The purpose of the document is to establish a uniform & general method for the detection of Aflatoxin
(B1, B2, G1, G2) in food products.
2.0 SCOPE
This procedure is applicable for the detection of Aflatoxin ( B1, B2, G1, G2) in food products.
3.0 RESPONSIBILITY
The expert and trained chemist are responsible for following this procedure. The technical manager is responsible for implementing this procedure.
4.0 REQUIREMENTS
4.1 Apparatus.
4.1.1 High-Performance liquid Chromatography System ( HPLC) Make and Model: Agilent Technologies, 1260 Infinity Series.
4.1.2 pH – meter
4.1.3 Ultrasonic bath
4.1.4 Analytical Balance
4.1.5 Vortex mixture
4.1.6 Syringe Vacuum manifold HPLC With FLD
4.1.7 Kobra cell
4.1.8 Immunoaffinity Column.
4.2 Glassware
4.2.1 Centrifuge tube 50ml
4.2.2 Amber Glass Vial
4.3 Reagent
4.3.1Sodium Chloride( NaCl)
4.3.2Anhydrous disodium Hydrogen Phosphate( Na2 HPO4)
4.3.3Potassium Chloride (KCl)
4.3.4Potassium Dihydrogen phosphate(KH2PO4)
4.3.5Methanol
4.3.6Potassium bromide(KBr)
4.3.7 Nitric acid
5.0 PROCEDURE
5.1. Preparation of phosphate buffer solution (PBS).
|
REAGENT |
QUANTITY |
|
Sodium Chloride( NaCl) |
8g |
|
Anhydrous
disodium Hydrogen Phosphate( Na2 HPO4) |
1.42 |
|
Potassium
Chloride (KCl) |
0.2 g |
|
Potassium
Dihydrogen phosphate(KH2PO4) |
0.24 g |
5.1.1. Make up the volume to 1000 ml with DM water.
5.2. Sample Preparation.
5.2.1. Weigh 25 g of ground sample and 2.5 g of sodium chloride into a 1-liter capacity jar.
5.2.2. Add 100 ml of 80% methanol and blend at high speed for 2 minutes.
5.2.3. Centrifuge at 4000 rpm for 10 minutes in 50 ml of the centrifuge tube.
5.2.4. Dilute 2 ml of filtrate with 14 ml of phosphate-buffered saline (PBS) solution in another centrifuge tube.
5.2.5. Set vacuum manifold assembly and Immunoaffinity Colum.
5.2.6. Pass the filtrate through the Immunoaffinity column at a flow rate of 2 ml per minute (or the sample can be allowed to pass through the column by gravity if preferred).
5.2.7. A slow, steady flow rate is essential for the toxins to bind with the antibodies.
5.2.8. Wash the column by passing 20 ml of PBS at a flow rate of approx. 5 ml per minute.
5.2.9. Pass air through the column to remove residual liquid.
5.2.10. Elute the toxins from the column at a flow rate of 1 drop per sec using 1 ml of 100% methanol and collect in an amber glass vial. Suck-back flushing is recommended.
5.2.11. Repeat the process 3-6 times.
5.2.12. Pass 1 ml of water through the column in the same vial to give a 2 ml total volume.
5.2.13. Suck back and repeat the process 2-3 times.
5.2.14. Inject the sample into the HPLC system.
5.3. Preparation of Mix Working Standard Solution of Aflatoxin.
5.3.1. Mix working standard Solution (Aflatoxin Mix containing B1, B2, G1, and G2 concentration as per the table given below.
|
Toxins |
Stock
Concn |
Prepared
Concn |
Vol.
Taken |
Makeup Vol. |
|
B1 |
25.1 ppm |
500 ppb |
199.20 µl |
10.0ml |
|
B2 |
25.0 ppm |
500
ppb |
200.00µl |
10.0ml |
|
G1 |
24.5 ppm |
500
ppb |
204.08µl |
10.0ml |
|
G2 |
25.1 ppm |
500
ppb |
199.20µl |
10.0ml |
5.3.2. Preparation of Mobile phase A :
5.3.2.1 Take 238 mg of Potassium bromide and 700µl of 4M Nitric Acid dissolved in 1000 ml milli-Q-water. Prepare fresh on the day of analysis.
5.3.2.2 Mobile phase B: Methanol 100 %
5.3.3. Precaution:
5.3.3.1 Mycotoxins are very hazardous substances. Only a laboratory equipped to handle toxic materials and solvents should perform analysis.
5.3.3.2 Suitable protective clothing, including gloves, safety glasses, and lab coats should be worn throughout the analysis.
5.4. Instrumental Conditions HPLC condition
|
|
6.0
CALCULATION
By using the below equation.
y=mx +c
Where,
y=Analyte area
x= Concentration of Analyte
m=Slope of the calibration curve
c= y-axis intercept value
7.0 REVISION/CHANGE HISTORY: NA
8.0 ATTACHMENTS
· Appendix-1: FLOW CHART FOR AFLATOXIN (B1, B2, G1, G2) IN FOOD PRODUCT
9.0 REFERENCES
· AOAC 999.07
You may like to read these links:
1. List of All SOPs and Documents for the Chemistry Laboratory
2. List of All SOPs and Documents for Quality Control
3. List of All SOPs and Documents For Quality Assurance Dept.
4. List of SOPs and Documents Required to Achieve NABL Accreditation
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