View Article Online / Journal Homepage / Table of Contents for this issue

Ce 25
le ye
br ar
at s
in
g
Journal of Analytical Atomic Spectrometry
Published on 15 April 2010. Downloaded by Universidad de Salamanca on 16/02/2017 12:04:43.

www.rsc.org/jaas Volume 25 | Number 6 | June 2010 | Pages 749–904

ISSN 0267-9477

COMMUNICATION
Huang et al. ASU REVIEW
Quantitative chemical extraction for Advances in atomic spectrometry and
arsenic speciation in rice grains related techniques 0267-9477(2010)25:6;1-C
 View Article Online

COMMUNICATION www.rsc.org/jaas | Journal of Analytical Atomic Spectrometry

Quantitative chemical extraction for arsenic speciation in rice grains†

Jen-How Huang,*a Gunter Ilgenb and Peter Fecherc
Received 2nd February 2010, Accepted 8th April 2010
First published as an Advance Article on the web 15th April 2010
DOI: 10.1039/c002306j
Published on 15 April 2010. Downloaded by Universidad de Salamanca on 16/02/2017 12:04:43.

Extraction using 0.28 M nitric acid at 95  C for 90 min recovers not
only arsenic quantitatively from rice grains independent of types but
also preserves arsenic speciation completely. It is simple, economic,
effective and reliable and is especially suitable for routine analysis of
large amounts of samples.
Aside from drinking, rice is the most important exposure route for
arsenic (As), a non-threshold class 1 carcinogen.1 Rice is an important
foodstuff for 3 billion people. World rice consumption has risen from
156 million in 1960 to 423 million metric tons in 2007.2 The nature of
paddy rice taking up As readily from waterlogged soils, where As is
liberated by anaerobic microbial activity, leads to As accumulation at
least of 10 fold higher concentrations than wheat and other cereals.1
The toxicity of As depends not only on total concentrations, but
also its chemical forms. In general, inorganic As species are more
toxic than organic ones. Arsenite (As(III)) has higher toxic effects than
arsenate (As(V)). Unlike sea food, in which As is dominately present
in its organic forms e.g. arsenobetaine, major parts of As found in rice
are in inorganic forms.1 Therefore, due to large differences of toxicity
among As(III), As(V) and organic As, accurate As speciation is
indispensable to access the impact of As toxicity in rice on human
health. To date, almost all existing methods are either not able to
satisfactorily recover As from rice grains independent of types3–7 or
As species undergo redox transformation during extraction4,8,9
(detailed discussion in ESI†). Today, a reliable method is still lacking.
Quantitative recovery of As from rice grains without species trans-
formation is very challenging, especially for As(III) and As(V): A
successful extraction is necessary to break As(III)-thiolate complexes6
and avoid any As redox transformation caused by an extraction
matrix, e.g. extractants and thiolate compounds released from rice
grains.
In order to develop an extraction method able to gain a full
spectrum of As speciation in rice grains, we first examined chemical
extraction based on dilute acids and bases at 95–100  C using either
heating blocks or microwave ovens (Fig. 1). The concentration of
0.07 M was chosen to simulate the condition in the stomach for acid
extraction and to minimize As transformation via mild extraction
conditions. The results indicated extraction with HCl and HNO3 with
and without addition of H2O2 recovered 90% of total As in whole
grain rice (Fig. 1). Nevertheless, partial reduction of As(V) was
observed during HCl extraction and the presence of H2O2 led to
complete oxidation of As(III). Additionally, large amounts of chlo-
rides adversely interfered with As(V) retention in chromatography
and caused an elevated ICP-MS background signal due to 40Ar35Cl+
formation. Monomethylarsonic acid (MMA) and dimethylarsinic
acid (DMA) were affected very little during most extraction proce-
dures. Tetramethyl ammonium hydroxide and H3PO4 extraction
caused swelling of the resulting rice matrix, which could not be
filtered. Accordingly, HNO3 was the most suitable extractant for our
purpose. Heating with heating blocks and microwave ovens seemed
to affect the extraction very little (Fig. 1). We preferred heating blocks
to microwave because heating blocks are available in most labora-
tories and are easily handled. Additionally, heating blocks provide
higher efficiency of sample treatment per time unit than microwave
ovens.
The extraction time modestly affected the total As recoveries from
whole grain rice with heating blocks or microwave ovens (ESI
Fig. S5†). However, the length of extraction time affects As specia-
tion remarkably (Fig. 2a). Nitric acid extracted more As(III) with
increasing time. The substantial time length seems to be necessary to
break As(III)-thiolate complexes with dilute HNO3.9 On the other
hand, the increased extraction time led to more reduction of As(V)

