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Arsenic Speciation in Water and Human Urine by HPLC–ICP-MS and HPLC–MO–HG-AAS
MICROCHEMICAL JOURNAL ARTICLE NO.
59, 89–99 (1998)
MJ971556
Arsenic Speciation in Water and Human Urine by HPLC–ICP-MS and HPLC–MO–HG-AAS Mariella Moldovan, M. Milagros Go´mez, M. Antonia Palacios,1 and Carmen Ca´mara Departamento de QuıB mica AnalıB tica, Facultad de Ciencias QuıB micas, Universidad Complutense de Madrid, 28040 Madrid, Spain Arsenite, arsenate, monomethylarsonate, dimethylarsinate, arsenobetaine, and arsenocholine have been successfully separated in one chromatographic run in a mixed mode column, Spherisorb ODS/NH2 , using 5.0 mmol liter01 phosphate buffer at pH 5.0 as mobile phase and final detection by inductively coupled plasma mass spectrometry (ICP-MS) or microwave-assisted oven (MO) coupled with hydride generation atomic absorption spectrometry (HG-AAS). The detection limits achieved with the HPLC–ICP-MS coupling (0.04–0.28 ng) were about 20 times lower than those achieved by HPLC–MO–HG-AAS, which makes it suitable for determining these species at their naturally occurring concentration levels. The chloride present in the samples was chromatographically separated from the arsenic species, and the interferent signal from the 40Ar35Cl formed on the ICP-MS system was insignificant. The proposed methods were successfully applied to the determination of six arsenic species in water and urine (after previous cleanup of the sample). q 1998 Academic Press Key Words: arsenic speciation; liquid chromatography; microwave digestion; atomic absorption spectrometry; inductively coupled plasma mass spectrometry; urine; water.
INTRODUCTION
Although the determination of total arsenic concentration in urine continues to be an important research task because urinary excretion is the major pathway by which arsenic compounds are eliminated from the body (1), these data do not provide sufficient information about intake or exposure to toxic or nontoxic doses of the element. It has been previously reported that after intake of toxic inorganic arsenic compounds, the element may be methylated in vivo and thus excreted in urine in the form of light-chain methylated compounds. In contrast, the less toxic or innocuous species of arsenic present in marine organisms and seafood, mainly arsenobetaine (AsB) and small amounts of arsenocholine (AsC), tetramethylarsonium ion (TMA/), and trimethylarsine oxide (TMAO), are not biotransformed but very rapidly excreted (2). The presence of arsenobetaine in urine is thus an indicator of the consumption of food (seafood, pork meat, etc.) with a relatively high content of this innocuous species. Therefore, the species appearing in urine can indicate the chemical form of arsenic intake and, through their identification, provide very valuable information. Also, the concentration of arsenic species in some mineral waters is usually higher 1
To whom correspondence should be addressed. 89 0026-265X/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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(mg liter01 level) than in other natural waters. In these waters, As species are usually present as toxic inorganic forms and their speciation is a mandatory task. Since the concentrations of arsenic compounds encountered in environmental and biological samples are rather low, their determination requires the use of very sensitive analytical techniques. High-performance liquid chromatography combined with hydride generation atomic absorption spectroscopy (HPLC–HG-AAS) or inductively coupled plasma mass spectrometry (HPLC–ICP-MS) combines the species separative power of HPLC with sensitive and specific detectors. The main limitation of these hyphenated techniques derives from problems associated with chromatographic separation of species, polyatomic interference in ICP-MS, and the different efficiency of hydride generation for the different species. For example, one problem associated with the direct determination of As by ICP-MS in samples with high chloride content is polyatomic interference from 40Ar35Cl/ ion, resulting from the combination of Ar and Cl in the plasma, which has the same nominal mass as the only arsenic isotope (75As/). In the optimum case this polyatomic interference can be overcome if the chloride is chromatographically separated from the arsenic species (3, 4). Careful evaluation of the optimum HPLC conditions for separating cationic and anionic arsenic species in a single run shows that anionic interaction or reverse-phase ion-pair chromatography is the most suitable method (5–12). The organic cationic species may also be retained in the anionic column by van der Waals forces or by the counterion in the resin. If there is no interaction, they elute in the dead volume. The retention of anionic species depends on their effective charge at the pH of the mobile phase. Also, a new method based on a vesicle-forming reagent such as didodecyldimethylammomium bromide (DDAB) has been recently proposed (13). However, quantitative separation of analytical peaks is a difficult task not yet satisfactorily solved, and the overlap of two or more species is usual when the main arsenic species [arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA), dimethylarsinate (DMA), arsenobetaine (AsB), and arsenocholine (AsC)] run together through the column. In this work we evaluate the performance of a mixed column, Spherisorb ODS/ NH2 , on which amino groups can be protonated by the use of an appropriate buffer in the mobile phase to separate the six arsenic species in one chromatographic run. This column combines different functional behaviors, such as anion exchange through the amino groups, reverse-phase mechanism through the octadecyldimethylsilyl groups (C18), and hydrogen bridge through the free silanol groups. The performance of this column coupled with microwave-assisted oxidation–HG-AAS or ICP-MS and the feasibility of its application to water and urine samples are evaluated in this work. EXPERIMENTAL
Instrumentation The HPLC–MO–HG-AAS system used, developed in a previous work (14), is illustrated in Fig. 1. The chromatographic system consisted of a Spherisorb ODS/NH2 column, which has a bonded phase of C18 and C3-NH2 (250 1 4.6 mm; i.d., 5 mm), a high-pressure solvent pump (Model 590, Waters), and a Rheodyne Model 7125 six-
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FIG. 1. HPLC-MO-HG-AAS manifold for arsenic speciation.
