Volume 57, Number 9, 2003 APPLIED SPECTROSCOPY 11510003-7028 / 03 / 5709-1151$2.00 / 0q 2003 Society for Applied Spectroscopy

Speciation Studies on DTPA Using the ComplementaryNature of Electrospray Ionization Mass Spectrometry andTime-Resolved Laser-Induced Fluorescence

CHRISTOPHE MOULIN,* BADIA AMEKRAZ, VALERIE STEINER,GABRIEL PLANCQUE, and ERIC ANSOBORLOCEA-Saclay, DEN/DPC/SECR/LSRM. 91191 Gif sur Yvette cedex, France (C.M., B.A., V.S., G.P.); and CEA Valrho,DEN/DRCP/CETAMA. 30207 Bagnols sur Ceze, France (E.A.)

Decorporation of radionuclides is of continuous interest in order toreduce doses in case of occupational or accidental human exposure.In the present study, insights into the non-covalent interactions thathold the well-known chelating agent DTPA (diethylenetriaminepen-taacetic acid) with inorganic elements of interest, such as europiumand strontium, and their ability to form stable complexes, are in-vestigated with two spectroscopic techniques, i.e., electrospray ion-ization mass spectrometry (ESI-MS) and time-resolved laser-in-duced � uorescence (TRLIF). First investigations are on DTPA andeuropium alone and end with a complete study of the Eu–DTPAsystem. The pH variation allows one to readily investigate whetherdifferent species (protonated, hydrolyzed, etc.) exist in the pH range2–9 and evaluate the stoichiometry and conditional stability con-stant for the Eu–DTPA complex. Additional experiments by ESI-MS are reported for Sr(II) in interaction with DTPA and EDTA.

Index Headings: Diethylenetriaminepentaacetic acid; DTPA; Elec-trospray mass spectrometry; ESI-MS; Europium; Laser-induced� uorescence; TRLIF; Speciation.

INTRODUCTION

Potential occupational or accidental human exposureto radionuclides is a relevant topic in the nuclear indus-try: in case of inhalation or wound contamination, therapid use of a strong chelating agent is required for de-corporation since complexation has been demonstrated asthe way to enhanced mobility of toxic metals. For de-cades, diethylenetriaminepentaacetic acid (DTPA) hasbeen demonstrated to be ef� cient in actinide Pu and Amdecorporation, poorly ef� cient in the case of contaminationby Th and Co, and inef� cient for U decorporation.1–5 Sincethe thermodynamic stability constants of the ligand werefound to be very high,6,7 this indicates that thermodynam-ics alone is not suf� cient to predict in vivo ef� ciency ofthe DTPA used as decorporant. Stability of the in vivodissociation of the complexes formed by the strongDTPA ligand is crucial in order to avoid the release oftoxic metals. At present, toxicity tests are still understudy, since it is not clear how far this stability dependson factors such as pH, metal-to-ligand ratio, and ex-change reactions with endogenous metal ions that couldcompete for ligand binding sites.1–5

Owing to the wide application found in the use of ami-nocarboxylate derivatives as ligands, signi� cant progresshas been made in the knowledge of their binding withmetal ions. Among spectroscopic methods such as nucle-

Received 21 November 2002; accepted 22 April 2003.* Author to whom correspondence should be sent. E-mail: cmoulin1@

cea.fr.

ar magnetic resonance (NMR), extended X-ray absorp-tion � ne structure (EXAFS), laser-induced photo-acousticspectroscopy (LIPAS), etc., time-resolved laser-induced� uorescence (TRLIF) is well suited to the study of suchinteractions and has been successfully applied for the de-termination of the stability constants of these ligands withlanthanide ions. This technique has the considerable ad-vantage of providing low limits of detection and reliablestability constant determinations, as well as � rst coordi-nation sphere environment through lifetime measure-ments. Different research groups have studied coordina-tion of Eu with DTPA by this powerful technique,8–11 butthese have been limited to � uorescent elements (mainlylanthanides (Ln) and actinides (U, Am, Cm)). On the oth-er hand, electrospray ionization mass spectrometry (ESI-MS)12–15 is complementary to this optical technique andincreases the possibility of obtaining speciation infor-mation since the resulting data provide quantitative andqualitative information on the free element, the organicligand, and the complex. Hence, working with an MSinstrument provides useful information about the chemi-cal form of the ligand and its binding to the metal when-ever it is charged. The viability of ESI-MS as a techniquefor the speciation of thermodynamically and kineticallylabile metal–ligand complexes in solution is at presentwell established. Direct speciation of dissolved metals,16–

20 actinides (U, Th),18,21,22 lanthanides (Eu),23,24 and � ssionproducts25,26 has already been achieved by this technique.Furthermore, ESI-MS has found wide application in thestudy of non-covalent complexes, but there have beenfew attempts to use ESI-MS to obtain information onmetal–polyaminocarboxylate ligand complexes. Hence,to date, most of the studies have dealt with the widelyused complexing agent EDTA (ethylenediaminetetraace-tic acid)27–30 in interaction with metals (Cu, Co, Cd, Fe,Sr, etc.). Surprisingly, no ESI-MS studies exist for metal–DTPA complexes in aqueous media, even though thismolecule is the only one used for decorporation in hu-mans.

In the present study, TRLIF and ESI-MS have beenused to investigate DTPA complexes with europium,which represents a good chemical analogue of trivalentactinides (such as Am and Cm) and is easier to handle.The purpose was, � rst, to examine the stability of theEu–DTPA complexes in wholly aqueous media by ESI-MS, either in the positive or negative mode, as a functionof pH and metal-to-ligand ratio. It was decided to � rstlook at each constituent separately and after that to study

1152 Volume 57, Number 9, 2003

both together. ESI-MS results are compared with thoseobtained by TRLIF, a well-known speciation techniquefor europium, using the same series of Eu–DTPA solu-tions. Additional experiments are reported on Sr(II) incombination with DTPA and EDTA. The results foraqueous Sr–EDTA complexes are in excellent agreementwith reported literature data and tend to con� rm that ESI-MS can provide speciation information regardless of theinstrumentation design.

