CN102621244A - Construction method for HPLC (high performance liquid chromatography) finger-print chromatogram of ginseng and astragalus strengthening injection and application of finger-print - Google Patents
Construction method for HPLC (high performance liquid chromatography) finger-print chromatogram of ginseng and astragalus strengthening injection and application of finger-printDownload PDFInfo
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明涉及一种参芪扶正注射液HPLC指纹图谱的构建方法，以及由此方法所得到的参芪扶正注射液HPLC标准指纹图谱，属于药物分析技术领域。The invention relates to a method for constructing the HPLC fingerprint of Shenqi Fuzheng injection and the HPLC standard fingerprint of Shenqi Fuzheng injection obtained by the method, belonging to the technical field of drug analysis.
背景技术Background technique
参芪扶正注射液是以扶正补气中药党参、黄芪为原料制成的中药大输液，具有益气扶正、提高机体免疫力等作用，主要用于各类肿瘤的辅助治疗，在临床实践中证实其与抗肿瘤药物联合使用时，有减毒增效的作用。同时，也是冠心病、心绞痛、中风患者较理想的治疗药物，具有广阔的临床应用前景。Shenqi Fuzheng Injection is a traditional Chinese medicine infusion made from Codonopsis pilosula and Astragalus membranaceus, which are traditional Chinese medicines for invigorating Qi and strengthening Qi. When it is used in combination with antitumor drugs, it has the effect of reducing toxicity and enhancing efficacy. At the same time, it is also an ideal therapeutic drug for patients with coronary heart disease, angina pectoris and stroke, and has broad clinical application prospects.
对中药注射液质量控制方法的深入研究始终是保证其稳定性和临床使用安全性的最重要的问题。在公开的200610098953.1号及200710136382.0的专利申请中均报道了有关参芪扶正注射液HPLC指纹图谱的报道。这两项专利申请公开中记载的方法的不足之处在于：（1）供试品要通过AB-8大孔树脂柱分离纯化或者用水饱和正丁醇萃取4次随后进行浓缩等步骤制备，复杂繁琐，高温浓缩步骤容易引起成分变化，影响方法的重现性。测定有紫外吸收的酚酸类成分与无紫外吸收的皂苷类成分要用同样繁琐的方法分别制备供试品溶液，方法存在耗时长，使用有机溶剂多的缺陷。（2）指纹图谱采用多达5个非线性梯度进行测定，要用同样的条件分别测定有紫外吸收的酚酸类成分与无紫外吸收的皂苷类成分，一次测定的时间长达166分钟，条件复杂、耗时太长、耗流动相多、不易重现。（3）指纹图谱测定方法全程分别紫外208nm、266nm检测和ELSD检测，得到3张图谱，色谱峰信息重叠较多，重复测定，给分析带来不便。（4）指纹图谱所提供的化学信息不够，且所提供的色谱峰归属较少，仅有毛蕊异黄酮-7-O-β-D葡萄糖苷、黄芪甲苷两个均归属于黄芪的成分得到指认，尚无指认归属于党参的色谱峰成分。In-depth research on the quality control methods of traditional Chinese medicine injections is always the most important issue to ensure its stability and safety in clinical use. In published patent applications No. 200610098953.1 and No. 200710136382.0, reports on the HPLC fingerprint of Shenqi Fuzheng Injection were reported. The disadvantages of the methods described in these two patent application publications are: (1) The test sample should be prepared through steps such as separation and purification of AB-8 macroporous resin column or extraction with saturated n-butanol for 4 times followed by concentration, which is complicated It is cumbersome, and the high-temperature concentration step is likely to cause composition changes and affect the reproducibility of the method. The determination of phenolic acids with UV absorption and saponins without UV absorption requires the same cumbersome method to prepare the test solution respectively. The method has the disadvantages of long time consumption and the use of many organic solvents. (2) Fingerprints are measured using up to 5 nonlinear gradients. The same conditions are used to measure phenolic acids with UV absorption and saponins without UV absorption. The time for one measurement is as long as 166 minutes. It is complicated, takes too long, consumes a lot of mobile phase, and is not easy to reproduce. (3) The determination method of the fingerprint spectrum was measured by ultraviolet 208nm, 266nm and ELSD detection respectively in the whole process, and three spectra were obtained. The chromatographic peak information overlapped a lot, and the repeated measurement brought inconvenience to the analysis. (4) The chemical information provided by the fingerprints is insufficient, and the chromatographic peaks provided are few. Only calycosin-7-O-β-D glucoside and astragaloside IV are identified as components of Astragalus , there is no chromatographic peak component assigned to Codonopsis pilosula.
综上所述，现有公开的参芪扶正注射液指纹图谱专利具有较多缺点。To sum up, the existing published patents on fingerprints of Shenqi Fuzheng Injection have many shortcomings.
