Post-aggression suicide under the influence of new psychoactive substances AMB–FUBINACA and U-47700

The objective of the present study was the development and validation of the method for determining AMB-FUBINACA and its metabolite - AMB-FUBINACA O-desmethyl acid – in blood samples, followed by verification of the method in toxicological judicial and forensic medicine practice employing the example of post-aggression suicide. Most likely in consequence of development of adverse effects resulting in psychotic symptoms, a male being under the influence of the synthetic cannabinoid AMB-FUBINACA and the new synthetic opioid U-47700, mortally wounded his female partner and subsequently committed suicide. Identification and determination of the afore-mentioned xenobiotics in blood samples collected from the male and female victims were performed employing high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The analytes were isolated from blood samples using the solid phase extraction (SPE) method. The blood samples collected from the male and female demonstrated respectively 110 and 196 ng/mL of AMB-FUBINACA O-desmethyl acid metabolite, 1935 and 357 ng/mL of U-47700, 250 and 200 ng/mL of N-desmethyl-U-47700, as well as 410 and 200 ng/mL of N,N-didesmethyl-U-47700. The concentration values of new psychoactive substances (NPS’s) in blood samples originating from the male and female were within the ranges encountered in cases of poisoning, including these resulting in death. Nevertheless, the evident signs of exsanguination proof that the woman was alive when she sustained lethal injuries. The presented cases illustrate the difficult to be anticipated effect exerted on the users by NPS’s.     

Forced Oxidative Degradation Pathways of the Imidazole Moiety of Daclatasvir

Daclatasvir hydrochloride (DCV) is the active pharmaceutical ingredient of Daklinza, a marketed product for the treatment of hepatitis C viral infection. The intrinsic stability of daclatasvir was evaluated via a forced degradation study. DCV was found to be stable in the solid state. In solution, its carbamate moiety is susceptible to basic hydrolysis, whereas its imidazole is liable to base-mediated autoxidation to form degradants 1 and 3, 7-8, respectively. The imidazole moiety can also be oxidized to form degradants 6-7 in the presence of hydrogen peroxide or azobisisobutyronitrile. The chloro-adduct degradant 9 was also observed in hydrogen peroxide solution. Furthermore, the imidazole moiety is sensitive to photodegradation in solution. Degradants 2-8 were observed in a solution of DCV exposed to high intensity light/UV light; the formation of degradants 2 and 5-8 was postulated through 4 degradation pathways. The degradants 3 and 4 were deemed to be secondary degradants of 7 and 5, respectively.     

UPLC-MS/MS测定人痰液中异烟肼、吡嗪酰胺和左氧氟沙星的浓度

目的建立超高效液相色谱串联质谱法(UPLC-MS/MS)同时检测痰液中3种药物异烟肼(isoniazid,INH)、左氧氟沙星(levofloxacin,LFX)和吡嗪酰胺(pyrazinamide,PZA)浓度的方法,冒在为实现临床个体化用药提供帮助。方法痰液样品离心后经甲醇/乙腈蛋白沉淀法预处理后进样液质联用仪进行分析。实验采用的流动相A为含0.1%甲酸的水溶液,流动相B为含0.1%甲酸的乙腈;流速0.35mL·min^-1;采用ACQUITY UPLAC HSS T31.8μm column(2.1mm×100mm,Waters公司)分离;采用电喷零电离源,采用多反应监测(multiple reaction monitoring,MRM)模式对待测物进行正离子扫描检测。此外,本试验盲态收集5例患者的痰液,用该法进行定量分析。结果测得痰液中INH、PZA和LFX分别在48~6000 ng·mL^-1(r=0.9988)、480~60000 ng·mL^-1(r=0.9993)、120~15000 ng·mL^-1(r=0.9995)内表现出良好的线性关系。INH、PZA和LFX 3种药物的日内和日间精密度均低于15%,且3种药物的提取回收率均处于97.21%~107.80%之间。7例受试者体内均检测到药物INH,质量浓度分别为44.74、120.1、301.5、481、595.5、1220及1570 ng·mL^-1;PZA的质量浓度分别为104.2、6273.34和3185 ng·mL^-1;1例受试者检出LFX,质量浓度为199.86 ng·mL^-1。结论本试验建立了痰液中INH、PZA及LFX的检测方法,具有灵敏度高、测定结果准确、快速等诸多优点,可以应用于临床治疗药物的监测。     

