Anal. Chem. 1988, 6 0 , 2587-2590
2587
Determination of Organomercury in Biological Reference Materials by Inductively Coupled Plasma Mass Spectrometry Using Flow Injection Analysis’ Diane Beauchemin,* K. W.M.Siu, a n d S. S. B e r m a n Analytical Chemistry Section, Chemistry Division, National Research Council of Canada, Ottawa, Ontario, Canada KIA OR9
Inductively coupled plasma mass spectrometry was used for the determination of organomercury in two marine biological standard reference materials for trace metals (dogflsh muscle tissue DORM-1 and lobster hepatopancreas TORT-1). I n most parts of thls study, the organomercury was extracted as the chloride from the materlai with toluene and back extracted Into an aqueous medium of cysteine acetate. Since the final extracts contained more than 4 % sodium, Isotope dilution and flow lnjectlon analysls were used to respectively counter the effect of concomttant elements and avold clogging the Interface. Comparison of resuns with gas chromatography shows that the only significant organomercury Is methyimercury. At least 93 % of mercury in DORM-1 and 39 % of mercury In TORT-1 exist as methylmercury.
There is an increasing demand for sensitive and rapid techniques that not only can determine the total concentration of individual elements but also can identify and quantify their chemical forms. This becomes especially important when the chemical forms involved are among the most dangerous substances in the environment. Organomercury (OrgHg) species belong to this group; not only are they toxic but they accumulate in marine organisms. It is thus important to develop analytical methods that can reliably determine OrgHg in biological materials. So far, however, only a few techniques are available. The most widely used are based on the conversion of the organically bound mercury to its halide derivative by acidification with hydrogen halide, isolation of the OrgHg halide by multiple extractions with benzene or toluene, and analysis using gas chromatography with electron capture detection (GC-ECD). These are thoroughly reviewed in ref 1. Although other techniques have been used, such as GC coupled to mass spectrometry (2) and GC with microwave-induced plasma emission detection (3),they are not as widespread as GC-ECD. Yet, results by more than one reliable technique are often required (e.g., for the certification of reference materials). This has led us to consider inductively coupled plasma mass spectrometry (ICP-MS) for the determination of OrgHg in extracts of the marine biological reference material DCRM-1 (dogfish muscle) and TORT-1 (lobster hepatopancreas). ICP-MS is a technique with many features, which have been summarized in four recent review articles (4-7).The features of main interest for the determination of Hg are its low detection limit, its freedom from spectral interferences and the possibility to do isotope dilution analysis. However, ICP-MS does not allow by itself the determination of a particular chemical species. It is also best suited for the analysis of *Author to whom correspondence should be sent. Present address: Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada. NRCC Publication No. 29615.
aqueous solutions. Procedures for extracting and separating OrgHg from inorganic mercury for GC-ECD determination are well established (1). Two of these were adapted for this study (8,9).However, most of the work was carried out with the procedure described in ref 21, in which the toluene extracts prepared for the analysis by GC-ECD were treated with a cysteine acetate solution (1)to extract the OrgHg back into an aqueous medium. These cysteine extracts had to be analyzed directly because of the low Hg level involved (the certified total Hg concentration in DORM-1 is 0.798 f 0.074 g / g ) , which raised a problem because the Na content in these extracts is about 4.3%. This is indeed much higher than the highest level recommended of 0.2%, if a solution is to be continuously nebulized for any length of time without inducing instrumental drift caused by solid deposition on the orifice (6). Flow injection analysis (FIA) was therefore considered because it can lessen the problem of solid sample deposition on the torch injector tip and on the mass spectrometer sampling interface (10,11). FIA was successfully used by Hutton and co-workers (12) for the introduction of 2% NaCl and alumina matrices into the ICP-MS for several hours without problem. Finally, effects of concomitant elements were expected to be caused by Na, as several workers have reported significant suppression or enhancement effects in the presence of Na (13-16). The