发明内容Contents of the invention
本发明的目的是克服上述现有技术中的不足，对参芪扶正注射液HPLC指纹图谱进行研究，提供一种操作简便、快速、重现性好的参芪扶正注射液的HPLC指纹图谱构建方法。该方法采用样品在线前处理、简单的HPLC非线性梯度洗脱，在一个流动相系统利用紫外检测波长切换及DAD -ELSD检测器串联的方式检测，一次进样分析得到一张完整的指纹图谱，即可包涵参芪扶正注射液中各类化学成分的信息。本发明所建立的参芪扶正注射液HPLC指纹图谱能全面地反映参芪扶正注射液的整体质量特征，可依据成品、半成品的指纹图谱变化来追根溯源寻找工艺操作中的问题，该指纹图谱化学成分首次得到99%(已知峰占共有峰峰面积比例)以上的指认，可进一步运用于建立参芪扶正注射液谱效学质量控制新模式的研究，从而全面控制产品的质量。因此，新建立的参芪扶正注射液HPLC指纹图谱方法具有普遍实用性。The purpose of the present invention is to overcome the deficiencies in the above-mentioned prior art, to study the HPLC fingerprint of Shenqi Fuzheng Injection, and to provide a method for constructing the HPLC fingerprint of Shenqi Fuzheng Injection that is easy to operate, fast and reproducible . The method uses online sample pretreatment, simple HPLC nonlinear gradient elution, and uses UV detection wavelength switching and DAD in a mobile phase system.- ELSD detectors are used in series for detection, and a complete fingerprint can be obtained from one sample injection analysis, which can contain the information of various chemical components in Shenqi Fuzheng Injection. The HPLC fingerprint of Shenqi Fuzheng Injection established by the present invention can fully reflect the overall quality characteristics of Shenqi Fuzheng Injection, and can be traced back to the source to find problems in the process operation according to the changes in the fingerprints of finished and semi-finished products. For the first time, the ingredients have been identified above 99% (the proportion of known peaks to the total peak area), which can be further used in the research of establishing a new spectral quality control model of Shenqi Fuzheng Injection, so as to fully control the quality of the product. Therefore, the newly established method for HPLC fingerprinting of Shenqi Fuzheng Injection has universal practicability.
本发明的目的可以通过以下技术方案实现：这种参芪扶正注射液HPLC指纹图谱的构建方法，可采用以下步骤：The object of the present invention can be realized through the following technical solutions: the construction method of this Shenqi Fuzheng Injection HPLC fingerprint collection can adopt the following steps:
取参芪扶正注射液样品经0.45μm微孔滤膜滤过，精密吸取75μl，注入双泵双梯度高效液相色谱仪测定。色谱条件为采用十八烷基硅烷键合硅胶为填充剂的色谱柱，柱温30℃；以乙腈-水为流动相，梯度洗脱，流速为0.6ml/min；以紫外检测器与蒸发光散射检测器串联检测，且紫外检测波长由266nm /208nm切换的方式检测；理论塔板数按毛蕊异黄酮-7-O-β-D-葡萄糖苷峰计应不低于3000。The sample of Shenqi Fuzheng Injection was filtered through a 0.45 μm microporous membrane, 75 μl was precisely drawn, and injected into a double-pump double-gradient high-performance liquid chromatograph for determination. The chromatographic conditions are a chromatographic column using octadecylsilane bonded silica gel as a filler, the column temperature is 30°C; acetonitrile-water is used as the mobile phase, gradient elution, the flow rate is 0.6ml/min; Scattering detectors are connected in series for detection, and the ultraviolet detection wavelength is switched from 266nm to 208nm; the number of theoretical plates should not be less than 3000 based on the peak of calycosin-7-O-β-D-glucoside.
取腺嘌呤、5-羟甲基糠醛对照品适量，加水制成每1ml含腺嘌呤、5-羟甲基糠醛各0.01mg的混合对照品溶液；取毛蕊异黄酮-7-O-β-D-葡萄糖苷、党参炔苷、黄芪甲苷对照品适量，加甲醇制成每1ml含毛蕊异黄酮-7-O-β-D-葡萄糖苷0.02mg、党参炔苷0.05mg、黄芪甲苷0.06mg的混合对照品溶液。对以上对照品溶液按前述方法检测得到对照品溶液的色谱图。Take an appropriate amount of adenine and 5-hydroxymethylfurfural reference substances, add water to make a mixed reference solution containing 0.01 mg of adenine and 5-hydroxymethylfurfural per 1 ml; take calycosin-7-O-β-D- Appropriate amount of glucoside, tangshenoside, and astragaloside IV reference substance, add methanol to make a mixture containing calycosin-7-O-β-D-glucoside 0.02mg, tangshenoside 0.05mg, and astragaloside IV per 1ml Reference substance solution. The above reference substance solution is detected by the aforementioned method to obtain the chromatogram of the reference substance solution.
对样品的前处理是运用双泵双梯度高效液相色谱仪，通过阀切换系统实现样品的在线自动净化。The pretreatment of the sample is to use the dual-pump dual-gradient high-performance liquid chromatography to realize the online automatic purification of the sample through the valve switching system.
所述色谱柱包括以十八烷基硅烷键合硅胶为填充剂的预柱和分析柱，通过柱切换技术，实现对参芪扶正注射液样品的自动化前处理及分析。The chromatographic column includes a pre-column and an analytical column with octadecylsilane bonded silica gel as filler, and realizes automatic pretreatment and analysis of Shenqi Fuzheng injection samples through column switching technology.
