Mikrochim.

Acta 122, 67-76 (1996)

Mikrochimica
Acta
9 Springer-Verlag 1996
Printed in Austria

Determination of Free Iodide in Human Serum:

Separation from Other I-Species
and Quantification in Serum Pools
and Individual Samples
B. Michalke*, P. Schramel, and S. Hasse
GSF-Forschungszentrum fiir Umwelt und Gesundheit, Institut fiir Okologische Chemie, Neuherberg,
D-85764 OberschleiBheim, Federal Republic of Germany

Abstract. A method for the determination of free iodide in human serum was
developed. For this purpose iodide from pooled serum samples was separated
from the organic manner by SEC. The iodide fraction subsequently was freezedried and analyzed by ion chromatography for quantification. Investigations for
recovery and precision were carried out and were found to show sufficient results.
For quality assurance ICP-MS was taken additionally as an total I-detector [1],
using native and iodide-spiked serum samples. The iodide results of ICP-MS as
well as those of IC were well corresponding. Iodine containing SEC-fractions
from iodide-spiked samples showed no increased I-values except that in the
iodide fractions, proving that there was no iodide conversion into other I-species
(and vice versa) during the whole procedure.
Free iodide from two serum pools of different healthy persons was determined as 2.25 and 2.43 gg I - / L , respectively. The values are related to total iodine
levels determined by ICP-MS. For comparative reasons a table of individual
iodine and iodide values is presented.
Key words: iodide, iodine, ion chromatography, quality control, human serum.

Iodine is an essential trace element [2, 31. I-containing hormones (thyroxine,

tri-iodothyronine [4, 5]) are strongly influencing an extended range of biochemical
reactions. Usually different iodine species (e.g. IOn-, I - ) are absorbed from the food
and reduced to iodide (or remains iodide) in the gastro-intestinal tract [5]. The
iodide pool in serum is considered as I-source for the complete synthesis of thyroxine
and tri-iodothyronine. The sufficiency of I-supplementation can be seen directly by
monitoring iodide in serum or secondarily by monitoring the urinary I--excretion
[6]. The latter is state of the art because the direct iodide determination in serum was
not possible at low physiological levels with desirable accuracy. A method (pub* To whom correspondence should be addressed

68

B. Michalke et al.

lished in 1966/67) u s i n g s e r u m w h i c h w a s labelled with 131I in v i v o in c o n s i d e r a b l e

a m o u n t s [-7, 8] is n o t p r a c t i c e d a n y longer. T h e r e , a q u a n t i t a t i v e s e p a r a t i o n of the
iodide f r o m o t h e r I-species is n o t realized [7].
T h e n e e d for a m e t h o d o f I - - d e t e r m i n a t i o n in s e r u m w a s the r e a s o n for
d e v e l o p i n g a m e t h o d w i t h the f o l l o w i n g features:
a) sufficiently l o w d e t e r m i n a t i o n limit; b) h i g h p r e c i s i o n a n d a c c u r a c y ; c) i n s t r u m e n t a t i o n a v a i l a b l e in clinical l a b o r a t o r i e s ; d) the possibility to e x t e n d the m e t h o d to
i o d i n e s p e c i a t i o n i n v e s t i g a t i o n s b y o n l y small m e t h o d i c a l c h a n g e s .

Material and Methods

1. Sampling and Sample Preparation

Blood was taken from healthy persons by "Vacutainers". The blood samples were stored for 30 rain at
room temperature and were then centrifuged with 3000 rpm for 20 min. The supernatant (serum) was
carefully collected, pooled and kept frozen at - 2 0 ~ until further use. In case of individual
I--determinations the serum samples were not pooled.

