Article

pubs.acs.org/JAFC

Bromine and Iodine Contents in Raw and Cooked Shrimp and Its
Parts
Marcia F. Mesko,*, Isis G. Toralles, Carla A. Hartwig, Gilberto S. Coelho, Jr., Aline L. H. Muller,
Cezar A. Bizzi, and Paola A. Mello

Centro de Ciencias Qumicas, Farmaceuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul
96160-000, Brazil

Departamento de Qumica, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul 97105-900, Brazil

ABSTRACT: The concentration of bromine and iodine was determined in shrimp and its parts (tissue and shells), and changes
in the analyte concentration were evaluated after the cooking procedure. Bromine and iodine concentrations were determined by
a method recently developed by our research group based on microwave-induced combustion for sample preparation and
inductively coupled plasma mass spectrometry for analyte determination. The accuracy was evaluated using a reference material
(NIST 8414) that was digested using the proposed method. No statistical dierence was observed between certied and
determined values (Students t test, 95% condence level). Suitable limits of detection (Br, 0.02 g g1 and I, 0.01 g g1) were
obtained for both analytes. Higher concentrations of both analytes were observed in shrimp shells in comparison to shrimp tissue
for raw and cooked samples. Moreover, losses of Br and I (between 24 and 43%) were observed after cooking.
KEYWORDS: bromine, iodine, shrimp analysis, cooking procedure

INTRODUCTION
Shrimp is an important crustacean among the species exploited
by sheries around the world. Just like other seafood, it
presents a high amount of nutrients, such as proteins, essential
fatty acids, and minerals. Thus, in many countries, this
crustacean is a shing product with a high value for
exportation.1,2
Among the minerals that must be controlled in general food,
including seafood, such as sh and crustaceans, iodine must be
highlighted mainly because it contributes to the synthesis of
thyroid hormones and can cause health problems when
ingested in an inadequate concentration. Iodine deciency or
its excess can cause human health disorders.3,4 Thus, according
to the World Health Organization (WHO), a daily intake of
150 g of I for adults for normal maintenance of the thyroid
gland is necessary.3 Bromine, in contrast, does not have a well-
established physiological function, because it presents an
unknown mechanism of action. The WHO recommended a
Br daily intake of 0.4 mg1 of body weight as acceptable.5,6
It is possible to nd several studies in the literature on the
chemical composition of seafood organisms.1,2,79 However,
there are few papers reporting Br and/or I determination in
marine products, such as oyster tissue, sh, and shrimp,1012
although it is known that the marine environment contains
relatively high amounts of these halogens.3,6,13 Specically for
shrimp, only one paper has reported the simultaneous
determination of Br and I in shrimp and respective distribution
in its parts (shells and tissue).12
With regard to the evaluation of mineral concentrations in
seafood after the application of dierent cooking procedures,
there have been few studies with this aim.1418 In this sense, it
is important to note that none of these studies that apply
cooking procedures for marine organisms have evaluated the Br
and I losses. Thus, a study related to the behavior of these
elements after cooking is very relevant.
Although no reports have evaluated the Br and I losses from
shrimp after cooking procedures, the loss of I from several
types of food following dierent cooking procedures has been
observed by other authors. A loss of I from Indian recipes
prepared using iodized salt and submitted to a cooking
procedure was observed. In the same way, losses of I from
chicken, peas, pasta, potatoes, and rice after a cooking process
have been reported.18
Many analytical techniques have been reported in the
literature for Br and I determination in biological sam-
ples.10,12,1923 In this sense, inductively coupled plasma mass
spectrometry (ICPMS) can be highlighted in view of the high
sensitivity and low limits of detection (LODs) for both
elements.12,24 However, an eective sample preparation step is
necessary previous to ICPMS determination because the
excessive residual carbon content in digests may cause
interferences. Moreover, the use of acids may enable the
formation of volatile substances in combination with halogen or
cause interference in the nebulization step, resulting in
unsuitable accuracy and impairing the precision of results.25
In this sense, studies have been developed using water26 or
organic solvents27 for extracting the halogens from dierent
matrices.28 However, the use of tetramethylammonium
hydroxide (TMAH) solution, water-soluble tertiary amines
(CFA-C) solution,29 or other alkaline solutions12 is considered
the most conventional way to extract halogens, mainly from
Received: December 5, 2015
Revised: January 24, 2016
Accepted: February 1, 2016
Published: February 1, 2016