EXPERIMENTAL

Materials. Standard solutions of DTPA (MW 5 393)and EDTA (MW 5 292) in ultra-pure water with andwithout metal have been prepared from Sigma products.Aqueous solutions of Eu(III) were obtained by dissolvingthe nitrate salt (Aldrich). Solutions of Sr(II) Spex (1 mg/mL) in HNO3 (2% or 5%) have been used. For pH rang-ing from 3 to 9, NH3 (1% or 5%) solutions have beenused. All chemicals used were reagent grade, and Milli-pore water was used throughout the procedure.

Apparatus. Electrospray Mass Spectrometry. The in-strument used to record positive and negative mass spec-tra was a Quattro II tandem mass spectrometer (Micro-mass, Manchester, UK) equipped with an electrosprayionization source. Samples were injected into the appa-ratus with a syringe infusion pump (Harvard apparatusCambridge, MA) delivering a constant � ow rate of 10mL/min. The voltage applied to the steel capillary wasaround 3 kV, and a nitrogen gas � ow rate of 250 L/h wasused to assist the nebulization process. When not varied,the cone voltage was set to 40 V and 20 V in the positiveand negative ion modes, respectively. The ion sourcetemperature was set to 80 8C. In all experiments, a verystable signal (with no discharge) was observed. Massspectra were recorded over a 45–1000 m /z range with ascan duration of 6 s and an acquisition time of 2 min.MS-MS experiments were used for the identi� cation ofcomplexes. Each experiment was carried out three timesto evaluate the reproducibility of the measurements. Formost studies, the signal reproducibility was around 10%.

Time-Resolved Laser-Induced Fluorescence . A Nd–YAG laser (model Minilite, Continuum) operating at 266nm (quadrupled) and delivering about 2 mJ of energy ina 4 ns pulse with a repetition rate of about 15 Hz wasused as the excitation source. The laser beam was di-rected into the 4 mL quartz cell of the spectro� uorometer‘‘FLUO 2001’’ (Dilor, France). The radiation comingfrom the cell was focused on the entrance slit of the po-lychromator (50 cm focal length and 1 mm slit widths).Taking into account dispersion of the holographic grating(300 grooves/mm; blaze, 500 nm) used in the polychro-mator, the measurement range extends to approximately200 nm into the visible spectrum with a resolution of 1nm. The detection was performed by an intensi� ed pho-todiode (1024) array cooled by the Peltier effect (2208C) and positioned at the polychromator exit. Recordingof spectra was performed by integration of the pulsedlight signal given by the intensi� er. The integration time,adjustable from 1 to 99 s, allows for variation in detectionsensitivity. Logic circuits, synchronized with the lasershot, allow the intensi� er to be active with a determined

time delay (from 0.1 to 999 ms) and during a determinedaperture time from 0.5 to 999 ms.

pH Measurement Procedure. The pH of the solutionwas measured with a conventional pH/ISE meter Orion710A equipped with a standard potassium chloride elec-trode. HClO4 and ammonia solutions were used to adjustthe pH.

RESULTS AND DISCUSSION

DTPA (and EDTA) Speciation by ESI-MS. TheDTPA ligand has � ve carboxylic groups and three nitro-gen atoms that can easily be protonated (one central andtwo terminals) as seen in the inset of Fig. 1a. The step-wise protonation constants and stability constants ofDTPA (and EDTA for comparison purposes) for Eu andSr are listed in Table I.6,7 The fractional proton populationfor each basic site has been estimated by NMR studies,allowing determination of the protonation sequence31–33

but still with some controversy. The � rst protonation (pKa

10.5) predominantly occurs on the central nitrogen atomand leads to a protonated form, HL42. The second pro-tonation (pK a 8.6) yields H2L32 species and has been sug-gested to involve one of the two terminal nitrogen atomsto reduce electrostatic repulsion. The third protonation(pKa 4.3) induces multi-hydrogen bond formation be-tween the protons and the other end nitrogen atom andcarboxylate donor sites. The latter � ve protonations areassigned to carboxylic groups (pK a 2.6, 2.0, 1.6, 0.7, and20.1).

Figure 1a presents the speciation diagram of DTPAobtained from the previously quoted data (Table I), whereat pH 0, DTPA is positively charged due to the proton-ation of the nitrogens and is progressively evolving to-wards anionic forms as the � ve carboxylates are pro-gressively activated. The main form between pH 5 to 8is H2L32 (5 carboxylates activated and 2 nitrogens stillprotonated).

The initial experiments were carried out with aqueoussolutions of 1023 M DTPA at pH 6.5 (pH chosen to beclose to the in vivo conditions). Figures 1b and 1c presentthe positive and negative electrospray mass spectra of thisDTPA solution. In the positive mode (Fig. 1b), the pro-tonated DTPA (H 5LH1), i.e., H6L1 (m /z 394), is the dom-inant peak and is accompanied by a smaller peak at m /z416, which is the ubiquitous sodium adduct H5LNa1. Un-der the soft conditions used, the fragmentation productsgenerated in the gas phase are weak and correspond to[H 5L–(C4H7O4N)]1 (m /z 261) with further CO2 elimina-tion leading to the m /z 217 ion. Further experiments atpH values from 3 to 10 have shown that the mass spectrawere dominated by H6L1 at m /z 394. At these pH values,DTPA exists under anionic forms (see Fig. 1a), and onemay assume that protonation occurs in the ES interface34

and that positive ions detected in this mode are not rep-resentative of species found in the solution phase.