port sample injection valve fitted with a 100-ml loop. Microwave-assisted oxidation was performed in a domestic microwave oven (Balay, Model BAHM-III) with a maximum power output of 700 W. A loop of PTFE tubing (3.0 m, 0.5 mm i.d.) was placed inside the microwave oven through the ventilation holes. A 250-ml beaker filled with water was used to prevent overheating. The thermo-oxidized effluent was run through an ice bath (PTFE tubing, 0.5 m 1 0.5 mm i.d.) before hydride generation to lower its high temperature and to avoid overpressure and decomposition of NaBH4 . The continuous manifold used to generate arsine consisted of PTFE tubing (0.5 mm i.d.), a four-channel peristaltic pump (Gilson HP4), and a U-tube gas–liquid separator (Philips). Hydrides were transported by intermediate argon flow to a quartz atomizing cell heated by an acetylene–air flame. An atomic absorption spectrometer (Perkin– Elmer 2380) equipped with an electrodeless discharge lamp was used. The ICP-MS spectrometer was a VG PlasmaQuad 3 (VG Elemental). For coupling the HPLC system to the ICP-MS, a PTFE capillary 10 cm long and with a 0.5-mm internal diameter was connected from the exit of the column to the entrance of the standard Meinhard nebulizer. For cleanup of the urines, a superspeed refrigerated centrifuge (Dupond RC-5), a rotavapor (Heidloph W 2000), and a deward task (Varila) were used. Reagents All reagents used were of analytical grade. Deionized water from a Milli-Q system was used throughout. Arsenic standard solutions (1000 mg liter01 as As) were obtained by dissolving appropriate amounts of NaAsO2 (Carlo Erba), Na2HAsO4r7H2O (Merck), CH3AsO3Na2r6H2O (Carlo Erba), and (CH3)2AsO2Nar3H2O (Sigma). AsB and AsC were reference standards from the Standard Measurements and Testing Institute (BCR, Brussels, Belgium). The standard stock solutions were stored in glass bottles kept at 47C in darkness. Fresh solutions were prepared daily with deionized water. The mobile phase for the separations was 5.0 mmol liter01 phosphate buffer at pH 5.0, prepared by mixing separate solutions of 5.0 mmol liter01 Na2HPO4 and NaH2PO4 (Panreac) until the desired pH was obtained. All solutions were filtered through a 0.45-mm filter and degassed before use. Absolute ethanol (Carlo Erba), acetone (Carlo Erba), and dry ice (CO2) were employed in the urine cleanup procedure.
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Sample Treatment Urine cleanup procedure. The dry residue from urines were obtained through a cleanup procedure based on that of Labardie et al. (15). First-void (morning) human urine samples were filtered and stored in the dark at 47C. No preservatives were added. To 2 ml urine, 20 ml absolute ethanol was added and the mixture chilled in a solid CO2 –acetone bath for 20 min. The chilled solution was centrifuged at 4000 rpm for 15 min at 0157C. At this temperature, high-molecularmass organic compounds and most salts are precipitated and the ethanol phase containing arsenic species separates. The residue was washed with another 10-ml aliquot of ethanol and the solution obtained by following the same procedure was added to the previous one. The ethanol extract was then dried by rotoevaporation at 407C and the resulting residue dissolved in 10 ml of 5 mmol liter01 NaH2PO4/Na2HPO4 (pH 5.0) eluant. One hundred microliters was injected into the HPLC–MO–HG-AAS or HPLC–ICP-MS system. Waters. Natural waters were filtered through a 0.45-mm membrane filter and degassed by sonication for 30 min. Procedures for On-line Determination of Arsenic Species by HPLC–MO–HG-AAS and HPLC–ICP-MS Chromatographic separation of As(III), As(V), MMA, DMA, AsB, and AsC was performed by injecting solutions containing the six compounds onto the column and isocratic elution was carried out with 5.0 mmol liter01 phosphate buffer of pH 5.0 at a flow rate of 1.5 ml min01. The peak height were recorded and the concentration of each species was obtained by extrapolation from the aqueous calibration curve. The chromatographic separation conditions of arsenic species are listed in Table 1. To generate the arsine from AsB and AsC species a step consisting of on-line 0 transformation of these species into As(V) using the oxidant mixture S2O20 8 /OH in a microwave oven was coupled between the HPLC and hydride systems. Optimization of the MO–HG-AAS system had been carried out in an earlier work (14) and the parameters used are also listed in Table 1. The operating conditions for ICP-MS determination are given in Table 1. Optimization of the mass spectrometer parameters was carried out before the chromatographic system was connected. The nebulizer gas flow rate, ion lens voltage, and sample depth were optimized for a maximum arsenic sensitivity using a 25 mg liter01 As standard solution. The analytical peaks were evaluated as peak height using the isotope 75As. RESULTS AND DISCUSSION
In a previous work (14), two different exchange columns, Hamilton PRP-X100 and IC PAK A, and a modified reverse-phase column, Spherisorb 5ODS, were tested for the six arsenic species under study. But As(III) and AsB peaks always overlapped below pH 9.0 and AsB and AsC above pH 10.0 in the exchange columns as well as the C18 column. At this point, the use of two columns, anionic and cationic, proposed by Larsen et al. (4), seems to be the best alternative, but this procedure involves the use of two different sets of chromatographic conditions, which is less practical for routine analysis.
