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Analyst, November 1994, Vol. 119
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248 1
Rapid Method for the Determination of Total
Mercury in Urine Samples Using Cold Vapour
Atomic Fluorescence Spectrometry
Warren T. Corns, Peter B. Stockwell and Mohammad Jameel
P S Analytical Ltd., Arthur House, B4 Chaucer Business Park, Watery Lane,
Kemsing, Sevenoaks, Kent, U K , TN15 6QY
A rapid method for the determination of total mercury in urine
samples is proposed. Samples are digested using a bromination
procedure at room temperature. Analysis is performed using
automated continuous flow vapour generation coupled to
atomic fluorescence spectrometry. This approach allowed the
analysis of 30 samples per hour and a limit of detection of 1
ng 1-1. The analytical procedure was assessed using certified
reference material NBS 2672a freeze-driedurine and two
batches of Seronorm trace elements in urine samples.
Keywords: Mercury, urine; bromination; atomic fluorescence
spectrometry; cold vapour generation
Introduction
Toxicological effects of mercury compounds on both plant and
animal life have long been recognized, but it was not until the
disaster at Minamata Bay in 1953 that the subject received
world-wide attention. 172
Exposure of mercury to the general population is mainly
through the diet and dental amalgam. In foodstuffs, mercury is
usually in the inorganic forms and of very low concentration.
The exemptions are fish and fish products, which are the main
sources of methylmercury in the diet. Recent experimental
studies3 have shown that mercury is released from amalgam
restoration in the mouth as a vapour and the rate of release
may be increased by certain foods or by the action of chewing.
Some therapeutic agents, such as teething powder, ointments
and laxatives, contain inorganic mercury. Mercury-containing
cream and soap has for a long time been used by dark skinned
people to obtain a light skin. English community health
authorities have identified several brands of soap containing
mercury iodide at 1-3% .4
There are several forms of metabolic transformation that
can occur after exposure to mercury. These can be sum-
marized as: oxidation of metallic mercury to divalent mercury,
reduction of divalent mercury to metallic mercury, methyla-
tion of inorganic mercury and conversion of methylmercury to
divalent inorganic mercury. Numerous papers have been
published in this areas-8 and these have been reviewed by
WHO.9 The faecal and urinary routes are the main pathways
for the elimination of inorganic mercury in humans, although
some elemental mercury is exhaled.
The urinary route dominates when exposure is high. After
exposure to metallic mercury vapour, a small fraction of the
mercury in the urine may be present as elemental mercury.10
Methylmercury is excreted in the urine to only a very limited
extent. When evaluating exposure to low concentrations of
inorganic mercury , interference from methylmercury expo-
sure can dominate blood analysis and therefore the analysis of
urine is preferred .9
There are numerous analytical techniques available to
determine total mercury in biological materials. The most
commonly used methods for measuring mercury are cold
vapour atomic absorption spectrometry (CVAAS) , and neut-
ron activation (NA). Although CVAAS has become the most
widely used technique, there are several disadvantages
associated with the use of AAS detection, such as limited
linear calibration range, spectral interferences resulting from
non-specific background absorption of volatile organics' and
difficulties with measurements at lower levels. Neutron
activation is generally regarded as being very accurate and
sensitive, but extremely expensive and time consuming. l2
Recently , atomic fluorescence spectrometry (AFS) has
been utilized for the detection of mercury in biological
materials.13 Mercury is a good element to determine by atomic
fluorescence because it absorbs and fluoresces at the same
wavelength, (i.e., resonance fluroescence) in the ultraviolet
(UV) region, intense mercury excitation sources are available
and no atomization is required when the technique is coupled
to vapour generation. The reported limit of detection using
this approach was 0.9 ng 1-1 or 2 pg absolute.13
Various digestion procedures are available for total mer-
cury in biological materials. A variety of combinations of
strong acids (HCI, H2S04, H N 0 3 , HC104) and oxidants
(H202, KMn04, K2Cr207, K2S208) have been used and
recommended. 14-17 The main concerns of the digestion stages
are related to loss of analyte during elevated temperature
digestion and interference from acid gases evolved during the
final reduction step. Determinations at low concentrations are
normally limited by blank contributions from reagents.
Fentons reagent (Fe" + H202) was utilized by Ping and
Dasgupta18 to minimize blank levels from reagent additives.
Vermeir et al. 13 have used a microwave digestion procedure
with nitric acid to mineralize a variety of biological materials.
Online microwave digestion for urine samples have been
reported. 19
Total mercury may also be determined by direct reduction
using a tin(r1) chloride-cadmium(I1) chloride reagent .20 Cad-
mium(r1) chloride actually displaces mercury from methyl-
mercury, thereby releasing mercury(i1) ions, which are
reduced by tin(r1) chloride. It is therefore possible to
differentiate between inorganic and methylmercury com-
pounds. Other workers have utilized sodium tetrahydroborate
in the presence of Cu(i1) ions t o determine total mercury,21 the
disadvantage of this approach being the possible interferences
from other gaseous species generated (e.g., As, Se, Sb and Te)
and dilution from excess hydrogen.
The use of the bromination digestion procedure for total
mercury determination has been used extensively for river,
raw and potable waters.22-23The approach is outlined by eqns.
(1) and (2).
Br03-+ 5Br- + 6H+ S 3Br2 + 3 H 2 0 (1)
2R-Hg + 5Br2 S 2R-Br + 2HgBr4*- (2)
Essentially acid bromate oxidizes bromide to bromine which

