Anal. Chem. 1988, 6 0 , 2209-2212
2209
Identification and Quantitation of Arsenic Species in a Dogfish Muscle Reference Material for Trace Elements’ Diane Beauchemin, M. E. Bednas, S. S. Berman, J. W. McLaren, K. W. M. Siu,* and R. E. Sturgeon Division of Chemistry, National Research Council of Canada, Montreal Road, Ottawa, Ontario, Canada K I A OR9 The arsenic species present in a dogflsh muscle reference material (DORM-1) have been identified by uslng hlgh-performance liquid chromatographyllnductlvelycoupled plasma mass spectrometry (HPLC/ICP-MS), thin-layer chromatography, and electron Impact mass spectrometry and quantified by uslng HPLC/ICP-MS and graphite furnace atomic absorptlon spectrometry. The major species is arsenobetalne (15.7 f 0.8 pg of As/g of DORM-I), constituting about 84% of the total arsenic. For this species, the HPLWICP-MS detection limit was 0.3 ng of As.
The need to identify and quantify actual chemical species, not just the elements, is well recognized. One of the reasons why elemental determination may not suffice is that different species of the same element may have very different chemical and toxicological properties. A good example is arsenic. Of the more environmentally important species, &(In) and &(V) are the most toxic; administration of 20 and 41 pg of As/g as arsenic trioxide and sodium arsenate was reported to cause 50% mortality in rats within 96 h (I). Monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are moderately toxic (2); the concentrations required to cause the same mortality rate are 1800 and 700 pg of As/g, respectively (1). Arsenobetaine (AB) and arsenocholine (AC) are relatively nontoxic; no signs of toxication were exhibited by rats, rabbits, and mice even when relatively large amounts of AB and AC (4-400 pg of As/g of body weight) were ingested (3, 4). Biomethylation of inorganic arsenic is regarded as a detoxification mechanism. The products are either excreted or stored (5). In many marine animals, including lobster (6, 7), fishes @-IO), sharks (7,11,12), crabs (9,13,14), and shrimps (9, 10, 15), the metabolic end product of arsenic has been identified as arsenobetaine. Various analytical techniques have been used: thin-layer chromatography (TLC) (6-9, 12-15), electrophoresis (8, 151, high-performance liquid chromatography (HPLC) coupled to graphite furnace atomic absorption spectrometry (GFAAS) (10, 14) or inductively coupled plasma atomic emission spectrometry (ICP-AES) (11, 13),nuclear magnetic resonance (14,15), X-ray crystallography (6, 7), and various forms of mass spectrometry (9,12,15,16). Often analytical confidence was raised by using several techniques to analyze one sample. Although AB was identified in previous studies, only one serious attempt to quantify it has been reported (10). This is understandable since complicated multistep sample manipulation was required in all cases to extract and purify arsenobetaine to a stage amenable to the particular analytical technique. Even in that case where quantitative analysis was performed (IO), the sample manipulation steps of solvent extraction, repeated column chromatography, and solvent evaporation rendered AB recovery nonquantitative. The reported recovery of 82 % is excellent in view of the complexity of AB isolation. In this paper, we report the identification of arsenobetaine as the principal arsenic species present in the dogfish muscle reference material for trace elements DORM-1 (National NRCC 29303. 0003-2700/88/0360-2209$01.50/0