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TABLE 1 System Parameters, Flow Rates, and Settings of HPLC–MO–HG–AAS and HPLC–ICP–MS Chromatography Anion exchange–reverse phase column Mobile phase Flow rate Injected volume Microwave oven Oxidizing agent Flow rate Microwave power Hydride generation Reducing agent Acid medium Flow rates Argon flow rate AAS Wavelength As EDL lamp Spectral bandwidth BG correction ICP-MS Forward power Reflected power Argon flow rates Coolant Auxiliary Carrier Nebulizer Spray chamber Data collection mode Dwell time Sweeps per replicate Integration mode
Spherisorb ODS/NH2 (25 cm 1 4.6 mm, i.d. 5 mm) NaH2PO4/Na2HPO4 (5 mmol liter01 pH 5.0) 1.5 ml min01 100 ml (water or urine) K2S2O8 5% (w/v) in 2.5% (w/v) NaOH 0.6 ml min01 700 W NaBH4 3% (w/v) in 1.5% (w/v) NaOH HCl, 4 mol liter01 1.9 ml min01 10 ml min01 193.7 nm 10 W 0.7 nm No 1340 W õ1.6 W 14 liters min01 0.8 liter min01 1.0 liter min01 Glass Meinhard concentric Double pass (Scott type) (77C) Single monitoring 75As 2000 ms 399 Peak height
Preliminary studies performed with a mixed mode resin, Spherisorb ODS/NH2 , showed that at the extreme pHs the overlap of species changes drastically from one species to another. Based on this initial experience, we tested the ability of this column to separate the six arsenic species in one chromatographic run. Optimization of Chromatographic Parameters The chromatographic parameters were optimized using an AAS detector because this technique is usually available in any routine laboratory. Three different separation mechanisms may occur in the Spherisorb ODS/NH2 column. First, a weak interaction may occur through the amino tertiary group protonated in acid, neutral, and slightly basic media. Second, in the reverse-phase mechanism,
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neutral analytes are retained on the column, probably by the nonpolar C18 support. Finally, another possible active group in the resin is the residual free silanol (SiOH), which could interact with analytes through hydrogen bridges or dipole–dipole interaction. In this column, the behavior of the different species depends on (i) their anionic/ cationic nature, (ii) the nature of their organic structure, and (iii) the nature, concentration, pH, and flow rate of the mobile phase. An experiment performed at pH 2.5 using 15 mmol liter01 phosphate buffer as the mobile phase and a flow rate of 1.0 ml min01 indicated that the elution order seems to be related more to the nature and size of the organic functional groups bonded to As than the apparent charge of the analyte, which is mainly in the neutral form. AsC, which is charged positively and has the biggest organic structure, is the least strongly retained, whereas arsenite and arsenate are eluted together in last place, which indicates the higher affinity of the OH0 groups of these arsenic species to the surface of the column packing. The big difference between the retention times of the organic and inorganic arsenic species allows arsenite and arsenate to be completely separated from the rest of the species, which is very important because of their toxic properties. At pH 8.0, As(III) and AsC are in their neutral form, AsB exhibits zwitterion behavior and the other species are anionic, with As(V) being doubled charged. Under the best chromatographic conditions, the six species appear in three clearly differentiated chromatographic peaks. The first peak corresponds to the overlap of AsB, MMA, and DMA; the second to As(III) and As(V); and AsC appears as an individual peak because it is the most strongly retained species. At this pH the amino groups cannot be protonated and this disqualifies the column as an anionic interactor. Under such conditions, arsenite and arsenate are able to form hydrogen bonds with the silanol groups, which explains their longer retention compared with the methylated species MMA, DMA, and AsB. At pH 8.0, the apparent neutral charge of AsC assists the retention observed experimentally. Since the elution order of the arsenic species changes significantly between pH 2.5 and 8.0, an exhaustive study was performed using phosphate buffer, the pH of which was varied between 4.5 and 7.0 at intervals of 0.5, the phosphate buffer concentration was varied between 5 and 15 mmol liter01 at intervals of 0.5, and the flow rates were varied between 1 and 2 ml min01 to achieve chromatographic separation at the intermediate pH. Figure 2 illustrates the retention times of species at the 1.5 ml min 01 flow rate and 5 mmol liter01 phosphate buffer concentration at different pHs. Migration of species in the column under the different conditions tested enabled us to establish the optimum conditions of chromatographic separation of species. At pH 5.0, 5 mmol liter01 of phosphate buffer, and a flow rate of 1.5 ml min01, the six arsenic species can be separated in one chromatographic run in 8.60 min. The elution order was AsC, arsenite, AsB, DMA, MMA, and arsenate. All measurements were made at 257C, controlling the column temperature with a water bath. In preliminary studies it was observed that an increase in temperature accelerates the separation kinetics for all species, whereas lower temperatures slow them down.
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FIG. 2. Retention times of AsC, As(III), AsB, DMA, MMA, and As(V) in a Spherisorb ODS/NH2 column as a function of pH of the mobile phase in the presence of 5 mmol liter01 phosphate buffer at 1.5 ml min01.
HPLC–ICP-MS Coupling Performance It is well known that the normal arsenic content of unpolluted samples is very low and that highly sensitive techniques such as ICP-MS are essential for its determination. Working under the chromatographic conditions previously optimized, the low concentration of the mobile phase (5.0 mmol liter01) prevented salt deposition and clogging of the sample cones, and the flow rate of the mobile phase (1.5 ml min01) was compatible with the ICP nebulizer rates. Under these conditions, good performance and stability of the plasma source were observed without a deterioration in sensitivity. Figure 3 is the chromatogram for the injection of the six arsenic species into the HPLC–ICP-MS system; good separation was achieved with this coupling. Owing to the fact that AsC elutes in the dead volume, other species not retained in the column, such as arsenosugars, could be eluted together with this species. This fact represents a major problem using the ICP-MS detector because of its capability to detect any arsenic species. On the contrary, it has not yet been demonstrated that arsenosugars could form the hydride under microwave-assisted oxidation, so that probably only AsC would be determined by atomic absorption. Interferences. It is known that the polyatomic species 40Ar35Cl/ interferes in the determination of arsenic by ICP-MS. This should result in the appearance of an additional peak corresponding to the elution of chloride. This additional peak could affect the arsenic quantification in the event of coelution of chloride with any arsenic compound of interest. It has previously been reported that 5% hydrochloric acid gives a mass peak equiva-
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FIG. 3. Chromatographic separation of arsenic species (50 mg liter01 of As) using the HPLC-ICP-MS system. Mobile phase, 5 mmol liter01 phosphate buffer at pH 5.0. Flow rate, 1.5 ml min01. (a) AsC, (b) As(III), (c) AsB, (d) DMA, (e) MMA, (f) As(V).
