A simple device for the extraction of TLC spots: direct coupling with an electrospray mass spectrometer

A new device is described which allows the recovery of compounds from thin layer chromatograms in short times, within a small volume, and without contamination. This apparatus can be coupled online to an electrospray mass spectrometer, but can also be used with other detectors or for micropreparations.     

Fabrication of low-melting-point alloy microelectrode and monolithic spray tip for integration of glass chip with electrospray ionization mass spectrometry

In this paper, a glass microchip-based emitter with a low-melting-point alloy (LMA) microelectrode and a monolithic tip for electrospray ionization mass spectrometry (ESI-MS) was described. So far, the fabrication of metal microelectrode achieving direct electrical contact in the microchannel of glass chip is still a challenge. A novel fabrication approach for LMA microelectrode in the glass chip was developed to achieve direct electrode-solution electrical contact in the microchannel. An electrode channel and a sample channel were firstly fabricated on a glass chip with a micropore connecting the two channels. The melted LMA was filled into the electrode channel under a pressure of ca. 100 kPa, forming a stable and nicely fitted interface at the micropore between the sample and the electrode channels due to surface tension effect. The melted LMA filled in the electrode channel was then allowed to solidify at room temperature. The channel geometries including the distance between the sample and the electrode channels on the mask and the turning angle of the electrode channel were optimized for fabricating the LMA electrode. In this work, an improved fabrication approach for monolithic emitter tip based on pyramid-shaped tip configuration and stepped grinding method was also developed to fabricate well-defined sharp tips with a smallest tip end size of ca. 15 μm × 50 μm. Two types of emitter tip end including puncher-shaped tip and fork-shaped tip were produced. The emitter with the fork-shaped tip showed better working stability (4.4% RSD, TIC) at nanoliter-scale flow rate of 50 nL/min. The fabrication approaches for the LMA microelectrode and emitter tip are simple and robust, and could be carried out in most of routine laboratories without the need of complicated and expensive instruments. The performance of the emitter was evaluated in the analysis of reserpine, angiotensin II and myoglobin. A continuous experiment over 6 h demonstrated good stability of the present system in long-term analysis.     

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography. Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramides than that achievable by thin-layer chromatography.     

Reduced matrix effects for anionic compounds with paired ion electrospray ionization mass spectrometry

It is well-known that matrix effects in high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) can seriously compromise quantitative analysis and affect method reproducibility. Paired ion electrospray ionization (PIESI) mass spectrometry is an approach for analyzing ultra-low levels of anions in the positive ion mode. This approach uses a structurally optimized ion pairing reagent to post-column associate with the anionic analyte, subsequently forming positively charged complexes. These newly formed complex ions are often more surface-active as compared to either the native anion or the ion pairing reagent. No studies have examined whether or not the PIESI approach mitigates matrix effects. Consequently, a controlled study was done using five analytes in highly controlled and reproducible synthetic groundwater and urine matrices. In addition, two different mass spectrometers (linear ion trap and triple quadrupole) were used. Compared to the negative ion mode, the PIESI-MS approach was less susceptible to matrix effects when performed on two different MS platforms. Using PIESI-MS, less dilution of the sample is needed to eliminate ionization suppression which, in turn, permits lower limits of detection and quantitation.     

Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono‐hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R² > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co‐existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.     

Improved compatibility of liquid chromatography with electrospray tandem mass spectrometry for tracing occurrence of barbital homologous residues in animal tissues

This work describes a liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure for multiplex screening, ultratrace quantification and reliable confirmation of barbital series residues in animal-derived food matrices. The method is developed based on a distinct dependency of the electrospray ionization (ESI) response of nine structural homologues on LC eluent properties and gas-phase ion chemistry during the ESI process. The “wrong-way-round” negative ionization aspect has been explored to optimize the compatibility of the hyphenated LC–MS/MS technique, which facilitates detection limits at 30–100-fold lower than 0.01 ppm without derivatization or post-column basification step. A mobile phase using methanol modified with 0.01% acetic acid is adopted to achieve an approximately 2–9-fold increase in signal-to-noise ratio over the results under suboptimal conditions. There is no significant differential matrix effects or deuterium isotope effects on chromatographic retention and ESI responsiveness at all levels across the different analyte–matrix pairs. Mean recoveries ranged from 79.6% (barbital) to 108% (secobarbital) at fortified levels of 0.5–20 ng/g within relative standard deviations less than 11%. Between-run repeatability and within-laboratory reproducibility were 3–11% and 5–13%, respectively. An ion ratio criterion for valid detection limit data for simultaneous screening of homologous multiresidues in complex sample matrices is proposed. The satisfactory applicability of the newly described procedure to 43 real samples including pork, poultry meat, swine liver, fish tissue and shrimp muscle demonstrated the LC–MS/MS technique with facile sample handling can serve as an attractive alternative analytical method accepted for regulatory purpose.     

