30 1982 - 2012 anniversary Assessing the fate of mercury ... · Assessing the fate of mercury in...
1
Assessing the fate of mercury in animal inoculaons: simultaneous determinaon of ethylmercury and methylmercury in pig livers and kidneys J. E. Creswell, A. Carter, M. Briscoe, M. Kennard-Mayer, B. Setzer Brooks Rand Labs, Seale WA USA ® 3 0 1982 - 2012 anniversary th Introduction Ethylmercury (EtHg) is a major component of thimerosal (50% by weight), a vaccine preservative that has been in use since the 1930s. 1 It has known neurotoxic effects that are similar to those of methylmercury (MeHg), but unlike methylmercury its tissue deposition and clearance rates in organisms are not well-understood. 2 In 1999, the U.S. Food and Drug Administration urged vaccine manufacturers to eliminate thimerosal from vaccines as soon as possible, 3 and almost all U.S. vaccines are free of thimerosal today; however, the preservative is still common in many vaccines used outside North America and Europe. Animal studies have shown that mammalian liver and kidney tissue are sites of accumulation of mercury originating in thimerosal-containing vaccines; 4–6 however, this topic has received relatively little research attention. In order to better understand the tissue deposition patterns of mercury originating from vaccines, a rapid, sensitive, simple analytical method for determining mercury species in biological tissues is required. In this project, we developed a method for the simultaneous determination of ethylmercury and methylmercury using the same analytical instrumentation widely used for methylmercury determination by U.S. EPA Method 1630. Unlike other analytical techniques for ethylmercury, this method can be readily implemented by a wide range of laboratories without purchasing new equipment or significant retraining of analysts. References 1. Pless, R.; Risher, J. F. The Journal of Pediatrics 2000, 136, 571–573. 2. Clarkson, T. W.; Magos, L. Critical Reviews in Toxicology 2006, 36, 609–662. 3. U.S. Food and Drug Administration Vaccine Safety & Availability > Thimerosal in Vaccines http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/VaccineSafety/UCM096228 (accessed Oct 17, 2012). 4. Rodrigues, J.; Serpeloni, J.; Batista, B.; Souza, S.; Barbosa, F. Archives of Toxicology 2010, 84, 891–896. 5. Burbacher, T. M.; Shen, D. D.; Liberato, N.; Grant, K. S.; Cernichiari, E.; Clarkson, T. Environmental Health Perspectives 2005, 113, 1015–1021. 6. Magos, L.; Brown, A.; Sparrow, S.; Bailey, E.; Snowden, R.; Skipp, W. Arch. Toxicol. 1985, 57, 260–267. 7. Rodrigues, J. L.; Alvarez, C. R.; Fariñas, N. R.; Berzas Nevado, J. J.; Barbosa Jr, F.; Rodríguez Martín-Doimeadios, R. C. Journal of Analytical Atomic Spectrometry 2011, 26, 436. 8. Gibičar, D.; Logar, M.; Horvat, N.; Marn-Pernat, A.; Ponikvar, R.; Horvat, M. Analytical and Bioanalytical Chemistry 2007, 388, 329–340. 9. Bloom, N. Can. J. Fish. Aquat. Sci. 1992, 49, 1010–1017. Conclusions • We have developed an effective method for the simultaneous determination of ethylmercury and methylmercury in liver and kidney samples that can be carried out using sample preparation equipment and analytical instrumentation common in laboratories that analyze methylmercury and total mercury by U.S. EPA methods. • Methods developed for the analysis of blood and other bodily fluids are not necessarily applicable to tissue analysis due to their inability to disperse solid particles. • There is a need for an animal tissue reference material certified for ethylmercury, in order to better validate this and other mercury speciation methods for biological tissues. • Substitution of sodium (tetra-n-propyl) borate for sodium tetraethyl borate is an effective way to extend analytical equipment designed for methylmercury analysis to the analysis of ethylmercury as well. Results of Sample Analyses We analyzed a large batch of cryogenically ground kidney samples and did not detect methylmercury in any of them. This is consistent with the finding of other researchers that the kidneys accumulate considerably more ethylmercury than methylmercury. Ethylmercury breaks down rapidly to inorganic mercury (Hg +2 ) in the body, and a large fraction of the resulting inorganic mercury is found in the kidneys. 