Quantification of Trace Elements in Natural Samples by Electrospray

Anal. Chem. 2009, 81, 6357–6363

Quantification of Trace Elements in Natural Samples by Electrospray Ionization Mass Spectrometry with a Size-Exclusion Column Based on the Formation of Metal-Aminopolycarboxylate Complexes Hiroki Hotta,*,† Takayuki Mori,† Akira Takahashi,† Yuta Kogure,† Keita Johno,† Tomonari Umemura,‡ and Kin-ichi Tsunoda† Department of Chemistry and Chemical Biology, Gunma University, Tenjin-cho, Kiryu 376-8515, Japan, and Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8603, Japan Metal ions were determined by ESI-MS in the negative ion mode as monovalent negative ions of their aminopolycarboxylic acid (APC) complexes, e.g., [Al(cydta)]-, [Pb(Hcydta)]-, where excess amounts of the APC agents were added to sample solutions. Among several APCs studied, we chose trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CyDTA) as the best chelating agent because of higher stabilities and higher sensitivities of the complexes. The ionization efficiency of these metal complexes was strongly affected by the presence of matrix salts, e.g., NaCl, KNO3, and etc. Thus, a size exclusion column (Sephadex G-10) was used for the online separation of the metal-APC complexes from other matrix salts. This method was successfully applied to the quantitative analyses for total amounts of Al, Ni, Cu, Zn, and Pb in the biological certified reference materials, olive leaves (BCR-062) and plankton (BCR-414). The detection limits of the present methods for these elements were several to several ten nanomolar levels. Moreover, this approach was extended to determine ultratraces of fluoride based on the formation of the ternary complex of aluminum, fluoride and nitrilotriacetic acid (NTA), i.e., [AlF(nta)]-. Its detection limit was 10 nM and was 2 orders of magnitude better than that of a fluoride ion selective electrode method. This method was applied to determine fluoride in tap water, river water, and green tea samples. Electrospray ionization mass spectrometry (ESI-MS) is one of the powerful tools for the detection of metal-organic or metal-inorganic ligand complexes because of its soft ionization; therefore, it is used for the speciation analyses for metals in biological1-4 and environmental samples,5 on which several review * To whom correspondence should be addressed. E-mail: hotta@ chem-bio.gunma-u.ac.jp. Fax: +81-277-30-1251. † Gunma University. ‡ Nagoya University. (1) McSheehy, S.; Mester, Z. Trends Anal. Chem. 2003, 22, 311–326. 10.1021/ac9006842 CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

articles have also been reported.6-8 ESI-MS analyses directly provide the information on molecular weight, structure, and oxidation state of the chemical species in a sample solution. This is an effective feature compared to other highly sensitive techniques such as inductively coupled plasma mass spectrometry (ICPMS), in which the analytes are completely destroyed by the high temperature of the plasma. On the other hand, we should pay great attention to the treatment of the ESI-MS signals especially in the case of quantitative analyses, because the ESIMS signal intensities are strongly affected by unfavorable effects of concomitant chemical species, i.e., so-called matrix effects. Numerous papers9-23 including several reviews24,25 dealt with the suppression of signal intensities of target ions by matrix effects (2) Gammelgaard, B.; Gabel-Jensen, C.; Stu ¨rup, S.; Hansen, H. R. Anal. Bioanal. Chem. 2008, 390, 1691–1706. (3) Umemura, T.; Asaka, K.; Sekizawa, K.; Odake, T.; Tsunoda, K.; Satake, K.; Wang, Q.; Huang, B. Anal. Sci. 2001, 17 (Supplement), i49–i52. (4) Hotta, H.; Wang, Q.; Fukuda, M.; Aizawa, S.; Umemura, T.; Sekizawa, K.; Tsunoda, K. Anal. Sci. 2008, 24, 795–798. (5) Szpunar, J.; Łobinski, R. Fresenius J. Anal. Chem 1999, 363, 550–557. (6) Stewart, I. I. Spectrochim. Acta, Part B 1999, 54, 1649–1695. (7) Łobin´ski, R.; Szpunar, J. Anal. Chim. Acta 1999, 400, 321–332. (8) Rosenberg, E. J. Chromatogr., A 2003, 1000, 841–889. (9) Olesik, J. W.; Thaxton, K. K.; Olesik, S. V. J. Anal. At. Spectrom. 1997, 12, 507–505. (10) Mollah, S.; Pris, A. D.; Johnson, S. K.; Gwizdala, A. B., III.; Houk, R. S. Anal. Chem. 2000, 72, 985–991. (11) Agnes, G. R.; Horlick, G. Appl. Spectrosc. 1994, 48, 649–654. (12) Barnett, D. A.; Horlick, G. J. Anal. At. Spectrom. 1997, 12, 497–501. (13) Urbansky, E. T.; Magnuson, M. L.; Freeman, D.; Jelks, C. J. Anal. At. Spectrom. 1999, 14, 1861–1866. (14) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem. 2003, 75, 3019–3030. (15) King, R.; Bonfiglio, R.; Fernandez-Metzler, C.; Miller-Stein, C.; Olah, T. J. Am. Soc. Mass Spectrom. 2000, 11, 942–950. (16) Avery, M. J. Rapid Commun. Mass Spectrom. 2003, 17, 197–201. (17) Souverain, S.; Rudaz, S.; Veuthey, J.-L. J. Chromatogr., A 2004, 1058, 61– 66. (18) Buhrman, D. L.; Price, P. I.; Rudewicz, P. J. J. Am. Soc. Mass Spectrom. 1996, 7, 1099–1105. (19) Liang, H. R.; Foltz, R. L.; Meng, M.; Bennett, P. Rapid Commun. Mass Spectrom. 2003, 17, 2815–2821. (20) Pascoe, R.; Foley, J. P.; Gusev, A. I. Anal. Chem. 2001, 73, 6014–6023. (21) Iavarone, A. T.; Udekwu, O. A.; Williams, E. R. Anal. Chem. 2004, 76, 3944–3950. (22) Sterner, J. L.; Johnston, M. V.; Nicol, G. R.; Ridge, D. P. J. Mass Spectrom. 2000, 35, 385–391.

