Development of HPLC methods for determination of ochratoxin A in pork kidney, pork meat and pork blood products F. Malir1, T. Roubal2, J. Skarkova3, V. Ostry3 1
University of Hradec Kralove, Faculty of Science, Department of Biology, Czech Republic 2
3
Institute of Public Health in Hradec Kralove, Czech Republic
National Institute of Public Health in Prague, Center for Health, Nutrition and Food in Brno, Czech Republic
[email protected]
Introduction Ochratoxin A (OTA OTA) is one of the toxic secondary metabolites of toxigenic microfungi. OTA is suspected to have immunosuppressive, nephrotoxic, nephrotoxic, hepatotoxic, hepatotoxic, genotoxic, genotoxic, carcinogenic and teratogenic effects on humans (Pfohl-Leszkowicz & Manderville, 2012). OTA was frequently detected above all in foodstuffs of vegetable origin, but also in foodstuffs of animal origin (e.g. pork blood products, pork kidney, pork liver, pork meat). The pigs are exposed to OTA through their contaminated feed (Pietri et al., 2006). According to recent knowledge about OTA carcinogenicity, information about new OTA exposure sources and recent results of urinary OTA excretion in men and women is realised new research project: „Ochratoxin A – health risk assessment for selected population groups in the Czech Czech Republic“ Republic . This study presents the development of validated liquid chromatography method with fluorescence detection for quantification of OTA in foodstuffs of animal origin.
Methodology
Validated HPLC methods for determination of OTA in pork kidney, kidney pork blood and meat products and pork meat were developed. OTA was determined in foodstuffs of animal origin by high performance liquid chromatography method with fluorescence detection (HPLC-FD) (Creppy et al., 1993; Zimmerli & Dick, 1995; Nicol et al., 1998; Dragacci et al., 1999; Petkova-Bocharova et al., 2005).
Preparation of OTA standards
A standard solution (10 10 µg.l-1) of OTA in methanol was prepared from the OTA crystalline substance. This solution was diluted to obtain working solutions (usually 40 ng.ml-1) of OTA in methanol. For building of the calibration curve is prepared a concentration line in the range of OTA from 0.125 to 4.0 ng.ml-1 in methanol.
Analytical conditions
The analytical column 8 μm x 300 mm x 4.6mm (SPHERISORB ODS 2, 10 µm), with guard cartridge 5μm x 4mm x 10mm was used at a temperature of 40 oC. Methanol:acetonitril:0.005 mol.l-1 sodium acetate:acetic acid (300 ml : 300 ml : 400 ml : 14 ml v/v/v/v) were used as a mobile phase with a flow rate 1.5 ml/min. Injection volume was 50 μl. Parameters of the fluorescence detector were excitation wavelenght at λ = 340 nm (333 nm), and emission wavelenght at λ = 465 nm, Gain = 1000 ( bandwidth EM: 18). nm Retention time under these chromatographic conditions was found between 6-9 min. min
The cleaning of OTA in samples
OTA was extracted three times from homogenised foodstuffs (5 5 g) g by acidified chloroform (20 20 ml, ml 20 ml and 10 ml for 10 minutes) minutes and the extracted solutions were further cleaned up by liquid-liquid extraction. OTA was consequently reextracted from the chloroform phase (20 20 ml) ml with 20 ml NaHCO3 (0.5 mol.l-1). The acquired extract was cleaned on the immunoaffinity chromatography (OCHRAPREP® columns, R-Biopharm, Germany). OCHRAPREP® immunoaffinity columns shoud be at ambient temperature before use. The extract (30 30 ml) ml was applied to the OCHRAPREP® column and the sample was allowed to pass through at a flow rate 2-3 ml.min-1. The column was washed by passing 20 ml Milli Q water at a flow rate 2-3 ml.min-1. The air was pushed trought the column to collect the last drops of water. The bound OTA was eluted slowly from the column using 5 ml methanolmethanol-acetic acid (98:2 v/v). The air was pushed trought the column to collect the last drops of eluate. The eluate was evaporated to dryness under nitrogen flow in EVATERM equipment by 45-50 oC, then dissolved in 1 ml methanol. methanol
Validation of methods Validation of these methods were performed in accordance with the protocol approved by the AOAC. The following parameters were assessed: calibration (range and linearity), accuracy of the method (trueness + precision), limit of detection (LOD), limit of quantification (LOQ). The certified reference materials (CRMs) for the determination of OTA in pork kidney and foodstuffs of animal origin are not yet available.
Safety Precautions
All glassware contaminated by OTA was decontaminated by soaking in a bath containing a solution of alkaline NaClO for at least 24 h, or in 4% RBS 35 (Fluka) for at least 30 min.
Parameter of validation
Pork kidney
Pork meat
Pork blood products
Salami
Limit of detection (LOD) (μg.kg-1)
0.1
0.1
0.1
0.1
Limit of quantification (LOQ) (μg.kg-1)
0.3
0.3
0.3
0.3
Max. determinable level (μg.kg-1)
200
200
200
200
Uncertainty of determination (%)
< 25
< 25
< 25
< 25
Recovery (%) RSDr (%)
80
83
83
88
5.66
3.77
5.62
4.61
Table The results of validation of methods
Conclusions
In summary, practical, cost effective, selective and accurate HPTC-FD methods for quantification of OTA in pork kidney, pork meat and pork blood and meat products were developed. It is sensitive to levels of 0.3 μg.kg-1 (limit of quantification - LOQ). This study was financially supported by the project (no. NT12051-3/2011) from the Czech Ministry of Health (IGA MZ CR) References: References: Creppy E.E., Castegnaro M., Grosse Y., Mériaux J., Manier C., Moncharmont P., Waller C., et al. " Etude de l ochratoxicose humaine dans trois régions de France : Alsace,Aquitaine et région Rhone - Alpes ", Human ochratoxicosis and its pathologies, Eds.Creppy E.E., Castegnaro M., Dirheimer G., Colloque INSERM (John Libbey Eurotext Ltd.,1993,Vol.231, pp.148 – 158. Dragacci, S., Frémy, J.M. Détermination de l´OTA dans les tissus des animaux. Méthode interne. CNEVA (Centre National d´Etudes Véterinaires et Alimentaires), Paris., 1999, 1-8 Nicol, B. et al.: Development of an extraction method for ochratoxin A in pig kidney using Ochraprep immunoaffinity columns : Revue Méd.Vét., 149, 1998, 512. Petkova-Bocharova, T., Castegnaro, M., Pfohl-Leszkowicz, A., Garren, L., Grosso, F., Scott, P.M. Biomarkers of Human Exposure to ochratoxin A. Food Additives and Contaminants, Suppl.1, 2005, 99-107 Pfohl-Leszkowicz, A., Manderville, R.A. An Update on Direct Genotoxicity as a Molecular Mechanism of Ochratoxin A Carcinogenicity. Chem. Res. Toxicol., 25, 2012, 252-262. Pietri, A., Bertuzzi, T., Gualla, A., Piva, G. Occurrence of ochratoxin A in raw ham muscles and in pork products from Northern Italy, Italian Journal of Food Science, 18 (2006), 99-106. Zimmerli, B., Dick, R.: Determination of ochratoxin A at the ppt level in human blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column cleanup: methodology and Swiss data, J. Chromatogr. B. 666, 1995, 85-99.