所述流动相的洗脱梯度如下，其中0～40分钟为指纹图谱采集及分析时间，40～60分钟为洗柱时间，记录40分钟色谱图：The elution gradient of the mobile phase is as follows, wherein 0 to 40 minutes is the fingerprint collection and analysis time, 40 to 60 minutes is the column washing time, and the chromatogram is recorded for 40 minutes:
所述参芪扶正注射液HPLC指纹图谱的构建方法中运用紫外检测波长266nm /208nm切换、紫外检测器(UVD或DAD)-蒸发光散射检测器(ELSD)、在同一流动相系统下、先后串联的方式检测，一次进样分析，得到参芪扶正注射液中的各类成分信息。其中，根据参芪扶正注射液成分的紫外吸收特征，1～9号共有峰在紫外266nm下检测；10～17号共有峰在紫外208nm下检测，18～21号皂苷类共有峰采用蒸发光散射检测器检测。以[9]号峰毛蕊异黄酮-7-O-β-D-葡萄糖苷为参照，各峰的保留时间与[9]号峰保留时间的比值分别为0.14、0.18、0.25、0.27、0.33、0.41、0.66、0.75、1.00、1.08、1.10、1.12、1.21、1.29、1.31、1.37、1.41、1.53、1.59、1.63、1.69。In the construction method of the HPLC fingerprint of Shenqi Fuzheng Injection, ultraviolet detection wavelength 266nm/208nm switching, ultraviolet detector (UVD or DAD)-evaporative light scattering detector (ELSD), under the same mobile phase system, successively connected in series The way of detection, one sample analysis, to obtain the information of various components in Shenqi Fuzheng Injection. Among them, according to the ultraviolet absorption characteristics of the components of Shenqi Fuzheng Injection, the common peaks of No. 1 to No. 9 were detected under ultraviolet 266 nm; the common peaks of No. 10 to 17 were detected under ultraviolet 208 nm; detector detection. Taking peak [9] as reference, the ratios of the retention time of each peak to peak retention time of [9] are 0.14, 0.18, 0.25, 0.27, 0.33, 0.41 , 0.66, 0.75, 1.00, 1.08, 1.10, 1.12, 1.21, 1.29, 1.31, 1.37, 1.41, 1.53, 1.59, 1.63, 1.69.
供试品指纹图谱采用《中药色谱指纹图谱相似度评价系统2009版》进行评价，相似度均大于0.90。The fingerprints of the test samples were evaluated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine 2009 Edition", and the similarities were all greater than 0.90.
本发明的目的还可以通过以下措施来达到：这种利用权利要求书1所述参芪扶正注射液HPLC指纹图谱的构建方法，通过对10批参芪扶正注射液进行检测所得到的HPLC指纹图谱并进行分析比较，找出其共有特征峰，得到具有21个共有峰的参芪扶正注射液HPLC标准指纹图谱。其中，归属于党参的特征峰为[1]、 [5]、[7]、[10]、[13] 5个峰；归属于黄芪的特征峰为[3]、 [8]、[9]、[11]、[12]、[14]、[15]、[16]、[17]、[18]、[19]、[20]、[21]13个峰，另有党参、黄芪共有的色谱峰3个，分别是 [2]、 [4]、[6]号峰。所述图谱的图形如说明书附图中的图1所示。The object of the present invention can also be achieved by the following measures: the method for constructing the HPLC fingerprints of the Shenqi Fuzheng Injection described in
利用对照品对照法确证3号峰为腺嘌呤、5号峰5-羟甲基糠醛、9号峰为毛蕊异黄酮-7-O-β-D-葡萄糖苷、13号峰为党参炔苷、14号峰为芒柄花苷、20号峰为黄芪甲苷。Using the reference substance control method, it was confirmed that peak No. 3 was adenine, peak No. 5 was 5-hydroxymethylfurfural, peak No. 9 was calycosin-7-O-β-D-glucoside, peak No. 13 was tangsheynoside, peak No. 14 The peak number is formononetin, and the
利用UFLC-DAD-MS/MS技术手段对共有峰进行色谱峰归属及峰纯度检查，紫外吸收曲线的比较，并通过质谱中分子离子峰、碎片离子峰的精确分子量匹配、保留时间匹配、同位素峰匹配等信息的比较指证：UFLC-DAD-MS/MS technology is used to check the chromatographic peak assignment and peak purity of the common peaks, the comparison of the ultraviolet absorption curve, and the precise molecular weight matching, retention time matching, and isotope peaks of the molecular ion peaks and fragment ion peaks in the mass spectrum Comparison evidence of matching and other information:
266nm：1号峰为胞苷、2号峰为尿嘧啶、3号峰为腺嘌呤、4号峰为鸟苷、5号峰为5-羟甲基糠醛、6号峰腺苷、7号峰为党参苷Ⅱ、9号峰为毛蕊异黄酮-7-O-β-D-葡萄糖苷；266nm:
208nm：10号峰为党参炔-二-葡萄糖苷、11号峰为异微凸剑叶莎醇-2`，7-二-O-葡萄糖苷、12号峰为樱花苷、13号峰为党参炔苷、14号峰为芒柄花苷、15号峰为5-羟基-4`-甲氧基-二氢黄酮-2-α-L-鼠李糖-β-D-葡萄糖苷、16号峰为9,10-二甲氧基紫檀烷-3- O-β-D-葡萄糖苷、17号峰为异微凸剑叶莎醇-7-O-葡萄糖苷；208nm: Peak No. 10 is Codonopsis-di-glucoside, Peak No. 11 is Isopicophyllol-2`, 7-di-O-glucoside, Peak No. 12 is Saranin, and Peak No. 13 is Codonopsis Codonopsis Alkyne glycoside, No. 14 peak is formononetin, No. 15 peak is 5-hydroxy-4`-methoxy-dihydroflavone-2-α-L-rhamnose-β-D-glucoside, No. 16 The peak is 9,10-dimethoxypteranane-3-O-β-D-glucoside, and the peak No. 17 is isopicophyllol-7-O-glucoside;
ELSD：18号峰为黄芪皂苷Ⅵ、19号峰为黄芪皂苷Ⅶ、20号峰为黄芪甲苷、21号峰为黄芪皂苷Ⅱ。ELSD: Peak No. 18 is astragaloside VI, peak No. 19 is astragaloside VII, peak No. 20 is astragaloside IV, and peak No. 21 is astragaloside II.
