化学计量学方法用于蛋白质组学质谱数据的特征筛选

提出了一种基于偏最小二乘判别分析和F-score的特征筛选方法,并将其用于蛋白质组学质谱数据分析。方法主要包含3个步骤:(1)用LIMPIC算法对原始数据进行预处理;(2)计算每个变量的F-score值并将所有变量按F-score值降底的顺序排列;(3)采用偏最小二乘判别分析交互检验按前向选择法选择最佳变量子集。用本方法对一组卵巢癌数据进行分析,最终从原始的15154个质荷比变量中选择了12个特征变量作为潜在生物标记物,它们在训练集上交叉检验的特异性和灵敏度分别为98.36%和98.15%,在独立测试集上的特异性和灵敏度分别为96.67%和100%。用筛选出的变量作PCA所得的结果显示这些变量能够较好地将样本分类,说明能够反映出样本的类别信息。所提出的方法可用于蛋白质组学质谱数据的特征筛选及样本分类。     

基于ProteinGoggle 2.0的组蛋白H4蛋白质变体的自上而下表征

由于大量可能蛋白质变体以及每一个翻译后修饰大量可能位点的存在,核心组蛋白上密集的组合式翻译后修饰的自上而下表征一直是一个巨大的分析挑战。结合高分辨串级质谱,基于同位素质荷比和轮廓指纹比对的整体蛋白质数据库搜索引擎ProteinGoggle 2.0在组蛋白翻译后修饰的自上而下鉴定方面拥有诸多独特的优势。该文报道ProteinGoggle 2.0对HeLa核心组蛋白H4的数据库搜索及蛋白质变体的鉴定结果。基于从UniProt网站下载的人类核心组蛋白H4的纯文本文件和“鸟枪法”注释,ProteinGoggle 2.0首先创建包含所有可能蛋白质变体的理论数据库;从纯文本文件中提取的信息主要是氨基酸序列、可能的翻译后修饰（单甲基化、二甲基化、三甲基化、乙酰化和磷酸化）及氨基酸变异（A77→P）。在控制质谱水平假阳性率低于1%的前提下,共鉴定到426个蛋白质变体,这是目前为止H4蛋白质变体的最全报道。这些ProteinGoggle 2.0鉴定到的H4蛋白质变体也与之前报道的ProSightPC 2.0的鉴定结果进行了肩并肩比较。总而言之,ProteinGoggle 2.0可以对具有复杂组合修饰及氨基酸变异的蛋白质组进行数据库搜索和蛋白质变体鉴定。     

基于ProteinGoggle 2.0的组蛋白H4蛋白质变体的自上而下表征（英文）

由于大量可能蛋白质变体以及每一个翻译后修饰大量可能位点的存在,核心组蛋白上密集的组合式翻译后修饰的自上而下表征一直是一个巨大的分析挑战。结合高分辨串级质谱,基于同位素质荷比和轮廓指纹比对的整体蛋白质数据库搜索引擎ProteinGoggle 2.0在组蛋白翻译后修饰的自上而下鉴定方面拥有诸多独特的优势。该文报道ProteinGoggle 2.0对HeLa核心组蛋白H4的数据库搜索及蛋白质变体的鉴定结果。基于从UniProt网站下载的人类核心组蛋白H4的纯文本文件和"鸟枪法"注释,ProteinGoggle 2.0首先创建包含所有可能蛋白质变体的理论数据库;从纯文本文件中提取的信息主要是氨基酸序列、可能的翻译后修饰(单甲基化、二甲基化、三甲基化、乙酰化和磷酸化)及氨基酸变异(A77→P)。在控制质谱水平假阳性率低于1%的前提下,共鉴定到426个蛋白质变体,这是目前为止H4蛋白质变体的最全报道。这些ProteinGoggle 2.0鉴定到的H4蛋白质变体也与之前报道的ProSightPC 2.0的鉴定结果进行了肩并肩比较。总而言之,ProteinGoggle 2.0可以对具有复杂组合修饰及氨基酸变异的蛋白质组进行数据库搜索和蛋白质变体鉴定。     