isotope dilution technique was thus used since it is the most efficient way of countering these effects (6). In summary, this work will describe the extraction of OrgHg from biological materials with the subsequent determination of Hg by isotope dilution ICP-MS and sample introduction by FIA. EXPERIMENTAL SECTION Instrumentation. The inductively coupled plasma mass spectrometer used for this work was the Perkin-Elmer SCIEX ELAN 250 (Thornhill, ON, Canada). Four modifications were made to the originally supplied instrument. A mass flow controller (Model 5850, Brooks Instrument Division, Emerson Electric, Hatfield, PA) was added to the nebulizer gas line and a peristaltic pump (Minipuls 11,Gilson Medical Electronics Inc., Middleton, WI) was used to maintain the sample delivery to the nebulizer at 1.1mL/min. Also, a conventional inductively coupled plasma atomic emission spectroscopy torch was used instead of the approximately 15 mm longer one that was provided with the instrument. Finally, a x,y,z translation stage (17) was installed under the torch box to allow precise and reproducible translation of the torch box in three dimensions. The operating conditions used throughout this work are summarized in Table I. Under these conditions, the sampling height (or sampling depth), defined as the distance between the tip of the sampler and the initial radiation zone (18)observed while aspirating a lo00 mg/L Y solution (19),was about 10 mm. The elutions were recorded in real time and stored on a hard disk with the “multiple elements” software provided with the instrument. The measurements were made while in the multichannel mode where the data were recorded by peak hopping rapidly from one mass to the other and staying only a short time (called dwell time) of 20 ma at each mass, until the total measurement time of 0.06 s was reached. Three measurements were
0003-2700/88/0360-2587$01.50/00 1988 American Chemical Society
2588
ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
Table I. ICP-MS Operating Conditions torch rf power reflected power plasma gas flow auxiliary gas flow nebulizer gas flow
Plasma Conditions conventional ICP-AES 1.2 kW
55 w 14 L/min 2.0 L/min 1.04 L/min
Mass Spectrometer Settings -6.1 to -7.7 v Bessel box stop Bessel box barrel 3.5 v Einzel lenses 1 & 3 -15.1 to -15.3 V Einzel lens 2 -130 V Bessel box end lenses -8.6 V sampler orifice diameter 1.14 mm skimmer orifice diameter 0.89 mm interface running pressure 0.8 Torr mass spectrometer running pressure (3.1-5.2) X Torr made per peak (one measurement being done at the central mass while the two others were done at A0.05 u from the assumed peak center). A resolution of 0.8 u (peak width) at 10% of peak height was used throughout. Two ions were continuously monitored (mlz 201 and 202). In these measuring conditions, a data point was generated every second. Flow Injection Setup. A sample injection valve (Rheodyne, Inc., Cotati, CA) was installed on the sample delivery l i e , between the peristaltic pump and the nebulizer. In the load mode, the sample was injected into the loop while either 0.1 M HN03 or deionized distilled water (carrier)was being continuously pumped to the nebulizer. In the injection mode, the carrier was routed through the loop and delivered the sample to the nebulizer. For most of the work, a 100-pL injection loop was used although a 250-pL loop was tried during the initial setup. Reagents. All acids were purified by subboiling distillation in a quartz still (20). The enriched 201Hgisotope used for the stable isotope dilution analysis was purchased from the Oak Ridge National Laboratory. It was dissolved as previously described (21) and its concentration was checked by reverse spiking. Toluene and acetone (Caledon, Georgetown, ON, Canada) were of “distilled in glass” grade. The marine biological reference materials DORM-1 and TORT-1 are partially defatted protein powders produced by spray drying and acetone extraction of homogenized dogfish muscle and lobster tomalley, respectively. (Complete information on the procurement of the marine biological reference material DORM-1 and other marine reference materials can be obtained from S. Berman, Marine Analytical Chemistry Standards Program, Division of Chemistry, National Research Council of Canada, Ottawa, Canada K1A OR6.) The 5 M HCl solution used to convert OrgHg to the chloride derivative was purified, immediately prior to use, by carrying out two extractions with toluene (see the extraction procedure below) and discarding the toluene extracts. To assess percent recovery, a 10 pg/mL Hg (as methylmercury (MeHg)) standard solution was used for spiking; this