a
Institute of Biogeochemistry and Pollutant Dynamics, Swiss Federal
Institute of Technology Zurich (ETH Zurich), CH-8092 Zurich,
Switzerland. E-mail: jenhow.huang@env.ethz.ch; Fax: +41 44 6331118;
Tel: +41 44 6328819
b
Central Analytic, Bayreuth Center for Ecology and Environmental
Research (BayCEER), University of Bayreuth, D–95440 Bayreuth,
Germany. E-mail: jenhow.huang@env.ethz.ch; Fax: +49 921 555709;
Tel: +49 921 555710
c
Bavarian Health and Food Safety Authority (LGL), D-91058 Erlangen,
Germany. E-mail: peter.fecher@lgl.bayern.de; Fax: +49 9131 764 401;
Tel: +49 9131 764 442
Fig. 1 Arsenic speciation in whole grain rice after different extraction
† Electronic supplementary information (ESI) available: Detailed
discussion about comparison of different extraction methods for treatments with heating blocks and microwave ovens for 90 min. Mean
arsenic speciation in rice grains, development of method and validation values of triplicate extraction are shown. S.D.s are all <10%. TMAH:
tests are available. See DOI: 10.1039/c002306j Tetramethyl ammonium hydroxide.

800 | J. Anal. At. Spectrom., 2010, 25, 800–802 This journal is ª The Royal Society of Chemistry 2010

Published on 15 April 2010. Downloaded by Universidad de Salamanca on 16/02/2017 12:04:43.
Fig. 2 Concentrations of arsenite (As(III)), dimethylarsinic acid (DMA),
monomethylarsonic acid (MMA) and arsenate (As(V)) in whole grain rice
extracted with 0.07 M HNO3 at 95  C with a microwave at different
lengths of time (a) and with 0.07 M HNO3 at different temperatures with
microwave ovens for 90 min (b). Mean values and S.D.s of triplicate
extraction are shown.
Fig. 3 Recoveries and redox transformation of individually spiked
arsenite (As(III)) and arsenate (As(V)) at 50 mg As L 1 during extraction of
whole grain rice using different concentrations of HNO3 at 95  C for 90
min. Mean values and S.D.s of triplicate extraction are shown.
(Fig. 2a). Ninety minutes was chosen as a compromise to possibly
recover more As and to minimize As(V) reduction. The extraction
efficiency for As(III), MMA and DMA increased with temperatures
This journal is ª The Royal Society of Chemistry 2010
View Article Online
based on the results from microwave-assisted extraction (Fig. 2b).
However, it is not feasible to conduct extraction at a temperature
higher than 100  C with heating blocks. 95  C was used for our
proposed method.
Preservation of As(III) and As(V) speciation during extraction of
rice grains was only allowed at a narrow range of HNO3 concen-
trations (Fig. 3). The released matrix led to significant As(V) reduction
and As(III) oxidation by hot HNO3 < 0.28 M and > 0.70 M,
respectively. This finding reveals the extreme sensitivity of the redox
transformation between As(III) and As(V) during extraction of rice
grains. The mild oxidation conditions provided by hot HNO3
between 0.28 and 0.70 M avoids As(V) reduction by thiolate
compounds in rice grains and, at the same time, As(III) oxidation
caused by the interaction between HNO3 and extraction in matrix
was still negligible. In comparison, a complete recovery of MMA and
DMA was fulfilled at all HNO3 concentrations tested (ESI Fig S6†).
Diluted HNO3 was often used to stabilize As redox transformation
in aqueous samples.10 Concentrated HNO3 was used to dissolve
organic materials at high temperatures for analysis of total As.11
Complete oxidation of As species in plant materials was described.11
Our preliminary experiments show little As redox transformation in
heated HNO3 solution up to 4.0 M (ESI Fig. S7†). This suggests that
As redox transformation is not caused by HNO3 itself but by the
resulting matrix during rice grain extraction. The rice starch consists
mainly of amylase and amylopectin, which are linear and branched
polymers of glucose, respectively.12 Therefore, acidic hydrolysis will
result in formation of glucose and glucose based polymers. We
examined the stability of As(III) and As(V) in 1.0 g L 1 of glucose,
fructose and lactose at 95  C for 90 min with and without 0.1 M HCl
solution (ESI Fig. S8†). However, the results revealed no As redox
transformation under these conditions, suggesting glucose like
compounds are not responsible for As redox transformation during
heated acid extraction.
The presence of thiolate containing compounds in rice grains such
as glutathione (GSH), phytochelatin and metallothionein has been
found in the literature.13,14 Lombi et al. (2009) indicated the dominate
As form in rice endosperm and bran are As(III)-thiolate complexes.14
Arsenite-thiolate complexes are usually unstable in acidic conditions
Fig. 4 Recoveries of arsenite (As(III)) and arsenate (As(V)) spiked at 3.75
mg As L 1 during treatments at 95  C for 90 min in the presence of 10%
acetic acid with different concentrations of HNO3. One hundred times
dilution was done for HPLC-ICP-MS analysis. Mean values and S.D.s of
triplicate extraction are shown.
J. Anal. At. Spectrom., 2010, 25, 800–802 | 801