Looking at the negative ion spectra (Fig. 1c), the basepeak occurs at m /z 195.4, corresponding to the H3L 22

anionic form. A secondary peak at m /z 392 of about 10%of the base peak intensity corresponds to H4L2. No evi-dence for gas-phase fragmentation of DTPA was ob-served in the mass spectrum. As shown on the speciationdiagram, at pH values from 5 to 8, the main DTPA form

APPLIED SPECTROSCOPY 1153

FIG. 1. (a) DTPA speciation diagram (with data from Table I). (b) Mass spectrum of DTPA (1023 M) in water in the positive ion mode, pH 6.5,cone voltage 40 V, T 5 80 8C. (c) Mass spectrum of DTPA (1023 M) in water in the negative ion mode, pH 6.5, cone voltage 20 V, T 5 80 8C.The inset shows the DTPA formula.

in solution would be H2L32 (m /z 130). This species is notdetected, which seems to indicate that the third proton-ation was particularly favored in the gas phase, so thatthe protonated H3L 22 species becomes the dominant peakin the spectrum. Decreasing the pH value towards 3 leadsto a slight increase of the H4L2 form, but not comparableto the speciation diagram, where only H4L2 and H3L 22

are present. On the other hand, when the pH is raised to9, the spectrum is almost identical to the one obtained atpH 6.5, which is again not representative since this timeHL42 should be the dominant species.

A correlation of these results with thermodynamic pro-

tonation parameters could be performed, since studies32,34

have shown that the third protonation step produces netnegative enthalpy and low positive entropy changes. DGand DS values are re� ective of the fact that the third pro-tonation, as previously shown by NMR studies, inducesmulti-hydrogen bond formation between the protons andthe terminal nitrogen/carboxylate donor sites through theformation of � ve-member rings.

Similar observations by ESI-MS (spectra not shown)were made with experiments carried out on aqueous so-lutions of 1023 M EDTA (taken for comparison purpos-es), which has four carboxylic groups and two nitrogen

1154 Volume 57, Number 9, 2003

TABLE I. Thermodynamic stability constants 6,7 of DTPA andEDTA (L) for ions of interest.

25 8C, 0.1 M

Ion EquilibriumDTPALog K

EDTALog K

H1 HL/H . LH2L/HL . HH3L/H2L . HH4L/H3L . HH5L/H4L . HH6L/H5L . HH7L/H6L . HH8L/H7L . H

10.58.64.32.62.01.60.7

20.1

10.26.12.72.01.50······

Sr21 ML/M . LMHL/ML . H

9.75.4

8.73.9

Eu31 ML/M . LMHL/ML . HM2L/ML . M

22.52.13.1

17.32.6

atoms (pKa values listed in Table I). Thermodynamicstudies indicate that, as expected, the protonation se-quence of the EDTA ligand is similar to DTPA. Again,the peak at [H 2L]22 m /z 145 remains the base peak evenat high pH values, and the intensity of the peak at m /z291 of the [H 3L]2 form, obtained by the third protonation,is around 30% of that of the base peak.

Europium Inorganic Speciation by TRLIF and ESI-MS. As previously mentioned, TRLIF is a very sensitiveand selective method for Eu studies in solution.8–11,35–39

Hence, europium possesses a characteristic � uorescencespectrum in the red, with speci� c peaks around 580, 593,618, 650, and 700 nm. In complexation studies, the main� uorescence wavelengths of interest are the one at 593nm (magnetic dipole transition) and the hypersensitiveone at 618 nm (electronic dipole transition), correspond-ing to the 5D0 ® 7F1 and 5D0 ® 7F2 transitions, respec-tively. The transition at 618 nm is particularly useful forthe characterization of the Eu complex, since its intensityis enhanced, leading to a modi� cation of the 618/593 nmratio. Moreover, � uorescence lifetime measurements giveinformation on the hydration number or the number ofwater molecules left around europium by using the fol-lowing formula:38 n 5 (1.05/t) 2 0.44.H O2

The aqueous speciation of Eu is mainly dominated (inthe absence of speci� c ligands) by carbonate and hydroxospecies, as seen Fig. 2a, which presents the speciationdiagram of Eu in water at 15 mg/L (1024 M).40 From thisdiagram, it can be seen that four different species existin aqueous solution, ranging from free aquo Eu31 at acid-ic pH to the different carbonate (coming from equilibriumwith air) complexes, Eu(CO 3)1 , Eu(CO 3)2

2 , andEu(CO3)3

32, at basic pH (the hydroxo species being veryminor). This time, the pH value and Eu concentrationwere chosen in order to get close to the biological pHwhile avoiding europium precipitation. The � rst hydro-lysis constant of Eu and the solubility product of hydro-lyzed Eu are 1026.1 and 10226.6, respectively,40 and there-fore, a signi� cant amount of Eu would hydrolyze andprecipitate at pH values around 6 since a Eu concentra-tion of 1024 M is used in order to obtain suf� cient signalin ESI-MS. Therefore, the pH was � xed at 5, and ac-cording to the speciation diagram, one may expect thatonly aqueous europium (Eu III(H 2O)n, n 5 8–9) exists insolution.

Figure 2b shows the TRLIF spectrum of a 1024 M Eusolution at pH 5, which is, as expected (Fig. 2a), char-acteristic of free europium, with the peak at 618 nm muchlower than the one at 593 nm (R(618/593) 5 0.3)41 anda narrow full width at mid-height (FWMH) of 7 nm. Themeasured europium � uorescence lifetime of 110 ms cor-responds (using the previously quoted equation) to thepresence of nine water molecules as expected 38 aroundeuropium.

Figure 2c shows the ESI-MS positive ion spectrum forthe same solution (1024 M Eu, pH 5), where a series ofpeaks in the range m /z 75–350 are present; most of themexhibit an isotope ratio that indicates the presence of Eu(48% 151Eu, 52% 153Eu). It should be mentioned that nospeci� c peaks were observed below 75 Da (for examplerelative to Eu31 around 51 Da). Assignments made forthe main observed species have been summarized in Ta-ble II. The principal ion observed is [Eu III(OH)2]1 at 185–187 m /z and the other ions belong to the two main fam-ilies [Eu III(OH)(H 2O)n]21 and [Eu III(OH)2(H 2O)n]1. Theseresults can be compared with previous studies on lantha-nides in various media by ESI–MS,23 where ions of type[Ln(OCH 3)(CH 3OH)n] 21 and [Ln(OCH 3)2(CH 3OH)n]1

(with n 5 2 to 10) in methanol and [Ln(CH3CN)x]31 inacetonitrile were also observed under gentle conditions.In this previous work, these differences were explainednot by solvent dielectric constant (« ; 36, «MeOH ;CH CN3

33), but by their difference in donicity (d ; 14, dMeOHCH CN3

; 19), leading to greater inner sphere coordination andto an overall ‘‘protection’’ of charge in the case of ace-tonitrile. In this study, the trend observed in water («H O2

; 80, d ; 33) is, as expected, very close to the oneH O2

observed in methanol. These results illustrate that lantha-nide ions behave as hard acids in the Pearson classi� ca-tion and bind most strongly to hard bases such as oxygen(HSAB theory); thereby, lanthanide–oxygen bonds arereadily observed as for the minor species [EuO]1 detectedat m /z 167–169.