2. SEC-Fraetionation

In principal, the $EC-fractionation was derived from the SEC-separation described in [9]. All sample
preparation steps were done in laminar flow benches to avoid iodide contamination from the air [1].
2.5 ml aliquots were taken from thawed serum for further investigations: 0.5 ml were used for total
iodine determination [1].
2 ml were given on a LPLC-system, equipped with a SEC-column for the separation of iodide from
the organic matrix. Fractions were taken by a fraction collector in 1 rain intervals. The fractions
between 38-42 rain (RT of iodide) were pooled, frozen at - 20 ~ and freezedried at a controlled sample
temperature of 10 ~
The dry powder was resuspended in 1.5 ml H20 and filtered through a 0.22 gm syringe filter. The
sample then was ready for two parallel I--determinations at the IC.

3. LC Parameters for SEC

An "Econo System" (BioRad, Munich, Germany) was used for SEC-separations. The system was
combined with a Beckman HPLC pump "114M" for a constant solvent delivery at 4 bar.
Eluent:
Column:
Stationary phase:
Fractionation:

0.02 M NaHCO3; flow: 3 ml/min; wavelength: 254 mm

500 x 20 mm, temperature controlled at 20 ~
TSK HW 40 F (Toso Haas, Stuttgart, Germany)
i min intervals = 3 ml by a "Fraction Collector 100" (Phamacia)

Table 1. A "3 step program" for the quality assurance during SEC-separation

Eluent
Flow
Time

Step 1

Step 2

Step 3

NaC1, 0.5 M
3 ml/min
120 min

H20

NazCO3, 0.02 M

3 ml/min
120 min

3 ml/min
120 min

Determination of Free Iodide in Human Serum

69

4. Quality-Assurance Concernin9 the SEC-Separation

Just before the separation of I - from the organic matrix the SEC column was rinsed, using
a "3-step-program" (Table 1) to get rid of organic molecules, probably adsorbed to the SEC material.
Further, a reproducible I - - R T and a quantitative separation is provided.

5. IC-Determination
The IC-determinations were carried out on a Dionex 4000 i ion chromatographic system, equipped
with a PED.
Parameters:

flow:
eluent:
PED-potential:
electrode:
reference electrode:
sample loop:
precolumn:
column:

2.0 ml/min
4 mM Na2CO3/1.5 mM NaHCO3, He degased
+ 0.05 V
Ag
Ag/AgC1
50 gl
AG 9-SC, Dionex
AS 9-SC, Dionex

6. Calibration
The IC was calibrated by two "5 Point calibration cu ryes" (0-10 gg I - / L or 10-50 pg I - / L ) for low level
or high level determinations.

7. Precision and Recovery

For reasons of quality control and for precision determinations, 2 ml of the pooled serum were mixed
with 50 ng iodide. The sample was allowed to stand for 15-30 minutes and then was injected into the
LPLC system. The procedure further was following 2. (SEC-separation) and 5. (IC-determination). The
whole procedure was repeated 10 times, each with 2 parallel IC-determinations.
The range of IC-determined values was taken for the precision in consecutive I - determinations.
The mean of determined values was used for recovery calculations.

8. Quality Control
Aliquots of the SEC-fractions (from native or spiked serum) were used for iodine determinations by
ICP-MS according to [1] in parallel to the IC-determinations. The ICP-MS is used as a total iodine
detector (independent on the I-species), whereas the IC (PED) is determining only free iodide (species
specific detection). The elution pattern of native and spiked samples were monitored and compared, to
get information about a possible conversion of free iodide into other I-species (or vice versa) during the
SEC separation.
Further, the "redox stability" of iodide was checked by an analytical scheme shown in Fig. 1. There,
mass balances were calculated for spiked serum after re-injection into the LPLC system. All fractions
were monitored for "isolated" iodide by ICP-MS, obtaining information about possible iodide conversion into other species, showing other retention times and being not detectable by IC at the I - potential.

