Mercury Speciation Determinations in
Asian Dietary Supplements
Terri Christison, Deepali Mohindra, Frank Hoefler, and Linda Lopez, Thermo Fisher Scientific, Sunnyvale, California, USA
Overview
Purpose: Demonstrate low cost solutions for use in food safety for mercury speciation
and counter anion determinations in solutions extracted from dietary supplements on
an integrated ion chromatography system.
FIGURE 1. Dionex AS-A
(Top Left) shown next t
Integrated IC Systems.
Methods: Mercury species (mg/L) were determined on a mixed anion-cation-exchange
column optimized for metal speciation using a cysteine-perchlorate eluent and detected
by absorbance at 210 nm. Inorganic anions and organic acids were determined on an
anion-exchange high-capacity column optimized for organic acids using electrolytically
generated hydroxide eluent and detected by suppressed conductivity detection.
Results: The Asian supplements had 0.05 to 0.1 wt% extractable amounts primarily of
less toxic inorganic mercury.
Introduction
Multiple patients had clinical symptoms of mercury poisoning and unusually high blood
concentrations (mg/dL) of total mercury after ingesting Asian dietary supplements
adulterated with mercury. Testing of the dietary supplements by Inductively Coupled
Plasma (ICP) showed they contained 10,000-fold higher concentrations of total
mercury (1–10 wt%) than measured in the patients’ blood. Because inorganic and
organic forms of mercury show disparate toxicity, mercury speciation was required to
establish the forms of mercury present in the supplement for determining food safety.
Counter-anion determinations were also needed to calculate mass balance. Ion
chromatography (IC) is well suited for ion separations and provides a well-established
method to determine and quantify metal species and anions.
Methods
Sample Preparation
The Asian dietary supplements were ground to a
coarse powder, extracted in 20 mL of eluent with
manual agitation for 5 min, filtered, diluted with
deionized water prior to injection for mercury speciation
determinations. The samples were treated in the same
manner and extracted with deionized water for counter
anion determinations.
Standard Preparation
The methylmercury and ethylmercury standards were prepared from their mercury
salts (Aldrich) in methanol, and diluted with water to the working standard
concentrations. All other standards were prepared in deionized water.
Instrument
Method 1: Ion Pair Ch
Columns:
Th
Eluent
1.
a
Flow Rate:
1.
Injection Volume:
25
Detection:
U
Method 2: Counter An
Columns:
D
Eluent Source:
Th
Gradient:
1.
15
28
Flow Rate:
1.
Injection Volume:
25
Detection:
S
A
Method 1: Mercury Speciation By Ion Pairing Chromatography
• Thermo Scientific Dionex ICS-1600 Standard Integrated or Dionex ICS-1100
Basic Integrated IC system
• Thermo Scientific Dionex ICS Series Variable Wavelength Detector (VWD)
• Thermo Scientific Dionex AS-AP Autosampler
Method 2: Counter Anion Determinations By Ion Chromatography
• Dionex ICS-2100 Integrated Reagent-Free™ IC (RFIC™) system
• Dionex AS-AP Autosampler
Software
• Thermo Scientific Dionex Chromeleon Chromatography Data System
2 Mercury Speciation Determinations in Asian Dietary Supplements
Safety Warnings
Mercury salts and orga
fatal. Additionally, perch
because it interferes w
safety data sheets for h
review local regulations
ns for use in food safety for mercury speciation
lutions extracted from dietary supplements on
em.
FIGURE 1. Dionex AS-AP Autosampler (Bottom Left), Dionex ICS Series VWD
(Top Left) shown next to Dionex ICS-1600 (Center), and ICS-2100 RFIC (Right)
Integrated IC Systems.
e determined on a mixed anion-cation-exchange
using a cysteine-perchlorate eluent and detected
ions and organic acids were determined on an
optimized for organic acids using electrolytically
ed by suppressed conductivity detection.
ound to a
luent with
ted with
ercury speciation
ted in the same
ater for counter
andards were prepared from their mercury
with water to the working standard
e prepared in deionized water.
