A novel method for integrating chromatographic fingerprint analytical units of Chinese materia medica:the matching frequency statistical moment method

Objective To facilitate the quality evaluation suitable for the unique characteristics of Chinese materia medica(CMM)by developing and implementing a novel approach known as the matching frequency statistical moment(MFSM)method.Methods This study established the MFSM method.To demonstrate its effectiveness,we applied this novel approach to analyze Danxi Granules(丹膝颗粒,DXG)and its constituent herbal materials.To begin with,the ultra-performance liquid chromatography(UPLC)was applied to obtain the chromatographic fingerprints of DXG and its constituent herbal materi-als.Next,the MFSM was leveraged to compress and integrate them into a new fingerprint with fewer analytical units.Then,we characterized the properties and variability of both the original and integrated fingerprints by calculating total quantum statistical moment(TQSM)parameters,information entropy and information amount,along with their relative standard deviation(RSD).Finally,we compared the TQSM parameters,information entropy and infor-mation amount,and their RSD between the traditional and novel fingerprints to validate the new analytical method.Results The chromatographic peaks of DXG and its 12 raw herbal materials were divided and integrated into peak families by the MFSM method.Before integration,the ranges of the peak number,three TQSM parameters,information entropy and information amount for each peak or peak family of UPLC fingerprints of DXG and its 12 raw herbal materials were 95.07−209.73,9390−183064μv·s,5.928−21.33 min,22.62−106.69 min^(2),4.230−6.539,and 50530−974186μv·s,respectively.After integration,the ranges of these parameters were 10.00−88.00,9390−183064μv·s,5.951−22.02 min,22.27−104.73 min^(2),2.223−5.277,and 38159−807200μv·s,respectively.Correspondingly,the RSD of all the aforementioned pa-rameters before integration were 2.12%−9.15%,6.04%−49.78%,1.15%−23.10%,3.97%−25.79%,1.49%−19.86%,and 6.64%−51.20%,respectively.However,after integration,they changed to 0.00%,6.04%−49.87%,1.73%−23.02%,3.84%−26.85%,1.17%−16.54%,and 6.40%−48.59%,respectively.The results demonstrated that in the newly integrated fingerprint,the analytical units of constituent herbal materials,information entropy and information amount were significantly reduced(P<0.05),while the TQSM parameters remained unchanged(P>0.05).Additionally,the RSD of the TQSM parameters,information entropy,and information amount didn’t show significant difference before and after integration(P>0.05),but the RSD of the number and area of the integrated analytical units significantly decreased(P<0.05).Conclusion The MFSM method could reduce the analytical units of constituent herbal mate-rials while maintain the properties and variability from their original fingerprint.Thus,it could serve as a feasible and reliable tool to reduce difficulties in analyzing multi-compo-nents within CMMs and facilitating the evaluation of their quality.

巴戟天质量评价研究进展

巴戟天为著名的“四大南药”之一,具有补肾阳、强筋骨、抗抑郁等多种药理作用。药材的真伪优劣直接影响到巴戟天在临床应用中的有效性与安全性,但目前巴戟天药材的质量评价标准尚不完善,建立科学全面的巴戟天质量评价体系对保证巴戟天用药安全有效至关重要。通过综述巴戟天药材的质量评价方法,针对巴戟天质量评价体系中存在的问题进行分析,并提出合理建议,以期为后续巴戟天的研究及其质量标准的完善提供参考。

痛风颗粒成型工艺研究及其物理指纹图谱建立

目的:优选痛风颗粒的成型工艺并建立其物理指纹图谱。方法:在单因素试验的基础上,以稠膏与辅料的比例、可溶性淀粉与微晶纤维素(MCC)的比例、稠膏与胶态二氧化硅的比例为考察因素,以成型率、休止角、吸湿率及软材性状的综合评分为评价指标,利用响应面法优选最佳成型工艺。采用物理指纹图谱对痛风颗粒的均一性、流动性、稳定性等物理属性指标进行综合表征,评价不同批次颗粒的质量一致性。结果:最佳成型工艺为稠膏与辅料比例为1∶0.99,可溶性淀粉与微晶纤维素(MCC)的比例为7∶2,稠膏与胶态二氧化硅的比例为1∶0.09,12批颗粒物理指纹图谱的相似度均高于0.9。结论:该工艺稳定可行,生产出的颗粒性质稳定,符合痛风颗粒实际大生产要求。

