quality standards of Lonicera japonica Thunb..

, correspAuthors=Zheng-Gang HUA, 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=Zhen-Zhen QI, Bing-Ya ZHU, Zheng-Gang HUA), CN=ArticleExt(id=1217529315080717152, articleId=1217529313067451125, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=一测多评法测定三大道地产区金银花中5种咖啡酰奎宁酸含量, columnId=1217529307954598811, journalTitle=食品安全质量检测学报, columnName=本期重点:现代分析仪器在食品检测中的应用, runingTitle=null, highlight=null, articleAbstract=

目的 建立一测多评法同时测定三大道地产区金银花药材中新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C 5种成分的含量。方法 采用Phenomenex Synergi Hydro-RP80A (250 mm×4.6 mm, 4 µm)色谱柱, 以乙腈(A)-0.1%磷酸水溶液(B)为流动相, 检测波长327 nm, 柱温20 ℃, 流速0.7 mL/min; 进样量2 µL。结果 新绿原酸、绿原酸、异绿原酸B、异绿原酸A、异绿原酸C的线性范围分别为0.41~16.40、7.22~289.00、0.21~8.50、1.65~66.00、0.37~14.80 μg/mL (r≥0.9993), 平均回收率为95.25%~100.42%, 精密度、重复性、稳定性相对标准偏差(relative standard deviation, RSD)均小于等于2.0%; 利用外标法测25批样品中新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C的含量, 其测定值与一测多评法计算值无显著性差异。结论 本方法同时测定金银花中5个化合物含量准确可靠且方法简便, 解决了金银花质量控制中缺乏对照品这一瓶颈问题。不同产地的金银花中咖啡酰奎宁酸类的含量差别较大。本研究为金银花的鉴别、品质评价、制定质量标准等提供了重要的依据。

, correspAuthors=华正罡, authorNote=null, correspAuthorsNote=
*华正罡(1983—), 男, 主任技师, 硕士, 主要研究方向为食品药品标准化研究。E-mail:
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祁珍祯(1989—), 女, 副主任技师, 硕士, 主要研究方向为食品药品检测与分析。E-mail:

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注: a. 新绿原酸; b. 绿原酸; c. 异绿原酸A; d. 异绿原酸B; e. 异绿原酸C。

, figureFileSmall=lpEkRRiR18maB/ccDLIhVQ==, figureFileBig=2bpd/5g/jDI5qzlFxczuHA==, tableContent=null), ArticleFig(id=1217901256740360914, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Fig.2, caption=High performance liquid chromatograms of 5 kinds of reference substances Lonicera japonica Thunb. sample solution, figureFileSmall=JYAzhaMK2pY6KRRr2R49Uw==, figureFileBig=F9z7Gb5DS8fd19jYG1d7fw==, tableContent=null), ArticleFig(id=1217901256845218526, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=图2, caption=5种对照品(a)和金银花样品溶液(b)的高效液相色谱图

注: 1. 新绿原酸; 2. 绿原酸; 3. 异绿原酸B; 4. 异绿原酸A; 5. 异绿原酸C。

, figureFileSmall=JYAzhaMK2pY6KRRr2R49Uw==, figureFileBig=F9z7Gb5DS8fd19jYG1d7fw==, tableContent=null), ArticleFig(id=1217901256958464744, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Table 1, caption=

Sources of Lonicera japonica Thunb. samples

, figureFileSmall=null, figureFileBig=null, tableContent=
样品号 产地 样品号 产地 样品号 产地
S1 山东平邑县1 S10 河南新密市1 S19 山东临沂市6
S2 山东平邑县2 S11 河南新密市2 S20 山东临沂市7
S3 山东平邑县3 S12 河南新密市3 S21 河南封丘县1
S4 山东平邑县4 S13 河南新密市4 S22 河南封丘县2
S5 山东平邑县5 S14 山东临沂市1 S23 河南封丘县3
S6 河北巨鹿县1 S15 山东临沂市2 S24 河南封丘县4
S7 河北巨鹿县2 S16 山东临沂市3 S25 河南封丘县5
S8 河北巨鹿县3 S17 山东临沂市4
S9 河北巨鹿县4 S18 山东临沂市5
), ArticleFig(id=1217901258371945207, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=表1, caption=

金银花样品来源

, figureFileSmall=null, figureFileBig=null, tableContent=
样品号 产地 样品号 产地 样品号 产地
S1 山东平邑县1 S10 河南新密市1 S19 山东临沂市6
S2 山东平邑县2 S11 河南新密市2 S20 山东临沂市7
S3 山东平邑县3 S12 河南新密市3 S21 河南封丘县1
S4 山东平邑县4 S13 河南新密市4 S22 河南封丘县2
S5 山东平邑县5 S14 山东临沂市1 S23 河南封丘县3
S6 河北巨鹿县1 S15 山东临沂市2 S24 河南封丘县4
S7 河北巨鹿县2 S16 山东临沂市3 S25 河南封丘县5
S8 河北巨鹿县3 S17 山东临沂市4
S9 河北巨鹿县4 S18 山东临沂市5
), ArticleFig(id=1217901258694906629, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Table 2, caption=

Comparison table of peak areas for different extraction solvents

, figureFileSmall=null, figureFileBig=null, tableContent=
成分 甲醇提取峰面积 乙醇提取峰面积 50%甲醇提取峰面积 75%甲醇提取峰面积 纯水提取峰面积
新绿原酸 30.82 26.35 37.84 36.57 18.25
绿原酸 803.25 789.52 873.84 876.31 543.16
异绿原酸B 20.48 20.83 21.41 18.25 17.35
异绿原酸A 563.27 594.62 606.45 601.43 487.90
异绿原酸C 75.46 79.35 83.32 83.11 53.99
), ArticleFig(id=1217901258812347150, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=表2, caption=

不同提取溶剂峰面积对照表

, figureFileSmall=null, figureFileBig=null, tableContent=
成分 甲醇提取峰面积 乙醇提取峰面积 50%甲醇提取峰面积 75%甲醇提取峰面积 纯水提取峰面积
新绿原酸 30.82 26.35 37.84 36.57 18.25
绿原酸 803.25 789.52 873.84 876.31 543.16
异绿原酸B 20.48 20.83 21.41 18.25 17.35
异绿原酸A 563.27 594.62 606.45 601.43 487.90
异绿原酸C 75.46 79.35 83.32 83.11 53.99
), ArticleFig(id=1217901258933981976, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Table 3, caption=

