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.