2482 Analyst, November 1994, Vol. 119
rapidly cleaves organomercury bonds. Inorganic mercury is
then converted to the extremely stable HgBr42- complex. The
procedure is rapid and occurs at room temperature, prevent-
ing losses of mercury owing to vaporization. All reagents used
can be obtained at extremely high purity, thus minimizing
blank contributions.
This paper assesses the suitability of this approach for urine
analysis. Determinations are performed using automated
continuous flow vapour generation coupled to AFS. The
analytical performance of the system is investigated and the
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accuracy is assessed using certified reference materials.
Experimental
Reagents
All reagents were of analytical-reagent grade unless stated
otherwise [BDH (now Merck), Poole, Dorset, UK]. A 2%
m/v tin(i1) chloride solution in 10% v/v hydrochloric acid was
used as a reductant and trace amounts of mercury were
removed from this solution by purging with argon at 1 1 min-1
for 30 min. This solution was prepared daily. Potassium
bromate-potassium bromide reagent was prepared by dilution
from a concentrated ‘Convol’ solution. The final concentra-
tion of this reagent mixture was 0.1 moll-’ KBr-O.01665 mol
1-1 KBr03 and this was also prepared daily. Hydroxylamine
hydrochloride (12% m/v) was used to remove free bromine
after digestion. This solution was prepared weekly and traces
of mercury were removed from this solution by purging with
argon.
Standard solutions were prepared by appropriate dilution of
a stock lo00 mg 1-1 mercury(i1) chloride solution (SpectrosoL,
Merck). Stock solutions of methylmercury(i1) chloride and
phenylmercury(i1) chloride were prepared by dissolution of
the salts (Fluorochem Ltd. ,Derbyshire, UK) with methanol.
Subsequent dilutions were carried out using 10% v/v hydro-
chloric acid. All glassware and autosampler vessels were
soaked in 50% v/v nitric acid overnight and then rinsed five
times with distilled water.
Preparation of Standards and Samples
The urine samples were digested using the bromination
procedure. A volume (5-10 ml) of urine sample was accurately
transferred into a clean 50 ml calibrated flask. Concentrated
hydrochloric acid (2.5 ml) was carefully added to the sample
followed by 2 ml of the potassium bromate (0.01665 moll-1)
bromide (0.1 mol 1-1) reagent ( i e . , 0.1 N bromate-bromide
solution).
The calibrated flask was stoppered and the sample was
allowed to stand for 15 min to ensure complete breakdown of
organomercury compounds. The bromide addition was always
adequate to maintain an excess of bromine for all of the urine
samples analysed. If more bromine is required, then addi-
Gas-liquid
separator
Fig. 1 Schematic diagram of the continuous flow vapour generator
shown in the blank position (dotted line represents the flow of the
sample solution).
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tional standards are required to compensate for the increased
bromide concentration. After this period, the free bromine
was removed by adding several drops of 12% m/v hydroxylam-
ine hydrochloride. The sample was then diluted to 50 ml using
distilled water and was analysed within 3 h.
A reagent blank solution containing the same concentration
of reagents as the samples was prepared. Standard solutions
were made by appropriate dilution of stock solution using the
reagent blank. Spiked sample solutions (Seronorm, Urine
batch 9024) were prepared by adding 50, 100 and 200 ng (as
Hg) of methylmercury(i1) chloride and 50 ng (as Hg) of
phenylmercury(r1) chloride to the sample. These were then
brominated as outlined in the above procedure.
Instrumentation
Continuous Flow Vapour Generator-Atomic Fluorimeter
A PS Analytical Ltd. (PSA, Kent, UK), automated continu-
ous flow vapour generation system (PSA 10.004) was used to
generate gaseous mercury. A schematic diagram is shown in
Fig. 1. Detection was achieved using an atomic fluorimeter
(PSA 10.023 Merlin). This instrumentation has been des-
cribed in detail elsewhere.24 The switching valve that alter-
nates between sample and reagent blank solutions was
computer controlled and typically 8 ml of sample was
introduced. Alternatively the valve may be activated quickly
so that small volumes can be introduced (e.g., 100 pl). This
approach can be utilized when only a small amount of sample
is available. The instrumental conditions are outlined in Table
1.
Instrumental Control
All of the instrumentation outlined in Fig. 1 was fully
automated and was controlled using the Touchstone Software
(M023T150). The different units are connected to an IBM A T
compatible computer through a D I O card.
Results and Discussion
The analytical performance of the continuous flow vapour
generation-atomic fluorimeter was assessed for linearity, limit
of detection and accuracy and precision of analytical measure-
ments.
The linearity of the detector was found to span over five
orders of magnitude (1 ng 1-1-100 pg 1-1). At high concentra-
tions (>1 mg I-*) the calibration deviates towards the
concentration axis; this is caused by the self absorption
process.25 The method detection limit (30,-~) based on ten
runs of the blank solution was found to be 1 ng I-’. The blank
solution was distilled water digested using the bromination
procedure. The detection limit was found to be dependant on
reagent purity rather than instrumental noise. All of the
Table 1 Operating conditions for the continuous flow vapour
generator
Parameter Value
Reductant flow rate/ml min-1 3.5
Reagent blank flow rate/ml min-1 8.0
Sample flow rate/ml min-1 8.0
Carrier gas flow rate/l min-1 Ar 0.3
Sheath gas flow rate/] min-1 Ar 0.3
Dryer gas flow ratdl min-1 Ar 2.6
Delay time/s 15
Rise time/s 30
Analysis timels 30
Memory wash time/s 50
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Analyst, November 1994, Vol. I 1 9 2483