2016 American Chemical Society 1817 DOI: 10.1021/acs.jafc.5b05769

J. Agric. Food Chem. 2016, 64, 18171822
Journal of Agricultural and Food Chemistry
complex matrices,29 even though occasionally the complete
extraction of halogens cannot be quantied.30
Combustion methods are important alternatives for prepara-
tion of biological samples prior to halogen determination,
especially because they allow to use alkaline solutions to absorb
analytes. In general, methods based on combustion enable
complete decomposition of the matrix with low residual carbon
content (RCC) in a relatively short time. In addition, to avoid
losses of analytes by volatilization, the combustion must be
carried out in closed vessels.31 In this sense, the microwave-
induced combustion (MIC) system may be considered as an
interesting alternative for preparing samples prior to Br and I
determination. In addition to the listed advantages, it allows a
reux step after sample combustion, which can improve analyte
recovery.32 Methods based on MIC have been used for further
halogen determination in food samples.12,23,3338
Thus, this work aimed to determine Br and I in whole shrimp
and its parts (shell and tissue) using MIC associated with ICP
MS to evaluate the content of analytes after cooking.
Concentrations of Br and I were determined in raw and
cooked shrimp to evaluate the distribution of these elements in
shrimp and estimate possible alterations of analyte concen-
trations after a cooking procedure. Furthermore, to characterize
the shrimp and their parts, the physicalchemical composition
of the samples was determined.
2. MATERIALS AND METHODS
2.1. Instrumentation. For the combustion of samples, a
microwave sample preparation system (Multiwave 3000, Anton Paar,
Austria) equipped with eight high-pressure quartz vessels was used.
The quartz vessels had an internal volume of 80 mL, and the maximum
operating temperature and pressure were 280 C and 80 bar,
respectively. Quartz holders were used to support the samples in the
quartz vessels to perform combustion by MIC.
Shrimp samples were freeze-dried using a lyophilizer (model L101,
Liotop, Brazil) and ground in a cryogenic mill (model 6750, SPEX
CertiPrep, Metuchen, NJ). The samples were prepared as pellets for
the MIC procedure using a hydraulic press (Manual Hydraulic Press in
15 Ton load conguration, Specac, U.K.). An analytical balance with
limitation from 0.01 to 220 g (model AY220, Marte, Brazil) was used
to weigh the samples.
Ultrapure water obtained from a water purier (MegaUp,
MegaPurity, South Korea) was used in all procedures. An ultrasonic
bath (Ultrasonic Cleaner 1440D Evolution, Odontobras, Brazil) and a
Class 100 laminar ow bench (CSLH-12, Veco, Brazil) were used to
clean and dry, respectively, the discs of paper used in the MIC
procedure. The same laminar ow bench was used to dry the
laboratory materials. A heating plate (RH Basic 2, IKA, China) was
used for the cooking procedure.
A digestion block (model 40S KB, C Gerhardt, Brazil) and a
nitrogen distiller (model MA 036 PLUS, Marconi, Brazil) were used to
determine the crude protein content. A furnace (model 1C.318.24,
Quimis, Brazil) and a conventional oven (model 400/2ND, DeLeo,
Brazil) were used to determine the ash and water contents,
respectively. A water bath (model Thermomix 18 BU, B. Braun
Biotech International, Germany) and a centrifuge (model Sigma 2-3,
Sigma, Germany) were used to determine the fat content.
An inductively coupled plasma mass spectrometer (model Elan
DRC II, PerkinElmerSCIEX, Waltham, MA), equipped with a
concentric nebulizer (Meinhard Associates, Golden, CO), a cyclonic
spray chamber (Glass Expansion, Inc., Australia), and a quartz torch
with a quartz injector tube (2 mm inner diameter), was used to
determine Br and I concentrations. Instrumental performance
optimization was carried out following the instructions of the
manufacturer.39 Operational conditions were conducted as described
in the literature.12 An inductively coupled plasma optical emission
spectrometer (Spectro Ciros CCD, Spectro Analytical Instruments,
1818
Article
Germany) was used to determine the dissolved carbon content in
digests, according to previous work.23
2.2. Reagents and Reference Material. Ultrapure water (18.3
M cm) and chemicals of analytical grade were used throughout this
study. The absorbing solution used in the MIC procedure was
prepared by dilution of commercial ammonium hydroxide (Synth,
Brazil) in water. Ammonium nitrate (Merck, Germany) solution (6
mol L1), which was used as an ignition aid in the MIC procedure, was
prepared by dissolving the solid reagent in water.
Discs of paper (12 mg, 15 mm of diameter) with a low ash content
(Black Ribbon Ashless, Germany) were used as an ignition aid for
sample combustion. The discs were cleaned by immersion in 20% (v
v1) ethanol solution in an ultrasonic bath, then rinsed with ultrapure
water, and dried in a class 100 laminar bench. The quartz vessels and