以上色谱峰构成了参芪扶正注射液的指纹图谱特征，可以完整的对参芪扶正注射液所含的2味植物药材作出色谱鉴定。The above chromatographic peaks constitute the fingerprint characteristics of Shenqi Fuzheng Injection, which can completely identify the two herbal medicinal materials contained in Shenqi Fuzheng Injection.
本发明相比现有技术具有如下优点：（1）样品前处理方法为在线自动前处理，方法简单、净化效果好、重现性好。与现有技术相比，节省前处理时间，减少了有机溶剂的使用，而且最大程度地保持了原有样品的品质，有助于更准确地对样品进行分析。（2）仅用3个HPLC非线性梯度洗脱在一个流动相系统下利用紫外检测波长切换技术，紫外检测器与蒸发光散射检测器串联技术，一次测定所得到的一张图谱即可同时检出复方中两味药材的各类成分，且分析时间仅为现有技术的1/4，流动相流速仅为0.6ml/min，方法更方便、快捷、环保。（3）该法建立的指纹图谱所提供的化学信息丰富，已归属的20个共有峰占所得到的参芪扶正注射液HPLC指纹图谱21个共有峰峰面积的99%以上，能够表达该产品的特征，具有良好的专属性。（4）该法针对成品所含有效成分的结构特点及理化特征，对样品的前处理方法、色谱柱、检测波长、流动相洗脱程序、柱温、流速等条件进行了筛选和优化，选用的方法易于推广、便于执行，具有良好的可行性。（5）在该完善的指纹图谱基础上，有助于开展指纹图谱信息与药效活性信息的相关性研究，从而深入地阐明产品的内在化学成分与该制剂疗效的相关性。Compared with the prior art, the present invention has the following advantages: (1) The sample pretreatment method is an online automatic pretreatment method, the method is simple, the purification effect is good, and the reproducibility is good. Compared with the prior art, the pretreatment time is saved, the use of organic solvents is reduced, and the quality of the original sample is maintained to the greatest extent, which helps to analyze the sample more accurately. (2) Using only 3 HPLC nonlinear gradient elutions, using the UV detection wavelength switching technology, the UV detector and the evaporative light scattering detector series technology under one mobile phase system, one spectrum obtained by one measurement can be detected simultaneously. Various components of the two medicinal materials in the compound prescription are produced, and the analysis time is only 1/4 of the prior art, and the flow rate of the mobile phase is only 0.6ml/min. The method is more convenient, fast and environmentally friendly. (3) The chemical information provided by the fingerprint established by this method is rich, and the assigned 20 common peaks account for more than 99% of the area of the 21 common peaks of the obtained Shenqi Fuzheng Injection HPLC fingerprint, which can express the product characteristics, with good specificity. (4) According to the structural characteristics and physical and chemical characteristics of the active ingredients contained in the finished product, the method screened and optimized the sample pretreatment method, chromatographic column, detection wavelength, mobile phase elution program, column temperature, flow rate and other conditions. The method is easy to promote, easy to implement, and has good feasibility. (5) On the basis of the perfect fingerprint, it is helpful to carry out the correlation research between the fingerprint information and the efficacy and activity information, so as to deeply clarify the correlation between the internal chemical composition of the product and the efficacy of the preparation.
综上所述，本发明制作该指纹图谱的方法简单、准确可靠、能更全面地反映参芪扶正注射液的物质基础与质量，适用于参芪扶正注射液的质量控制，也适用于进一步的质量研究，具有良好的实用性。In summary, the method for making the fingerprint of the present invention is simple, accurate and reliable, and can more comprehensively reflect the material basis and quality of Shenqi Fuzheng Injection, and is applicable to the quality control of Shenqi Fuzheng Injection, and is also applicable to further Quality research, with good practicality.
附图说明Description of drawings
图1是本发明的参芪扶正注射液标准指纹图谱，图中1～9号共有峰在紫外266nm下检测；10～17号共有峰在紫外208nm下检测；18～21号皂苷类共有峰采用蒸发光散射检测器检测；从左到右的分别是特征峰1至21。Fig. 1 is the standard fingerprint of Shenqi Fuzheng Injection of the present invention, among the figure No. 1～9 common peaks are detected under ultraviolet 266nm; No. 10～17 common peaks are detected under ultraviolet 208nm; No. 18～21 saponins common peaks adopt Detection by evaporative light scattering detector; from left to right are
图2是参芪扶正注射液、党参药材对照色谱图，图中从左到右的数字所指为参芪扶正注射液中归属于党参的色谱峰的峰号。Figure 2 is the chromatograms of Shenqi Fuzheng Injection and Codonopsis Codonopsis.