2,6 In order to quantify inorganic mercury, we made total mercury measurements, following U.S. EPA Method 1631 Appendix, and calculated the inorganic mercury concentrations by difference. Inorganic mercury made up the majority of the total mercury measured in these samples (Figure 7), with ethylmercury representing between 0.6%-30.1% of total mercury concentrations. The quality control results from this analytical batch are in Table 2. 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Concentraons (ng/g) Sample Number Hg +2 EtHg Figure 7: Sample Results QC Type Ethylmercury Methylmercury Mean NIST 955c SRM Recoveries 86.8% 84.0% Mean Matrix Spike Recoveries 106.9% 81.2% Mean Matrix Spike Duplicate RPD 6.0% 10.6% Mean Sample Duplicate RPD 12.7% non-detects Table 2: Quality Control Results Preparation Methods We analyzed homogenized pig liver and kidney samples, cryogenically ground pig kidney samples, and NIST Standard Reference Material 955c: Toxic Elements in Caprine Blood. We compared several methods of sample digestion as described in Table 1. Table 1: Sample Preparation Methods Method Name Step 1 Step 2 Step 3 TMAH Digestion 7 Add ~0.3 g sample and 2 mL 25% tetramethylammonium hydroxide (TMAH) solution to a glass vial. Bring volume to 10 mL with DI water. Seal the vial and heat to 65 °C in an oven for 4 hours. Cool to room temperature. Bring volume of samples up to constant level with DI water, to correct for volume loss. DCM Extraction 8 Add sample and 5% H 2 SO 4 + 18% KBr solution to a digestion vessel with a 1:4 (wt:v) ratio. Add 1 M CuSO 4 solution in a 1:5 (v:v) ratio with the H 2 SO 4 /KBr solution. Add dichloromethane in a 2:1 (wt:v) ratio with the H 2 SO 4 /KBr solution. Shake for 1 hour and let stand overnight. Centrifuge at 3,000 RPM for 15 mins. Separate the organic phase into a Teflon ® bottle and add DI water in a ~5:2 (v:v) ratio. Heat to 70 °C until dichloromethane evaporates. KOH/MeOH Digestion 9 Add ~0.1 g sample and 1 mL 25% KOH/Methanol solution (wt/v) to a Teflon ® digestion vial. Seal the vial and heat to 65 °C in an oven for 4 hours. Remove from the oven and bring the volume to 2.5 mL with methanol. Store at room temperature for 5 days prior to analysis. KOH/MeOH Digestion + Distillation Follow the KOH/MeOH Digestion procedure above. Transfer the digestate to a distillation vial. Distill following EPA Method 1630 for water samples. Results of Method Development TMAH Extraction Rejected due to matrix interference. Blank spike recoveries (Figure 3) were acceptable, but standard reference material and matrix spike recoveries (Figures 4 & 5) were unacceptably low. DCM Extraction Rejected due to incomplete digestion of liver and kidney matrix. Blank spike, standard reference material, and matrix spike recoveries (Figures 3-5) were generally within acceptable ranges for this method, but there was poor dispersion of tissue particles. Because there is no certified reference material for ethylmercury in a solid tissue matrix, we could not be certain that this method was extracting all ethylmercury from the solid samples. KOH/MeOH Digestion + DCM Extraction Rejected due to poor blank spike and matrix spike recoveries (Figures 3 & 5). KOH/MeOH Digestion + Distillation Accepted. This method produced acceptable blank spikes, standard reference material recoveries and matrix spike recoveries (Figures 3-5). It also produced the lowest consistent method detection limit (Figure 6). The detection limit for this method was calculated differently than for the others and may slightly exaggerate the precision. For the other methods, the detection limits were calculated using spiked liver samples, while for this method, it was calculated using blank spikes. However, the average precision of matrix spike duplicates for this method (6.0% for ethylmercury, 10.6% for methylmercury) is considerably better than the average precision of the other methods (range: 10.6%-50.3%). This sample digestion method selectively extracts organic mercury species from the matrix and leaves behind inorganic mercury. Figure 3: Blank Spike Recoveries 0% 20% 40% 60% 