3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P106 ARSENIC SPECIATION IN FISh
uSING hIGh PERFORMANCE LIQuID
ChROMATOGRAPhy COuPLED wITh
hyDRIDE GENERATION ATOMIC
FLuORESCENCE SPECTROMETRy
eVA VITOULOVá a , VEROnIKA HARKABUSOVá a and
BLAnKA MACHARáČKOVá b
a Department of Food Chemistry and Biotechnologies, Faculty
of Chemistry, Brno University of Technology, Purkyňova
118, 612 00 Brno, Czech Republic,
b Department of Biochemistry, Chemistry and Biophysics,
Faculty of Veterinary Hygiene and Ecology, University of
Veterinary and Pharmaceutical Sciences,Palackého 1–3,
612 42 Brno, Czech Republic,
vitoulova@fch.vutbr.cz
Introduction
Investigations of the chemical constituensts of aquatic
organisms can provide useful informations about the environment
as well as toxicologically relevant data about the composition
of biological species consumed by humans 1 . Arsenic
causes a variety of adverse health effects to humans after acute
and chronic exposures 2 . Toxicity, environmental mobility and
accumulation in living organisms usually depend on the form
in which the element is present. Information on the chemical
forms is important for understanding the role of the element
present as well as revealing its environmental cycle 3 . In the
environment arsenic can be found as several compounds ranging
from inorganic arsenite and arsenate to methylated compounds
like monomethylarsonic acid, dimethylarsinic acid,
trimethylarsine oxide, arsenobetaine, tetramethylarsonium
ion and arsenocholine. In biologic samples several arsenosugars
and arsenolipids can be present as well 4 .
The determination of arsenic compounds is mostly
carried out by hyphenated techniques, the main parts being
liquid chromatographic separation and element specific detection.
By far the most used systems are liquid chromatography
inductively coupled plasma mass spectrometry (LC-
ICP-MS) and liquid chromatography hydride generation
atomic fluorescence spectrometry (LC-HG-AFS) 5 . Atomic
fluorescence spectrometry (AFS) can be a good alternative
to inductively coupled plasma mass spectrometry (ICP-MS)
detector, with the advantage of a lower cost of investment
and handling 5,6 .
Experimental
S t a n d a r d s a n d R e a g e n t s
All solutions were prepared with deionized water
(18.2 MΩcm). Stock solutions of 1,000 mg(As)dm –3 were
prepared by dissolving naAsO 2 (Fluka, Switzerland),
na 2 HAsO 4 . 7H 2 O (Fluka, Switzerland), CH 4 AsnaO 3 . 1.5H2 O
(Chem. Service, USA), (CH 3 ) 2 AsHO 2 (Fluka, Switzerland)
and arsenobetaine (Fluka, Switzerland). All arsenic solutions
were stored in the dark at + 4 °C. These stock solutions were
s815
diluted with deionized water to the desired concentrations
before use.
For the analytical procedures the following chemicals
were used: potassium dihydrogen phosphate (Fluka, Switzerland),
potassium monohydrogen phosphate (Fluka, Switzerland),
hydrochloric acid (Analytika, Czech Republic), potassium
persulfate (Fluka, Switzerland), sodium borohydride
(Riedel-de Haën, Germany), sodium hydroxide (Riedel-de
Haën, Germany), nitric acid (Analytika, Czech Republic),
hydrogen peroxide (Lach-ner, Czech Republic), ascorbic
acid (Riedel-de Haën, Germany), potassium iodide (Analytika,
Czech Republic), magnesium nitrate (Merck, Germany).
All chemicals were at least of analytical grade.
I n s t r u m e n t a t i o n
A Gynkotek P508 (Gynkotek, Germany) HPLC pump
and six-port injection valve (Ecom, Czech Republic) were
used in conjunction with a strong anion-exchange column
Hamilton PRP X-100 (Hamilton, USA). An Excalibur atomic
fluorescence detector (PS Analytical, UK) equipped with a
boosted discharge hollow cathode lamp (Photron, Australia)
was used for detection. The measurements are carried out on
the resonance line of As 193.76 nm. For data collection and
evaluation was used CSW software – version 1.7 (Data Apex,
Czech Republic).
S a m p l e P r e p a r a t i o n
The muscle of commercially available fish samples were
homogenized and freeze-dried.
For the determination of total arsenic by AFS the microwave
digestion (using HnO 3 and H 2 O 2 ) was employed;
followed by the addition of Mg(nO 3 ) 2 to complete the
destruction.
Triplicate of samples were extracted with 10 ml of
deionized water using ultrasonic bath at 50 °C for 2 hours.
The extraction vessels were shaken every 15 min to ensure
dispersion of the sample. After extraction the samples were
centrifuged and extract was filtered through a 0.45 µm
membrane filter and kept at 4 °C till speciation analysis.
C o n d i t i o n s o f T o t a l A r s e n i c
D e t e r m i n a t i o n
A KI/ascorbic acid reducing solution was employed to
reduce arsenic species to As (III). The reducing solution was
added at least 30 minuts before analysis. Conditions for hydride
generation: 1 % naBH 4 in 1 % naOH at flow rate the
4.5 ml min –1 and 4.9 mol dm –3 HCl at flow rate 9 ml min –1 .
S p e c i a t i o n a n a l y s i s c o n d i t i o n s
The mobile phase was 10 mM K 2 HPO 4 /KH 2 PO 4 adjusted
to pH 6.1 at flow rate 1 ml min –1 . The hydride generation of
the arsines was carried out by adding 6 mol dm –3 HCl and
1.4 % naBH 4 in 1 % naOH, both at the 3 ml min –1 flow rate.
1 % K 2 S 2 O 8 in 1 % naOH at the flow rate 0.3 ml min –1 was
used as oxidation solution, oxidation was supported by UV.