Research Council of Canada) and the quantitation of arsenobetaine and other arsenic species after a simple methanol/chloroform extraction step. AB was identified by using electron impact mass spectrometry (EIMS),TLC, and HPLC. Quantitation was performed by using HPLC/inductively coupled plasma mass spectrometry (ICP-MS) and GFAAS. EXPERIMENTAL SECTION Sample. The preparation of DORM-1 has been described earlier (17). Briefly, spiny dogfish (Squalus acanthias) from the southwest coast of Nova Scotia was fiieted, minced, homogenized, spray dried, acetone extracted three times, screened, tumbled, bottled, and radiation sterilized. Reagents. All chemicals and solvents used were reagent grade, except as otherwise indicated. Aqueous stock solutions of As(III), As(V), MMA, and DMA were prepared by dissolving arsenic trioxide (Fisher), sodium arsenate (Baker), disodium methylarsonate hexahydrate (Pfaltz and Bauer), and dimethylarsinic acid (Pfaltz and Bauer), respectively. The latter three were standardized against As(II1) by using GFAAS and ICP-MS. Their species purity was also confirmed by high-performance anion exchange chromatography/ICP-MS. Aqueous stock solutions of AB and AC were prepared from arsenobetaine hydrate and arsenocholine iodide (synthesized by W. R. Cullen, Department of Chemistry, University of British Columbia). These were standardized against As(III), and their purity was checked by using HPLC/ICP-MS. Acids were purified in-house from reagent grade stock by subboiling distillation. Ammonia was purified by isothermal distillation of reagent grade stock. Deionized distilled water (DDW) was prepared by passing distilled water through a cartridge deionizer (Cole-Palmer). Isolation of Arsenobetaine. Quantitation and Chemical Characterization. A 2-g DORM-1 sample was placed in a 50-mL Pyrex centrifuge tube, followed by 20 mL of methanol and 10 mL of chloroform. The tube was placed in an ultrasonic bath, sonicated for 30 min, and centrifuged at 2000 rpm for 10 min. The orange colored liquid was pipetted into a 125-mL separatory funnel. The methanol/chloroform extraction was repeated and the extracts were combined. To the combined methanol/chloroform solution, a further portion of 20 mL of chloroform was added, followed by 20 mL of DDW. The separatory funnel was shaken vigorously and allowed to stand. Phase separation ensued; the lower chloroform phase contained little arsenic, most of which stayed in the upper methanol/water phase. The two phases were separately drained into beakers and the solvents evaporated overnight at room temperature in a d a s s 10 fumehood. The chloroform fraction residue and the methanol/chloroform-extracted DORM residue were subjected to nitric acid/magnesium nitrate digestion (18). The total arsenic concentration of these samples was measured by GFAAS and/or ICP-MS. The orange syrup from the methanol/water fraction was diluted to 50 mL with DDW and analyzed by HPLC/ICP-MS and GFAAS. TLC and EIMS Identification. The orange syrup from 25 extractions (50 g of DORM-1 total) was combined, dissolved in 300 mL of DDW, acidified to pH 2.0, and extracted with five 50-mL portions of liquid phenol (prepared by adding 8% (w/w) DDW to phenol). A 0.5-mL portion of the last extract was removed for analysis by GFAAS, which was used to monitor the progress throughout the purification. The combined phenol extracts were diluted with 1L of diethyl ether and back extracted with four 50-mL portions of DDW. The water extracts were combined, washed with 200 mL of diethyl ether, and allowed to evaporate at room temperature in a class 10 fumehood. The residue was dissolved in 10 mL of DDW and the solution was passed through a column of (3-18bonded silica gel (Waters As-
0 Published 1988 by the Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
sociates, preparative grade, 1 X 6 cm). The eluate was acidified to pH 2.0 and passed through a column of strong cation exchanger (Dowex 50W-X8, 1 X 6 cm). The column was washed with 20 mL of DDW, and the sorbed arsenic was eluted with 30 mL of 4 M ammonia. The ammonia was allowed to evaporate overnight, and the residue was redissolved in 20 mL of DDW. This solution was passed through a weak cation exchanger (Amberlite IRC-50, 1 X 6 cm), and the sorbed arsenic was eluted with 30 mL of DDW. The eluate was evaporated to approximately 10 mL. A 0.5-mL portion of this solution was reserved for TLC. The remainder was subjected to the three-stage (C-18, Dowex 50W-X8 and IRC-50) chromatographic separation