基于LC-MS的甘草炮制雷公藤降低肝毒性的代谢组学研究

目的 观察甘草炮制雷公藤Tripterygium wilfordii后对小鼠血清中代谢产物的影响，初步探讨其降低肝毒性的可能代谢通路。方法 将C57BL/6小鼠随机分为对照（Con）组、雷公藤（Raw）组、甘草炮制雷公藤（Pro）组，观察小鼠肝组织病理变化，检测小鼠肝功能生化指标、炎症因子水平；利用液质联用（LC-MS）技术结合代谢组学方法，对各组间的代谢差异进行表征。结果 与Raw组相比，Pro组小鼠肝组织损伤显著改善，肝功能生化指标、炎症因子水平显著降低，表明甘草炮制雷公藤后可以降低小鼠肝毒性；共筛选出脂肪酸、磷脂酸、甘油酯、磷脂酰胆碱、胆酸等12个潜在生物标志物，主要涉及脂肪酸生物合成、甘油磷脂代谢、花生四烯酸代谢等9个代谢通路。结论 甘草炮制雷公藤可有效降低小鼠的肝毒性，其机制可能与调节脂肪酸代谢等通路相关，为其临床合理应用提供参考依据。     

Simultaneous determination of LY3214996, abemaciclib,and M2 and M20 metabolites in human plasma,cerebrospinal fluid,and brain tumor by LC-MS/MS

A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of total and unbound concentrations of LY3214996, an extracellular signal-regulated kinase inhibitor; abemaciclib, a cyclin-dependent kinase 4/6 inhibitor; and abemaciclib active metabolites, M2 and M20, in human plasma, brain tumor, and cerebrospinal fluid samples. The method was validated over a concentration range of 0.2–500 nM within a total run time of 3.8 min using isocratic elution on a Kinetex™ F₅ column. Detection was performed on a Sciex QTRAP 6500+ mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization. The intra- and inter-batch accuracy as well as the precision of the method for all matrices was within ±20% and ≤20% at the lower limit of quantification, and within ±15% and ≤15% for other quality control levels for all analytes. The unbound fractions of drugs and metabolites in spiked and patient samples were determined using an optimized equilibrium dialysis. The validated method was successfully applied in a phase 0/2 clinical trial to assess the central nervous system penetration of LY3214996 and abemaciclib.     

利用电子顺磁波谱技术研究红参对羟自由基的清除作用

目的 探索电子顺磁波谱（electron paramagnetic resonance，EPR）技术检测红参清除自由基活性的适合产生体系，并研究红参对羟自由基（?OH）的清除作用及其物质基础。方法 采用电子顺磁共振波谱仪检测不同浓度的红参提取液对?OH的体外清除作用，并用液质联用技术（LC-MS）分析红参中的主要皂苷成分，然后研究这些人参皂苷成分对?OH的清除作用。结果 确定了羟自由基的生成体系为：Fe²⁺ 0.1 mmol?L^-1、H₂O₂ 500 mmol?L^-1、DMPO 225 mmol?L^-1；不同浓度的红参提取液对?OH具有明显的清除作用，且其清除?OH的效力与浓度成正相关；LC-MS分析显示该红参提取液中主要含有Rh1、Rf、Rb3、F1、F2、Rg3、Rg5等成分，其中人参皂苷Rg5的清除自由基活性最强。结论 本研究建立了用EPR技术研究红参清除自由基活性的适合产生体系，红参具有明显的清除?OH的能力，且人参皂苷Rg5是其清除自由基的活性成分之一，确定了红参抗自由基的主要物质基础，为红参及人参皂苷Rg5的开发、应用提供了科学依据和理论基础。     

