Characterization of the multiple components of Acanthopanax Senticosus stem by ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Acanthopanax Senticosus Harms. has been used widely in traditional Chinese medicine for the treatment of chronic bronchitis, neurasthenia, hypertension and ischemic heart disease. However, the in vivo constituents of the stem of Acanthopanax Senticosus remain unknown. In this paper, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry and the MarkerLynx^TM software combined with multiple data processing approach were used to study the constituents in vitro and in vivo. The aqueous extract from the Acanthopanax Senticosus stem and the compositions in rat serum after intragastric administration were completely analyzed. Consequently, 115 compounds in the aqueous extract from Acanthopanax Senticosus stem and 41 compounds absorbed into blood were characterized. Of the 115 compounds in vitro, 54 were reported for first time, including sinapyl alcohol, sinapyl alcohol diglucoside, and 1‐O‐sinapoyl‐β‐d ‐glucose. In the 41 compounds in vivo, 7 were prototype components and 34 were metabolites which were from 21 components of aqueous extract from Acanthopanax Senticosus stem, and the metabolic pathways of the metabolites were elucidated for first time. The results narrowed the range of screening the active components and provided a basis for the study of action mechanism and pharmacology.     

Qualitative analysis of major constituents from Xue Fu Zhu Yu Decoction using ultra high performance liquid chromatography with hybrid ion trap time‐of‐flight mass spectrometry

Xue Fu Zhu Yu Decoction, a famous formula that has been used for treating many blood stasis‐caused diseases for many centuries, comprises 11 kinds of traditional Chinese medicines. A convenient, efficient, and rapid analytical method was developed to simultaneously determine the major compounds in this decoction. An ultra‐high performance liquid chromatography with hybrid ion trap time‐of‐flight mass spectrometry method was used to rapidly separate and detect the major constituents of the decoction. Using this technique, we identified or tentatively identified 34 compounds, including 21 flavonoids, 5 terpenoids, 3 organic acids, 2 lactones, 1 alkaloid, 1 amino acid, and 1 cyanogenic glycoside. The MS analysis of these constituents was described in detail. Findings may contribute to future metabolic and pharmacokinetic studies of this medicine.     

Comparative analysis of the main bioactive components of Xin‐Sheng‐Hua granule and its single herbs by ultrahigh performance liquid chromatography with tandem mass spectrometry

Xin‐Sheng‐Hua granule, a representative formula for postpartum hemorrhage, has been used clinically to treat postpartum diseases. Its main bioactive components comprise aromatic acids, phthalides, alkaloids, flavonoids, and gingerols among others. To investigate the changes in main bioactive constituents in its seven single herbs before and after compatibility, a rapid, simple, and sensitive method was developed for comparative analysis of 27 main bioactive components by using ultrahigh‐performance liquid chromatography with triple quadrupole electrospray tandem mass spectrometry for the first time. The sufficient separation of 27 target constituents was achieved on a Thermo Scientific Hypersil GOLD column (100 mm × 3 mm, 1.9 μm) within 20 min under the optimized chromatographic conditions. Compared with the theoretical content, the observed content of each analyte showed remarkable differences in Xin‐Sheng‐Hua granule except thymine, p‐coumaric acid, senkyunolide I, senkyunolide H, and ligustilide; the total contents of 27 components increased significantly, and the content variation degrees for the different components were gingerols > flavonoids > aromatic acids > alkaloids > phthalides. The results could provide a good reference for the quality control of Xin‐Sheng‐Hua granule and might be helpful to interpret the drug interactions based on variation of bioactive components in formulae.     

超高效液相色谱-线性离子阱/静电场轨道阱质谱法快速检测化妆品中66种抗生素

采用超高效液相色谱-线性离子阱/静电场轨道阱高分辨质谱法同时测定化妆品中磺胺类、沙星类等66种抗生素类化合物,建立了快速筛查数据库和定量分析方法。待测物用乙腈超声提取,C18色谱柱（100 mm×2.1 mm,1.8 μm）分离,以0.1%（v/v）甲酸水溶液和乙腈为流动相进行梯度洗脱。在正离子模式下,以保留时间和一级母离子精确质量数进行快速筛查,以高能碰撞诱导解离获得的二级碎片离子精确质量数进行确证。结果表明,化合物的线性关系良好,线性相关系数（R²）>0.99;检出限（LOD）为2~4 μg/kg;定量限（LOQ）为5~10 μg/kg;3个添加水平（1LOQ、10LOQ、30LOQ）的平均回收率为58.2%~119.1%,相对标准偏差为1.03~11.9%。该方法简便快速、定性定量可靠,适用于化妆品中抗生素类化合物的快速筛查和定量检测。     

