Pulsed Large Volume Injection Gas Chromatography Coupled with Electron-Capture Negative Ionization Quadrupole Mass Spectrometry for Simultaneous Determination of Typical Halogenated Persistent Organic Pollutants

A pulsed large-volume injection gas chromatography coupled with electron-capture negative ionization quadrupole mass spectrometry (pLVI-GC/ECNI-qMS) was developed for the simultaneous determination of typical halogenated persistent organic pollutants (H-POPs). By monitoring the characteristic ions of large mass-to-charge ratio (m/z) for each of the H-POPs rather than the chlorine and/or bromine ions, this method avoided the possible interferences arising from the H-POPs themselves and from complex matrices encountered frequently in current GC/qMS methods; and allowed, on the other hand, the use of 13C-labeled and perdeuterated analogues as internal standards for reliable quantification. pLVI up to 120 μL improved the instrumental detection limits down to pg–fg mL−1, comparable to or lower than those obtained by the recognized GC/high-resolution MS methods reported so far. The H-POPs including 12 polybrominated diphenyl ethers, 1 polybrominated biphenyl, 10 polychlorinated biphenyls (PCBs), 4 hexachlorocyclohexane isomers, and hexachlorobenzene were involved in this study. The method developed demonstrated good linearity (r2 = 0.9904–0.9999) within 0.5 to 50,000 pg mL−1 for PCBs and 0.05 to 5000 pg mL−1 for other H-POPs, and was satisfactory in terms of both repeatability (0.07%–2.2%) and reproducibility (2.1%–8.4%). It was validated by analyzing a NIST standard reference material SRM-1946 of Lake Superior fish tissue with low 0.01 to 63 pg g−1 method detection limits, and successfully applied to the determination of the H-POPs in five reference materials of different matrices

Quantification of selenium-tagged proteins in human plasma using species-unspecific isotope dilution ICP-DRC-qMS coupled on-line with anion exchange chromatography

Inductively coupled plasma dynamic reaction cell-quadrupole mass spectrometry (ICP-DRC-qMS) coupled on-line with anion exchange chromatography (AEC) has been developed for the quantification of selenium-tagged proteins in human plasma. Methane was employed as a reaction gas in the dynamic reaction cell to achieve the determination of Se-80 free of spectroscopic interference from Ar-40(2)+ and (BrH+)-Br-79. Five selenium species including selenoprotein P (SelP), glutathione peroxidase (GPx), selenoalbumin (SeAlb), and two unknown selenospecies (U1 and U2) in a pooled plasma sample from five healthy people were separated using AEC, and the distribution of selenium in SelP, GPx, SeAlb, U1 and U2 (about 45.5%, 19.1%, 15.1%, 2.9% and 8.1%) was determined by ICP-DRC-qMS using species-unspecific isotope dilution (Se-80/Se-77). Based on the detection limit (DL) of selenium (0.54 ng mL(-1)), we estimated that the DLs for SelP and GPx were 0.59 pmol mL(-1) and 1.7 pmol mL(-1), respectively. Through the stoichiometry of the selenium atom in the selenium-tagged proteins, SelP (2.7 +/- 0.1 mu g mL(-1)) and GPx (5.4 +/- 0.2 mu g mL(-1)) were successfully quantified.National Natural Science Foundation of China [20535020, 20775062]; National 863 project [2006AA06Z404]; National Basic Research Program of China [2003CD415001

In vivo phytochelatins and Hg-phytochelatin complexes in Hg-stressed Brassica chinensis L.

In vivo phytochelatins (PCs) and their corresponding Hg-PC complexes were characterized using RPLC-ESI-MS/MS in the roots of Brassica chinensis L. under the stress of a mercury cysteine complex (HgCys(2)) and/or a mercury humic acid complex (Hg-HA). Results indicated that the presence of Cys and/or HA decreased the Hg uptake in both the roots and shoots of B. chinensis but increased the generation of PCs in the roots compared with those where only HgCl2 was in the culture solutions. A series of Hg-PC complexes were synthesized in vitro for predicting the possible Hg-PC formed in vivo in the HgCys(2) and/or Hg-HA stressed roots of B. chinensis. The discovery of in vivo oxidized PC2, PC3 and PC4 and their corresponding HgPC2, HgPC3, HgPC4 and Hg2PC4, which were confirmed by their specific isotope distribution, provided definite evidence for understanding the defense and accumulation mechanism of B. chinensis to Hg, in which the induced PCs play an important role not only in Hg detoxification through forming Hg-PC complexes but also for reducing the oxidative stress induced by Hg2+.National Natural Science Foundation of China [20535020, 20775062]; National 863 Hi-Tech Project [2006AA06Z404]; National Basic Research Program of China [2009CB416000

