基于UHPLC-Q-Orbitrap HRMS技术研究清肺排毒汤化学成分及小鼠组织分布

目的旨在开展国家卫生健康委员会和国家中医药管理局推荐治疗新型冠状病毒肺炎(COVID-19)的协定通用处方清肺排毒汤的主要化学成分及小鼠组织分布研究,为其药效物质研究提供参考依据。方法采用UHPLC-Q-Orbitrap HRMS对清肺排毒汤的主要化学成分进行分析,色谱分离采用Acquity UPLC?BEH C18色谱柱(100 mm×2.1 mm,1.7μm),流动相为甲醇-0.1%甲酸水,梯度洗脱,体积流量0.3mL/min。基于化合物的一级、二级质谱信息,结合对照品比对确认,实现复方主要化学成分的快速、精准识别。在此基础上,对小鼠ig清肺排毒汤后血及组织中的移行成分进行了鉴定,并建立了同步测定麻黄碱、伪麻黄碱、苦杏仁苷、野黑樱苷、甘草苷、金丝桃苷、橙皮苷、黄芩苷、次野鸢尾黄素的定量分析方法,对小鼠ig清肺排毒汤后上述9种入血成分的组织分布进行研究。结果从清肺排毒汤中共鉴定出39种化学成分,ig给药后,在小鼠血清、心、肝、脾、肺、肾中分别鉴定出12、9、9、8、10、10种化学成分。发现9种化学成分可快速吸收并分布到多个组织,在0.5 h时血清及各组织中除黄芩苷之外的8种化学成分的含量均达到峰值,黄芩苷含量峰值出现在2 h或4 h。重点关注的肺组织中0.5 h时各成分的暴露量依次为麻黄碱(2 759.11±784.39)ng/g野黑樱苷(1 819.7±427.28)ng/g伪麻黄碱(880.60±287.97)ng/g苦杏仁苷(304.43±234.70)ng/g橙皮苷(78.33±38.38)ng/g次野鸢尾黄素(8.62±4.66)ng/g黄芩苷(8.53±1.91)ng/g金丝桃苷(7.72±1.63)ng/g甘草苷(7.68±5.19)ng/g;2h时成分含量为麻黄碱(776.61±148.40)ng/g野黑樱苷(325.20±104.17)ng/g伪麻黄碱(212.21±44.63)ng/g黄芩苷(71.72±23.96)ng/g苦杏仁苷(46.39±36.45)ng/g橙皮苷(13.39±10.99)ng/g金丝桃苷(3.11±0.75)ng/g甘草苷(2.86±0.46)ng/g;4 h时各成分质量分数为麻黄碱(327.61±212.59)ng/g野黑樱苷(173.77±58.21)ng/g伪麻黄碱(84.68±59.04)ng/g黄芩苷(49.33±17.06)ng/g苦杏仁苷(1.26±0.26)ng/g。结论研究建立的UHPLC-Q-Orbitrap HRMS方法可实现清肺排毒汤中多种化学成分的快速、准确分析,并明确了ig给药后清肺排毒汤主要成分在小鼠的组织分布情况,为后续清肺排毒汤的药效物质基础研究及进一步的临床应用提供了药动学信息和指导。     

基于超高效液相色谱-四级杆-静电场轨道阱质谱的 胃复春片化学成分研究

目的:基于超高效液相色谱-四级杆-静电场轨道阱高分辨质谱（UPLC-Q-Orbitrap HRMS）技术对胃复春片的物质基础进行了全面的研究,对主要化学成分进行快速鉴定。方法:采用Thermo Hypersil GOLD C18色谱柱(4.6 mm×150 mm,3 μm),以0.05%甲酸水-乙腈为流动相的梯度洗脱进行分离。质谱采用正、负离子检测模式、全扫描及自动触发二级质谱扫描功能（Full MS/dd MS2）采集数据。通过化合物的最大紫外吸收波长、精确相对分子质量及二级碎片离子信息,与对照品的保留时间、mzVault 2.0质谱数据库及相关文献报道进行比对,鉴定胃复春片的主要化学成分。结果:共鉴定出164种化学成分,其中包括人参皂苷类42个、黄酮类97个、二萜类19个、其他6个。结论:UPLC-Q-Orbitrap HRMS技术能够快速、准确、较全面地鉴定胃复春片中化学成分,为明确药效物质基础研究及提升质量标准提供了理论依据。     

