ICP-MS review

focal point reviewDANIEL PROFROCK AND ANDREAS PRANGE

HELMHOLTZ ZENTRUM GEESTHACHT–ZENTRUM FUR MATERIAL UND

KUSTENFORSCHUNG,

DEPARTMENT MARINE BIOANALYTICAL CHEMISTRY,

MAX-PLANCK STR. 1, 21502

GEESTHACHT, GERMANY

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS)for Quantitative Analysis in

Environmental and Life Sciences:A Review of Challenges,

Solutions, and Trends

This focal point review provides an overview of

recent developments and capabilities of induc-

tively coupled plasma mass spectrometry (ICP-

MS) coupled with different separation tech-

niques for applications in the fields of quanti-

tative environmental and bio-analysis. Over the

past years numerous technical improvements,

which are highlighted in this review, have

helped to promote the evolution of ICP-MS to

one of the most versatile tools for elemental

quantification. In particular, the benefits and

possibilities of using state-of-the-art hyphenat-

ed ICP-MS approaches for quantitative analy-

sis are demonstrated with a focus on

environmental and bio-analytical applications.

Index Headings: Inductively couple plasma-

mass spectrometry; ICP-MS; Hyphenated tech-

niques; Isotope dilution; Quantification; Envi-

ronment; Proteomics; Chromatography; Trace

elements; Heteroelements.

INTRODUCTION

The accurate quantification ofbiologically relevant molecules,such as proteins or even hazard-

ous substances in the environment,becomes more and more an essentialprerequisite for monitoring and under-standing biological processes or com-paratively assessing our environment interms of, for example, contaminantconcentrations or the presence of novelemerging compounds. In particular, therecent developments within the promi-nent fields of genomics, proteomics, andmetallomics have enhanced the under-standing of the complex interplay ofgenes and proteins in cellular processes,which are often reflected by time-dependent changes in absolute proteinconcentrations or the degree of a site-specific post-translational modification.1

However, even though different tech-niques for the quantification of proteins

have been suggested during recent years,this still represents a challenging task.2

In addition, the quantitative analysis ofpriority hazardous substances in theenvironment becomes more and morechallenging because new legislationoften requires more sensitive methods,or even completely new approaches, forthe determination at very low concen-trations (pg/L levels) of already definedpriority compounds or newly emergingcontaminants that show up in theenvironment as substitutes for alreadybanned substances or as a result ofchanging industrial processes.3

Since its introduction in the 1980s,inductively coupled plasma mass spec-trometry (ICP-MS) has evolved tobecome arguably the most versatile,element-specific detection technique.4,5

In parallel, because of the fast develop-ments in the field of elemental specia-tion, the utilization concept of ICP-MShas undergone a significant change.

Received 10 April 2012; accepted 4 May 2012.* Author to whom correspondence should besent. E-mail: [email protected]: 10.1366/12-06681

APPLIED SPECTROSCOPY 843

Within this context, trace metals, metal-loids, semimetals, and hetero elementsplay important roles, since nature as wellas organic chemists have learned toskillfully combine these elements withhydrogen (H), carbon (C), nitrogen (N),and oxygen (O) to form an unimagin-ably large number of sometimes benefi-cial, but also sometimes hazardous,substances.

Although it was initially used for thetotal quantification of trace metals inliquid samples, ICP-MS has maturedinto a powerful chromatographic detec-tor and therefore an essential part ofstate-of-the-art hyphenated detectionschemes, allowing the detection of allkinds of compounds via their character-istic (hetero) element content. In partic-ular the development of hyphenatedtechniques has established the impor-tance of ICP-MS in the field of envi-ronmental speciation analysis duringrecent years,6,7 which continuouslyemerged to the field of bio-inorganicspeciation.8–10 Here the field of metal-lomics, which focuses on the globalanalysis of the entirety of all metal andmetalloid species within a cell or tissuetype, represents one of the most dynamicresearch areas that recently emergedfrom trace element speciation.11–13

Within this context, the utilization ofICP-MS has been changed, since for thefirst time this technique is now used todetect organic compounds,14 organome-tallic compounds,15 or even more com-plex bio molecules, such as nucleicacids,16–19 phospholipids,20 and metal–proteins9 via their specific and oftencharacteristic element content. As aresult the concept of hetero (element)tagged proteomics has been proposed, inwhich analytical information is generat-ed by the complementary application ofelemental and molecule-specific massspectrometry, as well as the utilizationof hetero atoms such as phosphorus,sulfur, and selenium for screening andquantification purposes.13,21–23

This progress was realized due to anumber of instrumental developmentsduring the last decade, which will behighlighted in the following sections.Their availability finally induced aparadigm shift, since it became possibleto analyze elements that are highlysusceptible to interference, which finally

allows the number of elements that areeasily detected by ICP-MS to be ex-tended to covalently bound hetero atomssuch as phosphorus, sulfur, selenium, oreven halogens, which show a wide-spread distribution in all classes ofchemical substances.24

More recently, labeling approachesthat utilize an ICP-MS detectable ele-ment have gained a great deal of interest,in particular in the life sciences, as suchapproaches can be used to allow thosemolecules that naturally contain nodetectable element tag to be detectableby ICP-MS.24–27 Meanwhile appropriatereaction chemistry has been developedthat allows the direct covalent labelingof a bio molecule with elements such asHg28–30 or I,31,32 both of which can bedetected with high sensitivity. In termsof flexibility, labeling with lanthanidesusing bi-functional chelating agents,which are covalently bound to thetargeted bio molecule, and which formhighly stable complexes with the metalion, represents the current state of the artwithin this field.33–35 Lanthanides areideal elements for such labeling ap-proaches because they can be detectedwith high sensitivity via ICP-MS due toa negligible background, as well as theabsence of interferences.

In consequence, all kinds of mole-cules that naturally contain covalentlybound (hetero) elements, or which havebeen chemically labeled with an ICP-MS detectable element, could be easilyquantified, whenever their final mole-cule-tag stoichiometry is known.22,24,36

However, in parallel this underlinesagain that a successful application ofsuch approaches strongly depends on thecomplementary application of molecule-specific detection techniques, such asmatrix-assisted laser desorption/ioniza-tion (MALDI) or electrospray ionizationmass spectrometry (ESI-MS), to gain thenecessary information related to the tagstoichiometry.21,24

The following sections of this focalpoint review will provide an overviewand critically discuss the recent progressmade in using ICP-MS hyphenated todifferent separation techniques for quan-titative analysis in environmental andlife science, via the utilization of specificelement tags. In addition, trends and thecurrent state of the art of suitable

analytical techniques will be highlight-ed. Whenever possible review articlesare referenced, which may provide agood starting point to gain more de-tailed, topic-related information.

ICP-MS FUNDAMENTALS

Ionization: The Potential of a Plas-ma Ion Source. In contrast to otherpopular mass spectrometric ionizationtechniques such as the so-called ‘‘soft’’electrospray (ESI) or MALDI, elementalMS utilizes a high-temperature plasmadischarge as source for mainly singly,positively charged ions.37 In conse-quence ICP-MS has matured to apowerful, important technique, allowingthe determination of most elementspresent in the periodic table (See Fig. 1).

Inductively coupled plasmas mostlyutilize noble gases, such as argon asplasma gas, in which the efficientvaporization, dissociation or atomiza-tion, excitation, and final ionization ofthe sample constituents to be analyzedtakes place. In addition, this high-temperature process leads to a completefragmentation of every sample mole-cule, leaving only their detectable,atomic constituents, namely metals,metalloids, or heteroatoms, which couldthen be used as surrogates to also detectcomplex molecules, such as proteins,nucleic acids, or even small organicmolecules, as illustrated in Fig. 2. Dueto the high temperature of the plasma,which may exceed 7000 K, ICP-MS isparticularly well suited to handle liquidsamples when hyphenated with intro-duction techniques such as flow-injec-tion analysis (FIA), high-performanceliquid chromatography (HP-LC), orcapillary electrophoresis (CE).9 Howev-er, gaseous or solid samples can also beanalyzed using gas chromatography(GC)38 or laser ablation (LA)39 assample introduction techniques, respec-tively. In summary ICP-MS provides anumber of attractive properties as adetector for quantitative analysis, suchas high sensitivity (ng L-1 range), widelinear dynamic detection range (up tonine orders of magnitude depending onthe instrument and the application), andspecificity for the accurate detection andquantification of metals, metalloids, andheteroelements (including non-metals,semi-metals, and halogens). In addition,

844 Volume 66, Number 8, 2012

focal point review

ICP-MS can be used to obtain preciseisotope ratio information for thoseelements that feature multiple stableisotopes. As a result, isotope dilutionanalysis has matured to the method ofchoice when the goal is the mostaccurate quantitative results.40 Despiteits current versatility, however, ICP-MSis still widely known as only a ‘‘metal’’detector and it is hoped that this articlewill change this state of affairs.

Sample Introduction: Current Sta-tus and Trends in Hyphenated Tech-niques. At first sight, and in comparisonto other techniques such as ESI orMALDI-MS, respectively, ICP-MSseems to be only interesting as a pureelemental detector, since all molecule-specific information is lost, due to thespecific properties of the plasma ionsource. However, the necessary molec-ular specificity can be obtained by thehyphenation of ICP-MS with different

state-of-the-art separation techniques,such as HPLC or GC as well aselectrophoretically driven techniquessuch as capillary or one- and two-dimensional gel electrophoresis (1D or2D GE).

High-Performance Liquid Chroma-tography–Inductively Coupled Plasma–Mass Spectrometry. The hyphenation ofLC to ICP-MS for the separation and(hetero)element specific detection isrelatively straightforward and can beeasily achieved via the direct connectionof the separation column and thenebulizer that is part of the spraychamber. Despite this simplicity, thecomposition of the liquid matrix oftencomplicates the detection of manyelements, due to the enhanced formationof interferences. The introduction ofhigh amounts of salts or organic solventsinto an ICP is known to change anumber of fundamental parameters, such

as plasma temperature, electron density,

aerosol generation or analyte transport,

as well as the overall ionization pro-

cesses that take place inside the plasma.

Plasma instabilities until its extinction,

carbon deposition on the cones and lens

system, or signal suppression are well-

known further detrimental effects related

to the introduction of high amounts of

organic solvents into an ICP, which

often limits the hyphenation of standard

reversed-phase (RP) HPLC setups with

ICP-MS detection, in particular when

working with solvent flow rates between

0.1 and 1 mL min-1. Membrane desol-

vation,41 oxygen addition,42 spray

chamber temperatures below 0 8C,20 or

reduced injector tubing inner diame-

ters42 are often-used tools to minimize

the organic load of the plasma, in

particular when utilizing RP-HPLC con-

ditions, which are necessary for many

FIG. 1. Illustration of the specific features of ICP-MS as a (hetero)element-specific detector. The instrumental progress in high-resolutionICP-MS, as well as the development of collision/reaction cells, during the last decade has minimized the interference problem that naturallydelimits the accurate detection and quantification of many elements. Meanwhile, most elements of the periodic table including metals,metalloids, semimetals, non-metals, or halogens can be accurately determined by ICP-MS, allowing their application as surrogate standardsfor the quantification of all kinds of molecules whenever the stoichiometry of the used element tag is known.


separations in environmental and lifescience applications.