Comparison table of peak area of different extraction methods and solvent dosage

, figureFileSmall=null, figureFileBig=null, tableContent=
成分 10 mL 50%甲醇提取峰面积 20 mL 50%甲醇提取峰面积 40 mL 50%甲醇提取峰面积 超声提取峰
面积
冷浸提提取峰
面积
回流提取峰
面积
新绿原酸 30.66 37.84 38.25 37.68 33.16 38.12
绿原酸 705.32 873.84 872.42 871.85 756.09 872.94
异绿原酸B 16.81 21.41 20.69 20.93 16.53 21.26
异绿原酸A 509.10 606.45 606.81 606.89 592.37 605.88
异绿原酸C 74.56 83.32 84.02 83.66 72.15 82.98
), ArticleFig(id=1217901259072394022, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=表3, caption=

不同提取方式及溶剂用量峰面积对照表

, figureFileSmall=null, figureFileBig=null, tableContent=
成分 10 mL 50%甲醇提取峰面积 20 mL 50%甲醇提取峰面积 40 mL 50%甲醇提取峰面积 超声提取峰
面积
冷浸提提取峰
面积
回流提取峰
面积
新绿原酸 30.66 37.84 38.25 37.68 33.16 38.12
绿原酸 705.32 873.84 872.42 871.85 756.09 872.94
异绿原酸B 16.81 21.41 20.69 20.93 16.53 21.26
异绿原酸A 509.10 606.45 606.81 606.89 592.37 605.88
异绿原酸C 74.56 83.32 84.02 83.66 72.15 82.98
), ArticleFig(id=1217901259185640242, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Table 4, caption=

Calibration curves and mass concentration ranges of 5 kinds of components

, figureFileSmall=null, figureFileBig=null, tableContent=
成分 校准曲线 相关系数(r) 质量浓度范围
/(μg/mL)
新绿原酸 Y=8.0348X+0.5741 0.9997 0.41~16.40
绿原酸 Y=8.2367X+6.4705 0.9999 7.22~289.00
异绿原酸B Y=8.9177X+2.6588 0.9995 0.21~8.50
异绿原酸A Y=10.44X+2.8075 0.9993 1.65~66.00
异绿原酸C Y=8.709X-1.8738 0.9998 0.37~14.80
), ArticleFig(id=1217901259298886461, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=表4, caption=

5种成分的校准曲线和质量浓度范围

, figureFileSmall=null, figureFileBig=null, tableContent=
成分 校准曲线 相关系数(r) 质量浓度范围
/(μg/mL)
新绿原酸 Y=8.0348X+0.5741 0.9997 0.41~16.40
绿原酸 Y=8.2367X+6.4705 0.9999 7.22~289.00
异绿原酸B Y=8.9177X+2.6588 0.9995 0.21~8.50
异绿原酸A Y=10.44X+2.8075 0.9993 1.65~66.00
异绿原酸C Y=8.709X-1.8738 0.9998 0.37~14.80
), ArticleFig(id=1217901259416326982, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Table 5, caption=

Results of intraday precision and daytime precision

, figureFileSmall=null, figureFileBig=null, tableContent=
成分 低浓度日内精密度 中浓度日内精密度 高浓度日内精密度 日间精密度
峰面积平均值 RSDs/% 峰面积平均值 RSDs/% 峰面积平均值 RSDs/% 峰面积平均值 RSDs/%
新绿原酸 7.35 1.66 34.68 1.56 131.16 1.48 34.84 1.37
绿原酸 306.50 1.29 1480.50 0.11 5918.30 0.08 1480.00 0.12
异绿原酸B 6.93 1.75 21.65 1.79 74.53 1.10 21.29 1.63
异绿原酸A 175.80 1.38 856.90 0.98 3464.00 0.09 858.40 0.51
异绿原酸C 31.28 1.81 162.20 0.94 648.10 0.21 161.80 0.79
), ArticleFig(id=1217901259542156108, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=表5, caption=

日内及日间精密度结果

, figureFileSmall=null, figureFileBig=null, tableContent=
成分 低浓度日内精密度 中浓度日内精密度 高浓度日内精密度 日间精密度
峰面积平均值 RSDs/% 峰面积平均值 RSDs/% 峰面积平均值 RSDs/% 峰面积平均值 RSDs/%
新绿原酸 7.35 1.66 34.68 1.56 131.16 1.48 34.84 1.37
绿原酸 306.50 1.29 1480.50 0.11 5918.30 0.08 1480.00 0.12
异绿原酸B 6.93 1.75 21.65 1.79 74.53 1.10 21.29 1.63
异绿原酸A 175.80 1.38 856.90 0.98 3464.00 0.09 858.40 0.51
异绿原酸C 31.28 1.81 162.20 0.94 648.10 0.21 161.80 0.79
), ArticleFig(id=1217901259688956754, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Table 6, caption=

Influence of various factors on the relative retention value

, figureFileSmall=null, figureFileBig=null, tableContent=
仪器 色谱柱 相对保留值
新绿原酸 绿原酸 异绿原酸B 异绿原酸A 异绿原酸C
Agilent 1260 Phenomenex Synergi Hydro-RP80A 0.73 1.00 2.53 2.75 2.93
InfinityLab Poroshell 120 EC-C18 0.71 1.00 2.55 2.78 2.91
Accucore™ XL C18 0.72 1.00 2.56 2.76 2.87
Waters e2695 Phenomenex Synergi Hydro-RP80A 0.71 1.00 2.52 2.68 2.89
InfinityLab Poroshell 120 EC-C18 0.70 1.00 2.55 2.72 2.92
Accucore™ XL C18 0.73 1.00 2.54 2.75 2.86
平均值 0.72 1.00 2.54 2.74 2.89
RSDs/% 1.69 0.00 0.62 1.18 0.91
), ArticleFig(id=1217901259827368797, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=表6, caption=

各种因素对相对保留值的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
仪器 色谱柱 相对保留值
新绿原酸 绿原酸 异绿原酸B 异绿原酸A 异绿原酸C
Agilent 1260 Phenomenex Synergi Hydro-RP80A 0.73 1.00 2.53 2.75 2.93
InfinityLab Poroshell 120 EC-C18 0.71 1.00 2.55 2.78 2.91
Accucore™ XL C18 0.72 1.00 2.56 2.76 2.87
Waters e2695 Phenomenex Synergi Hydro-RP80A 0.71 1.00 2.52 2.68 2.89
InfinityLab Poroshell 120 EC-C18 0.70 1.00 2.55 2.72 2.92
Accucore™ XL C18 0.73 1.00 2.54 2.75 2.86
平均值 0.72 1.00 2.54 2.74 2.89
RSDs/% 1.69 0.00 0.62 1.18 0.91
), ArticleFig(id=1217901259957392225, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Table 7, caption=