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for the determination of metal ions,9-11 inorganic anions,12,13 and organic chemicals.14-23 For example, more than a 20% decrease of signals in the presence of 1 mM or 90% decrease by 100 mM NaCl were observed for the detection of single metal ions (K+, Ni+, and so on),9 and the sensitivity decrease of 20% to over 90% was also reported by an addition of a certain amount of an internal standard for the analysis of drugs.19 These matrix effects usually provoke not only the S/N ratio reduction but also the lower reproducibility and lower accuracy of the assays. These suppressions of ionization were interpreted by a competition between analytes and coeluting species. Kebarle and his co-workers26-30 gave an explanation using a model in which the ion evaporation rate is proportional to the concentration of the ion in the droplet generated from the Taylor cone. Enke et al.31-34 proposed the equilibrium partitioning model, which says that the signal suppression could be attributed to the competition between analytes and coeluting species in the process of their accessing the droplet surface before gas phase emission of the ions. To minimize these matrix effects, various approaches, i.e., on-line or off-line sample pretreatment using chromatographic techniques14,16-19,35-38 or liquid-liquid extraction,17,18 two-dimensional liquid chromatography,20 the post column flow splitting,39 the addition of high concentration volatile salt,21 and uses of nanospray40 and Fourier transform mass spectrometry22 have been reported. The use of internal standard materials, which are usually stable isotope-labeled analogues or compounds having a similar structures with the analytes, is also effective to diminish the matrix effect,11,14,16,19,37 but it may not always be applicable.23 Nevertheless, the matrix effect is strongly dependent upon the chemical nature of each sample so that there is no technique which is always effective. Only a few attempts have been reported on the determination of total amounts of trace metals by ESI-MS so far.9-11,38,41,42 This situation may mainly be ascribed to the following two reasons, i.e., one is its severe matrix effect as discussed above and the other is that excellent methods such as atomic absorption spectroscopy (AAS), inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and ICPMS have already been available in conventional bases. However, we have been interested in this (23) Wang, S.; Cyronak, M.; Yang, E. J. Pharm. Biomed. Anal. 2007, 43, 701– 707. (24) Taylor, P. J. Clin. Biochem. 2005, 38, 328–334. (25) Jessome, L. L.; Volmer, D. A. LCGC 2006, 24, 498–510. (26) Tang, L.; Kebarle, P. Anal. Chem. 1991, 63, 2709–2715. (27) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654–3668. (28) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A–986A. (29) Kebarle, P.; Peschke, M. Anal. Chim. Acta 2000, 406, 11–35. (30) Kebarle, P. J. Mass Spectrom. 2000, 35, 804–817. (31) Enke, C. G. Anal. Chem. 1997, 69, 4885–4893. (32) Constantopoulos, T. L.; Jackson, G. S.; Enke, C. G. J. Am. Soc. Mass Spectrom. 1999, 10, 625–634. (33) Cech, N. B.; Enke, C. G. Anal. Chem. 2001, 73, 4632–4639. (34) Cech, N. B.; Enke, C. G. Mass Spectrom. Rev. 2001, 20, 362–387. (35) Niessen, W. M. A. J. Chromatogr., A 1999, 856, 179–197. (36) Sasaki, H.; Yonekubo, J.; Hayakawa, K. Anal. Sci. 2006, 22, 835–840. (37) Zuehlke, S.; Duennbier, U.; Heberer, T. J. Sep. Sci. 2005, 28, 52–58. (38) Collins, R. N.; Onisko, B. C.; Mclaughlin, M. J.; Merrington, G. Environ. Sci. Technol. 2001, 35, 2589–2539. (39) Gangl, E. T.; Annan, M.; Spooner, N.; Vouros, P. Anal. Chem. 2001, 73, 5635–5644. (40) Bahr, U.; Pfenninger, A.; Karas, M. Anal. Chem. 1997, 69, 4530–4535. (41) Baron, D.; Hering, J. G. J. Environ. Qual. 1998, 27, 844–850. (42) Sharp, B. L.; Sulaiman, A. B.; Taylor, K. A.; Green, B. N. J. Anal. At. Spectrom. 1997, 12, 603–609.

