HPLC Set-up

The flow sequence of LC system used in present study was from HPLC

column to PDA detector, to PHRED, to post column reactor, and then to

fluorescence detector. The reagent manager was connected to the post column

reactor and controlled by Empower software. The flow rate for derivatization

mixture was 0.5ml/min. The reagent manager was set to pump derivatization

mixture at 18.5min and stop at 23min in the section of event of Waters’ 2795

Separation Module. The column temperature was set to 30 ± 5ºC. The mobile

phase consisted of acetonitrile, methanol, and 0.1% phosphate acid in water; and

the flow rate was 1.0ml/min. Gradient elution, and the events of fluorescent

detector were shown in Table 1 and 2.

The derivatization of aflatoxins was performed by photochemical reactor

for enhanced detection. The derivatization of fumonisins was done according the

procedure described previously. Briefly, borate buffer was made by dissolving

19g of sodium tetraborate decahydrate in 1 liter purified water and filtering

through 0.45µm membrane, and stored in room temperature. Dissolve 150mg of

OPA in 5ml methanol, add to 500ml borate buffer, and add 500ul of 2-

mercaptoethanol, mix well, filter through 0.45µm membrane and place in a bottle

protecting from light. The mixture of fumonisin derivatization can be used for a

week.

Table 1. The gradient elution of mobile phase

Table 2. The event of fluorescent detector

Time (min) Flow Methanol (%) 0.1% H3PO3 (%) Acetonitrile (%) Curve1.00 0.0 85.0 15.0

4.00 1.00 0.0 85.0 15.0 65.00 1.00 25.0 60.0 15.0 616.00 1.00 25.0 60.0 15.0 617.00 1.00 30.0 40.0 30.0 630.01 1.00 0.0 85.0 15.0 11

Time (min)

Parameter Wavelength (nm)

Value

0.1 Ex 365Em 455

Gain 10Auto zero

11.0 Auto zero18.0 Ex 329

Em 46520.0 Auto zero23.0 Ex 276

Em 46026.5 Auto zero27.0 Gain 10027.9 Ex 329

Em 460Gain 10

Figure 1. Representative HPLC chromatogram of corn sample containing 11 mycotoxins. The spiking levels in this figure were: 300ppb for DON, 100ppb for HT-2 and T-2, 2.23ppb forAFG2, 6.45ppb for AFG1, 2.48ppb for AFB2, 12.93 for AFB1, 200ppb for FB1 and FB2, 150ppb for ZEA, and 50ppb for OTA.

DON

HT-2 T-2

G2 G2 B2 B2

FB1 FB2

ZEA OTA

Separation column:

Waters Symmetry C18, 3.5µm 3.9 x 150mm.

Order information:

• PCRM includes: the Temperature Control and the Heater, the

Waters part # is WAT 038039, please see the attachment.

• Reaction coil: it is a optional, part # is WAT 030844

• Reagent manager: 725000108, Japanese version is 725000109 see

the attachment.