CLIN. CHEM. 25/5, 769-772 (1979)
Direct Determination of Mercury in Blood by Use of Sodium
BorohydrideReduction and Atomic Absorption Spectrophotometry
Dinesh C. Sharma1 and Peter S. Davis
A method is presented for the rapid determination of total
mercury in blood. The reagent used is alkaline sodium
borohydride, and no digestionof the sample is needed. The
detection limit and sensitivity are 1.15 and 1.59 ng of Hg,
respectively. The method gives reproducible results (the
CV ranged from 5.3 to 6.7% for low and high mercury
blood samples, respectively), comparable to those obtained by the digestion method. The mean analytical recoveries of added mercuric chloride and methylmercuric
chloride were 106.65 and 99.02%, respectively. Other
advantages of the method are freedom from contamination
encountered with digestion methods and elimination of
matrix effects.
Additional Keyphrases:
trace elements
.
environmental
hazards
Mercuric
Clarkson
chloride.
Prepare
as described
by Magos and
(4).
Stock solution (500 mg of Hg per liter): Dissolve 676.7 mg
of mercuric chloride (B.D.H. Analar) in 50 g/L H2S04 and
dilute to 1 L with the same acid.
Dilute stock solution (500 zg of Hg per liter): Dilute 1 mL
of the stock solution to 1 L with water that contains 9.0 g of
NaCI, 754.5 mg of disodium dihydrogen
ethylenediaminetetraacetate, and 0.1 g of L-cysteine. The dilute stock solution
is stable for at least six months if stored refrigerated.
Working standard solution: Prepare further dilutions from
the dilute stock solution in fresh water, daily, as required.
Met hylmercuric
chloride. Prepare suitable dilutions from
methylmercuric
chloride
standard
solution
in water (Alfa
Products,
Ventron Corp., Danvers, MA 01923).
Antifoaming
agent. Tri-n-butyl
phosphate
(B.D.H.).
Apparatus
The estimation of mercury in blood has acquired increasing
importance
because of current concern about environmental
mercury contamination, and because blood is readily sampled
and reflects the extent of exposure (1).
Most of the published methods of blood mercury assay require wet digestion of the sample as a necessary preliminary
to cold-vapor atomic absorption spectrophotometry,
a technique surpassed for sensitivity only by neutron activation
analysis. All these methods suffer from disadvantages inherent
in wet digestion and oxidation procedures, such as high reagent blanks, and in the case of mercury the difficulties are
compounded
by the extreme volatility of the element itself
and many of its compounds (2, 3).
To our knowledge, the only published method capable of
directly determining mercury in blood without resort to prior
destruction of organic matter is that of Magos and Clarkson
(4).
We report another method for the direct determination
of
blood mercury, in which sodium borohydride is used as a reductant. The method is sensitive, simple,and rapid.
Materials and Methods
Reagents
Sodium borohydride.
Prepare
a 50 g/L solution of sodium
borohydride
(Merck) in 1 molfL NaOH just before use. The
manufacturer’s
instructions
for the safe handling of this
compound must be strictly observed.
The University of Adelaide Department
laide Hospital, Adelaide, South Australia
of Medicine,
5000.
1 Present address: Department
of Biochemistry,
College, Jaipur, India.
Received June 20, 1977; accepted Mar. 2, 1979.
Royal Ade-
S.M.S. Medical
An atomic absorption spectrophotometer
(Varian Techtron,
North Springvale,
3171 Australia), Model AA-6, was used. The
air-acetylene
burner was replaced by an absorption cell with
quartz end windows as described in the instrument
manual.
In practice, most commercially available atomic absorption
instruments
can be used for the cold-vapor
technique
as
suggested by Gilbert and Hume (5). The apparatus was assembled as shown in Figure 1. The reaction tube and the
drying tube were both 15 )< 2.5 cm hard-glass test tubes. The
air inlet to both these tubes had a fine jet tip. The inlet to the
reaction tube was connected to the air supply through a
flowmeter; the outlet of the drying tube was connected to the
absorption
cell. All connections
were made with polyvinyl
chloride tubing of minimum convenient length.
Procedure
Measure 2.0 mL of heparinized whole blood into the reaction tube. Add 3.0 mL of glass-distilled
water followed by 1
drop of antifoam. Then add 1.0 mL of sodium borohydride
reagent; immediately cap the tube and stir vigorously for 2 mm
on a vortex-type stirrer. Read the absorbance peak by turning
on the air supply (Test). When the absorbance reading has
almost declined to zero or background, turn off the air supply.