Chromatographic fingerprint analysis of Fructus Aurantii Immaturus by HPLC-DAD and chemometric methods

An efficient method for quality control of Fructus Aurantii Immaturus (FAI),a famous traditional Chinese medicine (TCM) was established. A simple and reliable high-performance liquid chromatography-photodiode array detector (HPLC-DAD) procedure coupled with chemometric methods was developed for fingerprint analysis,qualitative analysis and quantitative determination of this herb. In qualitative and quantitative analyses,heuristic evolving latent projection (HELP) method was employed to resolve the overlapping peaks of the tested samples. Two bioactive components,namely hesperidin and naringin,are confirmed and determined,together with four flavonoids compounds tentatively identified including two new ones. From fingerprint analysis,the fingerprint data were processed with correlation coefficients for quantitative expression of their similarity and dissimilarity. The developed method based on an integration of chromatographic fingerprint and quantitative analysis is scientific,and the obtained results can be applied to the quality control of herb medicine.

Box-Behnken response surface method combined with fingerprint to optimize the extraction process of total anthraquinone from Cassia seeds

Objective:The Box-Behnken response surface method combined with fingerprints was used to optimize the extraction process of total anthraquinone from Cassia seeds.Methods:A three-factor,three-level response surface test was conducted based on the single-factor test with comprehensive evaluation as the measurement index.The comprehensive evaluation indexes included the extraction rate of total anthraquinone of Cassia seeds or the equivalent amount of herbs per gram of total anthraquinone of Cassia seeds,the normalized value of peak areas of 5 index components such as aurantio obtusin in the fingerprint of each sample to 16 shared peaks and the similarity of fingerprints(the reference fingerprint was established by the extraction solvent for the determination of Cassia seeds content in the Chinese Pharmacopoeia 2020 edition)with the weights of 0.2,0.5 and 0.3,respectively.Results:The best extraction process was obtained:the liquid-to-material ratio was 20:1(mL·g-1),the extraction solvent was mixture of 60%ethanol-ethyl acetate(2:1),and the extraction time was 15.12 min.The results of five sets of validation experiments showed that the overall evaluation of total anthraquinone of cassia seeds by the best process was 0.528(RSD=0.45%),and the prediction result of response surface method model was 0.531,and the relative error with the prediction result was 0.531.The relative error of the predicted results was 0.56%,and the best extraction process was consistent with the model prediction,and the obtained best process could be used for the extraction of total anthraquinone from Cassia seeds.Conclusion:The Box-Behnken response surface method combined with the fingerprint technique can be used to find the best reaction conditions and examine the interactions among the factors in a comprehensive and accurate manner,which can provide reference for the optimization and evaluation of the extraction process of Chinese medicine.

Integrated evaluation of HPLC fingerprints for the quality control of Ginseng Yang Rong Wan by multi-wavelength fusion fingerprint method

The aim of this study was to develop comprehensive evaluation methods for the quality control of Ginseng Yang Rong Wan(RSYRW).Multi-wavelength fusion fingerprints were successfully developed by high-performance liquid chromatography,which avoided the one-sidedness of single fingerprinting.Characteristic fingerprints of thirty batches of samples were generated at five wavelengths and evaluated by quantified ratio fingerprint method(QRFM).The results showed that thirty batches of samples were classified into seven grades.The methods established in this paper were found suitable for the analysis of Ginseng Yang Rong Wan.

