高效液相色谱-串联质谱检测基因组DNA甲基化方法的建立

目的:建立高效液相色谱-串联质谱(HPLC-ESI-MS/MS)检测基因组DNA甲基化水平的方法。方法:以5-mdC和dG为标准品,采用全自动高效液相色谱系统进行分离,串联电喷雾质谱检测,选择多反应监测模式(MRM)测定标准品,绘制标准工作曲线。结果:在MRM模式下选取5-mdC(m/z 241.9→126.3)和dG(m/z 268.1→152.3)分别作为定量检测的母子离子对,各化合物能实现良好的基线分离;5-mdC和dG碰撞能均为15 eV,去簇电压分别为40和45 V,最低定量限分别为1.65和2.47 fmol;标准品的响应值比为90%~110%;5-mdC含量的天内相对标准偏差和天间相对标准偏差均小于8%。结论:HPLC-ESI-MS/MS是能应用于检测基因组DNA甲基化的一种高通量、高准确率、高分辨率、高灵敏度且重复性好的方法。     

液相串联质谱法同时定量检测生物样本中鞘脂类化合物

鞘脂类中的主要活性分子鞘氨醇1-磷酸（S1P）,可通过介导细胞增殖、分化和迁移等发挥广泛的生物学功能|同时,S1P可在相关酶的作用下转变为其它形式.本文旨在建立一种简便的鞘脂类的制备方法,并结合液相串联质谱（LC-MS/MS）快速检测生物样本中纳克水平的鞘脂类化合物. 采用甲醇沉淀或经典的脂质提取方法获得鞘脂类化合物,再采用LC-MS/MS在多反应监测模式（MRM）下进行定量分析. 实验结果表明,甲醇沉淀法可简便快捷地获得血浆或脂蛋白中鞘氨醇类化合物; S1P、二氢鞘氨醇（DH-S1P）和鞘氨醇（SPH）的定量限分别为110.5、215.6和44.3 pg;人血浆中S1P、DH-S1P和SPH的含量分别为257.8±49.4 nmol/L、93.5±17.3 nmol/L和44.6± 7.4 nmol/L, 鞘脂类化合物在人血浆脂蛋白上的分布存在显著性差异|在ApoE-/-小鼠血浆中S1P、DH-S1P和SPH的含量分别为590.1±78.2 nmol/L、197.8±60.6 nmol/L和35.4±16.7 nmol/L|每1×106个人脐静脉内皮细胞（EA.hy926）中,S1P和SPH的含量分别为103.7±21.8 pg和16.3±5.3 ng.甲醇沉淀法结合LC-MS/MS可简便快捷地对血浆和脂蛋白中的鞘脂类化合物进行定量检测. 该方法特别适用于大量生物样本中鞘脂类的快速定量分析.     

Quantitative Analysis of Ingenol in Euphorbia species via Validated Isotope Dilution Ultra‐high Performance Liquid Chromatography Tandem Mass Spectrometry

Introduction

Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol‐3‐angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis.

Objective

To develop a rapid and reliable method for quantification of ingenol in various plant extracts.

Methodology

Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra‐high performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) after methanolysis and solid‐phase extraction (SPE) purification. The ¹⁸O–labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation.

Results

The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates.

Conclusion

The newly established UHPLC–MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.     

Effects of Hexane in Supercritical Fluid Chromatography for the Separation of Enantiomers

Supercritical fluid chromatography (SFC), operated in conventional mode, is normally recognized as normal phase chromatography, and uses a solvent combination of supercritical CO₂ and alcohols to separate compounds. Hexane, a commonly used solvent in normal phase liquid chromatography (NP‐LC), is rarely used in SFC and, in some cases, is added to the organic modifiers to increase liquid content in order to achieve better efficiency in preparative SFC for poorly retained compounds. Although hexane is believed to have similar solvent strength to that of supercritical CO₂, its effects on the enantioseparation in SFC is largely unknown. To understand the chromatographic effects of an apolar solvent, such as hexane in SFC, we compared the chromatographic behaviors of 35 chiral compounds using a parallel SFC method under traditional SFC mode of only “pure” alcohol‐CO₂ to that of hexane‐assisted SFC (HA‐SFC), which uses mixtures of alcohol and hexane (as cosolvents) and CO₂. We observed that, in some cases, hexane behaves just like supercritical CO₂, where replacement of a portion of CO₂ with hexane does not significantly change retention times or resolution of the peaks. In many cases, however, addition of hexane in mobile phases does affect chromatographic behavior of one or both enantiomers. Such effects might provide opportunities for separation of some enantiomers. Chirality 28:192–198, 2016. © 2016 Wiley Periodicals, Inc.     