一种位点注释的蛋白质数据库用于磷酸化肽段的鉴定

磷酸化修饰的分析一直是蛋白质组学研究的热点之一.在鸟枪法的蛋白质组学研究中,通过在数据库检索中设定磷酸化为可变修饰可以直接鉴定磷酸化修饰的位点.但是翻译后修饰的引入会增加数据检索空间,造成鉴定灵敏度的降低.为了解决这一问题,我们构建了一种位点注释的数据库,这种数据库包含蛋白质的磷酸化位点信息,并开发了一种新的数据库检索策略用于磷酸化肽段的可靠鉴定.用不同类型的数据作为分析对象,通过Mascot检索软件对这种新的数据库检索策略进行了考察,证明了这种方法在保证鉴定结果可靠性的前提下提高了磷酸化肽段鉴定的灵敏度.     

高通量蛋白质组学分析研究进展

基于质谱的蛋白质组学技术已经日趋成熟,可以对细胞和组织中的成千上万种蛋白质进行全面的定性和定量分析,逐步实现“深度覆盖”。随着生物医学日益增长的大队列蛋白质组学分析需求,如何在保持较为理想的覆盖深度下实现短时间、快速的“高通量”蛋白质组学分析已成为当前亟需解决的关键问题之一。常规的蛋白质组学分析流程通常包括样品前处理、色谱分离、质谱检测和数据分析。该文从以上4个方面展开介绍近10年以来高通量蛋白质组学分析技术取得的一系列研究进展,主要包括:(1)基于高通量、自动化移液工作站的蛋白质组样品前处理方法;(2)基于微升流速液相色谱与质谱联用的高通量蛋白质组检测方法;(3)利用灵敏度高、扫描速度快的质谱仪实现短色谱梯度分离下蛋白质组深度覆盖的分析方法;(4)基于人工智能、深度神经网络、机器学习等的蛋白质组学大数据分析方法。此外,对高通量蛋白质组学面临的挑战及其发展进行展望。总而言之,预期在不久的将来高通量蛋白质组学技术将会逐步“落地转化”,成为大队列蛋白质组学分析的利器。     

基于质谱的单细胞蛋白质组学分析方法及应用

细胞是生命体的最小组成单位,遗传及外部环境等因素使单细胞异质性广泛存在于众多生物体中。传统的生物学实验获得的结果多是大量细胞的平均测量值,因此在单细胞层面开展研究对于精确理解细胞的生长发育以及疾病的诊断与治疗至关重要。而作为重要的细胞和生命活动的执行者,蛋白质由于其不具备扩增特性,且种类繁多、丰度低、动态分布范围宽,与核酸等其他生物大分子相比,其单细胞组学研究相对滞后。而在所有的检测手段中,荧光检测以及电化学分析方法具有极高的灵敏度,但是囿于其研究通量有限,以及电化学活性依赖,很难成为普适性的单细胞蛋白质组学研究方法。质谱分析作为传统蛋白质组学中最为核心的研究技术,由于其高灵敏、高通量、结构信息丰富等特点,在单细胞蛋白质组学研究中独树一帜。该文综述了近年来基于质谱的单细胞蛋白质组学研究中的代表性方法,根据质谱分析前蛋白质分离方式的差异,将其分为基于毛细管电泳分离、液相色谱分离和无分离手段的直接检测3类方法,在介绍研究现状的同时对这些方法在细胞通量、蛋白质鉴定数目、灵敏度以及方法应用方面进行了总结与比较。最后,基于目前研究中面临的挑战以及发展趋势对基于质谱的单细胞蛋白质组学的研究前景进行了展望。     

一种可用于评估肽质量指纹谱数据的方法——反转错位数据库

反转数据库常被用于估算大规模蛋白质组研究中串联质谱搜索数据库结果的可靠性。然而,对于经典的且现在依然在产出的肽质量指纹谱的数据,这种方法并不适用。为解决该问题,构建了另外一种随机数据库,称为反转错位数据库。这种数据库是在反转数据库的基础上,将序列中的K和R及其后的氨基酸交换位置(对于胰蛋白酶切割的结果)获得。这种处理避免了某些肽段因前后胰蛋白酶酶切位点氨基酸相同而在序列反转后质量依然不变,导致肽质量指纹谱法无法区分的问题。通过串联质谱和肽质量指纹谱测试数据的搜索结果,证明了这种方法同时适用于串联质谱和肽质量指纹谱的数据。这种方法扩大了经典反转数据库的适用范围,将对评估和整合串联质谱和肽质量指纹谱的数据具有重要意义。     

