^a^-nioar
Quantitation of perchlorate ion by electrospray ionization mass
spectrometry . (ESI-MS) using ^stable assbcialbn Complexes with
organic cations and bases to i enhance ^ selectivityt =- r
Edward T. Urbarisky,* Matthew L.Magnuspn? David Freemari arid Christopher Jelks : '
United States Environmental Protection Agency (EPA), Office bf Research : and Development - ' ' :;
National Risk Management Research Lahoratnrv Wntpr Simnh, n^A^^ta^ pjan,~Zii« '."':'' -""
^
V- ^'Analytical "";.",.
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,
Division '..'Treatment Technology Valuation 'Branch, 'Cincinnati
E-mail: urbansky.edward@epamail.epa.goy; magnuson.matthew@epamaiiepa.goy,
Received 14th July 1999, Accepted 28th September 1999
' ''--'' -'.'.--,'".. "i-i*-:." , ./. '«/ '.; 'r' """ J. '! -, ' :~r.^ " .* - ;'- "' : . " j f '
Quantitation of trace levels of perchlorate ion m^watef has become a key issue since this species was discovered
in water supplies around the United States. Although ion chromatographic methods presently offer the lowest ~
limit.pf detection; ^40, nk (4 rig ml ,1), c,hromatographic; retention times-are not considered to be unique
identifiers and.often cannot be used in legal-proceedings without confirmatory-testing. Mass spectromefery- can '"
provide such confirmation; However, detection capabilities can impbse a practical hmitatiori 6il;its^se;::' --' *
Moreover, quadrupole mass spectrometers 'cannot provide sufficient i accurac/ and precision ^ in w/z4o Identify" ' ' '
conclusively an ion as perchlofate! when samples are run directly without prior chromatographic or ' ' ;" "" '
electrophoretic separation: We report on the 'abilities of (1) tetralkylatnmoniuni cations an4 (2) minimally" ' : " " "
nucleophilic, sterica'lly hindered organic bases to increase selectivity iii the electrpspray ionization mass "'-"'""' ? ':>>>
spectrometric (ESr-MS) determination of perchlorate ion without concomitant loss of sensitivity. Selectivity ' "
anses from the formation of a stable association'cbmpiex between,a base^olecule and -a perchlorate ariion f
The best results were obtained using 10 UM chlorhexidine in methanohc solution; the lower limit of detection ' -'""
(LLOD) for S/NSS2 was less than or equalto 6.10 UM (10 ngml-1). This compares favorably with the LLOD '" ""
determined for perchlorate in the absence of any complexing agents ( « 0.05 UM = 5 ng mT '). For the other
bases, which were diazabicyclo compounds (DBN, DBU, DBO), sensitivity was lower by 90% or more The
chlorhexidine-perchlorate cdmplex-(w/z=<505) can be observed even in t&:Presehce ofequiformal nitrate" -''"" ' '
mtnte, hydrpgensulfate, chloride, bromide, 'bromate, and chlorate (all together) dowiTfo approximately 1 'JIM- ' ':
thus, themethpd is rugged;enbugh to fed appUcation to 'systems containing multiple morgamc anibris: ' " ' '"'"r
Introduction. ,.,''.: "'^.VL^ '"". '!? ";-;-;jEr-';i i:'-,"I-"r^';'''.',:
Perchlorate ion was identified in ground and surface waters of
the western United States in'l997, :arid has'since been found at
sites around 'the country: It may be found- in the 'ground or
surface" waters- near wherever- perchlorate salts -have been
manufactured;- stored,: o'r used. ;Such;sites -are ofteir associated
viith defense:pr aerospace prograins '(of supporiingiridu^tries)
since perchlorate salts find use- as ;s6lid 'bx'idants or energetics
boosters m'rotkets Mi ^missiles. The anaiytical "chemistry of
perchlorate and the significance of quantifying t'his coritami-^
nant have been reviewed and described in detail elsewhere!1'2"'
Because of the low concentrations of perchlorate found at
most of the contaminated :sites (5-50 ng ml"1),- the;=primary
technique used .for water analysis has been ion chroniatography
(1C): However, chromatographic 'retention time is not cpnT
sidered_to be a unique identifier, and additibnal confirmation "is
required to initiate legal actiori. Although 1C may be'used for
routine monitoring, periodic confirmatory testing must be
carried out. As, a consequence, techniques such as mass
spectrbmetry can be expected to find a role in "secondary
confirmation, even if the instrumentation is not generally
available on-site to potable water utilities, responsible parties,
or regulatory governmental agencies for primary environmen-
tal monitoring purposes. -:' -
There are essentially six different ways of assaying
perchlorate. Gravimetry and titrimetry only apply to :standar-
fUS Government copyright.