reagents used for the bromination procedure can be obtained
commercially with high purity.
Previous studies have shown that the cold vapour atomic
fluorescence approach is relatively free of interferences. 11.26
Chloride ions are known to interfere with permanganate
digestion procedures. This necessitates the use of additional
reagents in order to complete the digestion. The level of
chloride in urine samples can be quite high (100 mmol 1-1) so
potentially an interference exists. No chloride interference
was observed when using the bromination digestion proce-
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vapour-atomic absorption spectrometry. Two batches of this
material were analysed and the results obtained are reported
in Table 3. These correspond to six replicate analyses and the
uncertainty is based on 20,~-1. Good agreement was obtained
for both batches.
To test the recovery of organomercury from urine when
using the bromination procedure a batch of Seronorm control
standard (batch no. 9024) was spiked with various amounts of
methylmercury( 11) chloride and phenylmercury(i1) chloride.
These results are reported in Table 4. The recovery data

dure. This is because the half cell oxidation potential of the
BrdBr- couple is lower (1.087 V) than that of the CI*/CI-
couple (1.358 V). Sulfide ions are also known to interfere with
the cold vapour generation process, probably owing to the
formation of mercury sulfide complexes. Sulfide concentra-
tions as high as 24 mg 1-1 have been reported as not interfering
with the recovery of inorganic and organically bound mer-
cury.26
The accuracy of the method was assessed using certified
NBS SRM 2671a and Seronorm trace element freeze-dried
urine samples. The NBS 2672a is available at low and elevated
concentrations. The value for the low level is not certified, but
is given for information only. The results obtained for the NBS
materials are given in Table 2. The values obtained correlate
well with the certified values. The results reported correspond
to five replicate analyses and the uncertainties based on 20,~-
The elevated sample was also analysed 18 months after
purchase and a result of only 70% of the certified value was
obtained for total mercury. The expected storage time was 1y.
The Seronorm trace elements control standard is a lyophi-
lized human reference urine. The ‘recommended value’ is the
arithmetic mean of all values given by the participating
laboratories and the majority of laboratories utilized cold
Table 2 Determination of total mercury in NBS SRM 2672a
freeze-dried urine. Uncertainties based upon 20,,-~ ( n = 5 )
Certified Value
Material value/pg 1 - 1 obtainedjpg I-‘
Low level* 2 *
1.8 0.4
Elevated level 105 t 8 103 f 5
* Value for the low level (pg 1-1) is given for information only and is
not certified.
shown here was obtained after 15 min of bromination.
Although organomercury is excreted in the urine to only a
limited extent, these results indicate the effectiveness of the
digestion procedure.
The sampling and storage of urine samples can be critical,
since bactericidal growth can change the concentration of the
numerous forms of mercury that may be present. Addition of
hydrochloric acid, bactericidal substances and freezing the
sample are the usual methods of storage.9
One further advantage of bromination is that the samples
may be brominated at the time of collection. In the presence of
bromine, samples were found to be stable for at least 5 d.
Longer sample storage times were not investigated. Once the
bromine has been removed with hydroxylamine hydrochloride
the sample must be analysed within 3 h.
Conclusions
Automated continuous flow vapour generation coupled to
atomic fluorescence spectrometry has been utilized for the
determination of total mercury in urine samples. The bromi-
nation digestion procedure overcomes several interferences
for the urine analysis and takes place at room temperature in
15 min. Samples were found to be stable for at least 5 d in the
presence of bromine. Future work will involve a stability study
over a longer time period. A detection limit of 1ng 1-1 (30,~-1)
was obtained with the system described and this is significantly
lower than other methods available for urine analysis. The
speed of the digestion suggests that the bromination proce-
dure may be used for online digestion. This research is
currently being undertaken. The recoveries for certified
reference materials and quality control standards illustrate the
accuracy and precision obtainable with the technique.
The authors thank SpinOff Technical Systems for their
valuable assistance in software development.