A rapid method for simultaneous determination of 52 marker compounds in Xiao‐Qing‐Long‐Tang by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry†

Xiao‐Qing‐Long‐Tang (XQLT) is a classical Chinese medicine formula. It is generally used for the treatment of common cold, bronchial asthma, and allergic rhinitis in Asia. In this study, a multicomponent quantification fingerprinting approach based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry has been developed for the analysis of compounds in XQLT in 14.5 min. A total of 52 compounds were identified by co‐chromatography of sample extract with authentic standards and comparing the retention time, UV spectra, molecular ions and characteristic fragment ions with those of authentic standards, or tentatively identified by MS^E determination along with Mass Fragment software. Moreover, the method was validated for the simultaneous quantification of 16 components in XQLT commercial products. The method is practical for comprehensive standardization of XQLT and holistic comparison of its commercial products from different manufacturers.     

Development of a method to screen and isolate bioactive constituents from Stellera chamaejasme by ultrafiltration and liquid chromatography combined with semi‐preparative high‐performance liquid chromatography and high‐speed counter current chromatography

A simple and efficient method based on ultrafiltration with liquid chromatography and mass spectrometry was used for the rapid screening and identification of ligands in the extracts of Stellera chamaejasme. The bound ligands, i.e. daphnoretin, isopimpinellin, chamaechromone, neochamaejasmin A, and chamaejasmine (purity of 96.8, 90.75, 91.41, 93.98, and 98.91%, respectively), were separated by semi‐preparative high‐performance liquid chromatography combined with high‐speed counter‐current chromatography. To the best of our knowledge, this is the first study to report the detection of potent lipoxidase and lactate dehydrogenase inhibitors in Stellera chamaejasme extracts. The results demonstrate that our method of ultrafiltration with liquid chromatography and mass spectrometry combined with mixed chromatography can be used to screen and confirm the bioactivity of all isolated compounds. This method also eliminates the need for separation of inactive compounds, thereby improving efficiency when studying bioactive substances. For some complex mixtures, neither semi‐preparative high‐performance liquid chromatography nor high‐speed counter‐current chromatography can purify all the target active compounds with high purity in a one‐step separation. The combination of the two methods allow for efficient purification of target bioactive compounds with different polarities and physicochemical properties based on their complementary properties.     

Characterization and identification of the metabolites of Menthae Haplocalycis Herba water extracts in rat plasma,urine, and feces by ultra‐high performance liquid chromatography with linear ion trap‐Orbitrap mass spectrometry

Menthae Haplocalycis Herba has been utilized for food and medicinal purposes in China for thousands of years. It has various efficacies, including dispelling wind and heat and relieving sore throat. M. Haplocalycis Herba has been also widely used in food, cosmetics, spices, and other fields. Exploring the constituents and detecting the metabolites of M. Haplocalycis are of great significance to clarify the effective substances. However, the in vivo metabolites of M. Haplocalycis Herba water extract are still unclear. Herein, a sensitive and specific method, ultra‐high performance liquid chromatography with linear ion trap‐Orbitrap mass spectrometry, established in this assay was used to study the metabolism of M. Haplocalycis Herba water extract in rat plasma, urine, and feces. We characterized and identified 9, 50, and 34 metabolites in plasma, urine, and feces, respectively. Seven metabolic pathways, including phase Ⅰ (isomerization, demethylation, hydroxylation, and dehydration) and phase Ⅱ (sulfation and glucuronidation) were mainly involved in the metabolism. It is the first systematic study on the metabolism of M. Haplocalycis Herba water extract in vivo, which enrich current understanding of the metabolic behavior of M. Haplocalycis Herba water extract and provide a metabolic rationale for further in‐depth in vivo biotransformation and pharmacokinetic analysis.     