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As SPECIATION IN WATER AND URINE TABLE 2 Analytical Characteristics of the HPLC–ICP–MS and HPLC–MO–HG–AAS Systems k*
DL (ng)
RSD (%)
Species
ICP-MS
HG-AAS
ICP-MS
HG-AAS
ICP-MS
HG-AAS
AsC As(III) AsB DMA MMA As(V)
0.00 0.65 0.93 2.15 4.00 6.07
0.00 0.55 0.80 2.00 3.17 4.05
0.15 0.20 0.28 0.04 0.06 0.08
2.4 1.4 1.7 1.9 2.2 2.1
4 5 5 3 5 4
7 2 3 4 3 3
lent to a concentration of 6 mg liter01 arsenic (16). Human urine contains about 0.15 mol liter01 sodium chloride (or approximately 0.9% by mass) (17). Different methods have been proposed to reduce the 40Ar35Cl/ interference produced when the chloride in the sample combines with the argon in the plasma (18–20). To determine the influence of 40Ar35Cl/ interference on the 75As signal under the proposed experimental conditions, we recorded the signals of two solutions containing 0.5 and 2% of sodium chloride without As and spiked with 25 mg liter01 (as As) of the six arsenic species. No variation in peak area of any arsenic compound was observed for 0.5% NaCl. With 2.0% NaCl a small peak of 40Ar35Cl/ was detected after 510 s. This peak does not overlap with As species so that the Cl interference is discarded. Analytical Characteristics of the Methods The analytical characteristics of the HPLC–MO–HG-AAS and HPLC–ICP-MS methods were evaluated for the six arsenic compounds. The capacity factors (k*), detection limits, relative standard deviations (RSDs), and calibration parameters of each species are given in Table 2. k* was calculated considering that AsC is not retained [confirmed by ICP-MS by introducing into the column Li/ (ClLi), which overlapped with the AsC peak]. The chromatographic run time is about 10 min. The RSDs were within the 2–5 and 2–7% ranges for 25 and 100 mg liter01 with ICP-MS and HG-AAS, respectively. The detection limits were calculated following IUPAC rules (21) as three times the standard deviation of the blank signal (n Å 10). The detection limits achieved with HPLC–ICP-MS coupling, which were about 20 times lower than those obtained by HPLC–MO–HG-AAS, make it suitable for most environmental and biological applications. Applications To evaluate the suitability of the two systems developed, urine and mineral water samples were analyzed. Owing to the salt and organic content of urine, a cleanup was necessary to overcome the serious analytical problems that arise when it is directly injected into a HPLC system. It has been seriously reported that the urine cleanup method employed is able to remove the high content of salts from the sample without loss of any arsenic species (22).
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MOLDOVAN ET AL. TABLE 3 Speciation of As in Urine by HPLC–ICP–MS Urine (mg liter01) Species
Volunteer 1
Volunteer 2
Volunteer 3
AsB DMA
41.6 { 2.9 18.8 { 3.2
18.1 { 0.8 10.7 { 0.2
137.4 { 2.5 21.1 { 1.2
It has been checked that the chromatographic resolution achieved was independent of the nature of the urine matrix, being similar for water and for cleaned up urine working with the Spherisorb ODS/NH2 column. The urine samples were provided by three volunteers who abstained from eating seafood and pork meat for 3 days. On the fourth day the volunteers consumed a seafood-rich dinner. The following morning, first-void urine was collected for analysis and the cleanup procedure was applied. The analysis of three urine samples by HPLC– ICP-MS gave two significant peaks, attributed to AsB and DMA. To confirm these results the urines were spiked before and after their cleanup with 25 mg liter01 AsB and 25 mg liter01 DMA and a good fit was obtained, the recovery being about 100%. Table 3 lists the concentration obtained for each analyzed urine sample. Three different mineral waters from Cuenca, Granada, and Gerona were also analyzed. Only the Gerona water contained As(V). The high As content of this sample allows its determination by both methods, the As(V) concentration being about 60 { 5 mg liter01. The same concentration was found for this natural water in a previous work (6). CONCLUSIONS
The two analytical methods described, HPLC–MO–HG-AAS and HPLC–ICPMS, using the mixed mode column, Spherisorb ODS/NH2 , allow separation of the six arsenic species in one chromatographic run in a rather short time. The HPLC–ICP-MS method is considered the best alternative for monitoring arsenic at trace levels because it has detection limits about 20 times lower than those of the HPLC–MO–HG-AAS method. Furthermore, the use of an aqueous mobile phase with low buffer salt concentration minimizes the problems associated with the coupling of HPLC and ICP-MS. The chloride in urine was chromatographically separated from the arsenic analytes and the 40Ar35Cl signal formed was insignificant. The method was successfully applied to As speciation in mineral waters and cleaned up urines. Quantitative recovery was achieved by both techniques. ACKNOWLEDGMENTS The authors thank the Spanish Ministry of Education and DGICYT (Project PB95-0366-C02-01) for providing financial support.
REFERENCES 1. Pomroy, C.; Charbonneau, S. M.; McCullough, M.; Tam, G. K. H. Toxicol. Appl. Pharmacol., 1980, 53, 550.