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subject since we preliminarily obtained the sub-part per billion of the detection limit (DL) for Al as the Al-EDTA complex during our speciation study on Al.3,4 The installation of a simple quadrupole ESI-MS instrument is easier and less expensive than that of ICP instruments. The running cost is also less expensive in ESI-MS than in ICP instruments. Moreover, the ESI-MS may have potential as a mobile analytical technique used outside of the laboratory in the future. Thus, we have started to develop proper chemistry for this purpose and have picked up various aminopolycarboxylic acids (APCs) as chelating agents. Our tactics is that all the analyte metals in a sample are converted to their APC complexes with the addition of excess amounts of APCs, then the metal-APC complexes are detected by ESI-MS. As for the detection of metal-APC complexes, particularly EDTA (ethylenediamine-N,N,N′,N′-tetraacetic acid) complexes, by ESI-MS, several reports have been published.10,38,41-46 Baron and Hering41 tried to determine uncomplexed EDTA and Cu, Pb, Cd, Al, and FeIIIEDTA complexes by ESI-MS in the positive ion mode and obtained approximately 1-2 µM of the DLs for uncomplexed EDTA and for the CuEDTA and PbEDTA complexes. Sharp et al.42 also reported about 60 ppb of DL for Ni by the detection as a NiEDTA complex using ESI-MS. However, their methods were not applied to real samples probably because of the matrix effect. Their results suggested that the combination with a kind of separation technique such as chromatography and extraction should be necessary in order to eliminate the undesirable matrix effects. Collins et al.37 reported the use of an ion chromatography-ESI-MS to quantify some metal-EDTA complexes, and their method was applied to analyze soil solutions and plant xylem to which some metals and EDTA were artificially added. They reported the 0.1-1 µM of DLs for Mn, Zn, Al, Cd, and CuEDTAs. Moreover, Alvarez-Fernandes et al.45 recently reported the determination of various ferric chelates including some APC complexes in commercial fertilizers by LC-ESI-MS. Although their works are important to know the quantification ability of ESI-MS for stable metal APC complexes, it has not been fully evaluated particularly for the purpose of the total metal quantification. It will be necessary to develop the methodology (sample preparation, internal standard, and so on) to bring out the inherently high sensitivity of ESI-MS. In this study, we demonstrated to establish a noble quantification method of trace metals in biological samples using ESI-MS coupled with an online separation by a size-exclusion column. Target metals were detected as metal-APC complexes in the negative ion mode. Although APCs have poor selectivity, the chelating abilities are extremely high; therefore, most of metals can be detected at the same time. The effectiveness of the method has been demonstrated by the analyses of biological certified reference materials. Also, this approach was extended to the determination of trace amounts of fluoride using a ternary complex formation of fluoride with the Al-NTA complex. The analyses for fluoride in water samples, i.e., river water, tap water, and a tea sample, were performed by this method. (43) Dodi, A.; Monnier, V. J. Chromatogr., A 2004, 1032, 87–92. (44) Knepper, T. P.; Werner, A.; Bogenschu ¨ tz, G. J. Chromatogr., A 2005, 1085, 240–246. (45) A´lvarez-Fernańdez, A.; Orera, I.; Abadıá, J.; Abadıá, A. J. Am. Soc. Mass Spectrom. 2007, 18, 37–47. (46) Wang, H.; Agnes, G. R. Anal. Chem. 1999, 71, 3785–3792.

