Speciation of Alkyllead and Inorganic Lead by Derivatization with

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Anal. Chem. 2000, 72, 1788-1792

Speciation of Alkyllead and Inorganic Lead by Derivatization with Deuterium-Labeled Sodium Tetraethylborate and SPME-GC/MS Xiaomei Yu and Janusz Pawliszyn*

The GuelphsWaterloo Center for Graduate Work in Chemistry, Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada, N2L 3G1

A method for full speciation and determination of alkyllead and inorganic lead(II) in aqueous samples was developed. This was accomplished by in situ derivatization with deuterium-labeled sodium tetraethylborate NaB(C2D5)4 (DSTEB). The derivatization was carried out directly in the aqueous sample and the derivatives were extracted from the headspace by a solid-phase microextraction (SPME) fiber. The extracted analytes were then transferred to a GC/MS or a GC/FID for separation and detection. The research presented demonstrates that SPME and the derivatization reagent DSTEB can be used successfully for the speciation of Pb2+, Pb(CH3)3+, Pb(C2H5)3+, and Pb(C2H5)4 in water samples. All derivatives, Pb(C2D5)4, (CH3)3Pb(C2D5), (C2H5)3Pb(C2D5), and Pb(C2H5)4, are separated using an SBP-5 column. This method was applied to monitor degradation of tetraethyllead in water. This is the first report of ethylation by DSTEB for full speciation of methyllead, ethyllead, and inorganic lead compounds. This approach can be extended to other organometallic compounds as demonstrated for ethyltin speciation. This full speciation method will aid in monitoring occurrence, pathways, toxicity, and biological effects of these compounds in the environment. It is easily adopted for field analysis. The toxicity and bioavailability of organometallic compounds, in addition to their mobility and their impact on the environment, depends not only on their concentration but also significantly on their chemical form. In general, alkylmetals are more toxic than arylmetals. The toxic effects are maximal for the monopositive cations, i.e., the species derived by loss of one organic group from the neutral fully saturated organometalic, viz. R3Pb+, R3Sn+, and CH3Hg+.1 Recently, interest in the environmental pathways of organolead compounds has been growing in order to elucidate the biogeochemical cycle of lead. To understand occurrence, pathways, toxicity, and biological effects of these compounds in the environment, precise analytical methods are needed for determination and speciation of organolead species at ultratrace levels. The techniques most frequently applied for the speciation of organollead compounds are gas or liquid chromatography for the (1) Craig, P. J. Organometallic Compounds in the Environment Principles and Reactions; Wiley: New York, 1986; p 30.

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separation of species coupled to element-specific detection systems, such as atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP AES), microwave-induced plasma atomic emission spectrometry (MIP AES), or inductively coupled plasma mass spectrometry (ICPMS).2 Recent advances in instrumentation have enabled the detection of organolead at the subpicogram level on a routine basis, while in practice severe limitations still remained at the level of sample preparation. Most of the existing procedures are cumbersome, requiring a large amount of sample and several separation/ preconcentration steps.2,3 The well-accepted derivatization method using Grignard alkylation is a multistep and highly time-consuming procedure. The use of hydride generation using sodium tetrahydroborate (NaBH4) offers possibility, but is not suitable for the derivatization of organolead compounds. The hydrides generated tend to dismutate due to their poor stability.2 Ethylation by sodium tetraethyborate (STEB) has been reported for derivatization and determination of metal species such as tin, mercury, lead,4 and some of their organometallic compounds.5 For example, speciation of methyllead ions in water.6 This method, however, cannot be used for full speciation of ethyllead, because both ethyllead and inorganic lead compounds yield Pb(C2H5)4, thus eliminating species-specific information. In situ derivatization of organolead compounds with sodium tetrapropylborate (NaBPr4)7 and tetrabutylammonium tetrabutylborate2 offers an opportunity for the speciation analysis of all relevant organolead species. However, it requires extraction by organic solvent after derivatization and the addition of chelation reagent to first complex the inorganic lead. It cannot simultaneously determine inorganic and organolead compounds. GC-ICPMS and GC-MIP AES have also been used for these methods, although none of these methods is easily to adopted for field analysis. (2) Heisterkamp, M.; Adams, F. C. Fresenius J. Anal. Chem. 1998, 362, 489493. (3) £obinski, R.; Dirkx, W. M. R.; Szpunar-£obinska, J.; Adams, F. C. Anal. Chim. Acta 1994, 286, 381-390. (4) Liu, Y.; Lopez-Avlla, V.; Alcaraz, M. J. High Resolut. Chromatogr. 1994, 17, 527-536. (5) Rapsomanikis, S. Analyst 1994, 119, 1429-1439. (6) Rapsomanikis, S.; Donard, O. F. X.; Weber, J. H. Anal. Chem. 1986, 58, 35-38. (7) De Smaele, T.; Moens, L.; Dams, R.; Sandra, P. J. Chromatogr., A 1998, 793, 99-106. 10.1021/ac990699v CCC: $19.00