Investigation of zinc binding metallothioneins' polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry

Polymerization of metallothioneins (MTs) is one of the commonly encountered puzzles in researching the structure and function of metallothioneins. In this work, a method involving SEC coupled with negative ion electrospray ionization mass spectrometry (ESI-MS) detection has been developed for the study of zinc binding MTs’ polymerization in tris(hydroxymethyl)-aminomethane (TRIS) acetate buffer at physiological pH. This hyphenated technique allows separating the different polymeric states of MTs by SEC, followed by on-line identification of the individual MT subisoforms in each polymeric peak by ESI-MS detection. Purified MT subisoforms (MT-2d and MT-2a), MT-2d and MT-2a mixture and rabbit liver MT complexes were investigated in the experiments to confirm the results obtained. From the results, both oxidative polymerization and non-oxidative oligomerization were found. The cystein-dependent oxidation results in the tetrameric peak as shown in the chromatograms of oxidized MT-2d, and stable dimeric and monomeric of MT were detected in this peak by MS. For the dimeric and trimeric peaks, different MT subisoforms were detected. In the five major subisoforms detected in rabbit liver MT complexes, MT-2a and MT-2c exist primarily as trimer, while MT-2e, MT-2d and MT-1a exist mainly as dimer. Our results suggest that in the three kinds of polymers, dimer, trimer and tetramer that were found in samples, the tetramer comes from the oxidation of MT molecular; for the dimer and trimer resulting from cystein independent oligomerization, they are closely associated with the charge of subisoform.     

固相萃取-超高效液相色谱-电喷雾串联质谱法同时测定地表水中16种全氟有机化合物

建立了固相萃取-超高效液相色谱-电喷雾串联三重四极杆质谱联用技术分析水中16种全氟有机化合物的高通量检测方法。样品经WAX固相萃取柱富集和净化后,采用Waters ACQUITY UPLC^TM BEH C₁₈色谱柱,含2 mmol/L乙酸铵的甲醇和含2 mmol/L乙酸铵的水作为流动相进行梯度洗脱,质谱采用电喷雾负离子电离,多反应监测模式检测。16种全氟有机化合物在0.5~100 μg/L或1.0~100 μg/L浓度范围内线性关系良好,相关系数为0.9987~0.9999,方法的检出限为0.06~0.46 ng/L;高、中、低3个添加水平的回收率为67.6%~103%,相对标准偏差为2.94%~12.0%。结果表明,该方法灵敏、准确且检测范围广,分析速度快,是一种适于实际水样中全氟有机化合物检测分析的方法。     

Quantitative analysis of perchlorate in extracts of whole fish homogenates by ion chromatography: comparison of suppressed conductivity detection and electrospray ionization mass spectrometry

The perchlorate anion (ClO ₄ ^– ) is an anthropogenic contaminant of increasing concern in water supplies, and has been shown to disrupt thyroid activity. Most perchlorate analyses are currently carried out by ion chromatography (IC) with suppressed conductivity detection (SCD). While this procedure has been demonstrated to provide acceptable performance for analysis of water samples, the determination of perchlorate in high-conductivity aqueous extracts of plant or animal material is not readily accomplished by IC-SCD unless lengthy cleanup protocols are applied. With the addition of electrospray ionization mass spectrometry (ESI-MS) to IC, it was hypothesized that the interference imposed by various ionic species could be significantly reduced without the need for purification; however, the analysis of perchlorate in relatively unpurified extracts of biologically derived homogenates by IC-ESI-MS has not previously been described in the literature. The research presented here represents a comparison of the capabilities of IC-SCD and IC-ESI-MS to detect perchlorate in reagent water and in crude extracts of perchlorate-exposed fish (threespine stickleback, Gasterosteus aculeatus). ESI-MS was found to compare favorably to SCD for the detection of perchlorate in deionized water, and to exceed SCD performance in perchlorate analysis of fish-derived extracts.     

