Başak KÜÇÜKÇAKAN, Biljana DIMZOSKA STOJANOVSKA, Zehra MUSLIU HAJRULAI, Elizabeta STOJKOVIC IMITRIESKA, Risto UZUNOV, Katerina DAVCHEVA

Determination of Ochratoxin-A in Cattle Liver By HPLC-FD Method

Okratoksin- (OTA), Penicillium ve Aspergillus spp. tarafından üretilen bir mikotoksindir. OTA insanlar üzerinde karsinojenik, nefrotoksin ve immunosupresif etkilere sebep olmaktadır. Ayrıca toksin hayvan dokularında (böbrek, karaciğer, kas) birikerek, insan sağlığı için potansiyel tehlike kaynağı oluşturmaktadır. Bu çalışmadaki amaç, sığır karaciğerinde OTA varlığının HPLC-FD metodu ile araştırıldığı diğer çalışmaların sonuçlarını takiben, 2002/657/EC sayılı komisyon kararına uygun bir HPLC-FD metodu geliştirmektir. Bu amaç doğrultusunda; spesifite, doğrusallık, tespit limiti (LOD), tayin limiti, (LOQ), doğruluk ve kesinlik gibi parametreler değerlendirilmiştir. Doğrusallık; sekiz adet standart solüsyonun analizi ile belirlenmiştir. Korelasyon katsayısı (r2)>0.9999, tespit limiti 0.088 ng/ml ve tayin limiti 0.268ng/ml değerinde bulunmuştur. Doğruluk ve kesinlik analizleri üç farklı konsantrasyon seviyesi (1.0, 5.0 and 10.0 mg/kg) denk alınarak uygulanmıştır. Geri kazanım, %76.1-102.5 arasında bulunmuştur. Tekrarlanabilirlik relatif standart sapma (RSDr %) göre ifade edilerek; %3.7-14.28 arasında hesaplanmıştır. Tekrar üretilebilirlik sonuçları (RSDR%); birinci gün %0.16- 11.8, ikinci gün %2.15-17.88 arasında bulunmuştur. Metot oluşturulduktan sonra, OTA varlığı 15 adet doku örneğinde araştırılmıştır. Sadece 1 örnekte tayin limitine yakın oranda (0.2 mg/kg) OTA tespit edilmiştir. Sonuç olarak, üzerinde çalıştığımız bu HPLC-FD metodunun sığır karaciğerinde maksimum kalıntı limitleri altındabulunan OTA miktarlarının belirlemesinde kullanılmasını önermekteyiz.

Sığır Karaciğerinde Okratoksin-A Varlığının HPLC-FD Metodu İle Belirlenmesi

Ochratoxin-A (OTA) is a mycotoxin which is produced by several Penicillium and Aspergillus spp. OTA has carcinogenic, hepatogenic, nephrotoxic and immunosuppressive effects in human. The toxin accumulates in the animal tissues (kidney, liver and muscle) which could be potential harmful source for human health. Following results of previous investigations our aim was to validate HPLC-FD method for OTA determination in cattle liver which could be implemented in laboratory practise, complying with the European Union Commission Decision (2002/657/EC). Specificity, linearity of method, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision were evaluated. The linearity of the method was determined by preparation of eight standard solutions. The standard curve showed high correlation coefficient (r2>0.9999). LOD and LOQ were determined as 0.088 ng/ml and 0.268 ng/ml, respectively. Accuracy and precision were established in three concentration levels: 1.0, 5.0 and 10.0 mg/kg. Recovery was in the range of 76.1 to 102.5% and repeatability (RSDr%) was expressed through relative standard deviation which resulted in the range of 3.7 to 14.28%. Reproducibility results (RSDR%) were found in the range of 0.16-11.8% for the first day and for the second day in the range of 2.15-17.88%. (Post-hock analysis of samples (n=15), only in one sample OTA was detected (0.2 mg/kg) as close to the LOQ value. As a result, we recommend this method for OTA detection and quantification in cattle liver, for concentrations which are lower than the maximum residue limits (MRL).

