FluoroQuant

Afla IAC Test Kit

(FQ Afla IAC) for Aflatoxin Testing
Order #: COKFA4010, COKFA4020
Intended Use
The FQ Afla IAC test kit is a rapid, quantitative
fluorometric test for detection of total aflatoxins (B1, B2,
G1, and G 2) using an immunoaffinity column for sample
purification.
The FQ Afla IAC test was developed for use with corn,
corn screenings, distillers dried grains with solubles
(DDGs) and peanuts.
This product is simple enough for field use in grain, feed
and food handling facilities, but is also appropriate for
analytical laboratories.
Aflatoxin
Aspergillus flavus and Aspergillus parasiticus are the major fungi
associated with production of aflatoxins. Warm temperatures
with drought-like conditions cause grain to become more
susceptible to aflatoxin formation. Corn, cottonseed, peanuts,
and tree nuts are the main crops affected with aflatoxins.
Aflatoxins can cause liver disease in animals and humans. They
can cause a decrease in milk production and are
immunosuppressive, carcinogenic, and mutagenic.
Precautions
1.
Safety: It is recommended to use a lab coat, disposable
nitrile gloves, and safety glasses when running this test.
2.
Temperature: Optimal test results will occur when the test
is run in an environment where the temperature is
between 64 and 82F (18 and 30C).
3.
Storage: Test results may be adversely affected if the
storage instructions are not followed properly.
4.
Waste Disposal: Transfer sample extract solutions and
developer solutions into a liquid container for proper
disposal according to applicable regulations.
All other
materials may be disposed in the normal solid waste
system for your facility.
Procedure for all methods analyzed using a FluoroQuant
Afla Test Kit System
Periodically Calibrate Fluorometer
Power-up fluorometer and printer
For Romer Series III:
Wait for fluorometer to complete self
Set date/time and press continue
Press select and enter method number
For FQ-Reader:
Touch FluoroQuant Afla IAC
Touch IAC Method 1.
Completing Calibration for Both Fluorometers:
Follow instructions on screen to calibrate
fluorometer:
o
Insert High Calibrator, press enter
o
Insert Low Calibrator, press enter
Insert Control Calibrator
o
IF value is in range listed on box,
continue with samples
o
IF value is out of range, recalibrate
o
IF value is still out, call Technical
Services
Prepare Developer from Developer Concentrate (must be
made fresh a minimum of every twenty-four (24) hours)
1.
Prepare, store and dispense developer in a 50 mL
amber bottle with a 1 mL repipettor.
2.
Transfer the contents of one developer concentrate
vial into 25 mL deionized/distilled water using the
disposable transfer pipette. If the developer
concentrate is not yellow/brown when pulled into the
disposable pipette, discard and use another vial.
Please notify Technical Services at Romer Labs, Inc.
if you need to replace a vial.
3.
Rinse the empty vial three times with the prepared
developer, returning the rinse to the 50 mL amber
bottle each time.
Prepare 16% Methanol in Water Rinse Solution
1.
16% methanol rinse solution may be prepared by
adding 40mL of 80/20 extraction solution to 160mL
of distilled or deionized water. Other volumes may
be made, as long as this ratio is kept the same.
Note: 16% methanol rinse solution should be made fresh
after 24 hours if it is stored in the 2mL dispenser.

Method Procedures
Extract Sample
For all Approved Commodities Except Peanut Slurry
1.
Weigh 25 g of ground sample* into an extraction
container.
2.
Add 5 g of Sodium Chloride (salt).
3.
Add 100 mL 80/20 (v:v) methanol/water.
4.
Blend for 1 minute.
5.
Filter the sample extract into a glass or
polypropylene container.
For 1:2 Peanut Slurry
1.
Weigh 75 g of slurry into an extraction container.
2.
Add 5 g of Sodium Chloride (salt).
3.
Add 75 mL of HPLC grade Methanol (the total
solution will be 25g peanuts / 125 mL 60/40
methanol/water).
4.
Blend for 1 minute.
5.
Filter the sample extract into a glass or
polypropylene container.
For 1:1.5
1.
2.
3.
4.
5.

Peanut Slurry
Weigh 62.5 g of slurry into an extraction container.
Add 5 g of Sodium Chloride (salt).
Add 87.5 mL of 6/1 (v:v) Methanol/Water
Blend for 1 minute.
Filter the sample extract into a glass or
polypropylene container.

For 1:1 Peanut Slurry

1.
Weigh 50 g of slurry into an extraction container.
2.
Add 5 g of Sodium Chloride (salt).
3.
Add 100 mL of 3/1 (v:v) Methanol/Water
4.
Blend for 1 minute.
5.
Filter the sample extract into a glass or
polypropylene container.
*Grind sample such that 70% passes through a 20 mesh sieve and
mix well to assure homogeneity. Refer to specific industry or
regulatory guidelines where applicable. Other amounts of sample to
solvent may be used if ratio remains the same and sampling variability
is considered.