dization of fairly/concentrated' 'laboratory (5=0.01 M)":-"spite"
tibns. Ion-selective electrodes suffer from a number of anionic
interferences as well as lower1 limits of .detection (LLODs) of
ttl (11^=70ngml"1,1 \which is at least; twice; what fhe'no
observable /adverse effects level is predicted to'; be based on
current.reseafch.3 Although Hauser et al report a detection
limit of 10pgml"1 for an^ion-selebtive'-efectrode, this" was in
cPnjunctiph with a capillary electrophores'is (CE)'separation^
Barnett and Horlick used electrospray ionization mass spectrS-
metry (ESI-MS) to obtain an LLOD of 0.050 JIM (5 ng itnl"1)'.5
While spectrpphotometric determinations are reported to have
similar-LLODs to the; ESI-MS method,6 they are:'not
sufficiently?seleetive,'and dye purity can^be an issue. Without
priot analyte separation, quadrupole -mass Spectrometers
cannot measure mlz ratios accurately' and precisely enough
to' identify conclusively an ion Mthi a' mass of 99 u as
perchlorate. ;
Despite the selectivity generally regarded as associated with
mass spectrometric identification,-a large;number of small mass
(.< SOQ.u) hydrophilic inorganic, or organic anions can be found
in natural water sources as; well as disinfected potable water
supplies. -Mass spectrometry offers less ambiguity than 1C,
but with a reduction in "sensitivity. Introduction/ionizatioh
techniques for the involatile perchlorate ion are 'most likely
constrained to either electrospray, which was used by Barnett
and-Horlicfc,? or thermospray, which has not been reported for
perchlorate. Extraction of perchlorate salts from aqueous
solution is possible, but can be difficult to take advantage of
analytically. Use of large cationic organic dyes, e.g., Brilliant
Cresyl Blue or Brilliant Green, is reported for spectrophoto-
J.-Anal. At. Spectrom.,'1999,14, 1861-1866
This journal is © The Royal Society of Chemistry 1999
1861
-------
300 i
J] r
0 100 200 300 400 500
51 i i i I I' I I I I I I I I I I
0 100 200 ' 300
Perchlorate concentration/uM '-
Fig. 3 Calibration graphs (peak area versus perchlorate concentration)
for ESI-MS determination of perchlorate: (a) top to bottom, in (O)
10nM,(D)l HM,(A)0.1 UM chlorhexidine, MeOH solution; (b)(D,)
1 mM DBU, (0,) 10 mM DBN, (V, --) 1 HIM DBO. All lines are based
on least-squares regression.
to sec an unresolvable system of mixed anion complexes in the
mass spectrum or perhaps nothing at all, but this was not the
case. Instead, we found molecular ions of the form [chlorhex-
idine-X~] for perchlorate, chloride, nitrate, and bromide.