The recent development of newinterface systems that allow the hy-phenation of capillary or nano LC toICP-MS have helped to overcome theproblems related to the introduction ofreversed-phase gradients into the ICPduring (hetero)element specific detec-tion.43 First attempts have been madeby using conventional low-flow micro-concentric nebulizers, combined with alow volume spray chamber44 or modi-fied direct injection high efficiencynebulizer (DIHEN).45

More recently the utilization andmodification of a total consumptionmicro-concentric nebulizer, which was

originally developed and optimized forthe hyphenation of capillary electropho-resis (CETAC CEI 100, Omaha, Ne-braska) as an interface for capillary andnano HPLC to ICP-MS, has beendescribed.46,47 Due to its zero dead-volume design and the possibility todirectly connect standard chromato-graphic tubing to the nebulizer capillary,this device is well suited for the directhyphenation of capillary HPLC to ICP-MS.48

Recently a modified CEI-100 nebu-lizer, with a different nebulizer capillaryand a further reduced internal deadvolume, that allows the application ofsolvent flow rates between 0.5 and 6 lLmin-1 has been introduced.49

Giusti et al. even introduced a newnebulizer working at flow rates less than500 nL min-1, allowing the sheathlesshyphenation of nano-HPLC to ICP-MS.A nano-electrospray emitter needle hasbeen used as a nebulizer capillary toreduce the internal dead volume and toobtain a suitable back pressure, which isnecessary for stable nebulization.50 Ingeneral, these capillary or nano-LCinterface systems allowed for the firsttime the direct introduction of gradientswith up to 100% organic solvent into theICP, without the known negative effectssuch as carbon buildup or even plasmaextinction.

This evolution is particularly impor-tant because it facilitates the comple-

FIG. 2. The high-temperature process inside an ICP leads to complete fragmentation of every sample molecule, leaving only theirdetectable, atomic constituents, namely metals, metalloids, or heteroatoms that could be used as surrogates to detect complex moleculessuch as proteins, nucleic acids, or even small organic molecules.


focal point review

mentary application of electrospray-based MS techniques under exactly thesame chromatographic conditions,which is mandatory to elucidate the(hetero)element stoichiometry, especial-ly during quantitative studies of biomol-ecules.

Despite the advantages of such min-iaturized separation approaches, it has tobe kept in mind that ICP-MS (in contrastto ESI-based MS techniques) representsa mass-flow-dependent detection tech-nique. Therefore, the sample ion signalof an ICP-MS is proportional to the totalnumber of atoms detected per unit oftime. As a result, the gain in analyteconcentration obtained by using minia-turized separation columns occurs at theexpense of a reduced solvent flow thatenters the detector. In consequence, theimproved peak concentration due to theapplication of capillary or nano-HPLCdoes not necessarily result in an en-hanced response in the ICP-MS system,as observed for ESI-MS.51

Gas Chromatography–InductivelyCoupled Plasma–Mass Spectrometry.Gas chromatography as a sample intro-duction technique offers significant ad-vantages compared to standard liquidsample introduction approaches, since100% transport efficiency can be real-ized. In addition, an improved sensitiv-ity can be achieved due to the dryplasma conditions, since nearly noplasma energy is needed for sampledesolvation and vaporization, allowingalso the efficient analysis of interestinghigh heteroelements with a high ioniza-tion potential such as P, S, Cl, Br, or I.Furthermore, intensities of most plasma-and matrix-based polyatomic interfer-ences are negligibly small, due to theabsence of an aqueous liquid solvent,which provides outstanding sensitivityfor most elements. In consequence theinstrumental settings of GC-ICP-MSoften indicate strong differences incomparison to normal wet plasma con-ditions (e.g., lower plasma power oftenbelow 1000 W, highly negative extrac-tion lens voltages, introduction of addi-tional gases into the plasma such as Heor N2

14,52–54), which have to be consid-ered to obtain high sensitivity. Appro-priate interface technology has beenextensively developed in academic re-search, especially within the elemental

speciation community; however, in themeantime different companies such asAgilent Technologies and Thermo Fish-er have also released robust interfacesystems. Here two concepts have to bedistinguished. While most interface sys-tems benefit from the utilization of thedry plasma conditions, recently a dual-mode interface has been introduced.This sample introduction system facili-tates the simultaneous introduction ofboth liquid and gaseous samples into theICP-MS. Therefore, the dual sampleintroduction system allows the connec-tion of the GC transfer line to the torch,while a conventional nebulizer/spraychamber combination is mounted abovethe GC transfer line and connected to athird leg of the GC-ICP-MS torch. Thenebulizer is used for the continuousintroduction of a liquid matrix, which isutilized for calibration and to maintainwet plasma conditions. This setup mayhave the advantage to use unspecificstandards for a compound-independentcalibration and it allows the continuousintroduction of an internal standard;however, these wet plasma conditionsalso eliminate the advantages of GC as aseparation technique.

Maintaining a constant temperatureover the whole transfer line in order toprevent analyte condensation, as well aspreserving high peak resolution by usingcapillary GC are the most critical pointsin the hyphenation of GC and ICP-MS.Meanwhile, all commercially availableGC-ICP-MS interfaces allow transferline temperatures above 300 8C, therebyfacilitating the analysis of high-boiling-point compounds, such as multiplybrominated flame retardants like poly-brominated diphenyl ethers (PBDEs).55

Under such conditions, even such highlyinterfered isotopes as 32S can be mea-sured directly56,57 with high sensitivity,which is impossible under wet plasmaconditions.

Capillary Electrophoresis/Gel Elec-trophoresis–Inductively Coupled Plas-ma–Mass Spectrometry. In general,capillary electrophoresis hyphenated toICP-MS provides interesting capabilitiesas a separation technique for environ-mental and life science applications dueto its high separation efficiency (up to200 000 theoretical plates), the ability tohandle the smallest sample amounts (nL

range), and the absence of a packedstationary phase, which is prone tonegative interaction with the sam-ple.58,59 The electrophoretic movementof the analytes, which is overlaid by theelectroosmotic flow, allows the separa-tion of positively, neutral, and negative-ly charged ions and compounds in onerun. This makes it in particular interest-ing for the separation of the smallestsample amounts of labile complexes,such as metalloproteins,9,60 whose in-tegrity is often affected when using otherseparation techniques, such as LC.61

Despite these interesting features theapplication of CE-ICP-MS as well asrelated instrumental developments indi-cates a certain stagnation, so the readeris referred to some good reviewsfocusing on the hyphenation of CE toICP-MS.60,62,63 This stagnation may becaused in particular by the complexity ofsuch CE-ICP-MS systems, since differ-ent parameters have to be considered,such as the utilized buffer and make-upsolutions, which ideally should betotally decomposed in the plasma with-out leaving too much residue on thecones and the lens system. In addition,buffer concentrations should be kept aslow as possible to avoid any detrimentalcrystallization effects at the nebulizertip. Also, a high ionic strength of thebuffer should be avoided, which willresult in the production of excessiveJoule heat. This also helps to reduce therisk of air bubble production inside theinterface, which may lead to a break-down of the electrical current or insuf-ficient signal stability.

The interface itself represents a fur-ther critical issue, since it has tomaintain an effective electrical contactto the outlet of the CE capillary in orderto provide a stable electrical current forreproducible electrophoretic separations.Furthermore, any laminar flow inside theCE capillary due to the suction effect ofthe used nebulizer should be avoided, byadapting the flow rate of the electro-osmotic flow (EOF) (a few nL min-1

depending on the CE capillary i.d. andthe voltage) to those of the nebulizer(normally ranging from 1 to 1000 lLmin-1). Additionally, zero dead vol-umes are anticipated to reduce peakbroadening, which has a detrimental


effect on the high separation efficiencyof CE.

Up to now various setups have beensuggested to solve the above-mentionedproblems in coupling CE and ICP-MS.A conductive, coaxial sheath liquid(e.g., introduced via a cross piece),which is mixed with the CE capillaryeffluent, is frequently used to provide astable electrical connection at the CEcapillary outlet.46,47,64–72 Even though anumber of interface designs have beensuggested during the last decade, toaddress the mentioned challenges, mostrecent applications utilize commerciallyavailable interface systems.46,47,73 Insummary it must be said that despitethe exceptional separation capabilities ofCE and the high-level research studiesthat utilize CE-ICP-MS, more develop-ments are necessary to make CE-ICP-MS a technique suitable for routineanalysis.

Recently the application of laserablation (LA) ICP-MS as interface forthe utilization of one- or two-dimen-sional gel electrophoresis (GE) as aseparation technique has gained muchinterest for either the analysis of natu-rally74 or artificially75 tagged proteins.76

In particular, 2D GE represents anattractive tool for the separation ofcomplex protein mixtures. The combi-nation of an isoelectric focusing step,which separates the sample constituentsaccording to their isoelectric point, witha second separation dimension accord-ing to the molecular weight, allowshighly resolved maps of complex pro-teomes to be generated. Laser ablation isthen used to screen the different spotsfor the presence of specific covalentlybound (hetero)elements, e.g., phospho-rus, which may indicate the presence ofa phosphorylated protein species.74 Suchapproaches have also been used for theseparation of and screening for metalproteins; however, due to the need towork under native GE conditions, only apoor separation quality can be obtainedin comparison to the normally useddenaturating conditions.77 Since thepurity of many biochemical reagents interms of possible element contamina-tions is often not sufficient, elevatedbackground levels during the ablationprocess represent a further critical issue.To overcome this limitation, membraneblotting is frequently used to transfer theseparated species to a high purity, e.g.,

nitrocellulose, membrane, whose matrixis rather uncritical during the ablationprocess.78,79 Elemental fractionationprocesses caused by the ablation haveto be addressed, since they influence inparticular the accuracy of quantitativeLA-ICP-MS analysis.39

MASS ANALYZERS: STATUSAND RECENTDEVELOPMENTS

Depending on the analytical require-ments in terms of mass resolution,sensitivity, isotope ratio precision, ordata acquisition speed, several differentICP-MS platforms are commerciallyavailable, as summarized in Fig. 3. Formany years quadrupole ICP-MS hasrepresented the most frequently usedinstrumentation for multi-element anal-ysis or as a detector for hyphenatedapproaches. However, due to the pres-ence of interfering argides, hydrides,carbides, nitrides, and oxides, which arenaturally formed in an argon plasmaoperated under normal lab conditions ordue to the matrix constituents of atypical environmental or clinical sample,accurate multi-element determinationwas always challenging. Table I pro-vides an overview of some of the mostfrequently used element tags in classicalspeciation and metallomics analysis,elements that are in focus as covalenttags for absolute protein quantification(P, S, Se), and those showing potentialin particular for environmental analysis(P, S, Cl, Br, I) as well as their mostprominent interferences. The interfer-ence problems have been overcome bythe availability of high-resolution dou-ble-focusing sector field ICP-MS (HR-ICP-SF-MS) or more recently due to theintroduction of collision/reaction cell orthe dynamic reaction cell ICP-MS (CC-ICP-MS or DRC-ICP-MS, respectively).By utilizing a medium resolution of Dm/m = 4000 or by the application of cellgases such as H2, He, Xe, O2, or NH3

most polyatomic interferences can bephysically separated from the targetedions or minimized to an insignificantlevel utilizing different gas-phase mech-anisms.80 This includes collision-in-duced dissociation (CID),81 kineticenergy discrimination (KED),81,82

charge or proton transfer reactions,82 ormass shift reactions.83 Even though HR-

FIG. 3. Illustrative overview of the most recent commercially available ICP-MS platforms.


focal point review

ICP-SF-MS operated in the high-resolu-tion mode (Dm/m . 10 000) allowsmost target elements to be resolved frominterfering polyatomic ions, the en-hanced mass resolution also results in areduced ion transmission and in conse-quence a strong loss of sensitivity.Depending on the cell gas used, colli-sion/reaction cells allow the effectivesuppression of many interfering poly-atomic ions, leaving the ion transmissionmore or less unaffected.