图3是参芪扶正注射液、黄芪药材对照色谱图，图中从左到右的数字所指为参芪扶正注射液中归属于黄芪的色谱峰的峰号。Figure 3 is the chromatogram of Shenqi Fuzheng Injection and Radix Astragali.
图4是参芪扶正注射液成品与标准指纹图谱相似度示意图。Figure 4 is a schematic diagram of the similarity between the finished product of Shenqi Fuzheng Injection and the standard fingerprint.
具体实施方式Detailed ways
下面结合具体的实施例对本发明作进一步说明。 The present invention will be further described below in conjunction with specific examples.
实施例Example11：参芪扶正注射液: Shenqi Fuzheng InjectionHPLCHPLC指纹图谱的构建方法及参芪扶正注射液标准指纹图谱Construction method of fingerprints and standard fingerprints of Shenqi Fuzheng Injection
1 仪器与试药1 Instruments and reagents
1.1 仪器：美国戴安公司Dionex Ultimate 3000 DGLC高效液相色谱仪(DGP-3600SD双三元泵、SRD-3600脱气机、WPS-3000SL自动进样器、TCC3000-RS柱温箱、DAD检测器、Chromeleon6.8数据处理软件)、ELSD检测器(法国SEDERE公司，SEDEX 75型)；预处理色谱柱：戴安Dionex Acclaim® Polar Advantage C18(3μm，50 mm×3.0mm)；分析色谱柱：安捷伦Agilent ZORBAX Eclipse Plus C18 (3.5μm，150 mm×3.0mm)。1.1 Instrument: Dionex Ultimate from Dionex, USA3000 DGLC high performance liquid chromatography (DGP-3600SD double ternary pump, SRD-3600 degasser, WPS-3000SL autosampler, TCC3000-RS column thermostat, DAD detector, Chromeleon6.8 data processing software), ELSD detector (French SEDERE company, SEDEX 75 type); pretreatment chromatographic column: Diane DionexAcclaim® Polar Advantage C18 (3 μm, 50 mm×3.0mm); analytical chromatographic column: Agilent ZORBAX Eclipse Plus C18 (3.5 μm, 150 mm×3.0mm).
1.2 试药：10批成品及对照药材提取物，均由丽珠医药集团利民制药厂提供。实验中液相色谱所用试剂乙腈均为色谱纯，其余所用试剂均为分析纯，水为超纯水。1.2Test drug: 10 batches of finished products and control medicinal material extracts are provided by Limin Pharmaceutical Factory of Livzon Pharmaceutical Group. The acetonitrile reagent used in the liquid chromatography in the experiment is chromatographically pure, the rest of the reagents used are analytically pure, and the water is ultrapure water.
对照品Reference substance
2 方法与结果2 Methods and results
2.1 取参芪扶正注射液经0.45μm微孔滤膜滤过，精密吸取续滤液75μl，注入Dionex Ultimate 3000 DGLC双泵双梯度高效液相色谱仪测定；以Dionex Acclaim® Polar Advantage C18(3μm，50 mm×3.0mm)为预处理柱，以Agilent ZORBAX Eclipse Plus C18 (3.5μm，150 mm×3.0mm)为分析柱，柱温30℃；运用样品在线前处理模式，样品从进样器进样后，由装载泵将预处理流动相2%乙腈带入预处理柱，并于上样后1.2min将阀切换至与分析柱相接状态，分析泵用分析流动相将被测组分正冲(与预处理柱同向)入分析柱分离分析。采用DAD检测器-ELSD检测器串联的方式进行图谱采集，其中，0～33min采用DAD检测，检测波长0～23.5min为266nm，于23.5min后将波长切换为208nm；33～40min采用ELSD检测，ELSD检测参数为N2的压力为3.5bar，漂移管温度为60℃，增益Gain值为10；以乙腈为流动相A，以水为流动相B，按下表规定进行梯度洗脱，记录40分钟的色谱图，流速为0.6ml/min。理论板数按毛蕊异黄酮-7-O-β-D-葡萄糖苷计，应不低于3000。2.1 Take Shenqi Fuzheng Injection and filter it through a 0.45 μm microporous filter membrane, accurately absorb 75 μl of the filtrate, inject it into Dionex Ultimate 3000 DGLC dual-pump dual-gradient high-performance liquid chromatography; use Dionex Acclaim® Polar Advantage C18 (3 μm, 50 mm×3.0mm) as the pretreatment column, Agilent ZORBAX Eclipse Plus C18 (3.5μm, 150 mm×3.0mm) as the analytical column, the column temperature is 30°C; using the online sample pretreatment mode, the sample is injected from the injector After the sample is loaded, the pretreatment
2.2取参芪扶正注射液的党参提取液、黄芪提取液，分别用水稀释至与成品浓度相当后，经0.45μm微孔滤膜滤过，取续滤液，。按前述方法检测得到对照药材的色谱图。2.2 Take the extract of Codonopsis pilosula and Radix Astragali of Shenqi Fuzheng Injection, dilute with water to the concentration equivalent to the finished product, filter through a 0.45 μm microporous membrane, and take the subsequent filtrate. The chromatograms of the reference medicinal materials were detected by the aforementioned method.