Phytochemical analysis and comprehensive evaluation of pharmacological potential of Artemisia brevifolia Wall. ex DC

Multitude of diseases and side effects from conventional drugs have surged the use of herbal remedies. Thus, the current study aimed to appraise various pharmacological attributes of Artemisia brevifolia Wall. ex DC. Extracts prepared by successive solvent extraction were subjected to phytochemical and multimode antioxidant assays. Various polyphenolics and artemisinin derivatives were detected and quantified using RP-HPLC analysis. Compounds present in methanol (M) and distilled water (DW) extracts were identified using high resolution mass spectrometry (HRMS). Extracts were pharmacologically evaluated for their antibacterial, antifungal, antimalarial, antileishmanial and antidiabetic potentials. Moreover, cytotoxicity against Artemiasalina, human cancer cell lines and isolated lymphocytes was assessed. Genotoxicity was evaluated using comet, micronucleus and chromosomal aberration assays. Lastly, anti-inflammatory potential was determined through a series of in vitro and in vivo assays using BALB/c mice.Maximum extract recovery (5.95% w/w) was obtained by DW extract. Highest phenolics and flavonoids content, total antioxidant capacity, total reduction potential, percentfree radical scavenging, β-carotene scavenging and iron chelating activities were exhibited by M extract. RP-HPLC analysis revealed significant amounts of various polyphenolic compounds (vanillic acid, syringic acid, emodin and luteolin), artemisinin, dihydro artemisinin, artesunate and artemether in ethyl acetate (EA) extract. Total 40 compounds were detected through HRMS. A noteworthy antimicrobial activity (MIC 22.22 µg/ml) was exhibited by EA extract against A. fumigatus and several bacterial strains. Maximum antimalarial, antileishmanial, brine shrimp lethality and cytotoxic potential against cancer cells was manifested by EA extract. None of the extracts exhibited genotoxicity and toxicity against isolated lymphocytes. Highest α-amylase and α-glucosidase inhibition capacities were demonstrated by DW extract. Various in-vivo anti-inflammatory models revealed significant (p < 0.05) anti-inflammatory potential of M and DW extracts. In conclusion, present findings divulged theremarkable pharmacological potential of A. brevifolia and endorse its richness in artemisinin.     

UHPLC-Q-Orbitrap HRMS分析吉马酮大鼠体内代谢产物及代谢途径

目的 研究吉马酮在大鼠体内的代谢产物,探讨其可能的代谢途径。方法 采用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱技术（UHPLC-Q-Orbitrap HRMS）对灌胃给予吉马酮（10 mg·kg^-1）后大鼠的生物样品（血浆、尿液和粪便）进行分析,色谱柱采用Waters ACQUITY UPLC^® BEH C₁₈（2.1 mm×50 mm,1.7 μm）,流动相乙腈-0.1%甲酸水溶液,梯度洗脱,Q-Exactive高分辨质谱仪采用HESI离子源,辅助气温度300℃,离子传输管温度320℃,喷雾电压为+3.50 kV或者-2.80 kV,扫描方式Full MS/dd-MS²。分析比对给药组和空白组中高分辨质谱提供的准分子离子峰和碎片离子信息,使用Xcalibur 3.0、Mass Frontier 2.0和Compound Discovery 2.1软件进行数据处理,筛选并鉴定其可能的代谢产物。结果 从大鼠生物样品中共筛选鉴定了包括原型在内的23个代谢产物,推断吉马酮在大鼠体内发生的代谢类型主要有脱水反应、脱饱和反应、氧化反应、葡萄糖胺结合反应、葡萄糖醛酸化反应以及相关的复合反应等。结论 本研究较为详细地阐明了吉马酮在大鼠体内的代谢情况,剖析了其代谢途径,可为其进一步的药效学、药动学及毒理学研究提供理论依据。     