Relative correction factor and its RSDs

, figureFileSmall=null, figureFileBig=null, tableContent=
次数 新绿
原酸
绿原酸 异绿
原酸B
异绿
原酸A
异绿
原酸C
1 0.98 1.00 1.21 1.27 1.04
2 0.98 1.00 1.21 1.27 1.03
3 0.99 1.00 1.19 1.26 1.02
4 0.99 1.00 1.20 1.27 1.03
5 0.99 1.00 1.19 1.27 1.03
6 0.99 1.00 1.21 1.27 1.04
平均值 0.99 1.00 1.20 1.27 1.03
RSDs/% 0.52 0.00 1.52 0.32 0.73
), ArticleFig(id=1217901260087415657, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=表7, caption=

相对校正因子及其RSDs

, figureFileSmall=null, figureFileBig=null, tableContent=
次数 新绿
原酸
绿原酸 异绿
原酸B
异绿
原酸A
异绿
原酸C
1 0.98 1.00 1.21 1.27 1.04
2 0.98 1.00 1.21 1.27 1.03
3 0.99 1.00 1.19 1.26 1.02
4 0.99 1.00 1.20 1.27 1.03
5 0.99 1.00 1.19 1.27 1.03
6 0.99 1.00 1.21 1.27 1.04
平均值 0.99 1.00 1.20 1.27 1.03
RSDs/% 0.52 0.00 1.52 0.32 0.73
), ArticleFig(id=1217901260217439091, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=EN, label=Table 8, caption=

Comparison of the content of 5 kinds of caffeoylquinic acid in Lonicera japonica Thunb. Samples (%)

, figureFileSmall=null, figureFileBig=null, tableContent=
样品号 测定方法 新绿原酸 绿原酸 异绿原酸 B 异绿原酸A 异绿原酸C
S1 一测多评法 0.1143 - 0.0676 1.1255 0.3009
外标法 0.1154 2.1356 0.0694 1.1307 0.2996
RSDs 0.68 - 1.86 0.33 0.31
S2 一测多评法 0.1161 - 0.0546 0.8742 0.1880
外标法 0.1172 2.2900 0.0549 0.8765 0.1887
RSDs 0.67 - 0.39 0.19 0.26
S3 一测多评法 0.1391 - 0.0490 1.0091 0.2142
外标法 0.1408 1.9623 0.0488 1.0138 0.2146
RSDs 0.86 - 0.29 0.33 0.13
S4 一测多评法 0.1076 - 0.0495 0.7974 0.2472
外标法 0.1086 1.9099 0.0494 0.7457 0.2313
RSDs 0.86 - 0.14 4.74 4.70
S5 一测多评法 0.1197 - 0.0371 0.8305 0.2008
外标法 0.1209 2.1012 0.0354 0.8329 0.2015
RSDs 0.71 - 3.32 0.20 0.25
S6 一测多评法 0.1250 - 0.0256 1.0723 0.2011
外标法 0.1262 2.5859 0.0245 1.0756 0.2015
RSDs 0.68 - 3.11 0.22 0.14
S7 一测多评法 0.0801 - 0.0504 0.5833 0.1801
外标法 0.0805 1.9026 0.0503 0.5840 0.1812
RSDs 0.35 - 0.14 0.08 0.43
S8 一测多评法 0.1258 - 0.0623 1.4253 0.3425
外标法 0.1271 2.1696 0.0635 1.4332 0.3405
RSDs 0.73 - 1.35 0.39 0.41
S9 一测多评法 0.0890 - 0.0588 0.7642 0.1579
外标法 0.0896 2.0009 0.0597 0.7664 0.1593
RSDs 0.48 - 1.07 0.20 0.62
S10 一测多评法 0.0706 - 0.0390 0.8295 0.1628
外标法 0.0707 2.2636 0.0376 0.8316 0.1639
RSDs 0.10 - 2.58 0.18 0.48
S11 一测多评法 0.1012 - 0.0551 1.1803 0.2623
外标法 0.1019 2.6326 0.0555 1.1843 0.2613
RSDs/% 0.49 - 0.51 0.24 0.27
S12 一测多评法 0.1233 - 0.0486 1.0059 0.2523
外标法 0.1246 2.2548 0.0482 1.0095 0.2518
RSDs 0.74 - 0.58 0.25 0.14
S13 一测多评法 0.0862 - 0.0321 1.2871 0.2041
外标法 0.0866 2.7784 0.0307 1.2916 0.2044
RSDs 0.33 - 3.15 0.25 0.10
S14 一测多评法 0.0901 - 0.0595 1.1530 0.2418
外标法 0.0906 2.3354 0.0604 1.1576 0.2415
RSDs 0.39 - 1.06 0.28 0.09
S15 一测多评法 0.0791 - 0.0497 1.2249 0.2182
外标法 0.0795 1.9765 0.0494 1.2316 0.2185
RSDs 0.36 - 0.43 0.39 0.10
S16 一测多评法 0.0912 - 0.0426 1.1396 0.1931
外标法 0.0919 2.0855 0.0416 1.1451 0.1937
RSDs 0.54 - 1.68 0.34 0.22
S17 一测多评法 0.0968 - 0.0409 0.8777 0.2463
外标法 0.0976 2.0160 0.0398 0.8809 0.2461
RSDs 0.58 - 1.93 0.26 0.06
S18 一测多评法 0.1142 - 0.0620 0.8408 0.2809
外标法 0.1153 2.2413 0.0632 0.8430 0.2799
RSDs 0.68 - 1.36 0.18 0.25
S19 一测多评法 0.1106 - 0.0501 0.9756 0.2298
外标法 0.1116 2.0887 0.0500 0.9795 0.2299
RSDs 0.64 - 0.14 0.28 0.03
S20 一测多评法 0.0900 - 0.0594 0.8027 0.2211
外标法 0.0907 1.7333 0.0605 0.8062 0.2217
RSDs 0.55 - 1.30 0.31 0.19
S21 一测多评法 0.0696 - 0.0500 1.2694 0.2496
外标法 0.0698 2.0144 0.0499 1.2765 0.2493
RSDs 0.20 - 0.14 0.39 0.09
S22 一测多评法 0.1098 - 0.0554 0.8976 0.2630
外标法 0.1109 2.0111 0.0559 0.9010 0.2625
RSDs 0.70 - 0.64 0.27 0.13
S23 一测多评法 0.1058 - 0.0724 0.9439 0.3244
外标法 0.1067 2.1920 0.0748 0.9472 0.3226
RSDs 0.60 - 2.31 0.25 0.39
S24 一测多评法 0.0627 - 0.0320 0.6279 0.1228
外标法 0.0628 1.8482 0.0315 0.6291 0.1248
RSDs 0.11 - 1.11 0.14 1.14
S25 一测多评法 0.1007 - 0.0354 0.9582 0.1935
外标法 0.1015 2.0017 0.0335 0.9623 0.1943
RSDs 0.56 - 3.90 0.30 0.29
), ArticleFig(id=1217901260368434046, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1217529313067451125, language=CN, label=表8, caption=

