Microsoft PowerPoint - Technical Seminar on Aflatoxins Analysis-final.ppt []• under warm temperature and high humidity
• Toxic to plants, animals and human
In 1960, UK, more than 100,000 turkeys died
Duckling and young pheasants affectedg y g p
• At least 16 Aflatoxins identified
• Aflatoxins (B1, B2, G1 & G2) are highly toxic compounds 
(mutagens & carcinogens), Group 1 carcinogens by WHO.( g g ), p g y
• M1 and M2metabolites in milk or meat
• A. flavus produces B1, B2, 
• A parasiticus produces B1 B2 G1 &G2• A. parasiticus produces B1, B2, G1 &G2
• Order of toxicity: AFB1 > AFG1 > AFB2 > AFG2
• Toxicity > KCN (~10x)  ;  > Arsenic (~70x) > Melamine (~420x)
• Carcinogenicity > Dimethylnitrosamine (~70x), > benzene 
ML Level ug/kg Note
ML Level ug/kg
Peanut 15 The ML applies to products intended  for further processingAlmonds 15
Hazelnuts 15
Pistachios 15
Almonds 10 The ML applies to the product “read toeat”Hazelnuts 10
Pi t hi 10Pistachios 10
* ML – Aflatoxins total (B1+B2+B1+G2)
Product Alfatoxin M1  ML Level ug/kg
Note
Milk 0.5
US FDA A ti L lUS FDA Action Levels Commodity Level (μg/kg)
All products, except milk, designated for human 20
Milk 0.5
C f i t i l d d i ttl 20Corn for immature animals and dairy cattle 20
Corn for breeding beef cattle, swine and mature poultry 100
Corn for finishing swine 200Corn for finishing swine 200
Corn for finishing beef cattle 300
Cottonseed meal (as a feed ingredient) 300Cottonseed meal (as a feed ingredient) 300
All feedstuff other than corn 20
European Commission Regulation (EC)European Commission Regulation (EC) 
P d t Maximum level aflatoxin (μg/kg or ppb)
Product B1 B1 + B2 + G1 +G2 M1
Groundnuts, nuts and dried fruit and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs 2 4 -- Groundnuts to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs 8 15 -- Nuts and dried fruit to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs 5 10 -- Cereals and processed products thereof intended for direct human consumption or 2 4Cereals and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs 2 4 -- Cereals with the exception of maize, to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs 2 4 -- Maize to be subjected to sorting, or other physical treatment, before human 5 10 --consumption or use as an ingredient in foodstuffs 5 10 --
Milk -- -- 0.05 Following species of spices: -Capsicum spp., Piper spp., Myristica fragrans, Zingiber officinale, Curcuma longa 5 10 --p pp , p pp , y g , g , g
Baby foods and processed cereal-based foods for infants and young children 0.10 -- -- Infant formulae and follow-on formulae, including infant milk and follow-on milk -- -- 0.025 Dietary foods for special medical purposes intended specifically for infants 0.10 -- 0.025

: (B1) 20μg/kg (B1) 5μg/kg
Japan proposed to change the aflatoxin limit for p p p g food from current “Aflatoxin B1 at 10 ppb” to “Total aflatoxins at 10 ppb” (10 Aug 2010)pp ( g )
Harmful Substances in Food Regulation (Cap 132)
Schedule 1 Maximum Concentration of Certain Substances present in Specified Foods
Substance Description of substance Description of food Maximum  concentration
Group of bis Any food other than  15 μg/kg of the food
Aflatoxin
peanut or its  products
Peanuts or peanut 20 μg/kg of the food M1, M2, P1 and aflatoxicol
Peanuts or peanut  products
20 μg/kg of the food
H K Chi M t i M di St d dHong Kong Chinese Materia Medica Standards
Aflatoxin B1 ≤ 5μg/kg
≤ 10μg/kg≤ 10μg/kg
Low molecular mass
Soluble in methanol, acetone, acetonitrile, benzene and chloroform
Fluorescence under long UV illumination (ca. 365 nm) (B = Blue; G = Green)
Stable at high temperature: not destroyed under ordinary cooking conditions
Susceptible to alkaline hydrolysis and acid treatment B1→ B1A ; G1→ G2ASusceptible to alkaline hydrolysis and acid treatment B1 → B1A  ;   G1 → G2A
Metabolic products: M1 and M2
International Standard MethodsInternational Standard Methods
AOAC methods Chapter 49- Natural ToxinsAOAC methods Chapter 49- Natural Toxins, Subchapter 2 - Aflatoxins
TLC methods, Minicolumn methods, ELISA,LC methods.