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Irvin, T. R.; Irgolic, K. D. Appl. Organomet. Chem., 1988, 2, 509. Sheppard, B. S.; Caruso, J. A.; Heitkemper, D. T.; Wolnik, K. A. Analyst, 1992, 117, 971. Larsen, E. H.; Pritzl, G.; Hansen, S. H. J. Anal. At. Spectrom., 1993, 8, 557. Lo´pez, M. A.; Go´mez, M. M.; Palacios, M. A.; Ca´mara, C. Fresenius’ J. Anal. Chem., 1993, 346, 643. Lo´pez-Gonza´lvez, M. A.; Go´mez, M. M.; Ca´mara, C.; Palacios, M. A. J. Anal. At. Spectrom., 1994, 9, 291. Rauret, G.; Rubio, R.; Padro´, A. Fresenius’ J. Anal. Chem., 1991, 340, 157. Rubio, R.; Padro´, A.; Alberti, J.; Rauret, G. Mikrochim. Acta., 1992, 109, 39. Alberti, J.; Rubio, R.; Rauret, G. Fresenius’ J. Anal. Chem., 1995, 351, 415. Marin, P.; Amran, M. B.; Lakkis, M. D.; Leroy, M. J. Chromatographia, 1992, 33, 581. Beauchemin, D.; Siu, K. W. M.; McLaren, J. W.; Berman, S. S. J. Anal. At. Spectrom., 1989, 4, 285. Lamble, K. J.; Hill, S. J. Anal. Chem., 1996, 334, 261. Ming Liu, Y.; Ferna´ndez, M. L.; Blanco, E.; Sanz-Medel, A. J. Anal. At. Spectrom., 1993, 8, 815. Lo´pez-Gonza´lvez, M. A.; Go´mez, M. M.; Ca´mara, C.; Palacios, M. A. Chromatographia, 1996, 43, 507. Labardie, J.; Dunn, W. A.; Arouson, N. N. Biochem. J., 1976, 160, 85. Hutton, R. C. The Choice of Acid Matrix in ICP-MS. VG Isotopes, Winsford, UK, 1986. Low, G. K-C.; Batley, G. E.; Buchanan, S. J. Chromatographia, 1986, 22, 292. Branch, S.; Ebdon, L.; Ford, M.; Foulkes, M.; O’Neill, P. J. Anal. At. Spectrom., 1991, 6, 151. Branch, S.; Corns, W. T.; Ebdon, L.; O’Neill, P. J. Anal. At. Spectrom., 1991, 6, 155. Story, W. C.; Caruso, J. A.; Heitkemper, D. T.; Perkins, L. J. Chromatogr. Sci., 1992, 30, 427. Winefordner, J. D.; Long, L. G. Anal. Chem. 1983, 55, 713. Martin, I.; Lopez, M. A.; Gomez, M.; Ca´mara, C.; Palacios, M. A. J. Chromatogr. B, 1995, 666, 101.
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MJ971556
Arsenic Speciation in Water and Human Urine by HPLC–ICP-MS and HPLC–MO–HG-AAS Mariella Moldovan, M. Milagros Go´mez, M. Antonia Palacios,1 and Carmen Ca´mara Departamento de QuıB mica AnalıB tica, Facultad de Ciencias QuıB micas, Universidad Complutense de Madrid, 28040 Madrid, Spain Arsenite, arsenate, monomethylarsonate, dimethylarsinate, arsenobetaine, and arsenocholine have been successfully separated in one chromatographic run in a mixed mode column, Spherisorb ODS/NH2 , using 5.0 mmol liter01 phosphate buffer at pH 5.0 as mobile phase and final detection by inductively coupled plasma mass spectrometry (ICP-MS) or microwave-assisted oven (MO) coupled with hydride generation atomic absorption spectrometry (HG-AAS). The detection limits achieved with the HPLC–ICP-MS coupling (0.04–0.28 ng) were about 20 times lower than those achieved by HPLC–MO–HG-AAS, which makes it suitable for determining these species at their naturally occurring concentration levels. The chloride present in the samples was chromatographically separated from the arsenic species, and the interferent signal from the 40Ar35Cl formed on the ICP-MS system was insignificant. The proposed methods were successfully applied to the determination of six arsenic species in water and urine (after previous cleanup of the sample). q 1998 Academic Press Key Words: arsenic speciation; liquid chromatography; microwave digestion; atomic absorption spectrometry; inductively coupled plasma mass spectrometry; urine; water.
INTRODUCTION
Although the determination of total arsenic concentration in urine continues to be an important research task because urinary excretion is the major pathway by which arsenic compounds are eliminated from the body (1), these data do not provide sufficient information about intake or exposure to toxic or nontoxic doses of the element. It has been previously reported that after intake of toxic inorganic arsenic compounds, the element may be methylated in vivo and thus excreted in urine in the form of light-chain methylated compounds. In contrast, the less toxic or innocuous species of arsenic present in marine organisms and seafood, mainly arsenobetaine (AsB) and small amounts of arsenocholine (AsC), tetramethylarsonium ion (TMA/), and trimethylarsine oxide (TMAO), are not biotransformed but very rapidly excreted (2). The presence of arsenobetaine in urine is thus an indicator of the consumption of food (seafood, pork meat, etc.) with a relatively high content of this innocuous species. Therefore, the species appearing in urine can indicate the chemical form of arsenic intake and, through their identification, provide very valuable information. Also, the concentration of arsenic species in some mineral waters is usually higher 1
To whom correspondence should be addressed. 89 0026-265X/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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(mg liter01 level) than in other natural waters. In these waters, As species are usually present as toxic inorganic forms and their speciation is a mandatory task. Since the concentrations of arsenic compounds encountered in environmental and biological samples are rather low, their determination requires the use of very sensitive analytical techniques. High-performance liquid chromatography combined with hydride generation atomic absorption spectroscopy (HPLC–HG-AAS) or inductively coupled plasma mass spectrometry (HPLC–ICP-MS) combines the species separative power of HPLC with sensitive and specific detectors. The main limitation of these hyphenated techniques derives from problems associated with chromatographic separation of species, polyatomic interference in ICP-MS, and the different efficiency of hydride generation for the different species. For example, one problem associated with the direct determination of As by ICP-MS in samples with high chloride content is polyatomic interference from 40Ar35Cl/ ion, resulting from the combination of Ar and Cl in the plasma, which has the same nominal mass as the only arsenic isotope (75As/). In the optimum case this polyatomic interference can be overcome if the chloride is chromatographically separated from the arsenic species (3, 4). Careful evaluation of the optimum HPLC conditions for separating cationic and anionic arsenic species in a single run shows that anionic interaction or reverse-phase ion-pair chromatography is the most suitable method (5–12). The organic cationic species may also be retained in the anionic column by van der Waals forces or by the counterion in the resin. If there is no interaction, they elute in the dead volume. The retention of anionic species depends on their effective charge at the pH of the mobile phase. Also, a new method based on a vesicle-forming reagent such as didodecyldimethylammomium bromide (DDAB) has been recently proposed (13). However, quantitative separation of analytical peaks is a difficult task not yet satisfactorily solved, and the overlap of two or more species is usual when the main arsenic species [arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA), dimethylarsinate (DMA), arsenobetaine (AsB), and arsenocholine (AsC)] run together through the column. In this work we evaluate the performance of a mixed column, Spherisorb ODS/ NH2 , on which amino groups can be protonated by the use of an appropriate buffer in the mobile phase to separate the six arsenic species in one chromatographic run. This column combines different functional behaviors, such as anion exchange through the amino groups, reverse-phase mechanism through the octadecyldimethylsilyl groups (C18), and hydrogen bridge through the free silanol groups. The performance of this column coupled with microwave-assisted oxidation–HG-AAS or ICP-MS and the feasibility of its application to water and urine samples are evaluated in this work. EXPERIMENTAL
Instrumentation The HPLC–MO–HG-AAS system used, developed in a previous work (14), is illustrated in Fig. 1. The chromatographic system consisted of a Spherisorb ODS/NH2 column, which has a bonded phase of C18 and C3-NH2 (250 1 4.6 mm; i.d., 5 mm), a high-pressure solvent pump (Model 590, Waters), and a Rheodyne Model 7125 six-
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FIG. 1. HPLC-MO-HG-AAS manifold for arsenic speciation.