基于FTIR的黄芪药材质量评价研究

目的 建立黄芪药材的红外原始指纹图谱与量子指纹图谱,为其质量控制提供参考。方法 采用傅里叶变换红外光谱仪采集35批黄芪药材的红外指纹图谱,经数据处理建立黄芪的红外量子指纹图谱,采用系统指纹定量法以及t检验比较两种指纹图谱是否存在差异,并对量子指纹图谱的共有量子峰面积进行系统聚类分析。结果 黄芪药材的红外指纹图谱与量子指纹图谱间并不存在显著性差异,系统指纹定量法将35批黄芪分为8个质量等级,系统聚类分析表明35批黄芪的质量可聚为两类。结论 红外量子指纹图谱能够提供大量特征信息,系统指纹定量法能够全面、准确、直观地将黄芪药材分为8个质量等级,两者结合可以实现对黄芪等中药材质量的全面评价。

阿达木单抗还原肽指纹图谱鉴别方法的建立与验证

目的 建立阿达木单抗基于高分辨质谱的肽指纹图谱表征分析方法及用于质控放行的肽指纹图谱液相鉴别方法。方法 阿达木单抗经变性、还原、烷基化和酶解后,应用高分辨质谱测定阿达木单抗氨基酸序列覆盖率,并确证互补决定区(complementarity determining region, CDR)特征肽段。建立能够有效鉴别阿达木单抗肽指纹图谱的液相色谱分析方法,并进行方法学验证和应用评价。结果 阿达木单抗经胰蛋白酶(Trypsin)和胞内蛋白酶(Lys-C)酶解后,CDR肽段均由高分辨质谱完成确证。贝伐珠单抗和利妥昔单抗用于评估方法专属性,结果表明该方法专属性强。选择Fc段保守氨基酸序列作为参比峰,以CDR特征肽段的相对保留时间(RRT)和相对峰面积(RPA)考察方法的变异程度。Trypsin酶解的样品连续5次进样各特征肽段RRT的RSD在0.008%~1.097%之间,RPA的RSD在1.212%~7.951%之间;中间精密度考察各特征肽段RRT的RSD在0.010%~0.985%之间,RPA的RSD在2.306%~10.939%之间;酶切比例为1∶35~1∶45,酶切时间为3.5~4.5 h时,RRT和RPA的RSD均符合方法耐用性要求。Lys-C酶解的样品方法学验证结果也表明其精密度、耐用性均表现良好。不同色谱柱对肽段的选择性、保留强弱、分离效果以及峰强度等具有一定差异。结论 建立的基于Trypsin/Lys-C酶切、鉴定CDR特征肽段的高效液相肽指纹图谱鉴别方法可用于阿达木单抗的质量控制及批检验放行,同时可为企业建立单抗肽指纹图谱鉴别方法提供参考。

基于UPLC-Q-ExactiveMS构建麻痹性贝类毒素的特征指纹溯源技术

麻痹性贝类毒素污染是世界共同关注和重点管控的食品安全问题,其溯源一直是产业监管的难点和技术重点。基于指纹溯源技术理论,采用室内模拟3种产毒藻暴露紫贻贝后风险形成过程,开发基于超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UPLC-Q-Exactive MS)鉴定麻痹性贝类毒素区域特征的指纹溯源技术。结果表明,采用乙腈/甲醇/丙酮(体积比为1︰1︰1)混合溶液作为提取剂,C8色谱柱联合正离子模式,质荷比(m/z)100~600和Amide色谱柱联合正离子模式,m/z 600~1500作为色谱质谱检测条件下,提取的化合物数量较多且稳定性良好,覆盖了40.4%指纹信息;紫贻贝与产毒藻中的共检出11种毒素成分,且具有高度相关性。N-磺酰胺甲酰基类毒素(如C类)和膝沟藻毒素(GTX类)在暴露期发生了显著的代谢转化;将摄食3种产毒藻后紫贻贝的差异化合物与特征毒素组分进行主成分分析及正交偏最小二乘法判别分析,共筛选出13种复合指纹物质。构建6种特征物质的Fisher判别模型,交叉验证准确率为88.9%,可实现产毒藻种溯源。该研究建立了基于超高效液相色谱-四极杆-静电场轨道阱高分辨质谱UPLC-Q-Exactive MS紫贻贝特征指纹溯源技术,初步实现了从污染麻痹性贝类毒素的贝类到肇事藻种的逆向溯源,可应用于贻贝麻痹性贝类毒素风险溯源研究。