holders were decontaminated using concentrated 65% (w w1) nitric
acid in a microwave oven with irradiation of 1400 W/10 min (heating)
and 0 W/20 min (cooling), followed by decontamination with
ultrapure water under an irradiation program of 800 W/10 min
(heating) and 0 W/20 min (cooling).
For crude protein determination, the Kjeldahl method was used as
follows: (i) Digestion step: 98% sulfuric acid (Synth) and a catalytic
10:1 mixture of anhydrous sodium sulfate and pentahydrate copper
sulfate (Synth). (ii) Distillation step: 12.5 mol L1 sodium hydroxide
(Isofar, Brazil), 4% (w v1) boric acid (Vetec, Brazil), methyl red
(Synth) solutions, all dissolved in water, and 0.066% (w v1)
bromocresol green (Synth) solution, dissolved in 0.033% (w v1)
ethanol (Vetec) solution. (iii) Titration step: 0.01 mol L 1
hydrochloric acid (Synth) solution, prepared by dilution in water.
To determine the fat content, hydrochloric acid (Synth) diluted in
water (1:11), ethanol, and a mixture of ethylic ether and petroleum
ether (boiling point of <60 C) (Synth) were used.
Reference solutions used for dissolved C determination by
inductively coupled plasma optical emission spectrometry (ICP
OES) were prepared by dilution of citric acid (Merck) in 5% (v v1)
HNO3. Yttrium solution (1 mg L1, SPEX CertPrep, Metuchen, NJ)
was used as an internal standard for dissolved C determination. For
the determination of Br and I by ICPMS, standard solutions (110
g L1 for Br and 0.11 g L1 for I) were prepared by dilution of
stock solutions in 10 mmol L1 NH4OH. Stock solutions (1000 mg
L1) were prepared by dissolving potassium bromide and potassium
iodide salts (Merck, Germany) in water. A reference material (RM) of
bovine muscle powder from the National Institute of Standards and
Technology (NIST 8414) was used for accuracy evaluation.
Argon with a purity of 99.996% (White Martins, Brazil) was used
for plasma generation and nebulization and as an auxiliary gas in both
plasma-based instruments. Argon was also used to remove dissolved
CO2 from the digests for carbon determination by ICPOES.
2.3. Sampling and Preliminary Treatment. Fresh shrimp
samples (Litopenaeus vannamei) were obtained from a local market
(Pelotas, Rio Grande do Sul, Brazil) in two batches (A and B)
acquired in December of the years 2013 and 2014, respectively. The
samples of each batch were separated into three groups: whole shrimp,
shrimp tissue, and shrimp shells + head. The mixed shells and head
parts of the shrimp samples will be treated only as shells in this work.
After this separation, all of the groups of shrimp were washed with
ultrapure water.
Part of the whole shrimp and shrimp tissue samples belonging to
batches A and B were separated for the cooking procedure. Before
cooking, the samples were protected from light and stored in a
refrigerator (5 C) for a maximum of 24 h, according to the Technical
Rules of Practice for Food Services.40
The raw and cooked shrimp samples (whole shrimp and shrimp
tissue) were freeze-dried for 24 h (56 C) based on the
recommendation of previous work12 and comminuted in a cryogenic
mill (particle diameter below 80 m). Before the MIC procedure, all
samples were prepared as pellets in a hydraulic press. Figure 1 shows
the overall procedure of this study.
2.4. PhysicalChemical Characterization of the Samples.
The average weight and ratio of shells and tissue of raw shrimp
samples were obtained by weighing 20 units of whole shrimp (average
DOI: 10.1021/acs.jafc.5b05769
J. Agric. Food Chem. 2016, 64, 18171822
Journal of Agricultural and Food Chemistry
Figure 1. Schematic diagram of the experimental design to evaluate Br
and I distribution in shrimp samples and the inuence of a cooking
procedure on analyte concentrations.
size of 9 cm), followed by weighing the corresponding shells and
tissue. The water content was determined by drying the samples in a
conventional oven at 105 C up to a constant weight. The ash content
in shrimp was obtained by dry ashing in a furnace at 525 C. The
crude protein content was determined by the Kjeldahl method, and
the fat content was determined by the Mojonnier method. These
methods were conducted according to the ocial methods
recommended by AOAC International.41
2.5. Cooking Procedure. The cooking procedure was applied to
samples of whole shrimp and shrimp tissue. In this procedure, about
80 mL of water was added to a 250 mL glass beaker. The water was
heated to the boiling point using a hot plate. Each portion of 20 0.5
g of sample was added to each beaker and cooked for 10 min,
according to previous work.15 After the cooking procedure, the cooked
shrimps were separated by ltration and were stored for further
pretreatment, as described in section 2.3.
2.6. Decomposition by Microwave-Induced Combustion
and Br and I Determination by ICPMS. The MIC procedure
was applied to raw (whole shrimp, shrimp shells, and shrimp tissue)