three more times. At the end of the third separation cycle, the water was allowed to evaporate completely. The white residue was further purified by boiling/subliming off non-arsenic-containing compounds at Torr). The remaining 180 "C under reduced pressure (ca.5 X residue was dissolved in DDW and the solution was passed through '2-18 for the last time. The colorless eluate was subjected to EIMS. Determination of Arsenocholine. A 10-mL portion of the diluted orange syrup was passed through the C-18 column. The eluate was acidified to pH 2.0 and passed through the Dowex 50W-X8 column. Sorbed arsenic was eluted by using 30 mL of 4 M ammonia, 30 mL of DDW, and 30 mL of 4 M hydrochloric acid. This last fraction was analyzed for arsenocholine by GFAAS. A 2GmL portion of methanol was passed through the C-18 column. This was found to contain negligible amounts of arsenic. HPLC/ICP-MS. A HPLC system, consisting of a dual-piston reciprocating pump (Waters 6000), a valve injector (Rheodyne 7125, most work done with a 200-gL loop), and a 5-pm C-18 column (4.6 x 30 mm cartridge, Pierce), was directly interfaced via a Teflon tubing in. o.d., 0.25 mm i.d.) to the nebulizer of an inductively coupled plasma mass spectrometer (PerkinElmer SCIEX ELAN 250). The mobile phase was 10 mM sodium dodecyl sulfate solution containing 5% methanol (HPLC grade) and 2.5% glacial acetic acid. The usual flow rate was 3 mL/min. The presence of organic solvent necessitated the use of higher rf power, 1.4kW as opposed to the usual 1.2 kW to maintain the usual sensitivity. All other ICP-MS operating parameters were similar to those normally used for aqueous arsenic solutions. The mass spectrometer was operated in the single ion ( m / z 75) monitoring mode. Peak areas were measured with a custom software package developed in-house. Determinations were made by the method of standard additions; spikes of AB and other species were added to various DORM samples prior to injection. GFAAS. An atomic absorption spectrometer (Perkin-Elmer Zeeman 5000) equipped with an arsenic electrodeless discharge lamp and a graphite furnace (HGA 500 with Zeeman effect background correction) was used. Arsenic determination was performed by use of a L'vov platform with 20 pg of Pd as a matrix modifier (19). Calibration was made by using the method of standard additions. TLC. Separation was performed on preparative cellulose F plate (Merck)using a mobile phase of 1-butanol/aceticacid/DDW (60/15/25). Iodine was used to locate the arsenic-containingspots. Arsenic chromatographic profiles were obtained by scraping consecutive 1 cm diameter circles of cellulose off the plate, extracting each circle with methanol, diluting with weak nitric acid solution, and analyzing with GFAAS. EIMS. Samples were inserted into the ion source of the mass spectrometer (Finnigan 4000) with a solid probe, which was heated at a rate of 25 OC/min to 325 "C. RESULTS AND DISCUSSION The primary aim of this study was to develop analytical methods that would allow accurate quantitation of the arsenic species in DORM. To achieve this, sample handling was minimized and arsenic selective analytical techniques were used. One such technique was HPLC/ICP-MS. Figure l a shows a typical mass 75 chromatogram obtained from the methanol/water phase. Peak identification was made by coinjecting arsenic standards with the sample solution; peaks A and B were found to be As(II1) + As(V) + MMA and DMA, respectively, peaks C and D were arsenobetaine (see Figure lb), arsenocholine eluted very late. In contrast, an AB
140000
1
C
I
time (min)
Figure 1. m l z 75 chromatogram showing consecutive injections of (a) 200 pL methanoVwater fraction of DORM-1 and (b) 200 pL of the same solution spiked with 0.14 pg of As as arsenobetaine. Peak identification is as follows: A, As(II1) -I-As(V) -t MMA; B, DMA; C and D, arsenobetaine. The sum of the areas of peaks C and D is used for quantitation. The arrow shortly after 3 min indicates the point of
the second injection.