基于诊断离子策略的超高压液相色谱-线性离子阱-轨道离子阱质谱联用技术解析中药虎杖的化学成分

建立了基于超高压液相色谱-电喷雾-高分辨质谱联用技术(UHPLC-ESI-HRMS)的中药虎杖化学成分的快速定性分析方法.采用Waters UPLC C₁₈色谱柱,乙腈-甲酸水系统梯度洗脱,采用ESI-LTQ-Orbitrap高分辨杂交质谱技术负离子采集,线性离子阱质谱进行多级质谱碎裂,轨道离子阱高分辨检测,获取多维质谱数据.利用代表性成分的诊断离子进行搜寻,结合质谱偶电子规律、氮规律、不饱和度和同位素峰等信息,参阅文献,总结主要成分的裂解规律,对主要峰进行指认与鉴定.结果显示,整个分析过程在10 min内完成,该方法分离良好,灵敏度高.对虎杖根中的34种成分进行了结构分析,研究从中药虎杖中发现了多种酚酸取代二苯乙烯苷、酚酸取代蒽醌苷和蒽醌硫酸盐衍生物等成分.研究结果表明本方法为虎杖的后续化学成分研究、相关产品的质量控制和药效物质基础的研究提供了一种高效、可靠的检测手段.     

Systematic chemical profiling of a multicomponent Chinese herbal formula Huo Luo Xiao Ling Dan by ultra high performance liquid chromatography coupled with electrospray ionization quadrupoletime‐of‐flight mass spectrometry

Huo Luo Xiao Ling Dan, a Chinese herbal formula consisting of 11 different herbs, has been used in folk medicine for the treatment of arthritis and other chronic inflammatory diseases. However, the chemical compositions of Huo Luo Xiao Ling Dan are not completely characterized. In the present study, an ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry method in positive and negative ion modes was employed to identify biochemical constitutes in Huo Luo Xiao Ling Dan. As a result, a total of 76 compounds including alkaloids, monoterpene glycosides, iridoids, phenolic acids, and tanshinones, coumarins, lactones, flavones, and their glycosides, triterpenes, and triterpene saponins were characterized by comparing the retention time and mass spectrometry data with reference standards within 5 ppm error or by reference to the reference literature. These results would provide the basis for a further in vivo study of Huo Luo Xiao Ling Dan and information for potential new drug candidates for treating arthritis and other chronic inflammatory diseases.     

Multi‐constituent determination and fingerprint analysis of Scutellaria indica L. using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

An ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method integrating multi‐constituent determination and fingerprint analysis has been established for quality assessment and control of Scutellaria indica L. The optimized method possesses the advantages of speediness, efficiency, and allows multi‐constituents determination and fingerprint analysis in one chromatographic run within 11 min. 36 compounds were detected, and 23 of them were unequivocally identified or tentatively assigned. The established fingerprint method was applied to the analysis of ten S. indica samples from different geographic locations. The quality assessment was achieved by using principal component analysis. The proposed method is useful and reliable for the characterization of multi‐constituents in a complex chemical system and the overall quality assessment of S. indica.     

Targeted characterization of acylated compounds from Scrophulariae Radix using liquid chromatography coupled with Orbitrap mass spectrometry and diagnostic product ion‐based data analysis

Acylated compounds are often present in herbal medicines. In this study, a diagnostic product ion‐based strategy was established to comprehensively characterize acylated compounds in Scrophulariae Radix. After untargeted data acquisition using ultra‐high performance liquid chromatography coupled with Orbitrap mass spectrometry, the data were processed by three‐stage diagnostic product ions. First, diagnostic product ions corresponding to the acyl groups (cinnamoyl, p‐coumaroyl, feruloyl, and caffeoyl) were used to search 90 compounds. Second, these compounds were divided into three categories using diagnostic product ions for phenylethanoid glycosides, iridoid glycosides, and phenylpropanoids, respectively. Last, the linkage position of the acyl group to iridoid glycosides was discriminated via the third‐stage diagnostic product ions. As a result, 90 acylated compounds were characterized, and 37 of them were reported from Scrophulariae Radix for the first time.     