However, there was no evidence for chlorhexidine complexes
with nitrite, hydrogensulfate, acetate, bromate, or chlorate. It is
worth pointing out that we have never seen a peak that
corresponds to an association complex of acetate with any of
the reagents. We assume that a peak at m/z=586 corresponds
to a chlorhexidine-hydrogensulfite complex that formed from"
sulfate reduced by the electrospray process, and we suspect that
chlorate, bromate, and nitrite are', reduced as well. Further
exploration of these peaks and'the fate of the,Qther anions was
beyond the scope of this work' and was not pursued. .
The peaks for the chlorhexidine complexes of chloride,
nitrate, and bromide are larger than the" peak for the
perchlorate complex; nevertheless, Fig. 5 does show that the
perchlorate response remains visible even under competition
for the chlorhexidine. Although the sensitivity is reduced
relative to perchlorate" in the absence of the other inorganic
anions, a non-linear response curve can be generated, as shown
in Fig. 6. Attenuation of the signal at high'concentration is
probably due to reduced electrospray efficiency1 resulting from
ionic strength effects. "- -:-- - ''"- ' ' -'' -"'
Based on Fig. 3 and 6, we believe that this method could be
applied under a variety of conditions to both analytical
solutions and real water samples. Some variation may-be
needed for optimization, such as changing pH or chlorhexidine
concentration. Preconcentration may be advised .for some
samples, and we expect that standard approaches would work,
e.g., simple evaporation, lyophilization, or onVcolumn relent
tion (using a strong anion-exchange resin).: The suitability "of
these would depend on both analyte concentration, and matrix
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fig. 4 Calibratio^grapn (peaic area versus^^ perchlorate concentration)
for ' ESI-MS determination of perchlorate' iri 10 |M chlorhexidine,
MeOH solution. First six points used for least-squares regression line.
Presumably, points 7 and 8:suffer--from;loss of electrospray efficiency
due to ionic strength." Inset: .Calibration graph for perchlorate anipn
without any complexing agents,'MeOH solution, provided- for
comparison. Non-linear response.at .higher perchlorate concentration
is'observe'd with and without complexing agents. \' ; . ., , ,
584
,.-.80-
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" '-20;
567
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500
603 605
525
>550
.575.
"m/z
600
,625,
. .65,0-.
Fig.-5 Negative ion ESI;mass spectrum :for' I.UM' '(equifofmal)
ISTBUClOife NaClO3, NaCl,:,NaBrO3,,NaBr, NaND2,:.'NaNO3,,and
NaHSQ,} jn 10 ,UM chlorhexidine, MeQH solution. In this case, the only
perchlorate-chlorhexidine association complex .observed, is that con-
taining one chlorhexidme,niolecule. and one'perchiorate ion, .See, text
for'additional discussion:' , "'"' ,.,'',',~, r:, ,,',,,. ,-V-."
constituents'.'Given the ionic content of the test solutions, we
expect that matrix effects co,uld readily be accounted for in
drinking water by using the method of standard additions; but
this would require validation and optimization on/a per .case
basis. Fig. 5 suggests that .chlorhexidine might even prove
useful for selectively enhancing a number of different anions,
but further exploration ofl the phenomenon was beyond the
scope of this work. ; , ; . ' .'",'. ,, .