EXPERIMENTAL SECTION Materials. Nitrilotriacetic acid (NTA), N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid (HEDTA), ethylenediamineN,N,N′,N′-tetraacetic acid (EDTA), trans-1,2-diaminocyclohexaneN,N,N′,N′-tetraacetic acid (CyDTA). and diethylenetriamineN,N,N′,N′′,N′′-pentaacetic acid (DTPA) (Dojindo Laboratories, Kumamoto, Japan) were used as chelating agents. Two biological standard materials, olive leaves (BCR-062) and plankton (BCR414), were from Institute for Reference Materials and Measurements (IRMM) in Belgium. Metal standard solutions, potassium fluoride, and other reagents of analytical grade were obtained from Wako Pure Chemicals (Osaka, Japan). Water was purified by a Millipore Milli-Q system (Millipore Corp., Billerica, MA,). CoEDTA complex (K salt) was synthesized according to ref 47 and was used as an internal standard material. Instrumental Setup. The LC-ESI-quadrupole MS system, LCMS-2010 (Shimadzu Co., Kyoto, Japan), was used. An online size exclusion column (4.6 mm i.d. × 250 mm long) with Sephadex G-10 (an exclusion molecular weight limit of 700 from Sigma, St. Louis, MO) was used as a sample separation column in order to eliminate the interferences of concomitant salts. The column was packed in our laboratory as follows: dry G-10 particles were put into an empty stainless steel column for HPLC up to the top of the column, it was sealed tightly, and then pure water was sent by an HPLC pump to allow the G-10 gel to swell for 1 h. The retention times, the flow pressure, and the separation of target metal complexes and nitrate on the ESI-MS chromatogram were checked before every experiment. This column was very stable and has been used for more than half a year without any deterioration of the separation ability. The inlet pressure of the flow was nearly constant at around 1.5 MPa at 0.1 cm3 min-1. In our preliminary studies, both positive ion mode and negative ion mode were used for the detection of metal-APC complexes. ESI-MS spectra of several metal-EDTA complexes were shown in Figure S1 in the Supporting Information. Chemical species detected and those DLs using the scan mode were listed in Table S1 in the Supporting Information. Peak intensities in the positive ion mode were larger than those obtained by the negative ion mode; however, the negative ion mode gave a better DL because of higher background levels and their instabilities in the positive ion mode. Thus, the negative ion mode was used throughout in this study. The instrument conditions were as follows: electrospray voltage, -3.5 kV; Q-array voltage, -50 V; nebulizer gas (N2) flow rate, 1.5 dm3 min-1; drying gas (N2) pressure, 0.1 MPa; the curve desolvation line (CDL) temperature, 300 °C; and block heater temperature, 200 °C. Sample solutions were introduced by a flow injection method with a six-way loop injector whose sample loop volume was 30 mm3. A CH3COONH4 aqueous solution (1 mM, mmol dm-3) was used as a carrier solution (flow rate, 0.1 cm3 min-1). All quantitative measurements were performed in the selected ion monitoring (SIM) mode, where the signal intensities of some metal-APCs (maximum five channels) including the internal standard were sequentially monitored every 0.1 s. Thus, four metal ions could be determined in one sample injection by this method. (47) Dwyer, F. P.; Gyarfas, E. C.; Mellor, D. P. J. Phys. Chem. 1955, 59, 296– 297.

Figure 1. ESI-MS spectrum of a solution including 2 µM Al3+, 5 µM Mn2+, Cu2+, Cd2+, Zn2+, Pb2+ (these metals were dissolved as NO3salts), 400 µM EDTA, and 1 mM CH3COONH4. CoEDTA (20 µM) was also added as an internal standard.

The validity of the quantitative results obtained by ESI-MS was carefully checked by a conventional method using the ICP emission spectrometer (ICPS-1000III, Shimadzu Co.) for metals or by the fluoride ion selective electrode method (F-ISE, Orion, Thermo Fisher Scientific, Waltham, MA) for F- analysis. Sample Preparation. Standard metal test solutions were prepared by mixing the appropriate volumes of commercial metal standard solutions, APC stock solution, and CH3COONH4 aqueous solution (these final concentrations were 400 µM of APC and 1 mM of CH3COONH4). In preliminary studies, these ligands and buffer concentrations were optimized to show good performance for detecting metal-ligand complexes. Sample solution pH values were adjusted at around 4.0 by adding HNO3 or NH4OH aqueous solution. In the case of F- measurement, KF stock solution was mixed into AlNO3, NTA, and CH3COONH4 aqueous solutions (final concentration: 200 µM Al3+, 500 µM NTA, and 1 mM CH3COONH4). Sample solutions of biological standard materials were prepared by an acid digestion method. A 0.1 g of solid sample was put into PTFE cup (internal volume, 8 cm3), and the cup was set into a PTFE vessel (internal volume, 27 cm3), into which 2 cm3 of concentrated HNO3 was poured in advance. The vessel was tightly packed into a stainless steel container for high pressure and heated under 160 °C for 5 h. After the decomposition, the liquid sample was dried over the hot plate (180 °C). This residue was diluted to a proper volume (typically 50 cm3 with pure water), then, the appropriate volumes of APC solution, CH3COONH4 solution, and standard metal solutions were added to an aliquot of the sample solution for ESI-MS measurements. In the fluoride determination by F-ISE, a total ionic strength adjustment buffer (TISAB) solution (including 1 M NaCl, 1 M acetate buffer, and 1 mM citrate) was used for pH, ionic strength control, and also for the elimination of interfering metals. Equilibrium Calculation. A computer-based thermodynamic equilibrium model (MINEQL+, version 4.5, from Environmental Research Software, Hallowell ME) was used for equilibrium calculations. RESULTS AND DISCUSSION Detection of Metal-APC Complexes Using ESI-MS. Figure 1 shows an ESI-MS spectrum of a solution including M(NO3)n (M ) 2 µM Al3+, 5 µM Mn2+, Cu2+, Cd2+, Zn2+, Pb2+), 400 µM Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Table 1. Detection Limit of Each Metal-APC Complex (Units of nmol dm-3) metal 27