深圳市市售食品中二恶英、多氯联苯污染现状研究

目的了解深圳市市售食品中二恶英、多氯联苯本底水平和污染情况,为开展食品中二恶英类化合物污染状况的风险评估和预警监测提供科学依据。方法于2004年10月至2008年10月期间,采集深圳市市售鱼、猪肉、牛肉、羊肉、鸡肉、鸭肉、禽蛋、奶粉、植物油、豆制品、米面、蔬菜12种食品共123份样品,参照美国国家环保局EPA1613、1668A方法,采用索式抽提装置和FMS自动纯化系统分别对样品进行提取和净化,采用同位素稀释技术,用已建立的高分辨气相色谱/高分辨双聚焦磁式质谱联用(HRGC/HRMS)超痕量有机分析技术平台,对样品中的7个多氯二苯并-对-二恶英(PCDD s)、10个多氯二苯并-对-呋喃(PCDFs)、4个共平面的多氯联苯(PCBs)化合物、8个单邻位取代的PCBs化合物以及6个指示性的PCBs化合物共计35个单体进行定量分析检测和毒性当量浓度汇总分析和评价。结果共检测123份市售食品样品,总二恶英(包括PCDD/Fs和PCBs)污染毒性当量浓度水平,动物性食品由大至小依次是:鱼类>禽蛋>牛肉>奶粉>羊肉>鸡肉>鸭肉>猪肉,植物性食品以植物油>豆制品>粮谷类>蔬菜依次降低。其中动物性食品以鱼类二恶英含量最高,...     

Towards compound identification of synthetic opioids in nontargeted screening using machine learning techniques

The constant evolution of the illicit drug market makes the identification of unknown compounds problematic. Obtaining certified reference materials for a broad array of new analogues can be difficult and cost prohibitive. Machine learning provides a promising avenue to putatively identify a compound before confirmation against a standard. In this study, machine learning approaches were used to develop class prediction and retention time prediction models. The developed class prediction model used a naïve Bayes architecture to classify opioids as belonging to either the fentanyl analogues, AH series or U series, with an accuracy of 89.5%. The model was most accurate for the fentanyl analogues, most likely due to their greater number in the training data. This classification model can provide guidance to an analyst when determining a suspected structure. A retention time prediction model was also trained for a wide array of synthetic opioids. This model utilised Gaussian process regression to predict the retention time of analytes based on multiple generated molecular features with 79.7% of the samples predicted within ±0.1 min of their experimental retention time. Once the suspected structure of an unknown compound is determined, molecular features can be generated and input for the prediction model to compare with experimental retention time. The incorporation of machine learning prediction models into a compound identification workflow can assist putative identifications with greater confidence and ultimately save time and money in the purchase and/or production of superfluous certified reference materials.     

Liporetro-D-peptides - a novel class of highly selective thrombin inhibitors

Introduction

Plasma serine protease thrombin plays a key role in coagulation, haemostasis and thromboembolic diseases. Direct thrombin inhibitors could be beneficial for future anticoagulant therapy. We have synthesized and studied liporetro-D-peptides - efficient thrombin inhibitors resistant to enzymatic degradation.

Materials and Methods

Compounds X-D-Arg-D-Phe-OMe, where X = residue of lauric or myristic acid or 9-fluorenylmethoxycarbonyl, have been synthesized by conventional peptide synthesis in solution and their comparative inhibitory analysis in relation to thrombin, factor X, plasmin and trypsin has been conducted.

Results

Modification of the synthetic liporetro-D-peptides with the myristic acid residue was the most successful one. This modification has dramatically increased the inhibition efficacy (Ki = 0,17 μM) and selectivity toward the chosen target enzyme, thrombin, in comparison to factor X, plasmin and trypsin (more than 600, 900, and 5000-fold, respectively).

Conclusions

Our findings establish an important role of the fatty moiety in the structure of peptide inhibitors with regards to their potency and selectivity toward thrombin.     

