Capillary-assembled microchip as an on-line deproteinization device for capillary electrophoresis A capillary-assembled microchip -LRB- CAs-CHIP -RRB- , prepared by simply embedding square capillaries in a lattice polydimethylsiloxane -LRB- PDMS -RRB- channel plate with the same channel dimensions as the outer dimensions of the square capillaries , has been used as a diffusion-based pretreatment attachment in capillary electrophoresis -LRB- CE -RRB- . Because the CAs-CHIPs employ square-section channels , diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel . When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution , the low-molecular-weight species , which have larger diffusion coefficients than the high-molecular-weight species , can be collected in a buffer-solution phase . The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE . This type of system can be used for CE analysis in which pretreatment is required to remove proteins . In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed . Introduction On-line combination of sample pretreatment and electrophoretic separations , for example capillary electrophoresis -LRB- CE -RRB- and microchip-based capillary electrophoresis -LRB- MCE -RRB- , has been focused on by many researchers , because of the possibilities of integrating complicated pretreatment procedures . During the past decade a variety of approaches have been used to combine pretreatment with CE separations . The `` single capillary approach '' , involving position-selective immobilization of functional molecules or polymers inside a capillary , has been used to demonstrate preconcentration or enzymatic reaction before CE separation -LSB- 1 , 2 -RSB- , and in-capillary preconcentration during the electrophoretic process -LSB- 3 -- 8 -RSB- has provided a simple system enabling direct application of the capillary in commercial equipment . The `` microchip-based approach '' , recently reviewed by many authors -LSB- 9 -- 11 -RSB- , has enabled integration of a variety of complicated pretreatment processes with the separation channel on a single microchip . These approaches have different advantageous features for each purpose . From the standpoint of total system design , however , a `` combination approach '' involving the use of a flow-system or a functionalized capillary together with a separation capillary provides a more flexible system designed for particular analyte . Ye et al. reported connection of a trypsin-immobilized capillary with a separation capillary , by means of a home-built chip prepared by lamination of Lexan and Parafilm , and demonstrated on-line digestion and CE separation -LSB- 12 -RSB- . Kuban et al. and Fang et al. reported `` flow-injection capillary electrophoresis -LRB- FI-CE -RRB- '' comprising a flow-injection system and a separation capillary -LSB- 13 , 14 -RSB- . This system enabled various pretreatment processes to be combined with CE separation and has since been expanded to the use of MCE to achieve rapid separations . These systems have been reviewed by Chen et al. -LSB- 15 -RSB- . Vizioli et al. prepared a monolithic polymer capillary , for selection of histidine-containing peptides , connected it to a CE separation capillary -LSB- 16 -RSB- . Fan et al. recently reported combination of an FI-CE system with in-capillary preconcentration using a dynamic pH junction -LSB- 17 -RSB- . In all the examples mentioned above , combination of a flow-reaction system or chemically functionalized conduits with a separation capillary played an important role , and would provide an attractive integrated device enabling flexible design for combining pretreatment and CE separation . In contrast , we have recently reported a new concept for fabricating a chemically functionalized microchip , called a `` capillary-assembled microchip -LRB- CAs-CHIP -RRB- '' -LSB- 18 -RSB- . The microchip is fabricated simply by embedding chemically functionalized square capillaries in a lattice PDMS channel chip which has the same channel dimensions as the outer dimensions of the square capillaries . By using this technique , we have constructed a multi-ion sensing system based on combination of several different ion-sensing capillaries and a valving and sensing system based on thermo-responsive polymer-immobilized capillaries and an enzyme-immobilized capillary -LSB- 19 , 20 -RSB- . Because the CAs-CHIP enabled easy connection of different functional capillaries , we focused on this device as an on-line sample-pretreatment attachment for CE separation capillaries in a manner similar to the previously reported FI-CE system . In this instance the CAs-CHIP , prepared from a PDMS chip , served both as the device for pretreatment of the sample solution , by use of a variety of chemical processes , and the injection device . Interfacing the flow system and the separation capillary using a PDMS chip has already been reported by Bergstrom et al. -LSB- 21 , 22 -RSB- . Because the CAs-CHIP employs a square-shaped channel structure , however , the characteristic multilayer flow formed in the square section channel can also be used as a useful pretreatment process which can be integrated . The CAs-CHIP is , therefore , expected to enable the integration of universal pretreatment processes by using different kinds of chemically-functionalized capillary and multilayer flow-based chemical processes ; this is usually difficult to achieve using other reported techniques . In this paper , we report preliminary results concerning the preparation of a CAs-CHIP as a deproteinization attachment for CE separation . Deproteinization was achieved by use of the multilayer flow obtained in the PDMS microchannel , and the small molecules separated from the mixed protein sample were injected into the separation capillary connected directly to the CAs-CHIP , to be analyzed by CE . In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species , and feasibility study was performed . Experimental Square capillaries and reagents Square capillaries of 300 μm outer width -LRB- flat-to-flat -RRB- and 50 μm inner width were purchased from Polymicro -LRB- Phoenix , AZ , USA -RRB- . Before use of these capillaries the polyimide coating was removed by heating . Sylgard 184 silicone elastomer was purchased from Dow Corning -LRB- Midland , MI , USA -RRB- . Reagents of the highest grade commercially available were used to prepare the aqueous solutions . Rhodamine B -LRB- RB -RRB- , sulforhodamine -LRB- SR -RRB- , and bovine serum albumin fluorescein conjugate -LRB- F-BSA -RRB- were purchased from Sigma -LRB- St Louis , MO , USA -RRB- . All reagents were used without further purification . Distilled and deionized water had resistivity greater than 1.7 × 107 Ω cm − 1 at 25 °C . Fabrication of CAs-CHIP-CE system by embedding square capillaries on a PDMS plate and bonding of a PDMS cover Figure 1 shows the design of a CAs-CHIP-CE system . The PDMS plate for pretreatment was connected to the inlet of the separation capillary -LRB- left side of Fig. 1 -RRB- and another PDMS plate was used to fix the detection position -LRB- right side of Fig. 1 -RRB- . Square capillaries were cut into appropriate lengths and embedded in the lattice microchannel network fabricated on the PDMS plate . The general procedure for fabrication of a lattice microchannel on a PDMS plate has been reported elsewhere -LSB- 18 -RSB- . Briefly , a glass mold with a lattice structure was prepared by cutting a 300-μm depth with 1 mm pitch , using a dicing saw with an edge 300 μm wide . The conventional PDMS molding process using the glass mold was then performed to prepare a PDMS mold . The second molding process using this PDMS mold gave the lattice microchannel network on the second PDMS plate . Plugged capillaries were prepared by introduction of PDMS prepolymer into the square capillaries -LRB- inner width 50 μm -RRB- and cured at 70 °C for more than 5 h . These plugged capillaries were also cut and used to prepare the designed channel network . After embedding all the capillaries , a PDMS cover was bonded on top . For this , a spin-coated PDMS prepolymer on an acrylic plate -LRB- ca. 2 mm thick -RRB- was used as the cover plate , to fill the voids formed between the cover plate and the capillary-embedded PDMS plate -LSB- 18 -RSB- . PDMS prepolymer was spin-coated on the acrylic plate at 7000 rpm , then attached to the capillary-embedded PDMS plate before curing . Bonding was carried out by curing at 60 °C for 12 h. Figure 2 shows the final CAs-CHIP-CE system with a 12-cm separation capillary . Depending on the resolution required , 12-cm -LRB- effective length 10 cm -RRB- and 48-cm -LRB- effective length 33 cm -RRB- separation capillaries were used . To apply the high voltage , two plastic vials , as solution reservoirs , were connected at the outlets of the PDMS channel and the separation capillary . Fig. 1General concept for fabricating a diffusion-based pretreatment -- CE separation system using a capillary-assembled microchip -LRB- CAs-CHIP -RRB- . The plugged capillaries indicated as gray parts are actually square capillaries with 50 μm square-shaped conduits blocked with PDMS . In this figure , for simplicity , these conduits are not shownFig . 