was prepared by dissolving MeHg chloride (either I.C.N., Plainview, NY, or P & B, Waterbury, CT) in toluene. The cysteine acetate solution was prepared as in ref 22. Half a gam of cysteine hydrochloride monohydrate (Fisher Scientific, Fair Lawn, NJ), 0.338 g of sodium acetate (Anachemia, Montreal, PQ, Canada), and 6.25 g of anhydrous sodium sulfate (Anachemia), all reagent grade, were dissolved in 50 mL of deionized distilled water. The following was used for the second extraction procedure, which was tried in an attempt to quantify the inorganic Hg as well as MeHg (in TORT-1). The CuS04 solution was prepared by dissolving 12.5 g of CuS04.5H20 (Anachemia) in 500 mL of deionized distilled water (DDW). The sodium thiosulfate solution was prepared by dissolving 0.124 g of Na2&O36H20(Anachemia) in 100 mL of DDW and then adding 100 mL of absolute ethanol (National Research Council of Canada, Ottawa, ON, Canada). Extraction Procedure. Six different pairs of samples were prepared, each pair consisting of an unspiked sample, and of a sample spiked with 0.8 pg of Hg/g (as MeHg). This was done
to enable the calculation of the recovery of the extraction procedure. For the most parts of this study, the extraction procedure recommended by the Association of Official Analytical Chemists (8)was used with two modifications. They were as follows: toluene was used instead of benzene and the OrgHg concentration step by solvent evaporation was omitted. In each case, 2 g of material was washed 3 times with acetone and once with toluene; it was then acidified with 10 mL of the HCl solution (with or without the MeHg spike added) and extracted into three 20-mL aliquots of toluene, which were then combined and diluted to 100 mL with toluene. (This toluene solution was analyzed directly for OrgHg by GC-ECD.) Since ICP-MS is best suited for the analysis of aqueous solutions, OrgHg was back extracted into an aqueous solution by shaking a 20-mL aliquot of the toluene solution with 5 mL of cysteine acetate solution. Respectively 36.77 and 73.54 ng of 201Hgwere then added to 3-mL aliquots of the unspiked and the spiked extracts for their isotope dilution analysis by ICP-MS. Since the cysteine solution was prepared from reagent grade products, it served as a blank and was thus also analyzed by isotope dilution (with 18.78 ng of 201Hg being added per milliliter of cysteine solution). It should be noted that the addition of the 201Hgspike could not be made right at the beginning of the extractions because it was not in the form of OrgHg. Alternative Extraction Procedure. Another extraction procedure was also used, in an attempt to quantify inorganic Hg as well as OrgHg in TORT-I. It is based on a modified procedure the major difference being the reof Uthe and co-workers (9), placement of NaI with NaBr. This resulted in the formation of relatively more stable OrgHg bromide. Similar to the first procedure, several pairs of samples were prepared, each pair consisting of an unspike sample and a sample spiked with 0.2 pg of Hg/g (as MeHg). In each case, 2 g of material was shaken in a centrifuging tube with 8 mL of CuS04 solution and 4 mL of 2.5 M HBr and extracted with three 8-mL aliquots of toluene. Phase separation was accelerated by centrifugation at 2000 rpm for 5 min and the toluene aliquots were then combined. OrgHg was back extracted into an aqueous/ethanol solution by shaking the toluene extracts with 7 mL of sodium thiosulfate solution and centrifuging for 5 min at 2000 rpm. With this procedure, the inorganic Hg remained in the CuSo, phase, while the OrgHg was extracted into the sodium thiosulfate phase. GC-ECD showed that recovery of OrgHg was identical in both procedures (see Table IV). Only the CuSo, extracts were analyzed (for inorganic mercury), using isotope dilution ICP-MS (by spiking them with 19.1 ng of 201Hg). Analysis Procedure. Instrument Operating Conditions. Both the ion lens voltages of the instrument and the plasma operating conditions were chosen so as to maximize the sensitivity for Hg (see Table I). Data Treatment. The raw intensities were transferred to a VAX-11 computer (Digital Equipment Corp., Maynard, MA) and processed by using programs written in FORTRAN. The intensities recorded for the 100 pg/L Hg standard solution were f i t smoothed with a seven-point SavitzkyGolay moving window; background correction was performed by using points on both sides of the peak. The area and peak height of each peak were then measured. For the determination of the detection limit, the mean and standard deviation of several (typically 10) injections of 0.1 M HNO, were calculated. Finally, for the samples, the