TITLE: Procedure for Aflatoxin M1 and B1 in Liver by HPLC-Fluorescence Detection with Pre-column Derivatization
AUTHORS: duey

[DESCRIPTION]
The current procedure is for quantitative determination of aflatoxin M1 (AFM1) and B1(AFB1) in animal liver by HPLC-fluorescence detection at concentrations 0.2-10 ng/g (ppb). Note, sensitivity of the method greatly depends on sensitivity of the fluorescent detector. The method requires 1g of liver. Quantitation is based on matrix-matched calibration curve. Analytes (AfB1 and AfM1) are extracted, derivatized, cleaned up using Octadecyl (C18) material, injected into HPLC and chromatographed on C18 column for fluorescence detection.

Validation data (in-house and via collaborative studies such as Blinded Method Tests) are available in the following publications:

https://pubmed.ncbi.nlm.nih.gov/27638844/

https://pubmed.ncbi.nlm.nih.gov/30736868/

[GUIDELINES]
Each lab should perform a practice run to check the optimal incubation time and HPLC run time at three levels (low at 0.8 ppb, medium at 5 ppb, and high at 8 ppb). After incubation, inject samples at 20 h, 24 h, 28 h, and 32 h to check signal drift for each aflatoxin. The optimal incubation time is the time when minimal signal drift is observed.A signal drift of ± 15% is acceptable.

The HPLC run time is optimal as long as the last eluting peak has returned to the baseline (2-4 minutes) before the run has ended.The validated run time is 25 minutes, if needed the run time can be extended in 5-10 minutes increments until the run time has been optimized.

[STEPS]
SECTION: PREPARATION OF REAGENTS AND STANDARDS
1. The standard stock solutions of each aflatoxin is prepared by dissolving the pre-weighed standards in chloroform (AFM1) or methanol (AFB1) and stored in -20 °C when not in use.

2. A mixed standard solution (250 ng/mL for each aflatoxin) is prepared. Quantitatively transfer 1 mL of the AFM1 stock standard and 1 µLof the AFB1 stock standard to a7 mL scintillation glass vial.            

 
                                           
3. A working standard solution (25 ng/mL) is prepared by dilution of the 250 ng/mL standard solution using methanol. It is prepared on the day of use. 
3.1. Transfer0.5 mL of the 250 ng/mL mixed standard to a 7 mL vial, add 4.5 mLof methanol and vortex to mix.



4. 20% Citric acid solution is prepared by weighing 20.0 g of citric acid monohydrate and dissolving in de-ionized water to give a final volume of100 mL. This amount is good for about 80-90 samples.

5. Acetonitrile/water 90/10 (v/v) is prepared by adding 100 mLde-ionized water to 900 mLacetonitrile and mixed well. This amount is good for 80-90 samples.

6. Octadecyl (C18) LC packing / alumina neutral 50/50 (w/w) is prepared by mixing 70.0 galumina neutral and 70.0 gOctadecyl (C18) LC packing in a sealable plastic container. The mixture is shaken for five minutes. Shake for five seconds before each use. This amount is good for about 80-90 samples.

7. Clean-up columns are prepared by weighing 1.50 g premixed 50/50 (w/w) Octadecyl (C18) 40 µm Preparation LC packing/Alumina Neutral into 6 mL plastic syringes. The C18/alumina mixture is immobilized by 2 Whatman 740-E filter discs on both ends.                                                                                                                                                                                                                                                                                                                                                             
 


8. Derivatization reagent 35/10/5 (v/v) water/TFA/glacial acetic acid: 
SECTION: PREPARATION OF REAGENTS AND STANDARDS
1.1. Example:  A5 µg AFM1 standard is dissolved in 5 mL of chloroform to make a 1 ug/mLstock standard.

SECTION: PREPARATION OF REAGENTS AND STANDARDS
1.2. Example:  A5 mg AFM1 standard is dissolved in 5 mL of chloroform to make a 1 mg/mL stock standard.

8.1. Mix 10 mL TFA with5 mL glacial acetic acid and 35 mL de-ionized water

8.2. Store in dark or aluminum-foil-wrapped bottles

 

SECTION: Sample Preparation
9. Preparation of calibration curve in liver matrix: 
SECTION: Sample Preparation
9.1. Seven 1.0 gcontrol liver samples are weighed in 50 mL screw cap glass tubes.

SECTION: Sample Preparation
9.2. A series of volumes (8.0 μL, 20.0 μL, 40.0 μL, 80.0 μL, 200 μL, and 400 μL) of the 25 ng/mL working standard solution of aflatoxin M1 and B1 is spiked to 1.0 g of control liver to give a series of fortified concentration of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 ng/g. 
SECTION: Sample Preparation
9.3. The fortified samples are mixed thoroughly by vortexing for 10 s at maximum speed and subject to the following steps 7.2-7.4. 