2Photograph of a fabricated CAs-CHIP-CE device Operating procedures Channels -LRB- or capillaries -RRB- on the CAs-CHIP-CE system were washed sequentially with methanol , 0.1 mol L − 1 NaOH , 0.1 mol L − 1 HCl , and water , then rinsed and preconditioned with 50 mmol L − 1 phosphate-buffer solution -LRB- PBS -RRB- at pH 8.1 . Sample solution -LRB- RB , SR : 0.5 mmol L − 1 , F-BSA : 4 mg mL − 1 -RRB- and two buffer solutions for diffusion-based separation and sample injection were introduced by use of syringe pumps with appropriate flow rates -LRB- see figure captions -RRB- . Sample injection was achieved by on -- off switching of the syringe pump delivering buffer 2 , shown in Fig. 1 . Immediately after injection , high voltage was applied manually by use of a standard high-voltage power supply -LRB- Matsusada Precision , Shiga , Japan -RRB- . Capturing fluorescence images and laser-induced fluorescence measurement Fluorescence images of the microchannel were obtained by using an optical/fluorescence inverted microscope -LRB- Eclipse TS100-F , Nikon , Tokyo , Japan -RRB- . Photographs were captured using a 3CCD color camera -LRB- HV-D28S , Hitachi Kokusai Electric , Tokyo , Japan -RRB- installed at the front port of the microscope . Fluorescent images were collected using a mercury lamp as a light source and a filter block -LRB- G-2A and FITC , Nikon , Tokyo , Japan -RRB- . CE with laser-induced fluorescence -LRB- LIF -RRB- detection was performed on a home-built system based on an inverted fluorescence microscope -LRB- IX70 , Olympus , Tokyo , Japan -RRB- . Light at 488 nm from an argon ion laser -LRB- Newport Spectra Physics Laser Division , Mountain View , CA , USA -RRB- was introduced into the microscope . The laser beam was filtered through a 460 -- 490 nm band-pass filter , reflected by a 510 nm dichroic mirror , then focused on the detection point by means of a 20 × objective lens . Fluorescence was collected by use of the same objective lens , filtered through a 515 nm high-pass filter , and finally detected by use of a CCD camera -LRB- Model PMA-11 , Hamamatsu Photonics , Shizuoka , Japan -RRB- . To obtain electropherograms fluorescence at 600 nm was used throughout . Results and discussion Optimization of diffusion-based deproteinization Diffusion-based separation of chemical species by use of multilayer flow was first reported by Brody et al. -LSB- 23 -RSB- . They separated small molecules from a particle-containing sample , or proteins from a biological cell-containing sample -LSB- 23 , 24 -RSB- . Here we used this technique to separate small molecules from a mixed protein sample . When the sample solution containing small molecules and proteins made contact with the buffer-solution flow , the small molecules , which have larger diffusion coefficients than the proteins , diffused into the buffer solution . In this study , RB and SR were used as models for small molecules and F-BSA as that for a large molecule . Figure 3 shows the fluorescence images at the confluence point of two flows and the injection point . Fluorescence images for F-BSA and rhodamine molecules were captured using different optical filters . In this case , buffer 2 was not flowing . As can be seen , when the total flow rate of sample and buffer was very high , neither F-BSA nor rhodamine molecules could reach the separation capillary -LRB- Fig. 3a -RRB- . In contrast , selection of an appropriate total flow rate resulted in selective diffusion of small rhodamine molecules and negligibly small protein diffusion -LRB- Fig. 3b -RRB- . Figure 3b also shows the fluorescence intensity profiles obtained by analysis of the fluorescence images for the rhodamine derivatives . According to this , the fluorescence intensity of the rhodamine-based molecules at the injection point was approximately 40 % of the initial fluorescence intensity at the confluence point -LRB- see fluorescence intensity at position y at the injection point shown in the fluorescence intensity profile -RRB- . Because complete diffusion gives 50 % signal intensity for rhodamine-based molecules , these results suggest that diffusion of the rhodamine-based molecules was almost complete . In our experiments , surface adsorption of the solutes by the channel surface occurred , because of the hydrophobicity of the PDMS surface . Because these samples were continuously flowing for deproteinization , however , surface adsorption did not seriously affect the deproteinization procedure when sample injection was performed after steady flow was achieved . Fig. 3Fluorescence images obtained at the confluence and injection points at different flow rates . -LRB- a -RRB- Total flow rate 10 μL min − 1 -LRB- sample 5 μL min − 1 , buffer 1 5 μL min − 1 -RRB- . -LRB- b -RRB- Total flow rate 1 μL min − 1 -LRB- sample 0.5 μL min − 1 , buffer 1 0.5 μL min − 1 -RRB- . In -LRB- b -RRB- , fluorescence intensity profiles obtained by analysis of the fluorescence images obtained for rhodamine derivatives -LRB- SR , RB -RRB- are also shown . F-BSA and rhodamine-based molecules were detected by use of different optical filters In this study the molecular weights of RB and SR are 443 and 581 , respectively , and that for F-BSA is approximately 73,000 -LSB- 25 -RSB- . According to the literature , the diffusion coefficient for a small molecule with a molecular weight of 342 -LRB- sucrose -RRB- is 5.2 × 10 − 6 -LRB- cm2 s − 1 -RRB- and that for a large molecule with a molecular weight of 69,000 -LRB- HSA -RRB- is 6.1 × 10 − 7 -LRB- cm2 s − 1 -RRB- -LSB- 26 , 27 -RSB- . Because these molecular weights are very close to those of the molecules used in this study , an approximately tenfold difference between the diffusion coefficients of the small and large molecules enabled successful separation of small molecules from mixed protein sample solution . Although the difference between diffusion coefficients is a primary principle of diffusion-based separation , it should be noted that the optimum conditions may change , depending on the concentration ratio of the protein and small molecules . Therefore , careful optimization experiments may be required when the concentrations of the small molecules are very low . Sample injection and electrophoretic separation Sample injection was performed manually by on -- off switching of the syringe pump delivering buffer 2 . In the diffusion-based separation and the CE separation mode , three-layer flow composed of sample solution , collection buffer -LRB- buffer 1 -RRB- , and buffer 2 occurs in the 300 μm square-PDMS channel along the buffer 2 flow channel , as illustrated in Fig. 1 . Because buffer 2 prevents introduction of sample solution into the separation capillary , on-off switching of the syringe pump -LRB- buffer 2 -RRB- enables sample injection similar to the `` gated injection '' reported by Jacobson et al. -LSB- 28 -RSB- . Usually , stopping the syringe pump does not immediately stop flow of buffer 2 . In this work , therefore , it took several seconds to start injection . This causes diffusion of molecules at the front and rear ends of the sample plug . When sequential sample injections with injection times of 15 s were performed , however , fairly good reproducibility of 6.5 % RSD -LRB- peak height ; n = 5 -RRB- was obtained . Although this method of injection needs to be improved , good reproducibility led us to use this simple method for further investigation . Figure 4 shows detection signals obtained at 10 cm downstream of the injection point with and without voltage application . When the sample plug is introduced without voltage application , ca. 160 s , corresponding to a linear flow rate of 0.63 mm s − 1 , was required for detection . The approximate linear flow rate of the pressure-driven flow inside the separation capillary can be estimated from the total flow rate of sample and buffer flow -LRB- in total 2 μL min − 1 -RRB- , and the ratio of the cross sectional areas of the PDMS channel -LRB- 300 μm square -RRB- and the separation capillary -LRB- 50 μm square -RRB- . Under our experimental conditions the calculated linear flow rate was ca. 0.36 mm s − 1 . Although the order of the calculated value was the same as the experimental value , the difference may be because of the slight difference between the back pressures of the two waste reservoirs . When the high voltage of 5 kV -LRB- 417 V cm − 1 -RRB- was applied just after injection , the time required for detection was reduced to ca. 70 s and slight separation was observed . Because the net charge of SR was − 1 and that of RB was 0 , migration time of SR was slightly longer than that of RB . These results indicated that induction of electroosmotic flow and separation by electrophoresis , by voltage application , were confirmed . Fig. 4Detection of rhodamine-based molecules -LRB- RB and SR -RRB- downstream of the separation capillary with and without application of high voltage . Flow rates : sample , 1 μL min − 1 ; buffer 2 , 1 μL min − 1 . Injection time : 20 s Deproteinization and electrophoretic separation On the basis of these experiments , deproteinization and electrophoretic separation were achieved . Figure 5 shows the electropherograms obtained at the downstream of the separation capillary with and without diffusion-based deproteinization . When the sample solution containing RB , SR , and F-BSA was introduced into the separation capillary without deproteinization , a broad signal arising from F-BSA was observed after the appearance of the RB and SR peaks -LRB- Fig. 5a -RRB- . This broad signal may be because of the protein adsorption frequently observed in CE , or the multiple labeling of the F-BSA used in this work -LSB- 29 -RSB- . Commercially available F-BSA contains 7 -- 12 labeling molecules -LRB- average -RRB- per BSA molecule . Therefore , the electrophoretic mobilities of BSA species with different numbers of labels may cause the broad signals shown in Fig. 5a . In contrast , the electropherogram obtained after deproteinization by use of the multilayer flow had a completely flat baseline after the appearance of two peaks of the rhodamine molecules -LRB- Fig. 5b -RRB- . Thus our system enabled successful diffusion-based deproteinization and CE separation . Although interference from the protein was successfully removed , resolution of RB and SR is still not good in Fig. 5b -LRB- resolution : R = 1.4 -RRB- . When the longer , 48 cm , separation capillary -LRB- effective length : 33 cm -RRB- was connected to the CAs-CHIP instead of 12 cm capillary , however , baseline separation of these species after deproteinization was successfully achieved -LRB- resolution : R = 5.7 -RRB- . In addition , relative standard deviations of peak height and migration time for five sequential measurements were 11.4 and 11.5 % for RB and SR , and 1.5 and 1.4 % for RB and SR , respectively , indicating that reliable analysis with deproteinization pretreatment was successfully achieved by use of a longer separation capillary . Fig. 5Electropherograms obtained downstream of the separation capillary with and without diffusion-based deproteinization . Flow rates : sample , 0.5 μL min − 1 , buffer 1 0.5 μL min − 1 , buffer 2 1 μL min − 1 . Injection time 15 s. Applied voltage 5 kV -LRB- ca. 417 V cm − 1 -RRB- Conclusions We have demonstrated deproteinization and CE separation on a single device using CAs-CHIP technology . Diffusion-based deproteinization in a CAs-CHIP was successfully achieved by choosing an appropriate flow rate , and subsequent CE separation of small molecules was achieved by use of a separation capillary connected directly to the CAs-CHIP . Because diffusion-based separation is , in general , based on dilution of the sample solution , preconcentration before CE separation will be required in the next step . Because the CAs-CHIP is prepared by simply embedding square capillaries , however , further integration of the preconcentration process using chemically-functionalized capillaries , or the on-line preconcentration techniques reported to date , can be also used . These applications are currently under investigation .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

