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METHOD INVESTIGATION AND PARTIAL VALIDATION OF OCHRATOXIN A IN GROUND ROAST COFFEE,
DOMINIC LYNCH, DR PATRICE BEHAN AND DR JOHN KEEGANDT203T4, FORENSIC AND ENVIRONMENTAL CHEMISTRYSCHOOL OF CHEMICAL AND PHARMACEUTICAL SCIENCE
DOMINIC45@LIVE.IE Introduction: Mycotoxins are a secondary metabolites produced by a species of fungi that are capable of causing disease and death in humans and animals. Among various mycotoxins is Ochratoxin A (OTA), mainly produced by fungi Aspergillus genus (ochraeus and niger) . Optimum growth conditions for Aspergillus niger and Aspergillus ochraeus are 35-37 degC and 24-31 degC respectively. Its common contaminant of a variety of food including grain products, nuts, coffee beans, fruit, beer and wine. The aim of the method optimisation is to investigate different methods of OTA extraction, followed by detection and quantification by HPLC/FLD. These methods include a combination of various extraction solvents, solvent strengths, flow rates, mobile phases, clean-up methods and temperatures.
Fig 1. Structure of Ochratoxin A
Experimental:
Results and Discussion: • Two sets of blanks and 6 spikes were prepared (approximately 50g was
weighed out).• One set was spike with 0.5 ml of OTA stock standard and non-heated
extraction solution (ACN:UPW) was added and blended for 2 minutes by an Ultra Turrax Blender. • The second set was spiked with 0.5 ml of OTA stock standard, a heated
extraction solution was added, which was also blended for 2 minutes by an Ultra Turrax blender. • Heating the extracting solution (ACN:UPW) to 60 degC in a water bath
increases the solubility and increases the level of extraction of OTA from the coffee sample. • The flow rate of the diluted matrix passing through the IACs was
2ml/min. • The percentage recoveries set out in the legislation regarding
Ochratoxin A requires that the recoveries be in the region of 70-110% and the %RSD ≤ 20%. (Table 1.0)• From Table 1.1., the percentage recoveries for both heated and non-
heated comply with the current legislation.
Conclusion: • An optimised method for the extraction of OTA from ground roast
coffee was developed.• Heating of the extraction solution ACN:UPW (60/40) to 60 degC
was found to be the optimal method for the extraction of OTA.• The percentage recoveries for both the heated and non-heated
methods meet the legislation requirements for OTA.
Acknowledgements: Thanks to Dr Michael Sullivan, Dr John Keegan, Mr Patrick English and Mrs Nicola O Sullivan from the Public Analyst`s Laboratory for allowing me to carry out this project, also to Dr Patrice Behan who lent her help and advice during the project
Reference`s:• European Communities (sampling methods and the methods of
analysis for the official control of the levels of certain contaminants in foodstuff) (No.2), Regulations, 2006 (SI. 412 of 2006. • Cytotoxic effect of Ochratoxin A on renal corpuscles of rat kidney: could
Ochratoxin a Cause Kidney Failure. Suzan Aldu, Awatif Al and Shatha Ansari; Cell Biology and Histology, Faculty of Science, King Abdulaziz University Jeddah, Saudi Arabia.• Food control 46 (2014) 102-107, Occurrence of Ochratoxin A in roasted
and instant coffees in Chilean market, Oscar Galarce-Bustos, Maritza Alvarado, Mario Vega, Mario Aranda, Laboratory of Advanced Research on foods and Drugs, Department of Food Science and Technology, Faculty of Pharmacy, University of Concepcion, Concepcion, Chile.
Fig 2. example of a linear 6 point calibration curve achieving R2 ≥ 0.999
Fig 3. Chromatogram of a spike sample of ground roast coffee containing OTA. The sample contained heated extraction solution of ACN:UPW (60/40).
Original Method• 5g of ground roast coffee into
blending jar• Spike with 0.5ml of OTA
intermediate standard (103.8 µg/l)
• Add 100ml of NaHCO3, blend for 2 minutes
• Centrifuge and Clarify the matrix• Dilute 20ml of matrix in 20mls of
phosphate buffer saline (PBS)
Partial Validation• 50g of ground roast coffee
into blending jar • Spike with 0.5ml of OTA
stock standard (1038 µg/l)• Add 200ml of heated
extraction solution (60 degC) ACN:UPW (60:40), blend for 2 minutes
• Centrifuge and clarify the matrix
• Dilute 5ml of matrix in 43ml of PBS
Clean-up• Clean-up of diluted matrix is done using immunoaffinity columns (IACs)• Contains a gel bed with a toxin specific gel coupled to a gel bed• Antibodies will capture mycotoxin which are released through wash
step• After elution of the toxin, the toxin is quantified by HPLC/FLD• A calibration curve of working standards is run along side to determine
the concentration of an unknown substance (Fig 2)
Fig 4. Column chart showing percentage recoveries of heated and non-heated extraction solution of ACN:UPW.
1 2 3 4 5 60
10
20
30
40
50
60
70
80
90
100 93 92
83
94 9398
8783
69
8783 81
Heated Vs Unheated
% R
ecov
ery
Table.1.0; Required Performance Criteria for Ochratoxin A (Official Journal of the EU 401/2006)
Level ug/kg
Ochratoxin A
RSDr % Recovery %
< 1 ≤ 40 50 to 120
≥ 1 ≤ 20 70 to 110
Date
15/02/2016
SAMPLE TYPE Ground Roast Coffee
BLENDER Ultra Turrax
EXTRACTING SOLN ACN:UPW (60:40)
Time (mins) 2
% Recovery A B C D E F
Heated 93 92 83 94 93 98
Non-heated 87 83 69 87 83 81
Table 1.1; Percentage recoveries of heated and non-heated extraction solution of ACN:UPW.
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