基于香气与滋味物质特征分析不同干燥方式美味牛肝菌的风味差异

为了解不同干燥方式美味牛肝菌干制品的风味与滋味特征,探究不同干制方法之间的风味差异,对热风干燥、真空冷冻干燥及真空干燥美味牛肝菌的挥发性风味物质与非挥发性滋味物质进行测定分析。气相色谱-离子迁移谱(gas chromatography-ion mobility spectrometry,GC-IMS)和气相色谱-质谱(gas chromatography-mass spectrometry,GC-MS)分别定性64、85种挥发性有机物;指纹图谱分析结果表明,不同干制方法处理组间存在较大差异,真空干燥组的差异特征最为显著;正交偏最小二乘判别分析从GC-IMS结果中筛选出17个差异特征标记物,GC-MS中筛选出22个,结合相对气味活度值进一步分析可知,1-辛烯-3-醇为关键挥发性有机物。非挥发性滋味物质分析结果表明,游离氨基酸与5’-核苷酸在热风干燥组中含量最大,分别为234.57 mg/g和27.870 mg/g;16种游离氨基酸中谷氨酸是呈鲜味的主要成分。综上,热风干燥更有利于美味牛肝菌中滋味物质的积累,真空干燥更利于风味物质的产生和积累。本研究表征3种干制方法的风味特征,可为新鲜美味牛肝菌采后干制加工工艺的选择提供参考。

基于UPLC指纹图谱结合化学模式的党参质量标志物预测分析

目的:建立党参药材的UPLC指纹图谱,结合化学计量学方法进行分析,对党参的质量标志物(Q-Marker)进行预测分析,为党参的质量评价提供科学依据。方法:采用ACQUITY UPLC®BEH C18色谱柱(2.1 mm×100 mm,1.7μm),以乙腈-0.1%磷酸水溶液为流动相,梯度洗脱,流速0.3 mL·min-1,检测波长267 nm,柱温30℃,进样量2μL。采用高效液相色谱仪,使用Agilent ZORBAX SB-C18色谱柱和乙腈-0.1%磷酸水溶液进行梯度洗脱,建立21批党参样品的UPLC指纹图谱并进行相似度评价,采用聚类分析(HCA)、主成分分析(PCA)和正交偏最小二乘判别分析(OPLSDA)等化学计量学方法筛选出主要差异性成分,预测党参药材的Q-Marker。结果:建立了党参药材UPLC指纹图谱,确认了16个共有峰,通过对照品共指认出7个共有峰,分别为腺苷、色氨酸、紫丁香苷、绿原酸、党参苷Ⅰ、党参炔苷、白术内酯Ⅲ,指纹图谱相似度均大于0.85,经HCA、PCA和OPLSDA筛选出党参苷Ⅰ、党参炔苷、腺苷及色氨酸为不同产地党参药材的重要差异性成分,可作为党参潜在的Q-Marker。结论:通过指纹图谱结合化学计量学分析预测了党参药材潜在的Q-Marker,为党参质量评价提供一定的参考。

参莲草颗粒成型工艺优化及其理化指纹图谱建立

目的 优化参莲草颗粒成型工艺,并建立其理化指纹图谱。方法 湿法制粒后单因素试验筛选辅料,PB试验筛选潜在关键工艺参数。以辅(糊精)药比、乙醇体积分数、润湿剂(乙醇)用量为影响因素,成型率、吸湿率、休止角、溶化率的综合评分为评价指标,Box-Behnken试验结合AHP-CRITIC混合加权法优化成型工艺。采用物理指纹图谱、HPLC指纹图谱评价不同批次样品的质量一致性,并进行化学模式识别。结果 最佳条件辅药比0.98∶1,乙醇体积分数84%,润湿剂用量28%,在70℃下干燥60 min,综合评分为95.18分。10批样品物理指纹图谱相似度均大于0.95;HPLC指纹图谱中有18个共有峰,相似度均大于0.95。各批样品聚为3类,野黄芩苷、去乙酰基车叶草苷酸甲酯、木犀草素、芦丁、去乙酰基车叶草苷酸为关键质量差异物。结论 该方法稳定可行,可用于评价参莲草颗粒的质量一致性,为该制剂质量标准研究奠定基础,也为同类中药制剂相关研究提供思路。