Figure 2 shows the se
chromatograms show
methylmercury. Ethylm
FIGURE 2. Separatio
Method 1: Ion Pair Chromatography
Columns:
Thermo Scientific Dionex IonPac CG5A, CS5A, 4 x 250 mm
Eluent
1.0 mM acetic acid, 1.0 mM sodium perchlorate, 5 mM cysteine,
adjusted to pH = 4.0
Flow Rate:
1.75 mL/min
Injection Volume:
25 µL
Detection:
UV Absorbance, 210 nm
Variable Wavelength Detector (VWD)
sampler
ions By Ion Chromatography
45
mAU
1
Method 2: Counter Anion Determinations Using Reagent-Free Ion Chromatography
Columns:
Dionex IonPac™ AG11-HC, AS11-HC, 4 × 250 mm
Eluent Source:
Thermo Scientific Dionex EGC III KOH cartridge
Gradient:
1.0 mM KOH for 8 min, 1–15 mM KOH from 8 to 18 min,
15–20 mM KOH from 18 to 28 min, 30–60 mM KOH from
28 to 38 min, 1 mM KOH from 38 to 45 min
Flow Rate:
1.5 mL/min
Injection Volume:
25 µL
Detection:
Suppressed conductivity, Thermo Scientific Dionex ASRS 300
Anion Self-Regenerating Suppressor, recycle mode, 223 mA
Pairing Chromatography
andard Integrated or Dionex ICS-1100
Method 1: Ion Pairin
The mercury species
chromatography using
mode Dionex IonPac
cation-exchange prop
species. As the analyt
three mercury species
standards from 1 to 1
The ground samples w
throughout the chrom
.05 to 0.1 wt% extractable amounts primarily of
of mercury poisoning and unusually high blood
after ingesting Asian dietary supplements
dietary supplements by Inductively Coupled
0,000-fold higher concentrations of total
the patients’ blood. Because inorganic and
te toxicity, mercury speciation was required to
in the supplement for determining food safety.
so needed to calculate mass balance. Ion
on separations and provides a well-established
l species and anions.
Results
Safety Warnings
Mercury salts and organic mercury compounds are very toxic and sometimes can be
fatal. Additionally, perchlorate used in the eluent is classified as a hazardous chemical
because it interferes with iodide uptake thereby causing hypothyroidism. Review material
safety data sheets for handling precautions, safe storage, and disposal methods, and
review local regulations prior to ordering reagents or analyzing samples.
-5
5
0
Figure 3 shows the se
after extraction in 20 m
sample has moderate
other four samples. N
FIGURE 3. Determin
supplement Numbe
Free™ IC (RFIC™) system
50
Chromatography Data System
mAU
1
-10
0
Thermo Scientific Poster Note • PN70284_E 09/12S 3
5
Minu
(Bottom Left), Dionex ICS Series VWD
600 (Center), and ICS-2100 RFIC (Right)
Results
Method 1: Ion Pairing
The mercury species is separated as a mercury-cysteine complex by ion pair
chromatography using the cysteine perchlorate-based mobile phase on the mixed
mode Dionex IonPac CS5A column. The Dionex IonPac CS5A has both anion- and
cation-exchange properties making it ideal for the separation of transition metal
species. As the analytes elute from the column, they are detected by UV at 210 nm. All
three mercury species evaluated had linear responses replicate injections of four
standards from 1 to 10 mg/L with concentration (r2 > 0.999).
The ground samples were extracted with eluent to ensure that the ions were soluble
throughout the chromatographic separation.
Figure 2 shows the separation of methymercury and inorganic mercury standards. The
chromatograms show that inorganic mercury (Hg2+) is well resolved from
methylmercury. Ethylmercury is not shown.
FIGURE 2. Separation of mercury standards.
Dionex IonPac CG5A, CS5A, 4 x 250 mm
, 1.0 mM sodium perchlorate, 5 mM cysteine,
.0
mAU
1
10 nm
2
B
ons Using Reagent-Free Ion Chromatography
G11-HC, AS11-HC, 4 × 250 mm
Dionex EGC III KOH cartridge
A
-5
10
5
0
15
18
min, 1–15 mM KOH from 8 to 18 min,
om 18 to 28 min, 30–60 mM KOH from
M KOH from 38 to 45 min
ctivity, Thermo Scientific Dionex ASRS 300
rating Suppressor, recycle mode, 223 mA
pounds are very toxic and sometimes can be
e eluent is classified as a hazardous chemical
hereby causing hypothyroidism. Review material
ons, safe storage, and disposal methods, and
reagents or analyzing samples.