Remove the cap and add a known amount of mercuric chloride
solution by Eppendorf micropipet so as to give an absorbance
reading about equal to that of the test. Stir for 30s, then purge
the solution with air and note the absorbance peak height
(Standard).
Remove the rubber stopper and cap it onto another tube containing distilled water alone and pass air to
purge any residual mercury vapor from the system. Turn off
the air when the absorption reading returns to zero or to the
background level. Remove the stopper and the system is ready
for the next sample. For “blank” or “background” reading take
CLINICAL
CHEMISTRY,
Vol. 25, No. 5, 1979
769
air
flow
meter
_
-i-:.___ii
-
-+
-4
__
I
-4
‘Hi’
I
vent
-
.
crushed ice
-5
reaction tube
drying tube
Fig. 1. Diagram of the apparatus
5.0 mL of glass-distilled
water containing 1 drop of antifoaming agent and add 1.0 mL of sodium borohydride reagent,
then purge with air to obtain the peak reading (Blank). Calculate the total mercury from the following relationship:
Total blood mercury
= (test
X
blank)/(standard
- blank)
(ng Hg added/vol.ofblood,mL)
-
If many samples are to be run, a calibration curve can be
constructed and the values of the unknown samples can be
speedily determined by a reference to this curve.
The following instrument arrangements
were used: mercury
hollow-cathode lamp; lamp current, 3 mA; wavelength, 253.7
nm; slit width, 0.5 mm; scale expansion, Xl; and air flow rate,
3 L/min.
Notes:
1. All glassware must be adequately
cleaned to
minimize
contamination.
2. The peak
absorbance,
and hence
the sensitivity
of the
method, depends upon severalfactors,
some of which are the
final
volume and surface area of the liquid inthereaction
tube,
airflow,lengthofabsorptioncell,
and thedead volume ofthe
apparatus (6), so all of these variablesmust be kept constant.
3. The high air-flushing rate is advantageous,
as it causes
the peak to be attained rapidly with less residual mercury to
cause memory effects.
4. It is essential to flush the system efficiently to get rid of
residual mercury before introducing the next sample.
5. Because the rate of release of mercury from the reaction
tube is temperature dependent, all reagents should be allowed
to reach room temperature
before use.
6. Organomercurial compounds
other than methylmercury
compounds may or may not be measured by this method.
Results and Discussion
Analytical Conditions
Sodium borohydride, a powerful reducing agent, reduces
acids, esters, acid chlorides, disulfides, nitriles, and inorganic
anions. Although it reacts violently with water to liberate
770
CLINICAL
CHEMISTRY,Vol. 25, No. 5, 1979
flammable
gases,
the
aqueous
solutions
are stable
in the
presence of small amounts of sodium hydroxide and can be
kept for several days. Although reduction can be carried out
under neutral, basic, or acidic conditions, use of a pH between
9 and 10 results in a reaction rate suitable for most analytical
purposes (7).
It is now well established
that mercury
in blood is present
predominantly as methylmercury
and to a lesser extent as
inorganic mercury (8). The method reported here was based
on the knowledge that attempts to prepare a hydride of mercury by the reaction of an alkyl mercury compound with a
complex metal hydride have led to the rapid production of
mercury (9). Similarly, the reaction with diphenylmercury
results in the immediate separation of elemental mercury.
Apart from this, sodium borohydride is known to reduce a
variety of compounds containing sulfur-including
disulfides,
trisulfides, and tetrasulfides-to
mercaptans,
and mercury
salts to metal. Recently Cohen and Schrier (10) used this reagent to remove mercury from fish protein concentrates,and
Toffaletti and Savory (11) to determine the total mercury
content of urine. We have satisfactorily applied it to the direct
determination
of mercury in blood samples and have developed a method that is simple and rapid.
The calibration curve with mercuric chloride standards
containing 0 to 400 ng of mercury is given in Figure 2. The
curve is linear to 300 ng, but beyond this value it bends
slightly.
The stirring
methylmercuric
time
we
chloride
used
was
standards
1 mm. In the case of
it is essential
to create
conditions
by adding a few drops of 500 g/L sulfuric
acid; otherwise,
no reduction
takes place. In the presence of
acid there is an instantaneous
evolution of hydrogen, which
would release mercury and drive away some of the mercury
vapor, and soit is necessary to work in a closed system. Sodium borohydride solution was added to the reaction tube from
a syringe by passing the needle through the rubber stopper
of the reaction tube. Because the reduction in the acid medium
is very rapid, it is necessary to stir for only 30 s.
