Profiling primaquine metabolites in primary human hepatocytes using UHPLC‐QTOF‐MS with 13C stable isotope labeling

Therapeutic efficiency and hemolytic toxicity of primaquine (PQ), the only drug available for radical cure of relapsing vivax malaria are believed to be mediated by its metabolites. However, identification of these metabolites has remained a major challenge apparently due to low quantities and their reactive nature. Drug candidates labeled with stable isotopes afford convenient tools for tracking drug‐derived metabolites in complex matrices by liquid chromatography‐tandem mass spectrometry (LC‐MS‐MS) and filtering for masses with twin peaks attributable to the label. This study was undertaken to identify metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of ¹³C₆‐PQ/PQ with primary human hepatocytes. Acquity ultra‐performance LC (UHPLC) was integrated with QTOF‐MS to combine the efficiency of separation with high sensitivity, selectivity of detection and accurate mass determination. UHPLC retention time, twin mass peaks with difference of 6 (originating from ¹³C₆‐PQ/PQ), and MS‐MS fragmentation pattern were used for phenotyping. Besides carboxy‐PQ (cPQ), formed by oxidative deamination of PQ to an aldehyde and subsequent oxidation, several other metabolites were identified: including PQ alcohol, predictably generated by oxidative deamination of PQ to an aldehyde and subsequent reduction, its acetate and the alcohol's glucuronide conjugate. Trace amounts of quinone‐imine metabolites of PQ and cPQ were also detected which may be generated by hydroxylation of the PQ/cPQ quinoline ring at the 5‐position and subsequent oxidation. These findings shed additional light on the human hepatic metabolism of PQ, and the method can be applied for identification of reactive PQ metabolites generated in vivo in preclinical and clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Site-specific C content by quantitative isotopic C Nuclear Magnetic Resonance spectrometry: A pilot inter-laboratory study

Isotopic ¹³C NMR spectrometry, which is able to measure intra-molecular ¹³C composition, is of emerging demand because of the new information provided by the ¹³C site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic ¹³C NMR as a routine tool. This paper describes the first collaborative study of intra-molecular ¹³C composition by NMR. The main goals of the ring test were to establish intra- and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic ¹³C NMR was then assessed on vanillin from three different origins associated with specific δ¹³C_i profiles. The standard deviation was, on average, between 0.9 and 1.2‰ for intra-variability. The highest standard deviation for inter-variability was 2.1‰. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of δ¹³C_i in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results.     

Ultra high performance liquid chromatography-quadrupole-time of flight analysis for the identification and the determination of resveratrol and its metabolites in mouse plasma

Resveratrol is a polyphenol that has numerous interesting biological properties, but, per os, it is quickly metabolized. Some of its metabolites are more concentrated than resveratrol, may have greater biological activities, and may act as a kind of store for resveratrol. Thus, to understand the biological impact of resveratrol on a physiological system, it is crucial to simultaneously analyze resveratrol and its metabolites in plasma. This study presents an analytical method based on UHPLC-Q-TOF mass spectrometry for the quantification of resveratrol and of its most common hydrophilic metabolites. The use of ¹³C- and D-labeled standards specific to each molecule led to a linear calibration curve on a larger concentration range than described previously. The use of high resolution mass spectrometry in the full scan mode enabled simultaneous identification and quantification of some hydrophilic metabolites not previously described in mice. In addition, UHPLC separation, allowing run times lower than 10 min, can be used in studies that requiring analysis of many samples.     

Determination of 13C/12C-ratios of anthropogenic organic contaminants in river water samples by GC-irmMS

This study describes the application of a common analytical procedure adapted for compound-specific stable carbon isotope analyses of riverine contaminants. To evaluate the sensitivity of the analytical method and the precision of the isotopic data obtained, a set of numerous substances at different concentration levels were measured. For most of the anthropogenic contaminants investigated (including chlorinated aliphatics and aromatics, musk fragrances, phthalate-based plasticizers and tetrabutyl tin) acceptable carbon isotope analyses could be obtained down to amounts of approximately 5?ng absolutely applied to the gas chromatograph. These amounts correspond to concentrations in water samples at a natural abundance level of approximately 50–200?ng?L^?1 (low to medium contaminated river systems). However, it has to be considered that the precision and the sensitivity of the analytical method depend partially on the chemical properties of the substances measured. Five recovery experiments were conducted to assess changes in carbon isotope ratios during sample preparation and measurement. The compounds selected for these experiments are known riverine contaminants. Isotopic shifts or higher variations of the isotope ratios as a result of the analytical procedures applied were observed only for a couple of contaminants. Furthermore, compound-specific carbon isotope analyses were performed on eight water extracts of the Rhine river. By comparing the variation of the data of several individual compounds with the deviations obtained from the recovery experiments, it was possible to differentiate contaminants with unaffected isotope ratios and substances with significant alterations of the δ¹³C-values.     