and cooked (whole shrimp and shrimp tissue) samples. In the
procedure, after preliminary treatment, the samples were prepared as
pellets (0.5 g) and added to a quartz holder for combustion using a
disc of paper with 50 L of 6 mol L1 NH4NO3 solution as igniter.
The quartz holders with the samples were transferred to quartz vessels
previously charged with 6 mL of 50 mmol L1 NH4OH as an
absorbing solution for the analytes. After the vessels were closed and
xed in the rotor, they were pressurized with oxygen (20 bar) and
moved for combustion in a microwave oven. The microwave was
programmed to perform irradiation at 1400 W for 5 min and 0 W for
20 min (cooling step). The choice of the absorbing solution and the
microwave irradiation program was based on previous work published
by our research group.12
After decomposition, the solutions were diluted with water to 25
mL and analyzed by ICPMS. Calibration curves were always
prepared with the same matrix medium of solutions used in the MIC
method, considering the nal dilution of the digests. The results
obtained for the Br and I concentrations in the cooked samples were
compared to the results obtained for the raw samples to estimate the
changes in the concentrations of these elements.
The accuracy was evaluated by decomposition of 0.2 g of a RM
(NIST 8414) containing informed values for Br and I. This material
was digested under the same conditions of shrimp. Moreover, spike
recoveries were performed by the addition of a solution containing
1000 mg L1 Br and 20 mg L1 I (10, 50, or 100 L, depending upon
the analyte concentration in samples) on the sample pellet (0.5 g),
before the combustion procedure.
1819
Article
All statistical calculations were performed using GraphPad InStat
(GraphPad InStat Software, Inc., version 3.00) software, and a
condence level of 95% was used.
3. RESULTS AND DISCUSSION
3.1. PhysicalChemical Characterization. The average
weight obtained for shrimp belonging to the two evaluated
batches was around 12 g, and the mass percentages of shells
and tissue were about 45 and 55%, respectively.
With regard to the results obtained for the characterization of
samples, as shown in Table 1, it is possible to observe that the
Table 1. Water, Ash, Crude Protein, and Fat Contents in
Dierent Shrimp Parts (Results Are Expressed as the Mean
Standard Deviation; %; n = 3)
sample water ash fat crude protein
whole shrimp 73 3 3.2 0.3 <1a 13 1
shrimp tissue 73 4 1.1 0.1 <1a 14 1
shrimp shells 69 3 6.8 0.4 1.2 0.1 10 1
a
LOD.
water content obtained for the whole shrimp and shrimp tissue
(both about 73%) was not signicantly dierent from that
observed for the shrimp shells (about 69%). The ash content
values ranged from 1 to 7%, being higher in shrimp shells. The
higher percentage of ash obtained in this portion of shrimp can
be attributed to the fact that the inorganic composition of this
crustacean is concentrated mostly in the shell.
The fat content obtained was lower than 1.5% for all
analyzed samples, and the small dierence in the percentage
obtained in the analyzed shrimp parts can be explained because
the fat was not distributed evenly throughout the organism.
The values of crude protein obtained in this experiment were
between 10 and 14%, and the protein fraction was more
concentrated in shrimp tissue than in shrimp shells. It is
important to emphasize that variations in the chemical
composition of shrimp, as reported in other studies, can be
explained by factors, such as the species analyzed and the
season of sampling.1,2
Thus, the obtained results by physicalchemical character-
ization conrm that the shrimp samples have relatively complex
matrices, especially given the protein content. These character-
istics justify the use of appropriate methods of sample
preparation prior to determination of elements mainly at low
concentrations.
3.2. Distribution of Bromine and Iodine in Shrimp
Parts (Shells and Tissue). Shrimps can be consumed with or
without shells, and Br and I contents can be dierent in tissue
and shells.12 In this way, Br and I determination in shrimp parts
and the consequent evaluation of distribution of these elements
can be considered an important information for human
nutrition. Table 2 shows the results obtained for Br and I in
the samples of whole shrimp, tissue, and shells of the two
batches evaluated.
As seen in Table 2, the concentrations of Br and I were
higher in the shrimp shells than in the shrimp tissue (up to 7
and 16 times, respectively), in both evaluated batches.
Furthermore, Br concentrations were higher than I concen-
trations in all shrimp parts. Thus, considering the water content
(around 73%) and that the evaluated whole shrimps contained
approximately 45% shells and 55% tissue, it can be estimated
that Br and I concentrations in a whole shrimp are contained
DOI: 10.1021/acs.jafc.5b05769
J. Agric. Food Chem. 2016, 64, 18171822
Journal of Agricultural and Food Chemistry Article