© 2000 American Chemical Society Published on Web 03/14/2000

Solid-phase microextraction (SPME)8 is a solvent-free sample preparation and preconcentration method; it is a natural candidate for the extraction of tetraethyllead formed in the above reactions.9 It has been successfully used in the analysis of inorganic lead and tetraethyllead in water using direct10 and headspace extraction for water, blood, and urine samples.11,12 In this paper, we report that deuterium sodium labeled tetraethylborate NaB(C2D5)4 reagent (DSTEB) can be used to overcome these above-mentioned problems. Since the isotope labeled ethyl group does not occur in environmental samples, it can be used for distinguishing the original ethyl group from the introduced ethyl group. It is thus possible to distinguish inorganic lead, triethyllead, diethyllead, tetraethyllead, methyllead, and mixed methylethyllead species simultaneously. EXPERIMENTAL SECTION Instrumentation. SPME devices were obtained from Supelco, Inc. (Bellefonte, PA). Fibers coated with 100-µm-thick poly(dimethylsiloxane) (PDMS) were used. A Varian 3400 gas chromatograph (Varian Associates, Sunnyvale, CA) with a septum programmable injector (SPI) coupled to a Varian Saturn 4D ion trap MS system controlled by a computer with dedicated software was used. Both automatic gain control and fixed ionization time were used. A 15 m × 0.25 mm × 0.25 µm SPB-5 column (Supelco) and helium carrier gas, 1 mL/min, were used in these experiments. A VWR Dylastir magnetic stirrer (VWR Scientific of Canada, Ltd.) and PTFE-coated stir bars were used for stirring the sample. High-purity water was generated by a NANOpure ultrapure water system (Barnstead/Thermolyne, Dubuque, IA) and was used in all experiments. Materials and Methods. Analytical standard lead(II) nitrate was obtained from Aldrich Chemical Co. Deuterium sodium tetraethylborate (DSTEB; 98% by assay) was custom synthesized by Aldrich Chemical Co. Inc. (St. Louis, MO). Tetraethyllead was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Triethyllead, trimethyllead, tetraethytin, triethytin, and diethytin chloride were purchased from Alfa Aesar Chemical Co. (Ward Hill, MA). Acetic acid and sodium acetate were obtained from VWR Scientific (Toronto, ON). The standard lead solution was prepared from analytical reagent grade (99+%) lead nitrate. A stock 1 mg/mL solution was prepared by dissolving 79.9 mg of Pb(NO3)2 in 50 mL of distilled water. Standard solutions of 0.1, 0.01, and 0.001 mg/mL Pb2+ were prepared by appropriate dilutions of the stock solution. Stock solution were stored in 10-mL Teflon vials (Alfa Aesar). Stock 1 mg/mL organolead and organotin standard solutions were prepared separately by adding 10 mg of each compound to a preweighed 15-mL screw cap vial containing 10 mL of methanol. The vials were closed with a Mininter valve (Supelco). Standard solutions of 0.1, 0.01, and 0.001 mg/mL were prepared by appropriate dilutions of the stock solution. Working aqueous solutions were prepared by adding the appropriate volume of each (8) Pawliszyn, J., Ed. Applications of Solid-Phase Microextraction: Royal Society of Chemistry: Cambridge, U.K., 1999. (9) Bayona, J. In Applications of Solid-Phase Microextraction; Pawliszyn, J., Ed.; Royal Society of Chemistry: Cambridge, U.K., 1999; pp 284-295. (10) Tutschiku, S.; Mothes, S.; Wennrich, R. Fresenius’ J. Anal. Chem. 1996, 354, 587. (11) Go´recki, T.; Pawliszyn, J. Anal. Chem. 1996, 68, 3008. (12) Yu, X.; Yuan, H.; Go´recki, T.; Pawliszyn, J. Anal. Chem. 1999, 71, 2998.