Higher adducts containing up to nine water molecules(m /z 347–349) have also been observed but with verylow abundance (,5%). The peaks at m /z 75.5 Eu 21 (verylow level, not shown) and m /z 168–170 (also very minor)correspond to the [Eu II(OH)]1 ion, evidencing Eu III re-duction. In fact, the minor presence of these ions, char-acteristic of charge reduction reaction at the ESI inter-face, could be attributed to the low europium E3/2 redoxpotential value (E3/2 5 20.35 V),41 so a very small partof the solvated ions are likely converted to charged re-duced species by electrochemical reduction. Not surpris-ingly, the charge reduction process is enhanced at highcone voltage ($30 V). It has been shown that the ESionization process may yield charge balancing redox re-actions.43 In our experiments, special care has been takento overcome these dif� culties: a high � ow rate (10 mL/min) and low cone voltage were chosen, and a stainlesssteel 44 capillary was used to limit the charge reductionprocess.

This brief study is in agreement with previously ob-tained results in another solvent by ESI-MS 23 and showsthat under these gentle conditions, the proper oxidationstate of europium is conserved but that due to the desol-vation process, expected europium speciation in solution(Fig. 2a) is not observed. After studying each constituent

APPLIED SPECTROSCOPY 1155

FIG. 2. (a) Europium (1024 M) speciation diagram,40 P atm. (b) TRLIF spectrum of Eu (1024 M), pH 5. (c) Eu (1024 M) mass spectrum in theCO2

positive ion mode, pH 5, cone voltage 30 V, T 5 80 8C.

1156 Volume 57, Number 9, 2003

TABLE II. Mass assignment of europium positive ions in aqueoussolution (1024 M, pH 5, cone voltage 30 V); main peak observed isin bold.

m/z Eu speciesa

111–112120–121129–130

···168–170185–187203–205221–223

[EuIII(OH)(H2O)3]21

[EuIII(OH)(H2O)4]21

[EuIII(OH)(H2O)5]21

···[EuII(OH)]1

[EuIII(OH)2]1

[EuIII(OH)2(H2O)]1

[EuIII(OH)2(H2O)2]1

a Only species that are superior to 5% are mentioned.

(DTPA and Eu), it was decided to investigate the systemof interest, i.e., the complex between europium andDTPA, by both spectroscopic methods in order to reachconclusions on the potentiality of ESI-MS.

Eu–DTPA Speciation by TRLIF and ESI-MS. Forthe study of the system europium–DTPA, we decided towork with equal concentrations of both europium andDTPA in order to � rst identify, by the two spectroscopictechniques, all complexes using the data previously ob-tained for single elements. Secondly, the in� uence of pHhas been investigated and the DTPA concentration hasbeen varied to con� rm the stoichiometry and � nally reachthe complexation constant.

Identi� cation. A typical example of a TRLIF spectrumobtained at the stoichiometry (Eu 1024 M/DTPA 1024 M,pH 6.5) is presented in Fig. 3a. Complexation betweeneuropium and DTPA leads to a modi� cation of the � uo-rescence spectrum (as compared to the normalized freeeuropium spectrum added for comparison purposes) sincethe ratio 618/593 nm is now equal to 1 with the appari-tion of the band at 580 nm traducing a dissymmetricalenvironment for europium. Moreover, the two main peaksare split, with the peak at 593 nm giving rise to two peaksat 591 and 595 nm and the peak at 618 nm to two peaksat 616 and 622 nm, emphasizing this modi� cation of en-vironment. A single � uorescence lifetime of 550 ms wasobtained, indicating that one water molecule is left. Thisvarying information obtained by TRLIF shows the pres-ence of one single complex between europium andDTPA. It is well known, either experimentally by NMR9

or by modeling,45,46 that the DTPA forms a sort of cagemolecule where the europium, close to the oxygen andnitrogen, stands together with one water molecule. It isinteresting to compare these results with europium in car-bonate medium where, for the [Eu(CO3)3]32 complex, theratio is much stronger (618/593 nm $ 4), again withsplitting of the peaks but with a lifetime close to 450 ms,leaving two water molecules this time.41 These differenc-es can be directly related to a different environment foreuropium, i.e., a close shell arrangement for [Eu(CO3)3]32

with log K 5 13 and a cage arrangement for the Eu–DTPA with log K 5 22.5.

Spectra obtained by ESI-MS for the positive and neg-ative ion modes, respectively, are quite simple (Figs. 3band 3c) and are dominated as expected by Eu–DTPAcomplexes. It is important to remember that concerningthe formation of this complex (especially at neutral pH),the forward reaction is expected to be very fast while thereverse one is likely to be very slow compared to the

estimated lifetime of the droplets (1023 s). This has al-ready been observed for a similar system 29 (Sr–EDTA:k f 109 M21 s21, k r 1.6 s21) and should allow quantitativeidenti� cation of the complexes representative of the ther-modynamic equilibrium within the droplets.

In the case of the positive ion spectrum (Fig. 3b), themain peaks correspond to EuH3L1 complexes at m /z 542–544, which clearly illustrates that Eu–DTPA complexespresent in solution as anions are detected as protonatedspecies in the positive ion mode. No free europium oruncomplexed [Eu(OH)2]1 at m /z 185–187 is observed,but surprisingly, the uncomplexed DTPA [H5LH]1 at m /z394 is still present despite the fact that DTPA is knownto form very stable complexes (log K 5 22.5) with Euover a wide pH range. It is possible that the acidic con-ditions inherent in the ES positive ion mode favor theproton-assisted dissociation of Eu–DTPA complexes inthe droplets. Smaller peaks at m /z 580–582 of about 5%of the base peak intensities are observable and corre-spond to the hydrated species [EuH3L(H 2O)]1.