Table 2. Determination of Br and I in Whole Shrimp, Shrimp Tissue, and Shrimp Shell (Batches A and B) by ICPMS after
Decomposition by MIC (Results Are Expressed as the Mean Standard Deviation; g g1 of Dry Weight; n = 3)
batch A batch B
whole shrimp shrimp tissue shrimp shell whole shrimp shrimp tissue shrimp shell
Br 175 6 43.8 3.1 294 15 169 6 38.2 1.6 284 18
I 4.09 0.30 0.63 0.03 7.82 0.30 3.96 0.13 0.48 0.03 7.61 0.40

about 86 and 93%, respectively, in the shells. These higher Table 3. Concentrations of Br and I, Determined by ICP
concentrations of the analytes in the shells can be attributed to MS in Raw and Cooked Shrimp, after Sample Preparation by
the inorganic composition present in this part of shrimp (ash MIC (Results Are Expressed as the Mean Standard
content of about 7%). Deviation; g g1 of Dried Weight; n = 4)
When the individual concentrations of Br and I in shrimp
concentration
tissue and shrimp shells were compared to the corresponding
a
values obtained for whole shrimp, agreement of about 93 and batch sample Br I
98% for Br and I, respectively, was obtained in both evaluated A whole shrimp R 175 6 4.09 0.30
batches. This comparison was possible using the mass C 133 7 3.02 0.16
percentage of shrimp tissue and shrimp shells and the water shrimp tissue R 43.8 3.1 0.63 0.03
content in the samples, which were determined by physical C 25.1 1.7 0.38 0.02
chemical characterization procedures. B whole shrimp R 169 6 3.96 0.13
3.3. Changes in the Concentration of Br and I in C 128 6 2.89 0.21
Shrimp after the Cooking Procedure. The accuracy of the shrimp tissue R 38.2 1.6 0.48 0.03
MIC method using 50 mmol L1 NH4OH as an absorbing C 22.2 0.9 0.29 0.02
solution was evaluated by decomposing the RM (NIST 8414) a
R, raw samples; C, cooked samples.
under the same conditions as the sample preparation and
performing the same steps of determination as were used for
the shrimp samples. The agreement of the obtained results (Br,
1.15 0.07 g g1; I, 0.034 0.002 g g1) with the reference of the shrimp batches evaluated. These higher reductions in Br
values (Br, 1.1 0.5 g g1; I, 0.035 0.012 g g1) was 104 and I concentrations observed for shrimp tissue after the
and 97% for Br and I, respectively. This good accuracy was cooking procedure can be related to the absence of shells in this
conrmed by recoveries between 95 and 102% for both sample, which could acted protecting the tissue from water
analytes, achieved in spike recoveries. The relative standard leaching, minimizing the losses.
deviations (RSDs) for all measurements, including samples and Although there are no data in the literature about losses of Br
RM, were always below 7.5%, and the method used allowed for and I from shrimp after cooking, some authors have conducted
LODs in dry weight of 0.02 g g1 for Br and 0.01 g g1 for I, similar studies about losses of I from other foods. Rana and
which were suitable for shrimp analysis. Moreover, in this study, Raghuvanshi17 found similar results to that observed for shrimp
the blanks for Br and I were always negligible (<1.0 and <0.1 g tissue when evaluating dierent cooking procedures in six
L1, respectively), and no memory eects were observed during Indian cuisine recipes to assess the loss of I from the salt added.
analysis by ICPMS. In this experiment, authors observed a loss of 40.2% of I from