金银花样品中5种咖啡酰奎宁酸的含量测定比对(%)

, figureFileSmall=null, figureFileBig=null, tableContent=
样品号 测定方法 新绿原酸 绿原酸 异绿原酸 B 异绿原酸A 异绿原酸C
S1 一测多评法 0.1143 - 0.0676 1.1255 0.3009
外标法 0.1154 2.1356 0.0694 1.1307 0.2996
RSDs 0.68 - 1.86 0.33 0.31
S2 一测多评法 0.1161 - 0.0546 0.8742 0.1880
外标法 0.1172 2.2900 0.0549 0.8765 0.1887
RSDs 0.67 - 0.39 0.19 0.26
S3 一测多评法 0.1391 - 0.0490 1.0091 0.2142
外标法 0.1408 1.9623 0.0488 1.0138 0.2146
RSDs 0.86 - 0.29 0.33 0.13
S4 一测多评法 0.1076 - 0.0495 0.7974 0.2472
外标法 0.1086 1.9099 0.0494 0.7457 0.2313
RSDs 0.86 - 0.14 4.74 4.70
S5 一测多评法 0.1197 - 0.0371 0.8305 0.2008
外标法 0.1209 2.1012 0.0354 0.8329 0.2015
RSDs 0.71 - 3.32 0.20 0.25
S6 一测多评法 0.1250 - 0.0256 1.0723 0.2011
外标法 0.1262 2.5859 0.0245 1.0756 0.2015
RSDs 0.68 - 3.11 0.22 0.14
S7 一测多评法 0.0801 - 0.0504 0.5833 0.1801
外标法 0.0805 1.9026 0.0503 0.5840 0.1812
RSDs 0.35 - 0.14 0.08 0.43
S8 一测多评法 0.1258 - 0.0623 1.4253 0.3425
外标法 0.1271 2.1696 0.0635 1.4332 0.3405
RSDs 0.73 - 1.35 0.39 0.41
S9 一测多评法 0.0890 - 0.0588 0.7642 0.1579
外标法 0.0896 2.0009 0.0597 0.7664 0.1593
RSDs 0.48 - 1.07 0.20 0.62
S10 一测多评法 0.0706 - 0.0390 0.8295 0.1628
外标法 0.0707 2.2636 0.0376 0.8316 0.1639
RSDs 0.10 - 2.58 0.18 0.48
S11 一测多评法 0.1012 - 0.0551 1.1803 0.2623
外标法 0.1019 2.6326 0.0555 1.1843 0.2613
RSDs/% 0.49 - 0.51 0.24 0.27
S12 一测多评法 0.1233 - 0.0486 1.0059 0.2523
外标法 0.1246 2.2548 0.0482 1.0095 0.2518
RSDs 0.74 - 0.58 0.25 0.14
S13 一测多评法 0.0862 - 0.0321 1.2871 0.2041
外标法 0.0866 2.7784 0.0307 1.2916 0.2044
RSDs 0.33 - 3.15 0.25 0.10
S14 一测多评法 0.0901 - 0.0595 1.1530 0.2418
外标法 0.0906 2.3354 0.0604 1.1576 0.2415
RSDs 0.39 - 1.06 0.28 0.09
S15 一测多评法 0.0791 - 0.0497 1.2249 0.2182
外标法 0.0795 1.9765 0.0494 1.2316 0.2185
RSDs 0.36 - 0.43 0.39 0.10
S16 一测多评法 0.0912 - 0.0426 1.1396 0.1931
外标法 0.0919 2.0855 0.0416 1.1451 0.1937
RSDs 0.54 - 1.68 0.34 0.22
S17 一测多评法 0.0968 - 0.0409 0.8777 0.2463
外标法 0.0976 2.0160 0.0398 0.8809 0.2461
RSDs 0.58 - 1.93 0.26 0.06
S18 一测多评法 0.1142 - 0.0620 0.8408 0.2809
外标法 0.1153 2.2413 0.0632 0.8430 0.2799
RSDs 0.68 - 1.36 0.18 0.25
S19 一测多评法 0.1106 - 0.0501 0.9756 0.2298
外标法 0.1116 2.0887 0.0500 0.9795 0.2299
RSDs 0.64 - 0.14 0.28 0.03
S20 一测多评法 0.0900 - 0.0594 0.8027 0.2211
外标法 0.0907 1.7333 0.0605 0.8062 0.2217
RSDs 0.55 - 1.30 0.31 0.19
S21 一测多评法 0.0696 - 0.0500 1.2694 0.2496
外标法 0.0698 2.0144 0.0499 1.2765 0.2493
RSDs 0.20 - 0.14 0.39 0.09
S22 一测多评法 0.1098 - 0.0554 0.8976 0.2630
外标法 0.1109 2.0111 0.0559 0.9010 0.2625
RSDs 0.70 - 0.64 0.27 0.13
S23 一测多评法 0.1058 - 0.0724 0.9439 0.3244
外标法 0.1067 2.1920 0.0748 0.9472 0.3226
RSDs 0.60 - 2.31 0.25 0.39
S24 一测多评法 0.0627 - 0.0320 0.6279 0.1228
外标法 0.0628 1.8482 0.0315 0.6291 0.1248
RSDs 0.11 - 1.11 0.14 1.14
S25 一测多评法 0.1007 - 0.0354 0.9582 0.1935
外标法 0.1015 2.0017 0.0335 0.9623 0.1943
RSDs 0.56 - 3.90 0.30 0.29
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一测多评法测定三大道地产区金银花中5种咖啡酰奎宁酸含量
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祁珍祯 , 朱冰雅 , 华正罡 *
食品安全质量检测学报 | 本期重点:现代分析仪器在食品检测中的应用 2025,16(13): 144-154
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食品安全质量检测学报 | 本期重点:现代分析仪器在食品检测中的应用 2025, 16(13): 144-154
一测多评法测定三大道地产区金银花中5种咖啡酰奎宁酸含量
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祁珍祯 , 朱冰雅, 华正罡*
作者信息
  • 辽宁省疾病预防控制中心, 辽宁省空气雾霾与人群健康监测重点实验室, 沈阳 110005
  • 祁珍祯(1989—), 女, 副主任技师, 硕士, 主要研究方向为食品药品检测与分析。E-mail:

