Determination of carbamate insecticides in Chinese medicinal herbs by gas chromatography with a nitrogen-phosphorus detector

A simplified method for determining carbamate insecticides (including metolcarb, isoprocarb, fenobucarb, carbofuran, pirimicarb, and carbaryl) in Chinese medicinal herbs (White Peony Alba, Red Peony Root, and Baical Skullcap Root) is described. Standards were fortified into Chinese medicinal herbs at 3 levels (0.05-0.5 mg/kg). The carbamates were extracted with dichloromethane in a Soxhlet apparatus and analyzed by gas chromatography with a nitrogen-phosphorus detector. The results showed average recoveries between 80.77 and 104.56%. The method evidenced good robustness, accuracy, and precision for monitoring carbamates in Chinese medicinal herb samples, and it is a suitable alternative to replace the currently dedicated analytical systems. The minimum detectable amount ranged from 3.0 x 10(-10) to 5.0 x 10(-10)g, and the limit of quantification was 0.05 mg/kg. The method is rapid, simple, sensitive, and reproducible, and it can be conveniently used as a low-cost, rapid method for measuring the carbamate insecticide contamination of Chinese medicinal herbs.     

Quality transitivity of Danhong Huayu Koufuye: A study on chemical profiles of medicinal herbs,compound preparation and dosed rat plasma using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry

Danhong Huayu Koufuye (DHK), an effective Chinese medicine preparation, is mainly used for the treatment of blurred vision and sudden blindness caused by qi stagnation and blood stasis, as well as the absorption period of central retinal vein occlusion. However, the current quality standard is relatively low, only stipulating the content of protocatechualdehyde. Chemical transitivity is the basis for discovering quality markers and is used in quality process control of Chinese medicines. Herein, the chemical profiles of seven medicinal herbs, DHK and dosed rat plasma were comprehensively analyzed using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry. As a result, 134 chemical constituents were identified in seven medicinal herbs, including salvianolic acids, diterpene quinones, phenolic acids, phthalides, cyanogenic glycosides, flavonoids and triterpenoid saponins. Among them, 55 chemical constituents were transferred to DHK along with extraction and preparation, and 26 were further absorbed into blood and metabolized to produce 11 metabolites after oral administration. The transitivity of DHK from medicinal herbs to compound preparation and into blood was analyzed for the first time. This article will be valuable to ascertain the quality markers for quality process control and further pharmacokinetic studies.     

A novel approach to identify plant parasitic nematodes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Plant parasitic nematodes are difficult to identify because different species are morphologically similar, and this makes their control more difficult. The aim of this work was to develop a rapid, simple method to identify plant parasitic nematodes, based on analysis of protein profiles of nematodes generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Two methods have been used: grinding and direct analysis of intact nematodes. Both methods were standardised using the nematode Anguina tritici (wheat seed-gall nematode) as a model. Development of the approach involved optimisation of experimental parameters to generate reproducible diagnostic protein profiles for plant parasitic nematodes. With alpha-cyano-4-hydroxycinnamic acid (CHCA) as the matrix, the most effective solvent extraction was with 90% acetone. With sinapinic acid (SA) as matrix, 90% ethanol was most effective. When intact nematodes were analysed directly by mixing with the matrix solution, 40 min extraction with CHCA matrix solution generated the best protein profiles. The standardised methods were applied to analyse the seed-gall nematodes A. tritici and A. funesta and to the root-knot nematode, Meloidogyne javanica, which infects many horticultural crops. Typical protein profiles and diagnostic peaks were identified for these nematode species and for mixtures of Anguina species. The results provide 'proof-of-concept' that these nematode species can be identified by protein profiling using MALDI-TOFMS. This new approach could be extended to identify other plant and non-plant parasitic nematodes.     