Applicability to potable water analysis. Because of the high
ionic strength of potable.water; 'which includes sodium chloride
at considerably higher concentration than the analyte,
suppression of the electrospray signal" is to be expectecl: Two
different potable waters: were tested: Cincinnati tap water and
Tri-Township Water (TTW)7'The sdurce of Cincinnati tap
water is the Ohio River.;TTW.is supplied by a well in Dearborn
County, Indiana, and probably experiences some infiltration
from the Whitewater River. The TTW sample was collected
from a residential faucet in Logan Township. ." - - '. .>
A'20 ml sample of TTW water was evaporated to dryness at
60 °C and reconstituted in an 'eqiial volume of 10% v/v HaO-
MeOH:' Aqueoui*:;chlor'rie'xidrne'>was?::added'.; to giye':'i'"final
chlorhexidine concentration :<5'f. 1.6 IXM .iii ."the..'reconstituted
1864 /. Anal. At. Spectrom., 1999,14, 1861-186.6
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0.03
I I [ \Ll | I t i I 1 I I I I / /f\
5 10 15 20 50
[ClO4-]/uM
Pig. 6 Perchlorate response (peak area versus concentration) for ESI-
MS determination in 10 JJ.M chlorhexidine in the presence of seven other
equiformal anions, MeOH solution; see Fig. 5 caption. This is not
a true calibration graph, because the sample matrix (but not the
chlorhexidine) varies in concentration, too. Inset: Calibration graph for
perchlorate anion without any complexing agents, MeOH solution,
provided for comparison. Non-linear response at higher anion
concentration is observed, presumably due to ionic strength effects
on electrospray efficiency. -
sample. It is important to note that the mineralized residue
resulting from evaporation does not redissolve in the final
solution. In order to promote as much dissolution of
perchlorate salts as possible, the reconstituted samples were
permitted to sit for 20-40 min before decanting the solvent. A
signal distinguishable from that of unspiked samples was
obtained at 20 and 40 UM perchlorate with a response factor of
16 000 area units per 20 UM spiked into the sample. Much of the
chlorhexidine peak (m/z=5Q5 u) is lost from the supernatant,
presumably due to adsorption onto the mineralized residue.
For this reason, spiking the supernatant with additional
perchlorate after decanting from the evaporation vessel gives
no increase in signal. Nonetheless, if the chlorhexidine
concentration is returned to 10 JJ.M '(as indicated by the peak
at 7M/z=505u), further perchlorate spikes give appropriate
response.
Because of the electrospray suppression, large dilution
factors are required if the waters are to be run without
preconcentration. We found that a 5% v/v dilution of tap water
samples gave the best results. Of course, this would mean that
the perchlorate concentration in the water sample would have
to be 20 times greater than the post-dilution concentration. In
both TTW and Cincinnati samples, a post-dilution concentra-
tion of 250 nM perchlorate (1.25UM pre-dilution) gave a
response of about 9200-9500 area units above the unspiked
blank. We must point out that the response is not linear in these
matrices, and quantification of perchlorate under such cond-
itions would require careful construction of a calibration graph
in the matrix under study.
Simple dilution is adjudged not to be a generally suitable
approach for quantifying perchlorate in the presence of
multiple other inorganic anions. Ideally, a preconcentration
technique would select .for perchlorate over other anions. We
speculate that preconcentration using certain highly selective
resins, such as those recently developed by the Oak Ridge
National Laboratory,7 would dramatically improve the
performance of this method and could reduce the LLOD by
a factor of 100 or more. .-:'"--
Methanol versus propan-2-ol. We compared the signal of
50 ugmT1 C1O4~ standards with DBU and chlorhexidine in
propan-2-ol versus methanol. No measurable difference was
found. In addition, we were concerned that clusters of
(MeOH)2(H2O)2 might be responsible for units of A(mf
z)=+100 rather than HC1O4. Since these peaks are also
observed in the propan-2-ol solutions but not in perchlorate-
free methanolic blanks, we conclude that the perchlorate is in
fact responsible, rather than some cluster of solvent or
spectator species. -
Quaternary cations and other species <'.
Performance of the tetraalkylammonium ions as complexing
agents was so much poorer than the organic bases that it
suffices to say that these will be of little analytical utility, No
signal was detected for the molecular ions of the tetramethy-
lammonium, tetraethylammonium, or tetrapropylammonium
cations, [(NR4+)(C1O4~)2]. Triethanolarnmonium and betha-
nechol gave observable signals, but these did not compare
favorably with chlorhexidine or the diazabicyclo bases.
Tetrabutylammonium, tetrahexylammonium, and tetraocty-
lammonium gave signals whose magnitude increased with the
length of the .carbon chain for equiformal [NR4+]. However,
these salts are limited by their solubilities in water and
methanol (or combinations thereof), and did. not compare
favorably with the results obtained for the organic bases above.