80% 100% 120% EtHg MeHg Figure 4: NIST SRM 955c Recoveries 0% 20% 40% 60% 80% 100% 120% 140% EtHg MeHg Figure 5: Matrix Spike Recoveries 0% 20% 40% 60% 80% 100% 120% EtHg MeHg Figure 6: Detection Limits (ng/g) 0 0.1 0.2 0.3 0.4 0.5 0.6 EtHg MeHg Legend for Figures 3-6 TMAH Digestion DCM Extraction KOH/MeOH Digestion + DCM Extraction KOH/MeOH Digestion + Distillation Analytical Methods We analyzed all samples by pH buffering, aqueous phase propylation, purge and trap, chromatographic separation, thermal decomposition, and cold vapor atomic fluorescence spectrometry using a Brooks Rand MERX ® Methylmercury Analyzer (Figure 1). The MERX ® system is compatible with U.S. EPA Method 1630 and it should be possible to analyze samples by the method developed in this project on any Method 1630-compatible analytical system. This method differed from Method 1630 principally in its use of sodium (tetra-n-propyl) borate as a derivatizing reagent instead of sodium tetraethyl borate. It is not possible to quantify ethylmercury when derivatizing with sodium tetraethyl borate. With sodium tetraethyl borate, divalent mercury and ethylmercury derivatize as the same molecule and can’t be chromatographically separated. With sodium (tetra-n-propyl) borate, divalent mercury and ethylmercury derivatize as different molecules and can be separated (Figure 2). Other analytical changes included operating the gas chromatograph and a higher temperature and with a higher carrier gas flow rate than for methylmercury analysis and lengthening the run time to allow all species to elute. We found no difference in the performance of the methods analyzed between homogenized liver/kidney samples and cryogenically ground kidney samples.We did however find a difference in method performance between the tissue samples and the blood standard reference material. Figure 2: Ethylation vs. Propylation Ethylation Propylation Hg 0 elemental mercury Hg 0 elemental mercury Hg 0 elemental mercury Hg +2 divalent mercury CH 3 CH 2 HgCH 2 CH 3 diethylmercury dipropylmercury CH 3 CH 2 CH 2 HgCH 2 CH 2 CH 3 CH 3 Hg methylmercury CH 3 HgCH 2 CH 3 methylethylmercury CH 3 HgCH 2 CH 2 CH 3 methylpropylmercury CH 3 CH 2 Hg ethylmercury CH 3 CH 2 HgCH 2 CH 3 diethylmercury CH 3 CH 2 HgCH 2 CH 2 CH 3 ethylpropylmercury Species Figure 1: MERX ® Methylmercury Analyzer
Transcript of 30 1982 - 2012 anniversary Assessing the fate of mercury ... · Assessing the fate of mercury in...
Assessing the fate of mercury in animal inoculations: simultaneous determination of ethylmercury and
methylmercury in pig livers and kidneysJ. E. Creswell, A. Carter, M. Briscoe, M. Kennard-Mayer, B. Setzer
Brooks Rand Labs, Seattle WA USA
®
301982 - 2012
anniversaryth
Introduction
Ethylmercury (EtHg) is a major component of thimerosal (50% by weight), a vaccine preservative that has been in use since the 1930s.1 It has known neurotoxic effects that are similar to those of methylmercury (MeHg), but unlike methylmercury its tissue deposition and clearance rates in organisms are not well-understood.2
In 1999, the U.S. Food and Drug Administration urged vaccine manufacturers to eliminate thimerosal from vaccines as soon as possible,3 and almost all U.S. vaccines are free of thimerosal today; however, the preservative is still common in many vaccines used outside North America and Europe.
Animal studies have shown that mammalian liver and kidney tissue are sites of accumulation of mercury originating in thimerosal-containing vaccines;4–6 however, this topic has received relatively little research attention.
In order to better understand the tissue deposition patterns of mercury originating from vaccines, a rapid, sensitive, simple analytical method for determining mercury species in biological tissues is required.
In this project, we developed a method for the simultaneous determination of ethylmercury and methylmercury using the same analytical instrumentation widely used for methylmercury determination by U.S. EPA Method 1630.
Unlike other analytical techniques for ethylmercury, this method can be readily implemented by a wide range of laboratories without purchasing new equipment or significant retraining of analysts.