capillary electrophoresis
deproteinization
polydimethylsiloxane

capillary-assembled microchip
square capillary


Investigation

into

mercury

bound

to

biothiols

structural

identification

using

ESI

ion-trap

MS

and

introduction

of

a

method

for

their

HPLC

separation

with

simultaneous

detection

by

ICP-MS

and

ESI-MS

Mercury

in

plants

or

animal

tissue

is

supposed

to

occur

in

the

form

of

complexes

formed

with

biologically

relevant

thiols

-LRB-

biothiols

-RRB-

rather

than

as

free

cation

We

describe

a

technique

for

the

separation

and

molecular

identification

of

mercury

and

methylmercury

complexes

derived

from

their

reactions

with

cysteine

-LRB-

Cys

-RRB-

and

glutathione

-LRB-

GS

-RRB-

Hg

-LRB-

Cys

-RRB-

Hg

-LRB-

GS

-RRB-

MeHgCys

MeHgGS

Complexes

were

characterised

by

electrospray

mass

spectrometry

-LRB-

MS

-RRB-

equipped

with

an

ion

trap

and

the

fragmentation

pattern

of

MeHgCys

was

explained

by

using

and

calculations

showing

the

importance

of

mercury

amine

interactions

in

the

gas

phase

Chromatographic

baseline

separation

was

performed

within

min

with

formic

acid

as

the

mobile

phase

on

a

reversed-phase

column

Detection

was

done

by

online

simultaneous

coupling

of

ES-MS

and

inductively

coupled

plasma

MS.

When

the

mercury

complexes

were

spiked

in

real

samples

-LRB-

plant

extracts

-RRB-

no

perturbation

of

the

separation

and

detection

conditions

was

observed

suggesting

that

this

method

is

capable

of

detecting

mercury

biothiol

complexes

in

plants

Introduction

Mercury

is

one

of

the

most

important

elements

to

consider

when

environmental

pollution

is

concerned

and

ever

since

the

Minamata

disaster

in

the

organic

compound

methylmercury

has

been

in

focus

owing

to

its

enhanced

toxicity

and

its

biomagnification

properties

in

the

food

chain

-LSB-

-RSB-

Following

the

Minamata

disaster

the

differentiation

of

inorganic

mercury

in

the

form

of

from

organic

mercury

namely

in

the

form

of

methylmercury

has

become

a

major

task

Mercury

speciation

in

complex

matrices

is

not

easy

to

achieve

-LSB-

-RSB-

and

more

and

more

sophisticated

analytical

methods

have

been

developed

in

the

past

few

years

in

order

to

obtain

reliable

quantitative

results

In

particular

species-specific

isotope

dilution

mass

spectrometry

-LRB-

SSIDMS

-RRB-

employing

stable

mercury

isotopes

has

been

a

major

breakthrough

-LSB-

-RSB-

Otherwise

experiments

with

stable

isotope

tracers

introduced

into

the

environment

have

given

invaluable

information

on

the

distribution

and

transformation

behavior

of

mercury

in

the

environment

-LSB-

-RSB-

Mercury

has

also

been

identified

as

a

global

pollutant

and

the

importance

of

new

analytical

methods

towards

a

more

complete

understanding

of

species

distribution

pathways

and

global

impact

has

been

highlighted

recently

-LSB-

-RSB-

As

of

today

the

most

common

application

of

mercury

speciation

in

biota

is

focused

on

the

determination

and

distinction

between

methylmercury

and

inorganic

mercury

in

the

forms

of

MeHg

and

To

separate

the

mercury

species

from

their

matrix

usually

extraction

or

soft

digestion

methods

are

applied

which

conserve

the

C

Hg

bond

keeping

the

methylmercury

moiety

intact

However

it

is

more

than

unlikely

that

mercury

and

methylmercury

occur

as

free

cations

be

it

in

biota

or

in

the

environment

and

rather

they

are

likely

bound

to

sulfur-containing

biomolecules

e.g.

in

fish

-LSB-

-RSB-

or

present

as

chloride

complexes

e.g.

in

seawater

-LSB-

-RSB-

But

with

the

usual

practice

of

mercury

speciation

the

counterion

bound

to

the

mercury

species

is

lost

and

even

worse

completely

disregarded

Thus

the

information

on

the

molecular

entity

of

the

mercury

species

is

wasted

strictly

speaking

this

is

not

really

speciation

but

rather

a

fractionation

method

Now

in

spite

of

the

enormous

progress

made

in

mercury

speciation

many

questions

relating

to

mercury

and

its

behavior

in

biota

remain

unsolved

mysteries

Why

is

methylmercury

highly

bioaccumulated

while

is

not

Why

are

different

organs

targeted

by

methylmercury

as

opposed

to

and

how

can

the

enormous

latency

period

-LRB-

up

to

several

months

-RRB-

for

methylmercury

intoxication

be

explained

-LSB-

-RSB-

Little

is

known

today

about

the

mechanisms

that

enable

mercury

uptake

into

cells

and

its

subsequent

distribution

and

bioaccumulation

in

biota

But

mercury

and

methylmercury

are

well

known

to

bind

to

biothiols

abundant

in

biota

and

form

stable

complexes

via

Hg

S

bonds

Especially

in

a

model

system

mercury

complexes

with

cysteine

-LRB-

Cys

-RRB-

as

a

ligand

have

been

found

to

be

able

to

cross

cell

membranes

and

the

Hg

-LRB-

Cys

-RRB-

complex

has

been

defined

as

a

likely

form

in

which

is

present

Zalups

-LSB-

-RSB-

and

Bridges

and

Zalups

-LSB-

-RSB-

proposed

that

Hg

-LRB-

Cys

-RRB-

mimics

the

cystine

molecule

and

thus

may

use

the

active

cell

transport

systems

usually

used

for

cystine

transport

into

the

cell

Although

the

theory

of

molecular

mimicry

has

recently

been

questioned

using

structural

comparison

obtained

from

X-ray

absorption

studies

of

the

complexes

concerned

-LSB-

-RSB-

it

is

still

commonly

agreed

that

mercury

and

methylmercury

are

most

likely

bound

to

and

transported

as

complexes

formed

with

biothiols

abundant

in

biota

Other

studies

employing

direct

speciation

methods

in

biota

like

extended

X-ray

absorption

fine

structure

and

X-ray

absorption

near-edge

structure

underpin

the

assumption

that

methylmercury

and

mercury

are

bound

to

sulfur

in

a

complex

matrix

-LSB-

-RSB-

Although

mercury

biothiol

complexes

have

not

been

identified

in

vivo

so

far

mercury

biothiols

certainly

play

a

key

role

in

cell

uptake

distribution

and

subsequently

toxicity

of

mercury

in

biota

-LSB-

-RSB-

But

even

state-of-the-art

analytical

methods

for

mercury

speciation

fail

to

provide

us

with

all

the

necessary

information

i.e.