point-by-point ratioing of the intensity of 202Hgover that of zolHg was carried out, after which the mean of the ratios measured during each peak was calculated and used in the subsequent isotope dilution calculations. Isotope Dilution Calculations. The analyte concentration in the extracts was calculated by using the following formula: M&(A, - E$) C= V(ER - A) where C is the analyte concentration in the extract (nanograms per milliliter), M , is the mass of the stable isotope spike (nanograms), V is the volume of extract used (milliliters), A is the natural abundance of 202Hg(the reference isotope), which is 29.80% (23),B is the natural abundance of 201Hg(the spike isotope), which is 13.22% (23), A, is the abundance of the reference isotope in the mlHg spike, E, is the abundance of the spike isotope in the 201Hgspike, K is the ratio of the natural and spike atomic
ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
0.0
1.0
2.0
Flgure 1. Intensities of *"Hg (-) and mHg (...) as a function of time, during five consecutive 100-pL injections of a 100 pg/L Hg standard solution, obtained by FIA ICP-MS. Table 11. Sensitivity ((ions/s)/(ag/L)) and Detection Limit (pg/L) with and without FIA
(n = 126)
aolHgsensitivity 20.4 f 1.1" aolHgdetection limit 2.9 ao2Hgsensitivity 45.1 f 2.0 ao%Igdetection limit 1.9
5.0
6.0
7.0
8.0
10.0
9.0
Figure 2. Analysis of two cysteine acetate extracts (both with the appropriate amount of mlH added for their isotope dilution analysis), showing the intensities of "Hg (-) and "'Hg (.-.) monitored as a function of time, during three consecutive 100-pL injections of both an unspiked sample and a MeHgspiked sample.
Table 111. Concentration (ag/g) of OrgHg in DORM-1
FIA,
FIA,
100 p L
250 p L
(15 injections) (8injections) 9.9 f 1.1 4.8 22.1 f 2.7 2.7
12.0 f 1.4 4.0 27.1 f 3.2 2.2
" Precision expressed as the standard deviation. weights, and R is the ratio (reference isotope/spike isotope) measured after the addition of lolHg. The result of the isotope dilution analysis of the cysteine blank was subtracted from those of the extracts. The resulting concentration was finally converted to micrograms per gram by using the following formula:
c,
4.0
time (min)
tiny (rnin)
continuous run (mean over 2 min)
3.0
2589
CVfV, = v,w x 1000
where C, is the analyte concentration in the biological material (micrograms per gram), C was defined above, Vf is the volume of cysteine acetate solution (milliliters),Vi is the volume of toluene in which OrgHg was initially extracted (milliliters), V, is the volume of toluene extract that was extracted with cysteine acetate (milliliters), and W is the weight of biological material (grams).
RESULTS AND DISCUSSION Figures of Merit of FIA ICP-MS for Hg. The reproducibility of five 100-pL injections of 100 pg/L Hg standard solution is illustrated in Figure 1. The relative standard deviation (RSD) is about 7% for both zolHg and 20zHg. I t remained essentially the same throughout this work. Although a huge suppression was observed while running the samples, as will be seen later, the signal nonetheless came back to its original suppression-free value as soon as delivery of the cysteine extract ceased and a solution free of concomitant element was run. This behavior was also encountered in a previous work (14) and further supports the observation that a heavy analyte such as Hg is less affected by concomitant elements than a light one. The sensitivities and detection limits (based on 3a) for the two isotopes,are reported in Table 11, for measurements made with a 100-pL loop, with a 250-pL loop, and by continuously running the solution. It should be noted that this comparison was done while using exactly the same operating conditions and with the standard solution running through the flow injection system for the continuous mode. The detection limit in the continuous mode (for the major isotope of Hg) is degraded from its usual range of 0.1-0.9 pg/L because of the
mean f std dev
% re1 std
technique
dev
n
recovery
FIA ICP-MS GC-ECD" CV-AASb recommended valuec
0.721 f 0.033 0.730 f 0.035 0.728 i 0.061 0.74 f 0.06d
4.6 4.8 8.4
6 10 6
80f4 100 i 5 75h9
%