9. Chemicals
NazCO 3, NaHCO3, NaC1 and KI were purchased from Merck (Darmstadt, Germany). The "TSK 40
H W 40 F"-gel was bought from TosoHaas (Stuttgart, Germany) and packed into a glass column

70

B. Michalkeet al.

serum -~total iodide~--.~

2ml

50 ng I'spike

- -

i I'-recovery 9 75 %
[26. % retained /

~= 55

~ SEC

compute due[
to recovery

,I~ Quantification:

I C P - M S : 40 ng

54 ng

freeze drying .l~Quantification :

(-recovery- 75 % ~
25 % reta ned

~C

Fig. 1. Schemeof the analytical

ICP-MS:

56 ng
42 ng
[C :43ngrlg - - 57- ng

,I~ Quantification.
I C P - M S :31 ng

56 ng

[C :30 ng

54 ng

steps of the "re-injection experiments". Informationis provided

about the "redoxstability"ofiodide during SEC-separation as
wellas about speciesconversions.
Mass balancesare calculated

(500 x 20 ram) from Krannich (G6ttingen, Germany).Helium(5.0 quality)was obtainedfrom Messer

Griessheim(Munich,Germany).
Results

A. Methodical Aspects
1. Column stability/separations, sample pretreatment. The SEC-pretreatment of the
serum before IC analysis improved the possible IC-separations on the analytical
column from only few to appr. 400 runs as well as the reproducibility of I - quantification. This was true for a quantitative SEC-separation of I - from the
organic matrix, achieved by an appropriate column handling described in Table 1.
When using this procedure a sufficient SEC-separation was guaranteed for 50 runs.
Iodide was eluting reproducibly between 38-42 rain without organic interferences.
After more applications the I--fractions contained increasingly organic matter,
damaging the IC-column.
2. Recovery and precision. When using the described procedures the recovery was
found as 75% with a precision of _+ 3% at 50 LagI - / L in 10 consecutive investigations. The limit of determination of the whole method was calculated (_+ 3o-) as
0.1 ~ g I - / L serum. The recovery of the final determination (1C) was 102% _ 7%
using an aqueous solution of 1 lag I - / L . The detection limit (IC) was determined at
0.05 gg I - / L .

3. Quality control. For reasons of quality control the recovery and precision
investigations were monitored by ICP-MS in parallel to IC. Figure 2 shows the
UV-plot of the SEC pretreatment of spiked serum ( 5 0 n g I - ) compared with the
iodide-elution (IC determined) and the elution of all iodine species (ICP-MS
determined).

Determination of Free Iodide in Human Serum

71

UV
up to 2 AU
03
0,6
0,5
0,4

<

o,3

0,2

o,t

]
0

10

20

30

40

50

60

70

80

minutes

iodide (IC)
0,7
0,6
0,5
=L
0,3
0,2

~,! Ii

0,1
,

0
0

10

20

30

,,iitlliii~

4O

50

60

70

80

60

70

80

minutes

iodine (ICP-MS)
0,7
0,6
0,5
=1~ 0,4
=1.
0,3
0,2
0,1
0

10

20

30

40

50

minutes

Fig. 2. SEC-separation of 2ml spiked (50ng I-) serum: The UV-profile (top) is related to the IC
determined iodide elution (middle) and to a iodine monitoring (bottom) by ICP-MS

72

B. Michalke et al.

.=_

15

, m

10-

5-

0
13

'
16
19

, i
22
25

' q~--~
28
31
fraction

/
iodide elution

~ . F' ~ ' ~ i/ / - 4 3

2 ml native serum

49

Fig. 3. Comparison of the iodine elution from 2 ml native serum with 2 ml spiked (50 ng) serum. Only
the iodide fractions of spiked serum show an elevated I-level, proving that there is no I-conversion into
other I-species due to the additional iodide amount
Both, the I-values (ICP-MS) as well as the I--values (IC) were in accordance at
the iodide retention time (38-42 min).
Further, I containing SEC-fractions (other than 38-42 min, iodide !) showed the
same values in native and spiked samples by I C P - M S (e.g.: fractions 17-23: native:
4 5 n g / m l serum; spiked: 4 7 n g / m l serum). N o increased I-values were seen in
SEC-fractions of spiked samples except that at the iodide elution time (Fig. 3).
Re-injection of the SEC-separated iodide fraction again resulted in a iodide elution
only at the I - - r e t e n t i o n time. The species specific IC-detection proved this iodine
species totally to be iodide (Fig. 1),
B. Results About Native I-Values