port sample injection valve fitted with a 100-ml loop. Microwave-assisted oxidation was performed in a domestic microwave oven (Balay, Model BAHM-III) with a maximum power output of 700 W. A loop of PTFE tubing (3.0 m, 0.5 mm i.d.) was placed inside the microwave oven through the ventilation holes. A 250-ml beaker filled with water was used to prevent overheating. The thermo-oxidized effluent was run through an ice bath (PTFE tubing, 0.5 m 1 0.5 mm i.d.) before hydride generation to lower its high temperature and to avoid overpressure and decomposition of NaBH4 . The continuous manifold used to generate arsine consisted of PTFE tubing (0.5 mm i.d.), a four-channel peristaltic pump (Gilson HP4), and a U-tube gas–liquid separator (Philips). Hydrides were transported by intermediate argon flow to a quartz atomizing cell heated by an acetylene–air flame. An atomic absorption spectrometer (Perkin– Elmer 2380) equipped with an electrodeless discharge lamp was used. The ICP-MS spectrometer was a VG PlasmaQuad 3 (VG Elemental). For coupling the HPLC system to the ICP-MS, a PTFE capillary 10 cm long and with a 0.5-mm internal diameter was connected from the exit of the column to the entrance of the standard Meinhard nebulizer. For cleanup of the urines, a superspeed refrigerated centrifuge (Dupond RC-5), a rotavapor (Heidloph W 2000), and a deward task (Varila) were used. Reagents All reagents used were of analytical grade. Deionized water from a Milli-Q system was used throughout. Arsenic standard solutions (1000 mg liter01 as As) were obtained by dissolving appropriate amounts of NaAsO2 (Carlo Erba), Na2HAsO4r7H2O (Merck), CH3AsO3Na2r6H2O (Carlo Erba), and (CH3)2AsO2Nar3H2O (Sigma). AsB and AsC were reference standards from the Standard Measurements and Testing Institute (BCR, Brussels, Belgium). The standard stock solutions were stored in glass bottles kept at 47C in darkness. Fresh solutions were prepared daily with deionized water. The mobile phase for the separations was 5.0 mmol liter01 phosphate buffer at pH 5.0, prepared by mixing separate solutions of 5.0 mmol liter01 Na2HPO4 and NaH2PO4 (Panreac) until the desired pH was obtained. All solutions were filtered through a 0.45-mm filter and degassed before use. Absolute ethanol (Carlo Erba), acetone (Carlo Erba), and dry ice (CO2) were employed in the urine cleanup procedure.
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Sample Treatment Urine cleanup procedure. The dry residue from urines were obtained through a cleanup procedure based on that of Labardie et al. (15). First-void (morning) human urine samples were filtered and stored in the dark at 47C. No preservatives were added. To 2 ml urine, 20 ml absolute ethanol was added and the mixture chilled in a solid CO2 –acetone bath for 20 min. The chilled solution was centrifuged at 4000 rpm for 15 min at 0157C. At this temperature, high-molecularmass organic compounds and most salts are precipitated and the ethanol phase containing arsenic species separates. The residue was washed with another 10-ml aliquot of ethanol and the solution obtained by following the same procedure was added to the previous one. The ethanol extract was then dried by rotoevaporation at 407C and the resulting residue dissolved in 10 ml of 5 mmol liter01 NaH2PO4/Na2HPO4 (pH 5.0) eluant. One hundred microliters was injected into the HPLC–MO–HG-AAS or HPLC–ICP-MS system. Waters. Natural waters were filtered through a 0.45-mm membrane filter and degassed by sonication for 30 min. Procedures for On-line Determination of Arsenic Species by HPLC–MO–HG-AAS and HPLC–ICP-MS Chromatographic separation of As(III), As(V), MMA, DMA, AsB, and AsC was performed by injecting solutions containing the six compounds onto the column and isocratic elution was carried out with 5.0 mmol liter01 phosphate buffer of pH 5.0 at a flow rate of 1.5 ml min01. The peak height were recorded and the concentration of each species was obtained by extrapolation from the aqueous calibration curve. The chromatographic separation conditions of arsenic species are listed in Table 1. To generate the arsine from AsB and AsC species a step consisting of on-line 0 transformation of these species into As(V) using the oxidant mixture S2O20 8 /OH in a microwave oven was coupled between the HPLC and hydride systems. Optimization of the MO–HG-AAS system had been carried out in an earlier work (14) and the parameters used are also listed in Table 1. The operating conditions for ICP-MS determination are given in Table 1. Optimization of the mass spectrometer parameters was carried out before the chromatographic system was connected. The nebulizer gas flow rate, ion lens voltage, and sample depth were optimized for a maximum arsenic sensitivity using a 25 mg liter01 As standard solution. The analytical peaks were evaluated as peak height using the isotope 75As. RESULTS AND DISCUSSION
In a previous work (14), two different exchange columns, Hamilton PRP-X100 and IC PAK A, and a modified reverse-phase column, Spherisorb 5ODS, were tested for the six arsenic species under study. But As(III) and AsB peaks always overlapped below pH 9.0 and AsB and AsC above pH 10.0 in the exchange columns as well as the C18 column. At this point, the use of two columns, anionic and cationic, proposed by Larsen et al. (4), seems to be the best alternative, but this procedure involves the use of two different sets of chromatographic conditions, which is less practical for routine analysis.