GB methods
International Standard MethodsInternational Standard Methods BS 57667 1988.01.01 
(R 1992)  Method for Analysis of Animal Feeding Stuffs Part 7: Determination of Aflatoxin B Less Than  of 12 Greater Than 1
BS 576616 1999.05.15  Methods for Analysis of Animal Feeding Stuffs  Part 16: Determination of Aflatoxin B1  Content of Mixed Feeding Stuffs (HighPerformance Liquid Chromatographic Method)  ISO  14718: 1998;
BS EN 12955 1999.11.15  Foodstuffs  Determination of Aflatoxin B1, and the Sum of Aflatoxins B1, B2, G1 and G2 in  C l Sh ll F it d D i d P d t Hi h P f Li id Ch t hiCereals, ShellFruits and Derived Products  High Performance Liquid Chromatographic  Method with Post Column Derivatization and Immunoaffinity Column Clean up
BS EN 14123 2008.01.31  Foodstuffs — Determination of aflatoxin B1 and the sum of aflatoxin B1, B2, G1 and G2 in  hazelnuts, peanuts, pistachios, figs, and paprika powder — High performance liquid  chromatographic method with postcolumn derivatisation and immunoaffinity columnchromatographic method with post column derivatisation and immunoaffinity column  cleanup
BS EN 15851 2010.05.31  Foodstuffs — Determination of aflatoxin B1 in cereal based foods for infants and young  children — HPLC method with immunoaffinity column cleanup and fluorescence detection
BS EN ISO 14501 2008.03.31  Milk and milk powder — Determination of aflatoxin M1 content — Cleanup by S SO 50 008 03 3 a d po de ete at o o a ato co te t C ea up by immunoaffinity chromatography and determination by highperformance liquid  chromatography
BS EN ISO 14675 2003.04.29  Milk and milk products  Guidelines for a standardized description of competitive enzyme  immunoassays  Determination of aflatoxin M1 content  CORR 14482: May 15, 2003;
BS EN ISO 17375 2006.06.30  Animal feeding stuffs Determination of aflatoxin B
BS ISO 14674 2005.08.01  Milk and milk.. powder Determination of aflatoxin M1 content Cleanup by immunoaffinity chromatography and determination by.. thinlayer chromatography
M th d f A l i ELISAMethods of Analysis  ELISA
• Simple, rapid
• Lower cost
• Disadvantage – possible false positive
Methods of Analysis  TLC
smaller sample capacitys a e sa p e capac ty
matrix interference
Standards for Aflatoxins For aflatoxin standards received as dry films orFor aflatoxin standards received as dry films or 
solution:
benzene/ACN (98:2 v/v)
SpectrophotometerSpectrophotometer
S S i biliSystem Suitability UV Spectrophotometer Measurement range: 200 – 500 nmMeasurement range: 200 500 nm
1 cm quartz-face cell
Correction Factor (CF) 0 95 1 05Correction Factor (CF): 0.95 – 1.05
→ Determine absorbance at ca. 350 nm for K C O (0 25 0 125 d 0 0625 M)K2Cr2O7 (0.25, 0.125 and 0.0625 mM)
→ Calculate = (A 1000/conc. in mM)
→ CF = 3160/ av. K2Cr2O7
Ch ki Afl t i St d d C t tiChecking Aflatoxin Standard Concentration
Calculate the concentration of the individual aflatoxin standard.Calculate the concentration of the individual aflatoxin standard.