The negative ion spectrum (Fig. 3c) shows the metal–ligand complexes [ML]22 at m /z 269.5–270.5 and almostremoves the uncomplexed DTPA stable anionic form[H 3L]22 at m /z 195.7. Once again, the hydrated complex-es [ML(H 2O)] 22 at m /z 278.5–279.5 are only minor peakswith intensities about 5% of the base peak intensities.Looking at the higher mass, small peaks at m /z 540–542are detected in the spectrum and correspond to the pro-tonated complexes [MHL]2 with a single negative charge.These preliminary experiments, carried out with unmod-i� ed equipment, enabled us to choose the best experi-mental conditions (concentration and mass spectrometeroperating settings) to perform data acquisitions and showthat the negative mode ES can provide useful informationabout chemical forms of the metal–DTPA complexes in100% aqueous solution. The following experiment wasperformed in this mode.

In� uence of pH on Complexation Investigated byTRLIF and ESI-MS. In order to be sure that all euro-pium was complexed (assuming a 1–1 complex) and toavoid europium precipitation when working at high pHvalues, it was decided to work with an excess of DTPArelative to Eu. Figure 4a presents the different europium� uorescent spectra (normalized for comparison purposes)obtained for solutions of 5 3 1025 M Eu with 1024 MDTPA, at pH values of 2.1, 2.3, 2.6, 2.9, and 7.3. Specialattention was directed toward pH 2.6 since, as previouslymentioned, the carboxylic sites of the DTPA will be ac-tivated in this pH region (Fig. 1a). As the pH rises, aprogressive increase of the peak (618 nm) at the hyper-sensitive transition is observed, together with the splittingof both peaks, the apparition of the peak at 580 nm tra-ducing complexation, and, as previously mentioned, adissymmetrical environment for europium. The 618/593nm ratio evolved from 0.8 at pH 2.1, to 1.7 for pH 2.3,and � nally to 2 above pH 2.9, as seen in the inset of Fig.4. The same pattern is obtained by plotting the intensityat 580 nm versus pH. This means that the complexationis total above pH 3 since all carboxylic sites are activated(see Fig. 1a), which is in agreement with previous lu-minescence studies.8,47 Above this pH value, all spectraare nearly identical except for the splitting. Hence, thehypsochromic peaks (595 and 622 nm) increase with pH

APPLIED SPECTROSCOPY 1157

FIG. 3. (a) TRLIF spectrum of Eu (1024 M)/DTPA (1024 M), pH 6.5, compared to the free europium spectrum at pH 2 (time delay 10 ms, gatewidth 150 ms, E laser 2 mJ, integration time 1 s, and 10 accumulations); (b) mass spectrum of DTPA 1024 M/Eu 1024 M in the positive ion mode,cone voltage 30 V, T 5 80 8C; (c) mass spectrum of DTPA 1024 M/Eu 1024 M in the negative ion mode, cone voltage 20 V, T 5 80 8C.

relative to the bathochromic peaks (591 and 616 nm), andthis trend is under study using deconvolution procedures.

Concerning the � uorescence lifetime measurementsand associated hydration numbers38 (n 5 (1.05/t 2H O2

0.44)), the initial lifetime is equal to 110 ms (9 watermolecules) at pH 2, increases to 250 ms (4 water mole-cules left) for pH 2.3, then to 450 ms (2 water moleculesleft) for pH 2.6, and � nally the lifetime is constant around550 ms (1 water molecule left) for pH values above 3.Therefore, the Eu–DTPA complex can be described insolution as a nine-member structure, since the three-amine nitrogen and the � ve carboxylate groups of theDTPA ligand would occupy eight Eu coordination sites(the last one being occupied by the leftover water mol-

ecule). This is in agreement with luminescence studies onEu–DTPA complexes48,49 and modeling.45,46

Concerning ESI-MS experiments (in the negativemode), the main peak observed above pH 4 is the Eu–DTPA complex [EuL]22 at m /z 270 as already observedin the identi� cation section. Between pH 2.1 and 2.6, thepresence of free DTPA is observed, as well as free eu-ropium. Monitoring the ion abundance of [EuL]22 as afunction of pH allows us to con� rm the quantitativity ofthis complexation, as shown in the inset of Fig. 4. It isinteresting to observe that the sharp break occurs abovepH 3 where, as expected, the � nal complex [EuL]22 isformed, which is in good agreement with DTPA specia-tion (Fig. 1a).

1158 Volume 57, Number 9, 2003

FIG. 4. TRLIF spectra of Eu (5 3 1025 M)/DTPA (1024 M) solution; pH was varied from 2.1 to 8.4 (time delay 10 ms, gate width 150 ms, E laser

2 mJ, integration time 1 s, and 10 accumulations). (Inset) Evolution of I618 nm /I595 nm in TRLIF and variation of the [Eu/DTPA]22 complex intensitymeasured by ESI-MS vs. pH (cone voltage 25 V, T 5 80 8C).

Moreover, the difference observed between the twocurves (one pH unit) in TRLIF and ESI-MS is totally dueto phenomena that are taking place. The prior europium� uorescence intensity increases with pH and traduces theinteraction of europium with the sequentially activatedcarboxylate groups associated with the progressive elim-ination of the eight water molecules (due to complexa-tion) around europium, as observed with the lifetimemodi� cations, while in ESI-MS, the EuL 22 signal is onlymonitored when the complex is formed. Hence, takinginto account the DTPA pK a (0.1, 0.7, 1.6, 2.0, and 2.6)at pH 1.6, 50% of the three DTPA carboxylates are al-ready activated to interact with Eu(III) to form the neutralEu–DTPA complex that will be monitored by the changein the 618/593 nm ratio. On the other hand, the pH valuemust be 2.6 to have 50% of the � ve DTPA carboxylatesactivated to form the charged Eu–DTPA 22 complex.