Table 1 Concentrations of arsenic species (ng As g 1) and recoveries (%) of total arsenic in rice grains of different typesa
Rice type As(III) DMA MMA
Japonica 90.5 42.8 <DL
Round sticky 123 45.5 <DL
Long grain 85.0 28.3 <DL
Risotto 80.1 29.4 <DL
Wild rice 5.55 1.36 <DL
Whole grain 87.0 149 5.45
a
<DL: below detection limit (1.3 ng As g 1). b Sum of As(III), DMA, MMA and As(V). c Digested with 65% HNO3 at 280  C. d Including 2 unidentified
Published on 15 April 2010. Downloaded by Universidad de Salamanca on 16/02/2017 12:04:43.
As species (Fig. S1†).
e.g. As(III)-diethyldithiocarbamate (DDTC) and As(III)-pyrrolidine-
dithiocarbamate (APDC), so that acidic treatments may break
As(III)-thiolate complexes and release free As(III) ions and thiolate
containing compounds.15 The acidic hydrolysis of rice may release
additional free thiolate groups, which is potentially responsible for
As(V) reduction when HNO3 concentration is smaller than 0.28 M
(also during HCl extraction). For example, GSH, which is found in
rice grains, has been proved to reduce As(V), especially in acidic
conditions.16
3 H+ + AsO43 + 5 GSH / As(SG)3 + GS-SG + 4 H2O
In biological systems, a range of other thiols and dithiols, including
cysteine, mercaptoethanol, and dithiothreitol, may affect this reduc-
tion reaction.17 The slightly As oxidation with increasing HNO3
concentrations seems to be caused by interaction between the
resulting matrix and HNO3. We observed similar As(III) oxidation
with increasing HNO3 concentrations in the presence of 10% acetic
acid (Fig. 4). However, the exact mechanism can not be revealed only
with the data in this study.
We thus proposed a novel extraction method based on 0.28 M
HNO3 at 95  C for 90 min.18 The proposed method not only recovers
As from rice grains of different representative types (japonica, round-
sticky, long-grain, risotto, whole grain and wild rice) quantitatively
(89–106%) but also preserves all As speciation during extraction, as
demonstrated by satisfactory recoveries of spiked As(III) (101–105%),
As(V) (95–97%), DMA (97–100%) and MMA (96–101%) from
different rice grains (ESI Fig. S9† and Table 1). The recovery devi-
ations for each spiked As(III), DMA, MMA and As(V) were all
smaller than 5%. The analysis shown in Table 1 reveals the
predominance of As(III) in most rice grains, confirming rice as a bio-
accumulative plant for more toxic As(III) and supporting the
importance to detect the inorganic As in rice grains.
The resulting matrix based on HNO3 extraction was free of artifact
interference when using ICP-MS detection. Lower concentrations of
HNO3 (0.28 M) were favoured for the subsequent liquid chroma-
tography. The high acidity may shift retention times and damage
columns. A necessary dilution of extracts will lead to loss of
methodical sensitivity. The proposed method is based on acidic
hydrolysis of rice grains and is thus less sensitive to extraction
conditions than enzymatic methods. Additionally it has advantages