3+

a

Al (100% ) Mn2+(100%) Fe3+(92%) 60 Ni2+(26%) 63 Cu2+(69%) 66 Zn2+(28%) 114 Cd2+(29%) 208 Pb2+(52%) 55 56

a

HEDTA

NTA

EDTA

CyDTA

DTPA

10 61 2 29 4 15 71 7

2500 6 110 360 100 25 11 15

11 81 5 31 6 10 37 93

21 16 10 25 3 9 59 7

29 33 4 57 13 10 3 10

Isotope ratio. These were estimated as S/N ) 3 values.

-

Figure 2. Dependences of the ESI-MS signal intensities of [Al(edta)] (b) and [Cu(Hedta)]- (9) upon pH (solid lines) and the calculated abundances of these chemical species (dashed lines). These values were normalized by each maximum value.

EDTA, and 1 mM CH3COONH4. CoEDTA (20 µM), [59Co(edta)]- (m/z 347), was added as an internal standard material. These metals could be detected as EDTA complexes, [27Al(edta)]- (m/z 315), [55Mn(Hedta)]- (m/z 344), [63Cu(Hedta)](m/z 352), [66Zn(Hedta)]- (m/z 355), [114Cd(Hedta)]- (m/z 403), and [208Pb(Hedta)]- (m/z 497), and these isotope peaks were well observed. Free H3edta- (m/z 291) and its divalent form, H2edta2- (m/z 145), were also observed. These peak intensities of metal-EDTA complexes showed good reproducibility and linear responses against the metal concentrations from 0 (blank) to over 50 µM. Moreover, these linearities of the calibration curves were considerably improved, when an internal standard was used for the correction of signal intensities. In this study, [Co(edta)]-, which has an extremely high stability constant (KML ) 41.1),48 was used as an internal standard for all the quantitative measurements. The optimization of the solution pH was carried out. The pH dependences of the signal intensities of metal-EDTA complexes, [27Al(edta)]- and [63Cu(Hedta)]-, are shown in Figure 2 (solid lines) as examples, where the maximum values are obtained around pH 3.5-5.0 for both complexes. As for Al-EDTA, the experimental pH dependence showed good agreement with that of the calculated abundance of [Al(edta)]- monoanion in the solution (Figure 2, dashed line). However, a positive shift compared to that expected from equilibrium calculations was observed for [Cu(Hedta)]-. This might be due to the matrix effect of the acid, as the ion concentration increases in the lower pH solution. Therefore, pH 3.5-5.0 should be appropriate for the detection. Nearly the same pH dependences were obtained for other metal-APC complexes. The DLs for the several metal-APC complexes, which were calculated from 3 times the standard deviation of the background signal (S/N ) 3), were obtained by the SIM mode. The DLs of monoanions of the complexes were listed in Table 1. Several to several tens of nanomolar were obtained for all the metal-APC complexes except for the NTA complexes. The stability constants reported48 (KML) and the slopes of the calibration curves (i.e., sensitivities) obtained in this study were also listed in Table S2 in the Supported Information and Table 2, respectively. Although DTPA has higher KML values and low DLs, its (48) The Japan Society for Analytical Chemistry. Bunseki Kagaku Binran (Handbook of Analytical Chemistry), 5th ed.; The Japan Society for Analytical Chemistry: Maruzen, Tokyo, 2001; pp 651-655.