四种大宗常用中药饮片中黄曲霉毒素污染状况分布比较研究

目的 测定不同来源的4种大宗常用中药饮片中黄曲霉毒素（AFB₁、AFB₂、AFG₁和AFG₂）的含量，比较不同基质中药饮片中黄曲霉毒素的分布状况。方法 基于免疫亲和柱净化-高效液相色谱分离-荧光检测器（IAC-HPLC-FLD）方法，分析不同产地共75批中药样品。结果 柏子仁、薏苡仁、决明子及党参共计75批中药饮片中，阳性检出：26批柏子仁（AFs 1.22-46.67 μg·kg^-1，AFB₁ 1.22-31.40 μg·kg^-1）、4批薏苡仁（AFs 1.97-41.13 μg·kg^-1，AFB₁ 1.97-36.40 μg·kg^-1）、1批决明子（AFs 13.65 μg·kg^-1，AFB₁ 12.60 μg·kg^-1），阳性率41%，超标率15%。阳性样品经 LC-MS/MS确证，排除假阳性。4种大宗常用中药饮片柏子仁、薏苡仁、决明子、党参中黄曲霉毒素的污染水平依次降低，阳性检出率分别为77%、29%、7%、0%，表明中药材中黄曲霉毒素的污染状况与药材基质密切相关。结论 针对易污染AFs的中药品种，需进一步加强其污染状况的全面检测分析，为黄曲霉毒素的有效防控以及完善中药的质量标准提供科学依据，从而保障中药用药安全。     

Influence of harvest season on chemical composition and bioactivity of wild rue plant hydroalcoholic extracts

The rue (Ruta graveolens) copiousness in rural areas of the Campania Region based a thorough chemical and biological investigation aimed at exploring the seasonal variability of phenol constituents in rue leaves and its influence on their antioxidant, cytotoxic and anti-inflammatory capabilities. To this purpose, hydroalcoholic extracts were prepared from plant samples seasonally collected. LC-ESI-MS/MS techniques were employed to analyze qualitatively and quantitatively the seasonal rue phenol content, whereas different chemical antioxidant assays (by DPPH, ABTS, Fe³⁺ RP, ORAC, and FCR methods) and XTT redox metabolic activity assay were performed to screen the seasonal phenol complex-related antioxidant and cytotoxic power. The ability of the rue leaf extracts to counteract cyclooxygenase-2 (COX-2) expression was also evaluated. Data obtained highlighted that the adopted extraction procedure markedly pauperized the furanocoumarin content in all the prepared rue extracts. Flavonol glycosides, along with the flavone acacetin and two sinapic acid derivatives were the main constituents of the spring harvest-derived extract, which exerted the highest antioxidant capability in cell-free systems and was capable to inhibit COX-2 synthesis by 44% comparably to dexamethasone, used as positive control. Data provide new insights for developing a proper management of rue plants for new safe industrial purposes in herbal medicine field.     

LC-MS/MS法测定人血浆中布康唑浓度的不确定度评定

目的：评定LC-MS/MS法测定人血浆中布康唑浓度的不确定度。方法：分析测定过程中不确定度的来源，包括对照品的称量、仪器误差、标准溶液的配制、含药血浆样品的配制、血浆样品的处理、标准曲线的拟合、基质效应、重复性等，评定各来源分量的不确定度，计算合成不确定度和扩展不确定度。结果：人血浆中低（60.0 pg·mL^-1）、中（600.0 pg·mL^-1）、高（6 400.0 pg·mL^-1）浓度布康唑的扩展不确定度分别为5.62，63.90，626.26 pg·mL^-1（k=2，P=95%）。结论：LC-MS/MS法测定人血浆中布康唑浓度的不确定度主要由基质效应、血浆样品的处理（提取回收率），仪器误差、重复性（精密度）引入。