Simultaneous Measurement of Fluoroquinolones in Eggs by a Combination of Supercritical Fluid Extraction and High Pressure Liquid Chromatography

Simultaneous detection of the fluoroquinolone antibiotics ciprofloxacin, enrofloxacin, ofloxacin, and norfloxacin in eggs by a combination of supercritical fluid extraction (SFE) and high pressure liquid chromatography (HPLC) was studied. Lipid matrices that have been considered to result in poor extraction and isolation of fluoroquinolones in eggs were removed first by SFE with supercritical CO₂ alone, and then the fluoroquinolones were extracted by SFE with supercritical CO₂ containing 20% (v/v) methanol for HPLC analysis. A time-course study of the extraction of lipid matrices of eggs suggested that the SFE method successfully removed the matrices within 20 min. When the fluoroquinolones added to control eggs were extracted by SFE, the extraction efficiency was similar to that by the solvent extraction method, giving the recovery percentages from 83 to 96% in a 40 min-extraction time. The fluoroquinolones extracted from eggs by SFE were analyzed simultaneously by HPLC equipped with a fluorescence detector with detection sensitivity at about 10 ppb for the detection limit. The standard calibration profiles of fluoroquinolones showed linear responses to HPLC, showing more than 0.995 for the mean r ² value. This is the first report of the simultaneous measurement of fluoroquinolones in eggs by a combination of SFE and HPLC. Using the SFE method allowed us to avoid extensive sample preparation such as solvent extraction and chromatographic cleanup that are basically required in extraction of fluoroquinolones.     

Detection and Pharmacokinetics of Alginate Oligosaccharides in Mouse Plasma and Urine after Oral Administration by a Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) Method

A sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the detection of alginate oligosaccharides (AOs) in mouse plasma and urine after oral administration. In an AO mixture, dimer, trimer, and tetramer were detected by LC-MS/MS equipped with an anion-exchange column with extremely high sensitivity. By this method, we detected certain levels of AOs in samples prepared from mouse plasma and urine after a single oral administration of the AO mixture. Based on a calibration curve made with an AO trimer peak area as a standard, the maximum plasma and urine concentrations of AOs were estimated to be 24.5 μg/ml at 5 min and 425.5 μg/ml at 30 min, respectively. These results suggest that the LC-MS/MS method is well suited to pharmacokinetic analysis of AOs in an in vivo system, and that some of orally administered AOs, at least from dimer to tetramer, are absorbed by digestive organs promptly, and that unaltered, these oligomers were excreted into an urine after a single oral administration to a mouse.     

Shortlisting SARS‐CoV‐2 Peptides for Targeted Studies from Experimental Data‐Dependent Acquisition Tandem Mass Spectrometry Data

Detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a crucial tool for fighting the COVID‐19 pandemic. This dataset brief presents the exploration of a shotgun proteomics dataset acquired on SARS‐CoV‐2 infected Vero cells. Proteins from inactivated virus samples were extracted, digested with trypsin, and the resulting peptides were identified by data‐dependent acquisition tandem mass spectrometry. The 101 peptides reporting for six viral proteins were specifically analyzed in terms of their analytical characteristics, species specificity and conservation, and their proneness to structural modifications. Based on these results, a shortlist of 14 peptides from the N, S, and M main structural proteins that could be used for targeted mass‐spectrometry method development and diagnostic of the new SARS‐CoV‐2 is proposed and the best candidates are commented.     

C Isotopomer Analysis of Glutamate by Tandem Mass Spectrometry

Tandem mass spectrometry allows a compound to be isolated from the rest of the sample and dissociated into smaller fragments. We show here that fragmentation of glutamate mass isotopomers yields additional mass spectral data that significantly improve the analysis of metabolic fluxes compared to full-scan mass spectrometry. In order to validate the technique, tandem and full-scan mass spectrometry were used along with (13)C NMR to analyze glutamate from rat hearts perfused with three substrate mixtures (5 mM glucose plus 5 mM [2-(13)C]acetate, 5 mM [1-(13)C]glucose plus 5 U/L insulin, and 5 mM glucose plus 1 mM [3-(13)C]pyruvate). Analysis by tandem mass spectrometry showed that the enriched substrate contributed 98 +/- 2, 53 +/- 2, and 84 +/- 7%, respectively, of acetyl-coenzyme A while the rate of anaplerotic substrate entry was 7 +/- 3, 25 +/- 8, and 16 +/- 8%. Similar results were obtained with (13)C NMR data, while values from full-scan data had higher error. We believe that this is the first use of tandem mass spectrometry to determine pathway flux using (13)C-enriched substrates. Although analysis of the citric acid cycle by NMR is simpler (and more intuitive), tandem mass spectrometry has the potential to combine high sensitivity with the high information yield previously available only by NMR.