SEC-ICP-MS and ESI-MS/MS for analyzing in vitro and in vivo Cd-phytochelatin complexes in a Cd-hyperaccumulator Brassica chinensis

In this study, in vitro synthesized Cd-phytochelatin (PC) complexes and in vivo Cd -PC complexes in Cd-stressed Brassica chinensis, which has been identified as a Cd hyperaccumulator, were characterized using SEC-ICP-MS and ESI-MS/ MS. The PCs (n = 1 -5) obtained from Cd-stressed B. chinensis together with CdCl2 were used to synthesize the in vitro Cd -PC complexes, and the formation of CdGS(1-2), (CdGS)(2), Cd1-2PC2, Cd1-3PC3, Cd1-3PC4 and Cd1-3PC5 was observed. In addition, for the first time, in vivo CdPC3 and CdPC4 complexes, as well as Cd-free PCs (n = 2-5) and desGlu-PC3 were detected in the extracts of Cd-stressed B. chinensis and confirmed by means of their corresponding isotopic peak distribution and MS/ MS spectra. Nitrogen saturated ammonium bicarbonate buffer (pH 7.8), instead of Tris-HCl and phosphate buffer, was used as a suitable mobile phase in order to stabilize the Cd -PC complexes and e. ectively avoid possible oxidation of PC analogues during SEC fractionation. Results obtained in this study give definite evidence elucidating the important roles which PCs play in plant defensive mechanisms to Cd stress. SEC-ICP-MS and ESI-MS/ MS were demonstrated as powerful and promising techniques for screening and identifying the in vivo metallopeptides, with accurate isotopic distribution assignment, in metal toxicological studies

Vapour generation at a UV/TiO2 photocatalysis reaction device for determination and speciation of mercury by AFS and HPLC-AFS

In this study we report a method for direct vapor generation of mercury species on nano TiO2 under UV irradiation in the presence of a formic acid and sodium formate mixture as a hole scavenger. A novelly designed UV/TiO2 photocatalysis reaction device (UV/TiO2 PCRD) was used as an effective sample introduction unit and an interface for mercury species determination by atomic fluorescence spectrometry (AFS) and speciation by HPLC-AFS for the first time. The detection limits of 10, 20, 30 and 70 pg mL(-1) of mercury chloride, methylmercury chloride, ethylmercury chloride and phenylmercury chloride, respectively, were achieved by AFS using flow injection mode. Compared with the traditional KBH4/NaOH-HCl system, UV/TiO2 PCRD is a superior alternative for online vapor generation of Hg species

Strategy for absolute quantification of proteins: CH3Hg+ labeling integrated molecular and elemental mass spectrometry

Currently, molecular mass spectrometry is preferred by many for relative quantification but is not appropriate for "absolute" quantification of proteins. In this article we demonstrate a proof of concept for the absolute quantitative analysis of proteins via CH3Hg+ labeling and integrated application of molecular and elemental mass spectrometry. The smallest size of CH3Hg+ among monoalkyl mercurials and the specific and covalent interaction with sulfhydryl (-SH) in proteins results in forming a simple complex of CH3Hg+:-SH 1:1 when all -SH are exposed, as confirmed by ESI-MS. Based on the known number of -SH per protein, the absolute protein concentration can be obtained via Hg determination using ICP-MS, in which CH3HgCl could be simply used as an external standard. When bovine pancreatic ribonuclease A, lysozyme and insulin, which have an increasing number of various disulfide linkages in their molecules, were taken as model proteins, their corresponding absolute detection limits (3 sigma) reached 0.6, 1.2 and 0.4 pmol, respectively. These characteristics may be expected to provide an alternative approach for absolute protein quantification, especially specific biomarker determination, in the near future

Chemical interactions of mercury species and some transition and noble metals towards metallothionein (Zn7MT-2) evaluated using SEC/ICP-MS, RP-HPLC/ESI-MS and MALDI-TOF-MS