standard injected alone gave a single peak at a retention time slightly less than double that of peak D. The two different major ion-pair forms of AB were probably the results of concomitant ionic species in the DORM-1 extract interfering and competing with dodecyl sulfate anion. Similar phenomena were observed with different ion-pairing systems (13,14).The signifcant features that enabled thii present system to be used for quantitative analysis, however, were that (1)neither the retention times nor the peak shapes of peaks C and D changed with the amounts of coinjected AB for the purpose of standard additions analysis, (2) peaks C and D were relatively sharp, and (3) very impure extracts that had received minimal handling could be tolerated. T o confirm arsenobetaine identification, two other independent techniques, TLC and EIMS, as well as chemical characterization were employed. Complicated purification had to be performed before the methanol/water fraction could be analyzed by TLC and EIMS. For TLC, phenol ion-pair extraction/back extraction plus multistage column chromatography sufficed. The methanol/water fraction gave only one arsenic-containing spot whose retention (Rf = 0.51 f 0.03) matched that of an arsenobetaine standard. For EIMS, further purification using repeated chromatography and vacuum distillation/sublimation was necessary. The latter removed two major unresolved impurities that were more volatile than AB; one was trimethylamine and/or a compound that degraded to it, and the other was creatinine. The EIMS spectrum of the highly purified methanol/water fraction matched well with that of arsenobetaine, both ghown in Figure 2. The major ions attributed to fragmentation of AB were m / z 120,105, 103, and 89. Attempts a t chemical confirmation were less successful. Essentially all arsenic in the methanol/water extract of DORM-1 was inactive toward hydride generation atomic absorption spectrometry, characteristic of arsenobetaine (15, 20-22). However, attempts to convert this form as well as arsenobetaine to a form amenable to hydride generation by heating in an alkaline solution were only partially successful. Although several groups have reported that AB can readily be converted to trimethylamine oxide and/or DMA by heating in the presence of a base (15,21,22),the extent of AB conversion was found to be only 35% even after heating in 4 M NaOH for 72 h. In this case, the unreacted AB in the alkaline solution was recovered by acidification to p H 2.0, passing it through the strong cation exchange column (Dowex 50W-X8) and eluting it with base. Determination by GFAAS revealed that 64% of the AB was recovered, proving that the low
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
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3
a
58
.
b
150
I00
iis
58
288
I SR
zb0
mlz Figure 2. EIMS mass spectra: (a) arsenobetaine standard, (b) highly purified methanoVwater fraction of DORM.
Table I. Arsenic Species Concentrations in Various Fractions (Arsenic Concentration (rg/g)) fraction methanol/water
species
concentration
n
As(II1) + As(V) + MMA DMA
0.18 f 0.04 0.47 f 0.02 15.7 f 0.8 0.11 f 0.05
8
AB AC total As
sum = 16.5 f 0.8
3 10 3
method HPLC/ICP-MS HPLC/ICP-MS HPLC/ICP-MS" GFAAS
16.3 f 0.6
7
GFAAS
chloroform
total As
1.1f 0.2
6
ICP-MS and GFAAS
extracted DORM-1 residue
total As
1.2 f 0.2
5
ICP-MS and GFAAS
sum (DORM-1)
18.7 f 0.8'
"Both areas under Deaks C and D were used for auantitation. *Certifiedtotal As concentration in DORM-1 = 17.7 f 2.1 d a . conversion efficiency was not due to arsenic volatilization during prolonged heating. Despite the discrepancies in observed AB conversion efficiencies, it could be concluded that the major arsenic species in the methanol/water extract exhibited chemical properties no different from those of arsenobetaine. The results of HPLC/ICP-MS, TLC, EIMS, and chemical tests all showed that arsenobetaine is the major As species in the methanol/water fraction of DORM-1. To assess the recovery efficiency of the methanol/chloroform extraction, 35 of arsenic as arsenobetaine was spiked to 2 g of DORM-1, the