A general screening method for doping agents in human urine by solid phase extraction and liquid chromatography/time-of-flight mass spectrometry

A general screening method based on solid phase extraction (SPE) and liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) was developed and investigated with 124 different doping agents, including stimulants, -blockers, narcotics, -adrenergic agonists, agents with anti-estrogenic activity, diuretics and cannabinoids. Mixed mode cation exchange/C8 cartridges were applied to SPE, and chromatography was based on gradient elution on a C18 column. Ionization of the analytes was achieved with electrospray ionization in the positive mode. Identification by LC/TOFMS was based on retention time, accurate mass and isotopic pattern. Validation of the method consisted of analysis of specificity, analytical recovery, limit of detection and repeatability. The minimum required performance limit (MRPL), established by World Anti-Doping Agency (WADA), was attained to 97 doping agents. The extraction recoveries varied between 33 and 98% and the median was 58%. Mass accuracy was always better than 5 ppm, corresponding to a maximum mass error of 0.7 mDa. The repeatability of the method for spiked urine samples, expressed as median of relative standard deviations (RSD%) at concentrations of MRPL and 10 times MRPL, were 14% and 9%, respectively. The suitability of the LC/TOFMS method for doping control was demonstrated with authentic urine samples.     

Plasma-source mass spectrometry for speciation analysis: state-of-the-art

Current and emerging capabilities of plasma-source mass spectrometry (PS-MS) as it is employed for elemental speciation analysis are reviewed. Fundamental concepts and their advantageous aspects, experimental conditions, and analytical performance are described and illustrated by recent examples from the literature. Novel instrumentation, techniques, and strategies for inductively-coupled plasma mass spectrometry (ICP-MS), microwave-induced plasma (MIP) mass spectrometry, glow-discharge (GD) mass spectrometry, and electrospray ionization (ESI), among others, are described. The use of ionization sources that provide tunable ionization, others that can be modulated between different sets of operating conditions, and others used in parallel is also examined.     

啤酒中单糖的衍生化HPLC-ESI-MS测定方法研究

单糖类样品在溶液中非常稳定,难于离子化,不适合于进行ESI-MS检测。采用1-苯基-3-甲基-5-吡唑啉酮(PMP)将糖类物质衍生化,HPLC-ESI-MS在线联用,选择性离子扫描方式对几种啤酒样品中的5种单糖进行了分离检测。检出限可达到80pg。     

Coupling on-chip solid-phase extraction to electrospray mass spectrometry through an integrated electrospray tip

We report the integration of solid-phase extraction (SPE) with mass spectrometry (MS) through an on-chip electrospray tip for sample precleaning and preconcentration. An in situ polymerized alkylacrylate-based monolithic column was used as the stationary phase for the on-chip SPE. Each microchip consists of two sets of microchannels and their respective integrated electrospray tips, with a common gold electrode. After the microchip was fabricated from cycloolefin polymer by hot embossing, thermal bonding, and annealing steps, a mixture of monomers and porogenic solvents was pumped into the microchannels and certain areas of the main microchannels were exposed to UV irradiation through a mask. The resulting porous monolithic beds that were polymerized from different compositions of the mixture were characterized by scanning electron microscopy. The microchip containing the monolithic column was then interfaced to an ion trap (IT) mass spectrometer by modifying a commercially available interfacing system. Makeup solution from the side channel was infused concurrently with the solution flowing into the main channel, and the mixture of these two solutions was sprayed into the MS orifice. Both the adsorption and elution of a pharmaceutical test compound, imipramine, to and from the on-chip SPE columns were monitored by MS. The potential application of this device for sample cleanup was demonstrated by pretreatment of urine samples spiked with imipramine.     

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.     

Application of electrospray ionization tandem mass spectrometry for the rapid and sensitive determination of cobalt in urine

Recently, cobalt (Co) is reported to be taken as a supplement by athletes for improving anaerobic performance. For the diagnosis of abuse, the limit of detection (LOD) of Co in the analysis should be lower than the concentrations of Co in plasma and urine of normal persons. A simple, rapid and sensitive method has been developed for the determination of Co in urine. Co was complexed with diethyldithiocarbamate (DDC) and extracted with isoamyl alcohol in the presence of citric acid. The detection of Co was achieved by injecting a 1-μL aliquot of isoamyl alcohol containing Co-DDC complex directly into an electrospray ionization tandem mass spectrometric (ESI-MS-MS) instrument without chromatographic separation. The quantification was performed using selected reaction monitoring at m/z 291 of the product ion Co(C₄H₁₀NCS)₂⁺ which was produced by collision-induced dissociation from the precursor ion Co(DDC)₂⁺ at m/z 355. ESI-MS-MS data were obtained in less than 10 min with an LOD of 0.05 μg L⁻¹ and a linear calibration range of 0.1-100 μg L⁻¹ using 10 μL of urine. The procedure was validated with certified reference materials (SRM 2670a and SRM 1643e). This method is suitable for the analysis of Co in the laboratories already equipped with an ESI-MS-MS instrument.     