肽质量指纹谱鉴定蛋白质时生物信息学分析条件的优化

为了优化肽质量指纹谱(peptide mass fingerprint,PMF)鉴定蛋白质的生物信息学分析条件。将牛碳酸酐酶2(carbonic anhydrase-2,CAH2)和人热休克蛋白70s(Hsp70s)进行2-DE分离、酶解,肽段经过MALDI-TOFMS分析得到PMF数据。选择Swissprot、MSDB、NCBInr、Random等数据库和MASCOT与MS-Fit搜索引擎,以牛CAH2为模型优化搜索参数,结果表明:Swissprot是适合做蛋白PMF分析的数据库;主要参数最佳设置为:漏切位点数为1个,肽质量容错数为±1Da,同时肽质量类型选择平均分子质量比单同位素质量更便于候选蛋白的筛选。最后用人Hsp70s蛋白的PMF数据检验优化条件,结果表明,所选择的数据库及参数是可靠的。     

超高效液相色谱-四极杆飞行时间质谱分析不同加工何首乌中差异化学成分

建立了超高效液相色谱-四极杆飞行时间质谱(UPLC-QTOF-MS)结合多元统计分析技术对不同加工何首乌中化学成分差异的分析方法。何首乌样品采用甲醇在室温下超声提取后,采用UPLC-QTOF-MS进行分析,对采集的图谱通过峰匹配、峰对齐、滤噪处理等进行特征峰提取,然后用主成分分析(PCA)和偏最小二乘法-判别分析(PLS-DA)对数据进行分析。结果显示,不同加工何首乌样品间的化学组成存在显著性差异;根据一级质谱精确质荷比和二级质谱碎片信息,结合软件数据库搜索及相关文献进行成分鉴定,初步筛选并鉴定出33种不同加工何首乌间差异显著的化学成分,其中15种为共有差异化学成分,并呈现出不同的变化规律。研究结果可为揭示不同加工方法对何首乌代谢产物差异性的影响规律提供依据。     

基于生物质谱的定量蛋白质组学研究进展

随着蛋白质组研究和生物质谱技术的发展,大规模的蛋白质组相对定量和绝对定量已经成为了解生命活动进程、疾病发生发展过程以及生物标志物筛选和验证的重要策略,并形成蛋白质组学研究领域的一个重要分支：定量蛋白质组学.综述了近年来定量蛋白质组学的研究进展,并对其中的关键技术进行讨论.     

Study of tamoxifen urinary metabolites in rat by ultra‐high‐performance liquid chromatography time‐of‐flight mass spectrometry

Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug used for the treatment of breast cancer. It is also included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this work, the excretion of urinary metabolites of TMX after a single therapeutic dose administration in rats has been studied using ultra‐high‐performance liquid chromatography electrospray time‐of‐flight mass spectrometry (UHPLC‐TOFMS). A systematic strategy based on the search of typical biotransformations that a xenobiotic can undergo in living organisms, based on their corresponding molecular formula modification and accurate mass shifts, was applied for the identification of TMX metabolites. Prior to UHPLC‐TOFMS analyses, a solid‐phase extraction step with polymeric cartridges was applied to urine samples. Up to 38 TMX metabolites were detected. Additional collision induced dissociation (CID) MS/MS fragmentation was performed using UHPLC‐QTOFMS. Compared with recent previous studies in human urine and plasma, new metabolites have been reported for the first time in urine. Metabolites identified in rat urine include the oxygen addition, owing to different possibilities for the hydroxylation of the rings in different positions (m/z 388.2271), the incorporation of two oxygen atoms (m/z 404.2220) (including dihydroxylated derivatives or alternatives such as epoxidation plus hydroxylation or N‐oxidation and hydroxylation), epoxide formation or hydroxylation and dehydrogenation [m/z 386.2114 (+O –H₂)], hydroxylation of the ring accompanied by N‐desmethylation (m/z 374.2115), combined hydroxylation and methoxylation (m/z 418.2377), desaturated TMX derivate (m/z 370.2165) and its N‐desmethylated derivate (m/z 356.2009), the two latter modifications not previously being reported in urine. These findings confirm the usefulness of the proposed approach based on UHPLC‐TOFMS. Copyright © 2015 John Wiley & Sons, Ltd.     

