Structural elucidation of V‐type nerve agents by liquid chromatography/electrospray ionization mass spectrometry

V‐nerve agents present information‐poor spectra, both in GC‐EI‐MS and LC‐ESI‐MS/MS, with dominant fragments/product ions corresponding to the amine‐containing residue. Hence, derivatives/isomers with the same amine residue exhibit similar mass spectral patterns, leading to ambiguity in the phosphonate structure. We present a simple approach for their structural elucidation based on two complementary experiments: ESI‐MS/MS of the original compound, which provides information about the amine moiety, and ESI‐MS/MS of the phosphonic acid hydrolysis products generated by N‐iodosuccinimide, which provides ions' characteristic of the phosphonate structure. This approach enables the structural elucidation of the original V‐agents with a higher degree of certainty.     

Initiation of free‐radical polymerization by peroxypivalates studied by electrospray ionization mass spectrometry

Initiation by tert‐butyl peroxypivalate (TBPP), tert‐amyl peroxypivalate (TAPP), 1,1,3,3‐tetramethylbutyl peroxypivalate (TMBPP), or 1,1,2,2‐tetramethylpropyl peroxypivalate (TMPPP) of radical polymerization of methyl methacrylate in toluene solution at 90 °C was studied via polymer end‐group analysis using electrospray ionization mass spectrometry (ESI‐MS). Conclusive peak assignments allowed for measuring the type and concentration of the fragments that actually initiate macromolecular growth after thermal decomposition of these peroxypivalates. It was found that the pivaloyloxy radical moiety undergoes instantaneous decarboxylation to yield an initiating tert‐butyl radical. The alkoxy radical moiety, on the other hand, may generate, via β‐scission reaction, different types of carbon‐centered radicals (together with a ketone) or may undergo a 1,5‐H‐shift reaction, by which reaction an oxygen‐centered radical is transformed into a carbon‐centered hydroxy radical. This hydrogen shift reaction was found in case of TMBPP. Surprisingly, no evidence for initiating alkoxy radicals could be found, not even in case of initiation by TBPP, where the intermediate tert‐butoxy radical undergoes a rapid chain‐transfer reaction with the solvent toluene. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4266–4275, 2004     

Studying the mechanism of thioketone‐mediated polymerization via electrospray ionization mass spectrometry

The polymeric product spectrum generated in thioketone‐mediated free radical polymerization (TKMP) was analyzed via electrospray ionization mass spectrometry. Poly(n‐butyl acrylate) samples were synthesized in the presence of the (commercially available) thioketone 4,4‐bis(dimethylamino)thiobenzophenone under variable reaction conditions in toluene solution at 80 °C. To unambiguously assign the mass spectra, the samples are prepared under variation of the monomer (going from n‐butyl acrylate to ethyl acrylate) as well as by employing variable thermally decomposing initiators [i.e., 2,2′‐azoisobutyronitrile and azobis(cyclohexanecarbonitrile)]. In all mass spectra, significant amounts of the expected cross‐termination product, formed via bimolecular termination of propagating macroradicals with the dormant thioketone radical adduct (consisting of a propagating chain and the mediating thioketone) alongside conventional termination products can be identified. As the study was carried out on acrylate polymers, acrylate‐specific reaction products arising from intramolecular transfer reactions followed by β‐scission of the generated mid‐chain radicals are also identified in the mass spectra. In addition, a species congruent with the dormant thioketone radical adduct itself (oxidized to its cationic state) was identified. Products that could potentially be formed via a chain transfer mechanism cannot be identified. The results presented here thus support the earlier suggested TKMP mechanism involving a highly stabilized adduct radical which undergoes significant cross‐termination reactions. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1864–1876, 2009     

Targeted characterization of acylated compounds from Scrophulariae Radix using liquid chromatography coupled with Orbitrap mass spectrometry and diagnostic product ion‐based data analysis