Concentration in mg/mL = (A x MW x 1000 x CF)/ε
Aflatoxin Standard Vertification - B1, B2, G1 & G2
0.85
0.90
0.95
1.000
0.85
0.90
0.95
1.000
0.85
0.90
0.95
1.000
0.85
0.90
0.95
200.0 250 300 350 400.0 0.000
0.05
0.10
0.15
0.000
0.05
0.10
0.15
0.000
0.05
0.10
0.15
0.000
0.05
0.10
0.15
nm
Cheese and  Butter IcecreamFluid Milk Milk PowderNuts, peaneut
butter, etc
SPE clean upImmunoaffinity Column (IAC) clean up SPE cleanupImmunoaffinity Column (IAC) cleanup
HPLC with  Postcolumn derivation  system
HPLC 
y
Immunoaffinity Column (IAC)
Quick (< 10 mins) Handle variety/complex matrices High selectivity, low LOD (~ 0.1 ppb) Wide range (up to 300 ppb) Higher recovery Safe (less toxic solvent consumption) Shelf life ~ 6 months Storage in Refrigerator if necessary
Immunoaffinity Column (IAC) Performance Check
Check performance of IAC for each batch
– Aflatoxin Std. Recovery : B1, G1 > 90%, B2 > 80% and G2 > 60%
T f k i f d d i l d NTransfer a known portion of supernatant and reduce its volume under N2
Dilute with water to a known volume
Load the solution onto the IAC at 3 mL/minLoad the solution onto the IAC at 3 mL/min
Wash the IAC with water
Pass air through the IAC at 10 mL/ming
Elute with known volume of methanol and then pass air through
Transfer to volumetric. flask
Sample Preparation for Aflatoxins B1, B2, G1 G2
1. Weigh 25g sample into a blender jar 2. + 5g NaCl and 125 mL 70% MeOH 3. Blend for 2 min at high speed 4. Filter
f f5. Pipette 15 mL of filtrate 6. Add 30 mL water 7. Filter the diluted extract through glass microfiber filter paper
8. Prepare the immunoaffinity column * 9 Load 15 mL of filtrate and pass through the9. Load 15 mL of filtrate and pass through the
column at 6mL/min 10. Load 10 mL x 2 water to wash the column at
6mL/min6mL/min 11. Add 1.0 mL of HPLC grade of MeOH to
eluate the analytes 12 R tit t ith fi l l f 2 L ith12. Reconstitute with final volume of 2 mL with
water.
15 g sample in 500mL  centrifuge bottle
+ 1 mL Sat NaCl+5g
15 g sample in 250mL  centrifuge bottle
+ 1 mL Sat. NaCl+5g  Celite+100 mL CHCl3
Blend for 2 min
+ 10 mL Sat. NaCl+120  mL CHCl3
Shake for 2 minCentrifuge at 3000 rpm
Pass the CHC l3 layer over a bed of anhy. Na2SO4 into 100mL measuring cylinder 
Remove the organic solvent and redissolve the residue in 6 mL CHCl3
Silica SPE cleanup
50 g sample into a 50
10 g sample dissolved in  80 mL preheated water
mL centrifuge tube Mix until homogeneous
Add preheated water to  100 mL
Centrifuge at 3000 rpm
Separate the fat (top) later form the defatted (skim) layer
The defatted layer
IAC clean upIAC cleanup
IAC d SPE l f Afl i M1IAC and SPE cleanup for Aflatoxin M1 Silica SPE
Condition with 10 mL hexane
IAC SPE
Wash with 6 mL hexane
Wash with 10mL x2  water (~2.5mL/min)
Elute the analytes with  2.5 mL ACN:MeOH (3:2) 
(~1 mL/min)Wash with 2 mL x 5  hexane/ether (7:3)hexane/ether (7:3)