Table 1. All Methods and Instrumental Conditions

injector temperature column carrier oven program transfer line ion model mass range scan time segment length Fil/Mul delay peak threshold mass defect background mass

GC Parameters 250 °C 15 m × 0.25 mm × 0.25 µm SPB-5 5 psi, 1 mL/min helium isothermal, 70 °C ITMS Parameters 260 °C EI 204-315 0.500 s 15 min 1.0 min 10 counts 15 mu/100 u m/z 45

SPME Parameters SPME coating 100-µm PDMS sample volume 22 mL vial size 40 mL sample mode headspace stirring rate 1500 rpm reaction/extraction time 10 min desorption time (250 °C) 2 min DSTEB (2%) 400 µL

methanolic solution to 20 mL of purified water and 2 mL of 1 M acetate buffer (pH 4.0) solution. Samples were prepared in 40mL Amber vials with polypropylene hole cap screw tops and PTFE/Silicone septa (Supelco). Derivatization experiments were performed by adding 400 µL of 2% DSTEB to the solution. Samples were stirred at 1500 rpm using a digital hot plate/stirrer (PMC 720 series) with a 1-in. Teflon-coated stir bar (VWR) during extraction. Sampling was performed by exposing the SPME fiber to the headspace over vigorously stirred samples for a predetermined time. After sampling, the fiber was withdrawn into the needle, and the SPME device was transferred to a GC/MS or a GC/FID and desorbed at 250 °C for 2 min. After each analysis, the Teflon vials were rinsed in sequence using tap water and purified water and then baked for 15 min in an oven at 105 °C. All operations involving manipulation of dry DSTEB were performed in a glovebag under dry nitrogen due to its pyrophoric property. The DSTEB solution was stored under refrigeration (4 °C) to limit the degradation of the reagent and maintain the background that comes from the reagent. The reagent can be kept in a refrigerator for 2 days. All instrumental methods and conditions that were used in this study are listed in Table 1. Safety Considerations. Sodium tetraethylborate is hygroscopic, air/moisture sensitive, and flammable; therefore, it should be handled only in a glovebox or a glovebag under inert gas atmosphere. It may cause irritation or burns on contact with skin or eyes, so gloves, lab coat, and safety goggles should be worn when one is working with this compound. It should be stored in a tightly sealed container under inert atmosphere in a desiccator. Organollead and organotin compounds are highly toxic and, therefore, should be handled only in a fume hood, using appropriate protective clothing. Special care should be taken to avoid breathing tetraethyllead and tetraethyltin vapors. They should be stored in a tightly sealed container in a cool dry place. Lead salts are toxic; therefore, chemical protective gear should always be worn when they are handled. Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

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RESULTS AND DISCUSSION Overall reactions with DSTEB proceed as shown below, using ethyllead, methyllead, and inorganic lead as examples. Equations are unbalanced and incomplete because only organolead compounds are considered of interest here.