References
1. Pless, R.; Risher, J. F. The Journal of Pediatrics 2000, 136, 571–573.2. Clarkson, T. W.; Magos, L. Critical Reviews in Toxicology 2006, 36, 609–662.3. U.S. Food and Drug Administration Vaccine Safety & Availability > Thimerosal in Vaccines http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/VaccineSafety/UCM096228 (accessed Oct 17, 2012).4. Rodrigues, J.; Serpeloni, J.; Batista, B.; Souza, S.; Barbosa, F. Archives of Toxicology 2010, 84, 891–896.5. Burbacher, T. M.; Shen, D. D.; Liberato, N.; Grant, K. S.; Cernichiari, E.; Clarkson, T. Environmental Health Perspectives 2005, 113, 1015–1021.6. Magos, L.; Brown, A.; Sparrow, S.; Bailey, E.; Snowden, R.; Skipp, W. Arch. Toxicol. 1985, 57, 260–267.7. Rodrigues, J. L.; Alvarez, C. R.; Fariñas, N. R.; Berzas Nevado, J. J.; Barbosa Jr, F.; Rodríguez Martín-Doimeadios, R. C. Journal of Analytical Atomic Spectrometry 2011, 26, 436.8. Gibičar, D.; Logar, M.; Horvat, N.; Marn-Pernat, A.; Ponikvar, R.; Horvat, M. Analytical and Bioanalytical Chemistry 2007, 388, 329–340.9. Bloom, N. Can. J. Fish. Aquat. Sci. 1992, 49, 1010–1017.
Conclusions
• We have developed an effective method for the simultaneous determination of ethylmercury and methylmercury in liver and kidney samples that can be carried out using sample preparation equipment and analytical instrumentation common in laboratories that analyze methylmercury and total mercury by U.S. EPA methods.
• Methods developed for the analysis of blood and other bodily fluids are not necessarily applicable to tissue analysis due to their inability to disperse solid particles.
• There is a need for an animal tissue reference material certified for ethylmercury, in order to better validate this and other mercury speciation methods for biological tissues.
• Substitution of sodium (tetra-n-propyl) borate for sodium tetraethyl borate is an effective way to extend analytical equipment designed for methylmercury analysis to the analysis of ethylmercury as well.
Results of Sample Analyses
We analyzed a large batch of cryogenically ground kidney samples and did not detect methylmercury in any of them. This is consistent with the finding of other researchers that the kidneys accumulate considerably more ethylmercury than methylmercury. Ethylmercury breaks down rapidly to inorganic mercury (Hg+2) in the body, and a large fraction of the resulting inorganic mercury is found in the kidneys.2,6 In order to quantify inorganic mercury, we made total mercury measurements, following U.S. EPA Method 1631 Appendix, and calculated the inorganic mercury concentrations by difference. Inorganic mercury made up the majority of the total mercury measured in these samples (Figure 7), with ethylmercury representing between 0.6%-30.1% of total mercury concentrations. The quality control results from this analytical batch are in Table 2.
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Conc
entr
ation
s (ng
/g)
Sample Number
Hg+2
EtHg
Figure 7: Sample Results
QC Type Ethylmercury MethylmercuryMean NIST 955c SRM Recoveries 86.8% 84.0%Mean Matrix Spike Recoveries 106.9% 81.2%Mean Matrix Spike Duplicate RPD 6.0% 10.6%Mean Sample Duplicate RPD 12.7% non-detects
Table 2: Quality Control Results
Preparation Methods
We analyzed homogenized pig liver and kidney samples, cryogenically ground pig kidney samples, and NIST Standard Reference Material 955c: Toxic Elements in Caprine Blood. We compared several methods of sample digestion as described in Table 1.
Table 1: Sample Preparation Methods
Method Name Step 1 Step 2 Step 3
TMAH Digestion7
Add ~0.3 g sample and 2 mL 25% tetramethylammonium hydroxide (TMAH) solution to a glass vial. Bring volume to 10 mL with DI water.
Seal the vial and heat to 65 °C in an oven for 4 hours. Cool to room temperature.
Bring volume of samples up to constant level with DI water, to correct for volume loss.
DCM Extraction8
Add sample and 5% H2SO4 + 18% KBr solution to a digestion vessel with a 1:4 (wt:v) ratio. Add 1 M CuSO4 solution in a 1:5 (v:v) ratio with the H2SO4/KBr solution.
Add dichloromethane in a 2:1 (wt:v) ratio with the H2SO4/KBr solution. Shake for 1 hour and let stand overnight.
Centrifuge at 3,000 RPM for 15 mins. Separate the organic phase into a Teflon® bottle and add DI water in a ~5:2 (v:v) ratio. Heat to 70 °C until dichloromethane evaporates.
KOH/MeOH Digestion9
Add ~0.1 g sample and 1 mL 25% KOH/Methanol solution (wt/v) to a Teflon® digestion vial.