extraction was performed and arsenobetaine was determined by using HPLC/ICP-MS. The recovery of spike was found to be 101 f 4% (n = 3). The quantitative analysis results are summarized in Table I. Whenever comparison is possible, the agreement between results of ICP-MS and GFAAS is excellent. About 94% of the arsenic in DORM-1 is extractable with methanol/chloroform and about 88% is extracted into the methanol/water phase. Arsenobetaine (84% of total) is the most abundant species in DORM-1; As(III), As(V), MMA, DMA, and AC only constitute 4%. The exact nature of the arsenic fractions (each 6% of total) in the chloroform and the solid (DORM-1 residual) phase is unknown; however, it seems reasonable to presume that arsenic in the chloroform fraction may be lipid bound whereas arsenic in the residue may be associated with proteins. The total
Table 11. Detection Limits method HPLC/ICP-MS FIA"/ICP-MS FIA/ICP-MS GFAAS
species detection limit, pg of As AB AB As(II1)
300
30 30 20
" FIA flow injection analysis (HPLC column removed). arsenic concentration in DORM-1 obtained by summing contributions from all three fractions equals 18.7 f 0.8 pg/g, in good agreement with the certified value of 17.7 f 2.1 pg/g. In the production of DORM-1, the dogfish flesh was extracted with acetone to remove most of the lipids. To ascertain how much arsenic was removed by this process, samples of this extracted oil were digested by use of nitric acid/magnesium nitrate (18) and analyzed by GFAAS. It was found that 9 f 1% arsenic in the dogfish flesh was removed by the acetone extraction. As a result, we may further conclude that arsenobetaine is the predominant arsenic species in the flesh of spiny dogfish. HPLC/ICP-AES is an excellent arsenic speciation technique and quite a few reports testified to it. However, few studies involved real samples and of those even fewer (11,13, 23) involved arsenobetaine. The best limit of detection re-
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Anal. Chem. 1988, 60, 2212-2218
ported for AB was 7.5 ng of As (13),this is 25 times worse than the present AB HPLC detection limit of 300 pg reported in Table 11, largely due to the superior sensitivity of ICP-MS. With the column removed (flow injection mode), the limit of detection of AB improves to 70 pg, almost comparable to that of GFAAS. For AB, which was moderately retained, the detection limit of the HPLC mode was estimated to be about 10 times worse than that of the continuous nebulization mode, despite chromatographic band broadening, increased noise due to shorter measurement time, and the use of relatively high salt content in the mobile phase. ACKNOWLEDGMENT We thank W. R. Cullen, Department of Chemistry, University of British Columbia for synthesizing arsenobetaine and arsenocholine. LITERATURE CITED
Arsenic Symposium, Gaithersburg, MD; Van Nostrand Reinhokl: New York, 1983.
(6) Edmonds, J. S.; Francesconi, K. A.; Cannon, J. R.; Raston, C. L.; Skelton, B. W.; White, A. H. Tetrahedron Len. 1977, 18, 1543-1546. (7) Cannon, J. R.; Edmonds, J. S.; Francesconi, K. A.; Raston, C. L.; Saunders, J. B.; Skebn, B. W.; White, A. H. Aust. J. Chem. 1981, 34, 787-798. (8) Shiomi, K.; Shinagawa, A.; Azumi, M.; Yamananka, H.; Kikuchi, T. Compn. Biochem. Physiol., C: Comp. Pharmacol. 1983, 74C, 393-396. (9) Luten. J. B.; RiekweCBooy, G.; Greef, J. v. d.; ten Noever de Brauw, M. C. Chemosphere 1883, 12, 131-141. (10) Lawrence, J. F.; Michaiik, P.;Tam, G.; Conacher. H. B. S. J . Agic. food Chem. 1986, 34, 315-319. (11) Kurosawa. S.; Yasuda, K.; Taguchi, M.; Yamazaki. S.;Toda, S.; Morita, M.: Uehwo,T.; Fuwa, K. A N c . Bbl. Chem. 1980, 44, 1993-1994. (12) Kaise, T.; Watanabe. S.; Ito, K.; Hanaoka, K.; Tagawa, S.; Hirayama, T.; Fukul, S. Chemosphere 1987, 16, 91-97. (13) Francesconi, K. A.; Micks, P.; Stockton, R. A.; Irgoiic, K. J. Chemosphere 1985, 14, 1443-1453. (14) Matsuto, S.; Stockton, R. A.; Irgolic, K. J. Sci. Total Environ. 1986, 48, 133-140. (15) Norin, H.; Christakopouios. A. Chemosphere 1982, 1 1 , 287-298. (16) Lau, B. P. Y.; Michaiik, P.; Porter, C. J.; Kroiik, S. Biomed. Environ. Mess Spechom. 1987, 14 723-732. (17) Berman, S. S.; Sturgeon, R. E. Fresenius’ 2. Anal. Chem. 1987, 326, 712-715. (18) Siu, K. W. M.; Berman, S. S. Talsnta 1984. 31, 1010-1012. (19) Voth-Beach, L. M.; Shrader, D. E. Spectroscopy (Springfiekl, Oreg.) 1988, 1 , 49-59. (20) Lunde, G. EHP, Environ. Health Perspect. 1977, 19, 47-52. (21) Crecelius, E. A. EHP, Environ. Health Perspect. 1877, 19, 147-150. (22) Edmonds, J. S.; Francesconi, K. A. Nature (London) 1977, 265, 436. (23) Morita, M.; Uehiro, T.; Fuma, K. Anal. Chem. 1981, 53, 1606-1808. ~
