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 />
P33 ChIRAL SPECIATION OF IODINE IN<br />
ThyDOID hORMONES by uSING hPLC/MS/<br />
MS AND hPLC-ICP-MS<br />
ELISA GOMEZ DE LAS HERAS, JOSE LUIS GóMEZ-<br />
ARIZA and TAMARA GARCíA-BARRERA<br />
Universidad de Huelva, Departamento de Química y Ciencia<br />
de los Materiales, Facultad de Ciencias Experimentales,<br />
Campus de El Carmen, 21007-Huelv, Spain<br />
pippinheroe@hotmail.com<br />
Introduction<br />
Iodine is a key-element in the thyroid functions as active<br />
centre of the thyroid hormones, which activity depend on the<br />
number of iodine atoms into the molecule. These hormones<br />
are involved in the regulation of different important biological<br />
processes.<br />
Thyroxine (T 4 or 3,5,3’,5’-tetraiodothyronine) is the<br />
major hormone produced and secreted in the follicular cells<br />
of the thyroid gland by the protein thyroglobulin (Tg). Other<br />
compounds homologues have also been subsequently characterized.<br />
In addition, thyroid hormones have enantiomeric forms<br />
which affect their biological activities 1,2 . Therefore, these<br />
species have to be determined separately to know their real<br />
metabolic behaviour and action on living organisms.<br />
There are a number of problems associated with the<br />
analysis of thyroid hormones. The use of liquid chromatography/mass<br />
spectrometry presents several advantages over<br />
the currently used radio-immunoassay (RIA) and gas chromatography-mass<br />
spectrometry methods. The use of a UV<br />
detector to monitoring chromatographic separations allows<br />
a good sensitivity. There is an increasing interest for the use<br />
of HPLC coupled to MS, which increases the selectivity<br />
with acceptable sensitivity that is advisable for the complex<br />
biological matrices. However, the most sensitive analytical<br />
approach for iodinated hormones is HPLC-ICP-MS, which<br />
has been proposed by Michalke and other authors 3 .<br />
In the present study an analytical method for eight thyroid-related<br />
compounds has been performed. The coupling<br />
of HPLC-UV(PDA)-MS allows the identification and quantification<br />
of all the compounds and HPLC-ICP-MS the analysis<br />
of very low level of these hormones. A procedure for<br />
the analysis of these substances in human serum samples has<br />
been optimized and method reliability validated by recovery<br />
experiments.<br />
Experimental<br />
All the calibrants: L-thyronine – T 0 ; 3,5-diiodo-L-thyronine<br />
– L-T 2 ; 3,3’,5-triiodo-L-thyronine – L-T 3 ; 3,3’,5’-triiodo-L-thyronine<br />
– L-r-T 3 ; L-Thyroxine – LT 4 ; D-thyroxine<br />
– D-T4; 3,5-diiodo-L-tyrosine – DIT; 3-iodo-L-tyrosine<br />
– MIT were obtained from Sigma-Aldrich Chemie (Steinheim,<br />
Germany). Stock solutions were prepared at a concentration<br />
of 1 mg ml –1 in a mixture of MeOH and 10 nM naOH<br />
(1 : 1 (v/v)).<br />
s645<br />
C h r o m a t o g r a p h i c C o n d i t i o n s a n d<br />
I n s t r u m e t a t i o n<br />
A SpectraSYSTEM P4000 (HPLC) coupled in series<br />
with a SpectraSYSTEM UV6000LP photo diode array detector<br />
(PDA) and a LCQ Advantage mass spectrometer from<br />
Thermo-Finnigan (San José, CA, USA) with electrospray<br />
ionization (ESI) as ion source was used with a CHIRAL-<br />
PAK ® Qn-AX column. The mobile phase was 3% acetic<br />
acid in 40 % acetonitrile and 60 % water mixture; flow rate<br />
0.7 ml min –1 .<br />
H P L C - I C P - M S C o u p l i n g<br />
An ICP-MS (HP 7600e, Hewlett-Packard, USA) was<br />
used with the same chromatographic conditions of previous<br />
paragraph. Iodine was determined at m/z 127. The operational<br />
conditions were the followings: forward power 1500 W,<br />
plasma gas flow rate 15.0 ml min –1 , auxiliary gas flow rate<br />
1.0 ml min –1 , carrier gas flow rate 0.6 ml min –1 , optional gas<br />
(O 2 ) 6%, sampling depth 6.5 mm, sampling and skimmer<br />
cones of platinum. The dwell time was set to 0.3 s.<br />
H o r m o n e s E x t r a c t i o n f r o m S e r u m<br />
A ClinChek ® -Control serum (RECIPE, Chemicals+<br />
Instruments GmbH, Munich, Germany) serum was used for<br />
hormone extraction. A volume of sample between 300 and<br />
450 µl of serum was treated with a triple volume of 1% formic<br />
acid in acetonitrile. The mixture was vortexing for 5 min<br />
and centrifugated at 3,000 rpm for 10 min. The supernatant<br />
was filtered by 0.2 µm before to inject into the chromatographic<br />
column.<br />
Results<br />
The use of the in series coupling HPLC-PDA (UV-vis)-<br />
MS intents to combine the sensitivity of UV detector with the<br />
PDA system and the selectivity of MS. However, the very<br />
low levels of these hormones in healthy and hypothyroid people<br />
require more sensitive detection that can be reached with<br />
HPLC-ICP-MS.<br />
A chiral separation of these hormones was performed<br />
using a tert-butyl carbamoylated quinine stationary phase.<br />
Fig. 1. Tert-butyl carbamoylated quinine stationary phase<br />
Several solvent mixtures were tested as mobile phases.<br />
The optimum separation was obtained with 3 % of acetic in<br />
acetonitrile:water (40:60) at a flow rate 0.7 ml min –1 .<br />
In Fig. 2 is shown a typical chromatogram obtained<br />
with the HPLC-(PDA)UV system for 20 mg dm –3 of each<br />
compound. The different compounds are well resolved but