Testing an alternative search algorithm for compound identification with the ‘Wiley Registry of Tandem Mass Spectral Data,MSforID’

A tandem mass spectral database system consists of a library of reference spectra and a search program. State‐of‐the‐art search programs show a high tolerance for variability in compound‐specific fragmentation patterns produced by collision‐induced decomposition and enable sensitive and specific ‘identity search’. In this communication, performance characteristics of two search algorithms combined with the ‘Wiley Registry of Tandem Mass Spectral Data, MSforID’ (Wiley Registry MSMS, John Wiley and Sons, Hoboken, NJ, USA) were evaluated. The search algorithms tested were the MSMS search algorithm implemented in the NIST MS Search program 2.0g (NIST, Gaithersburg, MD, USA) and the MSforID algorithm (John Wiley and Sons, Hoboken, NJ, USA). Sample spectra were acquired on different instruments and, thus, covered a broad range of possible experimental conditions or were generated in silico. For each algorithm, more than 30 000 matches were performed. Statistical evaluation of the library search results revealed that principally both search algorithms can be combined with the Wiley Registry MSMS to create a reliable identification tool. It appears, however, that a higher degree of spectral similarity is necessary to obtain a correct match with the NIST MS Search program. This characteristic of the NIST MS Search program has a positive effect on specificity as it helps to avoid false positive matches (type I errors), but reduces sensitivity. Thus, particularly with sample spectra acquired on instruments differing in their setup from tandem‐in‐space type fragmentation, a comparably higher number of false negative matches (type II errors) were observed by searching the Wiley Registry MSMS. Copyright © 2013 John Wiley & Sons, Ltd.     

Differential characterization of biogenic amine‐producing bacteria involved in food poisoning using MALDI‐TOF mass fingerprinting

Histamine poisoning is caused by the consumption of fish and other foods that harbor bacteria possessing histidine decarboxylase activity. With the aim of preventing histamine formation, highly specific mass spectral fingerprints were obtained from the 16 major biogenic amine‐producing enteric and marine bacteria by means of MALDI‐TOF MS analysis. All bacterial strains analyzed exhibited specific spectral fingerprints that enabled its unambiguous differentiation. This technique also identified peaks common to certain bacterial groups. Thus, two protein peaks at m/z 4182±1 and 8363±6 were found to be present in all Enterobacteriaceae species analyzed except for Morganella morganii. Peaks at m/z 3635±1 and 7267±2 were specific to both M. morganii and Proteus spp. Biogenic amine‐forming Proteus spp. exhibited three genus‐specific peaks at m/z 3980, 7960±1 and 9584±2. The genus Photobacterium also showed three genus‐specific peaks at m/z 2980±1, 4275±1 and 6578±1. The two histamine‐producing Gram‐positive bacteria Lactobacillus sp. 30A and Staphylococcus xylosus exhibited a few protein peaks in the 2000–7000 m/z range and could be easily distinguished from biogenic amine‐forming Gram‐negative bacteria. Clustering based on MALDI‐TOF MS also exhibited a good correlation with phylogenetic analysis based on the 16S rRNA gene sequence, validating the ability of the MALDI‐TOF technique to establish relationships between microbial strains and species. The approach described in this study leads the way toward the rapid and specific identification of major biogenic amine‐forming bacteria based on molecular protein markers with a goal to the timely prevention of histamine food poisoning.     