Recently a collision/reaction cell ICP-MS-MS (ICP-QQQ-MS) has been intro-duced to the market.84 In comparison tothe already available DRC-ICP-MS,which utilizes a radio frequency/directcurrent (rf/dc) quadrupole reaction cell,which may be pressurized with areactive gas, in order to promote ion-molecule reactions, and which alsofeatures an adjustable DRC bandpass,allowing the suppression of new inter-ferences produced through sequentialreactions within the cell,85–87 this newtechnique includes two real independentquadrupole mass filters connected via anoctopole collision/reaction cell. In con-trast to the DRC approach, this device

shows comparable operation modes, asknown from the frequently used ESItriple quadrupole instrumentation, suchas neutral gain scan, product ion scan, orprecursor ion scan, which allows animproved reduction in interference aswell as better control of the gas-phasereactions or even completely new detec-tion schemes to handle specific interfer-ences.88

Also during the last decade multi-collector sector field ICP-MS (MC-ICP-MS) and more recently Mattauch-Her-zog (MH-ICP-MS)89–91 instrumentshave gained much attention, as theyallow, for example, the accurate deter-mination of isotopic ratios or the fullysimultaneous acquisition of the entire m/z range covered by the elements of theperiodic table. These technological de-velopments strongly promote applica-tions such as marine geochemistry,geochronology, cosmochemistry, orprovenance studies.

To make the picture complete, ICP-TOF-MS and ICP-IT-MS should also bementioned as possible analyzers; how-ever, from a commercial point of viewthey play an insignificant role. Recentlythe combination of an ICP source with

an ultra-high-resolution Orbitrap massanalyzer (Dm/m . 100 000) has beendemonstrated; however, such instrumen-tation is not currently commerciallyavailable and is still under furtherdevelopment.92

QUANTIFICATIONSTRATEGIES USING ICP-MS

As a mass-flow-dependent detectorutilizing an argon plasma for the totaldecomposition of the sample, as well asthe continuous generation of elementions, which finally reflect the composi-tion of the sample, ICP-MS opens someadvantages in terms of calibration andquantification. In particular its com-pound-independent response allows theapplication of simple element standardsto perform a calibration and finally toquantify virtually every compound aslong as it contains an ICP-MS detectableelement at a known, thermodynamicallystable stoichiometry.93,94

External Calibration or StandardAddition. Depending on the samplematrix complexity, the simplest way toquantify the targeted element speciesduring ICP-MS analysis is to useavailable, synthesized standards to gen-erate external calibration curves for eachcompound. Alternatively the standardaddition approach can be applied toconsider possible interferences originat-ing from the sample matrix. In manycases such standard addition calibrationscan be transferred to an external matrix-match calibration, which could be ap-plied to a larger set of samples withoutthe need to apply the time-consumingstandard addition procedure for everysample. This strategy has been exten-sively applied in the field of environ-mental speciation analysis of definedorganometallic compounds of mainlyanthropogenic origin and their degrada-tion products, such as methylmercury,alkyllead, butyl- and phenyltin com-pounds, or simple arsenic species.9

However, this concept fails, in particularin the field of quantitative bioanalysissuch as proteomics, due to the often highcomplexity of related samples andtherefore the lack of suitable, commer-cially available standards.

Compound-Independent Calibra-tion and Internal Standards. In con-trast to other ionization techniques, such

TABLE I. Selected metals, metalloids, and (hetero)elements utilized as tags for ICP-MS basedquantification in environmental and life sciences, their isotopes, and prominent polyatomicinterferences.

Isotope Abundance Accurate mass (Da) Most prominent polyatomic interferences

31P 100 30.97376 14N16O1Hþ32S 94.93 31.97207 16O2

þ34S 4.29 33.96787 16O18Oþ35Cl 75.78 34.96885 34S1Hþ37Cl 24.22 36.96590 36S1Hþ, 36Ar1Hþ51V 99.75 50.94396 35Cl16O, 37Cl14N, 40Ar11B52Cr 83.79 51.94051 36Arl6O, 40Ar12C, 35Cl16OH, 37Cl14NH54Fe 5.845 53.93961 40Ar14N, 38Ar16O, 37Cl16OH, 40Ca14N55Mn 100 54.93805 40Ar14NH, 39K16O, 23Na32S, 37Cl18O56Fe 91.75 55.93494 40Ar16O, 40Ca16O59Co 100 58.93320 36Ar23Na, 24Mg35Cl, 42Ca16OH, 23Na35ClH63Cu 69.17 62.92960 40Ar23Na, 40Ca23Na64Zn 48.63 63.92915 40Ar24Mg, 40Ar23NaH, 32S16O16O65Cu 30.83 64.92779 40Ar25Mg, 40Ar24Mg1H66Zn 27.90 65.92604 40Ar26Mg75As 100 74.92160 40Ar35Cl, 40Ca35Cl78Se 23.77 77.91730 40Ar38Ar, 40Ar37ClH, 38Ar40Ca79Br 50.69 78.91834 63Cu16Oþ, 40Ar39Kþ80Se 49.61 79.91652 40Ar40Ar, 40Ar40Ca, 79Br1H81Br 49.31 80.91629 40Ar40Ar1Hþ,82Se 8.73 81.91671 40Ar40Ar1H2

þ, 40Ar42Caþ111Cd 12.8 110.90418 79Br32S114Cd 28.73 113.90336 98Mo16O120Sn 32.59 119.90220127I 100 126.90448202Hg 29.80 201.97063


as electron ionization (EI), ESI, orMALDI, under certain conditions acomplete compound-independent ioni-zation can be obtained when using anICP as ion source. Under such condi-tions the instrumental sensitivity isproportional to the number of detectableatoms in the molecule investigated,independent of their chemical form.This allows a compound-independentcalibration and quantification (CIC) tobe performed based on non-sample-specific standards (e.g., inorganicsalts93,95 or small organic molecules96)that contain a known or even certifiedconcentration of the target element, thuseliminating the need for specific andoften expensive standards, which arestill rare for many applications. Suchcalibration strategies are in particularinteresting for the absolute quantifica-tion of biomolecules, e.g., in proteomicsor metallomics studies.

CIC approaches have been used, e.g.,for the quantification of metallothioneins(MT) via their sulfur and cadmiumcontent during CE-ICP-MS experimentsusing thiourea (as S standard) andcadmium nitrate, respectively,95 sulfur-labeled yeast,97 or more recently for thequantification of transferrin isoforms viause of simple, certified iron standards.98

CIC is in particular interesting for thequantification of monoisotopic elementtags such as phosphorus, which cannotbe quantified by isotope dilution analy-sis. Recently the application of anautomatic routine for flow-injectionanalysis (FIA) of a simple inorganicphosphorus standard implemented into achromatographic separation has beendeveloped, to obtain an instrumentalresponse factor for phosphorus, whichfinally allows absolute quantification ofthe separated phosphorylated peptidespecies whenever their tag stoichiometryis known.93

A further possibility to obtain thenecessary instrumental response factorsis to use internal standards, which areadded as a spike to the sample. Thesestandards are separated together with thesample under the same chromatographicconditions and again allow responsefactors to be computed; these responsefactors could then be used to perform anabsolute quantification of the targetedmolecules. The successful application of

such strategies has been demonstratedfor the quantification of sulfur-contain-ing proteins such as insulin99 or morerecently for the absolute quantificationof phosphorylated peptides96,100 usingthiamine (as the internal S standard) orbis(4-nitro-phenyl)phosphate (as the in-ternal P standard), respectively. Ingeneral such internal standardizationprovides a straightforward way to obtainthe necessary response factor for CIC;however, it is mandatory to find aninternal standard spike showing suitablechromatographic properties (no interfer-ence with the target analytes, no co-elution). This could be challenging, inparticular when analyzing complex, e.g.,biological, samples, which may show abroad elution window during theirchromatographic separation. Also, gra-dient effects during the separation haveto be taken into account and need to becompensated, since they will influencethe ionization behavior of the targetedelement and therefore result in gradient-dependent, changing response factors.96

Isotope Dilution Analysis (IDA).The utilization of isotopically labeledsurrogate standards, which incorporatevarying numbers of stable isotopes, suchas 2D, 13C, 15N, or 18O, has a longtradition and reflects the current state ofthe art, in particular within the field ofprecise, targeted quantitative environ-mental analysis, which often includescomplex sample extraction and pre-concentration schemes. Known amountsof these isotopically labeled standardsare added to each sample, ideallydirectly at the beginning of the samplepreparation process, to account for thepossible loss of the analytes during thewhole analytical process. Since thelabeled surrogate standards show thesame chemical properties as their nativecounterparts, both will co-elute duringtheir initial chromatographic separation,while showing a mass spectrum charac-terized by a specific mass shift of someneighboring peaks whose intensity ratiosare utilized for quantification.

Comparable approaches for an abso-lute quantification have also been devel-oped within the field of bioanalysis,utilizing proteotypic, isotopically labeledpeptides [AQUA peptides (absolutequantification peptides),101 PASTA pep-tides (phosphorus-based absolutely quan-

tified standard peptides),102 PolySIS(poly protein stable isotope standard),103

or QconCAT (Quantification conca-tamer)104] as internal standards for thetargeted proteins. While AQUA peptidesare quantified by classical amino acidanalysis, the recently developed PASTApeptides are quantified by LC-ICP-MS,utilizing their phosphorus tag, which ischemically or enzymatically cleavedbefore their final utilization. Such quan-tification approaches are particularlyinteresting for targeted quantitative stud-ies focusing on the study of a defined setof proteins, characterized by a number ofproteotypic peptides. In particular HPLC-ESI-MS-MS methods, utilizing the mul-tiple reaction monitoring (MRM) capa-bilities of such instrumentation, arefrequently used for such targeted ap-proaches.

In contrast the often used PolySIS orQconCAT proteins consist of severalisotopically labeled proteotypic pep-tides, which are synthesized in a con-catenated form as a single ‘‘non sense’’amino acid chain. Afterwards thesestandard proteins are purified and quan-tified via amino acid analysis. Suchinternal standard proteins are than addedto the sample after protein extraction.During the enzymatic cleavage reactiona set of labeled, internal standardpeptides is generated, allowing absolutequantification of their unlabeled coun-terparts, which in consequence allowsthe quantification of the targeted pro-teins. These approaches allow the pro-duction and quantification of multiplestandard peptides in a single step;however, such standards can only partlymimic the behavior of the targetedindividual intact proteins, which shouldbe reflected by the concatenated stan-dard, in particular during extraction andenzymatic cleavage.105 Recently it hasbeen demonstrated that fully traceableanalytical results can be achieved withsuch quantification approaches.106,107

Heumann and co-workers pioneeredthe development of comparable IDAapproaches for hyphenated ICP-MSsetups.108 In general two differentmodes, namely non-species-specificIDA as well as species-specific IDAhave to be differentiated.21,40,109

Non-Species-Specific Isotope Dilu-tion. The non-species-specific isotope


focal point review

dilution approach represents the mostflexible IDA method that can be used forICP-MS based quantification, in partic-ular for the analysis of biomolecules.

During non-species-specific IDA anisotopically enriched spike solution,which includes the targeted element, isadded post column with a constant massflow to the flow coming from theapplied separation technique (e.g.,HPLC, CE, or GC). This approach isin particular interesting for the accuratequantification of compounds for whichthere is a lack of suitable, stablestandards, or even unknown com-pounds. For non-species-specific IDA itis necessary to maintain a completemixing of the isotopically enrichedpost-column spike, which contains thetargeted elements as well as the sampleafter its elution from the column;however, this is easily achieved withspecific chromatographic hardware suchas mixing tees or knot reactors. Afterseparation the isotope ratio between thetarget element present in the sample witha natural isotopic abundance and theisotopically enriched spike is calculatedfor each data point of the chromatogram.The isotope dilution equation finallyallows the calculation of the mass flowof the targeted element during the wholeseparation process. The integration ofthe individual peaks present in the massflow chromatogram allows the directcalculation of the absolute amount of thecompounds reflected by the differentpeaks.