the

complete

molecular

structure

of

the

mercury

compounds

present

in

vivo

Therefore

our

interest

lies

in

the

development

of

an

analytical

method

that

allows

the

direct

detection

of

mercury

and

methylmercury

biothiol

complexes

in

biota

In

this

paper

we

describe

an

analytical

method

allowing

the

species

identification

of

mercury

and

methylmercury

biothiols

as

intact

molecules

As

example

species

Cys

and

glutathione

-LRB-

GS

-RRB-

complexes

with

mercury

and

methylmercury

were

synthesised

and

analysed

Cys

is

a

small

sulfur-containing

amino

acid

while

GS

is

a

small

peptide

comprising

only

three

amino

acids

γ-glutamyl

-LRB-

γ-Glu

-RRB-

Cys

and

glycine

-LRB-

Gly

-RRB-

Like

Cys

this

compound

is

usually

abundant

in

living

cells

in

millimolar

concentrations

and

it

is

a

key

compound

for

the

cell

's

redox

state

control

GS

is

the

smallest

building

block

for

the

synthesis

of

phytochelatins

which

are

produced

in

plants

as

a

response

to

metal

stress

and

are

believed

to

play

a

paramount

role

in

metal

detoxification

in

plants

-LSB-

-RSB-

Plants

are

well

known

to

accumulate

mercury

when

grown

on

polluted

soil

-LSB-

-RSB-

and

a

future

object

of

study

for

mercury

exposure

will

be

rice

-LRB-

Oryza

sativa

-RRB-

therefore

we

tested

the

method

and

the

species

stability

in

the

matrix

of

a

real

plant

extract

derived

from

rice

plants

Experimental

Instrumentation

The

instrumentation

used

for

this

work

consisted

of

an

Agilent

Technologies

-LRB-

USA

-RRB-

suite

comprising

an

series

high-performance

liquid

chromatography

-LRB-

HPLC

-RRB-

system

an

electrospray

mass

spectrometer

-LRB-

ES-MS

-RRB-

-LRB-

XCT

ion-trap

mass

spectrometer

-RRB-

and

an

inductively

coupled

plasma

mass

spectrometer

-LRB-

ICP-MS

-RRB-

-LRB-

c

series

-RRB-

The

HPLC

system

was

equipped

with

an

automatic

degasser

a

gradient

pump

a

thermostated

autosampler

tray

and

a

thermostated

column

device

For

the

chromatographic

separation

of

the

mercury

biothiols

an

HPLC

method

was

adapted

from

that

of

Raab

et

al.

-LSB-

-RSB-

Species

separation

was

carried

out

using

an

Agilent

Zorbax

Eclipse

XDB

-LRB-

mm

mm

μm

-RRB-

reversed-phase

-LRB-

RP

-RRB-

HPLC

column

using

a

gradient

elution

with

eluent

A

being

formic

acid

in

water

and

eluent

B

being

formic

acid

in

methanol

The

gradient

programme

went

from

eluent

A

to

eluent

B

in

min

with

a

flow

rate

of

mL/min

and

a

volume

of

μL

was

injected

via

the

autosampler

The

autosampler

tray

was

cooled

to

°C

for

all

experiments

Coupling

of

the

HPLC

instrument

to

the

ES-MS

and

ICP-MS

system

was

performed

either

individually

using

PEEK

capillary

tubing

-LRB-

outer

diameter

inner

diameter

-RRB-

or

simultaneously

via

a

micro

flow

splitter

-LRB-

Upchurch

UK

-RRB-

In

split

mode

for

simultaneous

coupling

of

the

HPLC

eluent

was

directed

into

the

ES-MS

system

while

went

into

the

ICP-MS

system

Hg

-LRB-

Cys

-RRB-

HgMeCys

Hg

-LRB-

GS

-RRB-

and

HgMeGS

solutions

were

prepared

as

described

above

in

concentrations

of

mg/L

-LRB-

as

Hg

-RRB-

each

in

formic

acid

From

these

solutions

a

mixture

containing

all

four

compounds

was

prepared

at

equal

concentrations

resulting

in

a

solution

with

a

concentration

of

mg/L

-LRB-

as

Hg

-RRB-

of

each

compound

A

continuous

internal

standard

-LRB-

Rh

μg/L

-RRB-

was

mixed

with

the

HPLC

effluent

prior

to

the

ICP-MS

nebuliser

via

a

Teflon

T-piece

This

internal

standard

enables

the

monitoring

of

overall

instrument

sensitivity

and

plasma

conditions

and

may

potentially

be

used

to

correct

the

mercury

signal

for

changes

in

intensity

due

to

matrix

effects

stemming

from

the

introduction

of

methanol

during

gradient

elution

-LSB-

-RSB-

The

ES-MS

system

was

also

used

in

direct

injection

mode

for

the

species

fragmentation

experiments

Here

the

sample

was

introduced

into

the

ES

source

via

a

syringe

pump

using

a

glass

syringe

The

ES-MS

and

ICP-MS

instrumentation

parameters

can

be

found

in

Table

Table

and

ES-MS

parametersInstrumentSettingsICP-MSInstrumentAgilent

Technologies

cTorchStandard

Ar

gas

flows

Cooling

L/min

Auxiliary

L/min

Nebuliser

L/min

Optional

gas

-LRB-

-RRB-

Spray

chamberScott

cooled

-LRB-

°C

-RRB-

NebuliserPFA

microconcentric

Internal

standardContinuous

aspiration

μg/L

Rh

in

ConesPlatinum

Isotopes

Technologies

XCT

ion-trap

mass

spectrometerIon

sourceElectrospray

ionisationCapillary

VNebuliser

pressureHPLC

psi

-LRB-

MPa

-RRB-

direct

injection

psi

-LRB-

MPa

-RRB-

Drying

gasHPLC

L/min

direct

injection

L/min

Gas

°C

Scan

windowm/z

fragmentation

window

m/z

perfluoroalkoxy

HPLC

high-performance

liquid

chromatography

Ion

trap

Reagents

All

reagents

used

were

of

analytical

grade

and

were

purchased

from

Sigma-Aldrich

UK

unless

mentioned

otherwise

The

water

used

for

all

the

preparations

was

of

suprapure

quality

-LRB-

MΩ

ELGA

water

system

UK

-RRB-

HPLC

eluents

were

prepared

from

formic

acid

at

in

and

in

methanol

An

inorganic

mercury

-LRB-

-RRB-

standard

stock

solution

at

approximately

mg/kg

-LRB-

as

Hg

-RRB-

was

prepared

by

dissolving

solid

in

water

A

methylmercury

-LRB-

MeHg

-RRB-

standard

stock

solution

at

approximately

mg/kg

-LRB-

as

Hg

-RRB-

was

prepared

from

solid

MeHgCl

in

methanol

For

the

preparation

of

mercury

and

methylmercury

biothiols

these

stock

solutions

were

further

diluted

in

formic

acid

The

water

for

the

preparation

of

standards

was

degassed

using

ultrasonication

-LRB-

min

-RRB-

followed

by

purging

with

-LRB-

min

at

mL/min

-RRB-

Stock

solutions

were

kept

in

a

freezer

at

°C

further

dilutions

were

kept

for

a

maximum

of

week

at

°C

in

the

dark

Reduced

l-cysteine

and

reduced

GS

were

dissolved

in

water

at

a

concentration

of

mg/mL

and

were

prepared

fresh

daily

Synthesis

of

HgMeCys

HgMeGS

Hg

-LRB-

Cys

-RRB-

and

Hg

-LRB-

GS

-RRB-

From

solutions

prepared

as

described

above

mercury

and

methylmercury

biothiols

were

synthesised

by

stoichiometrically

adding

the

respective

amounts

of

dissolved

biothiol

-LRB-

GS

or

Cys

-RRB-

to

a

defined

volume

of

mercury

or

methylmercury

solution

All

steps

were

carried

out

in

a

glove

bag

under

a

atmosphere

in

order

to

avoid

oxidation

of

the

thiols

Usually

mercury

thiol

compounds

were

prepared

fresh

daily

Modelling

the

MeHgCys

conformation

ab

initio

and

density

functional

theory

calculations

The

conformation

of

MeHgCys

might

involve

bonding

via

either

an

oxygen

atom

in

the

carboxylic

group

or

a

nitrogen

atom

from

the

amino

group

and

either

an

uncharged

or

zwitterionic

form

could

be

possible

In

an

attempt

to

predict

which

if

any

of

these

conformations/configurations

might

be

favoured

in

the

gas

phase

a

series

of

ab

initio

and

density

functional

theory

-LRB-

DFT

-RRB-

calculations

were

performed

on

a

variety

of

possible

structures

Geometry

optimisations

were

performed

using

the

electronic

structure

software

GAMESS-US

-LSB-

-RSB-

at

both

the

DFT

and

the

ab

initio

levels

of

theory

The

September

release

of

GAMESS-US

was

used

for

the

DFT

calculations

and

the

March

release

was

used

for

the

calculations

In

both

sets

of

calculations

the

-LRB-

d

p

-RRB-

basis

set

-LSB-

-RSB-

was

employed

for

all

atoms

with

the

exception

of

mercury

which

was

described

by

the

SBKJC

effective

core

potential

basis

-LSB-

-RSB-

A

subsequent

vibrational

analysis

at

the

optimised

geometries

showed

them

to

be

true

minima

and

also

provided

zero-point

energies

-LRB-

ZPEs

-RRB-

for

use

in

correcting

the

energies

obtained

from

the

minimisations

The

ZPEs

were

scaled

by

-LRB-

-RRB-

and

-LRB-

-RRB-

as

suggested

by

the

collected

data

available

online

from

the

CCCBDB

database

at

The

relative

populations

of

the

conformers

with

the

lowest

ZPE-corrected

energies

were

estimated

by

calculating

the

ratios

of

the

Boltzmann

factors

-LSB-

-RSB-

at

°C

which

is

the

gas

temperature

inside

the

ES

ion

source

Plant

preparation

Rice

-LRB-

Oryza

sativa

-RRB-

was

grown

from

seeds

in

vermiculite

medium

and

fed

with

regular

Hoagland

solution

-LSB-

-RSB-

three

times

a

week

After

months

of

growth

the

rice

plants

were

harvested

The

vermiculite

was

washed

off

the

roots

and

the

plants

were

sectioned

into

roots

and

shoots

cut

into

pieces

of

approximately

mm

length

Extracts

were

prepared

according

to

the

method

of

Raab

et

al.