" Reports MeHg value obtained by using the first extraction procedure (same as for FIA ICP-MS but without the back extraction step with cysteine acetate). bCold vapor atomic absorption spectrometry, with the first extraction procedure (same aa for FIA ICP-MS, including the back extraction step with cysteine acetate). CRecommendedvalue for MeHg in DORM-1. Confidence interval (95% level). short measurement time used in order to sample enough pointa to define each peak and to perform the measurement of the two Hg isotopes as simultaneously as possible for the isotope dilution analysis. Nonetheless, it can be seen that there is a loss in sensitivity and detection limit of less than a factor of 2 in switching from the continuous mode to FIA. Since there was little difference between the results for the two injection loops, the 100-pL loop was chosen in order to minimize solid deposition on the sampling interface. The MIHg/mHg ratio of the 100 pg/L Hg standard solution was also measured to assess mass discrimination, prior to the isotope dilution analysis. The mean ratio (n =4) for the 250-pL loop (used at the beginning of this work, before running any sample), was 2.263 f 0.027 (the precision is expressed as the standard deviation). For the 100-pL loop, ratios of 2.256 f 0.069 (n = 5) and 2.259 f 0.051 (n = 4) were obtained before and after running the samples, respectively. The expected value being 2.254 (231, it is obvious that the ratio was as expected in all cases and that masa discrimination is negligible. Concentration of OrgHg in DORM-1. A typical run (after polynomial smoothing) recorded as a sample was being delivered by FIA to the ICP-MS is shown in Figure 2. As can be seen, it is quite noisy, which is not surprising, considering the high concentration of dissolved solids involved. Furthermore, the extent of the effect of concomitant elements can be evaluated by making an estimate of the Hg concentrations in the unspiked and spiked samples, using the 100 pg/L standard solution run just before the samples. The concentrations found for the unspiked and the spiked samples are, respectively, 6.3 f 1.4 and 11.9 f 1.9 pg/L whereas they should be 45.6 and 93.2 pg/L (these values take into account a MeHg spike recovery of 8070,according to Table 111). The Hg signal is suppressed by a factor of about 8 by concomitant
2590 Table
ANALYTICAL CHEMISTRY, VOL. 60, NO.
IV.
C o n c e n t r a t i o n (pg/g) of
technique
23, DECEMBER 1, 1988
OrgHg in TORT-1 % recovery
mean f s t d dev
n
FIA ICP-MS GC-ECD" GC-ECDb
0.13 f 0.02 0.13 f 0.03 0.14 f 0.02
3
83 f
8 9
100 f 5 100 f 5
recommended valuec
0.13 f 0.02d
3
a Reports M e H g value obtained by using t h e f i r s t extraction procedure (same as f o r FIA ICP-MS but w i t h o u t t h e back extract i o n step w i t h cysteine acetate). *Reports M e H g value obtained by using t h e second extraction procedure (also used for t h e quant i t a t i o n o f inorganic Hg). cRecommended value f o r M e H g in DORM-I. dConfidence i n t e r v a l (95% level).
elements in the samples. Finally, it should be noted that only the mean of the 202Hg/201Hg ratios measured at the apex of each peak was used for the isotope dilution calculations. The concentration of OrgHg in DORM-1 obtained by FIA ICP-MS is compared to that found by other techniques practiced in this laboratory in Table 111,along with the number of determinations done and the recovery (which was used to scale up the corresponding concentration) obtained in each case. The agreement between the different techniques and the recommended value is excellent. As well, since the value reported for GC-ECD is that of methylmercury, it would appear that the only significant OrgHg species in DORM-1 is MeHg. Therefore, the recommended value (which includes the results of Table I11 along with those of oubide laboratories) issued for DORM-1 is for MeHg. Finally, since the certified value for the total Hg in DORM-1 is 0.798 f 0.074 pg/g, the percentage of MeHg in this material is approximately 93%. Concentration of OrgHg in TORT-1. The concentration of OrgHg in TORT-1 obtained by FIA ICP-MS is compared to that found by GC-ECD (using the two extraction procedures described earlier) in Table IV, along with the number of determinations done and the recovery (which was used to scale up the corresponding concentration) obtained in each case. As for DORM-1, the agreement between the two techniques and the recommended value is excellent. It is also obvious that the two extraction procedures yielded identical results. Since the certified value for the total Hg in TORT-1 is 0.33 f 0.06 pg/g, the percentage of MeHg in this material is only 39%. Unlike DORM-1, MeHg is not the principal species in TORT-1, which raised the question as to what the other
species may be. The isotope dilution ICP-MS analysis of the CuS04 extracts (from the second extraction procedure) gave a value of 0.13 f 0.01 pg of Hg/g as inorganic Hg. This made a total of 0.26 f 0.03 pg/g Hg (inorganic and MeHg), which compares reasonably well with the certified total Hg content of TORT-1. Work is currently under way to apply these procedures to other materials.