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TABLE 1 System Parameters, Flow Rates, and Settings of HPLC–MO–HG–AAS and HPLC–ICP–MS Chromatography Anion exchange–reverse phase column Mobile phase Flow rate Injected volume Microwave oven Oxidizing agent Flow rate Microwave power Hydride generation Reducing agent Acid medium Flow rates Argon flow rate AAS Wavelength As EDL lamp Spectral bandwidth BG correction ICP-MS Forward power Reflected power Argon flow rates Coolant Auxiliary Carrier Nebulizer Spray chamber Data collection mode Dwell time Sweeps per replicate Integration mode
Spherisorb ODS/NH2 (25 cm 1 4.6 mm, i.d. 5 mm) NaH2PO4/Na2HPO4 (5 mmol liter01 pH 5.0) 1.5 ml min01 100 ml (water or urine) K2S2O8 5% (w/v) in 2.5% (w/v) NaOH 0.6 ml min01 700 W NaBH4 3% (w/v) in 1.5% (w/v) NaOH HCl, 4 mol liter01 1.9 ml min01 10 ml min01 193.7 nm 10 W 0.7 nm No 1340 W õ1.6 W 14 liters min01 0.8 liter min01 1.0 liter min01 Glass Meinhard concentric Double pass (Scott type) (77C) Single monitoring 75As 2000 ms 399 Peak height
Preliminary studies performed with a mixed mode resin, Spherisorb ODS/NH2 , showed that at the extreme pHs the overlap of species changes drastically from one species to another. Based on this initial experience, we tested the ability of this column to separate the six arsenic species in one chromatographic run. Optimization of Chromatographic Parameters The chromatographic parameters were optimized using an AAS detector because this technique is usually available in any routine laboratory. Three different separation mechanisms may occur in the Spherisorb ODS/NH2 column. First, a weak interaction may occur through the amino tertiary group protonated in acid, neutral, and slightly basic media. Second, in the reverse-phase mechanism,
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neutral analytes are retained on the column, probably by the nonpolar C18 support. Finally, another possible active group in the resin is the residual free silanol (SiOH), which could interact with analytes through hydrogen bridges or dipole–dipole interaction. In this column, the behavior of the different species depends on (i) their anionic/ cationic nature, (ii) the nature of their organic structure, and (iii) the nature, concentration, pH, and flow rate of the mobile phase. An experiment performed at pH 2.5 using 15 mmol liter01 phosphate buffer as the mobile phase and a flow rate of 1.0 ml min01 indicated that the elution order seems to be related more to the nature and size of the organic functional groups bonded to As than the apparent charge of the analyte, which is mainly in the neutral form. AsC, which is charged positively and has the biggest organic structure, is the least strongly retained, whereas arsenite and arsenate are eluted together in last place, which indicates the higher affinity of the OH0 groups of these arsenic species to the surface of the column packing. The big difference between the retention times of the organic and inorganic arsenic species allows arsenite and arsenate to be completely separated from the rest of the species, which is very important because of their toxic properties. At pH 8.0, As(III) and AsC are in their neutral form, AsB exhibits zwitterion behavior and the other species are anionic, with As(V) being doubled charged. Under the best chromatographic conditions, the six species appear in three clearly differentiated chromatographic peaks. The first peak corresponds to the overlap of AsB, MMA, and DMA; the second to As(III) and As(V); and AsC appears as an individual peak because it is the most strongly retained species. At this pH the amino groups cannot be protonated and this disqualifies the column as an anionic interactor. Under such conditions, arsenite and arsenate are able to form hydrogen bonds with the silanol groups, which explains their longer retention compared with the methylated species MMA, DMA, and AsB. At pH 8.0, the apparent neutral charge of AsC assists the retention observed experimentally. Since the elution order of the arsenic species changes significantly between pH 2.5 and 8.0, an exhaustive study was performed using phosphate buffer, the pH of which was varied between 4.5 and 7.0 at intervals of 0.5, the phosphate buffer concentration was varied between 5 and 15 mmol liter01 at intervals of 0.5, and the flow rates were varied between 1 and 2 ml min01 to achieve chromatographic separation at the intermediate pH. Figure 2 illustrates the retention times of species at the 1.5 ml min 01 flow rate and 5 mmol liter01 phosphate buffer concentration at different pHs. Migration of species in the column under the different conditions tested enabled us to establish the optimum conditions of chromatographic separation of species. At pH 5.0, 5 mmol liter01 of phosphate buffer, and a flow rate of 1.5 ml min01, the six arsenic species can be separated in one chromatographic run in 8.60 min. The elution order was AsC, arsenite, AsB, DMA, MMA, and arsenate. All measurements were made at 257C, controlling the column temperature with a water bath. In preliminary studies it was observed that an increase in temperature accelerates the separation kinetics for all species, whereas lower temperatures slow them down.
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FIG. 2. Retention times of AsC, As(III), AsB, DMA, MMA, and As(V) in a Spherisorb ODS/NH2 column as a function of pH of the mobile phase in the presence of 5 mmol liter01 phosphate buffer at 1.5 ml min01.