-LSB-

-RSB-

Briefly

g

of

plant

material

was

ground

in

a

mortar

under

addition

of

liquid

the

sample

was

transferred

into

a

Greiner

tube

mL

of

formic

acid

was

added

and

the

mixture

was

left

standing

in

ice

at

°C

for

h.

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

Ion-trap

ES-MS

measurements

of

HgMeCys

HgMeGS

Hg

-LRB-

Cys

-RRB-

and

Hg

-LRB-

GS

-RRB-

using

direct

sample

injection

In

a

first

approach

of

identification

mercury

and

methylmercury

complexes

with

Cys

and

GS

synthesised

as

described

earlier

in

formic

acid

at

a

concentration

of

mg/kg

-LRB-

as

Hg

-RRB-

each

were

injected

as

single

standards

into

the

ES

source

of

the

ion-trap

MS

instrument

The

ES-MS

parameters

can

be

found

in

Table

The

species

identification

was

performed

by

using

information

obtained

from

the

ES-MS

and

ion-trap

MS

data

The

MH

molecule

clusterThe

characteristic

isotope

pattern

dominated

by

the

mercury

isotope

distributionIn-source

fragmentation

of

the

mercury

biomolecule

Cys

and

GS

moietiesIon

trap

MS

-LRB-

-RRB-

of

the

MH

molecular

ion

peak

In

Fig.

the

ES-MS

spectra

measured

for

the

molecular

ions

-LRB-

MH

-RRB-

of

the

four

compounds

are

shown

The

vertical

black

lines

represent

the

theoretical

isotope

ratio

pattern

of

the

molecules

according

to

their

molecular

structure

Fig.

-LRB-

ES

-RRB-

mass

spectrometry

-LRB-

MS

-RRB-

spectra

of

the

MH

peak

cluster

for

four

mercury

biothiol

compounds

-LRB-

direct

injection

of

single

standards

-RRB-

The

theoretical

isotope

pattern

is

shown

as

vertical

black

lines

Molecular

structures

as

ball-and-stick

models

are

shown

on

the

left

-LRB-

Hg

violet

S

yellow

O

red

N

green

C

grey

H

not

shown

-RRB-

Theoretical

isotope

abundance

is

matched

to

the

highest-abundance

peak

set

at

The

exact

mass

-LRB-

theoretical

-RRB-

is

given

for

the

peak

a

MeHgCys

b

MeHgGS

c

Hg

-LRB-

Cys

-RRB-

d

Hg

-LRB-

GS

-RRB-

Cys

cysteine

GS

glutathione

The

mercury

isotope

pattern

is

very

prominent

it

dominates

the

overall

isotope

pattern

of

the

molecules

and

can

therefore

be

used

as

a

characteristic

fingerprint

In

small

mercury

biomolecules

the

mercury

isotope

pattern

is

greatly

preserved

even

when

the

organic

molecular

structure

like

in

accounts

for

of

the

molecular

mass

The

mass

spectra

show

that

under

the

ionisation

conditions

applied

the

mercury

compounds

are

protonated

and

detected

by

the

ES-MS

instrument

as

a

molecule

cluster

The

experimentally

obtained

isotope

cluster

for

MH

match

well

with

the

theoretical

isotopic

molecule

patterns

calculated

for

each

compound

The

major

isotope

in

the

clusters

is

here

referred

to

as

the

abundance

isotope

reflecting

the

usual

practice

in

isotope

abundance

visualisation

by

defining

the

major

mass

as

and

the

less

abundant

isotopes

as

fractions

of

this

In-source

fragmentation

of

HgMeCys

HgMeGS

Hg

-LRB-

Cys

-RRB-

and

Hg

-LRB-

GS

-RRB-

Molecule

fragmentation

is

usually

observed

during

the

ionisation

step

in

the

ES

ion

source

The

fragmentation

pattern

obtained

in

this

process

gives

information

about

the

molecular

structure

of

a

compound

using

the

different

fragments

for

reconstruction

of

the

complete

molecule

However

for

our

experiments

we

are

interested

in

keeping

the

in-source

fragmentation

as

low

as

possible

in

order

to

get

the

main

peak

from

the

molecular

ion

MH

One

reason

for

this

is

that

we

try

to

obtain

optimum

detection

limits

for

mercury

biomolecules

in

real

samples

and

in-source

fragmentation

leads

to

a

diminished

MH

intensity

Moreover

the

MH

peak

gives

information

on

the

molecular

mass

and

enables

identification

via

the

characteristic

isotope

pattern

Finally

where

more

information

on

the

molecular

structure

is

needed

e.g.

to

elucidate

isomeric

forms

further

fragmentation

is

performed

on

selected

masses

within

the

ion-trap

MS

instrument

using

collision-induced

fragmentation

All

mercury

compounds

were

synthesised

in

solution

in

stoichiometric

amounts

-LRB-

i.e.

two

molecules

of

thiol

for

one

molecule

of

thiol

for

MeHg

-RRB-

In

order

to

determine

whether

the

mercury

compounds

had

reacted

quantitatively

with

the

organic

thiols

we

monitored

the

protonated

molecular

ions

-LRB-

MH

-RRB-

of

Cys

-LRB-

m/z

-RRB-

and

GS

-LRB-

m/z

-RRB-

As

only

the

reduced

thiols

are

able

to

bind

to

mercury

we

also

checked

for

the

oxidised

dimers

of

Cys

and

GS

with

m/z

and

m/z

The

ratio

between

the

molecular

ions

of

the

mercury

complexes

and

those

of

the

free

reduced

or

oxidised

thiols

gives

an

indication

for

the

reaction

yield

in

solution

as

well

as

the

stability

of

the

complexes

In

Fig.

the

mass

spectra

-LRB-

MS

-RRB-

of

the

four

mercury

compounds

investigated

are

shown

highlighting

the

in-source

fragments

observed

In-source

fragmentation

can

be

influenced

by

ionisation

parameters

such

as

capillary

voltage

Here

the

parameters

used

were

as

detailed

in

Table

for

the

direct-injection

mode

Fig.

spectra

-LRB-

MS

-RRB-

showing

the

in-source

fragmentation

for

four

mercury

biothiol

compounds

-LRB-

direct

injection

of

single

standards

-RRB-

a

MeHgCys

b

MeHgGS

c

Hg

-LRB-

Cys

-RRB-

d

Hg

-LRB-

GS

-RRB-

From

Fig.

it

is

evident

that

from

the

four

mercury

compounds

investigated

only

MeHgCys

-LRB-

Fig.

-RRB-

shows

extensive

in-source

fragmentation

resulting

in

two

clusters

of

approximately

the

same

peak

height

the

MH

cluster

with

m/z

as

the

abundance

isotope

and

a

second

cluster

with

m/z

as

the

abundance

isotope

showing

a

mass

loss

of

from

the

MH

molecular

ion

cluster

This

mass

loss

can

be

attributed

to

the

loss

of

either

OH

or

Apart

from

the

two

most

prominent

in-source

fragments

only

one

fragment

exhibiting

a

mercury-like

isotope

pattern

can

be

found

at

m/z

This

may

be

attributed

to

a

or

a

MeHgOH

moiety

and

accounts

for

just

of

the

combined

peak

areas

of

the

two

main

peaks

Similar

fragmentation

has

been

described

by

D'Agostini

et

al.

-LSB-

-RSB-

who

suggested

the

loss

of

from

the

MH

ion

and

hypothesised

that

two

isomeric

species

of

MeHgCys

are

present

in

solution

either

exhibiting

a

Hg

N

or

a

Hg

O

interaction

Here

we

show

that

the

Hg

N

interaction

is

the

most

likely

form

present

in

the

ion

source

and

that

the

in-source

fragment

at

m/z

is

indeed

most

likely

formed

through

loss

of

This

was

achieved

through

the

ion-trap

fragmentation

pattern

and

a

modelling

approach

in

which

the

most

stable

conformation

for

MeHgCys

was

calculated

in

the

gas

phase

These

experiments

are

highlighted

further

below

A

fragment

with

mass

may

be

attributed

to

free

Cys

or

cystine

-LRB-

doubly

charged

-RRB-

but

occurs

at

less

than

abundance

compared

with

the

two

main

clusters

The

absence

of

free

Cys

also

suggests

that

the

complex

is

formed

quantitatively

in

the

solution

Figure

shows

the

in-source

fragmentation

of

MeHgGS

during

direct

injection

clearly

showing

the

MH

molecular

peak

cluster

as

the

dominant

component

Two

more

fragment

peaks

can

be

identified

One

fragment

at

m/z

exhibiting

a

cluster

with

the

typical

mercury

isotope

pattern

can

be

attributed

to

a

loss

of

the

γ-Glu

moiety

from

GS

A

second

peak

appears

at

m/z

which

can

be

clearly

attributed

to

protonated

GS

The

spectrum

shows

the

MeHgGS

molecule

peak

prevailing

with

more

than

abundance

while

the

two

other

fragments

observed

correspond

to

approximately

MH

abundance

only

For

Hg

-LRB-

Cys

-RRB-

-LRB-

Fig.