Determination of energy metabolites in cancer cells by porous graphitic carbon liquid chromatography electrospray ionization mass spectrometry for the assessment of energy metabolism

A high performance liquid chromatography (HPLC) tandem mass spectrometric (MS/MS) method has been developed for the simultaneous determination of fifteen glucose, or acetate derived metabolites isolated from tumor cells. Glycolytic and tricarboxylic acid (TCA) cycle metabolites as well as acidic amino acids were separated on a HPLC porous graphitic carbon (PGC) column and simultaneously determined by means of triple quadrupole MS/MS using multiple reaction monitoring (MRM). Target compounds were eluted within 10 min with 8% v/v formic acid as an electronic modifier added to a 4:1 v/v methanol water mobile phase. The calibration is linear in the 1–100 μM concentration range for each analyte. The limit of detection ranges between 0.39 and 2.78 μM for the analytes concerned. To test the PGC–HPLC–MS/MS method in metabolomic studies, ZR-75.1 human mammary adenocarcinoma cells were labeled with U-¹³C glucose or 1-¹³C acetate. Applying the MRM mode, the incorporation of ¹³C into metabolites, isolated from the tumor cells, and derived from glucose or acetate, could be properly identified.     

Enzyme-assisted extraction and liquid chromatography mass spectrometry for the determination of arsenic species in chicken meat

Chicken is the most consumed meat in North America. Concentrations of arsenic in chicken range from μg kg⁻¹ to mg kg⁻¹. However, little is known about the speciation of arsenic in chicken meat. The objective of this research was to develop a method enabling determination of arsenic species in chicken breast muscle. We report here enzyme-enhanced extraction of arsenic species from chicken meat, separation using anion exchange chromatography (HPLC), and simultaneous detection with both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESIMS). We compared the extraction of arsenic species using several proteolytic enzymes: bromelain, papain, pepsin, proteinase K, and trypsin. With the use of papain-assisted extraction, 10 arsenic species were extracted and detected, as compared to 8 detectable arsenic species in the water/methanol extract. The overall extraction efficiency was also improved using a combination of ultrasonication and papain digestion, as compared to the conventional water/methanol extraction. Detection limits were in the range of 1.0–1.8 μg arsenic per kg chicken breast meat (dry weight) for seven arsenic species: arsenobetaine (AsB), inorganic arsenite (As^III), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), inorganic arsenate (As^V), 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone), and N-acetyl-4-hydroxy-m-arsanilic acid (NAHAA). Analysis of breast meat samples from six chickens receiving feed containing Roxarsone showed the presence of (mean ± standard deviation μg kg⁻¹) AsB (107 ± 4), As^III (113 ± 7), As^V (7 ± 2), MMA (51 ± 5), DMA (64 ± 6), Roxarsone (18 ± 1), and four unidentified arsenic species (approximate concentration 1–10 μg kg⁻¹).     

直接电喷雾离子阱质谱法快速表征烟叶

建立了一种快速、简单的同时检测烟叶中多酚类和非挥发性有机酸的电喷雾离子阱质谱方法。该方法采用水溶液萃取烟叶,萃取液无需色谱柱分离提纯,直接进入质谱检测,得到烟草质谱轮廓图。采用该方法对25种不同产地、不同等级的烟叶样品进行了测量,并结合模式识别的方法,对这25种烟叶进行了聚类分析,其结果基本符合烟草专家的等级划分。最后对各类烟叶进行了统计分析,得到了表征各类烟叶的平均质谱轮廓和各特征组分对区分烟叶贡献大小。表明该方法可以用于辅助烟叶等级划分及卷烟配方设计。     

Improved specificity of cardiolipin peroxidation by soybean lipoxygenase: a liquid chromatography - electrospray ionization mass spectrometry investigation

Peroxidation catalysed by Soybean Lypoxigenase was performed on tetralinoleyl-cardiolipin with the aim of generating selectively oxidized products, to be used subsequently as standards for studies on cardiolipin oxidation. The reaction products were characterized by LC-ESI-MS and MS/MS, and the process was found to link a hydroperoxylic group on one or more linoleic chains of cardiolipin, up to a total of four groups per molecule. Interestingly, the incidence of other oxidized products, like those arising from multiple hydroxylation or mixed hydroxylation-hydroperoxydation, previously observed after the chemical oxidation of the same cardiolipin, was found to be negligible. Moreover, evidences for the presence of the hydroperoxylic group(s) almost exclusively on carbon 13 of the linoleic chain(s) were obtained by MS/MS measurements. The enzymatic approach, integrated with a preparative separation step, which could be developed by adapting the chromatographic conditions adopted in the present work for analytical purposes, represents a promising strategy for the synthesis of highly specific mono- or multi-peroxidated derivatives of cardiolipins.