Altered Mascot search results by changing the <Emphasis Type="Italic">m</Emphasis>/<Emphasis Type="Italic">z</Emphasis> range of MS/MS spectra: analysis and potential applications

The Mascot search algorithm is one of the most commonly used tools for protein identification. Tandem mass spectrometry data searched against a protein sequence database is utilized for identifying peptides and proteins, each reported with a score. Higher Mascot scores are associated with lower chances of random hits. The process of peak selection performed by the search engine prior to the search is a critical aspect of the process. Here, we show that Mascot divides the MS/MS spectrum into fixed m/z regions for peak selection, starting at the lowest m/z value of the peak list. Therefore, modifying the m/z range of the peak lists by insertion of a dummy peak with low m/z value changes the ensemble of peaks used for searching. As a consequence, Mascot peptide scores and search results are altered significantly and a different subset of the peptides present in the sample is identified after processing. We further show that the effect can be exploited and additional proteins and peptides can be identified by repeating the search with a combined set of differently processed files, even when applying identical false-positive rates.     

Characterization of new metabolites from in vivo biotransformation of 2‐amino‐ 3‐methylimidazo[4,5‐f]quinoline in mouse by mass spectrometry

In studying the metabolic pathways underlying the mechanism of carcinogenesis of the heterocyclic amine of 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), we recently found a new metabolite which gave an [M + H]⁺ ion of m/z 217 when subjected to electrospray ionization (ESI) in positive‐ion mode. Following ip injection of this metabolite of m/z 217 (designated as m/z 217) to beta‐naphthoflavone‐treated mice, 57% of the total radioactivity was recovered in a 24‐h mouse urine sample. HPLC separation followed by MS analysis indicates that the urine sample contained m/z 217 (36 ± 3% of total recovered radioactivity) and two other peaks that gave rise to the [M + H]⁺ ions of m/z 393 (31 ± 4%, designated as m/z 393) and m/z 233 (14 ± 1%, designated as m/z 233). Beta‐glucuronidase treatment of m/z 393 resulted in a radioactive peak corresponding to m/z 217. ESI in combination with various mass spectrometry techniques, including multiple‐stage mass spectrometry, exact mass measurements and H/D exchange followed by tandem mass spectrometry, was used for structural characterization. The urinary metabolites of m/z 217, 393 and 233 were identified as 1,2‐dihydro‐2‐amino‐5‐hydroxy‐3‐methylimidazo[4,5‐f]quinoline, 1,2‐dihydro‐2‐amino‐5‐O‐glucuronide‐3‐methylimidazo[4,5‐f]quinoline and 1,2‐dihydro‐2‐amino‐5,7‐dihydroxy‐3‐methylimidazo[4,5‐f]quinoline, respectively. Our results demonstrated that m/z 217 is biotransformed in vivo to m/z 393 by O‐glucuronidation and to m/z 233 by oxidation. The observation of these more polar metabolites relative to IQ suggests that they may arise from a previously undescribed detoxicification pathway. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS‐PAGE and 2D gel electrophoresis

Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐TOF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS‐PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS‐PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐TOF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.     

Structure‐Based Design and Synthesis of the First Weak Non‐Phosphate Inhibitors for IspF,an Enzyme in the Non‐Mevalonate Pathway of Isoprenoid Biosynthesis

In this paper, we describe the structure‐based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non‐mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C₅ precursors to isoprenoids, isopentenyl diphosphate (IPP, 1 ) and dimethylallyl diphosphate (DMAPP, 2 ; Scheme 1). The non‐mevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non‐mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as potential drug‐like inhibitors of IspF protein, and synthesized (Schemes 2–5). Determination of the enzyme activity by ¹³C‐NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine‐2,5‐diamine derivatives in the upper micromolar range (IC₅₀ values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56′, which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub‐pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to ‘Pocket III’) and rings that occupy the flexible hydrophobic region of ‘Pocket II’. The proposed binding mode remains to be further validated by X‐ray crystallography.     

High‐throughput workflow for identification of phosphorylated peptides by LC‐MALDI‐TOF/TOF‐MS coupled to in situ enrichment on MALDI plates functionalized by ion landing