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1. European Food Safety Authority (EFSA): Opinion of the scientific panel on contaminants in the food chain on a request from the commission related ochratoxin-A in food. EFSA J, 365, 1-56, 2006.
2. El-Ghreeb W, Darwish W, Tharvat E, El-Deskoy K, Hussein A:Aflatoxin and Ochratoxin-A residues in some meat additives. Life Sci, 10 (4): 3411-3414, 2013.
3. European Food Safety Authority (EFSA): Opinion of the scientific panel on contaminants in food chain on a request from the commission related to ochratoxin-A (OTA) as undesirable substance in animal feed. EFSA J, 101, 1-36, 2004.
4. Asta C, Galaverna G, Bertuzzi T, Moseriti A, Pietri A, Dossena A, Marchelli R: Occurrence of ochratoxin- A in raw ham muscle, salami and dry-cured ham from pigs fed with contaminated diet. Food Chem, 120, 978-983, 2010. DOI: 10.1016/j.foodchem.2009.11.036
5. The Joint FAO/WHO Expert Committee on food Additives (JECFA): Ochratoxin A.JECFA Food Additive Series 47, Geneva, 2001. http://www. inchem.org/documents/jecfa/jecmono/v47je04.htm, Accessed: 09.01.2015.
6. International Agency Research on Cancer (IARC): Agents classified by the IARC monographs volumes 1-111. 2014. http://monographs.iarc.fr/ENG/Classification/ClassificationsAlphaOrder.pdf, Accessed: 09.01.2015.
7. Mobasar M, Hummal J, Blank R, Sudekum K: Ochratoxin-A in ruminants - Review on its degradation by gut microbes and effects on animals. Toxins, 2, 809-839, 2010. DOI: 10.3390/toxins204809
8. Reddy L, Bhoola K: Ochratoxins-food contaminants impact on human health. Toxins, 2, 771-779, 2010. DOI: 10.3390/toxins2040771
9. Stoev D: Complex etiology, prophylaxis and hygiene control in mycotoxic nephropathies in farm animals and humans. Int J Mol Sci, 9, 578-605, 2008.
10.Daurte C, Lino M, Angelina P: Food safety implications of ochratoxin-A an animal-derived food products. Vet J, 192, 286-292, 2012. DOI: 10.1016/j.tvjl.2011.11.002
11.Scientific Cooperation (SCOOP) Task Reports: Task 3.2.7. Assessment of dietary intake of ochratoxin-A by the population of EU member states. 27, Italy, 2002. http://ec.europa.eu/food/fs/scoop/3.2.7_en.pdf, Accessed:09.01.2015.
12.Codex Alimentarius Commission (CAC): Position paper on ochratoxin-A. Codex committee on food additives and contaminants 31thsession, 1-9, The Netherlands, 1999.
13.Dragacci S, Grosso FR, Bire R, Fremy M, Coulon S: A French monitoring programme for determining ochratoxin-A occurrence in pig kidneys. Nat Toxins, 7, 167-173, 1999. DOI: 10.1002/(SICI)1522-7189(199907/08)7:4_____167::AID-NT55>3.0.CO;2-Q
14.Matrella R, Monaci L, Milillo M, Tantillo G, Palmisano F: Ochratoxin-A determination in paired kidneys and muscle samples from swines slaughtered in southern Italy. Food Control, 17, 114-117, 2006. DOI: 10.1016/j.foodcont.2004.08.008
15.Guillamont ME, Lino CM, Baeta ML, Pena ASM, Silveira IN, Vinuesa J: A comparative study of extraction apparatus in HPLC analysis of ochratoxin-A in muscle. Anal Bioanal Chem, 383, 570-575, 2005. DOI: 10.1007/s00216-005-0051-4
16.Monaci L, Tantillo G, Palmisano F: Determination of ochratoxin-A in pig tissues by liquid-liquid extraction and clean-up and high-performance liquid chromatography. Anal BioanalChem, 378, 1777-1782, 2004. DOI: 10.1007/s00216-004-2497-1
17.Commission of the European Communities: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (2002/657/EC). Off J Eur Union, L 2218-2236, 2002.
18.Jorgensen K, Petersen A: Content of ochratoxin-A in paired kidney and meat samples from healthy Danish slaughter pigs, Food Addit Contam, 19, 562-567, 2002. DOI: 10.1080/02652030110113807
19.Singh R: HPLC method development and validation- on overview. J Pharm Educ Res, 4 (1): 26-33, 2013.
20.Taylor B, Kuyatt C: Guidelines for evaluating and expressing the uncertainty of NIST Measurement Results. In, NIST Editorial Review Boards. NIST Technical Note 1297, 1-20, US, 1994.
21.Commission of the European Communities: Commission Regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Off J Eur Union, L 70, 12-34, 2006.
22.Ceci E, Bozzo G, Bonerba E, Pinto A, Tantillo MG: Ochratoxin-A detection by HPLC in target tissues of swine and cytological and histological analysis. Food Chem, 105, 364-368, 2007.
23.Mili?evi? D, Stefanovi?S, Jankovi? S, Radi?evi? T: Risk analysis and exposure assessment of ochratoxin-A in Serbia. Vet World, 5, 412-416, 2012. DOI: 10.5455/vetworld.2012.412-416
24.Mili?evi? D, Juri? V, Dakovi? A, Jovanovi? M, Stefanovi? SM, Petrovic Z: Survey of slaughtered pigs for occurrence of ochratoxin-A and porcine nephropathy in Serbia. Int J Mol Sci, 9, 2169-2183,2008. DOI: 10.3390/ijms9112169
25.Mili?evi? D, Juri? V, Stefanovi? SM, Veskovi?- Moracanin SV, Jankovi? S: Analysis of ochratoxin-A in pig tissues using high pressure liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry as confirmative methods. Proc Nat Sci Matica Srpska Novi Sad, 117, 51-61, 2009. DOI: 10.2298/ZMSPN0917051M
26.Georgescu M, Constantin S, Dobrea M, Milca I, Georgescu D:Comparative assessment of ELISA and HPLC for ochratoxin-A detection in pork kidney samples. Bulletin UASVM Anim Sci Biotechnol,70 (2): 223-232, 2013.