运脾化痰通窍方提取工艺优化

目的实现运脾化痰通窍方提取工艺优选,并建立组方指纹图谱。方法应用总量统计矩法,对提取液指纹图谱进行宏观表征和相似度分析,以黄芩苷、汉黄芩苷、迷迭香酸、甘草苷、甘草酸、出膏率为关键质量属性,以提取时间、加水倍量、提取次数为关键工艺参数,采用AHP-独立性权重法进行综合评分,优选出最佳提取工艺,并进行验证。结果建立总量统计矩相似度评价方法,不同提取方法指纹图谱总量统计矩相似度均值为0.8807,工艺评价模型函数方差分析结果显示,P<0.0001、R2=0.9904,该模型具有统计学意义。优选得到最佳提取工艺为全方饮片加10倍量水,提取1.5 h,提取2次。结论优化的工艺稳定可靠,建立了符合该方提取液指纹图谱特点的总量统计矩分析方法,为该方后续工艺研究全过程质量控制奠定物质基础。

溶剂热法制备尼龙66基碳点及其指纹识别、荧光防伪、光线阻挡应用

以尼龙66(PA66)和植酸(IP6)为前驱体、乙酸为溶剂,采用溶剂热法制备了PA66基碳点(66CDs)。利用TEM、FTIR、XPS、荧光光谱对其进行了表征,对其光学性能、离子稳定性和时间稳定性进行了测试,探究了其指纹识别、荧光防伪、光线阻挡的应用。结果表明,将1.6 g PA66、1.1 g IP6加入20 mL乙酸中,于260℃下反应36h,制备的66CDs具有最大荧光强度。66CDs为球形结构,平均粒径4.00nm,表面含有羧基、羟基、氨基等官能团;66CDs的荧光为非激发波长依赖型,最佳激发波长和发射波长分别为360和490 nm,荧光量子产率可达11.69%,其荧光强度不受常见金属阳离子影响,30 d内具有稳定性。由66CDs与水溶性淀粉制备的荧光粉末可用于指纹识别,不仅可将66CDs制成油墨用于荧光防伪,还可将其制成防蓝光膜,用于蓝光防护。

铕-茚三酮纳米粉末的制备及在手印显现中的应用

采用溶胶凝胶法,以稀土Eu^(3+)为中心离子,水合茚三酮为配体,分别按配比1:1、1:2、1:4、1:8制成具有硅胶骨架结构的铕-茚三酮配合物纳米粉末。采用傅立叶红外变换光谱对材料进行了表征。考察了该纳米粉末不同配比、不同客体以及对陈旧手印的适用情况。结果表明,铕-茚三酮纳米粉末红外光谱变化规律符合稀土配合物合成规律,在302 nm~365 nm紫外光的照射下产生红色荧光,该纳米材料适用于常见非渗透性客体及纸张等渗透性客体上皮脂手印及汗潜手印的显现,对陈旧手印已表现出较好显现效果。对比传统粉末手印显现效果,该纳米材料在背景污染、纹线粘连、细节特征显现灵敏度低等问题上具有明显优势。

基于水质荧光指纹技术的复合污染河道溯源研究

复合污染水体的污染源组成解析是当前水污染溯源领域难题,基于三维荧光光谱的水质荧光指纹技术是解决该难题的关键方法。采用水质荧光指纹技术对苏南地区Y河开展溯源分析。通过平行因子分解与水质荧光指纹比对有效识别Y河中印染废水荧光信号来自上游河道异地输入。利用氨氮浓度划分重污染区域,依托水质荧光指纹对Y河流域内109个排口开展筛查,通过污染路径溯源法精准定位35家主要责任单位。落实整改要求后,Y河水质由劣Ⅴ类迅速提升至Ⅲ类,并持续稳定达标。Y河河道治理工程取消,降本增效效果显著。针对复合污染河道提出了以水质荧光指纹技术为抓手的精准溯源与微创治理新模式,对实现精准治污、科学治污、依法治污具有重要指导意义。

