How can I extract ochratoxin from these Aspergillus ochraceus cultures? Can you recommend a rapid and simple protocol for TLC or Spectrophometric procedure for quantification of ochratoxin?
it looks simple and effective way. But use acid washed glasware
Several efficient clean-up and preconcentration
procedures based on immunoaffinity columns (IAC)
Please if this protocol (Price from extraction of a liquid medium YES)
With the use of immunoaffinity colum (IAC) like Ochrarep or AflaOchrarep, you can achieve your aim. This IAC is very efficient in high throughput and ethier of these extraction solvent; methanol, methylene chloride/formic acid, and methanol/formic acid could be used
Also you can used a method of Extraction of OTA from YES media: OTA was extracted from YES media according to Tsubouchi et al. (1985) and determined by HPLC according to the AOAC [2000].
Determination of OTA by HPLC.
The fore-mentioned extracted were dissolve in 500 µl mobile phase consists of acetonitrile: water: acetic acid (99:99:2) and filter through 0.45 µm microfilter into 5 ml screw –cap vial for subsequent HPLC analyses. The separation was performed at ambient temperature at a flow rate of 1.0 ml/ min, with injection volume of 20 µl for both standard solutions and sample extracts. Equipped with a reverse phase analytical column packed with C18 material (Spherisorb 5 µm ODS2, 15cm×4.6nm) with a 20 μl loop;.The fluorescence detector was operated at an excitation wave length of 330 nm and an emission wave length of 460 nm (AOAC2000).
Ref:
AOAC Official Methods of Analysis of the Association of Official Analytical Chemists, Ochratoxin A in Corn and Barely Liquid Chromatography Method, published by Association of Official Analytical Chemists, Arlington VA. 2000, 17th Ed.Vol.II Sec, 991.44
H. Tsubouchi, H. Terada, K. Yamamoto, K. Hisada, Y. Sakabe. (1985) Caffeine degradation and increased ochratoxin A production by toxigenic strains of Aspergillus ochraceus isolated from green coffee beans. Mycopatho. 90 181–186.
Substrates can be autoclaved at 121 oC for 30 min before analysis for OTA to kill the spores and vegetative mycelia. OTA extracted with 50 mL of chloroform in laboratory shaker (50 cycles/min) for one hour at room temperature. The extract dried over anhydrous sodium sulphate and concentrated in a rotary evaporator to a volume 5 ml. OTA extract purified by colum chromatography and separeted by TLC
Hi Reddy,
For the quantification of OTA in ur liquid medium:
1- take 1 ml of the medium put it into a conic eppendorf tube.
2-add 1 ml of methanol (HPLC grade) to ur eppendorf.
3- make 3 times centrifugation at 10.000 rpm
4-Pass the total supernatent volume into a 0.45 micrometer filter and recuperate the filtrate into HPLC vial.
5-Quantify the concentration of OTA with HPLC.
the obtained concentration is ....microg OTA / 1 ml liquid medium so u can extrapolate it to your liquid medium volume.
Regards
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