通讯作者:

*华正罡(1983—), 男, 主任技师, 硕士, 主要研究方向为食品药品标准化研究。E-mail:
Determination of the content of 5 kinds of caffeoylquinic acid in Lonicera japonica Thunb. from Sandao real estate area by quantitative analysis of multi-components by single marker
Zhen-Zhen QI , Bing-Ya ZHU, Zheng-Gang HUA*
Affiliations
  • Liaoning Provincial Center for Disease Control and Prevention, Liaoning Provincial Key Laboratory of Air Haze and Population Health Monitoring, Shenyang 110005, China
出版时间: 2025-07-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250213007
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目的 建立一测多评法同时测定三大道地产区金银花药材中新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C 5种成分的含量。方法 采用Phenomenex Synergi Hydro-RP80A (250 mm×4.6 mm, 4 µm)色谱柱, 以乙腈(A)-0.1%磷酸水溶液(B)为流动相, 检测波长327 nm, 柱温20 ℃, 流速0.7 mL/min; 进样量2 µL。结果 新绿原酸、绿原酸、异绿原酸B、异绿原酸A、异绿原酸C的线性范围分别为0.41~16.40、7.22~289.00、0.21~8.50、1.65~66.00、0.37~14.80 μg/mL (r≥0.9993), 平均回收率为95.25%~100.42%, 精密度、重复性、稳定性相对标准偏差(relative standard deviation, RSD)均小于等于2.0%; 利用外标法测25批样品中新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C的含量, 其测定值与一测多评法计算值无显著性差异。结论 本方法同时测定金银花中5个化合物含量准确可靠且方法简便, 解决了金银花质量控制中缺乏对照品这一瓶颈问题。不同产地的金银花中咖啡酰奎宁酸类的含量差别较大。本研究为金银花的鉴别、品质评价、制定质量标准等提供了重要的依据。

金银花  /  一测多评  /  咖啡酰奎宁酸  /  质量控制

Objective To establish a method to simultaneously determine the content of 5 kinds of components of Lonicera japonica Thunb. herbs from Sandao real estate area (neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C) by quantitative analysis of multi-components by single marker. Methods Phenomenex Synergi Hydro-RP80A (250 mm×4.6 mm, 4 μm) column was used with acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) as mobile phase, the detection wavelength was 327 nm, the column temperature was 20 ℃, and the flow rate was 0.7 mL/min. The injection volume was 2 µL. Results The linear ranges of neochlorogenic acid, chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C were 0.41-16.40, 7.22-289.00, 0.21-8.50, 1.65-66.00 and 0.37-14.80 μg/mL (r≥0.9993), respectively. The average recovery rate was 95.25%-100.42%, and the relative standard deviation (RSD) of precision, repeatability and stability were less than 2.0%. The content of neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in 25 batches of samples was determined by external standard method, and there was no significant difference between the measured values and the calculated values of quantitative analysis of multi-components by single marker method. Conclusion This method is accurate, reliable and simple for the simultaneous determination of the content of 5 kinds of compounds in Lonicera japonica Thunb., which solves the bottleneck problem of lack of reference substances in the quality control of Lonicera japonica Thunb.. The content of caffeoylquinic acid in Lonicera japonica Thunb. from different origins varies greatly, and it is necessary to further strengthen the processing of Lonicera japonica Thunb.. This study provides an important basis for the identification, quality evaluation and formulation of quality standards of Lonicera japonica Thunb..