MFs ¼ csp 3 dsp 3 fsp 3Ms

Msp

3Ab

sp

Aas

3Rm-Rsp

1-Rm 3 Rs

� �ð1Þ

where MFs is the mass flow rate(normally ng min-1) of the targetedelement, csp is the concentration of thetargeted element in the spike solution,dsp is the density (g mL-1) of the spikesolution, fsp is the flow rate (mL min-1)of the spike solution, and Ms and Msp

are the atomic weights of the element inthe sample and in the spike, respective-ly. The isotope taken as reference in thesample is referred to as a and the usedstable enriched isotope in the spike isreferred to as b. Rm is the experimentalisotope ratio (a/b) measured in the

chromatographic peak of the targetedspecies after post-column mixing of thesample and the spike, and Rs and Rsp

are the elemental isotope ratio (b/a) and(a/b) in the sample and the isotopicspike, respectively.

The potential of non-species-specificIDA has been demonstrated in particularfor the quantification of proteins thatnaturally contain elements such assulfur59,110 or selenium111 in a covalent-ly bound form; however, the analysis ofstable metal-containing proteins such astransferrin via iron IDA has also beenrecently demonstrated.112,113 The non-species-specific IDA approach has beenfurther refined to improve the mass-flowstability, which is a critical parameterinfluencing the accuracy of the quanti-fication procedure.114,115

The main drawback of this IDAapproach is that any chemical or phys-ical loss of the analyte during the samplepreparation as well as the separationprocedure cannot be compensated. Inaddition, the thermodynamic and kineticstability of the targeted protein has to beensured during the entire analyticalprocess. It is also mandatory to knowthe (hetero)element stoichiometry, to beable to calculate the concentration of thetarget protein via the measured (hetero)element content.116

Species-Specific Isotope Dilution.The above-mentioned limitations canbe overcome by the application of thespecies-specific isotope dilution ap-proach, since it allows any losses duringthe analytical procedure to be compen-sated, assuming that the species-specificisotopically labeled spike compound andthe targeted analyte reach chemicalequilibrium prior to sample extraction.For species-specific IDA it is mandatorythat the targeted chemical species areknown and that a corresponding isoto-pically enriched spike material is avail-able or could be synthesized with highyields and simple reactions. Normallysuch spikes are added to the sample atthe beginning of the sample preparationprocedure. Such isotopically enrichedcompounds act as ideal internal stan-dards, since they show the same chem-ical structure as the target analyte,except that they are enriched with acertain isotope. In consequence, they co-elute with the target compound, which

also allows nebulization or ionizationeffects due to the sample matrix, orchanges in the mobile-phase composi-tion, to be compensated. Since thesespikes show a unique isotopic composi-tion that differs significantly from thenatural isotopic abundances of an ele-ment, it is not possible that they will beinterfered by other sample constituents.The concentration of the targeted speciescan then be calculated by using thespecies-specific isotope dilution equa-tion below.

cs ¼ csp 3msp

ms

3Ms

Msp

3Ab

sp

Aas

3Rm-Rsp

1-Rm 3 Rs

� �ð2Þ

where cs and csp are the concentrationsof the targeted element in the sampleand spike solution, respectively, msp andms are the masses taken from the sampleand the spike in the mixture, respective-ly, and Ms and Msp are the atomicweights of the element in the sample andin the spike, respectively. Isotope a istaken as reference in the sample and b isthe stable enriched isotope in the spike;Rm is the experimental isotope ratio(a/b) measured in the chromatographicpeak of the targeted species after post-column mixing of the sample and thespike, and Rs and Rsp are the elementalisotope ratios (b/a) and (a/b) in thesample and the isotopic spike, respec-tively.

Species-specific IDA has been fre-quently applied for environmental anal-ysis, in particular for the accuratequantification of different organometal-lic compounds such as tin,117 lead,118 ormercury119 species, due to the availabil-ity of suitable, highly enriched isotopesof the specific elements, as well as themore or less simple synthesis of thesespecies. Recently the synthesis of (het-ero)element labeled organic compounds,such as polybrominated flame retardants(PBDEs), and their application for thequantitative analysis of water samplesvia species-specific IDA has also beendemonstrated.120–122

The application of species-specificIDA for the quantification of selectedmetal-containing bio molecules repre-sents a more challenging approach. Theproduction of the isotopically enriched


metalloproteins is usually carried outunder in vitro conditions, via incubationof the apo protein (metal-free) with aspike solution, which contains the iso-topically enriched metal.123–125 Recentlythe in vivo synthesis of isotopicallyenriched metal proteins such as 57Fe-ferritin126,127 or 65Cu-plastocyanin128

has also been successfully demonstrated,just to give a few examples. Despite theinherent advantage of such species-specific standards it is important toensure that during the demetallationprocess, which is necessary for the invitro generation of the apo protein, nodegradation of the protein occurs. Also,the final storage environment, as well asthe chromatographic conditions, has tobe considered in order to avoid anyisotopic exchange, which will alter thesynthesized species-specific proteinstandard.

TRENDS IN APPLYING ICP-MSFOR QUANTITATIVEANALYSIS

Due to the ongoing progress in thefield of ICP-MS based quantification, itis almost impossible to provide a trulycomprehensive overview. The selectedexamples are intended to indicate theversatility of using ICP-MS as a tool forquantitative analysis, as well as chal-lenges related to the different applica-tions and hyphenation approaches.

Environmental Analysis. Since itsintroduction ICP-MS has been widelyused for environmental-orientated appli-cations, such as trace element determi-nation in a variety of sample matrices.This still represents the most commonarea for the routine application of ICP-MS. However, refined analytical strate-gies, new instrumentation, and thedevelopment of suitable interface sys-tems to combine chromatographic andelectrophoretic separation techniqueswith ICP-MS detection open a numberof new analytical possibilities, whichwill be highlighted in the followingsection. In particular, the potential ofusing (hetero)elements as surrogates forthe quantification of all kinds of chem-ical substances will be discussed.

Analysis of Halogenated VolatileOrganic Compounds. The transfer ofbrominated as well as iodinated orchlorinated volatile organic compounds

from different environmental compart-ments into the atmosphere, where theirdecomposition due to photochemicalreactions takes place, is strongly relatedto the ongoing reduction of the tropo-spheric ozone layer. Within this back-ground a two-dimensional on-linedetection scheme for brominated, iodin-ated, and chlorinated volatile organiccompounds has been developed usingGC hyphenated to an electron capturedetector (ECD) and ICP-MS.129 Underoptimized conditions ECD showed low-er detection limits for the measuredhalogens (below 1 pg); however, withonly ECD detection it was not possibleto distinguish between co-eluting spe-cies that contain different halogens.Seawater samples from the North Seahave been analyzed with respect toquantification of the different halogenat-ed volatile organic compounds, releasedinto the water phase or the atmosphereby seaweed or algae.

Pesticides. Since almost all pesticidescontain heteroatoms, element-specificdetection techniques such as atomicemission detectors (AED) are oftenutilized for pesticide detection aftercapillary GC separation.130–132 Its over-all low sensitivity as well as its matrixsusceptibility represent the main draw-backs of AED as a (hetero)elementspecific detector.133 Here ICP-MS rep-resents a powerful alternative due to itsspecial properties.

One of the first papers regarding theapplication of capillary GC and collisioncell ICP-MS for pesticide analysis waspublished by Vonderheide et al., inwhich they demonstrated the element-specific detection of organophosphoruspesticides such as Terbufos, Diazinon,Fonofos, Disulfoton, and Ronnel.54 He-lium has been used as cell gas at a flowrate of 1.25 mL min-1 to reducepolyatomic interferences on the massof phosphorus. In addition, nitrogen hasbeen added to the plasma for sensitivityenhancement, which unfortunately alsoresults in a substantially increasedphosphorus background due to theformation of nitrogen-containing inter-fering polyatomic ions such as14N16O1Hþ or 15N16Oþ. Instrumentallimits of detection in the mid ng L-1

range have been obtained for thedifferent pesticides measured.

To fully utilize the potential of ICP-MS, in particular its multi-elementcapability, Profrock et al. used GC-CC-ICP-MS for the simultaneous determi-nation of up to 23 phosphorus, sulfur,chlorine, bromine, and iodine containingpesticides.14 Low plasma power and hotextraction conditions have been used toimprove the overall sensitivity of theproposed setup. Even though the back-ground for all measured elements wasextremely low due to the dry plasmaconditions, helium at a flow rate of 2.5mL min-1 was added to the collisioncell to further reduce the background,especially on the sulfur isotope 32S. Incontrast to Ref. 54, helium was used asadditional plasma gas to further improvethe sensitivity without increasing the 31Pbackground. Depending on the element,detection limits in the low ng L-1 (P, Br,I) to lg L-1 (S, Cl) range have beenobtained. Pesticides in fruit and vegeta-ble samples have been quantified using astandard calibration as well as a com-pound-independent calibration ap-proach. In addition, retention timelocking has been applied by lockingthe retention time of selected com-pounds to those specified in a pesticidedatabase, allowing the identification ofadditional compounds without the needfor compound-specific standards. Figure4 shows the simultaneous separation and(hetero)element-specific detection of amulti-compound pesticide mixture (CUS3217, 0.1% in n-hexane, 1 lL pulsedsplitless injection) by GC-ICP-MS. Thisexample clearly indicates the highresolving power of GC and the out-standing multi-element capabilities ofcollision cell ICP-MS.

Since many pesticides are watersoluble and nonvolatile, which requirestheir error-prone derivatization prior toGC analysis, Sadi et al. used ion-pairingreversed-phase liquid chromatographycoupled to ICP-MS to quantify phos-phorus-containing herbicides, such as a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), Glufosinate,and Glyphosate.134 Phosphorus has beendetected as 31P using He as cell gas toreduce interference. Detection limits inthe low ng L-1 range for the differentherbicides have been obtained. Unfortu-nately, only spiked water samples havebeen analyzed so far to simulate an


focal point review

environmentally relevant matrix, so thepotential of the method for the detectionof trace amounts in real samples remainsunclear. Also, no comparison has beenmade of the detection limits of othermethods that can utilize an MRM toachieve outstanding sensitivity and se-lectivity and in parallel that may alsoallow the direct identification of un-known chromatographic peaks (e.g.,HPLC-MS).

Chemical Warfare Agents. Richard-son and Caruso published two papersdealing with the analysis of organophos-phorus nerve agent degradation prod-ucts, using either reversed-phase ionpairing HPLC135 or GC136 after trimeth-ylsilyl (TMS) and tert-butyldimethylsil-yl (TBDMS) derivatization for theirseparation. Phosphorus-specific detec-tion during HPLC experiments has beencarried out using collision cell ICP-MSand helium as the cell gas. In addition,phosphorus has been also detected as31P16Oþ at m/z 47. In contrast, no cellgas was needed for the GC experimentsbecause of the dry plasma conditions

obtained by using GC-ICP-MS, whichresults in an overall low backgroundcompared to HPLC-based approaches.The authors indicate a significant im-provement of the detection limits, espe-cially when using GC-ICP-MS aftersample derivatization. However, onlyspiked water and soil samples have beenanalyzed so far to simulate an environ-mentally relevant matrix, so again thepotential of the method for the detectionof trace amounts in real samples remainsunclear. Also no comparison to other,e.g., LC-MS based, methods in terms ofdetection limits is given. Recently thesame authors described the analysis oforganophosphorus nerve agent degrada-tion products spiked into pesticidemixtures using GC-ICP-MS.137

Flame Retardants. With the enact-ment of the 2000 European Community(EC) Water Framework Directive(WFD), the most significant piece ofEuropean water legislation for over 20years is coming into effect. Along with anumber of other compounds such as tinspecies, flame retardants such as PBDEs

have now become priority hazardoussubstances due to their persistent char-acter as well as their already widespreaddistribution within the environment.