We report an MS‐based workflow for identification of phosphorylated peptides from trypsinized protein mixtures and cell lysates that is suitable for high‐throughput sample analysis. The workflow is based on an in situ enrichment on matrix‐assisted laser desorption/ionization (MALDI) plates that were functionalized by TiO₂ using automated ion landing apparatus that can operate unsupervised. The MALDI plate can be functionalized by TiO₂ into any array of predefined geometry (here, 96 positions for samples and 24 for mass calibration standards) made compatible with a standard MALDI spotter and coupled with high‐performance liquid chromatography. The in situ MALDI plate enrichment was compared with a standard precolumn‐based separation and achieved comparable or better results than the standard method. The performance of this new workflow was demonstrated on a model mixture of proteins as well as on Jurkat cells lysates. The method showed improved signal‐to‐noise ratio in a single MS spectrum, which resulted in better identification by MS/MS and a subsequent database search. Using the workflow, we also found specific phosphorylations in Jurkat cells that were nonspecifically activated by phorbol 12‐myristate 13‐acetate. These phosphorylations concerned the mitogen‐activated protein kinase/extracellular signal‐regulated kinase signaling pathway and its targets and were in agreement with the current knowledge of this signaling cascade. Control sample of non‐activated cells was devoid of these phosphorylations. Overall, the presented analytical workflow is able to detect dynamic phosphorylation events in minimally processed mammalian cells while using only a short high‐performance liquid chromatography gradient. Copyright © 2015 John Wiley & Sons, Ltd.     

Flexible and powerful strategy for qualitative and quantitative analysis of valepotriates in Valeriana jatamansi Jones using high‐performance liquid chromatography with linear ion trap Orbitrap mass spectrometry

Valeriana jatamansi Jones is an important medicinal plant and its quality is closely related to its region of origin. In the current study, we utilized a flexible and powerful strategy for comprehensive evaluation of the quality diversity for 15 regions in China. The method was based on a hybrid linear ion trap‐Orbitrap mass spectrometry platform. For structure characterization, fragmentation patterns were detected by analyzing a series of standard compounds using data dependent multistage mass spectrometry acquisition. A fragment ion database for valepotriates was established, and the acquired data were high throughput filtered by fragment ion search for compound identification. For quantitative purposes, we normalized the mass spectrometry data of 15 samples using SIEVE 2.0 and the differences in composition were analyzed using principal component analysis combined with hierarchical clustering analysis. The results identified a total of 92 compounds from Valeriana jatamansi Jones. Samples from Dali, Kunming, and Baoshan have better qualities and concentrations of the main active constituents. To verify our strategy, we compared the valtrate, acevaltrate, and baldrinal contents using high‐performance liquid chromatography with diode array detector. We developed and validated a comprehensive qualitative and quantitative analytical method to achieve quality control of Valeriana jatamansi Jones.     

Energy‐resolved mass spectrometry for differentiation of the fluorine substitution position on the phenyl ring of fluoromethcathinones

A reliable method for structural analysis is crucial for the forensic investigation of new psychoactive substances (NPSs). Towards this end, mass spectrometry is one of the most efficient and facile methods for the identification of NPSs. However, the differentiation among 2‐, 3‐, and 4‐fluoromethcathinones (o‐, m‐, and p‐FMCs), which are ring‐fluorinated positional isomers part of the major class of NPSs referred to as synthetic cathinones, remains a challenge. This is mostly due to their similar retention properties and nearly identical full scan mass spectra, which hinder their identification. In this study, we describe a novel and practical method for differentiating the fluorine substitution position on the phenyl ring of FMCs, based on energy‐resolved mass spectrometry (ERMS) using an electron ionization‐triple quadrupole mass spectrometer. ERMS measurements showed that the three FMC positional isomers exhibited differences in relative abundances of both the fluorophenyl cation (m/z 95) and the fluorobenzoyl cation (m/z 123). The logarithmic plots of the abundance ratio of these two cations (m/z 95 to m/z 123) as a function of the collision energy (CE) followed the order of o‐FMC < p‐FMC < m‐FMC at each CE, which allowed the three isomers to be unambiguously and reliably differentiated. The theoretical dissociation energy calculations confirmed the relationship obtained by ERMS analyses, and additional ERMS measurements of methylmethcathinone positional isomers showed that the differences in abundance among the FMCs were attributed to the differences in their collision‐induced dissociation reactivities arising from the halogen‐induced resonance effects on the phenyl ring. Moreover, the method for differentiation described herein was successfully applied to the actual samples containing seized drugs. We expect that the described methodology will also contribute significantly to the reliable and accurate structural identification of NPSs in the fields of therapeutic, clinical, and forensic toxicology.