TITLE: Procedure for Detection of Aflatoxin B1 and M1 in Urine by High Performance Liquid Chromatography with Fluorescence Detection.
AUTHORS: x.du

[DESCRIPTION]
The purpose of this SOP is to describe how to determine the presence of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in urine with pre-column derivatization by high performance liquid chromatography (HPLC) with fluorescence detector.

Quantitation range: 0.5-15 ng/g (ppb). Note, sensitivity of the method greatly depends on sensitivity of the fluorescent detector. The method requires 2mL of urine. Quantitation is based on matrix-matched calibration curve. Analytes (AfB1 and AfM1) are extracted, derivatized, cleaned up using Octadecyl (C18) material, injected into HPLC, chromatographed on C18 column and quantitated using Fluorescence detector

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

 https://pubmed.ncbi.nlm.nih.gov/28985321/

https://pubmed.ncbi.nlm.nih.gov/36912688/

[BEFORE_START]
Checks Made Prior to Beginning Test Procedure

We run standard and make sure the retention time drift for each analyte should be within 15%.

[GUIDELINES]
1. The correlation coefficient R2 value for the standard curve must be >0.99. The back calculated deviation for each points 
must be within 20%.
.

2. The method for detection of AFB1 and AFM1 was validated in glassware. 7 mL borosilicate glass scintillation vials are also suitable for this step.

3. The method for detection of AFB1 and AFM1 was validated in glassware. 50 mL screw cap glass tube is also suitable for this step.

4. 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 10 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.

5. 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 33 minutes, if needed the run time can be extended in 5-10 minute increments until the run time has been optimized.

[STEPS]
SECTION: Preparation of Standard Stock Solutions and Reagents
2. 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.

 

SECTION: Preparation of Standard Stock Solutions and Reagents
2.3. A working mixed standard solution (250 ng/mL for each aflatoxin) is prepared. Quantitatively transfer 1 mL of the AFM1 stock standard and1 µL of the AFB1 stock standard to a 7 mL scintillation glass vial. Concentrate to dryness by nitrogen effusion and dilute in4 mL methanol. It is stored at -20 °C when not in use and is good for one year.

SECTION: Sample preparation
6. Urine samples were pooled into a beaker and stirred with a stir bar for 10 min. After homogenization, the urine was transferred into 50 mL centrifuge tubes and centrifuged at 3800 rpm for 10 min to remove precipitates.

SECTION: Sample preparation
7. The centrifuged urine sample was transferred into brown plastic bottles and stored at -80 oC before use.
SECTION: Preparation of Standard Stock Solutions and Reagents
2.1. Example: A 5 µg AFM1 standard is dissolved in 5 mL of chloroform to make a 1 ug/mL stock standard.

SECTION: Preparation of Standard Stock Solutions and Reagents
2.2. Example: A 5 mg AFB1 standard is dissolved in 5 mL of methanol to make a1 mg/mLstock standard.

SECTION: Preparation of Standard Stock Solutions and Reagents
2.4. 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 0.5 mL of the 250 ng/mL mixed standard to a 7 mL vial, add 4.5 mL of methanol and vortex to mix.

SECTION: Test Procedure
8. Preparation of calibration curve in urine matrix
SECTION: Test Procedure
8.1. 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 10 min

SECTION: Test Procedure
8.2. Seven2 mLcontrol 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 2.0 mL control urine to give a series of fortified concentration of 0.5, 1, 2, 5, 10, 15 ng/mL.

SECTION: Test Procedure
8.3. The fortified samples are mixed thoroughly by vortexing for 10 s at maximum speed and subject to the following steps 7-9.

SECTION: Test Procedure
9. Extraction
SECTION: Test Procedure
9.1. Add 4 mL 80/20 (v/v) methanol/water to the samples. Vortex at 2500 speed for5 minusing the Fisher Multi-tube Vortexer.

SECTION: Test Procedure
9.2. Pipet 2 mL solution into a 50 mL plastic conical-bottom centrifuge tube (See Appendix 3).

SECTION: Test Procedure
9.3. Dilute the 2 mL solution with 14 mL 1X PBS solution, vortex for5 s

SECTION: Test Procedure
10. Clean up
SECTION: Test Procedure
10.1. 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 16.0 mL 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).

SECTION: Test Procedure
10.2. 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.
SECTION: Test Procedure
10.3. Wash columns by passing 20 mL 1x PBS solution, at a flow rate of approximately 1 drop/sec (3 mL/min). Pass air through the column to remove residual liquid.

SECTION: Test Procedure
10.4. Elute the analytes from the column using 1.0 mL of methanol and collect the eluents in new tubes.
SECTION: Test Procedure
10.5. Pass 1.0 mL deionized water through the column.
SECTION: Test Procedure
10.6. Collect in the same tubes to give a 2 mL total volume. A backflush is needed to remove residual liquid.

SECTION: Test Procedure
10.7. 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.
SECTION: Test Procedure
11. Derivatization
SECTION: Test Procedure
11.1. The residue obtained in the previous step are reconstituted in 400 µL 35/10/5 (v/v/v) water/TFA/glacial acetic acid, vortexed for 10 s at maximum speed, then heated at 65 °C in heating block for 15 min

SECTION: Test Procedure
11.2. The solutions obtained from this step are incubated at least for 1200 min (This incubation time needs to be optimized under each lab’s conditions, see Appendix 5.3) at room temperature before HPLC analysis.

SECTION: Result Interpretation
16. 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.
SECTION: Reporting Results
17. See [9.3444, current version] Procedure for entering results in ISULIMS.
SECTION: HPLC Conditions
12. The optimized excitation and emission wavelengths for the fluorescence detector are 360 and 440 nm, respectively.
SECTION: HPLC Conditions
15. Retention time: A total running time of 33 min is normally used.
SECTION: HPLC Conditions
14. An injection volume of 20 µLis used.
SECTION: HPLC Conditions
13. 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)
SECTION: Preparation of Standard Stock Solutions and Reagents
5. Derivatization reagent 35/10/5 (v/v) water/TFA/glacial acetic acid: Mix10 mL TFA with 5 mL glacial acetic acid and 35 mLL deionized water. The mixture is mounted on Roto rack for rotate-mixing for 30 min, 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

SECTION: Preparation of Standard Stock Solutions and Reagents
4. 1x Phosphate-buffered saline (PBS) solution is prepared by adding   NaCl, 0.20 g KCl,  1.44 gNa2HPO4, and 0.24 g KH2PO4 in 800 mL deionized water and adjust the pH to 7.4 with 1 M HCl, then diluted to 1.0 L with deionized water. This amount is good for about 25-28 samples.

SECTION: Preparation of Standard Stock Solutions and Reagents
3. 1 M HCl solution is prepared by adding 8.3 mL of concentrated HCl and diluting it in a volumetric flask to 100 mL.

SECTION: Preparation of Standard Stock Solutions and Reagents
1. Methanol/water 80/20 (v/v) is prepared by adding80 mLdeionized water to 320 mL methanol. This amount is good for about 75-80 samples.