Procedure for Detection of Aflatoxin B1 and M1 in Urine by High Performance Liquid Chromatography with Fluorescence Detection.

Methanol/water 80/20 (v/v) is prepared by adding80mLdeionized water to 320mL methanol. This amount is good for about 75-80 samples.

The standard stock solutions of each aflatoxin is prepared by dissolving the pre-weighed standard in methanol (AFB1) or chloroform (AFM1) and stored in -20 °C when not in use.

Example: A 5µg AFM1 standard is dissolved in 5mL of chloroform to make a 1ug/mL stock standard.

Example: A 5mg AFB1 standard is dissolved in 5mL of methanol to make a1mg/mLstock standard.

A working mixed standard solution (250 ng/mL for each aflatoxin) is prepared. Quantitatively transfer 1mL of the AFM1 stock standard and1µL of the AFB1 stock standard to a 7 mL scintillation glass vial. Concentrate to dryness by nitrogen effusion and dilute in4mL methanol. It is stored at -20 °C when not in use and is good for one year.

A working mixed standard solution (25 ng/mL) is prepared by dilution of the 250 ng/mL mixed standard solution using methanol. It is prepared on the day of use. Transfer 0.5mL of the 250 ng/mL mixed standard to a 7 mL vial, add 4.5mL of methanol and vortex to mix.

1 M HCl solution is prepared by adding 8.3mL of concentrated HCl and diluting it in a volumetric flask to 100 mL.

1x Phosphate-buffered saline (PBS) solution is prepared by adding Sample NaCl, 0.20g KCl, 1.44gNa2HPO4, and 0.24g KH2PO4 in 800mL deionized water and adjust the pH to 7.4 with 1 M HCl, then diluted to 1.0L with deionized water. This amount is good for about 25-28 samples.

Derivatization reagent 35/10/5 (v/v) water/TFA/glacial acetic acid: Mix10mL TFA with 5mL glacial acetic acid and 35mLL deionized water. The mixture is mounted on Roto rack for rotate-mixing for 0h 30m 0s, then stored in dark or aluminum-foil-wrapped bottles. This amount is good for about 100-120 samples. We suggest this is good for 3 months

Urine samples were pooled into a beaker and stirred with a stir bar for 0h 10m 0s. After homogenization, the urine was transferred into 50 mL centrifuge tubes and centrifuged at 3800 rpm for 0h 10m 0s to remove precipitates.

The centrifuged urine sample was transferred into brown plastic bottles and stored at -80 oC before use.

Preparation of calibration curve in urine matrix

Thaw samples stored in -80 °C freezer in a water bath and centrifuge the blank urine samples in plastic centrifuge tubes at 3800 rpm for 0h 10m 0s

Seven2mLcontrol urine samples are weighed into 15 mL Falcon plastic polypropylene conical tubes (See Guideline 2). A series of volumes (4 μL, 8 μL, 16 μL, 40 μL, 80 μL and 120 μL ) of the 250 ng/mL working standard solution of AFB1 and AFM1 is added to the 2.0mL control urine to give a series of fortified concentration of 0.5, 1, 2, 5, 10, 15 ng/mL.

The fortified samples are mixed thoroughly by vortexing for 0h 0m 10s at maximum speed and subject to the following steps 7-9.

Extraction

Add 4mL 80/20 (v/v) methanol/water to the samples. Vortex at 2500 speed for0h 5m 0susing the Fisher Multi-tube Vortexer.

Pipet 2mL solution into a 50 mL plastic conical-bottom centrifuge tube (See Appendix 3).

Dilute the 2mL solution with 14mL 1X PBS solution, vortex for0h 0m 5s

Clean up

Immuno-affinity columns (stored at 5 C) are mounted on the vacuum container with the other end of the column connected with 30 mL syringes through adaptors before use. Ensure that the column has not dried out and contains buffer above the gel when loading the 16.0mL PBS solution. (It is important to note that the antibody included in the immuno-affinity column can be denatured by extreme temperature or pH change).

Pass solutions through the columns at a steady slow rate of 0.25 - 0.5 mL/min under vacuum or by gravity (This has to be followed exactly). A slow, steady flow rate is essential for the capture of the aflatoxins by the antibody.

Wash columns by passing 20mL 1x PBS solution, at a flow rate of approximately 1 drop/sec (3 mL/min). Pass air through the column to remove residual liquid.

Elute the analytes from the column using 1.0 mL of methanol and collect the eluents in new tubes.

Pass 1.0 mL deionized water through the column.

Collect in the same tubes to give a 2mL total volume. A backflush is needed to remove residual liquid.

The eluates collected in last two steps are transferred to 7 mL glass scintillation vial. The tubes are washed with 2 x 0.5 mL methanol. The solutions are concentrated to dryness under gentle nitrogen stream at ambient temperature.

Derivatization

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

The solutions obtained from this step are incubated at least for 20h 0m 0s (This incubation time needs to be optimized under each lab’s conditions, see Appendix 5.3) at room temperature 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 mL/min. A gradient elution is used to give the optimized separation. The details of the gradient program are as follows: 1 min isocratic step at 100% A; 1 min linear gradient (1-2 min) to 84% A and 16% B; an isocratic step from 2 to 16 min at 84% A and 16% B; a 1 min linear gradient (16-17 min) to 80% A and 20% B; an isocratic step from 17 to 27 min at 80% A and 20% B; 1 min linear gradient (27-28 min) to 100% A; and a final isocratic step at 100% A to the end (28-33 min)

An injection volume of 20µLis used.

Retention time: A total running time of 0h 33m 0s is normally used.

The calibration curve is established by plotting the fluorescence intensity (in peak area, fluorescence unit) versus the injected mass of standard (in ng) by linear regression. The concentration of the unknown sample is calculated based on this standard curve.

See [9.3444, current version] Procedure for entering results in ISULIMS.