2.3取腺嘌呤、5-羟甲基糠醛对照品适量，加水制成每1ml含腺嘌呤、5-羟甲基糠醛各0.01mg的混合对照品溶液A；取毛蕊异黄酮-7-O-β-D-葡萄糖苷、党参炔苷、黄芪甲苷对照品适量，加甲醇制成每1ml含毛蕊异黄酮-7-O-β-D-葡萄糖苷0.02mg、党参炔苷0.05mg、黄芪甲苷0.06mg的混合对照品溶液B。按前述方法检测得到对照品溶液的色谱图。2.3 Take an appropriate amount of adenine and 5-hydroxymethylfurfural reference substances, add water to make a mixed reference solution A containing 0.01mg each of adenine and 5-hydroxymethylfurfural per 1ml; take calycosin-7-O-β- Appropriate amount of D-glucoside, tangshenoside, and astragaloside IV reference substances are made by adding methanol. Each 1ml contains calycocetin-7-O-β-D-glucoside 0.02mg, tangshenoside 0.05mg, and astragaloside IV 0.06mg The mixed reference solution B. Detect and obtain the chromatogram of reference substance solution by the aforementioned method.
2.4共有峰确定：对10批参芪扶正注射液进行分析，所得供试品溶液的指纹图谱应检出与党参、黄芪2味对照药材指纹图谱相同的色谱峰。采用《中药色谱指纹图谱相似度评价系统2009版》进行评价，共出现21个共有峰，列表如下。2.4 Determination of common peaks: Analyze 10 batches of Shenqi Fuzheng Injection, and the fingerprints of the obtained test solution should detect the same chromatographic peaks as the fingerprints of Codonopsis Codonopsis and Radix Astragali. Using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2009 Edition" for evaluation, a total of 21 common peaks appeared, the list is as follows.
所述参芪扶正注射液HPLC指纹图谱共检测出21个共有峰。以[9]号峰毛蕊异黄酮-7-O-β-D-葡萄糖苷为参照，各峰的保留时间与[9]号峰保留时间的比值分别为0.14、0.18、0.25、0.27、0.33、0.41、0.66、0.75、1.00、1.08、1.10、1.12、1.21、1.29、1.31、1.37、1.41、1.53、1.59、1.63、1.69。其中，归属于党参的特征峰为[1] 、 [5]、[7]、[10]、[13]号峰，共5个峰；归属于黄芪的特征峰为 [3]、[8]、[9]、[11]、[12]、[14]、[15]、[16]、[17]、[18]、[19]、[20]、[21]号峰，共13个峰，两者共有的有 [2]、 [4]、[6]号峰，共3个峰。所述图谱的图形如说明书附图中的图1所示。A total of 21 common peaks were detected in the HPLC fingerprint of the Shenqi Fuzheng Injection. Taking peak [9] as reference, the ratios of the retention time of each peak to peak retention time of [9] are 0.14, 0.18, 0.25, 0.27, 0.33, 0.41 , 0.66, 0.75, 1.00, 1.08, 1.10, 1.12, 1.21, 1.29, 1.31, 1.37, 1.41, 1.53, 1.59, 1.63, 1.69. Among them, the characteristic peaks belonging to Dangshen are [1], [5], [7], [10], [13], a total of 5 peaks; the characteristic peaks belonging to Astragalus are [3], [8] , [9], [11], [12], [14], [15], [16], [17], [18], [19], [20], [21], a total of 13 peaks There are three peaks [2], [4], and [6] in common between the two. The graph of the atlas is shown in Figure 1 in the accompanying drawings.