References
Table 3 Determination of total mercury in Seronorm trace elements
urine. Uncertainties based on 20,-~ ( n = 6) 1 Smith, W. E . , and Smith, A. M.. Minamata, Holt, Rinehardt
and Winston, New York, 1975.
Batch Recommended* Analytical Value 2 Tsuchiya, K . . Am. J. h d . Med., 1992, 21, 275.
number value/pg 1-l va1uedp.g 1-1 obtainedlyg I - ’ 3 Aronsson, A . M., Lind, B., Nylander, M., and Nordberg, M.,
Biol. Met., 1989, 2, 25.
115 35 34.3,35.5,35.6 35.3 t 0.2
9024 48 49.7,47.5,47.5 47.0 0.8 * 4 Mercury soaps and skin lightening cream, Directorate of
Environmental and Consumer Services, Lambeth, London
* The ‘recommended value’ is the arithmetic mean of all values Borough, London, 1988.
given by the participating laboratories. 5 Hursh, J . B., Sichak, S. P . , and Clarkson, T . W., Pharmacol.
Toxicol. Engl. Transl., 1988,63, 266.
6 Ogata, M., Kenmotsu, K., Hirota, N., Meguro, T . , and Aikoh,
H., Arch. Environ. Health, 1987. 42, 26.
Table 4 Spike recovery data for organomercury compounds (Sero- 7 Heintze, U., Edwardsson. S . , Derand, T., and Birkhed, D..
norm trace elements urine, batch no. 9024) Scand. J . Dent. Res., 1983, 91, 150.
8 Lind, B., Friberg, L., and Nylander, M., 1. Trace Elem. Exp.
Amount Med., 1988, 1 , 49.
Mercury compound addedlng Hg Recovery (%) 9 WHO, Inorganic Mercury, Environmental Health Criteria 118,
Methylmercury( 1 1 ) chloride Geneva, World Health Organisation, 1991.
CH3HgCI 50 94.6 10 Yoshida, M., and Yamamura, Y., Int. Arch. Occup. Environ.
100 95.3 Health. 1982, 51, 99.
200 94.8 11 West, C. D., Anal. Chem., 1974, 46, 797.
Phenylmercury( 11) chloride 50 98.0 12 Rottschafer, J . M., Jones, J . D., and Mark, Jr. H. B., Environ.
Sci. Technol., 1971, 5, 336.
 View Article Online

2404 Analyst, November 1994, Vol. 119

13 Vermeir, G., Vandecasteele, C., and Dams, R., Trace Elements
in Health and Disease, ed. Aitio, A., Aro, A., Jarvisalo, J., and
Vainio, H., Royal Society of Chemistry, Cambridge, 1991, pp.
29-36.
14 Baxter, D. C., and Frech, W., Anal. Chim. Acta, 1990,236,377.
15 Goto, M., Shibakawa, T., Arita, T., and Ishii, D., Anal. Chim.
Acta, 1982, 140, 179.
16 Iverfeldt, A., Mar. Chem., 1988, 23, 441.
17 Freimann, P., and Schmidt, D., Fresenius’ Z. Anal. Chern.,
1982, 299, 97.
18 Ping, L., and Dasgupta, P. K., Anal. Chem., 1989, 61, 1230.
19 Welz, B., Tsalev, D. L., Sperling, M., Anal. Chim. Acta, 1992,
Published on 01 January 1994. Downloaded by University of Chicago on 28/10/2014 03:51:22.
21 Margel, S., and Hirsh, J., J. Clin. Chem., 1984, 30,243.
22 Farey, B. J., Nelson, L. A., and Rolph, M. G., Analyst, 1978,
103, 656.
23 Yorkshire Water Authority Methods of Analysis, 1988,5th Ed.,
Yorkshire Water, Leeds, Section 9.
24 Stockwell, P. B., and Corns, W. T., J. Autom. Chem., 1993,15,
79.
25 Corns, W. T., Ebdon, L. C. Hill, S. J., and Stockwell, P. B.,
J. Autom. Chem., 1991, 13, 267.
26 Farey, B. J., and Nelson, L. A., Anal. Chem., 1978,50, 2147.
Paper 4100029C

261, 91. Received June 22, 1994

20 Magos, L., Analyst, 1971,96, 847. Accepted August 15, 1994