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AFFINISEP
Bd Sonopa| Pôle d’innovation des Couronnes |76650 Petit couronne | FRANCE
E-mail : [email protected] | Website : www.affinisep.com
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Application Note AFFINIMIP® SPE 0chratoxin A - SPICES Spices
New regulation for Ochratoxin A in spices
Ochratoxin (OTA) is a mycotoxin produced as a secondary
metabolite of various Aspergillus and Penicillium fungis. It
can be found on several commodities (e.g. cereals and
cereal-based products, coffee, beer, grape juice, wine,
cacao products and spices).
Ochratoxin A exhibits toxicity in animals and mankind,
including nephrotoxic, hepatotoxic, immunotoxic,
teratogenic and carcinogenic effects and represents
therefore a serious health risk to livestock and population.
In order to limit these effects, European Regulation (EC)
594/2012 sets maximum levels for Ochratoxin A in spices
(e.g. 15μg/kg in pepper or nutmeg, 30μg/kg until
31/12/2014 (15µg/kg as from 1.1.2015) in chilli or
cayenne).
N
OH O
Cl
COOH
O
O
H
Figure 1. Chemical structure of Ochratoxin A, CAS N° 303-47-9.
How to solve this?
AFFINISEP has developed a specific method based on
Molecularly Imprinted Polymer (MIP) for solid-phase
extraction, clean-up and pre-concentration of Ochratoxin
A in spices.
AFFINIMIP® SPE Ochratoxin A - Spices is designed
especially for a simple, fast, sensitive and selective
extraction of ochratoxin A from spices. This application
note demonstrate a reliable quantification of Ochratoxin
A from spices at low concentrations where we obtained a
high recovery yield (> 89%) with a low background, even
with UV detection.
Results
High recovery
Table 1. Recoveries of Ochratoxin A after AFFINIMIP® SPE
Ochratoxin A – Spices Clean-up in different matrices and Relative
Standard Deviation under reproducibility conditions.
Matrix C° (µg/kg) Recoveries % % RSDR
White
pepper (n=7) 15 89.0 2.9
Nutmeg
(n=3) 15 94.2 4.5
Figure 2. Chromatogram obtained after purification of nutmeg
(spiked at 15µg / kg (red) or not contaminated (blue)) with
AFFINIMIP® SPE Ochratoxin A- Spices
Figure 3. Chromatogram obtained after purification of white
pepper (spiked at 15µg / kg (red) or not spiked (blue)) with
AFFINIMIP® SPE Ochratoxin A- Spices
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
mV
olts
0,0
2,5
5,0
7,5
10,0
12,5
15,0
17,5
20,0
22,5
25,0
27,5
30,0
32,5
35,0
Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
mVolts
- 4 - 2 0 2 4 6 8
10 12 14 16 18 20 22 24
Selective Solid Phase Extraction of Ochratoxin A from Spices Products Using Molecularly Imprinted Polymers
Ochratoxin A
Ochratoxin A
AFFINISEP
Bd Sonopa| Pôle d’innovation des Couronnes |76650 Petit couronne | FRANCE
E-mail : [email protected] | Website : www.affinisep.com
Pag
e2
Application Note AFFINIMIP® SPE 0chratoxin A - SPICES Spices
Experimental conditions
Purification procedure of Ochratoxin A from pepper and nutmeg matrices with NaHCO3 1% extraction solution
Preparation of samples prior to SPE
10g of finely ground spices are mixed with 100mL of extraction solvent (1% NaHCO3 in water) during 30 minute using magnetic stirring. The extract is filtered through a filter paper. Then, 20mL of the extract is diluted with 20mL of HCl solution pH=1, 0.1M. After a filtration through a filter paper, this solution is used as the loading solution.
Solid phase extraction (SPE) protocol
The details of each step are as follows:
Condition the SPE Cartridge with 4mL of
acetonitrile (ACN), then with 4mL of deionized
water
Load 20mL of the loading solution (eq. 1g of
sample)
Wash the cartridge with 12mL of 60/40 HCl
solution pH=1, 0.1M /ACN (v/v)
Wash the cartridge with 5mL of buffer ammonium
formate pH=7.4, 30mM
Dry 10 min or force the water down into the
cartridge and out the bottom
Wash the cartridge with 6mL of ACN
Elute Ochratoxin A with 2 x 4mL of methanol
(MeOH) containing 2% of acetic acid (v/v)
The elution fraction is then evaporated and dissolved in
the mobile phase. Alternatively, the elution may be diluted
to a known volume by addition of water for further
analysis.
Materials
All reagents and chemicals were ACS grade quality or
better. Ochratoxin A was obtained from AFFINISEP.
Samples were purchased in different supermarkets.
The SPE procedure used 6mL AFFINIMIP® SPE Ochratoxin
A – Spices Cartridges.
Analysis
HPLC was performed on a ThermoFinnigan Spectra System
with a Thermo Hypersil Gold C18 column (150mm x
2.1mm) protected by a Hypersil Gold (10×2.1mm) guard
column. Separation was carried out using a mobile phase
of deionized water/acetic acid/MeOH (47/1/52, v/v) at a
flow rate of 0.2mL/min. The detection system was a Jasco
Model FP-2020 Fluorescence detector set to
excitation/emission wavelengths of 333 and 460nm,
respectively. The injection volume was 20µL.
Conclusion
The use of AFFINIMIP® SPE Ochratoxin A – Spices
cartridge is a simple, fast, sensitive and selective tool for
the extraction of Ochratoxin A.
The use of AFFINIMIP® SPE Ochratoxin A - Spices enables
to obtain recoveries above 85% for 15µg / kg with
reproducibility relative standard deviation below 5%. This
method is well-suited for the analysis of Ochratoxin A in
spices.
Product reference
AFFINIMIP® SPE Ochratoxin A – Spices
FS101PP-02 for 25 cartridges 6mL
FS101PP-03 for 50 cartridges 6mL