Acylated compounds are often present in herbal medicines. In this study, a diagnostic product ion‐based strategy was established to comprehensively characterize acylated compounds in Scrophulariae Radix. After untargeted data acquisition using ultra‐high performance liquid chromatography coupled with Orbitrap mass spectrometry, the data were processed by three‐stage diagnostic product ions. First, diagnostic product ions corresponding to the acyl groups (cinnamoyl, p‐coumaroyl, feruloyl, and caffeoyl) were used to search 90 compounds. Second, these compounds were divided into three categories using diagnostic product ions for phenylethanoid glycosides, iridoid glycosides, and phenylpropanoids, respectively. Last, the linkage position of the acyl group to iridoid glycosides was discriminated via the third‐stage diagnostic product ions. As a result, 90 acylated compounds were characterized, and 37 of them were reported from Scrophulariae Radix for the first time.     

Quality assessment of Cortex Phellodendri by high‐performance liquid chromatography coupled with electrospray ionization mass spectrometry

A simple method based on liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC‐DAD‐ESI‐MS) was developed for the quality assessment of Cortex Phellodendri (CP), which was mainly derived from two species of Phellodendron chinense Schneid and Phellodendron amurense Rupr. Total 41 compounds, including 14 phenols, 24 alkaloids and three liminoidal triterpenes were identified or tentatively characterized from the 75% methanol extract of CP samples by online ESI‐MSⁿ fragmentation and UV spectra analysis. Among them, two phenols and six alkaloids were simultaneously quantified using HPLC‐DAD method. The validated HPLC‐DAD method showed a good linearity, precision, repeatability and accuracy for the quantification of eight marker compounds. Furthermore, the plausible fragmentation pathway of the representative compounds were proposed in the present study. The differences of the chemical constituents content and the comprehensive HPLC profiles between the two CP species using LC‐DAD‐ESI‐MS method are reported for the first time, indicating that the CP drugs from different resources should be used separately in the clinic. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural elucidation of diglycosyl diacylglycerol and monoglycosyl diacylglycerol from Streptococcus pneumoniae by multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

The cell wall of the pathogenic bacterium Streptococcus pneumoniae contains glucopyranosyl diacylglycerol (GlcDAG) and galactoglucopyranosyldiacylglycerol (GalGlcDAG). The specific GlcDAG consisting of vaccenic acid substituent at sn‐2 was recently identified as another glycolipid antigen family recognized by invariant natural killer T‐cells. Here, we describe a linear ion‐trap multiple‐stage (MSⁿ) mass spectrometric approach towards structural analysis of GalGlcDAG and GlcDAG. Structural information derived from MSⁿ (n = 2, 3) on the [M + Li]⁺ adduct ions desorbed by electrospray ionization affords identification of the fatty acid substituents, assignment of the fatty acyl groups on the glycerol backbone, as well as the location of double bond along the fatty acyl chain. The identification of the fatty acyl groups and determination of their regio‐specificity were confirmed by MSⁿ (n = 2, 3) on the [M + NH₄]⁺ ions. We establish the structures of GalGlcDAG and GlcDAG isolated from S. pneumoniae, in which the major species consists of a 16:1‐ or 18:1‐fatty acid substituent mainly at sn‐2, and the double bond of the fatty acid is located at ω‐7 (n‐7). More than one isomers were found for each mass in the family. This mass spectrometric approach provides a simple method to achieve structure identification of this important lipid family that would be very difficult to define using the traditional method. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolite identification of tanshinol borneol ester in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry

Tanshinol borneol ester (DBZ) is a potential drug candidate composed of danshensu and borneol. It shows anti‐ischemic and anti‐atherosclerosis activity. However, little is known about its metabolism in vivo. This research aimed to elucidate the metabolic profile of DBZ through analyzing its metabolites using high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry. Chromatographic separation was performed on an Agilent TC‐C₁₈ column (150 × 4.6 mm, 5.0 μm) with gradient elution using methanol and water containing 0.2% (v/v) formic acid as the mobile phase. Metabolite identification involved analyzing the retention behaviors, changes in molecular weights and MS/MS fragment patterns of DBZ and its metabolites. As a result, 20 potential metabolites were detected and tentatively identified in rat plasma, urine and feces after administration of DBZ. DBZ could be metabolized to O‐methylated DBZ, DBZ‐O‐glucuronide, O‐methylated DBZ‐O‐glucuronide, hydroxylated DBZ and danshensu. Danshensu, a hydrolysis product of DBZ, could further be transformed into 12 metabolites. The proposed method was confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of DBZ and providing valuable information on its druggability.     