over existing methods due to low-cost and low requirement of
infrastructures, so that it can be easily performed in almost every
802 | J. Anal. At. Spectrom., 2010, 25, 800–802
View Article Online
As/V Total Asb Total Asc Recoveries
<DL 132 147 91
3.06 170 184 93
22.6 134 149 91
10.4 120 135 89
<DL 6.91 6.50 106
9.34 257d 260 99
laboratory and is especially suitable for analysis of large amounts of
samples. Using HPLC-ICP-MS for the quantification of dissolved As
species in the extracts, the detection limit may reach 1.3 ng As g 1. In
conclusion, the novel extraction presented in this study opens a new
approach for determination of total As and As speciation in rice
grains.
Notes and references
1 R. Stone, Science, 2008, 321, 184–185.
2 USDA, 2008 http://www.irri.org/science/ricestat/data/may2008/-
WRS2008-Table17-USDA.pdf.
3 I. Pizarro, M. G omez, C. Camara and M. A. Palacios, Anal. Chim.
Acta, 2003, 495, 85–98.
4 K. Baba, T. Arao, Y. Maejima, E. Watanabe, H. Eun and
M. Ishizaka, Anal. Chem., 2008, 80, 5768–5775.
5 E. Sanz, R. Mu~ noz-Olivas and C. Camara, Anal. Chim. Acta, 2005,
535, 227–235.
6 J. L. Guzman Mar, L. H. Reyes, G. M. Mizanur Rahman and
H. M. Skip Kingston, J. Agric. Food Chem., 2009, 57, 3005–3013.
7 T. Narukawa, K. Inagaki, T. Kuroiwa and K. Chiba, Talanta, 2008,
77, 427–432.
8 R. A. Schoof, L. J. Yost, J. Eickhoff, E. A. Crecelius, D. W. Cragin,
D. M. Meacher and D. B. Menzel, Food Chem. Toxicol., 1999, 37,
839–846.
9 D. T. Heitkemper, N. P. Vela, K. R. Stewart and C. S. Westphal,
J. Anal. At. Spectrom., 2001, 16, 299–306.
10 D. A. Polya, P. R. Lythgoe, F. Abou-Shakra, A. G. Gault,
J. R. Brydie, J. G. Webster, K. L. Brown, M. K. Nimfopoulos and
K. M. Michailidis, Mineral. Mag., 2003, 67, 247–261.
11 F. A. Austenfeld and R. L. Berghoff, Plant Soil, 1982, 64, 267–271.
12 R. F. Tester, J. Karkalas and X. Qi, J. Cereal Sci., 2004, 39, 151–165.
13 S. Ida and Y. Morita, Agr. Bio. Chem.Tokyo, 1971, 35, 1550.
14 E. Lombi, K. G. Scheckel, J. Pallon, A. M. Carey, Y. G. Zhu and
A. A. Meharg, New Phytol., 2009, 184, 193–201.
15 M. Georgiadis, Y. Cai and H. M. Solo-Gabriele, Environ. Pollut.,
2006, 141, 22–29.
16 N. Scott, K. M. Hatlelid, J. N. E. MacKenzie and D. E. Carter, Chem.
Res. Toxicol., 1999, 6, 102–106.
17 W. R. Cullen and K. J. Reimer, Chem. Rev., 1989, 89, 713–764.
18 All rice grains were grounded or obtained as flours. A 1.5 g rice flour
was extracted with 15 mL of different extractants in 50 mL
polypropylene tubes at 95  C for 1.5 h using heating blocks (Digi
PREP Jr, SCP Science) or microwave ovens (Multiwave 3000,
Anton Paar). A part of the resulting suspensions were filtered to
0.45 mm (Teflon) for As speciation using HPLC-ICP-MS. Another
part of suspensions was 1 : 1 mixed with 65% HNO3 and digested in
a High Pressure Asher (HPA, Anton Paar, Austria) by heating to
280  C in 60 min and lasting for 90 min. The resulting clear
supernatants were analysed for total As by ICP-MS.
This journal is ª The Royal Society of Chemistry 2010