Quantitative Determination Of Ochratoxin Research Articles

Buğday ve Çeltikte Okratoksin A’nın Kantitatif Olarak Tespiti ve Validasyon Çalışması

Bu çalışmada, buğday ve çeltik Okratoksin A (OTA) varlığı yönünden değerlendirilmiştir. Buğday örnekleri Mersin, çeltik örnekleri ise Edirne ve Çanakkale illerindeki çiftçilerden temin edilmiştir. OTA yüksek performanslı sıvı kromatografisi (HPLC) yöntemi ile tespit edilmiştir. Buğdayörneklerinin 3'ünde (%15) OTA tespit edilmiştir. Konsantrasyon, <ölçüm limiti (LOQ)-6.93µg kg-1 arasında değişmekte olup, pozitif numunelerin iki tanesi, doğrudan insan tüketimine zararlı olacağı tahmin edilen OTA için 5 µg kg-1 maksimum limiti geçmiştir. Çeltik örneklerinin 2’sinde (%10) OTA tespit edilmiştir. Konsantrasyon, <ölçüm limiti (LOQ)-6.93µg kg-1 arasında değişmekte olup, pozitif numunelerin iki tanesi, doğrudan insan tüketimine zararlı olacağı tahmin edilen OTA için 5 µg kg-1 maksimum limiti geçmiştir. Çeltik örneklerinin 2’sinde (%10) OTA tespit edilmiştir. Konsantrasyon, <LOQ-5.44µg kg-1 arasında değişmekte olup, pozitif numunelerin bir tanesi, doğrudan insan tüketimine zararlı olacağı tahmin edilen OTA için 5 µg kg-1 maksimum limiti aşmıştır. Genel olarak elde edilen sonuçlar, Türk Gıda Kodeksi (TGK) ve Avrupa Birliği (AB) Maksimum Kalıntı Limitleri (MRL)’nin altında değerlerdir.

Development of a new format of competitive immunochromatographic assay using secondary antibody–europium nanoparticle conjugates for ultrasensitive and quantitative determination of ochratoxin A

In this study, to enhance the sensitivity of conventional immunochromatographic assay (ICA) two strategies including using a high sensitive label and changing the assay format, were simultaneously applied to develop an ultrasensitive format of ICA. In new format, primary monoclonal antibody against ochratoxin A (OTA) was used without any labeling, and a secondary polycolonal antibody was labeled with europium fluorescent nanoparticles (EuNPs). Detection was performed in a single step by inserting the test strip into a microtube containing all reagents. The results were obtained within 12 min and read by a portable fluorescent strip reader. Salient features of the new format of ICA compared with conventional format include: (1) A 100-fold decrease in limit of detection (LOD) due to application of two amplification strategy; (2) Reduction in expensive monoclonal antibody consumption. The established method was evaluated for the quantitative determination of OTA with LOD as low as 0.4 pg mL−1.

Quantitative determination of ochratoxin A in wine after the clarification and filtration

Through the use of the analytical method known as HPLC-FD and previously used the method for extraction of ochratoxin A by immunoaffinity columns, we have analyzed the possible effect of the clarification and filtration process on 54 samples of 2013 newly fermented wine, which have finished alcoholic fermentation process and racking, without knowing in advance whether there and how is the amount of OTA in wine. The racking of the wine before clarification and filtration followed by adequate clarification and filtration process during winemaking seems to be crucial in the reduction or complete elimination of the analyzed mycotoxin (OTA).The results of all analyzed samples have been below the limit allowed by the EU for ochratoxin A i.e. 2 ng/ml, and as such does not represent a risk to human health.

Development of an Electrochemical Immunosensor for Ochratoxin A

A direct, competitive electrochemical enzyme‐linked immunosorbent assay (ELISA) has been developed for the quantitative determination of ochratoxin A (OTA) using polyclonal antibodies. The assay is carried out on carbon‐based screen printed electrodes (SPE). Optimisation of the ELISA competitive conditions allowed us to realise an assay with improved analytical behaviour compared to the classical spectrophotometric ELISA based assay. The performance was comparable to a published monoclonal based assay. The assay gave a detection limit of 180 pg mL−1 and sensitivity of 6.1 ± 0.1 ng mL−1. The immunosensor was challenged with wine to assess a matrix effect. Recoveries obtained were in the 70–118% range. The method appears to be suitable for OTA contamination screening in food samples.

Quantitative determination of ochratoxin A by liquid chromatography/electrospray tandem mass spectrometry.

Mass spectrometry of ochratoxin A (OTA) and B (OTB) under electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) was studied. ESI offers higher sensitivities and less fragmentation than APCI. A sensitive LC/MS/MS method for the determination of ochratoxin A (OTA) in human plasma samples was developed. The absolute minimum detection limit was around 10-20 pg per injection, corresponding to 0.5 ppb in an injection equivalent to 20-40microg of human plasma. Ochratoxin B (OTB) was used as an internal standard and its absence in real-life samples was carefully checked before samples were spiked with the internal standard. It was found that these two ochratoxins are susceptible to sodium adduct formation. Fragment ions from the [M + H](+) and [M + Na](+) ions of both OTA and OTB were monitored in the multiple reaction monitoring mode. Three quantitative approaches, standard addition method, internal standard method (using ochratoxin B as an internal standard) and external standard method, were compared in the analysis of human blood plasma. Results from the mass spectrometric method were comparable to those from a conventional LC/fluorescence method. The LC/MS/MS method was also applied to the analysis of contaminated coffee samples.

Determination of Ochratoxin A by Monoclonal Antibody-Based Enzyme Immunoassay

A simple and rapid indirect enzyme-linked immunosorbent assay was developed for the quantitative determination of ochratoxin A in barley after the successful production of a high affinity, specific monoclonal antibody. A rapid sample cleanup was achieved by extracting ochratoxin A from barley with chloroform and partitioning the toxin into bicarbonate buffer; the buffer solution was then added directly to the assay plate and ochratoxin A content was assessed. Recoveries were greater than 85% and detection limits were 5 micrograms ochratoxin A/kg barley.