利用对照品对照法确证3号峰为腺嘌呤(Adenine)、5号峰为5-羟甲基糠醛(5-Hydroxymethyl- furaldehyde)、9号峰为毛蕊异黄酮-7-O-β-D-葡萄糖苷(Calycosin-7-O-β-D-glucopyranoside)、13号峰为党参炔苷(Lobetyolin)、14号峰为芒柄花苷(Ononin)、20号峰为黄芪甲苷(Astragaloside IV)。利用UFLC-DAD-MS/MS技术手段对共有峰进行色谱峰归属及峰纯度检查，紫外吸收曲线的比较，并通过质谱中分子离子峰、碎片离子峰的精确分子量匹配、保留时间匹配、同位素峰匹配等信息的比较指证：Use the reference substance control method to confirm that peak No. 3 is adenine (Adenine), peak No. 5 is 5-Hydroxymethylfurfural (5-Hydroxymethyl-furaldehyde), and peak No. 9 is calycosin-7-O-β-D-glucose Glycoside (Calycosin-7-O-β-D-glucopyranoside), No. 13 peak is Lobetolin, No. 14 peak is Ononin, No. 20 peak is Astragaloside IVIV). UFLC-DAD-MS/MS technology is used to check the chromatographic peak assignment and peak purity of the common peaks, the comparison of the ultraviolet absorption curve, and the precise molecular weight matching, retention time matching, and isotope peaks of the molecular ion peaks and fragment ion peaks in the mass spectrum Comparison evidence of matching and other information:
266nm：1号峰为胞苷(Cytidine)、2号峰为尿嘧啶(Uracil)、3号峰为腺嘌呤(Adenine)、4号峰为鸟苷(Guanosine)、5号峰为5-羟甲基糠醛(5-Hydroxymethyl- furaldehyde)、6号峰腺苷(Adenine nucleoside)、7号峰为党参苷Ⅱ(Tangshenoside II)、9号峰为毛蕊异黄酮-7-O-β-D-葡萄糖苷(Calycosin-7-O-β-D-glucopyranoside)；266nm: Peak No. 1 is Cytidine, Peak No. 2 is Uracil, Peak No. 3 is Adenine, Peak No. 4 is Guanosine, and Peak No. 5 is 5-hydroxymethyl 5-Hydroxymethyl-furaldehyde, 6th peak adenosine (Adeninenucleoside), peak No. 7 is Tangshenoside II, peak No. 9 is Calycosin-7-O-β-D-glucoside (Calycosin-7-O-β-D-glucopyranoside);
208nm：10号峰为党参炔-二-葡萄糖苷(Lobetyolinin)、11号峰为异微凸剑叶莎醇-2`，7-二-O-葡萄糖苷(Isomucronulatol-7,2'-di-O-glucoside)、12号峰为樱花苷(Sakuranin)、13号峰为党参炔苷(Lobetyolin)、14号峰为芒柄花苷(Ononin)、15号峰为5-羟基-4`-甲氧基-二氢黄酮-2-α-L-鼠李糖-β-D-葡萄糖苷(5-Hydroxy-2-O-deoxy-α-L-mannopyranosyl-β-D-glucopyranoside)、16号峰为9,10-二甲氧基紫檀烷-3- O-β-D-葡萄糖苷(9,10-Dimethoxypterocarpan-3-O-β-D-glucoside)、17号峰为异微凸剑叶莎醇-7-O-葡萄糖苷(Isomucronulator 7-O-glucoside)；208nm: Peak No. 10 is Lobeyolinin, and Peak No. 11 is Isomucronulatol-2`, 7-di-O-glucoside (Isomucronulatol-7,2'-di- O-glucoside), No. 12 peak is cherry blossom glycoside (Sakuranin), No. 13 peak is lobetyolin (Lobetolin), No. 14 peak is formononetin (Ononin), No. 15 peak is 5-hydroxy-4`-formazan 5-Hydroxy-2-O-deoxy-α-L-mannopyranosyl-β-D-glucopyranoside, peak 16 9,10-Dimethoxypterocarpan-3-O-β-D-glucoside (9,10-Dimethoxypterocarpan-3-O-β-D-glucoside), No. 17 peak is Isoconvex Alcohol-7-O-glucoside (Isomucronulator7-O-glucoside);
ELSD：18号峰为黄芪皂苷Ⅵ(Astragaloside VI)、19号峰为黄芪皂苷Ⅶ(Astragaloside VII)、20号峰为黄芪甲苷(Astragaloside IV)、21号峰为黄芪皂苷Ⅱ(Astragaloside II)。ELSD: Peak No. 18 is Astragaloside Ⅵ (AstragalosideVI), No. 19 peak is Astragaloside VII (AstragalosideVII), peak No. 20 is Astragaloside IV, peak No. 21 is Astragaloside II (Astragaloside II).
2.5指纹图谱精密度试验：取同一份参芪扶正注射液供试品溶液，连续进样6次，检测指纹图谱，采用《中药色谱指纹图谱相似度评价系统2009版》进行评价。结果显示仪器的精密度很好，相似度均大于0.99。相似度结果如下：2.5 Fingerprint precision test: Take the same Shenqi Fuzheng Injection test solution, inject 6 times continuously, detect the fingerprint, and use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2009 Edition" for evaluation. The results show that the precision of the instrument is very good, and the similarity is greater than 0.99. The similarity results are as follows:
DAD部分DAD section
ELSD部分ELSD section
2.6 指纹图谱稳定性试验：取同一份参芪扶正注射液供试品溶液，分别于制备后的0、2、4、8、10、12小时进样，检测指纹图谱，采用《中药色谱指纹图谱相似度评价系统2009版》进行评价。稳定性良好，相似度均大于0.99，表明供试品溶液在放置12小时内稳定。相似度结果如下：2.6 Fingerprint Stability Test: Take the same Shenqi Fuzheng Injection test solution, inject samples at 0, 2, 4, 8, 10, and 12 hours after preparation, and detect the fingerprints. Similarity Evaluation System 2009 Edition" for evaluation. The stability is good, and the similarity is greater than 0.99, showing that the test solution is stable within 12 hours. The similarity results are as follows:
DAD部分DAD section
ELSD部分ELSD section
2.7 指纹图谱重复性试验：取同一批参芪扶正注射液6 份制备成供试品溶液，分别进样，采用《中药色谱指纹图谱相似度评价系统2009版》进行评价。相似度均大于 0.99，方法重复性好。相似度结果如下：2.7 Fingerprint repeatability test: 6 parts of the same batch of Shenqi Fuzheng Injection were prepared into the test solution, injected separately, and evaluated using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2009 Edition". The similarities were all greater than 0.99, indicating good repeatability of the method. The similarity results are as follows:
DAD部分DAD section
ELSD部分ELSD section