Qualitative and quantitative determination of Atractylodes rhizome using ultra‐performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometry with data‐dependent processing

A quick and effective workflow based on ultra‐performance liquid chromatography coupled with electron spray ionization and LTQ‐Orbitrap mass spectrometry (UPLC‐LTQ‐Orbitrap MS) was established for compositional analysis and screening of the characteristic compounds of three species of Atractylodes rhizome for quality evaluation. This technique was employed to determine the seven main components in Atractylodes rhizome samples. Ultimately, 78 constituents were identified; of these, seven characteristic compounds were selected for species discrimination, comprising atractylodin (63), atractylenolide I (43), atractylenolide II (49), atractylenolide III (53), atractylon (69), methyl‐atractylenolide II (54) and (4E,6E,12E)‐tetradecadecatriene‐8,10‐diyne‐1,3‐diacetate (59). The seven main compounds, including six characteristic compounds, were simultaneously determined in 29 batches of Atractylodes rhizome samples. Thus, the method validation showed acceptable results. Quantitative analysis showed significantly different contents of the seven main components among the three species of Atractylodes rhizome, which indicates possible distinctions in the pharmacological effects. This established method can simultaneously provide qualitative and quantitative results for compositional characterization of Atractylodes rhizomes and for quality control.     

Rapid separation and characterization of diterpenoid alkaloids in processed roots of Aconitum carmichaeli using ultra high performance liquid chromatography coupled with hybrid linear ion trap‐Orbitrap tandem mass spectrometry

The lateral root of Aconitum carmichaeli, a popular traditional Chinese medicine, has been widely used to treat rheumatic diseases. For decades, diterpenoid alkaloids have dominated the phytochemical and biomedical research on this plant. In this study, a rapid and sensitive method based on ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap tandem mass spectrometry was developed to characterize the diterpenoid alkaloids in Aconitum carmichaeli. Based on an optimized chromatographic condition, more than 120 diterpenoid alkaloids were separated with good resolution. Using a systematic strategy that combines high resolution separation, highly accurate mass measurements and a good understanding of the diagnostic fragment‐based fragmentation patterns, these diterpenoid alkaloids were identified or tentatively identified. The identification of these chemicals provided essential data for further phytochemical studies and toxicity research of Aconitum carmichaeli. Moreover, the ultra high performance liquid chromatography with linear ion trap‐Orbitrap mass spectrometry platform was an effective and accurate tool for rapid qualitative analysis of secondary metabolite productions from natural resources.     

Ultra‐fast liquid chromatography with tandem mass spectrometry determination of ochratoxin A in traditional Chinese medicines based on vortex‐assisted solid–liquid microextraction and aptamer‐affinity column clean‐up

A rapid, selective, and sensitive ultra‐fast liquid chromatography with tandem mass spectrometry method was developed for the determination of ochratoxin A in traditional Chinese medicines based on vortex‐assisted solid–liquid microextraction and aptamer‐affinity column clean‐up. Through optimizing the sample pretreatment procedures and chromatographic conditions, good linearity (r² ≥ 0.9993), low limit of detection (0.5–0.8 μg/kg), and satisfactory recovery (83.54–94.44%) expressed the good reliability and applicability of the established method in various traditional Chinese medicines. Moreover, the aptamer‐affinity column, prepared in‐house, showed an excellent feasibility owing to its specific identification of ochratoxin A in various kinds of selected traditional Chinese medicines. The maximum adsorption amount and applicability value were 188.96 ± 10.56 ng and 72.3%, respectively. The matrix effects were effectively eliminated, especially for m/z 404.2→358.0 of ochratoxin A. The application of the developed method for screening the natural contamination levels of ochratoxin A in 25 random traditional Chinese medicines on the market in China indicated that only eight samples were contaminated with low levels below the legal limit (5.0 μg/kg) set by the European Union. This study provided a preferred choice for the rapid and accurate monitoring of ochratoxin A in complex matrices.     