Pb2+ + NaB(C2D5)4 f (C2D5)4Pb

(1)

Pb(C2H5)22+ + NaB(C2D5)4 f (C2D5)2Pb(C2H5)2 (2) Pb(C2H5)3+ + NaB(C2D5)4 f (C2D5)Pb(C2H5)3

(3)

Pb(CH3)3+ + NaB(C2D5)4 f (C2D5)Pb(CH3)3

(4)

Tetraethyllead formed in reactions 1-3 and trimethylethyllead formed in reaction 4 are volatile compounds that can be easily analyzed by GC. However, they must first be extracted from the aqueous phase. This can be accomplished by purge and trap,13 or by liquid/liquid extraction.6 Considering the volatility of tetraethyllead, the extract is not easily concentrated before injection; therefore, in the latter case, the sensitivity is adversely affected because of the fact that only a small aliquot of the extract can be injected to a GC column. Headspace SPME is ideally suited for preconcentration and convenient introduction. Figure 1a presents the total ion chromatogram for the separation of tetraethyllead (TEL) and derivatives of trimethyllead (TrML), dimethyllead (DiML), inorganic lead (Pb2+), and triethyllead (TrEL) after ethylation with deuterium-labeled sodium tetraethylborate. DiML is an impurity originating from the trimethyllead standard. This chromatogram was obtained from a Varian Saturn (II) GC/MS/MS. From the chromatogram, the total separation time is ∼12 min. Peaks for derivatives of TrML, DiML, and Pb2+ are well separated. Peaks for the TrEL derivative and TEL are poorly resolved. There are two possibilities why these compounds can be separated by a GC column. We first consider the principle of “like dissolves like”, where “like” refers to the polarities of the solute and the immobilized liquid in the column. Polarity is the electrical field effect in the immediate vicinity of a molecule and is measured by the dipole moment of the species. One possible reason may be because the dipole moment of the carbon-hydrogen bond is changed when hydrogen is replaced by deuterium. After the ethyl group of the tetraethyllead is changed to a deuterated ethyl group, the polarity of the molecule changes. Therefore, species with different numbers of deuteriumlabeled ethyl groups show different degrees of affinity toward the stationary phase. Thus, the retention time is changed. Second, we consider the report that deuteration at methyl or methylene, and also carboxyl, will increase the vapor pressure of these compounds.14 If the same concept is applied for deuteration at the ethyl group, the vapor pressure of these species should be (C2D5)4Pb > (C2H5)3Pb(C2D5) > Pb(C2H5)4. The order of the elution should be (C2D5)4Pb, (C2H5)3Pb(C2D5), and then Pb(C2H5)4. This assumption matches the experimental result after these compounds pass through a SPB-5 column. However, further studies are needed to verify these postulates. This chromatogram (13) Rapsomanikis, S. Analyst 1994, 119, 1429. (14) Rock, P. A., Ed. Isotopes and Chemical Principles; ACS Symposium Series 11; American Chemical Society: Washington, DC, 1975; p 112.

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Figure 1. (a) Total ion chromatogram of trimethyllead (TrML), dimethyllead (DiML), inorganic lead (Pb2+), triethyllead (TrEL), and tetraethyllead (TEL) after derivatization with deuterium sodium tetraethylborate. (b) Single-ion chromatogram of inorganic lead derivative (Pb2+, m/z 309). (c) Single-ion chromatogram of triethyllead derivative (TrEL, m/z 299). (d) Single-ion chromatogram of tetraethyllead derivative (TEL, m/z 294). Experimental conditions are described in Table 1.

also shows the possibility of separating some other methyl- or mixed methylethyllead compounds under the same experimental conditions, since the peaks TrML and Pb2+ are well separated. Peaks TrEL and TEL are not completely separated on a 15-m column (see Figure 1a). Using triangulation, the calculated resolution between these two peaks is ∼0.8. This resolution can be improved by the optimization of the separation conditions. However, by plotting single-ion chromatograms, the individual peaks may be distinguished as shown in Figure 1, panels b-d. In these chromatograms, m/z ) 309 is [M]+ - C2D5, and the base peak used for the quantification of Pb2+; m/z ) 299 is [M]+ - C2H5, and the base peak used for the quantification of TrEL; m/z ) 294 is [M]+ - C2H5, and the base peak used for the quantification of TEL. The same experiment was also performed with a Varian 3500 GC using a 60 m × 0.25 mm × 0.25 µm SPB-5 column (Supelco), hydrogen carrier gas at 1 mL/min, and FID as a detector. Experimentally, it was found that better resolution was achieved between peaks TrEL and TEL, but the separation time was 25 min and the sensitivity not as good as GC/MS. This method simplifies the instrument requirements for speciation compared to other methods. It can be easily adopted for field analysis, since transportable GCs are commonly used instruments