It may appear to be paradoxical that mercury from a pure
acidic
-
/
Table 1. Analytical Recovery of Mercury Added
to Blood8
Hg added,
HOCI2
MMC1’
HOCI2
MMCb
10
20
50
100
200
82.35
79.00
89.20
87.67
89.21
66.22
58.51
61.20
74.88
62.89
118.70
123.90
104.70
100.30
97.10
100.00
117.30
103.70
94.30
96.60
300
101.86
93.05
88.90
70.46
72.96
66.73
99.10
102.80
106.65
ng/2 mL blood
400
Mean
MERCURY
(,g)
Fig. 2. Calibration curve
standard solution of a methylmercury
salt is not released by
alkaline sodium borohydride
without the prior addition of
acid, whereas reduction does take place in the case of blood
or methylmercury
added to blood. The phenomenon
can be
explained
on the basis of the amphoteric
character
of the
proteins of plasma and blood cells. In alkaline medium these
proteins behave as protein anions, which furnish the protons
essential for reduction
of methylmercury
to metallic mercury.
This is supported
by the fact that polarographic
reduction
of
R-Hg-X compounds
proceeds in two distinct steps (12) with
formation
of an intermediate
free radical.
R-Hg-X
R-Hg.
It is not possible
+ e
+ e
-
+ H
to distinguish
R-Hg.
-
+ X
R-H + Hg
between
organic
and inor-
ganic mercury in the presence of blood proteins, because alkalone sodium borohydride releases mercury from both forms.
In the absence of a proton donor, only inorganic mercury salts
liberate mercury by this reagent. Thus this method cannot be
extended to distinguish between industrial and environmental
exposure.
Analytical
Recovery, % (mean of duplicate determinations)
1-mm stIrring
2-mm stirrIng
92.70
88.60
99.02
B
Blood Hg in 15 unexposed persons was 3.004 ± 2.012 gIL.
b
Methylmercuric chloride.
the time course of mercury
release from this compound
is
considered
(Figure 3). Stirring for 1 mm resulted in the release
of only 75% of the added mercury; 2 mm of stirring was necessary to complete the release of mercury. On the other hand,
the release of mercury from mercuric chloride was complete
even after 30 s of stirring. Accordingly,
a 2-mm stirring time
is suggested.
In this connection
it is pertinent
to mention that Kubasik
et al. (13) recovered
only some 45% of mercury
when
[203Hg]methylmercuric
chloride was added to whole blood and
digested
overnight
with sulfuric
acid and potassium
permanganate;
in contrast,
the recovery
of added
inorganic
mercury exceeded
96%.
Precision.
The precision or reproducibility
of the method
was tested by replicate
analysis of blood samples from two
individuals
known to have high and low blood-mercury
values,
respectively,
and calculating
the standard
deviation
of the
results of nine replicate analyses of each. The standard deviations were 0.18 (mean value 2.70) tg of Hg per liter in the
low range and 0.77 (mean value 14.59) g of Hg per liter in the
high blood-mercury
range. Precision was slightly less when
the calibration curve was used, the corresponding values being
0.30 (mean 3.03) and 0.83 (mean 14.67) zg of Hg per liter.
Detection
limit. In atomic absorption spectrophotometry,
the detection limit is defined as that quantity of the element
that gives a reading equal to twicethe standarddeviationof
a series of at least 10 determinations
of a near-blank
concen-
Variables
Analytical
recovery
of added
mercuric
chloride
and
met hylmercuric
chloride.
Known amounts
of mercuric
chloride and methylmercuricchloridewere added to blood
samples.
The peak absorbance
was compared with the absorbance of the corresponding
standard
in aqueous solution.
The mean recovery
of inorganic
mercury
was 88.9%, thus
confirming
the observations
of Magos and Clarkson
(4) that
the peak height obtained when a standard amount of mercury
is added to blood is always less than for the same amount in
water if all other conditions
(such as stirring time) are kept
the same. This fact illustrates
the importance
of running the
calibration
standard
in the same matrix as the sample. Of
course, use of an internal
standard
would automatically
overcome this difficulty.
The analytical
recovery of methylmercury
was even lower
than that of mercuric
chloride. This is to be expected
when
z
C.,
90
120
STIRRING TIME
Fig. 3. Time course of mercury release
chloride ( 400 ng Hg) added to blood
CLINICAL CHEMISTRY,
ISO
SECONOS
from methylmercuric
Vol. 25,
No. 5, 1979 771
tration
(14).
This was found to be 1.15 ng of Hg for this
Work
is now in progress to extend the use of sodium borohyof mercury in hair and in fish after
method.
dride to the determination
Sensitivity.