SECTION: Sample Preparation
10. Extraction
SECTION: Sample Preparation
10.1.  1.0 gof liver samples are weighed in 50 mL screw cap glass tubes.

SECTION: Sample Preparation
10.2. 1.0 mL of 20% citric acid solution is added to each sample and calibrant.   Samples and calibrants are subsequently mixed thoroughly by vortexing for approximately 10 s at maximum speed. Samples are allowed to sit for 5-10 minutes and mixed again. 

SECTION: Sample Preparation
10.3. 0.20 g CeliteTM is added to each sample and followed by vortexing for10 sat maximum speed.                                                                                                                                                                                                                                     

 

SECTION: Sample Preparation
10.4. 20.0 mL of dichloromethane is then added to each sample and calibrant.   Vortex for 10 s

SECTION: Sample Preparation
10.5. The mixture is mounted on Roto rack for rotate-mixing for 35 min, followed by centrifuge at 2000 rpm for 10 min. 

SECTION: Sample Preparation
10.6. After centrifugation, the bottom clear layer is carefully aspirated and passed through sodium sulfate (7.20 g) housed in a set of glass fiber filter circle and plastic funnel. The filtrates are collected in clean 50 mL tubes. 
SECTION: Sample Preparation
10.7. Sodium sulfate is then washed with 6 mL dichloromethane. The wash is combined with the solution obtained from step 10.6

SECTION: Sample Preparation
10.8. The solutions are subsequently concentrated to dryness by gentle nitrogen effusion at ambient temperature.
SECTION: Sample Preparation
11. Clean-up 
SECTION: Sample Preparation
11.1. The dried extracts obtained in the previous step are reconstituted in5.0 mL 90/10 (v/v) acetonitrile/water and vortexed for 10 sat maximum speed. 

SECTION: Sample Preparation
11.2. Clean-up columns are pre-conditioned by 5.0 mL 90/10 (v/v) acetonitrile/water before use. The acetonitrile/water is discarded.

SECTION: Sample Preparation
11.3. The dissolved extracts are then loaded on clean-up columns. The solution flowing through the columns is collected in a new 7.0 mL scintillation vial. Solutions are allowed to flow through by gravity. After all the solution flows through the column, the plungers are pushed down to the barrel of the syringe to drive out the residual liquid in the packing. The residual liquid is received in the same 7.0 mL vials for each sample. 
SECTION: Sample Preparation
11.4. The solutions are subsequently concentrated to dryness by gentle nitrogen effusion at ambient temperature.
SECTION: Sample Preparation
12. Derivatization
SECTION: Sample Preparation
12.1. The residue obtained in the previous step are reconstituted in 400 µL 35/10/5 (v/v) water/TFA/glacial acetic acid, vortexed for 10 s at maximum speed, then heated at 65°C in heating dry bath for 15 min.

SECTION: Sample Preparation
12.2. The solutions obtained from this step are incubated at ambient temperature for 16-20 hours (This incubation time needs to be optimized under each lab’s conditions, see Guidelines) before HPLC analysis.
SECTION: HPLC Conditions
13. The optimized excitation and emission wavelengths for the fluorescence detector are 360 and 440 nm, respectively.
SECTION: HPLC Conditions
14. The mobile phase consisting of water (A) and acetonitrile (B) is pumped at a flow rate of 1.0 mL/min. A gradient elution is used to give the optimized separation. The details of the gradient program is as follows: 0-7.5 min isocratic step at 86% A, 14% B; 0.5 min linear gradient (7.5-8.0 min) to 85% A, 15% B, an isocratic step from 8.0 to 20.0 min at 85% A, 15% B; a 1.0 min linear gradient (20.0-21.0 min) to 86% A, 14% B; and a final isocratic step at 86% A, 14% B to the end. A total running time is 25 min. (This needs to be optimized according to each lab’s HPLC system, see Guidelines).
SECTION: HPLC Conditions
15. An injection volume of 20 µLis used. 

SECTION: HPLC Conditions
16. Retention time: based on the above-mentioned HPLC conditions, aflatoxin M1 chromatographs at approximately 4.0 minutes, aflatoxin B1 chromatographs at approximately 18.2 min

SECTION: Result Interpretation
17. The calibration curve is created by plotting the blank-subtracted fluorescence intensity (the peak are of fluorescence unit) versus the injected mass of standard (in ng) by linear regression.