Introduction 1 2+ 2 3 4 5 7 8 + 2+ 9 10 2+ 2+ 1 11 2 2+ 12 13 2 14 9 1 11 13 Therefore, our interest lies in the development of an analytical method that allows the direct detection of mercury and methylmercury biothiol complexes in biota. 15 16 17 Oryza sativa Experimental Instrumentation The instrumentation used for this work consisted of an Agilent Technologies (USA) suite comprising an 1100 series high-performance liquid chromatography (HPLC) system, an electrospray mass spectrometer (ES-MS) (XCT ion-trap mass spectrometer) and an inductively coupled plasma mass spectrometer (ICP-MS) (7500 c series). The HPLC system was equipped with an automatic degasser, a gradient pump, a thermostated autosampler tray and a thermostated column device. 18 Coupling of the HPLC instrument to the ES-MS and ICP-MS system was performed either individually using PEEK capillary tubing (1.6-mm outer diameter, 0.3-mm inner diameter), or simultaneously via a micro flow splitter (Upchurch, UK). In split mode for simultaneous coupling, 80% of the HPLC eluent was directed into the ES-MS system, while 20% went into the ICP-MS system. 2 2 19 1 Table 1 ICP-MS and ES-MS parameters Instrument Settings ICP-MS Instrument Agilent Technologies 7500 c Torch Standard  Ar gas flows    Cooling gas 16 L/min  Auxiliary gas 1 L/min   Nebuliser gas 0.95 L/min 2 5%   Spray chamber Scott, cooled (2 °C)   Nebuliser PFA, microconcentric    Internal standard 3   Cones Platinum   Isotopes monitored 200 202 103 34 32 16 ES-MS Instrument Agilent Technologies XCT ion-trap mass spectrometer Ion source Electrospray ionisation Capillary voltage 4,500 V Nebuliser pressure HPLC 50 psi (0.345 MPa); direct injection 20 psi (0.138 MPa) Drying gas HPLC 12 L/min; direct injection 5 L/min  Gas temperature 350 °C Scan window m/z 100–900 2 m/z 4 PFA HPLC MS 2 Reagents 2 2+ 2 + 2 Reduced l-cysteine and reduced GS were dissolved in water at a concentration of 1 mg/mL and were prepared fresh daily. 2 2 2 Modelling the MeHgCys conformation: ab initio and density functional theory calculations 20 21 22 http://srdata.nist.gov/cccbdb/ 23 Plant preparation Oryza sativa 24 25 2 Results and discussion The objective of this work was to develop an analytical method for the separation and identification of mercury and methylmercury bound to Cys and GS. Structural MS carried out here using an ES mass spectrometer equipped with an ion trap was used as a tool for the identification and structural characterisation on a molecular level. The information gained via the fragmentation pattern obtained from the ion-trap measurements proves invaluable for the identification of unknown molecules, along with some information on the molecules’ conformation, while the simultaneous element-selective detection of mercury via ICP-MS is the key to identifying mercury-containing biomolecules in a complex matrix amidst a variety of other organic molecules. More detailed information on the conformation of the HgMeCys molecule was obtained by a modelling approach, which underpins the findings of ion-trap ES-MS. In this paper, we focus on the structural identification of the selected mercury and methylmercury biomolecules via ion-trap ES-MS after their separation using RP HPLC. Here, all mercury compounds were measured under the same detection conditions, and all ES-MS parameters were kept constant for all four species during direct injection as well as in HPLC injection mode. Simultaneous detection with ICP-MS was applied with HPLC separation for mercury-selective determination. 2 2 1 + The characteristic isotope pattern dominated by the mercury isotope distribution In-source fragmentation of the mercury biomolecule Cys and GS moieties 2 + 1 + Fig. 1 + vertical black lines left violet yellow red green grey a b c 2 d 2 Cys GS  2 + 2 2 + + + 2+ + + m z m z m z m z 2 + 1 Fig. 2 + a b c 2 d 2  1 1 + m z m z + 3 m z \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$ {\text{MeHg}} - {\text{NH}}^{ + }_{3}  $$\end{document} 26 3 + m z 3 2 A fragment with mass 119.5 may be attributed to free Cys or cystine (doubly charged), but occurs at less than 1% abundance compared with the two main clusters. The absence of free Cys also suggests that the complex is formed quantitatively in the solution. 