Table 1. Summary of m
Sample
Mea
(m
Control
1
3
2
1
3
4
4
5
5
11
Column:
45
Dionex IonPac CG5A, CS5A,
4 mm
Eluent:
1 mM Acetic acid,
1 mM Sodium perchlorate,
5 mM Cysteine, pH 4
Flow Rate:
1.75 mL/min
Column Temp.: 30 °C
Detection:
UV Absorbance, 210 nm
Inj. Volume:
25 µL
Preparation:
A: Deionized water
B: Methanol, working standards
diluted in deionized water
The results of the mercu
Sample 2 had the highes
concentrations of inorgan
Although the results are
devastating if ethylmercu
Peaks:
1.Hg2+
2. Methylmercury
2.5 mg/L
9.3
Method 2: Counter An
Counter anion determin
speciation in the dietary
IonPac AS11-HC anionthis application because
are uncharacterized. Ino
separated using a shallo
possible. The eluent wa
adding water. The chrom
shown in Figure 4. The
with < 50 mg/kg of nitra
FIGURE 4: Determinat
extract of dietary supp
Figure 3 shows the separation of inorganic and organic mercury in dietary supplement 1
after extraction in 20 mL of the acetic acid, sodium perchlorate, and cysteine eluent. This
sample has moderately high concentrations of inorganic mercury as compared with the
other four samples. No methylmercury or ethylmercury was detected.
FIGURE 3. Determination of organic and inorganic mercury in dietary
supplement Number 1.
Dionex IonPac CG5A, CS5A,
4 mm
Eluent:
1 mM Acetic acid,
1 mM Sodium perchlorate,
5 mM Cysteine, pH 4
Flow Rate:
1.75 mL/min
Column Temp.: 30 °C
Detection:
UV Absorbance, 210 nm
Inj. Volume:
25 µL
Sample Prep.: 1:5 dilution with deionized water
20
Column:
50
mAU
1
Peaks:
-10
0
5
10
Minutes
4 Mercury Speciation Determinations in Asian Dietary Supplements
15
18
1. Total Hg2+
3.69 mg/L
52 µg/tablet
0.0586 wt%
µS
1
2
-2
0
10
M
mercury-cysteine complex by ion pair
chlorate-based mobile phase on the mixed
he Dionex IonPac CS5A has both anion- and
deal for the separation of transition metal
column, they are detected by UV at 210 nm. All
near responses replicate injections of four
entration (r2 > 0.999).
th eluent to ensure that the ions were soluble
ation.
ymercury and inorganic mercury standards. The
ercury (Hg2+) is well resolved from
own.
andards.
8
Dionex IonPac CG5A, CS5A,
4 mm
Eluent:
1 mM Acetic acid,
1 mM Sodium perchlorate,
5 mM Cysteine, pH 4
Flow Rate:
1.75 mL/min
Column Temp.: 30 °C
Detection:
UV Absorbance, 210 nm
Inj. Volume:
25 µL
Preparation:
A: Deionized water
B: Methanol, working standards
diluted in deionized water
The results of the mercury speciation determinations are summarized in Table 1.
Sample 2 had the highest concentration and sample 1 the lowest. Only very high
concentrations of inorganic mercury were found; no organic mercury was detected.
Although the results are very high (400 to 2200 mg/kg), the effect could be more
devastating if ethylmercury and methylmercury were present at these concentrations.