(1) Lewis, R. J.; Tatken, R. L. Reg&try of Toxic Effects of Chemical Substances; U.S. Department of Heatth, Education and Welfare: Cincinnati, OH, 1978. (2) Peoples, S. A. Review of Arsenical PestlCMes; Woolson, E. A., Ed.; ACS Symposium Series 7; American Chemical Society: Washington, DC, 1974 pp 1-12. (3) Vahter, M.; Marafante, E.; Dencker, L. Scl. Total Environ. 1983, 30, 197-21 1. (4) Marafante, E.; Vahter, M.; Dencker, L. Sci. Total. Environ. 1984, 34, 223-240. (5) Zingaro, R. A.; Bottino. N. R. Biochemistry of Arsenlc: Recent Developments; Lederer, W.H., Fensterhein, R. J., Eds.; Proceedings of the
RECEIVEDfor review March 14,1988. Accepted July 1,1988.
Anomalous Intensities of Apodized and Unapodized Magnitude Spectra Judy P. Lee, Kim H. Chow, and Melvin B. Comisarow*
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V 6 T 1 Y6
While the valley helght between the peaks of two overlapping spectral lines could be expected to decrease monotonically as the frequency difference between the two lines Increases, In actuality, the valley helght between two overlapping peaks in a Fourier transform magnltude spectrum oscllletes as the frequency between the peaks increases. This phenomenon can cause the apparent magnitude resoiutlon to Increase as the llne separation decreases. A resolution criterion based upon the helght of a valley between two overiapplng peaks is pointless when the phenomenon Is operatlve. When the phenomenon Is present, the apparent position of the spectral lines can be dlsplaced from the true spectral positlon. The severity of the phenomenon Is a function of the windowlng function, the amount of relaxation In the time domain signal, and the peak separation. The presence of the phenomenon can be predlcted from the llne separatlon and can be confirmed by changing the windowlng functlon and by computer matching to the experimental spectrum.
INTRODUCTION Resolution Criteria. Spectral resolution is conventionally defined for a single spectral line as the line width at some chosen fraction of the peak height divided by the spectral
position. For a spectral line located at frequency f , whose line width at some fraction of the peak height is Af, the resolution for the single line could be defined as resolution = f / A f
(1)
An alternate criterion of resolution involves two overlapping lines, where resolution is defined in terms of the height of the valley between the lines. For example, in mass spectrometry it is conventional to define resolution in terms of the peak separation required to give a 10% valley between the peaks (11, whereas in optical spectroscopy it is conventional to define resolution by the Rayleigh criterion (2),which corresponds to a peak separation having an 81.057% ( 8 / 9 )valley between the peaks. Figure 1 illustrates the definitions of resolution discussed above for a single peak (Figure 1a) and two overlapping peaks (Figure Ib,c). It seems intuitively obvious that the valley height, V in Figure 1,would montonically decrease as the separation between the peaks increases. In this work we show that for Fourier transform magnitude spectra this intuition is erroneous and that the valley height, V ,oscillates with increasing peak separation. Moreover, the apparent individual positions of the component peaks are displaced from the true spectral position. Both the displacement and the intensities are also functions of the phase of the time signals that lead to the magnitude spectra. The oscillation phenomenon, which to
0003-2700/S8/0360-2212$01.50/00 1988 American Chemical Society