The sensitivity of an atomic absorption method
as the concentration,
in solution, of the element to
be determined
that will result in 1% absorption
of the incident
radiation at the wavelength used (14). Using this definition,
we found the sensitivity of the method here reported to be 0.26
tg of Hg per liter and the absolute
sensitivity
1.59 ng of
Hg.
Accuracy. The most commonly used method for estimating
the accuracy or reliability of a method is to compare the results
obtained
by the method with those obtained with some wellestablished
independent
method.
Accordingly,
15 different
blood samples were analyzed by this method as well as by the
nitric acid digestion procedure (15). The coefficient of correlation was calculated for the paired values and the value of
r was 0.91, which was significant at the 1% level.
Interference.
As the method depends upon the reducing
property of sodium borohydride, all oxidizing substances
such
as nitric acid must be absent and oxidizing conditions should
not be allowed to prevail. Acids should be absent from standards because they decompose the reagent with evolution of
hydrogen, which may be hazardous. Interference
from liberated hydrogen
in the atomic absorption
of mercury is negligible. In a medium sufficiently
acid to decompose the whole
of the added sodium borohydride,
the liberated hydrogen
accounted
for 1% absorption.
the sample is dissolved in sodium hydroxide.
is defined
Advantages
The present
method
offers several advantages
over the
methods already published.
It is very simple. It is rapid; the
time required for the analysis itself is only 2.5 mm. It is easy
to carry out, as no pretreatment
of the blood sample is required. Because the same sample acts as test and standard,
matrix effects are avoided. A further advantage is that even
commercial grades of sodium borohydride
contain no metal
impurities, whereas such impurities
in acids and reagents used
in sample-digestion
methods can appreciably
affect blank
values.
We conclude that sodium borohydride
can be used safely
and effectively
for the rapid and accurate determination
of
blood mercury and that the method is well suited to (e.g.)
epidemiological
studies in which many samples are processed.
772
CLINICAL CHEMISTRY,
Vol. 25, No. 5, 1979
References
I. Study Group on Mercury Hazards. Hazards of mercury. Environ.
Res. 4, 1(1971).
2. Gorsuch, T. T., The Destruction
of Organic Matter,
Pergamon
Press, Oxford, 1971, pp 79-84.
3. Ure, A. M., The determination
of mercury by non-flame atomic
absorption arid fluorescence spectrometry. A review. Anal. Chim. Acta
76, 1(1975).
4. Magos, L., and Clarkson, T. W., Atomic absorption determination
of total, inorganic, and organic mercury in blood. J. Assoc. Off. Agr.
Chem. 55, 966 (1972).
5. Gilbert, T. R., and Hume, D. N., Improved apparatus for determination
of mercury by flameless atomic absorption. Anal. Chim.
Acta 65, 461 (1973).
6. Hawley, J. E., and Ingle, J. D., Jr., Improvements
in cold vapor
atomic absorption determination
of mercury. Anal. Chem. 47, 719
(1975).
7. Sullivan, E. A., Sodium Borohydride:
Handling,
Uses, Properties
and Analytical
Procedures, Chemical Division, Ventron Corp.,
Beverley, MA, 1973.
S. MacGregor, J. T., and Clarkson, T. W., Distribution, tissue binding
and toxicity of mercurials. Adv. Exp. Med. Biol. 48, 463 (1974).
9. Gaylord, N. G., Reduction with Complex Metal Hydrides, Interscience, New York, N.Y., 1956, p 73.
10. Cohen, G. B., and Schrier, E. E., Removal of mercury from fish
protein concentrate
by sodium borohydride reduction. J. Agr. Food
Chem. 23, 661 (1975).
11. Toffaletti,
J., and Savory, J., Use of sodium borohydride
for determination
of total mercury in urine by atomic absorption spectrometry. Anal. Chem. 47, 2091 (1975).
12. Reutov, 0. A., and Beletskaya, I. P., Reaction Mechanisms
of
Organometallic
Compounds,
(Translated
by A.M.A. Mincer),
North-Holland
Publishing Co., Amsterdam, 1968, pp 405-415.
13. Kubasik, N. P., Volosin, M. T., and Sine, H. E., Rapid analysis
of total mercury in biological fluids by flameless atomic absorption
spectrophotometry.
Clin. Chem. 18, 716 (1972).
14. Varian-Techtron,
Basic Atomic Absorption
Spectroscopy,
A
Modern Introduction,
Varian-Techtron
Pty. Ltd., Springvale, Victoria, 3171 Australia, 1975, pp 79-80.
15. Dennis, C. A. R., and Fehr, F., Mercury levels in whole blood of
Saskatchewan residents. Sci. Total Environ. 3, 267 (1975).