Control
Calculated
(wt %)
Calculated
(µg/tablet)
Chloride
Bromide
--
--
--
1
3.69
0.0586
52
ND
Nitrate
2
15.6
0.2265
387
ND
Sulfate
3
4.82
0.0437
117
ND
Oxalate
4
5.31
0.0473
123
ND
5
Phosphate
11.3
0.0708
243
ND
Citrate
Column:
Peaks:
1.Hg2+
2. Methylmercury
2.5 mg/L
9.3
Formate
Methylmercury /
Ethylmercury
--
Method 2: Counter Anion Determinations
Counter anion determinations were needed to conduct a mass balance of the mercury
speciation in the dietary supplement samples. We selected the high capacity Dionex
IonPac AS11-HC anion-exchange column optimized for organic acid determinations for
this application because this column provides the best separations when the samples
are uncharacterized. Inorganic anions and organic acids in these samples were
separated using a shallow potassium hydroxide gradient to resolve as many peaks as
possible. The eluent was electrolytically generated without solution preparation by just
adding water. The chromatographic separation of a water extract from Sample 5 is
shown in Figure 4. The sample has high amounts of chloride and sulfate (~ 700 mg/kg)
with < 50 mg/kg of nitrate, oxalate, phosphate, and citrate.
Conclusion

These tests ide
supplements wa
amounts found

IC with Inductiv
speciation with
 ICP analysis
when only o
element whe
 IC analysis s
speciation w
FIGURE 4: Determinations of inorganic anions and organic acids in a water
extract of dietary supplement Number 5.
and inorganic mercury in dietary
Dionex IonPac CG5A, CS5A,
4 mm
Eluent:
1 mM Acetic acid,
1 mM Sodium perchlorate,
5 mM Cysteine, pH 4
Flow Rate:
1.75 mL/min
Column Temp.: 30 °C
Detection:
UV Absorbance, 210 nm
Inj. Volume:
25 µL
Sample Prep.: 1:5 dilution with deionized water
20
Column:
Peaks:
1. Total Hg2+
3.69 mg/L
52 µg/tablet
0.0586 wt%
3
µS
7
6
45
1
2
-2
0
10
20
Minutes
8
Dionex IonPac AG11-HC,
AS11-HC, 4 mm
Eluent Source: Dionex EGC III KOH
Gradient:
1 mM from 0–8 min,
1–15 mM from 8–18 min,
15–30 mM from 18–28 min,
30–60 mM from 28–38 min
Temperature:
30 °C
Flow Rate:
1.5 mL/min
Inj. Volume:
25 µL
Detection:
Suppressed conductivity,
Dionex ASRS™ 300, 4 mm,
recycle mode
Sample Prep.: 5 min extraction in deionized
water, settle 5 min, filter,
dilute 1:5
total wt %
Peaks:
1. Acetate
—
2. Formate
—
3. Chloride
0.716
10
4. Bromide
—
9
5. Nitrate
0.142
6. Carbonate
—
7. Sulfate
0.662
8. Oxalate
0.043
9. Phosphate
0.025
30
35
10. Citrate
0.039

Mercury specia

Inorganic anion
easily and accu
Reagent-Free I
•
For more inform
references 1–4
Columns:
anic and organic mercury in dietary supplement 1
cid, sodium perchlorate, and cysteine eluent. This
tions of inorganic mercury as compared with the
or ethylmercury was detected.
8
Acetate
Inorganic Mercury
Measured
(mg/L)
Table 2. Summary of
Counter Anion
(wt %)
Table 1. Summary of mercury speciation determinations.
Sample
The counter anions re
primarily chloride and
References
1. Sarzanini, C.; S
Chromatograph
Spectrometric D
Mercury, Anal. C
2. Dickson, H.R; P
Levels of Mercu
Cambridge, UK
3. Christison, T.; H
Synergies of Io
Mercury Contam
Sunnyvale, CA,
4. Dionex Applicat
Levels in High P
Fisher Scientific
For research purposes only,
All trademarks are the prope
This information is not intend
intellectual property rights of
PO70284_E 09/12S
Thermo Scientific Poster Note • PN70284_E 09/12S 5
rminations are summarized in Table 1.
nd sample 1 the lowest. Only very high
ound; no organic mercury was detected.
2200 mg/kg), the effect could be more
cury were present at these concentrations.
Table 2. Summary of counter anion results.
Counter Anion
(wt %)
n determinations.
Acetate
Mercury
ated
%)
The counter anions results are summarized in Table 2. The counter anions are
primarily chloride and sulfate. Sample 2 has the highest results.
