3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P08 QuANTITATIVE DETERMINATION OF
SuLFONAMIDE RESIDuES IN ChICKEN
MEAT by A NEw SOLID PhASE ExTRACTION
AND hPLC-uV FOR DETECTION
COnSTAnTIn BELE a , OCTAVIAn nEGREA a , ADELA
PInTEA a , FRAnCISC VASILE DULF a , DAn LUPU b and
ALEXAnDRU R. BIRIS b
a University of Agricultural Sciences and Veterinary Medicine,
R- 400372 Cluj-Napoca, Romania,
b National Institute for Research and Development of Isotopic
and Molecular Technologies, R-400293 Cluj-Napoca,
Romania,
cbele2002@yahoo.com
Introduction
Sulfonamides (SAs) are a group of synthetic antibiotics
that have been used in food-producing animals for therapeutic
and prophylactic purposes 1 . There is a risk of occurrence
of unwanted residues in animal products if these antimicrobials
have been improperly administered or if the proper
withdrawal period has not been observed. To safeguard
human health, the European Community has adopted for SAs
safe maximum residue limits (MRLs) at the total level of
100 μg kg –1 in food of animal origin 2 .
Many methods such as LC and LC-MS, GC and GC-
MS, ELISA, biosensor immunoassay (BIA) and high performance
capillary electrophoresis (HPCE) have been developed
for the determination of SA residues in food 3 . Solid phase
extraction (SPE) was used as clean-up or enrichment method
for SAs in tissues. The main sorbents used for the extraction
of SAs are C18, aluminium oxide, strong cation-exchange 4 .
Multi-walled carbon nanotubes (MWCnTs) are a novel
carbon material used as an effective solid phase adsorbent for
organic compounds (including SAs) 5 .
In this paper, a sensitive method was developed for the
determination of six SAs in chicken meat using MWCnTs
and aluminium oxide as solid phase adsorbents followed by
HPLC with UV detector.
Experimental
M a t e r i a l s a n d R e a g e n t s
Chicken muscle tissues were purchased from local food
market and deep frozen until analysis. Organic solvents such
as acetonitrile, acetic acid and 1- propanol were all pesticide
residue grade , commercially available from Merck . Anhydrous
sodium sulfate was analytical grade (Bucarest, Romania).
Deionized and redistilled water was prepared from Milli-Q
Plus (Millipore). Sodium acetate trihydrate (Merck) was used
as buffer for HPLC mobile phase. Sulfadiazin (SDZ), sulfamerazine
(SMR), sulfapyridine (SPY), sulfisoxazole (SIO),
sulfamethoxazole (SMO) and sulfadimethoxine (SDM) were
purchased from Sigma.
MWCnTs were purchased from Institute for Research
and Development of Isotopic and Molecular Technologies
Cluj-napoca, Romania.
s589
Standard stock solutions were prepared by accurately
dissolving approximately 10 mg of SAs in 10 ml of acetonitrile
LC grade and stored at 4 °C. Working standards were
prepared weekly by appropriate dilution in acetate buffer at
pH 4.5.
C h r o m a t o g r a f i c S y s t e m a n d
C o n d i t i o n s
All experiments were carried out by using Shimadzu VP
Series liquid chromatograph equipped with a UV-VIS detector.
The chromatographic separation was accomplished in 30 min
with gradient elution on a C 18 (250 mm × 4.6 mm, 5 μm)
analytical column (Alltima) with a mobile phase 0.01M acetate
buffer pH 4.5 (A) and acetonitrile (B). Flow 1 ml min –1
was used for the separation of analytes at the following program:
20 % B to 50 % within 22 min, back to 20 % in 3 min,
equilibration for 5 min. The injection volume was 20μl and
the detection of SAs was conducted at 266 nm.
S a m p l e P r e p a r a t i o n a n d S a m p l e
C l e a n - u p
Ten grams of minced chicken tissue was placed into a
50 ml polypropylene tube. 20 ml acetonitrile and 5 grams
baked anhydrous sodium sulfate was then added. The sample
was homogenized with an Ultraturax for about 1 min.,
and then centrifuged at 5,000 rpm for 5 min.The residue was
extracted by sonication with 20 ml acetonitrile and then centrifuged
at 5000 rpm for 5 min.The extracts were combined
and the solvent was concentrated to 5 ml.The solution was
passed through the Alumina n SPE cartridge preconditioned
by 10 ml acetonitrile.The analytes were eluted with 5 ml acetonitrile
: water (80 : 20, v/v).The eluent and loading solvents
were combined and transferred to concentration bottle.Then
5 ml 1-propanol was added and the solution was evaporated
to near dryness. The residue was dissolved by ultrasonication
for 1 min with 30 ml acetate buffer (pH 5). A second SPE
cartridge (200 mg MWCnTs) was utilized to remove potential
interferences. The SPE was conditioned with acetonitrile
(5 ml) and water (5 ml) prior to loading the sample. The
analytes were eluted with a mixture of 2 ml acetate buffer
(pH 4.5) and 4 ml acetonitrile. The eluate was evaporated to
1 ml under nitrogen stream in a 40 °C block heater and filtered
through a 0.45 μm disposable syringe filter.
Results
The present procedure uses two SPE cartridges for
clean-up because acetonitrile extracted a lot of endogenous
compounds from meat sample.The first one, Sep-Pak Alumina
n, is a polar sorbent SPE cartridge. The second SPE
cartridge (MWCnTs) is a non-polar sorbent and was utilized
to further cleanse the extract.
The calibration graphs obtained by plotting peak area
versus drug concentration in 0.1–10 μg ml –1 range are
reported in Table I.