Reversible addition–fragmentation chain‐transfer polymerization: Unambiguous end‐group assignment via electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry was performed to identify the structure of polymeric methyl acrylates generated via the cumyl dithiobenzoate (CDB), cumyl p‐fluorodithiobenzoate (CPFDB), and 1‐phenylethyl dithiobenzoate (PEDB) mediated reversible addition–fragmentation chain‐transfer (RAFT) polymerizations. The relatively simple spectra clearly demonstrate the end groups of this living free‐radical polymerization technique. Only polymeric chains carrying one leaving group of the RAFT agent and the dithiobenzoate end group as the active RAFT center were discovered. Multiple‐stage mass spectrometric experiments and oxidation of the dithioester end group confirmed the structure of the generated polymers. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 4032–4037, 2002     

Characterization of forsythoside A metabolites in rats by a combination of UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques

Forsythoside A (FTA), the main active constituent isolated from Fructus Forsythiae, has various biological functions including anti‐oxidant, anti‐viral and anti‐microbial activities. However, while research on FTA has been mainly focused on the treatment of diseases on a material basis, FTA metabolites in vivo have not been comprehensively evaluated. Here, a rapid and sensitive method using a UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques including high‐resolution extracted ion chromatograms, multiple mass defect filters and diagnostic product ions was developed for the screening and identification of FTA metabolites in rats. As the result, a total of 43 metabolites were identified in biological samples including 42 metabolites in urine, 22 metabolites in plasma and 15 metabolites in feces. These results demonstrated that FTA underwent a series of in vivo metabolic reactions including methylation, dimethylation, sulfation, glucuronidation, diglucuronidation, cysteine conjugation and their composite reactions. The research enhanced our understanding of FTA metabolism and built a foundation for further toxicity and safety studies.     

Sheathless capillary electrophoresis electrospray ionization‐mass spectrometry interface based on poly(dimethylsiloxane) membrane emitter and thin conducting liquid film

This study develops a sheathless CE‐MS interface using a robust PDMS membrane emitter and liquid‐film electric conduction. A 3D mold was constructed for casting the device by using a one‐step casting procedure. The interface consisted of a 125 μm‐thick triangular emitter with a 50 μm‐diameter microchannel, a conducting reservoir, and a 375 μm‐diameter channel for assembling the separation capillary. The separation capillary was inserted into the 375 μm channel and connected to the emitter through the conducting reservoir. The electric contact for the CE outlet was established through a conductive liquid film in the space between the capillary terminus and the 375 μm channel. The one‐step casting procedure and using a membrane emitter instead of a tapered emitter produced an easily fabricated and robust interface. A stable electrospray was obtained from 30 to 350 nL/min. Analyzing a five‐peptide mixture in low‐EOF (60 nL/min) and high‐EOF (210 nL/min) conditions demonstrated the utility of the interface.     

Characterization and identification of iridoid glucosides,flavonoids and anthraquinones in Hedyotis diffusa by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MSⁿ). The optimized separation condition was obtained using an Agilent ZorBax SB‐C₁₈ column (4.6×150 mm, 5 μm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC‐ESI‐MSⁿ method.     

