3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P33 ChIRAL SPECIATION OF IODINE IN
ThyDOID hORMONES by uSING hPLC/MS/
MS AND hPLC-ICP-MS
ELISA GOMEZ DE LAS HERAS, JOSE LUIS GóMEZ-
ARIZA and TAMARA GARCíA-BARRERA
Universidad de Huelva, Departamento de Química y Ciencia
de los Materiales, Facultad de Ciencias Experimentales,
Campus de El Carmen, 21007-Huelv, Spain
pippinheroe@hotmail.com
Introduction
Iodine is a key-element in the thyroid functions as active
centre of the thyroid hormones, which activity depend on the
number of iodine atoms into the molecule. These hormones
are involved in the regulation of different important biological
processes.
Thyroxine (T 4 or 3,5,3’,5’-tetraiodothyronine) is the
major hormone produced and secreted in the follicular cells
of the thyroid gland by the protein thyroglobulin (Tg). Other
compounds homologues have also been subsequently characterized.
In addition, thyroid hormones have enantiomeric forms
which affect their biological activities 1,2 . Therefore, these
species have to be determined separately to know their real
metabolic behaviour and action on living organisms.
There are a number of problems associated with the
analysis of thyroid hormones. The use of liquid chromatography/mass
spectrometry presents several advantages over
the currently used radio-immunoassay (RIA) and gas chromatography-mass
spectrometry methods. The use of a UV
detector to monitoring chromatographic separations allows
a good sensitivity. There is an increasing interest for the use
of HPLC coupled to MS, which increases the selectivity
with acceptable sensitivity that is advisable for the complex
biological matrices. However, the most sensitive analytical
approach for iodinated hormones is HPLC-ICP-MS, which
has been proposed by Michalke and other authors 3 .
In the present study an analytical method for eight thyroid-related
compounds has been performed. The coupling
of HPLC-UV(PDA)-MS allows the identification and quantification
of all the compounds and HPLC-ICP-MS the analysis
of very low level of these hormones. A procedure for
the analysis of these substances in human serum samples has
been optimized and method reliability validated by recovery
experiments.
Experimental
All the calibrants: L-thyronine – T 0 ; 3,5-diiodo-L-thyronine
– L-T 2 ; 3,3’,5-triiodo-L-thyronine – L-T 3 ; 3,3’,5’-triiodo-L-thyronine
– L-r-T 3 ; L-Thyroxine – LT 4 ; D-thyroxine
– D-T4; 3,5-diiodo-L-tyrosine – DIT; 3-iodo-L-tyrosine
– MIT were obtained from Sigma-Aldrich Chemie (Steinheim,
Germany). Stock solutions were prepared at a concentration
of 1 mg ml –1 in a mixture of MeOH and 10 nM naOH
(1 : 1 (v/v)).
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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
I n s t r u m e t a t i o n
A SpectraSYSTEM P4000 (HPLC) coupled in series
with a SpectraSYSTEM UV6000LP photo diode array detector
(PDA) and a LCQ Advantage mass spectrometer from
Thermo-Finnigan (San José, CA, USA) with electrospray
ionization (ESI) as ion source was used with a CHIRAL-
PAK ® Qn-AX column. The mobile phase was 3% acetic
acid in 40 % acetonitrile and 60 % water mixture; flow rate
0.7 ml min –1 .
H P L C - I C P - M S C o u p l i n g
An ICP-MS (HP 7600e, Hewlett-Packard, USA) was
used with the same chromatographic conditions of previous
paragraph. Iodine was determined at m/z 127. The operational
conditions were the followings: forward power 1500 W,
plasma gas flow rate 15.0 ml min –1 , auxiliary gas flow rate
1.0 ml min –1 , carrier gas flow rate 0.6 ml min –1 , optional gas
(O 2 ) 6%, sampling depth 6.5 mm, sampling and skimmer
cones of platinum. The dwell time was set to 0.3 s.
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
A ClinChek ® -Control serum (RECIPE, Chemicals+
Instruments GmbH, Munich, Germany) serum was used for
hormone extraction. A volume of sample between 300 and
450 µl of serum was treated with a triple volume of 1% formic
acid in acetonitrile. The mixture was vortexing for 5 min
and centrifugated at 3,000 rpm for 10 min. The supernatant
was filtered by 0.2 µm before to inject into the chromatographic
column.
Results
The use of the in series coupling HPLC-PDA (UV-vis)-
MS intents to combine the sensitivity of UV detector with the
PDA system and the selectivity of MS. However, the very
low levels of these hormones in healthy and hypothyroid people
require more sensitive detection that can be reached with
HPLC-ICP-MS.
A chiral separation of these hormones was performed
using a tert-butyl carbamoylated quinine stationary phase.
Fig. 1. Tert-butyl carbamoylated quinine stationary phase
Several solvent mixtures were tested as mobile phases.
The optimum separation was obtained with 3 % of acetic in
acetonitrile:water (40:60) at a flow rate 0.7 ml min –1 .
In Fig. 2 is shown a typical chromatogram obtained
with the HPLC-(PDA)UV system for 20 mg dm –3 of each
compound. The different compounds are well resolved but