高效液相色谱-线性离子阱/静电场轨道阱高分辨质谱筛查和确证纺织品中的致癌染料

建立了高效液相色谱-线性离子阱/静电场轨道阱高分辨质谱(HPLC-LTQ/Orbitrap MS)筛查和确证生态纺织品中致癌染料的方法。样品在水浴(95 ℃)中用吡啶/水(1/1, v/v)振荡(150 r/min)提取,上清液过聚四氟乙烯(PTFE)滤膜后,CAPCELL PAK C₁₈色谱柱(100 mm×2.0 mm, 5 μm)分离,以乙腈和5 mmol/L乙酸铵水溶液(含0.01%甲酸)、乙腈和5 mmol/L乙酸铵水溶液分别作为正、负电喷雾离子化(ESI)模式的色谱流动相梯度洗脱。在m/z 200~800范围内进行一级质谱全扫描。以准分子离子峰的精确质量数和提取的色谱图峰面积进行筛查分析和定量,以保留时间和数据依赖扫描(data-dependent scan)模式获得的子离子质谱图进行定性确证。9种染料的质量准确度小于5×10^-6(5 ppm),线性良好,相关系数大于0.99,方法检出限为0.125~25 mg/kg。3个添加水平的回收率范围为62.13%~116.28%,相对标准偏差小于15%。应用该方法检测了棉、涤纶及混纺纤维等20余件纺织品样品中的致癌染料残留。该方法准确、可靠。     

基于精确质量数筛选的超高效液相色谱-线性离子阱/静电场轨道阱质谱法快速鉴别化妆品中的邻苯二甲酸酯

采用超高效液相色谱-线性离子阱/静电场轨道阱组合式高分辨质谱联用(UPLC-LTQ Orbitrap MS),结合邻苯二甲酸酯(PAEs)精确质量数,建立了快速筛选、定性识别化妆品中PAEs的分析方法。不同种类的化妆品样品经甲醇提取,Waters ACQUITY UPLC BEH C18色谱柱(100×2.1mm,1.7μm)分离,以乙腈-水(含5mmol/L乙酸铵和0.1%甲酸)为流动相梯度洗脱。通过UPLC-LTQ/Orbitrap MS的正离子模式全扫描分析,获得提取物中PAEs化合物母离子和主要碎片离子精确质量数,实现对化妆品的快速筛选。以保留时间和数据依赖扫描(Data Dependent Scan)模式获得的子离子质谱图进行定性确证。所发展的UPLC-LTQ Orbitrap MS方法分离度高、灵敏度好,所考察的14种常见PAEs的精确质量数相对偏差小于5.0×10-6,线性良好,相关系数大于0.99,14种PAEs的检出限在0.05~0.5mg/kg范围内,能满足化妆品实际样品的分析要求。对50种化妆品实际样品进行筛选,结果良好,说明该方法是化妆品中PAEs快速筛选、定性识别的有效方法。     

Determination and Fingerprint Analysis of Steroidal Saponins in roots of Liriope muscari (Decne.) L. H. Bailey by ultra high performance liquid chromatography coupled with ion trap time‐of‐flight mass spectrometry

Liriope muscari (Decne.) L. H. Bailey is a well‐known traditional Chinese medicine used for treating cough and insomnia. There are few reports on the quality evaluation of this herb partly because the major steroid saponins are not readily identified by UV detectors and are not easily isolated due to the existence of many similar isomers. In this study, a qualitative and quantitative method was developed to analyze the major components in L. muscari (Decne.) L. H. Bailey roots. Sixteen components were deduced and identified primarily by the information obtained from ultra high performance liquid chromatography with ion‐trap time‐of‐flight mass spectrometry. The method demonstrated the desired specificity, linearity, stability, precision, and accuracy for simultaneous determination of 15 constituents (13 steroidal glycosides, 25(R)‐ruscogenin, and pentylbenzoate) in 26 samples from different origins. The fingerprint was established, and the evaluation was achieved using similarity analysis and principal component analysis of 15 fingerprint peaks from 26 samples by ultra high performance liquid chromatography. The results from similarity analysis were consistent with those of principal component analysis. All results suggest that the established method could be applied effectively to the determination of multi‐ingredients and fingerprint analysis of steroid saponins for quality assessment and control of L. muscari (Decne.) L. H. Bailey.     

Characterization of the chemical constituents in Da‐Huang‐Gan‐Cao‐Tang by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry

In this work, the chemical constituents in Da‐Huang‐Gan‐Cao‐Tang, a traditional Chinese formula, were studied by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry for the first time. Among the 146 compounds detected in Da‐Huang‐Gan‐Cao‐Tang, 104 compounds were identified unambiguously or tentatively based on their accurate molecular weight and multistage MS data, including one potential novel compound and two reported in Glycyrrhiza genus for the first time. The possible fragmentation pathways were proposed and fragmentation rules of the major types of compounds were concluded. This study provided an example to facilitate the tedious identification of chemical composition in traditional Chinese medicine, and maybe a promising reference approach to research the analogous formulae.