Structural characterization of a vegetable oil‐based polyol through liquid chromatography multistage mass spectrometry

A commercial vegetable oil‐based polyol for rigid polyurethane foams has been characterized by liquid chromatography‐electrospray ionization‐quadrupole ion trap mass spectrometry (LC‐ESI‐QIT‐MS). The absolute molecular weight (MW = 960) was measured by gel permeation chromatography (GPC) equipped with both refractive index (RI) detector and static laser light‐scattering detector (SLSD), which allowed further analysis by LC‐MS. The oligo‐polyol mixture was first separated in two elutes and then investigated by a deep multistage mass spectrometry (MSⁿ) study and completed using NMR. The major constituents identified were regioisomers of propoxylated sucrose (n_PO = 6–12), and the related esters of C16:0, C18:1, and C18:2 fatty acids had a mass ratio of 6:3:1. A comparison of fatty acids composition between the sample and palm oil demonstrated that the sample was initially prepared from the mixture of sucrose and palm oil by direct propoxylation. The MSⁿ fragmentation studies validated the structure of propoxylated sucrose and the related fatty acids derivatives. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 255–262     

高效液相色谱-电喷雾质谱法分离和鉴别枯草芽孢杆菌产生的脂肽类化合物

枯草芽孢杆菌JA因产生多种脂肽类化合物而具有广阔的开发前景。JA发酵液经过离心、酸沉淀、甲醇抽提等步骤得到脂肽类化 合物的粗提物。将粗提物溶于流动相,采用反相高效液相色谱分离,对收集的洗脱峰组分进行电喷雾质谱(ESI-MS)分析。根据质荷比推 断JA菌株产生的脂肽类化合物属于3个家族,分别为surfactin, iturin和fengycin,是枯草芽孢杆菌合成的重要生物表面活性素。对一 级质谱中的主成分进行串联质谱分析,进一步确定了3种脂肽类化合物的分子结构。实验证明ESI-MS是一种鉴定脂肽类化合物及其同系 物的可靠方法。     

Isolation and characterization of flavonols from blackcurrant by high-performance counter-current chromatography and electrospray ionization tandem mass spectrometry

Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties of other minor flavonoids constituents have not yet been investigated due the difficulties related to their isolation. Multiple steps of high-performance counter-current chromatography in combination with ESI tandem mass spectrometry (MS(n)) were successfully used for the preparative isolation of flavonols from blackcurrant extract, to study their electrospray ionization mass spectrometry fragmentation behavior. Seven flavonols, namely myricetin-3-O-rutinoside (145.5 mg), myricetin-3-O-hexoside (79.7 mg), myricetin-3-O-(6″-malonyl)-glucoside (17.4 mg), kaempferol-3-O-glucoside (20.5 mg), quercetin-3-O-rutinoside (55.1 mg), quercetin-3-O-hexoside (25.8 mg), and myricetin (129.1 mg) have been successfully isolated and their multistage MS(n) data were used for detailed structure characterization. The results of these experiments demonstrated that high-performance counter-current chromatography along with ESI-MS(n) is a sensitive, selective, and effective technology for isolation and characterization of minor constituents from a complex mixture.     

液相色谱-电喷雾串联质谱法测定生姜中的215种农药残留

建立了生姜中215种农药多残留测定的液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)方法。样品用1%醋酸-乙腈溶液均质提取,经Sep-Pak Vac固相萃取柱净化,乙腈-甲苯(3:1, v/v)洗脱,旋转蒸发浓缩至约0.5 mL后,于室温氮气吹干,用乙腈-水(3:2, v/v)溶解,以电喷雾电离串联质谱在正离子多反应监测(MRM)模式下进行测定。在定量限水平进行添加回收率实验,方法的回收率范围为68.1%～132.6%,其中回收率在70%～120%的占94.4%,相对标准偏差(RSD)范围为0.4%～25.0%。方法的检出限(S/N=3)和定量限(S/N=10)范围分别为0.01～70.45 μg/L和0.04～234.84 μg/L。该方法操作简便,灵敏度、准确度和精密度均符合农药多残留检测技术要求,适用于生姜中215种农药多残留的快速测定。     

Simultaneous analysis of ten phytohormones in Sargassum horneri by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry

Phytohormones have attracted wide attention due to their important biological functions. However, their detection is still a challenge because of their complex composition, low abundance and diverse sources. In this study, a novel method of high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous determination of ten phytohormones including indole‐3‐acetic acid, isopentenyladenine, isopentenyl adenosine, trans‐zeatin riboside, zeatin, strigolactones, abscisic acid, salicylic acid, gibberellin A3, and jasmonic acid in Sargassum horneri (S. horneri). The phytohormones were extracted from freeze‐dried S. horneri with methanol/water/methanoic acid (15:4:1, v/v/v) analyzed on a Hypersil Gold C₁₈ column and detected by electrospray ionization tandem triple quadrupole mass spectrometry in the multiple reaction monitoring mode. The experimental conditions for the extraction and analysis of phytohormones were optimized and validated in terms of reproducibility, linearity, sensitivity, recovery, accuracy, and stability. Distributions of the phytohormones in the stems, blades, and gas bladder of the S. horneri in drift, fixed, and semi‐fixed growing states were investigated for the first time. The observed contents of the phytohormones in S. horneri range from not detected to 5066.67 ng/g (fresh weight). Most phytohormones are distributed mainly in the stems of S. horneri in drift and semi‐fixed states.     

Structural identification of the metabolites of ganoderic acid B from Ganoderma lucidum in rats based on liquid chromatography coupled with electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry

Ganoderic acid B (GAB), a representative triterpenoid in Ganoderma lucidum, possesses various pharmaceutical effects and has been used as a chemical marker in quality control of G. lucidum and related products. The metabolites of GAB in vivo after its oral administration to rats were investigated by liquid chromatography coupled with electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry. A total of 14 metabolites of GAB in rat plasma, bile and various organs were detected and identified by direct comparison with the authentic compounds and their characteristic mass fragmentation patterns. The results showed that oxidization and hydroxylation were the common metabolic pathways for GAB in rats. Moreover, some reduction metabolites of GAB were detected in rat kidney and stomach and glucuronidation only appeared in rat bile. This is the first report on the metabolites of GAB in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

超高效液相色谱-串联质谱法测定浓缩苹果汁中的熊果苷

建立浓缩苹果汁样品中熊果苷的固相萃取-超高效液相色谱-串联质谱(SPE-UPLC-MS/MS)检测方法。浓缩苹果汁样品用水溶解、过滤后,用聚苯乙烯-二乙烯基苯共聚物(PS-DVB)固相萃取柱净化,外标法定量。测定时用Eclipse Plus C18色谱柱(100 mm×2.1 mm, 1.8 μm)分离,甲醇-水系统梯度洗脱;MS测定采用多反应监测(MRM)模式。熊果苷的检出限为0.02 mg/L,在0.04～2.0 mg/L的范围内标准溶液的峰面积与质量浓度呈良好的线性关系,回收率为75.2%～102.7%,相对标准偏差(RSD)低于8.9%。该方法简便、快速、灵敏,可用于浓缩苹果汁样品中熊果苷的检测和确证。     

超高效液相色谱-电喷雾串联质谱法分离鉴定烟草中5种糖苷类香味前体物质

采用超高效液相色谱-电喷雾串联质谱法(UPLC-ESI MS/MS)并结合气相色谱-质谱法分离鉴定了烟草中5种主要的糖苷类香味前体物质。烟样经甲醇提取、XAD-2柱净化,得到初步纯化的糖苷,在pH 5条件下将其酶解,释放出糖苷配基。采用气相色谱-质谱分析并通过标准谱库检索确定了5种挥发性苷元;然后通过电喷雾质谱(负离子模式)确定糖苷母离子并作碎片离子扫描(MS2),确定了5种糖苷类香味前体物质的存在形式;最后采用UPLC-ESI MS/MS,以甲醇和乙酸-乙酸铵水溶液为流动相,通过RP-C18柱分离,在多反应监测(MRM)模式下,鉴定了烟草中5种主要的糖苷类香味前体物质,为应用液相色谱-质谱分析缺乏标准样品的糖苷类香味前体物质奠定了基础。