Lonicera japonica Thunb.  /  quantitative analysis of multi-components by single marker  /  caffeoylquinic acid  /  quality control
祁珍祯, 朱冰雅, 华正罡. 一测多评法测定三大道地产区金银花中5种咖啡酰奎宁酸含量. 食品安全质量检测学报, 2025 , 16 (13) : 144 -154 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250213007
Zhen-Zhen QI, Bing-Ya ZHU, Zheng-Gang HUA. Determination of the content of 5 kinds of caffeoylquinic acid in Lonicera japonica Thunb. from Sandao real estate area by quantitative analysis of multi-components by single marker[J]. Journal of Food Safety & Quality, 2025 , 16 (13) : 144 -154 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250213007
金银花为忍冬科植物忍冬Lonicera japonica Thunb.的干燥花蕾或带初开的花[1], 是我国传统中药材和药食同源植物的典型代表[2-5]。金银花具有抗菌、抗病毒、抗炎、免疫、保肝、止血等功效[6-12], 有中药抗生素之称[13]。金银花的人工栽培的传统产区主要在河北巨鹿、河南封丘、山东平邑等地[14]。现代研究表明, 金银花的药理活性与其所含的多种活性成分密切相关[15-16], 绿原酸是金银花的主要抗菌成分[17], 也是金银花质量控制的重要指标[18]
绿原酸是由奎宁酸和咖啡酸通过酯化反应形成的缩酚酸, 是植物体在有氧呼吸过程中产生的一种苯丙素类化合物[19], 根据酯化位点的不同, 可形成多种同分异构体, 包括新绿原酸(5-咖啡酰奎宁酸)、隐绿原酸(4-咖啡酰奎宁酸)、异绿原酸A (3,5-二咖啡酰奎宁酸)、异绿原酸B (3,4-二咖啡酰奎宁酸)及异绿原酸C (4,5-二咖啡酰奎宁酸)等[20]。文献报道咖啡酰奎宁酸类化合物的检测方法主要包括紫外分光光度法[21]、高效液相色谱[22]、薄层色谱法[23]和毛细管电泳法[24]等。但上述文献报道定量方法均采用外标法, 对照品的种类和数量需求均较大。一测多评法由王智民等[25]于2006年首次提出, 核心原理是通过已知对照品与目标成分之间的响应值关系(如色谱峰面积、吸收度等), 计算校正因子, 建立相对校正因子或数学模型, 实现对其他同类成分的间接定量分析, 无需每种成分都配备对照品。一测多评技术已被广泛应用于中药多组分同步含量测定领域[26-29]。但目前一测多评法用于金银花中咖啡酰奎宁酸类化合物含量的测定研究较少。
因此本研究采用一测多评法测定我国三大道地产区样品中5种咖啡酰奎宁酸类化合物含量, 通过廉价易得的绿原酸对照品与其余成分之间的响应值关系建立相对校正因子, 通过方法学和交叉验证, 使其计算值与实测值符合定量方法学的要求。解决了因部分标准品昂贵而导致外标法不能广泛应用的问题, 实现了“以一测多”“以易测难”“以廉测贵”的多指标质量控制方案, 并比较分析不同产地金银花的品质差异。研究成果不仅可为金银花的质量评价提供新思路, 也将为其在医药、食品等领域的深度开发应用奠定基础。
绿原酸对照品(质量分数为99.3%, 批号110753-202119) (中国食品药品检定研究院); 异绿原酸A对照品(质量分数为98.0%, 批号J0202AS)、异绿原酸B对照品(质量分数为98.0%, 批号D1018AS)、异绿原酸C对照品(质量分数为98.0%, 批号D0507AS)、新绿原酸对照品(质量分数为98.0%, 批号N0805AS)(大连美仑生物技术有限公司); 乙腈(色谱纯)、甲醇(色谱纯)[赛默飞世尔科技(中国)有限公司]; 磷酸(分析纯, 天津市大茂化学试剂厂)。
试验用25份金银花, 均经沈阳药科大学中药学院路金才教授鉴定为忍冬科植物忍冬Lonicera japonica Thunb.的干燥花。样品来源信息见表1
Agilent 1260高效液相色谱仪[配二极管阵列检测器(型号G1315C)]、InfinityLab Poroshell 120 EC-C18色谱柱(250 mm×4.6 mm, 4 μm)(美国Agilent公司); Waters e2695高效液相色谱仪(美国Waters公司); Phenomenex Synergi Hydro-RP 80A色谱柱(250 mm×4.6 mm, 4 μm)(美国Phenomenex公司); Accucore™ XL C18色谱柱(250 mm×4.6 mm, 4 μm)(美国Thermo Fisher Scientific公司); AE240型电子分析天平(精度0.01 mg, 梅特勒-托利多上海有限公司); GZX-9070MBE型电热鼓风干燥箱(上海博迅实业有限公司医疗设备厂); 800Y型高速粉碎机(永康市铂欧五金制品有限公司); KQ-100DE型超声波清洗器(昆山市超声仪器有限公司)。
精密称定过完筛的金银花粉末约100.00 mg, 置于具塞锥形瓶中, 精密加入50%甲醇20.00 mL, 盖好塞子涡旋混合5 min, 置于分析天平上称定重量, 超声波提取(功率300 W, 频率40 kHz) 40 min, 冷却后, 用分析天平称定重量, 用50%甲醇补足减少的重量, 盖好塞子涡旋混合1 min, 取上清液过0.22 μm滤膜于进样小瓶中, 上安捷伦高效液相色谱仪。
分别精密称取对照品新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C适量, 置于10.00 mL容量瓶中, 甲醇溶解并稀释至刻度, 摇匀, 得新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C质量浓度分别为0.164、2.890、0.660、0.085、0.148 mg/mL的混合溶液。
色谱柱: Phenomenex Synergi Hydro-RP80A (250 mm× 4.6 mm, 4 µm), 柱温为 30 ℃, 流动相体系: A: 乙腈, B: 0.1%磷酸水溶液; 流速0.7 mL/min; 梯度洗脱条件为: 0~14 min, 19% A; 14~35 min, 19%~31% A; 35~40 min, 90% A, 检测波长327 nm; 流速0.7 mL/min; 进样量2 µL。
依次精密吸取1.3.2项下混合对照品溶液1.00、0.50、0.25 mL分别置于10.00 mL容量瓶中, 用甲醇稀释至刻度, 摇匀, 标号Ⅰ、Ⅱ、Ⅲ。依次精密吸取Ⅰ号混合对照品溶液1.00、0.50、0.25 mL分别置于10.00 mL 容量瓶中, 用甲醇稀释至刻度, 摇匀, 标号Ⅳ、Ⅴ、Ⅵ。各对照品待测液新绿原酸含量为0.41、0.82、1.64、4.10、8.20、16.40 μg/mL, 绿原酸含量为7.225、14.45、28.90、72.25、144.50、289.00 μg/mL, 异绿原酸A含量为1.65、3.30、6.60、16.50、33.00、66.00 μg/mL, 异绿原酸B含量为0.2125、0.4250、0.8500、2.1250、4.2500、8.5000 μg/mL, 异绿原酸C含量为0.37、0.74、1.48、3.70、7.40、14.80 μg/mL, 按照1.3.3项液相色谱条件进行进样分析, 以峰面积为纵坐标(Y), 进样质量浓度为横坐标(X, μg/mL), 绘制标准曲线。