Antibacterial activity of crinane alkaloids from Boophone disticha (Amaryllidaceae)

Ethnopharmacological relevance

Boophone disticha (Amaryllidaceae) is one of the most common bulbous plants used for medicinal purposes by the indigenous people of southern Africa. Its use as a narcotic substance by the Khoi/San tribes has been known for several centuries, while the Sotho, Xhosa and Zulu people are known to use the plant to treat a host of ailments, including inflammation, wounds, gynaecological conditions and psychosis.

Aim of the study

Much of the pharmacological work on the plant, such as affinity to the serotonin transporter, has been based on its reputed usage for narcotic purposes. However, its widespread use to treat wounds and infections has not been linked to a specific chemical entity. In this regard, Boophone disticha was here examined for its phytochemical composition which could shed light on the use of the plant for such purposes.

Materials and methods

The known crinane alkaloids buphanidrine and distichamine were isolated via column chromatography of the ethanolic extract of bulbs of Boophone disticha. Structural details of the compounds were determined by high field 2D NMR and mass spectroscopic techniques. Microbial activity against selected Gram-positive and Gram-negative bacteria was ascertained according to the micro-dilution assay.

Results

Both buphanidrine and distichamine were uncovered as novel, broad spectrum moderately active, antibacterial agents with the best MIC value detected at 0.063 mg/ml for Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. MIC values for Bacillus subtilis were two-fold less than that observed for the other three bacteria, suggesting that the extract and pure compounds were selective in their interaction with the bacterial pathogens.

Conclusion

Phytochemical investigation of Boophone disticha has led to the identification of two known crinanes, buphanidrine and distichamine. Based on the reputed traditional use of the plant for wounds and infections, both compounds were screened for antibacterial activity which revealed them to be novel, broad spectrum antibacterial agents with the best MIC value set at 0.063 mg/ml. Their close structural similarity may have bearing on their similar activity profiles.     

院内制剂消炎抗菌片的UPLC-LTQ-Orbitrap HRMS分析

目的：采用UPLC-LTQ-Orbitrap HRMS方法鉴定消炎抗菌片中的主要成分。方法：选用Kromasil C₁₈色谱柱（250 mm×4.6 mm，5 μm），流动相为甲醇和水，梯度洗脱，流速为1 mL·min^-1，进样量1 μL，柱温为35℃，采用正、负离子检测模式。结果：通过相关文献及质谱数据，共鉴定出30个成分，分别为16个黄酮类化合物、10个鞣质类化合物、1个三萜类化合物，2个有机酸类化合物和1个酚类化合物。结论：本研究首次建立UPLC-LTQ-Orbitrap HRMS技术对消炎抗菌片的主要成分进行鉴定，为消炎抗菌片的药效成分研究提供科学依据。     

Use of split‐free nano‐liquid chromatography–mass spectrometry/high resolution mass spectrometry interface to improve the detection of α‐cobratoxin in equine plasma for doping control

Cobra (Naja naja kaouthia) venom contains a toxin called α‐cobratoxin (α‐Cbtx) containing 71 amino acids (MW 7821 Da) with a reported analgesic power greater than morphine. In 2013, the first analytical method for the detection of α‐Cbtx in equine plasma was developed by Bailly‐Chouriberry et al, allowing the confirmation of the presence of α‐Cbtx at low concentrations (1–5 ng/mL or 130–640 fmol/mL) in plasma samples. To increase the method sensitivity and therefore to improve the detection of α‐Cbtx in post‐administration plasma samples, a nano‐liquid chromatography–mass spectrometry/high resolution mass spectrometry (nLC–MS/HRMS) method was developed. This new method allowed us to confirm the presence of α‐Cbtx in plasma samples spiked at 100 pg/mL (12.8 fmol/mL) and the detection of α‐Cbtx was obtained in plasma samples collected 72 hours post‐administration (50 pg/mL or 6.4 fmol/mL) which was defined as the limit of detection (LOD). The presented method is 20‐fold more sensitive compared to the method previously described.