For PBDEs, high-performance liquidchromatography with mass spectromet-ric detection via an atmospheric pressurechemical ionization source (HPLC-AP-CI-MS) is the method of choice. How-ever, GC-MS has also been widely usedfor PBDE analysis.138,139 The currentmain challenges for these methods arethe required lower detection limits (pg/Lrange) and the extension of the numberof detectable congeners due to theimproved chromatographic resolution.Here ICP-MS indicates some advantagessince in comparison to GC-MS setups,the interface outlet is operated at atmo-spheric pressure, which reduces possibleband broadening effects due to thevacuum of the mass analyzer. In partic-ular the commercial availability ofrobust GC-ICP-MS interface systemsthat allow transfer line temperaturesover 300 8C opens this field of analyticsfor the ICP-MS.

Vonderheide et al. described for thefirst time the application of GC-ICP-MSfor the analysis of different PBDEcongeners.55 A normal quadrupoleICP-MS has been used for brominespecific detection. Again the dry plasmaconditions result in an overall lowbromine background, allowing detectionlimits in the medium parts per trillionrange. The optimized method has beenused for the quantification of selectedbrominated flame retardants in sewagesludge samples from a local watertreatment plant. In a more recent paperthe same author used GC-ICP-MS forthe analysis of the breakdown of bromi-nated flame retardants by microorgan-isms in soil samples140 or to quantifythem in different marine referencematerials.141 Recently the synthesis andapplication of bromine labeled PBDEsas species-specific standards has beendemonstrated for the accurate quantifi-cation of priority PBDEs in watersamples.120–122

Shah et al. used solid-phase micro-extraction (SPME) and GC-ICP-MS toovercome the limitations of other meth-ods, such as liquid-liquid extractioncombined with GC-MS, for the analysisof phosphorus-based flame retardants in

FIG. 4. Separation and (hetero)element-specific detection of a multi-compound pesticidemixture (CUS 3217, 0.1% in n-hexane, 1 lL pulsed splitless injection) by GC-collision cell-ICP-MS. Peak assignment and retention order: (a) 1,2,3-Trichlorobenzene, (b) Dichlobenil,(c) 4,4-Dibromooctafluorobiphenyl, (d) Ethoprop, (e) 2,4,6-Tribromoanisole, (f) Phorate, (g)Silvex, (h) Pentachloronitrobenzene, (i) Terbufos, (j) Diazinon, (k) Ioxynil-Methyl, (l)Malathion, (m) Chlorphyrifos, (n) Triphenylphosphate, and (o) Decachlorobiphenyl. (Takenfrom Ref. 14, reprinted with permission from Journal of Analytical Atomic Spectrometry2004, 19, 623-631. Copyright, The Royal Chemical Society 2004.)


human plasma samples, which oftenrequires large sample amounts.142 Ex-traction parameters such as salt content,pH, temperature, and extraction time andtheir effects on the recovery of thedifferent phosphorus compounds havebeen evaluated to obtain a maximumextraction efficiency. Helium at a flowrate of 1.2 mL min-1 has been used ascell gas to reduce the phosphorusbackground during analysis, allowingdetection limits down to 17 ng L-1 forselected compounds such as tributylphosphate.

In a comparable paper Ellis et al.extended the approach published byShah et al. by combining a microwave-assisted extraction protocol with apreviously described SPME procedurefor the extraction of organophosphorustriesters and their analysis using GC-ICP-MS.143 Wastewater samples havebeen analyzed with the optimized meth-od. Unfortunately an extraction efficien-cy of only 40% has been achieved,which complicates accurate quantifica-tion. In addition, GC-TOF-MS has beenused to confirm the presence of thedifferent species.

Petroleum Products. Environmentalconcerns about the effects resulting fromthe combustion of sulfur-containingfuels has led to a drastic reduction ofthe legal limits for sulfur in fuels withinthe last years.144 The effective removalof sulfur species from crude oil requiresaccurate quantitative knowledge aboutthe sulfur species composition of thedifferent oil fractions during the refineryprocess. This requires robust analyticalmethods that are able to provide specificinformation on sulfur-containing com-pounds present in petroleum products atthe ng g-1 range. In comparison to othertechniques often used for sulfur analysissuch as atomic emission or chemilumi-nescence detection, ICP-MS ideallycombines high sensitivity and robust-ness, which allows the complex hydro-carbon matrix to be handled.

Bouyssiere et al. showed for the firsttime the application of GC-ICP-MS forpetroleum analysis. The on-line cou-pling of capillary GC with ICP-collisioncell-MS was proposed for the speciationof sulfur compounds such as differentthiophenes in hydrocarbon matrices. Thetechnique showed an absolute sulfur

detection limit of 0.5 pg for a 1 lLsample injected in the splitless mode,which is about two orders of magnitudelower compared to currently used tech-niques.145

Recently, the development of a non-species-specific isotope dilution GC-ICP-MS method for the quantificationof sulfur species in petroleum productsusing a single 34S labeled spike com-pound has been described. In additionEI-IT-MS has been applied for structuralcharacterization of the sulfur species.Detection limits for sulfur of 9 ng g-1

were obtained. Different reference ma-terials such as BCR107 or SRM-2296have been analyzed with the developedmethod with respect to their sulfurspecies composition. Results were ingood agreement with the specifiedvalues, indicating the high accuracyobtainable by non-species-specificIDMS.56

Tao et al. developed a GC-ICP-MSmethod for sulfur analysis in petroleumliquids. The operating conditions of theICP-MS and their effects on the back-ground intensities at m/z 32 and 34 wereinvestigated to decrease possible con-tamination and interference. The detec-tion limit was around 0.6 ng S mL-1,corresponding to 0.05 pg S. The presentmethod was successfully applied topetroleum liquids, such as naphtha,gasoline, kerosene, and light oil.146

Miscellaneous Environmentally Re-lated Applications. Caruso and co-workers published two papers dealingwith the analysis of iodinated phenols inwater samples, which are byproductsproduced during drinking-water disin-fection and whose chemistry and toxi-cology are not well understood. EitherGC or CE hyphenated to ICP-MS, inboth cases combined with solid-phasemicroextraction, has been used for theanalysis of the iodinated com-pounds.147,148 Carboxen poly(dimethyl-siloxane) SPME fibers provide the bestextraction rates and recoveries for theiodophenol species analyzed. Detectionlimits around 0.1 ng L-1 have beenobtained for the different compoundsusing GC-ICP-MS, while CE-ICP-MSprovides detection limits around 40 ngL-1. In comparison, the GC-basedapproach indicates a much higher sensi-tivity in comparison to CE, also allow-

ing the detection of low-abundant,iodine-containing species. Even thoughthe CE approach has been optimized forshort separation times, no advantage wasgained in comparison to GC, which alsoallows baseline separation of the differ-ent iodophenols within 6 minutes. Fromthe analytical point of view the utiliza-tion of CE-ICP-MS for this applicationis quite interesting. However, bearing inmind the extraordinary detection limitsobtained by GC-ICP-MS and its robust-ness, this approach seems to be moresuited for real-life samples.

More recently Shah et al. used SEC,IC, and RP-HPLC hyphenated to ICP-MS to separate and specifically detectiodinated compounds extracted fromcommercially available seaweeds.149

Three extraction procedures have beenoptimized, followed by the applicationof different chromatographic approachesto separate various molecular-weightfractions of iodinated compounds. Peakidentification has been carried out onlyby retention time matching, using anumber of known iodine containingstandards. Unfortunately, the authorsonly provide information on analyticalparameters such as detection limits forthe applied IC-ICP-MS setup. Here adirect comparison with techniques suchas GC and CE-ICP-MS, which havebeen utilized in some previous papers,could be interesting.147,148

Pharmaceuticals. The application ofICP-MS during drug development andquality control as well as to study theirbehavior in the environment represents afurther future field of application thatmight also benefit from its particularproperties with respect to specificallydetecting and quantifying (hetero)ele-ments.

Quantitative information on the drugitself and possible metabolites, as wellas the number, nature, and concentrationof impurities, will provide valuableinformation to improve drug safety andproduct quality. In particular chlorine,bromine, and iodine are essential partsof various pharmaceuticals, which facil-itates the application of ICP-MS.

Iodine represents an essential part ofthe thyroid hormones 3,30,5,50-tetraio-dothyronine (T4) and 3,50,5-triiodothy-ronine (T3), which are synthesized bythe thyroid gland. Both are essential


focal point review

parts of the thyroglobulin proteins. In1993 Takatera and Watanabe usediodine as a (hetero)element tag to detectand quantify the content of iodinatedamino acids in an enzymatic digest ofbovine thyroglobulin.150 Absolute de-tection limits in a range from 35 to 130pg as iodine have been obtained, whichis an order of magnitude lower thanthose in conventional methods using thestable isotope of iodine.

One of the first papers dealing withthe (hetero)element-specific detection ofpharmaceuticals was published by Ax-elsson and co-workers in 2001. Theyused LC-ICP-MS for the analysis ofimpurities in different iodine-containingcontrast agents, such as OmnipaqueTM

(Iohexol) or VisipacTM (Iodixanol).94

Membrane desolvation, as well as oxy-gen addition, have been utilized toreduce the acetonitrile vapor, whichmay cause plasma instabilities andcarbon buildup. Detection limits of 40lg L-1 were obtained, which corre-sponds to 0.4 ng of iodine.

Jensen et al. used reversed-phaseHPLC hyphenated with ICP-MS toanalyze a number of halogen-containingdrugs, such as furosemid, diclofenac, orbromazepam.151 Membrane desolvationhas been used for sample introduction toreduce the organic solvent load of theplasma during isocratic and gradientseparation of selected compounds. Theyobserved the partial loss of differentchlorine, bromine, and iodine containingcompounds in the desolvation system,which complicates the quantitative anal-ysis of analytes with unknown proper-ties and for which standards are notavailable. Absolute detection limits forchlorine, bromine, and iodine of 8 ng, 2pg, and 2 fg, respectively, have beenobtained.

HPLC-ICP-MS was utilized by Kan-namkumarth et al. for the determinationof levothyroxine, an iodine-containinghormone, and its degradation productsin different batches of pharmaceuticaltablets.152 Isocratic separation condi-tions using 22% (v/v) acetonitrile in atrifluoroacetic acid solution allowedcomplete resolution of the differentiodine species without compromisingthe quantification, due to gradient-relat-ed effects on the elemental response for127I. Instrumental detection limits below

200 ng L-1 have been reported for thedifferent forms of the hormone, alsoallowing the detection of low abundantdegradation products that could not bedetected by UV detection.

Recently gadolinium (Gd) chelatesused in magnetic resonance imaging(MRI) and their behavior during sewagetreatment, as well as their final distribu-tion in the environment, have beenanalyzed via the coupling of hydrophilicinteraction chromatography (HILIC)with ICP-MS to account for the specialchemical properties of such com-pounds.153,154

Life Sciences Applications. In gen-eral life science related applications thatutilize ICP-MS for quantification can beseparated into three different maindirections: (i) applications that use non-covalently bound metals (e.g., Cu, Zn,Cd, Fe, . . . .), (ii) applications based onthe utilization of covalently bound(hetero)elements such as P, S, Se, andI, and (iii) applications that includechemical labeling with ICP-MS detect-able elements (e.g., I, Hg, lanthanides,etc.)