Seal the vial and heat to 65 °C in an oven for 4 hours. Remove from the oven and bring the volume to 2.5 mL with methanol.
Store at room temperature for 5 days prior to analysis.
KOH/MeOH Digestion + Distillation Follow the KOH/MeOH Digestion procedure above.
Transfer the digestate to a distillation vial.
Distill following EPA Method 1630 for water samples.
Results of Method Development
TMAH ExtractionRejected due to matrix interference. Blank spike recoveries (Figure 3) were acceptable, but standard reference material and matrix spike recoveries (Figures 4 & 5) were unacceptably low.
DCM ExtractionRejected due to incomplete digestion of liver and kidney matrix. Blank spike, standard reference material, and matrix spike recoveries (Figures 3-5) were generally within acceptable ranges for this method, but there was poor dispersion of tissue particles. Because there is no certified reference material for ethylmercury in a solid tissue matrix, we could not be certain that this method was extracting all ethylmercury from the solid samples.
KOH/MeOH Digestion + DCM ExtractionRejected due to poor blank spike and matrix spike recoveries (Figures 3 & 5).
KOH/MeOH Digestion + DistillationAccepted. This method produced acceptable blank spikes, standard reference material recoveries and matrix spike recoveries (Figures 3-5). It also produced the lowest consistent method detection limit (Figure 6). The detection limit for this method was calculated differently than for the others and may slightly exaggerate the precision. For the other methods, the detection limits were calculated using spiked liver samples, while for this method, it was calculated using blank spikes. However, the average precision of matrix spike duplicates for this method (6.0% for ethylmercury, 10.6% for methylmercury) is considerably better than the average precision of the other methods (range: 10.6%-50.3%). This sample digestion method selectively extracts organic mercury species from the matrix and leaves behind inorganic mercury.
Figure 3: Blank Spike Recoveries
0%
20%
40%
60%
80%
100%
120%
EtHg MeHg
Figure 4: NIST SRM 955c Recoveries
0%
20%
40%
60%
80%
100%
120%
140%
EtHg MeHg
Figure 5: Matrix Spike Recoveries
0%
20%
40%
60%
80%
100%
120%
EtHg MeHg
Figure 6: Detection Limits (ng/g)
0
0.1
0.2
0.3
0.4
0.5
0.6
EtHg MeHg
Legend for Figures 3-6
TMAH Digestion
DCM Extraction
KOH/MeOH Digestion + DCM Extraction
KOH/MeOH Digestion + Distillation
Analytical Methods
We analyzed all samples by pH buffering, aqueous phase propylation, purge and trap, chromatographic separation, thermal decomposition, and cold vapor atomic fluorescence spectrometry using a Brooks Rand MERX® Methylmercury Analyzer (Figure 1).
The MERX® system is compatible with U.S. EPA Method 1630 and it should be possible to analyze samples by the method developed in this project on any Method 1630-compatible analytical system.
This method differed from Method 1630 principally in its use of sodium (tetra-n-propyl) borate as a derivatizing reagent instead of sodium tetraethyl borate.
It is not possible to quantify ethylmercury when derivatizing with sodium tetraethyl borate. With sodium tetraethyl borate, divalent mercury and ethylmercury derivatize as the same molecule and can’t be chromatographically separated. With sodium (tetra-n-propyl) borate, divalent mercury and ethylmercury derivatize as different molecules and can be separated (Figure 2).
Other analytical changes included operating the gas chromatograph and a higher temperature and with a higher carrier gas flow rate than for methylmercury analysis and lengthening the run time to allow all species to elute.
We found no difference in the performance of the methods analyzed between homogenized liver/kidney samples and cryogenically ground kidney samples.We did however find a difference in method performance between the tissue samples and the blood standard reference material.
Figure 2: Ethylation vs. Propylation
Ethylation Propylation
Hg0
elemental mercuryHg0
elemental mercuryHg0
elemental mercury
Hg+2
divalent mercury
CH3CH2HgCH2CH3diethylmercury dipropylmercury
CH3CH2CH2HgCH2CH2CH3
CH3Hgmethylmercury
CH3HgCH2CH3methylethylmercury
CH3HgCH2CH2CH3methylpropylmercury
CH3CH2Hgethylmercury
CH3CH2HgCH2CH3diethylmercury
CH3CH2HgCH2CH2CH3ethylpropylmercury
Species
Figure 1: MERX® Methylmercury Analyzer