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Table 2. Slope of the Calibration Curve for the Detection of Metal-APC Complex by ESI-MS (Units of cps dm3 nmol-1) metal 27

3+

a

Al (100% ) Mn2+(100%) Fe3+(92%) 60 Ni2+(26%) 63 Cu2+(69%) 66 Zn2+(28%) 114 Cd2+(29%) 208 Pb2+(52%) 55 56

a

HEDTA

NTA

EDTA

CyDTA

DTPA

54 16 62 16 13 19 9 19

0.02 7 0.11 7 0.4 6 3 33

150 28 1400 14 11 13 4 7

110 36 90 20 18 18 9 16

32 14 21 11 8 9 11 18

Isotope ratio.

sensitivities were slightly lower than those obtained by EDTA, CyDTA, and HEDTA. Taking advantage of the separation by the size-exclusion column as stated in the next section, the higher molecular weight ligand was preferred. From these considerations, we have selected CyDTA as the best chelating agent for our purpose. These DL values are one to two orders smaller than those reported earlier for the detection of metal-EDTA complexes using ESI-MS38,41,42 and are almost the same or slightly smaller than those of ICP-AES and AAS, although they are still 3 orders larger than those of ICPMS. Influences of Concomitant Salts and Online Separation by Size-Exclusion Column. The signal intensities of the metal-APC complexes were drastically decreased by adding NaCl or other alkaline metal halides or nitrates into the sample solutions. As an example, the signal intensity change of [Al(cydta)]- and [Pb(Hcydta)]- against NaNO3 concentration was shown in Figure 3. Different concentrations of NaNO3 (0-1 mM) were added to the sample solutions including 100 ppb of Al3+ (3.7 µM) and Pb2+ (0.5 µM), 400 µM CyDTA and 1 mM CH3COONH4, and the intensities were normalized by those without NaNO3 addition. The relative intensities were drastically decreased by the addition of NaNO3. When the [NaNO3] increased from 0.2 to 0.5 mM, the plot for [Al(cydta)]- showed an increase, although the reason is unclear at present. The signal suppression of not only [Al(cydta)]- but also [Pb(Hcydta)]- could not be corrected by the addition of an internal standard ([Co(edta)]-), although its response against the NaNO3 addition looks similar to the behavior of the PbCyDTA complex. This was partly because the baseline shift occurred at the same time. This difference of the behavior against the concomitant salts between the metal-APC complexes and the internal standard was a possible source of error. The similar

Table 3. Determination of Trace Metals in Biological Certified Reference Materials by ESI-MS with a Size-Exclusion Columna (a) BCR-062 olive leaves Al Cu Zn Pb a

Figure 3. Interference of concomitant NaNO3 (0-1 mM) with signal intensities of [Al(cydta)]- (O), and [Pb(Hcydta)]- (b) without any sample preseparations. Al3+ and Pb2+ (100 ppb), 200 µM of CyDTA, and 1 mM CH3COOH were dissolved in the sample solutions.

Figure 4. Mass chromatograph of m/z 409 ([Zn(Hcydta)]-), m/z 457 ([Cd(Hcydta)]-), m/z 551 ([Pb(Hcydta)]-), m/z 347 ([Co(Hedta)]-), and m/z 62 (NO3-). KNO3 (1 mM) was added into 200 ppb Zn2+, Cd2+, Pb2+(these were dissolved as NO3- salts), 200 µM CyDTA, and 1 mM CH3COONH4 aqueous solution. Online separation with the size exclusion column (Sephadex G-10) was applied.

results were observed for all the other APC complexes. These results suggested that the preseparation of the APC complexes from the sample matrixes is needed before the electrospray. In our preliminary studies, we had tried several off-line separation methods as follows: (i) the elimination of small molecules by a dialysis membrane which has a molecular weight cut off of 100, (ii) the separation of alkali metals by a cation-exchange pretreatment column (Presep-CM, Wako). However, these off-line sample preparation techniques could not eliminate the matrix effect. Then, separation with a size exclusion column, in which Sephadex G-10 was packed, was tried for an online pretreatment of the sample solution. Figure 4 shows an example of the ESI-MS chromatogram of m/z 409 ([Zn(Hcydta)]-), 457 ([Cd(Hcydta)]-), 551 ([Pb(Hcydta)]-), 347 ([Co(edta)]-), and 62 (NO3-) which was obtained when 1 mM NaNO3 was added into 200 ppb Zn(NO3)2, Cd(NO3)2, Pb(NO3)2, 20 µM K[Co(edta)], 400 µM CyDTA, and 1 mM CH3COONH4 aqueous solution. The higher molecular weight complexes were eluted faster than the lower molecular weight species (NO3-). All of the metal-CyDTA complexes, internal standard, and free CyDTA anion ([H3cydta]- (the data was not shown in the figure) were virtually eluted simultaneously. Therefore, the unfavorable dissociation of the metal-APC complexes and the error of the ionization efficiency against the elution time could be neglected. As a result, the reproducible quantification

measured

certified

447 ± 23 49.8 ± 2.9 17.6 ± 3.0 28.2 ± 3.9

450 ± 20 46.6 ± 1.8 16.0 ± 0.7 25.0 ± 1.5

(b) BCR-414 plankton Ni Cu Zn Pb

measured

certified

19.6 ± 1.6 30.3 ± 1.8 107 ± 9.6 4.3 ± 1.0

18.8 ± 0.8 29.5 ± 1.3 112 ± 3 3.97 ± 0.19

Standard addition method was used for the determination.