Establishment of GC-MS fingerprint of fresh Houttuynia cordata

Fresh Houttuynia cordata THUNB. is a Chinese materia medica generally used in Chinese medicine therapy. It possesses the actions of clearing heat, eliminating toxins, reducing swelling, discharging pus and relieving stagnation. However, dry H. cordata has traditionally been used in clinical application instead of the fresh counterpart. In this paper, the chemical profiles of H. cordata were established using fingerprinting techniques. A modified GC-MS method was developed in the comparison of fingerprints among fresh and dry herbs of H. cordata. It was shown that the varieties, as well as relative levels of chemical components, in the fresh herb were more abundant than in the dry counterpart. Fingerprinting profiles were found to be consistent for fresh herbs acquired from various production areas, but the relative abundance of peaks were varied. Besides, the chemical components among different medicinal portions of fresh herbs were found to be inconsistent. The developed fingerprint can be successfully applied to distinguish between fresh and dry herbs, as well as determining differentiation among different medicinal portions.     

植物类中药中微量元素的因子分析和聚类分析

尝试利用化学计量学方法探讨微量元素含量与中药药性的相关性。对１０５味植物类中药４２种微量元素测定数据用因子分析和聚类分析进行了多因素分析。因子分析证实了一个１０因子模型合理解释这些微量元素间的相关关系；样本聚类分析证明了１０５株中药合理地聚类成不同组；     

Differentiation of herbs linked to "Chinese herb nephropathy" from the liquid chromatographic determination of aristolochic acids

A HPLC method was developed and applied to analyze aristolochic acids (AA-I and AA-II) in Chinese medicinal herbs. The herb samples were extracted by using ultrasonication with the extraction efficiency of better than 82%. Extracts were then filtered and injected onto a C18 column eluting under a gradient program using methanol and water-containing 0.5% acetic acid. The method with the detection limits of 1.33 ng for AA-I and 7.29 ng for AA-II per injection was successfully applied for the analysis of traditional Chinese medicine (TCM) and related products and differentiation of Chinese medicinal herbs that have previously been misused and caused toxicological effects. The developed protocol provided an example that analysis of selected component markers could serve for health security and quality control of TCM consumption.     

气相色谱-串联质谱法快速筛查测定中药材中144种农药残留

利用气相色谱-串联质谱(GC-MS/MS)检测技术,采用QuEChERS法作为样品前处理方法,建立了能应用于11种中药材中144种农药残留的检测方法。探究了样品前处理过程中提取溶剂、缓冲盐体系、净化剂组成和用量对样品提取、净化等方面的影响,最终确定了用乙腈提取,甲苯复溶,以混合净化剂净化,过有机膜后经GC-MS/MS测定,外标法定量。144种农药在10~2000 μg/kg之间线性关系良好,相关系数(r²)>0.983;除乙酰甲胺磷、灭虫威、西玛津、克菌丹、异狄氏剂、异菌脲外,其余农药的定量限(LOQ)均低于20 μg/kg;在20、50、200 μg/kg的添加水平下,除乙酰甲胺磷、艾氏剂和双甲脒回收率偏低外,其余141种农药的平均回收率在74.3%~111.8%之间,相对标准偏差(RSD)为0.5%~14.6%。与已有的标准方法对比,此方法不仅检测结果一致,而且高效、快速,准确性好,灵敏度高,适用于中药材中144种农药残留的快速筛查与定量分析。     

Hydrophilic interaction liquid chromatography with tandem mass spectrometry for the determination of underivatized dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) in Panax medicinal plant species

Dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) is a haemostatic agent present in important Chinese medicinal herbs such as Panax notoginseng, as well as other Panax species. It is also a reported neurotoxic agent found in Lathyrus sativus (grass pea seed). A selective analytical method incorporating hydrophilic interaction chromatography with positive electrospray ionization tandem mass spectrometry (HILIC/ESI-MS/MS), for the analysis of dencichine in Panax plant species, was developed. Using multiple reaction monitoring (MRM) mode, underivatized dencichine, a small and highly polar compound, was selectively detected and quantified. The contents of dencichine in raw and steamed Panax notoginseng roots, 11 pairs of raw and steamed P. notoginseng herbal products, Panax ginseng roots, and Panax quinquefolium roots, were analyzed and compared. Optimal sensitivity of 0.3 ppm (detection limit) and 1.5 ppm (quantification limit) was achieved. The method was rapid (< or =5 min), with the HILIC peak eluting at about 1 min. Steamed P. notoginseng samples were found to contain less dencichine than the corresponding raw samples, and there were also differences among the three Panax species; raw P. ginseng and P. quinquefolium contained less dencichine than the raw P. notoginseng species. This rapid and specific method may be applied to the quantification of dencichine in complex medicinal plants and their products.     