2.8 中间精密度：精密称取同一批参芪扶正注射液，分别在不同日期、不同分析人员等变动因素条件下，依法测定，检测指纹图谱，采用《中药色谱指纹图谱相似度评价系统2009版》进行评价。表明该方法中间精密度好，参芪扶正注射液HPLC指纹图谱质量控制方法可行。相似度结果如下：2.8 Intermediate precision: Precisely weigh the same batch of Shenqi Fuzheng Injection, and measure it according to the law under the conditions of variable factors such as different dates and different analysts, and use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2009 Edition" Make an evaluation. It shows that the method has good intermediate precision and the quality control method of HPLC fingerprint of Shenqi Fuzheng Injection is feasible. The similarity results are as follows:
不同分析日期DAD部分Different analysis date DAD part
不同分析日期ELSD部分Different analysis date ELSD part
不同分析人员DAD部分Different analysts DAD section
不同分析人员ELSD部分Different analysts ELSD section
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| WO2014067478A1 (en)* | 2012-11-02 | 2014-05-08 | 丽珠集团利民制药厂 | Method for establishing shenqi fuzheng injection fingerprint spectrum |
| CN103913533A (en)* | 2013-01-09 | 2014-07-09 | 丽珠集团利民制药厂 | Method for determining chemical compositions in Shenqi Fuzheng injection and application thereof |
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| CN107941927A (en)* | 2017-10-18 | 2018-04-20 | 罗颂平 | A kind of method of UPLC/Q TOF MS measure lobetyolin content |
| CN107974491A (en)* | 2017-12-13 | 2018-05-01 | 湖北省农业科学院中药材研究所 | A kind of method using ISSR fingerprint identification Radix Codonopsis germ plasm resources |
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| CN108333280A (en)* | 2018-01-26 | 2018-07-27 | 上海上药第生化药业有限公司 | The separation method of nucleosides material and its application in a kind of Pericarpium Trichosanthis injection |
| CN109187791A (en)* | 2018-09-15 | 2019-01-11 | 丽珠集团利民制药厂 | A kind of method of ucleosides content in measurement ginseng and astragalus injection for strengthening body |
| CN109580858A (en)* | 2019-01-31 | 2019-04-05 | 中山大学 | The extraction of 5 kinds of gradient elutions and its content assaying method in a kind of wide dragon |
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| CN112147242A (en)* | 2020-08-14 | 2020-12-29 | 贵州师范大学 | Construction method of fingerprints of semi-finished and finished preparations of Zhenqi Fuzheng Capsules |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101352478A (en)* | 2007-07-26 | 2009-01-28 | 丽珠集团利民制药厂 | Quality control method of ginseng and astragalus injection for strengthening body |
- 2012
- 2012-03-27CNCN201210083789.2Apatent/CN102621244B/ennot_activeExpired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101352478A (en)* | 2007-07-26 | 2009-01-28 | 丽珠集团利民制药厂 | Quality control method of ginseng and astragalus injection for strengthening body |
Non-Patent Citations (1)
| Title |
|---|
| 盛龙生 等: "参芪扶正注射液成分分析", 《中国处方药》, vol. 8, no. 29, 31 December 2004 (2004-12-31)* |
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| CN107941927B (en)* | 2017-10-18 | 2020-11-17 | 广州中医药大学第一附属医院 | Method for determining lobetyolin content by UPLC/Q-TOF-MS |
| CN107991424A (en)* | 2017-12-07 | 2018-05-04 | 吉林师范大学 | The detection method of grub |
| CN107991424B (en)* | 2017-12-07 | 2019-10-01 | 吉林师范大学 | The detection method of grub |
| CN107974491A (en)* | 2017-12-13 | 2018-05-01 | 湖北省农业科学院中药材研究所 | A kind of method using ISSR fingerprint identification Radix Codonopsis germ plasm resources |
| CN108333280A (en)* | 2018-01-26 | 2018-07-27 | 上海上药第生化药业有限公司 | The separation method of nucleosides material and its application in a kind of Pericarpium Trichosanthis injection |
| CN109187791A (en)* | 2018-09-15 | 2019-01-11 | 丽珠集团利民制药厂 | A kind of method of ucleosides content in measurement ginseng and astragalus injection for strengthening body |
| CN109187791B (en)* | 2018-09-15 | 2021-09-21 | 丽珠集团利民制药厂 | Method for determining nucleoside content in Shenqi Fuzheng injection |
| CN109580858A (en)* | 2019-01-31 | 2019-04-05 | 中山大学 | The extraction of 5 kinds of gradient elutions and its content assaying method in a kind of wide dragon |
| CN110554127A (en)* | 2019-09-26 | 2019-12-10 | 山东大学 | method for constructing sweet dream oral liquid fingerprint and application thereof |
| CN110554127B (en)* | 2019-09-26 | 2021-02-02 | 山东大学 | Method for constructing sweet dream oral liquid fingerprint and application thereof |
| CN112147242A (en)* | 2020-08-14 | 2020-12-29 | 贵州师范大学 | Construction method of fingerprints of semi-finished and finished preparations of Zhenqi Fuzheng Capsules |
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