HPLC–ICP-MS Coupling Performance It is well known that the normal arsenic content of unpolluted samples is very low and that highly sensitive techniques such as ICP-MS are essential for its determination. Working under the chromatographic conditions previously optimized, the low concentration of the mobile phase (5.0 mmol liter01) prevented salt deposition and clogging of the sample cones, and the flow rate of the mobile phase (1.5 ml min01) was compatible with the ICP nebulizer rates. Under these conditions, good performance and stability of the plasma source were observed without a deterioration in sensitivity. Figure 3 is the chromatogram for the injection of the six arsenic species into the HPLC–ICP-MS system; good separation was achieved with this coupling. Owing to the fact that AsC elutes in the dead volume, other species not retained in the column, such as arsenosugars, could be eluted together with this species. This fact represents a major problem using the ICP-MS detector because of its capability to detect any arsenic species. On the contrary, it has not yet been demonstrated that arsenosugars could form the hydride under microwave-assisted oxidation, so that probably only AsC would be determined by atomic absorption. Interferences. It is known that the polyatomic species 40Ar35Cl/ interferes in the determination of arsenic by ICP-MS. This should result in the appearance of an additional peak corresponding to the elution of chloride. This additional peak could affect the arsenic quantification in the event of coelution of chloride with any arsenic compound of interest. It has previously been reported that 5% hydrochloric acid gives a mass peak equiva-
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FIG. 3. Chromatographic separation of arsenic species (50 mg liter01 of As) using the HPLC-ICP-MS system. Mobile phase, 5 mmol liter01 phosphate buffer at pH 5.0. Flow rate, 1.5 ml min01. (a) AsC, (b) As(III), (c) AsB, (d) DMA, (e) MMA, (f) As(V).
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As SPECIATION IN WATER AND URINE TABLE 2 Analytical Characteristics of the HPLC–ICP–MS and HPLC–MO–HG–AAS Systems k*
DL (ng)
RSD (%)
Species
ICP-MS
HG-AAS
ICP-MS
HG-AAS
ICP-MS
HG-AAS
AsC As(III) AsB DMA MMA As(V)
0.00 0.65 0.93 2.15 4.00 6.07
0.00 0.55 0.80 2.00 3.17 4.05
0.15 0.20 0.28 0.04 0.06 0.08
2.4 1.4 1.7 1.9 2.2 2.1
4 5 5 3 5 4
7 2 3 4 3 3
lent to a concentration of 6 mg liter01 arsenic (16). Human urine contains about 0.15 mol liter01 sodium chloride (or approximately 0.9% by mass) (17). Different methods have been proposed to reduce the 40Ar35Cl/ interference produced when the chloride in the sample combines with the argon in the plasma (18–20). To determine the influence of 40Ar35Cl/ interference on the 75As signal under the proposed experimental conditions, we recorded the signals of two solutions containing 0.5 and 2% of sodium chloride without As and spiked with 25 mg liter01 (as As) of the six arsenic species. No variation in peak area of any arsenic compound was observed for 0.5% NaCl. With 2.0% NaCl a small peak of 40Ar35Cl/ was detected after 510 s. This peak does not overlap with As species so that the Cl interference is discarded. Analytical Characteristics of the Methods The analytical characteristics of the HPLC–MO–HG-AAS and HPLC–ICP-MS methods were evaluated for the six arsenic compounds. The capacity factors (k*), detection limits, relative standard deviations (RSDs), and calibration parameters of each species are given in Table 2. k* was calculated considering that AsC is not retained [confirmed by ICP-MS by introducing into the column Li/ (ClLi), which overlapped with the AsC peak]. The chromatographic run time is about 10 min. The RSDs were within the 2–5 and 2–7% ranges for 25 and 100 mg liter01 with ICP-MS and HG-AAS, respectively. The detection limits were calculated following IUPAC rules (21) as three times the standard deviation of the blank signal (n Å 10). The detection limits achieved with HPLC–ICP-MS coupling, which were about 20 times lower than those obtained by HPLC–MO–HG-AAS, make it suitable for most environmental and biological applications. Applications To evaluate the suitability of the two systems developed, urine and mineral water samples were analyzed. Owing to the salt and organic content of urine, a cleanup was necessary to overcome the serious analytical problems that arise when it is directly injected into a HPLC system. It has been seriously reported that the urine cleanup method employed is able to remove the high content of salts from the sample without loss of any arsenic species (22).
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MOLDOVAN ET AL. TABLE 3 Speciation of As in Urine by HPLC–ICP–MS Urine (mg liter01) Species
Volunteer 1
Volunteer 2
Volunteer 3
AsB DMA
41.6 { 2.9 18.8 { 3.2
18.1 { 0.8 10.7 { 0.2
137.4 { 2.5 21.1 { 1.2
It has been checked that the chromatographic resolution achieved was independent of the nature of the urine matrix, being similar for water and for cleaned up urine working with the Spherisorb ODS/NH2 column. The urine samples were provided by three volunteers who abstained from eating seafood and pork meat for 3 days. On the fourth day the volunteers consumed a seafood-rich dinner. The following morning, first-void urine was collected for analysis and the cleanup procedure was applied. The analysis of three urine samples by HPLC– ICP-MS gave two significant peaks, attributed to AsB and DMA. To confirm these results the urines were spiked before and after their cleanup with 25 mg liter01 AsB and 25 mg liter01 DMA and a good fit was obtained, the recovery being about 100%. Table 3 lists the concentration obtained for each analyzed urine sample. Three different mineral waters from Cuenca, Granada, and Gerona were also analyzed. Only the Gerona water contained As(V). The high As content of this sample allows its determination by both methods, the As(V) concentration being about 60 { 5 mg liter01. The same concentration was found for this natural water in a previous work (6). CONCLUSIONS
The two analytical methods described, HPLC–MO–HG-AAS and HPLC–ICPMS, using the mixed mode column, Spherisorb ODS/NH2 , allow separation of the six arsenic species in one chromatographic run in a rather short time. The HPLC–ICP-MS method is considered the best alternative for monitoring arsenic at trace levels because it has detection limits about 20 times lower than those of the HPLC–MO–HG-AAS method. Furthermore, the use of an aqueous mobile phase with low buffer salt concentration minimizes the problems associated with the coupling of HPLC and ICP-MS. The chloride in urine was chromatographically separated from the arsenic analytes and the 40Ar35Cl signal formed was insignificant. The method was successfully applied to As speciation in mineral waters and cleaned up urines. Quantitative recovery was achieved by both techniques. ACKNOWLEDGMENTS The authors thank the Spanish Ministry of Education and DGICYT (Project PB95-0366-C02-01) for providing financial support.
REFERENCES 1. Pomroy, C.; Charbonneau, S. M.; McCullough, M.; Tam, G. K. H. Toxicol. Appl. Pharmacol., 1980, 53, 550.
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