ACKNOWLEDGMENT The authors thank P. Maxwell and V. P. Clancy for the extractions of OrgHg from the biological material DORM-1 and determinations by GC-ECD and CV-AAS.
LITERATURE CITED (1) Crompton, T. R. Gas Chromatographyof Organometallic Compounds;
Plenum: New York, 1982; pp 37-73. (2) West%, G.; Johansson, E.; Rykager, R. Acta Chim. S c a d . 1970, 2 4 , 2349. (3) Decadt, Ghlslain; Baeyens, Wllly; Bradley, Dennis; Goeyens, Leo Anal. Chem. 1985, 5 7 , 2788-2791. (4) Douglas, Donald J.; Houk, Robert S. Prog. Anal. At. Spectrosc. 1985, 8 , 1-18. (5) Gray, Alan L. Spectrochim. Acta, Part8 1985, 408. 1525-1537. (6) Houk, Robert S. Anal. Chem. 1988, 5 8 , 97A-105A. (7) Gray, A. L. Fresenius' 2.Anal. Chem. 1986, 324, 561-570. (8) OM&/ Methods of Anaksis, 14th ed.;Wllllams, S.. Ed.; Association of Official Analytical Chemists: Arlington, VA, 1984; pp 472-473. (9) Uthe, J. F.; Solomon, J.; Grift, 8. J. Assoc. Off. Anal. Chem. 1972, 5 5 , 583. (IO) McLeod, Cameron W. J. Anal. At. Spectrom. 1987, 2 , 549-552. (11) Thompson, J. J.; Houk, R. S. Anal. Chem. 1988, 5 8 , 2541-2548. (12) Hutton, R. C.; Gordon, J. S.; Tye, C. T. XXV Colloquium Spectroscopicum Internationale, Toronto, ON, Canada, June 1987; Paper E1.8. (13) Olivares, J. A.; Houk. R. S. Anal. Chem. 1988, 5 8 , 20-25. (14) Beauchemin, Diane; McLaren, J. W.; Berman S. S. Spectrochlm. Acta, Part 8 1987, 428, 467-490. (15) Gregoire, D. C. Spectrochlm. Acta Part 8 1987, 428. 985-907. (16) Tan, S. H.; Horlick, G. J. Anal. At. Specfrom. 1987, 2 , 745-763. (17) Beauchemin, Diane: McLaren, J. W.; Berman, S. S. J. Anal. At. Spectrom., In press. (18) Kolrtyohann, S. R.; Jones, J. S.; Jester, C. P.; Yates, D. A. Spectrochim. Acta, Part 8 1981, 368,49-59. (19) Beauchernin, Diane: McLaren, James. ICP Inf. Newsl. 1985, 7 7 , 441-446. (20) Dabeka, R. W.; Mykytiuk, A. P.; Berman, S. S.; Russel, D. S. Anal. Chem. 1976. 48, 1203-1207. (21) McLaren, J. W.; Beauchemin, Diane; Berman, S. S. Anal. Chem. 1987, 5 9 , 610-613. (22) Kamps, L. R.; McMahon, E. J. Assoc. Off. Anal. Chem. 1972, 55, 590-595. . . ~ . ~ . (23) De BiBvre, Paul; Gallet, Marc; Hoklen, Norman E.; Barnes, I . Lynus J . Phys. Chem. Ref. Data 1984, 13, 809-891.
RECEIVED for review June 6, 1988. Accepted September 1, 1988.