(1)仪器精密度考察
精密吸取1.3.2项下的各混合对照品溶液高质量浓度(绿原酸含量为16.40 μg/mL、绿原酸含量289.00 μg/mL、异绿原酸A含量66.00 μg/mL、异绿原酸B含量8.500 μg/mL、异绿原酸C含量14.80 μg/mL)、中质量浓度(绿原酸含量为4.10 μg/mL、绿原酸含量72.25 μg/mL、异绿原酸A含量16.50 μg/mL、异绿原酸B含量2.125 μg/mL、异绿原酸C含量3.70 μg/mL)、低质量浓度(绿原酸含量为0.82 μg/mL、绿原酸含量14.45 μg/mL、异绿原酸A含量3.30 μg/mL、异绿原酸B含量0.4250 μg/mL、异绿原酸C含量 0.74 μg/mL)混合对照品2 µL, 在同一天内连续进样6次, 计算各成分峰面积的相对标准偏差(relative standard deviation, RSD)考察日内精密度; 每天进样3次, 连续进样3 d, 计算各成分峰面积的RSD考察日间精密度。
(2)重复性考察
取同一批次金银花样品(编号: S13), 按照1.3.1项下方法平行制备6份供试品, 按照1.3.3项下方法进行处理与测定, 以外标法计算新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C的含量RSD值。
(3)稳定性考察
取S13金银花样品溶液, 分别于0、2、4、6、8、10、12、24 h按照1.3.3项下方法进行分析测定, 分别记录色谱峰面积, 计算新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C的含量RSD值。
(4)回收率考察
精确称取已知含量的S13金银花样品6份, 每份约0.10 g, 各平行6份, 分别加入相应低、中、高量(分别相当于金银花样品中各被测成分含量的80%、100%、120%)的新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C对照品, 制成供试品溶液, 测定其峰面积, 按照公式(1)计算加样回收率。
回收率/%=(实测量-原始量)/加入量×100%
计算得新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C平均回收率及RSD。
用Agilent液相色谱仪自带色谱工作站采集的数据进行定性、定量分析; 采用WPS 365进行数据分析, 分析试验结果。
查阅文献[30]确定了咖啡酰奎宁酸类化合物最大吸收波长分别为327 nm。采用二极管阵列检测器对5个成分进行了全波长扫描, 结果如图1, 在327 nm波长附近均有明显吸收, 且各待测成分色谱峰分离度良好, 从整体结果分析选择327 nm作为检测波长。
按照1.3.3项下方法, 分别考察流动相的有机相与水相。
有机相的考察分别为乙腈(A)-0.1%磷酸溶液(B)和甲醇(A)-0.1%磷酸溶液(B), 比较样品溶液中各成分的峰面积, 峰之间的分离度, 以及峰形。结果显示, 当选用乙腈为有机相, 0.1%磷酸溶液为水相, 各成分峰形较好, 分离度更高, 所以选择流动相为乙腈(A)-0.1%磷酸溶液(B)。
水相的考察分别为纯水、0.1%甲酸、0.3%甲酸、0.1%磷酸、0.2%磷酸, 比较样品溶液中各成分的峰面积, 峰之间的分离度, 以及峰形。结果显示, 流动相加酸分离效果更好; 加入磷酸后, 各成分经色谱柱分离后峰形更好, 分离度更高, 且0.2%磷酸和0.1%磷酸分离效果无明显差别, 考虑到色谱柱的耐酸性有限, 酸性过强会对色谱柱造成损害, 所以选择水相中加入0.1%磷酸。
分别考察色谱柱填充粒径为4 µm和5 µm的色谱柱, 比较样品溶液中各成分的峰面积, 各成分峰形以及分离度。结果显示, 粒径4 µm色谱柱对于咖啡酰奎宁酸类化合物分离效果更好, 各组分峰面积更大, 且峰形更佳, 所以选择粒径为4 µm的色谱柱作为咖啡酰奎宁酸类分析的色谱柱。
按照1.3.1项下方法, 分别以20.00 mL甲醇、乙醇、50%甲醇、75%甲醇和纯水为提取溶媒制备样品溶液。按照确定的色谱条件进行分析, 在单一波长(327 nm)处同步测定样品溶液中5个成分的峰面积, 比较不同溶媒提取的样品的峰面积。结果显示, 以50%甲醇为溶媒测得的各待测成分峰形和分离度较好, 总峰面积较大, 结果见表2, 故最终确定50%甲醇为提取溶剂。
分别以10、20、40 mL 50%甲醇为提取溶媒制备样品溶液。按照确定的色谱条件进行分析, 在单一波长(327 nm)处同步测定样品溶液中5个成分的峰面积, 比较不同溶媒用量提取的样品溶液的峰面积。结果显示, 10 mL的50%甲醇的峰面积低于20 mL和40 mL, 20 mL与40 mL的峰面积差异不大, 结果见表3, 故最终确定提取溶媒50%甲醇的用量为20 mL。
按照1.3.1项下方法, 分别采用超声、冷浸提和回流法进行提取, 制备样品溶液。按照确定的色谱条件进行分析, 在单一波长(327 nm)处同步测定样品溶液中5种成分的峰面积, 比较不同提取方式提取的样品溶液的峰面积。结果显示, 除浸提法外, 其余两种提取方式峰面积差异不大, 结果见表3, 因超声波提取法简便快速, 故最终确定提取方法为超声提取法。
金银花包含多种主要成分, 其中咖啡酰奎宁酸类(约占3.8%)是金银花中主要的生物活性物质。一测多评具有诸多优势, 能更全面的反应药材质量情况, 选取主要的5种咖啡酰奎宁酸类化合物(新绿原酸、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C), 其中绿原酸是金银花中含量最高的咖啡酰奎宁酸类化合物, 具有化学性质稳定, 对照品价格廉价易得等优点, 由于其他几种咖啡酰奎宁酸类化合物的标准品纯化难度较高, 价格昂贵, 极大地增加了常规分析的成本。故绿原酸符合一标多测方法中一标的选择原则, 故以绿原酸为内标物对金银花进行多指标同步含量测定分析。
分别将混合对照品溶液、样品溶液按照1.3.3项下色谱条件进样分析。色谱图见图2, 结果显示异绿原酸A、异绿原酸B、异绿原酸C、新绿原酸、绿原酸5种成分与其相邻色谱峰的分离度均大于1.5, 拖尾因子在0.98~1.20之间, 理论塔板数以各色谱峰计均在10000以上, 以上化合物含量较高、分离效果好且互不干扰。
各对照准品利用外标法的线性回归方程、相关系数以及对照品的线性范围如表4所示。从表4中数据可知, 新绿原酸、绿原酸、异绿原酸B、异绿原酸A、异绿原酸C的线性范围分别为0.41~16.40、7.22~289.00、0.21~8.50、1.65~66.00、0.37~14.80 μg/mL, 5种咖啡酰奎宁酸类成分进样量与峰面积之间具有良好的线性关系, 其相关系数r均大于0.9993。各分析物与其相邻峰的分离度均大于1.5, 拖尾因子在1.00~1.10之间, 理论塔板数均在10000以上。
按照1.3.5(1)方法进行分析, 结果见表5。计算得得新绿原酸、绿原酸、异绿原酸B、异绿原酸A、异绿原酸C其日内精密度RSDs分别是1.57%、0.49%、1.55%、0.82%、0.99%, 日间精密度RSDs分别是1.37%、0.12%、1.63%、0.51%、0.79%, 结果显示, 该分析方法的日内日间精密度良好。