Due to the importance of (hetero)ele-ments such as phosphorus and sulfur forbiological systems, in particular life andbio-science orientated applications orig-inated the ongoing development andimprovement of ICP-MS as a (hetero)element-specific detection technique.The analysis of phospholipids, DNA/RNA, protein phosphorylation, and sul-fur-based absolute protein quantificationcan be identified as the main researchdirections that will be emphasized in thefollowing section.

Phospholipids. Despite their impor-tance for biological systems and thepossible advantages of using ICP-MSfor phospholipid analysis over otherapproaches in terms of detection sensi-tivity and method linearity, only a fewexamples of the application of ICP-MSfor phospholipid analysis can be foundin the recent literature. Phospholipidsare the main building blocks for all pro-and eukaryotic cell membranes. Alsovarious biological processes, such ascellular signaling cascades, are based onphospholipids. However, neither thequalitative nor quantitative analysis ofphospholipids is straightforward due totheir complexity and special chemical

properties, such as hydrophobicity or theability to form micellar structures inaqueous solutions. In particular, theapplication of ICP-MS for phospholipidanalysis represents a challenge becausephospholipids are only soluble in organ-ic solvents and high organic solventloads are necessary for their chromato-graphic separation. Membrane desolva-tion, oxygen addition, low-flow LCsystems, or flow splitting have beenused to reduce the carbon load of theplasma during ICP-MS-assisted phos-pholipid analysis.

Axelsson et al. published the firstreports on phospholipid analysis, byliquid chromatography hyphenated to ahexapole collision/reaction cell ICP-MSsystem via an ultrasonic nebulizer and amembrane desolvator, using phosphorusas an element tag.94 The authors clearlyshowed the advantages of this genericdetection approach over other tech-niques such as ultraviolet, refractiveindex, evaporative light scattering de-tection, or MS in terms of sensitivity andlinearity.94

More recently Kovacevic et al. de-scribed the application of normal-boreHPLC hyphenated to an octopole reac-tion cell ICP-MS system for the separa-tion and phosphorus-specific detectionof different phospholipid standards, aswell as lipid extracts from the yeastSacharomyces cerivisiae.20 Instead ofmembrane desolvation, solvent splitting,spray chamber chilling down to -5 8C,and the addition of oxygen were utilizedto reduce the carbon load of the plasma.To reduce polyatomic interferences onthe mass of phosphorus, helium wasused as cell gas. Absolute detectionlimits for phosphorus between 0.21 and1.2 ng phosphorus were obtained.20

Nucleic Acid Analysis. In one of thefirst papers on the application of natural(hetero)element tags, Siethoff and co-workers used phosphorus for the quan-titative determination of in vitro gener-ated adducts of styrene oxide and thefour nucleotides 20-deoxyguanosine-50-monophosphate (dGMP), 20-deoxythy-midine-5 0-monophosphate (dTMP), 20-deoxycyt idine-5 0-monophosphate(dCMP), and 2 0-deoxyadenosine-5 0-monophosphate (dAMP). High-resolu-tion ICP-MS and ESI-MS coupled tonormal-bore RP-HPLC has been used


for the identification and quantificationof the different adducts. The applicationof a mathematical correction function tocompensate for changes of the instru-mental phosphorus response during theHPLC gradient was necessary to obtainaccurate quantitative results. In addition,oxygen has been added to the spraychamber to reduce the carbon build-upwithin the system. Inorganic phosphorusstandards were used for the quantifica-tion of the different adducts, allowingthe detection of one modified nucleotidewithin 3.5 3 105 un-modified nucleo-tides, which is comparable to standardradioactive post-labeling techniques.155

More recently Edler et al. used ahexapole collision cell ICP-MS com-bined with a membrane desolvationsystem and a microbore HPLC setupfor the quantification of DNA adductsgenerated by reaction with either styreneoxide or Melphalan.16 In comparison tothe work of Siethoff et al., no mathe-matical corrections were needed tocompensate for changes in the instru-mental response, due to the membranedesolvator. As an example Fig. 5 showsthe LC-ICP-MS chromatograms moni-tored at m/z 31 of Melphalan adducts ofsingle nucleotides. Gradient elution andbis(4-nitrophenyl)phosphate (BNPP) asinternal standard were used for theseparation and quantification of thedifferent modified nucleotides. Eventhough quadrupole-based instrumenta-tion has been used, comparable analyt-ical figures of merit to those presentedby Siethoff et al. have been ob-tained.16,155,156

As an alternative to HPLC, differentauthors demonstrated the use of capil-lary electrophoresis (CE) hyphenated toICP-MS for the separation and phos-phorus-specific detection of mono-phos-phorylated deoxynucleotides or RNAnucleotides.17,157 In parallel, HPLC-ESI-MS has been used to identify theseparated compounds.17

Recently Bruchert and Bettmer devel-oped an interface for the online couplingof 1D slab gel electrophoresis and ICP-MS that allows the separation andquantification of DNA strands.158 De-tection limits of 1 ng DNA absolutehave been achieved, which correspondsto 96 pg of phosphorus. In comparisonto CE, relatively poor separation effi-

ciency can be obtained with a 1Dagarose gel, which represents the mainlimitation of this setup. To indicate theversatility of the interface, the authorsrecently applied their approach forphosphorylation analysis of selectedmodel proteins.159

SEC-ICP-MS has been used to inves-tigate the interaction of chromium withDNA extracted from metal-contaminat-ed soil samples. Helium was used as cellgas to reduce polyatomic interferenceson the masses of phosphorus (31P) andchromium (52Cr). In this case phospho-rus has been monitored to trace theDNA-containing fraction during theSEC separation.160

Protein Phosphorylation Analysis.As already mentioned, a number ofinitial studies focusing on the applica-tion of ICP-MS as a (hetero) element-specific detector have been conducted inthe field of protein phosphorylationanalysis.

The reversible phosphorylation ofproteins at their serine, threonine, andtyrosine residues is one of the mostimportant post-translational modifica-tions in eukaryotic organisms that reg-ulate cell signaling as well as theenzymatic activity, localization, com-plex formation, and degradation ofproteins.161,162

Phosphorylation is regulated by thecomplex interaction of kinases andphosphatises that catalyze protein phos-phorylation and de-phosphorylation, re-spectively.163 However, despite theoutstanding methodological and instru-mental developments, especially withinthe field of mass spectrometry, theanalysis of protein phosphorylation isstill not straightforward.

ESI-based tandem MS systems pro-vide various scan modes that can beused for qualitative phosphorylationanalysis. These include neutral lossscanning in the positive ion mode aftercollision-induced dissociation (CID) ex-periments for H3PO4 (-98 Da) or HPO3

(-80 Da), which is characteristic for thepresence of phosphoserine or phospho-threonine containing peptides. Thescreening for a diagnostic immoniumion at m/z 216.043 is characteristic forphosphotyrosine-containing peptides.Precursor ion scanning in the negativeion mode for fragments that generate the

loss of PO3- (-79 Da) represents a

further sensitive mode for the selectivedetection of phosphorylated pep-tides.164–166

The quantitative determination ofphosphorylation, e.g., in selected signal-ing proteins, will permit a true systemsbiology approach, which will providevaluable data on regulatory pathwaysand networks based on phosphoryla-tion.165 However, the accurate quantifi-cation of phosphorylation eventsespecially in signal transduction path-ways is still not straightforward andrequires synthetic internal phosphopep-tide standards. ICP-MS represents avaluable complementary approach forprotein phosphorylation analysis withrespect to the fast screening of complexsamples for phosphopeptides, as well astheir absolute quantitative determina-tion.

With a number of papers, Wind andco-workers pioneered the field of ICP-MS assisted protein phosphorylationanalysis.41,44,45,99 In this context theyalso introduced the successful applica-tion of capillary HPLC hyphenated toICP-MS under the same chromatograph-ic conditions, which is a prerequisite forthe real complementary application ofESI and ICP based MS techniques in lifesciences related research.

As already stated, the application ofnano or capillary HPLC dramaticallyreduces the problems related to theintroduction of the frequently used RPorganic gradient solvents, such as ace-tonitrile or methanol, into the ICP.

Wind et al. used capillary LC hy-phenated to either high-resolution ICP-MS and a hexapole collision-reactioncell ICP-MS or ESI-MS-MS for thephosphorus-specific detection of differ-ent synthetic phosphopeptides and tryp-tic digests of b-casein or activated MAPkinase. Membrane desolvation was usedto reduce the organic solvent load withinthe plasma, especially during the hexa-pole ICP-MS experiments. However,incomplete peptide recovery from thedesolvation systems has been observed,especially for late eluting compounds.41

A further paper focused on thedemonstration of phosphorylation de-gree determination of different modelproteins via simultaneous measurement


focal point review

of the phosphorus and sulfur content ofthe eluting peptide fractions.44

Minimized dead volumes are essentialfor highly resolved nano or capillary LCseparation of phosphopeptides. Withinthis background, Wind et al. modified adirect injection nebulizer (DIHEN) tominimize the dead volume of the device.In comparison with their previouslyused spray chamber nebulizers, theDIHEN was slightly less sensitive butshowed better chromatographic resolu-tion, superior signal stability, and morerobustness with respect to changinggradient conditions.45

In a number of further papers the

same authors extensively demonstratedthe complementary application of ele-mental and molecule specific MS for theinvestigation of protein phosphorylationof real samples, such as the so-calledpolo-like kinases Plx1 and Plx2, humanfibrinogen and fetuin subunits, or thephosphorylation state at His-48 of thechemotaxis protein CheA, which influ-ences the stability of this protein.167–169

To overcome some of the limitationsrelated to the different described inter-face systems, Profrock and co-workersintroduced a new interface system forthe successful direct hyphenation ofcapillary and nano HPLC to a collision

cell ICP-MS. This interface allows thedirect introduction of organic gradientsinto the ICP-MS, without the need forusing membrane desolvation or oxygenaddition.49 Helium was used as cell gasto minimize polyatomic interferences atthe mass of phosphorus, while main-taining a good overall instrumentalsensitivity. With this new interface,100% transport efficiency, good nebuli-zation stability, and minimized deadvolumes at capillary as well as nanoHPLC flow rates (below 1 lL min-1)have been realized.

The well-known protein b-casein wasused as a model to demonstrate the

FIG. 5. LC-ICP-MS (31P) chromatograms of Melphalan adducts in the nucleotide mixture (top trace) and of single nucleotides; all withinternal standard (last signal). The phosphate adducts (first signal) and the base adducts (smaller following signals) are clearlydistinguishable. (A) dAMP, (B) dCMP, (C) dGMP, and (D) dTMP. (Taken from Ref. 16, reprinted with permission from Journal of MassSpectrometry 2006, 41, 507-516. Copyright 2006 John Wiley & Sons Ltd.)


potential of the setup for the phosphor-ylation profiling of tryptic protein di-gests. In addition capillary LC-ESI-MS-MS was used to further characterize thepre-selected peptides, due to their phos-phorus content. Four hundred femto-moles (400 fmol) of the singlyphosphorylated peptide with the se-quence FQpSEEQQQTEDELQDK de-rived by the tryptic digestion of b-caseincould be detected, while detection limitsof 1.95 lg L-1 (1.95 pg absolute) forphosphorus were obtained.49

More recently Kruger et al. analyzedprotein phosphorylation levels of Arabi-dopsis thaliana and the algae Chlamy-domonas reinhardtii as representativesfor multicellular and unicellular green,photosynthetically active organismswith the aim of quantifying differencesin the cellular protein phosphorylationlevel of both species.170 Therefore,

capillary HPLC and high-resolutionICP-MS in the medium resolution modewere employed for the separation andelement-specific detection of phosphor-ylated peptides after enzymatic digestionof the protein extracts. In parallel, thecorresponding sulfur traces were alsomonitored, which allows the conversionof the molar phosphorus-to-sulfur ratiointo a stoichiometric protein phosphor-ylation degree.