National Natural Science Foundation of China [21035006, 21275120]; National Basic Research Program 973 Project [2009CB421605]The chemical affinities of three submetallomes ("Mn, Co, Ni, Cu, Cd''; "Pd, Pt, Au''; and "Hg, CH3Hg, C2H5Hg-THI'') towards metallothionein-2 (Zn7MT-2) in a physiological solution environment were evaluated using SEC/ICP-MS together with RP-HPLC/ESI-IT-MS and MALDI-TOF-MS, and followed the order: "Zn2+ Ni2+ > Mn2+, Co2+''; "Pd2+ > Au3+ > Pt2+''; and "Hg2+ > CH3Hg+ > C2H5Hg-THI''. Besides these, the structural change and composition of the CH3Hg-MT-2 complexes formed during the CH3Hg+ titration process were further investigated using CD spectroscopy, RP-HPLC/ESI-MS and MALDI-TOF-MS, indicating that linear and more hydrophobic (CH3Hg)(x)MT-2 (x = 12, 13, 14, 15, 16, 17, 18, 19 and 20) were formed. This information is important in understanding the interactions of the metals with Zn7MT-2 and their corresponding biological functions and toxicities

Synergistic defensive mechanism of phytochelatins and antioxidative enzymes in Brassica chinensis L. against Cd stress

Brassica chinensis L. was chosen and exposed to different concentrations of Cd exposure to evaluate its Cd-accumulating capacity and its potential cellular defensive mechanisms. Cd accumulation in the shoots and roots of B. chinensis was up to 1348.3 +/- 461.8 and 3761.0 +/- 795.0 mg per killogram of dry weight, respectively, under 200 mu mol/L of Cd exposure. Increasing Cd accumulation in the plant was accompanied by rapid accumulation of phytochelatins (PCs), and the sequestration of Cd by PCs provided a primary cellular mechanism for Cd detoxification and tolerance of B. chinensis. Furthermore, malondialdehyde formation, hydrogen peroxide content and antioxidative enzyme activities such as superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase were observed in the shoots of Cd-stressed B. chinensis. Increasing enzyme activities in response to concentrations of 5 to 50 mu mol/L Cd showed an efficient defense against oxidative stress, suggesting that the antioxidative system was a secondary defensive mechanism. These resulted in reduced free Cd damage and enhanced Cd accumulation and tolerance. Glutathione plays a pivotal role in these two detoxification pathways. In general, these results suggested that PCs and the antioxidative system are synergistic in combatting Cd-induced oxidative stress and that they play important roles in Cd detoxification of B. chinensis, and also give a deep understanding of the natural defensive mechanisms in plants under heavy metal stress

Distribution of lanthanum among the chloroplast subcomponents of spinach and its biological effects on photosynthesis: location of the lanthanum binding sites in photosystem II

The effects of lanthanum at different concentrations on the related photosynthetic activities of Hill reaction, Mg2+-ATPase and Ca2+-ATPase in spinach chloroplast were studied. Experimental results showed that lanthanum can increase all the activities at suitable concentration (1530 mg center dot L-1), however, it behaves toxically on them when over used (60 mg center dot L-1). To get an improved understanding of the mechanism of lanthanum effects on the photosynthesis of spinach, the different subcomponents in the chloroplast of the cultured spinach were isolated, and the content of lanthanurn in each subcomponent was determined by ICP-MS. The results obtained indicated that among these different subcomponents, about 90% out of the total chloroplast lanthanum was located in photosystem II (PS II) while there was little lanthanum in photosystem I (PS I). Moreover, size exclusion high performance liquid chromatography (SE-RPLC) coupled with online UV and ICP-MS detections was novelly used for locating lanthanum binding sites in PS II proteins for the first time. It was found that lanthanum has two binding sites in PS II: La associates with chlorophyll together with magnesium in PS II by partly replacing magnesium and also shares the common binding sites of PS II proteins together with the inorganic cofactors of calcium and manganese, influencing the process of photosynthesis

Preliminary study of the enrichment and fractionation of REEs in a newly discovered REE hyperaccumulator Pronephrium simplex by SEC-ICP-MS and MALDI-TOF/ESI-MS

Concentrations of rare earth elements (REEs) were determined in the laminae of 10 species of ferns and their acetone-extractable pigments, as well as their host soil and soil extract, by ICP-MS. A new REE hyperaccumulator, Pronephrium simplex, was discovered which could accumulate REEs up to 1.2 mg g(-1) dry mass under natural growth conditions. Three typical species of ferns chosen were divided into lamina, petiole, stem and root for the study of REE translocation and fractionation. A hyphenated technique, size exclusion HPLC coupled with online UV/ICP-MS, was developed to provide reliable evidence of the existence of REE-binding proteins in the fern's lamina. A new REE-binding protein was discovered and separated from the lamina of natural grown P. simplex. Further characterization of the protein showed that its molecular mass is 5068.4 Da by MALDI-TOF-MS and ESI-MS. Amino acid composition analysis by RP-HPLC indicated that the protein has relatively high contents of proline and glycin