-RRB-

MH

is

the

most

prominent

peak

in

the

spectrum

An

in-source

fragment

obtained

from

a

mass

loss

of

is

also

observed

but

accounts

for

less

than

of

the

MH

peak

only

Another

fragment

with

the

typical

mercury

isotope

pattern

and

m/z

the

abundance

peak

may

be

attributed

to

a

moiety

similar

to

the

fragment

observed

for

MeHgCys

a

Cys

group

can

obviously

be

cleaved

from

the

molecule

leaving

a

Hg

N

bond

behind

Two

more

peaks

can

be

distinguished

at

m/z

and

m/z

corresponding

to

protonated

cystine

-LRB-

Cys

Cys

-RRB-

and

Cys

Cystine

at

m/z

seems

quite

abundant

but

accounts

for

only

of

the

MH

cluster

of

Hg

-LRB-

Cys

-RRB-

Only

traces

of

free

Cys

at

m/z

-LRB-

less

than

of

the

mercury

molecular

cluster

-RRB-

were

found

The

spectrum

of

Hg

-LRB-

GS

-RRB-

-LRB-

Fig.

-RRB-

shows

two

mercury-containing

fragments

at

m/z

and

m/z

besides

the

most

prominent

peak

for

MH

at

m/z

While

m/z

can

clearly

be

attributed

to

a

protonated

HgGS

fragment

after

loss

of

one

GS

group

the

cluster

at

m/z

can

not

be

clearly

identified

but

might

be

a

Hg

GS

cluster

with

two

water

molecules

associated

A

peak

at

m/z

corresponds

to

the

doubly

charged

molecule

peak

Two

more

peaks

can

be

distinguished

m/z

corresponding

to

protonated

oxidised

GS

and

m/z

corresponding

to

free

reduced

GS

The

two

mercury-containing

fragments

correspond

to

-LRB-

m/z

-RRB-

and

-LRB-

m/z

-RRB-

respectively

of

the

overall

abundance

while

the

protonated

oxidised

and

reduced

GS

only

account

for

approximately

each

and

accounts

for

less

than

MH

is

found

to

occur

at

more

than

The

ionisation

efficiencies

differ

substantially

between

the

four

different

compounds

While

MeHgCys

exhibits

the

lowest

intensities

with

counts

the

MeHgGS

signal

is

tenfold

higher

Hg

-LRB-

Cys

-RRB-

and

Hg

-LRB-

GS

-RRB-

each

with

around

counts

show

intensities

between

these

two

Taking

into

account

that

MeHgCys

forms

an

abundant

in-source

fragment

with

the

same

intensity

as

the

MH

ion

these

three

molecules

appear

to

have

similar

ionisation

behaviour

leading

to

comparable

ratios

of

protonation

in

the

ES

source

under

the

conditions

applied

Conformation

of

HgMeCys

ion-trap

fragmentation

and

conformation

modelling

Collision-induced

fragmentation

was

performed

using

the

ion-trap

device

of

the

ES-MS

instrument

The

in-source

fragmentation

of

MeHgCys

showed

an

important

fragment

with

a

mass

loss

of

amu

with

abundance

as

high

as

the

MH

molecular

ion

This

mass

loss

can

be

explained

by

either

loss

of

OH

or

from

MH

and

both

possibilities

have

been

postulated

before

-LSB-

-RSB-

Here

we

use

the

ion-trap

information

on

the

two

prevailing

peaks

obtained

combined

with

a

modelling

approach

to

find

the

most

probable

conformation

of

MeHgCys

which

may

explain

the

fragmentation

behaviour

observed

As

shown

in

Fig.

the

spectrum

of

the

MeHgCys

molecular

ion

-LRB-

m/z

-RRB-

shows

a

predominant

fragment

at

m/z

due

to

loss

of

A

second

fragment

at

m/z

can

be

attributed

to

either

or

MeHg

O

formed

through

rearrangement

of

the

molecule

under

loss

of

the

residual

Cys

moiety

and

corresponds

to

the

fragmentation

pattern

obtained

in

source

This

finding

is

an

indication

that

the

Hg

S

bond

is

not

the

only

interaction

between

Cys

and

HgMe

but

that

either

Hg

O

or

Hg

N

bonding

is

involved

i.e.

either

the

carboxylic

or

the

ammonia

group

forms

a

bond

to

the

central

metal

Fig.

trap

MS

-LRB-

-RRB-

spectra

as

obtained

from

collision-induced

fragmentation

in

the

ion-trap

device

from

the

two

main

in-source

fragments

obtained

from

the

direct

injection

of

a

single

standard

of

MeHgCys

a

of

MH

at

m/z

-LRB-

-RRB-

b

of

MH

at

m/z

-LRB-

OH

or

-RRB-

A

different

picture

is

seen

when

is

performed

on

the

in-source

fragment

ion

with

m/z

-LRB-

formed

through

loss

of

OH

or

from

at

m/z

-RRB-

Here

we

can

distinguish

several

fragments

m/z

-LRB-

loss

of

-RRB-

m/z

and

m/z

formed

through

the

successive

loss

of

parts

of

the

Cys

moiety

and

m/z

corresponding

to

MeHg

In

contrast

to

the

of

MH

-LRB-

m/z

-RRB-

the

fragment

at

m/z

is

not

formed

This

is

an

indication

that

the

group

forming

this

fragment

has

been

lost

during

in-source

fragmentation

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

The

final

conformations

obtained

from

the

geometry

optimisations

at

the

level

are

shown

in

Fig.

-LRB-

conformers

-RRB-

Those

obtained

from

the

calculations

differed

only

by

small

deviations

in

bond

lengths

and

angles

and

are

therefore

not

shown

Two

of

the

geometry

optimisations

were

started

in

zwitterionic

configurations

with

either

a

carboxylic

oxygen

-LRB-

conformer

-RRB-

or

the

amino

nitrogen

-LRB-

conformer

-RRB-

positioned

close

to

the

mercury

atom

The

zwitterionic

form

of

MeHgCys

was

found

to

be

unstable

and

optimisations

led

to

the

neutral

form

regardless

of

the

initial

protonation

state

Of

the

remaining

calculations

conformer

and

were

started

with

the

amino

nitrogen

close

to

the

mercury

whereas

conformers

and

were

started

with

a

carboxylic

oxygen

in

this

position

In

all

of

these

optimisations

the

initial

Hg

X

-LRB-

X

is

O

or

N

-RRB-

interaction

was

retained

throughout

suggesting

that

this

is

generally

favoured

in

the

conformational

behaviour

of

MeHgCys

Fig.

