3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
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Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology<br />
P106 ARSENIC SPECIATION IN FISh<br />
uSING hIGh PERFORMANCE LIQuID<br />
ChROMATOGRAPhy COuPLED wITh<br />
hyDRIDE GENERATION ATOMIC<br />
FLuORESCENCE SPECTROMETRy<br />
eVA VITOULOVá a , VEROnIKA HARKABUSOVá a and<br />
BLAnKA MACHARáČKOVá b<br />
a Department of Food Chemistry and Biotechnologies, Faculty<br />
of Chemistry, Brno University of Technology, Purkyňova<br />
118, 612 00 Brno, Czech Republic,<br />
b Department of Biochemistry, Chemistry and Biophysics,<br />
Faculty of Veterinary Hygiene and Ecology, University of<br />
Veterinary and Pharmaceutical Sciences,Palackého 1–3,<br />
612 42 Brno, Czech Republic,<br />
vitoulova@fch.vutbr.cz<br />
Introduction<br />
Investigations of the chemical constituensts of aquatic<br />
organisms can provide useful informations about the environment<br />
as well as toxicologically relevant data about the composition<br />
of biological species consumed by humans 1 . Arsenic<br />
causes a variety of adverse health effects to humans after acute<br />
and chronic exposures 2 . Toxicity, environmental mobility and<br />
accumulation in living organisms usually depend on the form<br />
in which the element is present. Information on the chemical<br />
forms is important for understanding the role of the element<br />
present as well as revealing its environmental cycle 3 . In the<br />
environment arsenic can be found as several compounds ranging<br />
from inorganic arsenite and arsenate to methylated compounds<br />
like monomethylarsonic acid, dimethylarsinic acid,<br />
trimethylarsine oxide, arsenobetaine, tetramethylarsonium<br />
ion and arsenocholine. In biologic samples several arsenosugars<br />
and arsenolipids can be present as well 4 .<br />
The determination of arsenic compounds is mostly<br />
carried out by hyphenated techniques, the main parts being<br />
liquid chromatographic separation and element specific detection.<br />
By far the most used systems are liquid chromatography<br />
inductively coupled plasma mass spectrometry (LC-<br />
ICP-MS) and liquid chromatography hydride generation<br />
atomic fluorescence spectrometry (LC-HG-AFS) 5 . Atomic<br />
fluorescence spectrometry (AFS) can be a good alternative<br />
to inductively coupled plasma mass spectrometry (ICP-MS)<br />
detector, with the advantage of a lower cost of investment<br />
and handling 5,6 .<br />
Experimental<br />
S t a n d a r d s a n d R e a g e n t s<br />
All solutions were prepared with deionized water<br />
(18.2 MΩcm). Stock solutions of 1,000 mg(As)dm –3 were<br />
prepared by dissolving naAsO 2 (Fluka, Switzerland),<br />
na 2 HAsO 4 . 7H 2 O (Fluka, Switzerland), CH 4 AsnaO 3 . 1.5H2 O<br />
(Chem. Service, USA), (CH 3 ) 2 AsHO 2 (Fluka, Switzerland)<br />
and arsenobetaine (Fluka, Switzerland). All arsenic solutions<br />
were stored in the dark at + 4 °C. These stock solutions were<br />
s815<br />
diluted with deionized water to the desired concentrations<br />
before use.<br />
For the analytical procedures the following chemicals<br />
were used: potassium dihydrogen phosphate (Fluka, Switzerland),<br />
potassium monohydrogen phosphate (Fluka, Switzerland),<br />
hydrochloric acid (Analytika, Czech Republic), potassium<br />
persulfate (Fluka, Switzerland), sodium borohydride<br />
(Riedel-de Haën, Germany), sodium hydroxide (Riedel-de<br />
Haën, Germany), nitric acid (Analytika, Czech Republic),<br />
hydrogen peroxide (Lach-ner, Czech Republic), ascorbic<br />
acid (Riedel-de Haën, Germany), potassium iodide (Analytika,<br />
Czech Republic), magnesium nitrate (Merck, Germany).<br />
All chemicals were at least of analytical grade.<br />
I n s t r u m e n t a t i o n<br />
A Gynkotek P508 (Gynkotek, Germany) HPLC pump<br />
and six-port injection valve (Ecom, Czech Republic) were<br />
used in conjunction with a strong anion-exchange column<br />
Hamilton PRP X-100 (Hamilton, USA). An Excalibur atomic<br />
fluorescence detector (PS Analytical, UK) equipped with a<br />
boosted discharge hollow cathode lamp (Photron, Australia)<br />
was used for detection. The measurements are carried out on<br />
the resonance line of As 19<strong>3.</strong>76 nm. For data collection and<br />
evaluation was used CSW software – version 1.7 (Data Apex,<br />
Czech Republic).<br />
S a m p l e P r e p a r a t i o n<br />
The muscle of commercially available fish samples were<br />
homogenized and freeze-dried.<br />
For the determination of total arsenic by AFS the microwave<br />
digestion (using HnO 3 and H 2 O 2 ) was employed;<br />
followed by the addition of Mg(nO 3 ) 2 to complete the<br />
destruction.<br />
Triplicate of samples were extracted with 10 ml of<br />
deionized water using ultrasonic bath at 50 °C for 2 hours.<br />
The extraction vessels were shaken every 15 min to ensure<br />
dispersion of the sample. After extraction the samples were<br />
centrifuged and extract was filtered through a 0.45 µm<br />
membrane filter and kept at 4 °C till speciation analysis.<br />
C o n d i t i o n s o f T o t a l A r s e n i c<br />
D e t e r m i n a t i o n<br />
A KI/ascorbic acid reducing solution was employed to<br />
reduce arsenic species to As (III). The reducing solution was<br />
added at least 30 minuts before analysis. Conditions for hydride<br />
generation: 1 % naBH 4 in 1 % naOH at flow rate the<br />
4.5 ml min –1 and 4.9 mol dm –3 HCl at flow rate 9 ml min –1 .<br />
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<br />
The mobile phase was 10 mM K 2 HPO 4 /KH 2 PO 4 adjusted<br />
to pH 6.1 at flow rate 1 ml min –1 . The hydride generation of<br />
the arsines was carried out by adding 6 mol dm –3 HCl and<br />
1.4 % naBH 4 in 1 % naOH, both at the 3 ml min –1 flow rate.<br />
1 % K 2 S 2 O 8 in 1 % naOH at the flow rate 0.3 ml min –1 was<br />
used as oxidation solution, oxidation was supported by UV.