of metals without interference of concomitant salts could be achieved by a standard addition method, even when over 1 mM concomitant salt was present in the sample. The DL values of the present method for metal ions were 70, 68, 48, 69, 40, and 24 nM for Al, Ni, Cu, Zn, Cd, and Pb, respectively. Regrettably, we did not apply this method to the simultaneous determination of FeIII and MnII, because the monovalent FeIII-CyDTA complex has the same m/z value as MnII-CyDTA. Determination of Trace Metals in Biological Samples. The effectiveness of the method has been verified by the real sample analyses. Trace amounts of metals in two certified reference materials (olive leaves (BCR-062) and plankton (BCR-414)) were determined by ESI-MS with the G-10 column where a standard addition method was used. The results listed in Table 3 showed good agreement within experimental error with their certified values. These values were the average of three measurements. The larger errors of our results were probably ascribed to the lower reproducibility of electrospray ionization. The other certified metals could not be determined because of their lower contents. Except for Fe and Mn, we could establish the quantification method for the total amounts of trace metals in biological samples containing various matrix components. Determination of F- Using Ternary Complex Formation [AlF(nta)]-. This approach was extended to the determination of nonmetallic elements. Several papers have been published on quantification analyses for nonmetallic inorganic anions by ESIMS so far.12,13,49-51 Direct detection of halide anions with 1 ppb levels of DL was reported but was severely affected by the sample matrixes.12 Detection of ionic pairs of organic dications and perchlorate ion13,49,50 and other inorganic anions51 were intensively studied. Dasgupta and co-workers were interested in the determination of perchlorate ion in environmental samples by LC-MS.49,50 In this paper, fluoride determination was studied using a ternary complex formation with the metal-APC complex. The fluoride ion is well-known as a strong Lewis base, and it binds strongly with aluminum in the aqueous solution (first stability constant, log β1 ) 6.3052). In the ESI-MS spectrum of a solution including F- and AlCl3, many peaks related to the Al3+ and Fcomplexes were observed. On the contrary, when NTA was added into the solution, these peaks were all converted into the single peak assigned as the [AlF(nta)]- ternary complex (49) Martinelango, P. K.; Anderson, J. L.; Dasgupta, P. K.; Armstrong, D. W.; Al-Horr, R. S.; Slingsby, R. W. Anal. Chem. 2005, 77, 4829–4835. (50) Martinelango, P. K.; Dasgupta, P. K. Anal. Chem. 2007, 79, 7198–7200. (51) Soukup-Hein, R. J.; Remsburg, J. W.; Dasgupta, P. K.; Armstrong, D. W. Anal. Chem. 2007, 79, 7346–7352. (52) Kragten, J. Atlas of Metal-Ligand Equilibria in Aqueous Solution; John Wiley & Sons: New York, 1978.


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Table 4. Slope of the Calibration Curve and Detection Limit (DL) for the Detection of the AlF-APC Complex by ESI-MS analyte

m/z

slope (cps dm3 nmol-1)

DLa (nmol dm-3)

KML of Al-APCb

[AlF(nta)][AlF(hedta)][AlF(Hedta)][AlF(Hcydta)]-

234 321 335 389

38 21 12 0.9

10 5.8 12 170

10.53 14.4 16.5 18.9

a

Figure 5. ESI-MS spectrum of a solution containing 10 µM KF, 200 µM Al3+, 500 µM NTA, and 1 mM CH3COONH4. CoEDTA was added to the sample as an internal standard.