It should also be noted that whatever the pH (espe-cially neutral or basic), no hydrolyzed species such as[Eu(OH)L]2 or carbonate species are observed, con� rm-ing that the Eu–DTPA complex is strong enough to avoidcompetition phenomena. This feature will be particularlyuseful for metals whose chemical forms change with pH,especially in in vivo experiments. In conclusion, resultsobtained by both spectroscopic techniques (TRLIF andESI-MS) are in good agreement with one another, as wellas with solution chemistry. This result is very importantsince it con� rms several speciation studies that show thatthe ESI process is likely to be suf� ciently gentle so thatthe fundamental information concerning the solutionphase composition should be maintained in the gas phase.

Titration of Eu with DTPA at Constant pH. Monitor-ing metal ion complexation vs. ligand concentration read-ily establishes the stoichiometry composition of the Eu–

DTPA complexes at a given pH. Solutions at pH 6.5 wereprepared containing a 1024 M solution of Eu with DTPAconcentration ranging from 1025 M to 1023 M. ForTRLIF, intensity of the peak at 618 nm was directly re-corded, while for ESI-MS, the ion abundances of the Eu–DTPA complexes were monitored in the mass spectrumobtained with each solution. Looking at the ESI-MSspectra, again, the 1:1 complexes are observed at m /z268–270 [EuL]22 and m /z 540–542 [EuHL]2. The resultsare plotted as a function of the DTPA/Eu ratio in Fig. 5.Both titration curves obtained show a strong binding witha sharp break in the titration curve at a 1:1 stoichiometry.Above equimolar ratio, the � uorescence peak intensityand the [Eu–DTPA] 22 remain constant, indicating that thecomplexation is total. Furthermore, as the uncomplexedDTPA species becomes more important, the appearanceof [H 5L 1 H4L]2 dimer ions can be noticed, and no peaksin the mass spectra that would indicate the formation ofclusters with more than one metal ion per DTPA mole-cule are present. This indicates that a single 1:1 compo-sition for the Eu–DTPA complex exists in solution phase,whatever the ligand concentration and the pH (above 3).These results are in agreement with the reported stoichi-ometry for europium–DTPA.8,9 It was also veri� ed thatthe Eu–DTPA ion abundance was linear within the con-centration range studied, with a detection limit (3s) closeto 4 3 1027 M. However, it should be noted that signalsaturation (with no impact on our study) occurs in theupper concentrations, showing that great care should betaken during quantitative determination by ESI-MS, butthis could be overcome by the use of internal standards,as shown in previous studies.16,50

Concerning the TRLIF data, the strong slope between0 and 1 [DTPA]/[Eu] ratios cannot be exploited for the

APPLIED SPECTROSCOPY 1159

FIG. 5. Evolution of the Eu–DTPA complexes by TRLIF ( M : I593 nm) and ESI-MS ( m : Im /z268–270) as a function of the [DTPA]/[Eu] ratio. [Eu] 1024

M, pH 6.5. Operating conditions are as for Fig. 4.

europium–DTPA complexation constant determinationsince the equilibrium is totally displaced, as already seenat this neutral pH when taking into consideration theDTPA pKa values (0.1, 0.7, 1.6, 2.0, and 2.6). However,as mentioned and described by Horrocks et al.,10 workingat lower pH values leads to a titration curve (If vs. [Eu])with some curvature that can be analyzed. Hence, thistime, hydrogen ions compete with europium for the li-gand, and by knowing the different pKa values, it is pos-sible to reach the complexation constant. TRLIF titrationcurves (not shown) at pH 2.5 and 3 (I 5 0.1 M) haveallowed us, by using nonlinear regression treatment, toreach a complexation constant of 21.2 6 0.5, in goodagreement with literature data.7

Sr(II), Eu(III)/DTPA, and EDTA Speciation. TheSr–EDTA system was chosen to determine whether thespeciation information is disguised by the instrumentaldesign characteristics, since this system was successfullystudied by Agnes et al.,29,30 as well as to compare thedifferences in behavior between a divalent (Sr) and a tri-valen t (Eu) towards polyaminocarboxylate ligands(EDTA, DTPA), knowing the thermodynamic stabilityconstants (Table I). Furthermore, comparing Sr–EDTAvs. Sr–DTPA complexation provides insights into thestructural effects of the increasing number of carboxylatearms.

Electrospray ionization mass spectromety experimentshave been performed in the negative ion mode with aque-ous solutions of 5 3 1025 M alkaline earth metal ion Sr21

and 1024 M each of EDTA and DTPA, from pH 2 to 9.The resulting ESI-MS spectra for each Sr–DTPA solutionare not shown, but the observations are almost identicalwith those obtained for the Eu–DTPA solutions. TheEDTA and DTPA complexes with Sr were present en-tirely as 1–1 metal–ligand complexes. For the Sr–EDTAsystem, the major complex above pH 4 is [SrL]22 at m /z188, while the protonated form [SrHL]2 at m /z 377 ap-peared at low levels. For the Sr–DTPA system, the majorcomplex is [SrHL]22 at m /z 238, and the other complexbarely observed is [SrH 2L]2 at m /z 478.

By using the different equations for the interaction be-tween the metal M (Sr or Eu) and the ligand L (EDTAor DTPA), as well as the stepwise protonation constant(Km

H ) of the ligand L with m 5 1 to 6 for EDTA and m5 1 to 8 for DTPA, it is possible to express the differentconcentrations of Sr–EDTA, Sr–DTPA, and Eu–DTPA asa function of pH with the different data given in Table I.