Figure 4 shows the I C - c h r o m a t o g r a m s of two individual samples c o m p a r e d to an

iodide standard of 1 I~g/L as an example. The iodide and total iodine values of serum
pools are c o m p a r e d with individual values of 7 persons in Table 2, each corrected for
recovery.
The two different pools (1 and 2) of healthy and not supplemented persons show
identical values for total iodine and free iodide. The iodide level is about 3% of total
iodine for both pools.

Determination of Free Iodide in Human Serum

73

Standard IHglL
0,035
0,03
0,025
0,02
1= 0,015
0,01
0,005
0
5

minutes

Person "A"
0,035
0,03
0,025
1~ 0,02
~" 0,015
0,01
0,005
0
5

minutes

Person "D"
0,035

o,03

~
~

o,o25

.~

0,02
~r 0,015
0,01
0,005
5

minutes
Fig. 4. IC-chromatogram of a 1 ~g/L standard compared to two individual samples with a "normal"
iodide value (middle) and a slightly decreased one (bottom). The determined values are 1.6 gg/L ("A")
and 0,75 gg/L (D), which have to be corrected for recovery (75%). The corrected values are given in
Table 2

74

B. Michalke et al.

Table 2. Iodine and iodide values of different serum pools and of individual samples

Sample source

Sex
male/
female

Pool 1
Pool 2

m/fro
m/fro

82.2 + 8
81.3 + 7

2.43 _+0.3
2.25 _+0.25

2.96
2.77

Pool 3

m/fro

92.4 + 7

9.0 __+0.53

9.74

"A"
"B"
"C"
"D"

m
fm
m
fm

78.7
76.1
45.5
99.0

2.1
4.9
3.3
1.0

2.67
6.44
7.25
1.01

Person "E"

fm

68.5

42.4

61.9

Person "F"

fm

103.8

13.6

13.1

Person "G"

fin

128.5

4.2

Person
Person
Person
Person

Total iodine
[pg/L]

Free iodide
[pg/L]

I-/I
[%]

3.27

Remarks

From healthy persons

From healthy persons
other than from pool 1
Pooled from resting serum
samples of persons "A, C, F"
Healthy
Diet slightly I supported
An increased thyroid gland
activity is known
Strongly I supported 2
times per day.
Last supplementation:
3 h before blood sampling
Using I supplemented salt
for cooking
I supplementation 1 times
per week.
Last supplementation:
2 days before blood
sampling

Discussion

The methods developed here provide an iodide determination at physiological levels

with high reproducibility and precision. The determination limit of the whole
procedure (0.1 gg/L) is more than 20 times lower than "normal" physiological
I--values enabling monitoring of even decreased I--values (e.g. person "D") with
sufficient accuracy. The methodical check by a second, independent method for final
determination like ICP-MS gives further evidence of a high accurracy. The stability
of iodide during the whole determination method is proven by a) the correspondence
of the I-values in SEC-fractions from native and spiked samples (except iodide
fractions !), b) the correspondence of iodide results of IC and ICP-MS and c) the
"re-injection experiment". There is no iodide conversion into other I-species or vice
versa.

The total I - recovery of 75% is sufficient, especially when looking at the low SD
(+ 3 %). When using the "high level" calibration curve, elevated iodide levels can be
easily determined (e.g. persons "E, F').
The total correspondence of total iodine and free iodide of serum pools from
different persons give strong evidence for a marked regulatory mechanism for both,
total I and free I-. This is in accordance to the literature [-5].