Table 2. Method Performance of Analysis Alkyllead and Inorganic Lead in Water Sample

RSD (%; n ) 5) LOD (ppt) R2

Pb2+

TrML

TrEL

TEL

6.58 95 0.9987

3.92 130 0.999

4.61 83 0.9831

5.17 90 0.9982

for field analysis and most transportable GCs are equipped with FID. Since the SPME extraction process is an equilibrium process, the extraction time is an important parameter. Therefore, different extraction/reaction times were investigated using the headspace SPME extraction mode.11 The results (not shown) indicated that after a 5-min extraction/reaction, the equilibrium was reached for TEL, but for TrML, TrEL, and Pb2+, the equilibrium time was longer, requiring ∼10 min. This is because TEL is directly extracted by the PDMS fiber without reaction. The other compounds require reaction with DSTEB prior to extraction. In our experiments, a 10-min extraction/reaction time was used. The blank value of the derivatization reagent and water was studied using 10 min of extraction/reaction time. Experimentally it was found that no peaks corresponding to organollead compounds were extracted from water alone with the derivatization reagent. However, the inorganic lead blank is relatively high. Most likely this is related to derivatization of lead leached from the glass walls11 of the sample vials. This problem was overcome by using custom-made quartz vials. Method performance of the analysis of alkyllead and inorganic lead in water samples is listed in Table 2. The precision of the results for extraction/reaction was estimated for a 80 ppb solution of each compounds and n ) 5 analyses under conditions described previously. RSD values are less than 7%. This precision is good considering the relatively complex character of the speciation. Duplicate five-point calibration curves of organollead and inorganic lead compounds were obtained from 0.1 to 100 ppb, using GC/ MS. A good linear relationship was obtained for each of these compounds. The obtained limits of detection were based on the total ion area count signal-to-noise ratio (S/N) equal 3. These limits of detection reach the requirements for most environment and biological samples. If single-ion area counts were chosen, the limits of detection would be 5-10 times lower. An interesting application of this method is investigation of the decomposition of tetraethyllead. Experimentally, 2 µL of 0.78 mg/mL TEL was spiked into 20 mL of water contained in a 40mL screw top vial with a polypropylene hole cap and PTFE/ Silicone septum (Supelco). Three of these samples were exposed to light at room temperature. After 10 min, 12 h and 24 h, 2 mL of acetate buffer (1 M pH 4) and 400 µL of 2% DSTEB were added followed by SPME extraction/reaction. The experimental results are shown in Figure 2. Figure 2a is the chromatogram of after 10-min exposure, Figure 2b is the chromatogram of after 12-h exposure, and Figure 2c is the chromatogram after 24-h exposure. Figure 2a shows two peaks that are a derivative of Pb2+ after ethylation with DSTEB and the peak of TEL. Thus, there is no decomposition of TEL during 10 min. The lead ion peak comes from the background. However, after 12 h, triethyllead and diethyllead were found, which are decomposition products of TEL.

Figure 2. Chromatograms of light-decomposed tetraethyllead sampled at different times after exposure, after derivatization with deuterium sodium tetrethylborate: (a) 10-min exposure; (b) 12-h exposure; (c) 24-h exposure. Sampled from headspace using 100µm PDMS fiber and analyzed by ITMS. Experimental conditions as described in Table 1.