of m/z 234.53,54 Figure 5 shows an ESI-MS spectrum of a solution including 10 µM KF, 200 µM Al3+, 500 µM NTA, and 1 mM CH3COONH4 (pH 7). Although the [Al3+]/[NTA] ) 1:2 complex ([Al(nta)(H2nta)]-, m/z 405) was the largest peak, the target complex, [AlF(nta)]- (m/z 234), was definitely observed. Free NTA (H2nta-, m/z 190), [AlNO3(nta)]- (m/z 277), and [AlOH(nta)]- (m/z 232) were also observed. We could not detect F- and its hydrated ions directly by ESI-MS because of high baseline signals in the low m/z range. A 20 µM CoEDTA (m/z 347) was also dissolved as an internal standard. Preliminary studies showed that too much Al3+ induced the reduction in the detection sensitivity, and the interferences by the concomitant salts (several part per million of other metals or various anions) could be prevented by an excess amount of NTA (300-500 µM). There was no interference by 50 µM of Fe3+, which has also high stability constant against F- (log β1 ) 5.2052). The signal intensity of m/z 234 was proportional to [F-] from 0 to 50 µM (0.95 ppm). Its’ DL was 10 nM (0.2 ppb). This value was 1 or 2 orders smaller than those of known analytical methods for fluoride such as the F-ISE method,55 ion chromatography (IC),56,57 or AlF molecular absorption spectrometry.58 The comparable DL value was reported by an indirect detection of AlF2+ ion using IC-ICPMS.59 Because the Al-NTA complex has a neutral charge in the wide pH range, its detection efficiency on ESIMS analysis was fairly lower than those of other Al-APC complexes studied in this study (see Tables 1 and 2); however, its ternary complex with F- showed high detection efficiency. Other APCs could also form similar ternary complexes of [AlF(hedta)]-(m/z321),[AlF(edta)]-(m/z335),and[AlF(cydta)]-(m/ z 389) under the similar conditions. The detection sensitivities (slopes of the calibration curves) and DLs at pH 7 were shown (53) Yuchi, A.; Hotta, H.; Wada, H.; Nakagawa, G. Bull. Chem. Soc. Jpn. 1987, 60, 1379–1382. (54) Yuchi, A.; Hokari, N.; Terao, H.; Wada, H. Bull. Chem. Soc. Jpn. 1996, 69, 3173–3177. (55) Moody, G. J.; Thomas, J. D. R. Selective Ion Sensitive Electrodes; Merrow Publishing: Watford, England, 1971. (56) Chen, Y.; Ye, M.; Cui, H.; Wu, F.; Zhu, Y.; Fritz, J. S. J. Chromatogr., A 2006, 1118, 155–159. (57) Amin, M.; Lim, L. W.; Takeuchi, T. J. Chromatogr., A 2008, 1182, 169– 175. (58) Tsunoda, K.; Fujiwara, K.; Fuwa, K. Anal. Chem. 1977, 50, 2035–2039. (59) Bayoń, M. M.; Garcia, A. R.; Alonso, J. I. G.; Sanz-Medel, A. Analyst 1999, 124, 27–31.

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The DLs were estimated as S/N ) 3 values. b Reference 48.

Table 5. Determination of F- in Green Tea, River Water (Kiryu River), and Tap Watera

green tea river water tap water a

this method

F-ISE

38.4 ± 0.66 9.60 ± 0.17 4.53 ± 0.38

34.8 ± 2.95 8.40 ± 0.15 4.63 ± 1.76

Standard addition method was used for the determination.

in Table 4. The smaller the KML value of Al-APC, the ternary complex was formed easier and thus the higher sensitivity could be achieved by the addition of NTA. Nearly the same results were also observed at pH 3. The present method was applied to determine fluoride ion in green tea, river water, and tap water samples as shown in Table 5. In these measurements, the application of proper dilution of samples and a standard addition method was enough to avoid the matrix effects of the samples. The results were compared to those measured by the commonly used F-ISE method (a standard addition technique for F-ISE was referred to in the literature60). These two methods showed good agreement with each other as shown in the table. This methodology has naturally been applied to the determination of other halides: the ternary complexes of indium, NTA and Cl-, Br-, and I- (i.e., [InCl(nta)]-, [InBr(nta)]-, and [InI(nta)]-, respectively) provided the extreme sensitivities, and their DLs were 0.3 µM for Cl-, 3 nM for Br-, and 2 nM for I-, respectively. The detail of these methods will be described elsewhere. These DL values were comparable to reported values for the ion-association experiments using dicationic reagent.51 This approach may also be useful to determine other Lewis bases such as CN- or SCN- coordinating to the metal-APCs. CONCLUSION Trace amounts of metals and fluoride in biological and environmental samples could be determined by ESI-MS with an online size-exclusion column for sample matrix separation. The DLs of the present method for trace elements studied were of 10-12 mol level (30 mm3 sample). At present, four metal ions could be determined simultaneously by this method. Although severe matrix effects on ESI-MS measurements were observed, they could be eliminated by applying a size-exclusion separation, an addition of internal standard, and standard addition technique. As discussed above, this method is rapid and sensitive and is based on rather simple chemistry. Moreover, (60) Rix, C. J.; Bond, A. M.; Smith, J. D. Anal. Chem. 1976, 48, 1236–1239.

it is applicable to the determination of not only metal ions but also inorganic anions. The sensitivity improvement could be expected by the instrumental development of an ESI mass spectrometer in the future. Thus, we believe there is a great potential for ESI-MS not only in organic and bioanalyses but also in inorganic trace analysis.

Masanobu Mori, Gunma University, for his helpful discussions on the detection of F-.

ACKNOWLEDGMENT We thank Hitomi Ito, Emi Kimura, Rie Ide, and Masaomi Kitazume for their technical assistance. And we also thank Dr.

Received for review April 1, 2009. Accepted June 18, 2009.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AC9006842


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Quantification of Trace Elements in Natural Samples by Electrospray

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