Elute the analytes with  7 mL chloroform/MeOH
Add 2.5 mL of distilled  water and push all to 7 mL chloroform/MeOH
(9:1)
HPLC Analysis for y Aflatoxin B1, B2, G1 and G2
Column: Suplcosil LC18 DB column p 5μm, 4.6 mm x 250 mm
Mobile Phase: H2O:ACN:MeOH = 3:1:1
Post column reagent: Iodine  
Coil: 610 cm x 0.5mm id Teflon  tubing
0 CTemperature: 70oC
Emission Wavelength: 450nm
Injection Volume: 50μL
– AFB1 → B2a ; AFG1 → G2a
– not stable in methanol
– Fluorescence of AFB1 and/or AFG1
Optimization for Post-column Derivatization
Flow rate (0.3 mL/min)
Coil temperature (70 oC)
b dCoil tubing dimension
Aflatoxins Real Sample
HPLC Analysis for Aflatoxin M1
Column: Suplcosil LC18 DB column  5μm, 4.6 mm x 250 mm
M bil Ph H O ACN M OH 68 24 8Mobile Phase: H2O:ACN:MeOH = 68:24:8
Column flow: 1mL/min
Excitation Wavelength: 360nm
Emission Wavelength: 440nm
Injection Volume: 50μL
Structural Confirmation: LC MS/MS Conditions for Aflatoxins B1 B2 G1 and G2Structural Confirmation: LCMS/MS Conditions for Aflatoxins B1, B2, G1 and G2
System : Waters 2695 HPLC withHPLC system : Waters 2695 HPLC system or equivalent System  : Waters 2695 HPLC with  MicroMass Quattro Micro
Ionization  Mode
: Electrospray positive ionization
HPLC system : Waters 2695 HPLC system or equivalent Injection volume : 20 µL Flow rate : 200 µL/min Column : Aglient Zorbax 300SB-C18, 150 mm x 2.1 mm,
5 μm or equivalent
5 μm or equivalent
Column temperature : Room temperature Mobile Phase : Solvent A: Water with 2 mM ammonium
formate and 3.6 mM formic acid
RF lens : 0.5V
: 100oC
Solvent B: 95% acetonitrile with 2 mM ammonium formate and 3.6 mM formic acid
Solvent program : Time min
min % v/v % v/v
Desolvation  Gas Flow
Aflatoxins
(m/z) Daughter ion
B1 313.3 269.3 40 30 0.1 285.3 40 25 0.1
B2 315 3 259.3 40 30 0.1
B2 315.3 287.3 40 25 0.1
G1 329.3 243.3 45 25 0.1 311.3 45 18 0.1
G2 331 3 313.3 47 25 0.1
G2 331.3 245.3 47 27 0.1
Relative intensity (% of Base Ion) Tolerance, R
>50% ± 20 % > 20% to 50% ± 25 %> 20% to 50% ± 25 % >10% to 20% ± 30 %
less than or equal to 10% ± 50 %
STD4STD4
100
%
331.3 > 313.3 6.20e3
3 83 3 10.36
G2
%
0
100
%
101013_STD4 3: MRM of 2 Channels ES+ 315.3 > 287.3
8.19e3 10.65
100
%
315.3 > 259.3 7.71e3
1.32e4 10.62
B2
%
0
100
%
2.12e4 10.63
G1
0
100
%
313.3 > 285.3 4.66e4
2.82e4 10.88
B1
%
0
100
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 0
101013_STD4 1: MRM of 2 Channels ES+ TIC
7.29e4 10.90
Structural Confirmation: LCMS/MS Conditions for Aflatoxin M1Structural Confirmation: LCMS/MS Conditions for Aflatoxin M1
System : Waters 2695 HPLC withHPLC system : Waters 2695 HPLC system or equivalent System  : Waters 2695 HPLC with  MicroMass Quattro Micro
Ionization  Mode
: Electrospray positive ionization
HPLC system : Waters 2695 HPLC system or equivalent Injection volume : 20 µL Flow rate : 200 µL/min Column : Aglient Zorbax 300SB-C18, 150 mm x 2.1 mm,
