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

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.

Example:  A5µg AFM1 standard is dissolved in 5mL of chloroform to make a 1ug/mLstock standard.

Example:  A5mg AFM1 standard is dissolved in 5mL of chloroform to make a 1mg/mL stock standard.

A mixed standard solution (250 ng/mL for each aflatoxin) is prepared. Quantitatively transfer 1mL of the AFM1 stock standard and 1µLof the AFB1 stock standard to a7mL scintillation glass vial. 

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.

Transfer0.5mL of the 250 ng/mL mixed standard to a 7 mL vial, add 4.5mLof methanol and vortex to mix.

20% Citric acid solution is prepared by weighing 20.0g of citric acid monohydrate and dissolving in de-ionized water to give a final volume of100mL. This amount is good for about 80-90 samples.

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

Octadecyl (C18) LC packing / alumina neutral 50/50 (w/w) is prepared by mixing 70.0galumina neutral and 70.0gOctadecyl (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.

Clean-up columns are prepared by weighing 1.50g 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.

Derivatization reagent 35/10/5 (v/v) water/TFA/glacial acetic acid:

Mix 10mL TFA with5mL glacial acetic acid and 35mL de-ionized water

Store in dark or aluminum-foil-wrapped bottles

Preparation of calibration curve in liver matrix:

Seven 1.0gcontrol liver samples are weighed in 50 mL screw cap glass tubes.

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.

The fortified samples are mixed thoroughly by vortexing for 0h 0m 10s at maximum speed and subject to the following steps 7.2-7.4.

Extraction

1.0gof liver samples are weighed in 50 mL screw cap glass tubes.

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 0h 0m 10s at maximum speed. Samples are allowed to sit for 5-10 minutes and mixed again.

0.20g Celite^TM is added to each sample and followed by vortexing for0h 0m 10sat maximum speed.

20.0mL of dichloromethane is then added to each sample and calibrant. Vortex for 0h 0m 10s

The mixture is mounted on Roto rack for rotate-mixing for 0h 35m 0s, followed by centrifuge at 2000 rpm for 0h 10m 0s.

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.

Sodium sulfate is then washed with 6mL dichloromethane. The wash is combined with the solution obtained from step 10.6

The solutions are subsequently concentrated to dryness by gentle nitrogen effusion at ambient temperature.

Clean-up

The dried extracts obtained in the previous step are reconstituted in5.0mL 90/10 (v/v) acetonitrile/water and vortexed for 0h 0m 10sat maximum speed.

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

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.

The solutions are subsequently concentrated to dryness by gentle nitrogen effusion at ambient temperature.

Derivatization

The residue obtained in the previous step are reconstituted in 400µL 35/10/5 (v/v) water/TFA/glacial acetic acid, vortexed for 0h 0m 10s at maximum speed, then heated at 65°C in heating dry bath for 0h 15m 0s.

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.

The optimized excitation and emission wavelengths for the fluorescence detector are 360 and 440 nm, respectively.

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).

An injection volume of 20µLis used.

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

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.