Development of direct microwave desorption/gas chromatography mass spectrometry system for rapid analysis of volatile components in medicinal plants

A new direct microwave desorption–gas chromatography‐mass spectrometry method was developed for the analysis of the essential oils of medicinal plants. A homemade direct microwave desorption system was fabricated and used for the desorption of volatile components of medicinal herbs. The desorbed volatiles are transferred directly into the gas chromatography injector for analysis in a one‐step process. Approximately 0.3 g of the herb was needed for the desorption of samples in 60 s. In this study, more than 53 volatile compounds were identified and quantified for Echinophora platyloba DC as model herb sample. The results were found to be in good agreement with the conventional hydrodistillation extraction data. The described results show that direct microwave desorption is fast, simple, and easy to automate and requires only a small amount of sample. The results indicate that essential oil components valuable for varietal identification and characteristic of each variety analyzed when direct microwave desorption–gas chromatography‐mass spectrometry was used for analysis.     

Application of mid-infared spectroscopy with multivariate analysis for the discrimination of toxic plant,Gelsemium elegans

Gelsemium elegans is a commonly used herb to treat different kinds of diseases. However, the indole alkaloid present in the plant might cause serious side effects. In this research, the infrared spectroscopic identification approach including Fourier transform infrared spectroscopy (FT-IR), Second derivative infrared spectra (SD-IR) and two-dimensional correlation infrared spectra (2D-IR) was used to develop a simple and rapid method to discriminate the stem, leaf and root of the Gelsemium elegans plant. This is because the stem, leaf and root contained different amount of indole alkaloid that contributed to the toxicity. Through this study, all the three parts were successfully identified and discriminated through the infrared spectroscopic identification method. The identification approach was also validated by comparing the samples of the mixture of both stem and root (SR) to the stem and root, respectively and also by comparing different plants with Gelsemium elegans plant. Besides that, all the samples of different parts of the Gelsemium elegans were analyzed with the Principal Component Analysis (PCA) and Soft Independent Modelling of Class Analogy (SIMCA) pattern recognition technique to test and verify the experimental results. The SIMCA model was validated by comparing 70 standard herbs to the model. From the results, macroscopic IR fingerprint method and the classification analysis successfully discriminate not only between Gelsemium elegans samples and standard herbs but also successfully distinguished the three different parts of Gelsemium elegans plant.     

中药中砷镉汞铅ICP-MS测定值的聚类分析

聚类分析又称集群分析,它是研究“物与类聚”的一种数理统计方法,聚类分析可将一些观察对象依据某些特征加以归类。已经在生物学和医学分类问题[1]以及中药的鉴别与质量评价[2]中获得广泛应用。祁俊生等[3]利用因子分析和聚类分析探讨了微量元素含量与中药药性的相关性。梁逸曾     

MALDI tissue profiling of integral membrane proteins from ocular tissues

MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this article, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed age related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods.     

Comprehensive quality evaluation of the lateral root of Aconitum carmichaelii Debx. (Fuzi): Simultaneous determination of nine alkaloids and chemical fingerprinting coupled with chemometric analysis

Amino alcohol alkaloids are the active components in the lateral root of Aconitum carmichaelii Debx. (Fuzi), and they have a variety of pharmacological activities. However, the chemical fingerprints of the ester alkaloids reported to date were mainly obtained from high‐performance liquid chromatography coupled with ultraviolet detection, and it is difficult to obtain information about amino alcohol alkaloids in Fuzi from such chromatograms. In this paper, a comprehensive fingerprinting method was established using high‐performance liquid chromatography coupled with an evaporative light‐scattering detector for the simultaneous quantitative analysis of both the amino alcohol alkaloids and ester alkaloids. A total of 42 samples of Fuzi from four production areas were analyzed by constructing high‐performance liquid chromatography fingerprints. Then, the quantitative results of the chemical fingerprints combined with chemometrics methods were employed to reveal the factors affecting the geo‐authentic Fuzi and to determine characteristic components that can be used to identify these samples. The results indicated distinct differences in the alkaloid contents among samples from the four regions; the geographical origin may be the primary factor affecting the geo‐authentic Fuzi, and 15 major components (including songorine, neoline, and hypaconitine, which were quantitatively determined) were found to be characteristic components for the discrimination of Fuzi samples from various regions. Neoline might be a critical component for identifying geo‐authentic Fuzi. This approach is convenient, reproducible and provides a promising method for the quality evaluation of Fuzi.     