the

MeHgCys

conformation

final

conformations

of

MeHgCys

obtained

from

geometry

optimisation

at

the

level

Model

numbers

-LRB-

-RRB-

as

used

in

Table

The

pattern

of

relative

conformational

energies

-LRB-

Table

-RRB-

is

basically

the

same

at

both

levels

of

theory

with

the

most

energetically

favoured

conformations

displaying

the

Hg

N

interaction

Those

conformations

displaying

Hg

O

interaction

make

up

the

second-most

thermodynamically

stable

grouping

whereas

the

extended

conformer

where

the

mercury

atom

is

free

from

nonbonded

interaction

is

considerably

higher

in

energy

than

all

the

others

The

energy

differences

are

more

pronounced

at

the

level

than

at

the

level

and

this

is

probably

due

to

the

fact

that

the

perturbation

theory

treatment

is

capable

of

describing

dispersion

effects

whereas

DFT

is

not

Dispersion

effects

are

responsible

for

mediating

nonbonded

interactions

such

as

hydrogen

bonds

and

van

der

Waals

bonds

and

therefore

it

would

be

expected

that

these

would

be

important

in

this

particular

case

For

this

reason

the

results

are

expected

to

be

more

reliable

than

those

obtained

with

DFT

since

the

former

provide

a

more

complete

physical

picture

of

the

MeHgCys

system

Table

energies

obtained

for

the

two

different

models

G

-LRB-

d

p

-RRB-

ΔE

-LRB-

kJ/mol

-RRB-

G

-LRB-

d

p

-RRB-

ΔE

-LRB-

kJ/mol

-RRB-

.418.0212.730.330.00.047.66.9525.821.0613.721.4

Scale

factors

obtained

from

the

CCCBDB

database

at

energies

scaled

by

energies

scaled

by

The

results

of

the

Boltzmann

factor

calculations

based

on

the

corrected

energies

indicate

that

at

the

experimental

temperature

approximately

of

the

MeHgCys

would

be

present

as

the

form

displaying

the

Hg

N

interaction

with

the

remaining

consisting

almost

exclusively

of

the

Hg

O

forms

Using

the

energies

these

calculations

suggest

that

the

slightly

lower

figure

of

for

the

Hg

N

forms

with

one

of

the

Hg

O

conformers

making

up

most

of

the

remaining

population

As

the

energies

are

thought

to

be

more

accurate

on

the

basis

of

physical

considerations

it

is

reasonable

to

assume

that

the

figure

of

for

the

Hg

N

population

is

similarly

more

accurate

however

even

at

the

level

the

figures

clearly

point

to

this

being

the

form

of

the

molecule

expected

to

be

dominant

in

the

gas

phase

at

°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

Assuming

that

the

Hg

N

form

is

dominant

in

the

ion

source

it

is

reasonable

to

assume

that

the

in-source

fragment

obtained

at

m/z

-LRB-

-RRB-

corresponds

to

a

-LRB-

protonated

-RRB-

ion

This

is

confirmed

by

the

occurrence

of

the

same

fragment

in

at

m/z

-LRB-

-RRB-

The

in-source

fragment

at

m/z

-LRB-

-RRB-

does

not

exhibit

the

m/z

fragment

during

but

loses

the

Cys

completely

with

MeHg

remaining

as

the

smallest

mercury-incorporating

fragment

This

is

suggestive

of

the

possibility

that

the

in-source

fragment

with

m/z

has

lost

rather

than

OH

a

MeHg

N

fragment

can

not

be

formed

from

this

mother

ion

However

it

should

be

noted

that

besides

the

Hg

N

interaction

a

small

contribution

of

Hg

O

is

possible

Ion-trap

of

the

MH

cluster

of

Hg

-LRB-

Cys

-RRB-

HgMeGS

and

Hg

-LRB-

GS

-RRB-

Figure

depicts

the

ion-trap

fragmentation

patterns

obtained

for

the

three

remaining

mercury-containing

biomolecules

The

m/z

values

of

the

spectra

shown

here

in

Fig.

are

each

defined

for

the

isotope

However

the

chosen

trap

window

ion

width

of

amu

ensures

that

a

major

part

of

the

ion

cluster

is

trapped

and

undergoes

collision-induced

fragmentation

Thus

easier

identification

of

mercury-containing

fragments

is

possible

as

the

mercury

pattern

is

greatly

conserved

However

the

fragment

incorporating

will

be

trapped

with

the

highest

abundance

Fig.

spectra

as

obtained

from

collision-induced

fragmentation

of

the

MH

molecular

cluster

in

the

ion-trap

device

from

three

mercury

biothiols

a

MeHgGS

b

Hg

-LRB-

Cys

-RRB-

c

Hg

-LRB-

GS

-RRB-

Hg

-LRB-

Cys

-RRB-

The

ion-trap

collision-induced

fragmentation

of

Hg

-LRB-

Cys

-RRB-

exhibits

a

most

abundant

fragment

at

m/z

corresponding

to

the

loss

of

and

a

second

important

fragment

at

m/z

which

corresponds

to

the

loss

of

one

Cys

molecule

leaving

a

nitrogen

atom

bound

to

mercury

-LRB-

CysHg

N

-RRB-

This

behaviour

is

similar

to

what

was

found

for

the

fragmentation

of

MeHgCys

and

seems

to

be

typical

for

mercury

bound

to

a

Cys

moiety

No

other

fragments

containing

mercury

can

be

observed

and

no

unbound

Cys

molecules

were

detected

MeHgGS

The

of

MeHgGS

shows

one

high-abundance

fragment

at

m/z

corresponding

to

the

loss

of

a

Glu

moiety

and

OH

plus

two

low-abundance

fragments

at

m/z

-LRB-

loss

of

-RRB-

and

m/z

-LRB-

loss

of

Glu

-RRB-

GS

is

not

lost

during

this

fragmentation

but

rather

the

Glu

moiety

is

chopped

off

the

GS

Interestingly

a

MeHg

ion

fragment

that

it

is

formed

during

of

HgMeCys

can

not

be

detected

either

hence

mercury

is

probably

stabilised

by

the

Gly/Cys

moiety

Hg

-LRB-

GS

-RRB-

The

of

Hg

-LRB-

GS

-RRB-

shows

two

main

fragments

with

m/z

-LRB-

loss

of

Glu

-RRB-

and

m/z

-LRB-

loss

of

GS

-RRB-

plus

four

less

abundant

fragments

m/z

-LRB-

loss

of

-RRB-

and

m/z

m/z

and

m/z

formed

by

successive

loss

of

GS

amino

acids

HPLC

separation

of

HgMeCys

HgMeGS

and

with

simultaneous

ES-MS

and

ICP-MS

detection

For

the

chromatographic

separation

of

the

mercury

biomolecules

HPLC

and

the

simultaneously

coupled

ES-MS

and

ICP-MS

system

was

used

as

described

in

Instrumentation

Hg

-LRB-

Cys

-RRB-

HgMeCys

Hg

-LRB-

GS

-RRB-

and

HgMeGS

solutions

were

prepared

accordingly

as

well

as

a

mixture

of

four

compounds

with

a

concentration

of

mg/L

-LRB-

as

Hg

-RRB-

each

Single

injections

of

each

standard

revealed

the

retention

times

for

each

compound

and

these

injections

were

followed

by

injection

of

the

mixture

of

the

compounds

The

elution

order

obtained

was

Hg

-LRB-

Cys

-RRB-

HgMeCys

Hg

-LRB-

GS

-RRB-

and

HgMeGS

with

HgMeGS

being

eluted

at

approximately

min

All

compounds

were

eluted

at

a

gradient

composition

of

less

than

eluent

B

showing

that

relatively

soft

elution

conditions

can

be

applied

Figure

shows

the

combined

ICP-MS

-LRB-

-RRB-

and

ES-MS

traces

for

the

extracted

ion

chromatograms

of

m/z

for

MH

for

the

four

mercury

species

Fig.

ICP-MS

and

ES-MS

spectra

from

high-performance

liquid

chromatography

injection

of

MeHgCys

MeHgGS

Hg

-LRB-

Cys

-RRB-

and

Hg

-LRB-

GS

-RRB-

The

ES-MS

traces

of

the

extracted

ion

chromatograms

show

distinct

peaks

of

the

protonated

molecular

ion

masses

For

each

compound

the

same

isotope

cluster

was

found

as

was

obtained

in

the

flow-injection

experiments

with

the

single

species

Moreover

the

MS

spectra

-LRB-

in-source

fragmentation

-RRB-

are

identical

to

those

obtained

in

the

direct-injection

experiment

as

are

the

spectra

of

the

MH

molecular

ions

-LRB-

data

not

shown

-RRB-

All

ion

traces

in

ES-MS

are

on

the

same

y-scale

and

it

is

evident

that

the

intensities

measured

in

ES-MS

are

species-dependent

like

in

the

direct-injection

experiments

MeHgGS

shows

the

highest

intensity

approximately

times

higher

than

MeHgCys

Although

a

methanol

gradient

was

used

in

the

HPLC

experiment

-LRB-

methanol

at

min

retention

time

-RRB-

the

relative

peak

intensities

are

comparable

with

those

obtained

with

the

direct

injections

A

different

picture

is

seen

for

the

ICP-MS

signal

for

mercury

In

ICP-MS

all

compounds

should

be

completely

destroyed

by

the

hard

ionisation

conditions

in

the

plasma

Therefore

the

signal

intensities

for

the

different

compounds

should

only

be

governed

by

the

absolute

mercury

concentration

As

all

the

compounds

were

made

up

in

the

same

concentration

-LRB-

as

Hg

-RRB-

the

ICP-MS

response

should

be

very

similar

for

all

compounds

Figure

shows

that

for

the

four

compounds

the

intensities

obtained

are

much

more

similar

than

those

obtained

by

ES-MS

but

are

not

ideally

equal

the

peaks

corresponding

to

the

first

and

the

last

species

eluted

-LRB-

and

MeHgGS

-RRB-

are

approximately

twice

the

peaks

obtained

for

MeHgCys

and

Several

reasons

can

be

responsible

for

this

finding

Overall

intensity

changes

due

to

the

input

of

methanol

into

the

plasma

As

shown

in

Fig.