Application of a new focused microwave technology with species-specific isotope dilution analysis for the quantitative extraction of organometallic contaminants in solid environmental matrices

A new self-tuning single-mode-focused microwave technology has been evaluated in this work to perform the quantitative routine extraction of organometallic species from solid matrices of environmental interest. Species-specific isotope dilution analysis has been employed to better investigate the real influence of the microwave-assisted extractions on the final results. The advantages of such methodology in comparison with other established microwave units for the routine speciation analysis of organomercury and organotin compounds are discussed (such as the capability of using disposable glass vials, a self-tuning mode to provide an accurate control of the temperature and pressure inside of the vials, and the possibility of performing automated sequence of extractions with low sample size). The results obtained in this work demonstrated that such technology provides a fast and reliable quantitative extraction of the organometallic species in a wide range of extraction conditions even when the multi-elemental (Sn and Hg) species-specific determination is carried out.     

18O同位素标记定量肽段串联体蛋白质结合同位素稀释-多反应监测质谱的蛋白质绝对定量新方法

建立了定量肽段串联体蛋白质(concatamers of Q peptides, QconCATs)结合¹⁸O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组蛋白质的纯度在99%以上,相对分子质量约为63.4 kDa。对QconCAT重组蛋白质酶切后的肽段混合物进行质谱分析,并经pFind和pLabel软件处理,验证了目标肽段。还考察了QconCAT重组蛋白质的酶切效率和¹⁸O标记效率,并对QconCAT蛋白质结合¹⁸O标记-同位素稀释-多反应监测质谱方法进行了评价。实验结果表明,采用该方法对腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis, TTE)中选定蛋白质的肽段进行绝对含量测定时,相对标准偏差小于20%,准确度较高,说明该方法可用于复杂生物样本中蛋白质的绝对定量。更重要的是所建方法不仅解决了细胞培养氨基酸稳定同位素标记(SILAC)技术的重标试剂价格昂贵的问题,也为定量蛋白质组学提供了一种新的方法。     

Muon Spin Rotation/Resonance (μSR) for Studying Radical Reactivity of Unsaturated Organophosphorus Compounds

The positive muon (μ⁺) can be regarded as a light isotope of proton and has been an important tool to study radical reactions of organic compounds. Recently, muons have been applied to produce short-lived paramagnetic species from the heavier unsaturated organic molecules including the p-block elements. This article overviews recent muon spin rotation/resonance (μSR) studies on the phosphorus analogs of alkenes, anthracenes, and cyclobutane-1,3-diyls together with the fundamentals of μSR. The acyclic phosphaalkene of P=C and phosphasilenes of P=Si can accept muonium (Mu=[μ⁺e⁻]) at the heavier double bonds, and the corresponding radicals have been characterized. The phosphorus atom in 9-phosphaanthracene, whose P=C double bond is stabilized by the peri-substituted CF₃ groups, predominantly captures muonium to provide the corresponding paramagnetic fused heterocyclic system. The peri-trifluoromethyl groups are functional to promote the unprecedented light isotope effect of muon providing the planar three-cyclic molecular structure to consume the increased zero-point energy. The formally open-shell singlet 1,3-diphosphacyclobutane-2,4-diyl unit can accept muonium at the (ylidic) phosphorus or the skeletal radicalic carbon, and the corresponding paramagnetic phosphorus heterocycles can be characterized by μSR. The findings on these muoniation processes to the unsaturated phosphorus-containing compounds will contribute not only to development of novel paramagnetic functional species but also to progress on muon science.     

加速溶剂提取-同位素稀释-高分辨气相色谱-高分辨质谱法测定生物样品中82种多氯联苯

我国水产品中多氯联苯(PCBs)的检测方法,主要以6种指示性PCBs和12种二噁英类共平面PCBs为主,仅涵盖有限的PCBs。为更全面地获得生物体中PCBs的浓度水平,深入探讨PCBs在生物体内的代谢和富集特征,进而准确评价PCBs对人类的暴露水平及风险,以鱼和贝类作为生物样品代表,建立了加速溶剂提取-同位素稀释-高分辨气相色谱-高分辨质谱(ASE-ID-HRGC-HRMS)测定生物样品中82种PCBs的方法。比较了振荡提取和加速溶剂提取两种提取方式的回收率和重复性,最终采用正己烷-二氯甲烷(1∶1, v/v)对PCBs进行加速溶剂提取。考察了各流分淋洗液对PCBs的回收率,确定了样品提取液经8 g 44%酸性硅胶层析柱(内径15 mm), 90 mL正己烷洗脱的净化方式。样品提取液净化浓缩后进行HRGC-HRMS分析,色谱柱采用DB-5MS超低流失石英毛细管柱(60 m×0.25 mm×0.25 μm)。通过优化后的升温程序对化合物进行分离,以保留时间和两个特征离子精准定性,采用同位素内标法定量。结果表明,在0.1~200 μg/L范围内,平均相对响应因子(RRF)的相对标准偏差值(RSD, n=7)均≤20%,相关系数(r²)>0.99。生物样品中PCBs的方法检出限为0.02~3 pg/g;鱼类中PCBs平均加标回收率为71.3%~141%, RSD(n=7)为2.1%~14%;贝类中PCBs平均加标回收率为76.9%~143%, RSD为1.4%~11%。该方法灵敏、准确、可靠,可以更加全面具体地分析鱼和贝类等水产品受PCBs的污染情况,为国内外开展生物监测提供有效的技术支持,从而服务于相关生态环境管理及履行《斯德哥尔摩公约》。