In addition, metal oxide affinitychromatography (MOAC) has been usedto specifically enrich phosphoproteinsfrom the different plant extracts. Unfor-tunately the authors did not presentadditional ESI data with respect tophosphoprotein/peptide identification,even though the ICP-MS based screen-ing of the samples indicated the pres-ence of a number of phosphorylatedpeptides. As an example Fig. 6 shows

the LC–ICP-MS analysis of digestedprotein extracts, before and after phos-phoprotein enrichment by MOAC.170

The quantification of unknown phos-phorylated peptides requires the correc-tion of the well-known gradient impacton the instrumental phosphorus re-sponse, in particular during reversed-phase separations, e.g., by the applica-tion of mathematical correction func-tions.

To overcome the need for a mathe-matical correction of the phosphorusresponse, Pereira Navaza et al. intro-duced a new strategy for the accuratequantification of protein phosphoryla-tion using simple organic compounds asa phosphorus standard.96 They describedthe utilization of a constant post-columnsheath flow with a constant acetonitrilecontent to buffer gradient compositionchanges that influence the ionization

FIG. 6. LC–ICP-MS analysis of digested protein extracts before and after phosphoprotein enrichment by metal oxide affinitychromatography (MOAC). Left row: A. thaliana seeds (A) before and (B) after MOAC enrichment; right row: C. reinhardtii (C) before and (D)after MOAC enrichment. (Taken from Ref. 170, reprinted with permission from Biochemical and Biophysical Research Communications2007, 355, 89-96. Copyright Elsevier 2007)


focal point review

efficiency of phosphorus within theplasma, even at capillary LC or nano-LC flow rates. They achieved a constantelemental response over the used capil-lary LC gradient (10–50% B), allowingthe application of phosphorus-contain-ing compounds, such as bis(4-nitro-phenyl) phosphate (BNPP) as internalstandards for accurate phosphopeptidequantification.96

Recently a new approach for thecompensation of gradient-related chang-es of the instrumental response forphosphorus during reversed-phase gra-dient separation of phosphorylated pep-tides has been developed. Instead of aconstant sheath flow, a second precisecapillary LC pump has been used togenerate a countercurrent reversed gra-dient that is mixed post-column with theRP column outflow before entering thecapillary and nano-LC-ICP-MS inter-face. The experimental design allowsthe application of gradient separations,while the element-specific detection iscarried out under isocratic conditionswith a constant organic solvent intakeinto the plasma during the wholeseparation. This helps to eliminate anychanges in the elemental response dur-ing reversed-phase separations, which isa general prerequisite for the applicationof ICP-MS for absolute quantification ofproteins and peptides via their (hetero)element content, especially when nocorresponding high-purity standards areavailable. Highly reproducible separa-tions have been obtained, with retentiontime and peak area RSDs of 0.05% and7.6% (n = 6), respectively. Detectionlimits for phosphorus of 6.24 lg L-1

(6.24 pg absolute) have been realized. Inaddition, an automatic routine for flowinjection analysis (FIA) at the end ofeach chromatographic separation hasbeen developed to calibrate each chro-matographic separation. This makesabsolute quantification of the separatedspecies possible whenever their tagstoichiometry is known. As proof ofconcept, phosphorylated peptides havebeen used as model compounds formethod development and to demonstratethe applicability of the proposed setupfor phosphopeptide quantification (seeFig. 7) on the basis of simple inorganicphosphorus standards.93,171 Beside theapplication of capillary LC, laser abla-

tion ICP-MS (LA-ICP-MS) as an inter-face to combine high-resolutionseparation techniques such as 1D or2D gel electrophoresis has also gainedmuch interest, especially for proteinphosphorylation analysis.

The first report with respect to theutilization of LA-ICP-MS for proteinphosphorylation analysis was publishedby Marshall et al., who described theinvestigation of membrane-blotted phos-phorylated proteins by LA-ICP-MS.172

b-casein was used as model protein anddetection limits of 16 pmol were ob-tained.

Also a direct analysis of gels contain-ing the destained protein spots has beenperformed. However, due to the highphosphorus background of the gelmatrix it was not possible to determinethe location of the phosphorylatedproteins.172

To overcome the contamination prob-lem, Wind et al. improved this approachby adding a washing step after theblotting process into their strategy forthe LA-ICP-MS based analysis of gel-separated phosphoproteins.173 Ga(NO3)3

was used as complexing agent to removeany non-covalently bound phosphatefrom the membrane, which is known to

result in non-specific interactions withother non-phosphorylated sample con-stituents, leading to false positive re-sults. Detection limits of 5 pmolphosphorus were obtained. The authorsalso presented quantitative data indicat-ing the possibility of using this approachfor the quantification of gel-separatedphosphoprotein species.173

A new strategy for the analysis of gel-separated phosphoproteins by combin-ing whole gel elution with flow-injectionICP-MS detection has been published byElliott and colleagues. Phosphorus wasmeasured as 31P16Oþ at m/z 47. Resultswere compared with the phosphorus-specific screening of 1D PAGE gels byLA-ICP-MS.174 Along with the contam-ination problem, the relatively small sizeof the standard ablation cell represents afurther limitation of LA-ICP-MS, sincegels or blot membranes had to be cutinto pieces to facilitate their wholeanalysis.

To overcome this shortcoming, Feld-mann and co-workers developed andoptimized a new laser ablation cell forthe detection of phosphoproteins blottedon to nitrocellulose membranes.175 Incomparison to other ablation cells, theirnew device allowed the direct analysis

FIG. 7. Quantification of an IR10 Insulin Receptor [1142–1153] standard sample using asimple inorganic phosphorus standard and translation of the phosphorus content intomolar peptide amounts, based on the known phosphorus stoichiometry of the peptide.(Taken from Ref. 93, reprinted with permission from Journal of Chromatography A 2009,1216, 6706–6715. Copyright Elsevier 2009).


of whole blot membranes with dimen-sions up to 8.5 cm height and 10 cmlength. Detection limits for the modelproteins pepsin and b-casein down to 5and 3 pmol, respectively, were obtained.The quantitative results calculated for acommercially available protein markerstandard on the basis of a previouslyacquired calibration function were alsoin good agreement with the concentra-tions specified by the manufacturer.Overall this approach is mainly limitedby the possibility of additional sampleloss and the additional time needed forthe blotting process.175

Based on the ablation cell describedby Feldmann and co-workers, Venkata-chalam et al. introduced a new calibra-tion approach for the quantitativedetection of phosphorylated proteins,blotted onto membranes.78 For thecalibration procedure different standards(b-casein, pepsin, phosphate solution)containing different known amounts ofphosphorus were dotted directly onto theblotting membrane. The results indicatethat the dotting procedure leads torecoveries of the test proteins of onlyaround 55%. As an alternative, thestandards were separated and blottedtogether with the test sample. A totalamount of 126 pmol of phosphorus wasdetermined with the second approach.These results were in good agreementwith the known calculated total phos-phorus amount of 139 pmol of the testsample. Additionally, a higher dynamicrange in comparison to previous workhas been obtained. The standard devia-tion of the whole method (electropho-retic separation, blotting process, laserablation procedure) was 6%.78

In a recent publication Kruger et al.showed for the first time the protein andproteome phosphorylation stoichiometryanalysis of the cytoplasmatic proteomeof bacterial cells (Corynebacterium glu-tamicum) and eukaryotic cells (Musmusculus) by a combined approachbased on one-dimensional gel electro-phoresis, in-gel digestion, and capillaryreversed-phase LC-ICP-MS, as well asan additional strategy that includes theapplication of 1D gel electrophoresis,protein blotting, and LA-ICP-MS.176

Consistent quantitative results have beenobtained with both approaches; howev-er, higher sensitivities were achieved

with the capillary LC based setup. Inthis study the eukaryotic proteome ofMus Musculus was found to be signif-icantly more phosphorylated in compar-ison to the bacterial proteome (around0.8 mol of P/mol of protein versusaround 0.01 mol of P/mol of protein).This study demonstrated for the firsttime the direct and rapid determinationof a global phosphorylation degree of acomplex protein mixture by using dif-ferent ICP-MS methodologies.176

The examples given above clearlyshow the possibilities that arise becauseof the complementary application ofICP-MS in protein phosphorylationstudies. In addition, ICP-MS can helpto replace standard procedures, e.g., thein vivo or in vitro incorporation ofradioactive 32P for phosphorylationanalysis, which is still restricted toselected laboratories.177,178 A very valu-able review article also dealing with thistopic was recently published by Navazaet al.36

Absolute Protein and Peptide Quan-tification. The accurate absolute quanti-fication of proteins and peptides is still achallenge. Knowledge about the dynam-ic change of protein abundance directlyreflects the status of biological systemsand therefore can provide valuableinformation in particular for life scienceorientated research.

Wind et al. proposed sulfur as a keyelement for the absolute quantificationof proteins and peptides.99

Recent calculations on the basis of thehuman proteome have revealed that26.6% and 25.5%, respectively, of theresulting tryptic peptides include at leastone of the sulfur-containing amino acidscysteine or methionine within theirsequence.2 These peptides represent96.1% and 98.9%, respectively, of allhuman proteins. This facilitates thequantitative determination of the majorityof proteins on the basis of their naturalsulfur content, once the sulfur stoichiom-etry is clarified by the complementaryapplication of ESI or MALDI based MSapproaches.99

Insulin has been used as a modelprotein to demonstrate its absolutequantification, while thiamine has beenused as an internal sulfur standard. Theauthors also showed the complementaryapplication of capillary LC HR-ICP-MS

and capillary LC ESI-MS-MS for thequantification and characterization oftryptic protein digests of two functionaldomains of the bacterial chemotaxisprotein cheAH (3-137) and cheA-C(257-513) over-expressed in E. co-li.99,179

Over the last few years Heumann andco-workers have promoted the applica-tion of isotope-dilution approaches foraccurate quantitative elemental specia-tion analysis using either non-species-specific or species-specific strate-gies.108,180–182 Based on this develop-ment, Prange and Schaumloffel et al.pioneered the field of sulfur isotope-dilution analysis by combining post-column non-species-specific isotope di-lution and capillary electrophoresis hy-phenated to sector field ICP-MS for thehighly resolved separation of differentmetallothionein (MT) isoforms, whichallowed determination of the metalstoichiometry of the different isoformsas well as their absolute quantifica-tion.183,184 A non-species-specific ele-ment spike containing 34S, 65Cu, 68Zn,and 116Cd introduced via the make-upflow of the CE interface was used foron-line isotope ratio measurement of32S/34S, 63Cu/65Cu, 64Zn/68Zn, and114Cd/116Cd. Reversed isotope dilution,using a standard solution containing S,Cu, Zn, and Cd with natural isotopicabundances, has been used for massflow calibration of the different ele-ments. Based on the known MT-to-sulfur stoichiometry, molar ratios be-tween sulfur and the different metals, aswell as the absolute amounts of thedifferent isoforms were quantified.183,185

This initial study has been furtherimproved by Wang and Prange by usingsurface-modified CE capillaries to ob-tain a better separation of the differentMT isoforms, which can be found in thecommercially available preparations.186

Polec-Pawlak et al. demonstrated theapplication of the CE-ICP-IDSF ap-proach developed by Prange andSchaumloffel for the analysis of metal-lothionein complexes in rat liver ex-tracts. In addition, ESI-MS was used asa complementary technique to furtheridentify the separated protein species.187

A comparable setup has been used byVan Lierde et al. for the quantificationand determination of the stoichiometric


focal point review

Zn/protein ratio for the Aeromonashydrophila (AE036) metallo-beta-lacta-mase.188 However, in this study simplenon-species-specific element standardssuch as albumin (for S) and ZnCl2 havebeen utilized for quantification. Proteindetection limits of 8 ng calculated on thebasis of sulfur were obtained.188

The introduction of collision andreaction cell technology induced aparadigm shift since it became possibleto analyze highly interfered elementssuch as sulfur with quadrupole-basedinstrumentation either when using GC,LC, or CE for sample introduction.