M 1 L . ML

K 5 [ML]/[M][L]ML

M 1 HL . MHL

K 5 [M][HL]/[MHL]MHL

(n2m)2 1 (n2 (m11))2H L 1 H . H Lm m11

H (n2m11))2 (n2m)2 1K 5 [H L ] /[H L ][H ]m11 m11 m

Figure 6a shows the theoretical evolution of the Sr–EDTA 22 complex (from previously quoted equations andthe data of Table I), together with experimental data ob-tained by ESI-MS in this study and the data obtained byAgnes et al.29,30 (brought back to the same scale andshown for comparison purposes). Several commentsshould be made: � rst, the good agreement between thetwo sets of experiments in ESI-MS demonstrates that dif-ferent instrumental con� gurations (source design, use ofO2 � ow to minimize electrical discharge, etc.) could worksimilarly for kinetically labile species detection; second,and more importantly, these ions detected in gas phaseby ESI-MS are very close (less than 0.3 pH units) to thetheoretical solution-phase concentration. It should be not-ed that the shift in pH is in the reverse direction, as ex-pected, since in the negative mode, the droplet is likelyto be enriched in OH2, leading to a pH increase. On theother hand, for the Sr–DTPA system (as seen later), theshift is in the expected direction and shows that the elec-trolytic shift mentioned in ESI-MS can be considered aswithin the experimental error for these systems.

It is interesting to note that it was previously shownby NMR on similar systems (Ln–DOTA and Ln–DTPA–

1160 Volume 57, Number 9, 2003

FIG. 6. (a) Evolution of the [Sr, EDTA]22 ( m ), [Sr, EDTA]22 ( l ) ions,29 and theoretical concentration (plain curve) with pH. (b) Evolution of the[Eu, DTPA]22 ( m ), [Sr, DTPA]22 (3) ions, and theoretical concentrations (plain curves) with pH. [Sr] 5 [Eu] 5 5 3 1025 M, [EDTA] 5 [DTPA]5 1024 M.

BMA) for extracellular pH in vivo measurements that thewater exchange rate was slow for the early members ofthe Ln series, including europium, and then increaseswith Ln size.51,52 It is also known in the framework ofESI-MS investigations that metals with low water ex-change rates will not undergo extensive change in speciesdistribution in the evaporating droplet prior to desorp-tion.30

This differing information is certainly a key point tothe proper agreement between solution chemistry andESI-MS results, as shown in Fig. 6b, which presents thetheoretical evolutions (from chemical equations usingvalues of Table I) of Eu–DTPA 22, Sr–DTPA 22, and Sr–DTPA32 complexes, together with experimental data

points obtained by ESI-MS. Again, an appropriate matchis obtained in the case of Eu–DTPA between ions de-tected in the gas phase and the theoretical solution-phaseconcentration (less than 0.5 pH units).

Concerning the Sr–DTPA system, the main complexobserved by ESI-MS (i.e., [Sr–DTPA]22) appears at theexpected pH (Fig. 6b, around 5) but is not the expectedone in solution (i.e., [Sr–DTPA]32). This is not totallysurprising, since proton addition at the non-coordinatedbinding sites of the additional amino carboxylate arm inDTPA vs. EDTA is likely to be favored in the gas phase.It is thus reasonable to assume that the measured ionabundance of [Sr–DTPA]22 complexes is likely to comefrom the contribution of both the [Sr–DTPA]32 complex

APPLIED SPECTROSCOPY 1161

that becomes protonated in the gas phase and the [Sr–DTPA] 22 complex found in solution phase. This lastpoint con� rms the importance of knowing both the so-lution- and gas-phase behaviors for proper interpretationof ESI-MS data and that each system under investigationis speci� c.

It is also important to note that this speciation studyon Sr–Eu/polycarboxylates by ESI-MS has investigateda very large pH range (from 2 to 9), with nearly no orvery little expected electrolytic effect. The proper agree-ment between most ions detected in gas phase and chem-ical equilibria con� rm the capacity of ESI-MS for spe-ciation studies.

CONCLUSION

This study has shown � rst that the two different spec-troscopic techniques are complementary in tackling theproblem of speciation in the particular case of DTPA,which is a strong chelating agent. Concerning europiuminorganic speciation, TRLIF is more appropriate thanESI-MS, which gives rise to results certainly biased bythe desolvation process. Concerning Eu–DTPA specia-tion, results obtained by both techniques are consistentwith known thermodynamic data (1–1 complex, largecomplexing constant), as well as with solution-phasechemical equilibria. Such comparative studies are of greatinterest to show the capabilities of ESI-MS for furtherspeciation studies on non-� uorescent elements or in com-plex matrices. Even though results obtained by ESI-MSshould always be analyzed with care and related toknown chemistry and, whenever possible, be con� rmedby other spectroscopic data, the striking results obtainedon the DTPA–Eu systems con� rm the potential of ESI-MS as a speciation technique. Further experiments are inprogress for monitoring the evolution of such systems inbiological media.

ACKNOWLEDGMENTS

The authors would like to thank the CEA-HC project (98T12), theNuclear Toxicology program (DSV), DOB/DDIN (Risk Assessment),and DSOE (Research and Development) for funding. The authors wouldalso like to thank Dr. V. Moulin for helpful comments.

1. D. M. Taylor, G. N. Stradling, and M.-H. Henge-Napoli, Radiat.Prot. Dosim. 87, 11 (2000).

2. G. N. Stradling, M.-H. Henge-Napoli, F. Paquet, J. L. Poncy, P.Fritsch, and D. M. Taylor, Radiat. Prot. Dosim. 87, 19 (2000).

3. G. N. Stradling, M.-H. Henge-Napoli, F. Paquet, J. L. Poncy, P.Fritsch, and D. M. Taylor, Radiat. Prot. Dosim. 87, 29 (2000).

4. G. N. Stradling, D. M. Taylor, M.-H. Henge-Napoli, R. Wood, andT. J. Silk, Radiat. Prot. Dosim. 87, 41 (2000).

5. R. Wood, C. Sharp, P. Gourmelon, B. Le Guen, G. N. Stradling, D.M. Taylor, and M.-H. Henge-Napoli, Radiat. Prot. Dosim. 87, 51(2000).

6. L. G. Sillen, A. E. Martell, and J. Bjerrum, in Stability Constantsof Metal-Ion Complexes, sup. 1 (Chemical Society, London, 1971).