Analysis Step 1 Purify Sample Using the IAC Column

1.
Pipet 500 L of sample extract (use 600 L for
peanut slurry) into a clean cuvette and discard
pipette tip.
2.
Add 2000 L of distilled or deionized water.
3.
Pierce the top seal of an AflaStar IAC column with a
pointed filter column and remove the bottom cap
from the IAC column.
4.
Place entire 2500 L diluted sample into the barrel
of the pointed filter column.
5.
Insert syringe plunger and stopper assembly in top
of column.
6.
Push extract completely through column, at a rate of
about 1 2 drops per second. (Alternate method:
Use manual or powered vacuum column stand to
push extract through column.)
7.
Place 2 mL of distilled or deionized water into the
cuvette that was used for diluting the sample. Pour
this water onto the pointed filter column and push
completely through the IAC column.
8.
Remove the pointed filter column from the IAC
column and discard.
9.
Rinse the IAC column with 2 mL of 16% methanol
in water. Repeat this procedure once, so that a
total of 4mL of the 16% methanol have been
passed over the column.
10. Place a clean cuvette under the IAC column.
11. Elute the aflatoxin by adding 2 mL of methanol
(HPLC grade) to the IAC column.
12.

Push the methanol through the IAC column at a rate

of about 1 2 drops per second.

Analysis Step 2 Add Developer (Derivatize) and Read

1.
Add 1000 L of prepared developer to purified
extract.
2.
Cap the cuvette and vortex for 5 seconds.
3.
Wipe cuvette with lint-free paper and insert into
calibrated fluorometer.
4.
After a preprogrammed 30 second delay, the results
will be displayed and printed in parts per billion.
Note: Once the developer is added to the purified sample,
it must be mixed and analyzed immediately.

2008 by Romer Labs , Inc. All Rights Reserved. This document is the property of Romer Labs , Inc.
FQ-AFLA-IAC-COKFA4010-COKFA4020-PI-000295-5-GB Finalized 14 Aug 2008

Romer Labs Methods

General Information
Notice
The FluoroQuant Afla IAC Test Kit System has been validated
for use in a wide range of commodities; however; specific
commodities; processed feed and food samples may vary widely
based on variety, environmental influences, and processing
factors. It is recommended that the users validate kit
performance using their own samples to ensure that the kit is
suitable for use. Action limits should be developed using your
own risk profile with a clear understanding of the Measurement
Uncertainty for the range and method being considered for use.

all other remedies, warranties, guarantees or liabilities,

expressed or implied, arising by law or otherwise, and it shall
have no liability for any lost profits or damage, direct, indirect or
otherwise, to person or property, in connection with the use of
any of its products or services. This warranty shall not be
extended, altered or varied except by a written instrument
signed by an authorized representative of Romer Labs, Inc.

Reagents Supplied with Kit

Salt (Sodium Chloride)
Developer concentrate vials
Elution solvent (USA only)
Materials Supplied with Kit
12X75 mm cuvettes with caps
Pipette tips
Filter paper
Disposable transfer pipettes
IAC Aflatoxin Columns
Pointed filter columns
Additional Materials Required (available from Romer)
80/20 (v:v) methanol/water extraction solvent.
Distilled or Deionized Water
High, Low, and Control Fluorometer Calibrator
Ampoules (COKFA2040)
Apparatus Required (available from Romer)
Romer Series III Fluorometer (Model RL100 or
equivalent, EQFFM1010) or FQ-Reader with printer
(EQFFM3010)
Blender (EQOLE1020)
Blender jars pint (8 oz.) (EQOLE1025)
Extraction jars or containers (EQOLE1130)
Vortex mixer (EQOLE1335)
Scale (EQOLE1010)
Syringe Plunger and stopper assembly (COKFA1081)
or optional plunger stand (COKFA1085)
Repipettor (EQOLE1241)
2mL repipettor for 16% methanol solution
(EQOLE1247)
Pipettes
o
Fixed 500 L (EQOLE1160)
o
Fixed 1000 L (EQOLE1050)
Test tube rack (EQOLE1210)
Graduated cylinder 100 Ml (EQOLE1050)
Funnel (EQOLE1350)
Timer (EQOLE1301)
Technical Support
Tel:
(636) 583-8600
Fax:
(636) 583-2340
e-mail:
techserv@romerlabs.com
website:
www.romerlabs.com
Address:
Romer Labs, Inc.
1301 Stylemaster Drive
Union, MO 63084-1156
Shelf Life
Shelf life of test kit is nine months from date of manufacture.
Mixing components from different lots of the kits or use of kits
after expiration is not covered by any warranty.
Exclusion of Warranty Statement
The user assumes all risk in using Romer Labs, Inc. products
and services. Romer Labs, Inc. will warrant that its products
and services meet all quality control standards set by Romer
Labs, Inc., and Romer Labs, Inc. will, at its option, repair or
replace any product, components, or repeat services which prove
to be defective in workmanship or material within product
specific warranty periods or expiration dates and which our
examination shall disclose to our satisfaction to be defective as
such. This warranty is expressly in lieu of all other warranties,
expressed or implied, as to description, quality, merchantability,
fitness for any particular purpose, productiveness, or any other
matter. Romer Labs, Inc. shall be in no way responsible for the
proper use of its products. Romer Labs, Inc. hereby disclaims

2008 by Romer Labs , Inc. All Rights Reserved. This document is the property of Romer Labs , Inc.
FQ-AFLA-IAC-COKFA4010-COKFA4020-PI-000295-5-GB Finalized 14 Aug 2008