It is also important to emphasize that the use of MIC to foods after baking. Goindi et al.18 observed interim losses
prepare the samples provided low dissolved carbon content between the results obtained for shrimp tissue and whole
(<13.4 mg L1) in the nal digests. The obtained results were shrimp when assessing the losses of I (between 28 and 32%)
in agreement to those reported in the literature on shrimp and from chicken, peas, pasta, potatoes, and rice.
other samples submitted to sample preparation by MIC.12,23,33 Thus, the analytical method used in this study employing
Determination of Br and I concentrations was conducted in MIC and ICPMS was suitable and robust for the purpose of
samples of whole shrimp and shrimp tissue (batches A and B) evaluating the distribution of Br and I in shrimp samples and
after a cooking procedure to estimate possible changes in the estimating possible alterations in their concentrations after a
analyte concentrations in comparison to the raw samples. It is cooking procedure. In the determination by ICPMS, no
important to emphasize that, as expected, the characteristics of interferences or memory eects were observed for the analytes
the raw shrimp changed after the cooking process, possibly as a and good accuracy and precision were obtained. Moreover, the
result of dehydration by evaporation of water or protein method was suitable for the determination of Br and I
coagulation by water loss. However, considering that all of the concentrations in shrimp, with low LODs and negligible blank
samples (raw and cooked) were freeze-dried before the analyte values.
determination and the results were compared in terms of dry With regard to the distribution of Br and I in shrimp, higher
weight, the water loss is not considered relevant in this study. concentrations of both analytes in the shrimp shells in
The results obtained for Br and I concentrations in the raw and comparison to the shrimp tissue were observed in the two
cooked samples are shown in Table 3. batches evaluated. In addition, it is important to emphasize that
According to Table 3, it was possible to observe a reduction the Br concentration is always higher than the I concentration,
of about 25% in the Br and I concentrations when the whole regardless of the part of the shrimp evaluated. Br and I losses
shrimp of both evaluated batches (A and B) were cooked. were about 25% in the whole shrimp and 40% in the shrimp
However, for shrimp tissue, the reduction of Br and I tissue submitted to the cooking procedure, conrming the
concentrations in the cooked samples (about 40%) was higher occurrence of changes in the analyte concentrations after this
than those obtained in the raw samples (about 15%), for both culinary treatment.
1820 DOI: 10.1021/acs.jafc.5b05769
J. Agric. Food Chem. 2016, 64, 18171822
Journal of Agricultural and Food Chemistry
AUTHOR INFORMATION
Corresponding Author
*E-mail: marcia.mesko@pq.cnpq.br. Telephone: +55-53-3275-
7387.
Funding
The authors are grateful to Coordenacao de Aperfeicoamento
de Pessoal de N vel Superior (CAPES), Conselho Nacional de
Desenvolvimento Cient co e Tecnologico (CNPq), and
Fundacao de Amparo a Pesquisa do Estado do Rio Grande
do Sul (FAPERGS) for supporting this study.
Notes
The authors declare no competing nancial interest.
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1822 DOI: 10.1021/acs.jafc.5b05769

J. Agric. Food Chem. 2016, 64, 18171822