Profrock et al. demonstrated the firstapplication of a quadrupole-based colli-sion/reaction cell ICP-MS with xenon ascell gas to reduce the interferences at themain isotope 32S.95 Xenon as cell gashelped to decrease the spectral back-ground by about six orders of magni-tude, which is a prerequisite for thedetermination of sulfur using its mainisotope 32S. Instrumental detection lim-its of 1.3 ng mL-1 (34S) and 3.2 ngmL-1 (32S) were obtained. The authorsalso demonstrated the sulfur specificdetection and quantification of metallo-thionein isoforms isolated from non-incubated fish samples after on-line CE-ICP-MS, using non-species-specific ele-ment standards.95

To overcome the 16O2þ interference

problem Bandura and co-workers ap-plied quadrupole ICP-MS with a dy-namic reaction cell for the determinationof sulfur by using oxygen as cell gas togenerate 32S16Oþ ions. Due to theoxidation of 32Sþ a mass shift of þ16was generated, allowing the detection ofsulfur at m/z 48, which suffers lessinterference.83

Yeh et al. used this strategy for thedetermination of sulfur-containing ami-no acids by using capillary electropho-resis coupled to dynamic reaction cellICP-MS.189

In general CE-based approaches pio-neered the application of sulfur as anelement tag for absolute protein quanti-fication; however, this technique is stilllimited by the small sample amountsused in CE, the relatively high detectionlimits, and the reduced long-term stabil-ity of most interface systems.

In a very recent paper Wang et al.used normal bore HPLC coupled to

hexapole collision cell ICP-MS for thequantitative analysis of intact proteins(BSA, SOD, MT-II) via their sulfurcontent. Interferences have been mini-mized by using oxygen as cell gas. Post-column isotope dilution analysis hasbeen used to quantify the differentmodel proteins. Detection limits of 8,31, and 15 pmol have been obtained.190

SEC coupled to either ICP-DRCMS(oxygen as cell gas) or ICP-SFMS(medium resolution) has been used byHann et al. for the determination ofmetal sulfur ratios in selected metallo-proteins, such as myoglobin or Mnsuperoxide dismutase.191 Detection lim-its for sulfur of 4.3 lg L-1 wereobtained with the ICP-DRCMS ap-proach in comparison to 14 lg L-1

obtained with ICP-SFMS instrumenta-tion. Non-species-specific calibrantssuch as Fe3þ, Mn2þ, or SO4

2- weretested for quantification; however, interms of measurement uncertainty, thebest results were obtained by usingmetalloprotein standards. Unfortunatelythis application may be restricted to pureor purified protein samples due to thelimited chromatographic resolution ob-tainable with SEC.191

Both LC-ICP-MS and LC-ESI-TOF-MS have been used by the same authorfor the investigation of the metalstoichiometry of native and recombinantcopper proteins derived from the cyano-bacterium Synechocystis. Sulfur wasmeasured as 32S16Oþ using oxygen asthe cell gas. An altered sulfur/metal ratiocaused by the removal of an N-terminalmethionine indicates the heterogeneousexpression of two of the recombinantproteins. Mainly influenced by thechromatographic separation method, de-tection limits for sulfur between 4.6 lgL-1 (SEC-ICP-MS) and 16 lg L-1 (IC-ICP-MS) have been obtained.128

Recently Ellis and colleagues demon-strated the complementary molecule-and element-specific detection of thio-arsenicals by using collision cell ICP-MS and xenon to enable a sulfur-specificdetection. Spiked, NIST, freeze-driedurine was used as sample matrix. Sulfurdetection limits below 0.05 mg kg-1

have been obtained.192

Newly developed molecule complex-es allow the cross-membrane transportof gadolinium complexes, which are

widely used as paramagnetic contrastagents for the imaging of intracellularcompartments. Kruger et al. used acombined approach based on elementaland molecular mass spectrometry for thecharacterization of a gadolinium-taggedmodular contrast agent for MRI.193

SEC-ICP-MS has been used to investi-gate the Gd saturation of the synthetictransporter complex via the measure-ment of the sulfur-to-gadolinium ratio.The contrast agent was further charac-terized by static nano-ESI-QTOF-MS.193

In 2008 Zinn et al. reported thecoupling of capillary LC to high-resolu-tion ICP-MS for the absolute quantifi-cation of intact proteins (humanapolipoprotein A1, a-fetoprotein) viapost-column sulfur isotope dilution anal-ysis.194 Oxygen has been added to thespray chamber to improve the signalstability, due to oxidation of the carbonoriginating mainly from the organicsolvents used. The sulfur blank derivedfrom the instrument itself, the solvent,and the used gases restricted the sensi-tivity of the setup; however, absolutedetection limits for sulfur of 350 pg havebeen realized.

Garijo Anobe and colleagues usedsulfur as an additional natural tag duringthe investigation of different iron-con-taining metalloprotein standards.195

They introduced a new interface com-parable to those described by Bruchertand Bettmer for on-line isotope-dilutionGE-ICP-MS. Iron–sulfur ratios weremeasured to monitor possible iron lossduring the sodium dodecyl sulfate–polyacrylamide gel electrophoresis sep-aration.158,195,196

In summary it is fair to say that mostexamples regarding the absolute quanti-fication of a biomolecule via its sulfurcontent have been carried out withproteins. In consequence, currently onlyprotein samples of low complexity canbe quantified with a sulfur-based ap-proach due to the problems related to thechromatographic separation of intactproteins. In particular, column recover-ies have to be considered for reliablequantification, since this parameterstrongly depends on the chemical prop-erties of the investigated proteins, suchas molecular weight, amino acid com-position, pI, or hydrophobicity.

To overcome this problem, a protein


can also be quantified on the peptidelevel. Recently Schaumloffel et al. usedpre-column isotope dilution analysis andnano-HPLC-ICP-MS for the absolutequantification of sulfur-containing pep-tides.197 In parallel, nano-HPLC-ESI-QTOF-MS has been utilized to identifyand elucidate the sulfur stoichiometry ofthe separated peptides, which is manda-tory for the precise, absolute quantifica-tion based on the natural sulfur tag,especially when working with unknownproteins. As introduced by Profrock etal., they also utilized xenon as cell gas toovercome the sulfur interference prob-lem. Different sulfur-containing pep-tides, as well as tryptic digests ofhuman serum albumin (HSA) and thesalt-induced protein (SIP 18), isolatedfrom selenium-rich yeast, were used asmodel compounds for method develop-ment and validation. A detection limitfor sulfur of 45 lg L-1, which corre-sponds to 1–2 pmol of the individualpeptide species, has been reported.Recovery rates of 103% of the individ-ual peptides and good precision of thequantification with an RSD of 2.1%were obtained.

In summary the selected examplesclearly indicate the variety of possibili-ties that arise due to the quantification ofproteins or peptides using their naturalsulfur tag. Unfortunately the overallnumber of examples that can be foundin the literature is still quite limited.Currently most applications are focusedon the quantification of selected, intact,model proteins.

(Hetero)element-Labeled Com-pounds. Despite the widespread distri-bution of ICP-MS detectable (hetero)elements such as phosphorus or sulfurwithin biomolecules, a multiplicity ofmolecules may still not contain thementioned elements. However, selectedchemical reactions can be utilized toderivatize selected amino acids with theaim to covalently attach a (hetero)ele-ment tag, which makes the moleculedetectable and therefore visible for ICP-MS. Different good reviews are mean-while available, indicating the potentialof this approach.24,27

Early labeling studies used mercuryspecies for the specific labeling of thiolresidues. More recently such approacheshave been used for the successful

quantification of proteins such as insulinor ovalbumin, respectively.28,29

Cartwright et al. described the appli-cation of tris(2,4,6-trimethoxyphenyl)-phosphonium propylamine bromide(TMPP) to label compounds containingcarboxylic acid residues with phospho-rus.198 HPLC hyphenated to high-reso-lution sector field ICP-MS operated atmedium resolution has been used todetect the labeled compounds via theirphosphorus content. Absolute detectionlimits for phosphorus ranging from 1.4to 7.8 ng have been obtained. Theproposed approach helps to make com-pounds detectable by ICP-MS; however,the selected element tag is not ideallysuited due to the well-known limitationsrelated to the detection of phosphorususing ICP-MS. In addition chemicalactivation of the carboxylic acids isrequired, which complicates the overallprocedure.

Unfortunately only 10–20% of thecarboxylic acids were derivatized evenunder optimized reaction conditions,which is insufficient especially whenattempting a real quantitative analysis ofcarboxylic-acid-containing compoundssuch as amino acids in peptides.

More recently Venkatachalam et al.used stable iodine isotopes for thelabeling of antibodies, which specifical-ly targets different members of thecytochrome P450 enzyme family. Laserablation ICP-MS has been used for thedetection of membrane-blotted micro-somal cytochromes via specific, iodinat-ed antibodies.199 A similar approach hasbeen recently used for the absolutequantification of iodinated peptides us-ing capillary LC hyphenated to ICP-MS.32

During the last few years labelingusing lanthanides and bi-functional che-lating agents gained much interest andalso represents the latest trend in ICP-MSbased quantitative analysis.24,27 Theirapplication for absolute protein quantifi-cation,35 multiplexed immunohistochem-ical detection of tumor markers,200

peptide quantification,201 or multiplexedbioassays202 indicate the future potentialof such labeling approaches.

CONCLUSION

The outstanding instrumental devel-opments during the last decade in the

field of elemental mass spectrometry andits application as a (hetero)element-specific detector, as well as the contin-uous progress in the development ofnew hyphenated techniques, has resultedin a growing interest in such technolo-gies and their unique analytical proper-ties. As indicated by the broad range ofexamples ranging from environmentalanalysis of emerging compounds toproteomics related topics, such as pro-tein phosphorylation studies or absoluteprotein quantification, ICP-MS has thepotential to become a key technology forquantification, especially because of itsunique characteristics, such as com-pound-independent ionization behavior,sensitivity, and robustness, which repre-sent the main strengths of ICP-MS.However, it has to be kept in mind thatICP-MS can be used for quantificationonly when suitable standards for cali-bration are available. In the case of acompound-independent calibration,knowledge about the (hetero)elementtag stoichiometry is always mandatory.This clarifies that ICP-MS has to be apart of an integral concept that alsoincludes detection techniques such asESI or MALDI-MS that can provide theessential molecule-specific informationin terms of molecular weight, structure,or (hetero)element composition, whichare necessary for the successful applica-tion of ICP-MS for quantification inboth environmental as well as lifescience research disciplines.

From the above summary of thepublished literature, it can be seen thatthe coupling of GC to ICP-MS has greatpotential for (hetero)element-based ap-plications due to the resulting dryplasma conditions and its robustness.However, the recent improvements inthe hyphenation of capillary and nano-LC to ICP-MS have helped to overcomesome of the well-known problemsrelated to ‘‘normal’’ LC-ICP-MS setups.As recently reviewed, elemental labelingstrategies can help to overcome thelimitations of natural (hetero)elementtags in terms of occurrence and detect-ability.24,27 This technique shows inter-esting potential, especially for the designof accurate multiplexed quantificationschemes as needed for future biomedicalapplications such as high-throughputbiomarker screening and quantification.


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ICP-MS review

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