Comprehensive quality evaluation of Fructus Schisandrae using electrospray ionization ion trap multiple-stage tandem mass spectrometry coupled with chemical pattern recognition techniques

Electrospray ionization ion trap multiple-stage tandem mass spectrometry (ESI-MS(n)) was used to evaluate Fructus Schisandrae of similar species (Schisandra chinensis (Turcz.) Baill. fruits and Schisandra sphenanthera Rehd. et Wils. fruits) and different growth characteristics (color, shape, etc.). The application of chemical pattern recognition in the ESI-MS(n) data analysis was carried out by principal component analysis (PCA), hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA). Then the antioxidant activity of different Fructus Schisandrae samples were determined by an LC-ESI-MS method and ferric reducing antioxidant power (FRAP) assay. Using the ESI-MS(n) method coupled with chemical pattern recognition analysis and correlated with the antioxidant activity evaluation, the two similar species were successfully distinguished, thus improving the therapeutic safety and effectiveness. The superior characteristics of Schisandra chinensis (Turcz.) Baill. fruits were obtained and made the selection and breeding of Chinese medicine materials more scientific. This study indicates that ESI-MS(n) is a valuable tool for the authentication of botanical origin and can also be useful for the quality control of Chinese medicinal herbs.     

Identification of gamma-irradiated Chinese herbs by thermoluminescence analysis

The feasibility of thermoluminescence (TL) to differentiate irradiated Chinese medicinal herbs from non-irradiated was investigated. Thirty different dried Chinese herbs were tested, including root, flower, ramulus, rhizome, cortex, and whole plant samples. Irradiation of Chinese herbs was associated with strong TL peaks at ~150–250 °C, while TL curves of non-irradiated herbs had very low intensities above 250 °C, which was also confirmed by the TL ratio (non-irradiated, TL₁/TL₂ < 0.1). The ability to determine the irradiation dose by the TL method was influenced by the amount and types of minerals in the samples. All levels of irradiation doses could be detected when between 0.1 and 1.0 kGy, except for three herbs at 0.1 kGy dose. Different blends with small quantities (0.1–10 %) of irradiated herbs were also tested in this study. Samples with powder mixtures containing 1 % irradiated components could be differentiated (TL₁/TL₂ > 0.1) except for sterculia lychnophora, semen cassia, flos inulae, and anemone root. TL ratios of some herbs indicated irradiation (TL₁/TL₂ > 0.1) even if the irradiated components were as low as 0.1 %. Thus we demonstrated that TL analysis had excellent sensitivity and reliability for the identification of irradiated Chinese herbs.     

Computational network pharmacological research of Chinese medicinal plants for chronic kidney disease

The interaction between drug molecules and target proteins is the basis of pharmacological action. The pharmacodynamic mechanism of Chinese medicinal plants for chronic kidney disease (CKD) was studied by molecular docking and complex network analysis. It was found that the interaction network of components-proteins of Chinese medicinal plants is different from the interaction network of components-proteins of drugs. The action mechanism of Chinese medicinal plants is different from that of drugs. We also found the interaction network of components-proteins of tonifying herbs is different from the interaction network of components-proteins of evil expelling herbs using complex network research approach. It illuminates the ancient classification theory of Chinese medicinal plants. This computational approach could identify the pivotal components of Chinese medicinal plants and their key target proteins rapidly. The results provide data for development of multi-component Chinese medicine.     