5 μm or equivalent
5 μm or equivalent
Column temperature : Room temperature Mobile Phase : Solvent A: Water with 2 mM ammonium
formate and 3.6 mM formic acid
RF lens : 0.1V
: 100oC
Solvent B: 95% acetonitrile with 2 mM ammonium formate and 3.6 mM formic acid
Solvent program : Time min
min % v/v % v/v
Desolvation  Gas Flow
Analyte
273 30 25 0 2273 30 25 0.2
Relative intensity (% of Base Ion) Tolerance, R
>50% ± 20 % > 20% to 50% ± 25 %> 20% to 50% ± 25 % >10% to 20% ± 30 %
less than or equal to 10% ± 50 %
10G1453D
329.13 > 273.35 5.86e3
8.57
8.62
8.65
%
100
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 100607_10G1453D MRM of 2 Channels ES+
329.13 > 229.04 3.10e3
8.59 8.61
8.62 8.64
%
%
8.70 8 72
Time 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
0
RSD
Aflatoxin B2 87% 8.2%
Aflatoxin G2 69% 13.8%
fl i ( l ) % %Aflatoxin M1 (SPE cleanup) 95% 5.3%
Aflatoxin M1 (IAC cleanup) 91% 6.0%
Safety Aspects of Testing  1
Personal Protective Equipment – laboratory coat – safety goggles – disposable gloves – hand-washing facilities
H dli d St (S lid Afl t iHandling and Storage (Solid Aflatoxin Standard)
– stored at 2 - 8 oCstored at 2 8 C – tightly closed, air and light sensitive
Safety Aspects of Testing 2
Inhalation – remove to fresh air
Ingestion – wash out mouth with water
Skin Contact flush with large amount of water for 15Skin Contact – flush with large amount of water for 15
mins, remove contaminated clothes and shoes
Eye Contact – flush with large amount of water for 15 mins
Safety Aspects of Testing 3Safety Aspects of Testing 3 Conduct testing in fume hood
Injecting solvent through septum to dissolve solid aflatoxins standard
Decontamination
– Laboratory equipment/surface, swabbed with 5% hypochlorite solution
– Contaminated glassware, immersed in 10% hypochlorite solution for at least 10 hrs, then rinse with acetone
C d M t i A l t A il bl U it Si
FAPAS Quality Control Samples Code Matrix Analytes Available Unit Size
T04117 Brazil Nut Aflatoxins B & G &/or total
30-Oct-2014 100g
17-Dec-2015 100g (water/fruit
slurry) &/or total
17-Dec-2015 100g
slurry) &/or total
13-Aug-2016 100g
11-Nov-2016 100g
slurry) &/or total
ERMAC057 Aflatoxin B1 in acetonitrile
ERM AC058 Aflatoxin B2 in acetonitrileERMAC058 Aflatoxin B2 in acetonitrile
ERMAC059 Aflatoxin G1 in acetonitrile
ERMAC060 Aflatoxin G2 in acetonitrile
ERMBD282 Whole Milk Powder (zero level)
ERMBD283 Whole Milk Powder (low level)
ERMBD284 Whole Milk Powder (high level)
ERMBE376 Compound Feedingstuff (High level)
N tNotes: R ti Li itReporting Limits:
1 ppb for total aflatoxins (B1+B2+G1+G2)
0.02 ppb for aflatoxin M1 for SPE clean-up
0.01 ppb for aflatoxin M1 for IAC clean-up
Detected Levels:Detected Levels: 1 to 7 ppb for total aflatoxins in peanut butter, mooncake, samples etc. 0 03 to 0 06 ppb in some milk samples0.03 to 0.06 ppb in some milk samples.
Thank youThank you