按照1.3.5(2)方法进行分析, 结果测得供试品各成分含量的RSDs分别是1.43%、0.43%、1.94%、0.56%、1.32%, 表明该方法的重复性良好。
按照1.3.5(3)方法进行分析, 结果测得供试品各成分含量的RSDs分别是1.49%、0.09%、1.79%、0.18%、1.62%, 表明样品溶液在24 h内是稳定的。
按照1.3.5(4)方法进行分析, 按照按1.3.1项下方法前处理, 按1.3.3项下方法进行检测, 经过计算得到新绿原酸、绿原酸、异绿原酸B、异绿原酸A、异绿原酸C 5个化合物的平均回收率依次为96.90%、95.25%、98.93%、97.26%、100.42%, 其RSDs依次为1.68%、2.28%、3.99%、2.94%、3.98%, 表明该方法的准确度良好。
相对保留值的建立: 根据绿原酸的保留时间, 利用待测成分(新绿原酸、异绿原酸B、异绿原酸A、异绿原酸C)相对于绿原酸的相对保留时间(RRTx)对待测峰进行定位, 计算公式如公式(2)所示:
RRTx=RTx/RTs
式中: RTs、RTx分别表示内标化合物和待测化合物的保留时间, min。
相对校正因子的建立: 根据绿原酸的保留时间和峰面积, 分别计算咖啡酰奎宁酸类待测成分相对于绿原酸的相对校正因子, 计算公式如公式(3)所示:
fk/s=$\frac{{{W}_{k}}\times {{A}_{k}}}{{{W}_{s}}\times {{A}_{s}}}\times 100%$
式中: fk/s为内参物与其他组分之间的相对校正因子; As为内标化合物峰面积; Ws为内标化合物的质量浓度, μg/mL; Ak为某待测化合物峰面积; Wk为某待测化合物的质量浓度, μg/mL。
测得的fk/s可作为某一色谱条件下某一组分的相对校正因子。通过使用一个对照品标准物和相对校正因子计算其他组分的量, 计算公式如公式(4)所示:
$W_{s}^{'}=\frac{W_{_{k}}^{'}\times A_{s}^{'}}{{{f}_{k/s}}\times A{{_{k}^{'}}_{{}}}}$
式中: $W_{s}^{'}$为样品中待测化合物的质量浓度, μg/mL; $W_{k}^{'}$为用峰面积一外标法测得的样品中标准物质量浓度, μg/mL; fk/s为内参物与其他组分之间的相对校正因子; $A_{k}^{'}$$A_{\text{s}}^{'}$分别为样品中标准物和待测组分的峰面积。
因此一测多评法可同时测定样品中n个组分, 其中一个组分(标准物)用外标法测定, n-1个组分按式(3)~(4)计算, 即得。
分别考察了Agilent 1260和Waters e2695两套高效液相色谱系统和Phenomenex Synergi Hydro-RP80A (250 mm× 4.6 mm, 4 μm)、InfinityLab Poroshell 120 EC-C18 (250 mm× 4.6 mm, 4 μm)、Accucore™ XL C18 (250 mm×4.6 mm, 4 μm) 3种均以十八烷基硅烷键合硅胶为填充剂的色谱柱, 考察不同色谱系统和色谱柱对相对较正因子的影响。结果显示不同仪器对化合物的相对保留时间稍微有影响, 不同色谱柱对相对保留时间影响均较小, 重现性良好, RSDs值均小于5%(表6)。
精密吸取混合对照品溶液2 μL进样分析, 以绿原酸为参照物计算相对校正因子, 所得的相对校正因子及其相对标准差, 见表7, 结果显示该相对校正因子fk/s均比较稳定, 均满足检测要求, RSDs值均小于5%。
取各个产地金银花样品粉末各约100.00 mg, 精密称定, 按照前述1.3.1项下所示方法制备不同产地金银花供试品, 各平行3份, 按照1.3.3项色谱分析方法进行进样分析, 测定25批不同产地金银花中5种成分含量。外标法采用2.4所示线性回归方程计算含量, 一测多评相对校正因子采用表7的平均值计算含量。结果如表8所示, 采用一测多评法与外标法分别检测25批次金银花中5种成分的含量, 计算结果之间并无明显差异(RSDs<5%)表明该方法能够用于金银花中5种咖啡酰奎宁酸类成分含量的同时测定。
结果表明, 5种咖啡酰奎宁酸的相对校正因子较为稳定准确, 一测多评法与外标法测定结果之间不存在显著性差异。说明该一测多评方法可以测定金银花药材中5种咖啡酰奎宁酸类成分含量, 且方法简便、准确, 解决了金银花药材质量控制中缺乏对照品这一瓶颈问题。同时对数据进行分析, 可以看出河南新密产区的5种咖啡酰奎宁酸总量高于河南封丘县产区及河北山东产区; 几大产区的异绿原酸B与异绿原酸C含量差别不大, 山东平邑县的新绿原酸含量显著高于其他产区, 而河南新密市的绿原酸含量显著高于其他产区, 此种差异可能源于土壤、气候、光照等多种原因, 还需进一步探究其原因, 为我国金银花药材及其制剂有效成分的统一性, 实用性提供依据。
本研究曾考察过不同方法提纯5种成分, 如用超声、冷浸提和回流法等方式, 结果发现超声波提取法简便高效。提取溶剂选择纯水、甲醇、乙醇、50%甲醇、75%甲醇等, 结果以50%甲醇提取5种成分的溶出率最高。而本研究发现采用50%甲醇超声40 min提取后, 以乙腈-0.1%磷酸水溶液的梯度体系作为流动相检测结果分离度良好, 基线稳定。本研究结果表明以绿原酸为参照物, 建立的5种咖啡酰奎宁酸的校正因子较为稳定准确, 计算25批金银花样品中5种成分, 并与外标法检测结果进行分析, RSD 值均小于5%, 证明所建立的一测多评方法可靠准确。采用外标法以及本研究所建立的一测多评法计算结果无明显不同, 且检测方法更便捷。
本研究成功建立了分析不同道地产区金银花中咖啡酰奎宁酸类成分一测多评含量测定方法。该检测方法可解决因对照品缺乏而导致外标法难以在实际工作中广泛应用的问题, 利用相对校正因子实现对金银花中多种咖啡酰奎宁酸的含量测定的方法是可行的, 可显著的降低检测成本。不同产地和不同采收期的金银花中咖啡酰奎宁酸类的含量差别较大, 这种差异的产生可能与生长环境, 如光照时间, 降雨量, 种植土壤的成分因素有关。
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2025年第16卷第13期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250213007
  • 接收时间:2025-02-13
  • 首发时间:2026-01-12
  • 出版时间:2025-07-15
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  • 收稿日期:2025-02-13
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    辽宁省疾病预防控制中心, 辽宁省空气雾霾与人群健康监测重点实验室, 沈阳 110005

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*华正罡(1983—), 男, 主任技师, 硕士, 主要研究方向为食品药品标准化研究。E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

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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