7. R. M. Smith, A. E. Martell, and R. J. Motekaitis, NIST CriticalStability Constants of Metal Complexes Database (U.S. Dept. ofCommerce, Washington, D.C., September 1993).

8. S. L. Wu and W. D. Horrocks, Jr., Anal. Chem. 68, 394 (1996).9. E. N. Rizkalla, G. Choppin, and W. Cacheris, Inorg. Chem. 32, 582

(1993).

10. S. L. Wu and W. D. Horrocks, Jr., J. Chem. Soc., Dalton Trans.1497 (1997).

11. C. Granberg, K. Blomberg, I. Hemmila, and T. Lovgren, J. Im-munol. Methods 114, 191 (1988).

12. M. Yamashita and J. B. Fenn, J. Phys. Chem. 88, 4451 (1984).13. M. Dole, L. L. Mach, L. Hines, R. C. Mobley, L. P. Fergusson, and

M. B. Alice, J. Chem. Phys. 49, 2240 (1968).14. B. A. Thomson and J. V. Iribarne, J. Chem. Phys. 71, 4451 (1979).15. P. Kebarle, Int. J. Mass Spectrom. 200, 313 (2000).16. I. I. Stewart, D. A. Barnett, and G. Horlick, J. Anal. At. Spectrom.

11, 877 (1996).17. A. R. S. Ross, M. G. Ikonomou, K. J. Orians, and J. J. A. Thomp-

son, Anal. Chem. 70, 2225 (1998).18. G. R. Agnes and G. Horlick, Appl. Spectrosc. 46, 401 (1992).19. R. Colton, A. D’Agostino, and J. C. Traeger, Mass Spectrom. Rev.

14, 79 (1995).20. C. C. Raymond, D. L. Dick, and P. K. Dorhout, Inorg. Chem. 36,

2678 (1997).21. C. Moulin, N. Charron, G. Plancque, and H. Virelizier, Appl. Spec-

trosc. 54, 843 (2000).22. C. Moulin, B. Amekraz, S. Hubert, and V. Moulin, Anal. Chim.

Acta 441, 269 (2001).23. I. Stewart and G. Horlick, Anal. Chem. 66, 3983 (1994).24. S. Colette, B. Amekraz, C. Madic, L. Berthon, G. Cote, and C.

Moulin, Inorg. Chem. 41, 7031 (2002).25. F. Allain, H. Virelizier, C. Moulin, C. Jankowski, J. F. Dozol, and

J. C. Tabet, Spectroscopy 14, 127 (2000).26. C. Lamouroux, C. Moulin, J. C. Tabet, and C. Jankowski, Rapid

Commun. Mass Spectrom. 14, 1869 (2000).27. B. L. Shrap, A. B. Sulaiman, K. A. Taylor, and B. N. Green, J.

Anal. At. Spectrom. 12, 603 (1997).28. D. Baron and J. G. Hering, J. Environ. Qual. 27, 844 (1998).29. H. Wang and G. R. Agnes, Anal. Chem. 71, 3785 (1999).30. H. Wang and G. R. Agnes, Anal. Chem. 71, 4166 (1999).31. J. L. Sudmeier and C. N. Reilley, Anal. Chem. 36, 1698 (1964).32. H. Lammers, A. M. Van der Heidjen, H. Van Bekkum, C. F. Ger-

aldes, and J. A. Peters, Inorg. Chim. Acta 277, 193 (1998).33. T. J. Manning and E. D. Gravley, Spectrosc. Lett. 28, 291 (1995).34. G. Wang and R. Cole, in Electrospray Ionization Mass Spectrom-

etry, R. B. Cole, Ed. (John Wiley and Sons, New York, 1997),Chap. 4.

35. T. Kimura, G. R. Choppin, W. P. Cacheris, L. A. de Laerie, T. J.Dunn, and D. H. White, Inorg. Chem. Acta 258, 227 (1997).

36. S. Lis, J. Konarski, Z. Hnatejko, and M. Elbanowski, J. Photochem.Photobiol., A 79, 25 (1994).

37. E. Soini and T. Lovgren, Crit. Rev. Anal. Chem. 18, 109 (1987).38. T. Kimura and Y. Kato, J. Alloys Compd. 275, 806 (1998).39. C. Moulin, J. Wei, P. Van Iseghem, I. Laszak, G. Plancque, and V.

Moulin, Anal. Chim. Acta 396, 253 (1999).40. J. Van der Lee, CHESS, Another Speciation and Surface Complex-

ation Computer Code, Technical report, LHM/RD/93-39 (1994).41. G. Plancque, V. Moulin, P. Toulhoat, and C. Moulin, Anal. Chim.

Acta 478, 11 (2003).42. L. J. Nugent, R. B. Bayrbaz, J. L. Burnett, and J. L. Ryan, J. Phys.

Chem. 77, 1528 (1973).43. G. J. Van Berkel, F. Zhou, and J. T. Aronson, Int. J. Mass Spectrom.

Ion Proc. 162, 55 (1997).44. P. Kebarle and L. Tang, Anal. Chem. 65, 972 (1993).45. A. Borel, L. Helm, and A. Merbach, Chem. Eur. J. 3, 600 (2001).46. J. L. Fleche, G. Plancque, and C. Moulin, Chem. Eur. J., paper to

be published (2003).47. H. G. Brittain, G. R. Choppin, and P. Barthelemy, J. Coord. Chem.

26, 143 (1992).48. S. L. Wu, S. J. Franklin, K. N. Raymond, and W. D. Horrocks, Jr.,

Inorg. Chem. 35, 162 (1996).49. E. Bovens, M. A. Hoefnagel, E. Boers, H. Lammers, H. van Bek-

kum, and J. A. Peters, Inorg. Chem. 35, 7679 (1996).50. G. R. Agnes and G. Horlick, Appl. Spectrosc. 48, 649 (1994).51. D. Pubanz, G. Gonzaloz, D. H. Powell, and A. E. Merbach, Inorg.

Chem. 34, 4447 (1995).52. S. Zhang, K. Wu, and A. D. Sherry, J. Am. Chem. Soc. 124, 4226

(2002).