Sample #
1
2
3
4
5
0.012
0.389
0.014
0.008
--
Calculated
(µg/tablet)
Methylmercury /
Ethylmercury
Formate
0.002
0.019
--
--
--
Chloride
0.080
1.624
0.128
0.254
0.716
--
--
Bromide
--
--
0.093
0.192
--
52
ND
Nitrate
0.102
0.507
0.061
0.201
0.142
0.240
1.818
0.196
0.554
0.662
0.003
0.026
0.005
0.011
0.043
86
65
387
ND
Sulfate
37
117
ND
Oxalate
73
123
ND
Phosphate
--
0.005
--
0.070
0.025
08
243
ND
Citrate
--
0.050
0.018
0.042
0.039
ns
ed to conduct a mass balance of the mercury
ples. We selected the high capacity Dionex
optimized for organic acid determinations for
des the best separations when the samples
d organic acids in these samples were
roxide gradient to resolve as many peaks as
enerated without solution preparation by just
ation of a water extract from Sample 5 is
mounts of chloride and sulfate (~ 700 mg/kg)
hate, and citrate.
Conclusion

These tests identified that the majority of the soluble mercury in the dietary
supplements was inorganic mercury (Hg2+), corresponding to the sub-percent
amounts found in the patients.

IC with Inductively Coupled Plasma (ICP) provides a total solution by combining
speciation with total elemental content.
 ICP analysis screens samples for unknown metal contamination especially
when only one species is present, but also provides the total amount of an
element when more than one species is present.
 IC analysis separates metal species thereby providing important mercury
speciation when multiple species are present.
anions and organic acids in a water
5.
Dionex IonPac AG11-HC,
AS11-HC, 4 mm
Eluent Source: Dionex EGC III KOH
Gradient:
1 mM from 0–8 min,
1–15 mM from 8–18 min,
15–30 mM from 18–28 min,
30–60 mM from 28–38 min
Temperature:
30 °C
Flow Rate:
1.5 mL/min
Inj. Volume:
25 µL
Detection:
Suppressed conductivity,
Dionex ASRS™ 300, 4 mm,
recycle mode
Sample Prep.: 5 min extraction in deionized
water, settle 5 min, filter,
dilute 1:5
total wt %
Peaks:
1. Acetate
—
2. Formate
—
3. Chloride
0.716
10
4. Bromide
—
9
5. Nitrate
0.142
6. Carbonate
—
7. Sulfate
0.662
8. Oxalate
0.043
9. Phosphate
0.025
30
35
10. Citrate
0.039

Mercury speciation determinations define the potential toxicity in food sources.

Inorganic anions and organic acids needed for mass balance calculations are
easily and accurately determined by anion-exchange chromatography with a
Reagent-Free IC system.
•
For more information on this method and mercury analysis methods, see
references 1–4.
Columns:
References
1. Sarzanini, C.; Sacchero, G.; Aceto, M.; Aboilino, O.; Mentasti, Ed. Ion
Chromatographic Separation and On-Line Cold Vapour Atomic Absorption
Spectrometric Determination of Methylmercury, Ethylmercury and Inorganic
Mercury, Anal. Chim. Acta, 1993, 00, 1–7.
2. Dickson, H.R; Price, R. Application Note AN40992 Accurate Analysis of Low
Levels of Mercury in Fish by Vapor Generation AA, Thermo Fisher Scientific,
Cambridge, UK, 2010.
3. Christison, T.; Hoefler, F.; Lopez, L. Application Note AN43130 Combining the
Synergies of Ion Chromatography and Inductively Coupled Plasma to Identify
Mercury Contamination in Herbal Medicines, Thermo Fisher Scientific,
Sunnyvale, CA, 2012.
4. Dionex Application Note AN 131, Determination of Transition Metals at PPT
Levels in High Purity Water and SC-2 (D-Clean) Baths, LPN 1058, Thermo
Fisher Scientific, Sunnyvale, CA, 1998.
For research purposes only, not for clinical diagnostics.
All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
PO70284_E 09/12S
6 Mercury Speciation Determinations in Asian Dietary Supplements
www.thermofisher.com/dionex
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