Determination of Free Iodide in Human Serum

75

This strong regulation may be further proved by the results of person "E". In spite of
very high I doses per day (50 tag iodate), the hormone bound I values were
conspicuously lower than the (normal) pool values. This is surprising as free I - is
available in big amounts. Obviously, here the regulatory mechanism cannot be
sufficiently influenced by the increased serum iodide level. Only about 26 lag I/L
serum (38%) are bound to I-species of high molecular weight, being probably
thyroid gland hormones [5]. This agrees with [11]. There, the I-supplementation is
excreted nearly quantitative (92%) via urine.
When looking at person "D", only 1% of total iodine appears to be free iodide.
The remaining 99% are obviously hormone bound [5]. This slight decrease of the
iodide value may be insignificant, but it principally agrees with the total iodine value
being in the upper range and the increased thyroid gland hormone concentration,
known from this person. The total iodine values (organic bound + free iodide) and
the values of free iodide alone are in good accordance with the literature. [1, 10]
report about 80gg iodine/L serum and [4] and [11] give iodide serum values of
7.2_+ 5.8 lag/L (+ 80%) and below 10lag/L, respectively. [8] reports about total
serum iodine in the 10 to 100 gg/L range and [7, 8] found 2.8% or 1.8%, respectively,
I - from total iodine.
The work of [4] gives iodide values in the physiological range, but the method
described there does not fulfill some of our requirements sufficiently. So the detection
limit of 1 lag/L (20 pg in 20 lal) was too high for serum samples from the Munich area
for an exact determination (e.g. person "D": 1 lag/L). Additionally, our aim was to
develop an I--determination method, which can be easily extended for an iodine
speciation work. But the sample preparation and the specific chemical mechanism of
the post column reaction in [4] avoid the possibility for speciation investigations.
Summarizing, the method described here provides the possibility for monitoring
both, physiological ("normal") iodide vahles in serum as well as increased ones.
Thus, a more direct information is now available about the sufficiency of iodine
supply compared to urinary iodide determinations. The use of ion chromatography
for final iodide determination makes the method principally available for clinical
laboratories and provides a species specific I--detection. In future, the method may
be a further tool for diagnosis purposes, as well as a basis for iodine speciation
investigations, when using ICP-MS as the final I-detector.
Abbreviations
IC = ion chromatography,ICP-MS = inductivelycoupled plasma mass spectrometry,LPLC = low
pressure liquid chromatography, PED = pulsed electrochemicaldetector, SEC= size exclusion
chromatography,RT = retention time.
References
[I] P. Schramel,S. Hasse, Mikrochim. Acta 1994, 116, 205.
1-2] E.J. Underwood, Trace Elements in Human and Animal Nutrition, 4th Ed. AcademicPress, New
York, 1977.
1-3] A. S. Prasad, Trace Elements and Iron in Human Metabolism, Plenum, New York, 1978.
[4] W. Buchberger,J. Chromat. 1988, 439, 129.
[5] H. Keller,Klinisch-chemische Labordiagnostikf~ir die Praxis, Thieme,Stuttgart, 1991.

76
[6]
[7]
[8J
[9]
[10]
[11]
[12]

Determination of Free Iodide in Human Serum

J.T. Dunn, H. E. Crutchfield, R. Gutekunst, A. D. Dunn, Thyroid, 1993, 3, 119.
K. Miiller, Clin. Chim. Acta 1967, 17, 21.
E. Makowetz, K. Miiller, H. Spitzy, Microchem. J. 1966, 10, 194.
B. Michalke, D. C. Mfinch, P. Schramel, J. Trace Elem. Electrolytes Health Dis. 1991, 5, 251.
G. V. Iyengar, Elemental Analysis of Biological Systems, Vol. 1, CRC, Boca Raton, 1987.
W. Buchberger, U. Huebauer, Mikrochim. Acta 1989, 3, 137.
S. K. Nath, B. Moinier, F. Thuillier, M. Rongier, J. F. Desjeux, Internat. J. Vitamin Nutr. Res.
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Received February 10, 1995. Revision June 12, 1995.