Ultra-performance LC/TOF MS analysis of medicinal Panax herbs for metabolomic research

In this study, metabolite profiling of five medicinal Panax herbs including Panax ginseng (Chinese ginseng), Panax notoginseng (Sanchi), Panax japonicus (Rhizoma Panacis Majoris), Panax quinquefolium L. (American ginseng), and P. ginseng (Korean ginseng) were performed using ultra-performance LC-quadrupole TOF MS (UPLC-QTOFMS) and multivariate statistical analysis technique. Principal component analysis (PCA) of the analytical data showed that the five Panax herbs could be separated into five different groups of phytochemicals. The chemical markers such as ginsenoside Rf, 20(S)-pseudoginsenoside F11, malonyl gisenoside Rb1, and gisenoside Rb2 accountable for such variations were identified through the loadings plot of PCA, and were identified tentatively by the accurate mass of TOFMS and partially verified by the available reference standards. Results from this study indicate that the proposed method is reliable for the rapid analysis of a group of metabolites present in herbal medicines and other natural products and applicable in the differentiation of complex samples that share similar chemical ingredients.     

Metabolomics study on Fuzi and its processed products using ultra-performance liquid-chromatography/electrospray-ionization synapt high-definition mass spectrometry coupled with pattern recognition analysis

The lateral root of Aconitum carmichaelii Debx is named "Fuzi" which is widely distributed across Asia and North America and has been used to relieve joint pain and treat rheumatic diseases for over two thousand years. However, it has very narrow therapeutic ranges and despite the toxicological risk, its usage remains very high. A traditional Chinese processing approach (Paozhi, detoxifying measure) is necessary to remove the poisonous Aconitum alkaloids mainly deriving from the diester diterpene alkaloids (DDAs) including aconitine, mesaconitine and hypaconitine. They can be decomposed into less or non-toxic derivatives through Paozhi that plays an essential role in detoxification. Processed Fuzi is mainly focused on the three main forms of Yanfuzi (YFZ), Heishunpian (HSP) and Baifupian (BFP) which are highly desirable in order to guarantee the clinical safety and their low toxicity in decoctions. The difference in metabolomic characters between Fuzi and its processed preparations is still completely unclear. Therefore, this paper was designed to investigate a comprehensive metabolome of Fuzi and its processed products by ultra-performance liquid-chromatography/electrospray-ionization synapt high-definition mass spectrometry (UPLC-Q-TOF-HDMS) combined with pattern recognition methods. The difference in metabolic profiles between Fuzi and its processed preparations was well observed by the principal component analysis (PCA) of the MS spectra. Significant changes of 19 metabolite biomarkers were detected in the Fuzi samples and three preparations. The underlying regulations of Paozhi-perturbed metabolic pathways were also discussed according to the identified metabolites. The present study proves that UPLC-Q-TOF-HDMS based metabolomic analysis greatly contributes to the investigation of Fuzi metabolism through Paozhi techniques, and provides useful information to further comprehensively understand the pharmacological activity and potential toxicity of processed Fuzi in a clinical environment.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A2 and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase A2 and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained. In addition to the dominant protein [M + H]+ ions, multimeric and multiply charged ions were also observed in the mass spectra. The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected. Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes. Mass accuracies of 0.1 and 0.3% were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants.     

Protein profiles and identification of high performance liquid chromatography isolated proteins of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used to rapidly profile the protein content of human cell lysates from MCF-10 cell and variant lines. The method was used to study the protein profiles of these cells as they progressed from normal breast epithelium to fully malignant cells. Distinct differences in the protein profiles were observed with progression, and specific proteins associated with carcinogenesis (p53, c-myc, and c-erbB-2) were heavily expressed in these cells as detected by MALDI-TOFMS. These proteins were also isolated using non-porous reversed-phase high performance liquid chromatography (NP-RP-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight information without interference from other proteins in the whole cell lysates, and to avoid suppression effects in mixtures of proteins detected by MALDI-TOFMS. In order to confirm the identity of these oncoproteins, the cell lysates were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for further analysis. Trypsin and cyanogen bromide digestions were performed on these proteins eluted from excised PVDF bands which were then analyzed by MALDI-TOFMS. The identity of these proteins was confirmed by database matching procedures.