the

signal

for

the

internal

standard

is

constant

up

to

approximately

min

of

runtime

afterwards

it

decreases

to

about

of

the

initial

value

at

runtime

when

the

last

mercury

compound

is

eluted

If

the

mercury

intensities

are

influenced

by

this

process

only

the

signals

for

the

first

compounds

eluted

-LRB-

both

less

than

min

retention

time

-RRB-

should

be

equal

whereas

the

signals

for

the

GS

complexes

should

be

only

about

half

This

is

not

the

case

in

fact

the

last

compound

eluted

MeHgGS

exhibits

a

signal

equal

to

the

first

compound

eluted

However

the

behaviour

of

mercury

with

its

high

ionisation

potential

-LRB-

eV

-RRB-

is

greatly

influenced

by

organic

matter

in

the

plasma

and

the

mercury

signal

intensity

may

change

in

a

different

way

from

the

intensity

of

rhodium

The

dependence

of

the

mercury

intensity

on

the

methanol

content

of

the

eluent

must

be

determined

but

this

was

not

performed

during

the

experiments

reported

here.The

volatilities

of

the

compounds

may

be

different

so

different

amounts

of

each

compound

may

be

introduced

into

the

plasma

during

pneumatic

nebulisation

this

effect

is

well

known

for

volatile

species

like

and

has

to

be

investigated

further

for

the

compounds

used

in

this

experiment.MeHgCys

and

may

partly

decompose

on

the

column

so

not

all

of

the

compound

injected

may

reach

the

plasma

in

this

case

we

would

expect

broad

tailing

peaks

and

substantial

amounts

of

free

Cys

and

reduced

GS

However

these

have

not

been

observed.MeHgCys

and

are

not

present

to

in

the

standard

and

thus

the

concentrations

of

the

mercury

reaching

the

plasma

are

diminished

in

this

case

signals

for

unbound

Cys

and

GS

should

show

also

significant

intensity

In

fact

some

oxidised

Cys

and

oxidised

GS

can

be

distinguished

in

the

ES-MS

but

is

not

clear

whether

the

amounts

may

sum

up

to

explain

the

intensity

loss

in

MeHgCys

and

However

the

corresponding

mercury

part

of

the

molecules

-LRB-

and

MeHg

-RRB-

should

show

up

in

the

ICP-MS

trace

and

this

is

not

the

case

As

shown

further

later

for

the

spiking

experiments

-LRB-

Fig.

-RRB-

and

HgMe

are

eluted

from

the

column

with

distinct

retention

times

differing

from

the

retention

times

of

the

other

complexes.Fig

chromatograms

for

injections

of

free

and

biothiol-complexed

mercury

species

in

a

plant

extract

spiked

with

different

Hg

and

HgMe

biothiol

complexes

Peak

assignment

Hg

-LRB-

Cys

-RRB-

MeHgCys

MeHg

MeHgGS

Hg

-LRB-

GS

-RRB-

Thus

a

final

explanation

for

the

difference

in

intensity

of

the

four

compounds

can

not

be

given

at

this

point

Compound

stability

during

spiking

experiments

of

plant

extracts

The

mercury

biomolecules

synthesised

and

characterised

here

shall

be

detected

and

identified

in

real-world

samples

One

future

application

is

the

determination

of

such

compounds

in

plants

exposed

to

mercury

contamination

Mercury

can

readily

be

taken

up

by

plants

can

accumulate

in

the

roots

and

can

be

translocated

into

shoots

and

fruits

-LSB-

-RSB-

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

The

plant

extracts

prepared

as

described

earlier

were

spiked

with

the

four

compounds

and

the

signals

obtained

were

compared

with

the

corresponding

signals

when

the

compounds

were

prepared

in

formic

acid

Simultaneous

ES-MS

and

ICP-MS

detection

was

applied

using

the

same

setup

as

described

earlier

Additionally

plant

extracts

were

spiked

with

free

and

HgMe

in

order

to

determine

their

retention

behaviour

on

the

column

and

to

see

whether

any

complexes

would

form

in

situ

with

any

biothiols

possibly

present

in

the

plant

extracts

In

Fig.

the

traces

monitored

by

ICP-MS

are

shown

for

MeHgCys

Hg

-LRB-

Cys

-RRB-

MeHg

Hg

-LRB-

GS

-RRB-

and

MeHgGS

spiked

into

the

shoot

extract

of

a

rice

plant

at

a

concentration

of

mg/kg

-LRB-

as

Hg

-RRB-

each

It

is

evident

that

the

elution

order

of

the

four

mercury

biothiol

complexes

matches

very

well

the

one

obtained

in

a

standard

mixture

-LRB-

Fig.

-RRB-

Surprisingly

is

eluted

later

than

whereas

MeHg

is

eluted

with

a

broad

peak

with

a

retention

time

of

approximately

min

between

the

Cys

and

GS

complexes

The

elution

order

and

the

retention

times

are

practically

the

same

for

the

standard

root

and

shoot

extracts

and

this

also

remains

over

time

a

second

injection

of

all

samples

done

h

after

the

first

run

showed

no

significant

change

in

retention

times

A

similar

picture

is

seen

for

the

peak

areas

Except

for

and

MeHgCys

the

peak

areas

measured

after

h

are

more

than

of

the

initial

value

-LRB-

peak

areas

corrected

for

instrumental

drift

using

the

internal

standard

signal

-RRB-

Surprisingly

MeHgCys

turns

out

to

be

more

stable

in

the

extracts

than

in

the

standard

solution

This

might

be

due

to

the

presence

of

some

unidentified

chelating

agents

The

peak

area

for

varies

considerably

showing

a

rise

for

the

standard

in

formic

acid

whereas

a

decrease

is

observed

in

the

plant

extract

While

no

explanation

can

be

given

here

for

the

intensity

rise

of

the

peak

in

the

standard

in

the

shoot

extract

the

formation

of

other

unidentified

mercury

complexes

may

be

proposed

The

results

for

retention

time

and

peak

intensity

reproducibility

are

summarised

in

Tables

and

Table

times

for

consecutive

injection

of

six

mercury

compounds

with

h

MeHgCysMeHg

Standard

Roots

extract

Roots

extract

Shoots

extract

Shoots

extract

σ2

RSD

-LRB-

-RRB-

not

available

RSD

relative

standard

deviationTable

area

-LRB-

as

percentage

of

first

measurement

-RRB-

recoveries

for

consecutive

injection

of

six

mercury

compounds

with

h

MeHgCysMeHg

The

addition

of

MeHg

to

the

extracts

did

not

result

in

any

additional

peaks

in

the

ICP-MS

trace

while

for

the

addition

of

a

small

very

broad

peak

can

be

distinguished

around

the

retention

time

of

HgMeGS

An

elevated

mercury

background

is

seen

after

the

introduction

of

to

the

standard

and

the

extract

pointing

to

some

kind

of

exchange

on

the

HPLC

column

possibly

through

formation

of

unidentified

labile

complexes

However

the

mercury

counts

are

at

the

normal

background

level

again

prior

to

injection

of

the

next

sample

Regarding

the

ES-MS

measurements

-LRB-

data

not

shown

-RRB-

the

same

MH

ion

cluster

patterns

are

found

for

the

four

spiked

mercury

complexes

as

in

the

standards

As

already

seen

from

the

ICP-MS

traces

addition

of

or

MeHg

did

not

result

in

the

formation

of

any

other

distinguishable

mercury

biothiol

compounds

In

the

unspiked

extracts

only

trace

amounts

of

free

Cys

or

GS

were

found

but

there

was

some

oxidised

and

thus

inactive

GS

Conclusion

and

outlook

In

this

work

we

could

successfully

show

the

synthesis

structural

identification

and

HPLC

separation

of

mercury

and

methylmercury

complexes

with

Cys

and

GS

An

ES-MS

system

equipped

with

an

ion

trap

for

measurements

revealed

characteristic

MH

and

fragmentation

patterns

for

these

compounds

enabling

the

identification

of

these

or

similar

complexes

in

biota

samples

The

simultaneous

coupling

of

HPLC

with

ES-MS

and

ICP-MS

with

its

invaluable

combined

information

on

molecular

structure

and

quantitative

mercury

detection

proved

to

be

suited

for

this

kind

of

compounds

The

method

described

seems

to

be

acceptably

robust

and

reproducible

and

also

suited

for

real

samples

This

was

shown

through

spiking

experiments

of

plant

extracts

However

the

quantification

approach

via

the

ICP-MS

trace

needs

further

investigation

especially

in

terms

of

mercury

intensity

changes

due

to

the

gradient

introduction

of

methanol

into

the

plasma

and

also

in

terms

of

decomposition

on

column

as

has

been

described

for

As

-LRB-

GS

-RRB-

-LSB-

-RSB-

The

work

presented

here

is

the

first

step

in

the

determination

of

mercury

species

in

biota

on

the

molecular

level

Ongoing

experiments

are

aimed

at

plant

exposure

to

mercury

and

methylmercury

Here

we

can

also

expect

mercury

complexes

with

larger

biothiols

especially

phytochelatins

phytochelatins

are

small

peptides

of

the

general

structure

-LRB-

γ-Glu

Cys

-RRB-

n

Gly

-LRB-

n

-RRB-

which

can

bind

to

metal

ions

and

play

an

important

role

in

metal

detoxification

and

translocation

processes

in

plants

Other

biota

samples

e.g.

fish

will

be

targeted

as

well

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

O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O B O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O O O O O O O O O O O B B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O B O B B O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B B O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O B O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B B O O O O O O O O O O O O O O O O B O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O

mercury
methylmercury
biothiols
electrospray mass spectrometry
conformation modelling

inductively coupled plasma mass spectrometry