No signal was found at the mlz ratio predicted for the
molecular ion of tetraphenylarsonium cation associated with a
perchlorate anion and either another perchlorate anion or a
chloride anion. No signal was detected for a protonated nitron
associated with perchlorate anion; neutral nitron is zwitter-
ionic, and it is usually protonated with acetic acid to solubilize
it and to form the precipitant cation. In positive ion mode ESI-
MS, however, all of the expected cations could be identified;
Consequently, we feel confident in saying that there is a lack of
the complexation behavior characteristic of the other bases
rather than an inability of .the cations to be carried into the
vapor phase. ....... ; . . "
Cationic organic dyes ,
We did not observe any evidence for the presence of complexes
of perchlorate with cationic dyes in the ESI mass spectra when
directly injecting the methanolic dye-perchlorate solutions or
the reconstituted extracts. While we are uncertain whether
the extractions were successful,, we suspect that the failure Of the
direct injection is probably due to the inability of the
electrospray apparatus to nebulize and ionize these species
effectively. Regardless, this particular strategy does not appear
to be worth pursuing further, even though it may be suitable for
the reported spectrophotometric determinations.6
Conclusions
Chlorhexidine gave the greatest improvement in selectivity
without loss of sensitivity for perchlorate detection and
quantification, and it was the only base to show resistance to
matrix effects, even at much lower base concentration.
Although the non-nucleophilic organic bases (i.e., DBO,
DBN, DBU) gave acceptable increases in selectivity, losses of
sensitivity were rather high. Surprisingly, cationic precipitants
used in perchlorate gravimetry, i.e., tetraphenylarsonium and
nitron, gave no signal at all. Because the organic bases
substantially elevate the massby a factor of 3-8the peak is
completely separated from the low mass area where other
anions are commonly found. In addition, complexes with water
or methanol molecules are not seen in the mass spectrum; thus,
peaks of species such as H2O;81Br~ (m/z=99), which was'seen
by Barnett and Horlick,5 are conveniently absent. The Use of
complexing reagents therefore permits ESI-MS analysis with-
out separation on account of its increased selectivity. None-
theless, use of 1C or CE is not precluded, and hyphenated
techniques (i.e., LC-MS or CE-MS) may find a use for such
reagents.The thoughtful use of complexing agents such as
chlorhexidine will perhaps provide a valuable tool in terms of
; J. Anal-At. Spectrom., 1999, 14, 1861-1866 1865
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increased selectivity without diminished sensitivity in the
analytical determination of the perchlorate anion.
Acknowledgements
The financial support of EPA's summer mentoring program is
recognized by D.F. and C.J.
References
1 E. T. Urbansky, Biorem. J., 1998,2,81, and references cited therein.
2 E. T. Urbansky and M. R. Schock, J. Environ Manag., 1999,56,79.
3 US Environmental Protection Agency, Perchlorate Environmental
Contamination: Toxicological Review and Risk Characterization
Based on Emerging Information, Review Draft, December 31, 1998,
Document No. NCEA-1-503.
4 P. C. Hauser, N. D. Renner and A. P. C. Hong, Anal. Chim. Acta,
1994, 295, 181.
5 D. A. Barnett and G. Horlick, /. Anal. At. Spectrom., 1997,12, 497.
6 A. A. Ensafl and B. Rezaei, Anal. Lett., 1998, 31, 167.
7 B. Gu, G. M. Brown, S. D. Alexandratos, R. Ober and V. Patel,
Selective Anion Exchange Resins for the Removal of Perchlorate
CIO4~ from Groundwqter, Oak Ridge National Laboratory, Oak
Ridge, TN, February, 1999, Doc. No. ORNL/TM-13753, Environ-
mental Sciences Division Publication No. 4863.
Paper 9105721H
.A~
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