傅里叶变换红外光谱指纹图谱鉴别艾绒等级

提出了傅里叶变换红外光谱指纹图谱鉴别艾绒等级的方法,通过8种光谱预处理方法(去噪处理、高斯滤波、多元散射校正、标准正态变换、一阶导数+Savitzky-Golay(SG)平滑、二阶导数+SG平滑、一阶导数+Norris Gap、二阶导数+Norris Gap)和5种模式识别方法[反向传播神经网络(BP-NN)算法、遗传优化支持向量机(SVM-ga)、粒子群优化支持向量机(SVM-pso)、随机森林(RF)算法、K-最近邻(KNN)算法]的结合对比,得到鉴别艾绒等级的最佳模型。结果表明,艾绒的指纹图谱中有11个共有峰,对其进行主成分分析,得到9个主成分,累计方差贡献率达到99.67%。标准正态变换结合SVM-pso算法的鉴别效果最好,其训练集的鉴别正确率为100%,测试集的鉴别正确率为93.3%。

基于荧光指纹的土门西沟小流域农业面源溯源研究

于2022—2023年采集密云水库的典型农业小流域土门西沟小流域内源样品构建指纹库,采集7、8月河道6个重点断面水样进行三维荧光光谱测定,通过平行因子分析解析小流域氮素来源。结果表明:研究区氮平均质量浓度为7.92 mg/L,主要形态为硝态氮;通过结合荧光激发发射矩阵与平行因子分析共解析出4种荧光组分,主要包括两种类腐殖质组分(C1、C2)及类蛋白组分(C3、C4),C1与农业有机肥施用有关,C2与堆肥有关,C3、C4主要来自农村生活污水或未分解的尿素化肥;研究区农村生活污水荧光指数平均值约为2.16,土壤浸提液荧光指数平均值约为1.83,农村生活污水在8月对农业面源约贡献35%;小流域丰水期水质波动主要与类腐殖质C1、C2组分相关,应对研究区有机肥带来的农业面源给予关注并加强管控。

乌梢蛇醇溶蛋白质提取工艺优化及其指纹图谱表征

目的优化乌梢蛇醇溶蛋白质提取工艺,并对其进行指纹图谱表征。方法在单因素试验基础上,以料液比、乙醇体积分数、提取温度、提取时间为影响因素,提取率为评价指标,响应面法优化提取工艺,再进行FTIR、SDS-PAGE分析。结果最佳条件为料液比1∶43.15,乙醇体积分数46.35%,提取温度37.53℃,提取时间250min,提取率为0.4072%。醇溶蛋白质在4000~400cm-1波数范围内有10个吸收峰,并且其种类丰富,分子量在11.19~158.10kDa范围内,其中26.48、19.67kDa为指纹图谱主峰。结论本实验可为乌梢蛇开发利用及分析鉴定提供依据。

基于QbD理念的油菜花粉颗粒制备工艺及物理指纹图谱研究

目的基于质量源于设计(quality by design,QbD)理念对油菜花粉颗粒进行制备工艺研究及质量控制。方法以油菜花粉颗粒成型率(%)、休止角(°)、平衡吸湿率(%)为评价指标,通过G1-熵权法组合赋权得到综合评价指标,单因素试验结合Box-Behnken响应面法优化油菜花粉颗粒制备工艺。采用物理指纹图谱评价不同批次油菜花粉颗粒的物理质量一致性并测定临界相对湿度。结果油菜花粉颗粒最佳制备工艺为:淀粉、甘露醇作为填充剂,药辅比1∶2、乙醇浓度55%、辅料配比7∶3。10批油菜花粉颗粒物理指纹图谱相似度均大于0.99。结论基于QbD理念的油菜花粉颗粒制备工艺稳定可行,可用于油菜花粉颗粒的制备。