Figure 2b shows the 12-h chromatogram; all peaks were identified by MS. After 24 h, the peak area counts of triethyllead, diethyllead, and lead ions were increased compared with 12-h exposure. (See Figure 2c). As the decomposition process continues, more TEL is degraded to TrEL. TrEL is then further decomposed to DiEL, DiEL to monoethyllead, and finally to lead ion. The peak area count of TrEL at 24 h was increased by 38% compared to the peak at 12-h exposure. The peak area count of diethyllead did not change much between 12 and 24 h, because diethyllead is less stable in water. It is just an intermediate form of decomposition between TrEL and Pb2+. In these experiments, monoethyllead was not found. As monoethyllead is very unstable in water, the evidence for its presence is only circumstantial.3 Experimentally, the decomposition process of TEL is as follows: tetraethyllead (relatively stable) f triethyllead (relatively stable) f diethyllead (less stable) f inorganic lead. (stable) To demonstrate the versatility of this approach we investigated derivatization and separation of tetraethyltin (TET), triethyltin (TrET), and diethyltin (DiET). Figure 3 shows the total and selected ion chromatograms of these compounds. m/z ) 215 is [M]+ - C2H5, and the base peak used for the quantification of DiET. m/z ) 210 is [M]+ - C2H5, and the base peak used for the quantification of TrET. m/z ) 205 is [M]+ - C2H5, and the Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

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Figure 3. Total and signal-ion chromatograms of a mixture of 140 ppb diethyltin (DiET, m/z 215), 300 ppb triethyltin (TrEL, m/z 210), and 400 ppb tetraethyltin (TET, m/z 205) after derivatization with deuterium-labeled sodium tetraethylborate. Sampled from headspace over an aqueous sample with a 100-µm PDMS fiber and analyzed by ITMS. The experimental conditions as described in Table 1.

base peak used for the quantification of TET. As indicated in the Figure 3, the resolution of these three peaks was not as good as for organolead. The resolution may be improved by optimization of separation conditions. CONCLUSION The research presented here demonstrates that deuteriumlabeled sodium tetraethylborate can be successfully used as a derivatization reagent for simultaneous speciation of inorganic and alkyllead species in water at sub-ppb levels. The derivatives can be extracted by SPME fiber and separated/detected by GC/FID or GC/MS. The detection limits obtained for inorganic lead and

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its alkyllead compounds with the simple experimental setup and instrumentation presented are comparable to those obtained using more sophisticated instrumentation. The precision of the results is good. The method can be easily adopted for field measurement,11 as most transportable GCs are equipped with FID, and the only additional pieces of equipment necessary to perform the experiment are a magnetic stirrer and an SPME holder. This method is also easily adopted for biological sample determinations, such as from urine, blood, and even sediment samples.9,12,15 The SPME method is well suited for the speciation of inorganic and organometallic compounds. Headspace SPME extraction overcomes effects from biological sample matrix and eliminates the potential adverse effects that nonvolatile organic compounds may have on the extraction and chromatographic separation. Sturgeon16 has found that large excesses of Ca2+, Na+, Mg2+, Fe3+, Cr6+, Ni2+, Mn2+, Al3+, or Zn2+ do not affect the derivatization process and that it is only slightly affected by a 1000-fold excess of Cu2+. It has been shown that SPME has the potential for speciation of ethyltin compounds. This approach has potential as a general method for speciation of inorganic and organometallic compounds. This method will be useful in monitoring occurrence, pathways, toxic, and biological effects of organometallic compounds in the environment and biological samples since it can be used to differentiate among all possible species that can be generated in natural systems. ACKNOWLEDGMENT The authors thank Supelco Canada, Varian Inc., the Natural Sciences and Engineering Research Council of Canada, and Institute for National Measurement Standards, Ottawa, Ontario, for financial support. Special thanks is given to Aldrich Chemical Co. Inc. for synthesis of the deuterium-labeled sodium tetraethylborate. Thanks are also given to Drs. Tadeusz Go´recki and Zoltan Mester for their discussions and encouragement. Received for review June 24, 1999. Accepted January 19, 2000. AC990699V (15) Millan, E.; Pawliszyn, J. J. Chromatogr., A 2000, 873, 63. (16) Sturgeon, R. E.; Willie, S. N.; Berman, S. S. Anal. Chem. 1989, 61, 18.