2 + m z m z + 2 2 + + m z \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$ {\text{Cys}} - {\text{Hg}} - {\text{NH}}^{ + }_{3}  $$\end{document} m z m z m z + 2 m z 2 2 m z m z + m z m z m z m z 2+ m z m z m z m z 2+ + 5 2 2 5 + 2 + 3 + 26 2 3 2 m z 200 m z 2 m z \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$ {\text{MeHg}} - {\text{NH}}^{ + }_{2}  $$\end{document} + Fig. 3 2 a 2 + m z 200 b 2 + m z 200 200 3  2 m z 3 200 + m z m z 2 m z m z m z + 2 + m z m z Modelling the MeHgCys conformation using ab initio and DFT calculations The conformation of MeHgCys can involve either bonding via an oxygen atom in the carboxylic group or a nitrogen atom from the ammonia group, and can carry the proton at either group. A modelling approach was used to determine the total energies for the different possible conformations of HgMeCys, including correction for the ZPE. 4 Fig. 4 1 6 2  2 Table 2 Conformational energies obtained for the two different models used Conformer E a E b 1 7.4 18.0 2 12.7 30.3 3 0.0 0.0 4 7.6 6.9 5 25.8 21.0 6 13.7 21.4 http://srdata.nist.gov/cccbdb/ a b The results of the Boltzmann factor calculations based on the corrected MP2 energies indicate that at the experimental temperature approximately 98% of the MeHgCys would be present as the form displaying the Hg–N interaction, with the remaining 2% consisting almost exclusively of the Hg–O forms. Using the B3LYP energies, these calculations suggest that the slightly lower figure of 87.5% for the Hg–N forms, with one of the Hg–O conformers making up most of the remaining population. As the MP2 energies are thought to be more accurate on the basis of physical considerations, it is reasonable to assume that the figure of 98% for the Hg–N population is similarly more accurate; however, even at the B3LYP level the figures clearly point to this being the form of the molecule expected to be dominant in the gas phase at 350 °C. The use of larger and/or more flexible basis sets might lead to alterations in these results owing to improved description of the Hg–X interactions but it is felt that this would be unlikely to significantly alter the distribution of energies in this case. m z 202 \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$ {\text{MeHg}} - {\text{NH}}^{ + }_{3}  $$\end{document} 2 m z \documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym} 
\usepackage{amsfonts} 
\usepackage{amssymb} 
\usepackage{amsbsy}
\usepackage{mathrsfs}
\usepackage{upgreek}
\setlength{\oddsidemargin}{-69pt}
\begin{document}$$ {\text{Me}}^{{200}} {\text{Hg}} - {\text{NH}}^{ + }_{2}  $$\end{document} m z 200 m z 2 + m z 3 2 + 2 2 5 m z 2 5 200 200 Fig. 5 2 + a b 2 c 2  2 2 m z 2 m z + MeHgGS 2 m z m z 2 m z + 2 2 2 2 m z m z m z 2 m z m z m z 2 2 2 2 2 2 6 202 103 m z + Fig. 6 2 2  + 2 + y 6 2 2 6 2 2 2 2 2+ 7 2+ + Fig. 7 1 2 2 2+ 3 4 + 5 6 2  Thus, a final explanation for the difference in intensity of the four compounds cannot be given at this point. Compound stability during spiking experiments of plant extracts 16 17 27 28 For the determination of such complexes in plant material, the latter has to be extracted using a minimally invasive method which does not destroy or transform the compounds we are looking for. Here, we tested the stability of the four example compounds in extracts from roots and shoots of rice plants. 2+ + 7 202 2+ 2 + 2 6 2+ 2 + 2+ 2+ 2+ 3 4 Table 3 Retention times for consecutive injection of six mercury compounds with 7 h difference Sample 2 2+ MeHgCys + 2 MeHgGS Standard 1 120.7 152 213.5 302 458.4 529.1 Standard 2 120.7 152 218.5 303 454.1 527.7 Roots extract 1 125.7 NA 213.5 286 457.8 529.8 Roots extract 2 126 NA 212.8 284.9 452.7 526.9 Shoots extract 1 125.7 150.6 213.5 295.6 457.7 529.1 Shoots extract 2 125 151.4 214.2 292.8 455.5 526.2 Average 123.9 151.5 214.3 294.1 456.0 528.1 σ 2.5 0.66 2.1 7.7 2.3 1.4 RSD (%) 2.0 0.4 0.9 2.6 0.5 0.3 NA RSD Table 4 Peak area (as percentage of first measurement) recoveries for consecutive injection of six mercury compounds with 7 h difference Sample 2 2+ MeHgCys + 2 MeHgGS Standard  91 159 10 98 92 83 Roots extract 93 NA 67 89 87 86 Shoots extract 96 77 49 95 86 86 + 2+ 2+ + 2+ + Conclusion and outlook 2 + 2 3 18 The work presented here is the first step in the determination of mercury species in biota on the molecular level. n n Finally, this study presents the first identification of mercury and methylmercury biothiols in spiked plant extracts; hence, this method is a novel tool to investigate whether mercury and methylmercury indeed form complexes with biothiols such as Cys and GS, or if they rather bind to larger entities such as phytochelatins in plants or proteins in fish and other biota.