Page 74 - Application Handbook - Liquid Chromatography

LAAN-A-LC-E261

Application High Performance Liquid Chromatography

News Analysis of Nivalenol and Deoxynivalenol in Wheat

Using Prominence-i
No.L484

Nivalenol and deoxynivalenol (DON, vomitoxin) are types of
mycotoxins produced by Fusarium fungi. In Japan, the N"6
provisional reference value for deoxynivalenol was set at
1.1 ppm in May, 2002 (Notification No. 0521001 issued by ˙ 1FBLT
Department of Food Safety, Pharmaceutical and Food Safety /JWBMFOPM
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Bureau, Ministry of Health, Labour and Welfare of Japan).
Previously, in Application News No. L362, the analysis using
an ultra high-performance LC system was introduced, but
here, referring to the test method for deoxynivalenol
specified in the Notification No. 0717001 (issued by
Department of Food Safety, Pharmaceutical and Food Safety
Bureau, Ministry of Health, Labour and Welfare of Japan, in
July 2003), a washing process for the analytical column is
included.
The detector component of the new Prominence-i
integrated high-performance liquid chromatograph
incorporates a temperature control function for both the
flow cell component and the optical system. Here, good
repeatability was obtained despite the susceptibility of UV
detection in the short wavelength region due to NJO
environmental temperature fluctuations.

Fig. 1 Chromatogram of a Standard Mixture (4.0 ppm each)
n Analysis of Standard Mixture
Fig. 1 shows the results of analysis of a standard mixed
solution of nivalenol and deoxynivalenol (each at
4.0 ppm) using a 10 µL injection. Table 1 shows the N"6

analytical conditions used. The test method specifies ˙ 1FBLT
the use of isocratic analysis, but a column washing /JWBMFOPM
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process after elution of the deoxynivalenol was added.
As the Prominence-i is equipped with a low-pressure
gradient unit as standard, a mobile phase with a high
organic solvent ratio can easily be pumped through the
system following elution of the target component.
Six repeat analyses of a 0.1 ppm standard solution were
conducted, corresponding to about one-tenth the
provisional reference value. The relative standard
deviation (% RSD) of peak area and retention time
obtained for the two substances are shown in Table 2,
and the chromatogram is shown in Fig. 2.

Table 1 Analytical Conditions
Column : Shim-pack GIS C18 NJO
(250 mm L. × 4.6 mm I.D., 5 µm)
Mobile Phase A : Water / Acetonitrile / Methanol = 90/5/5 (v/v/v)
Mobile Phase B : Acetonitrile / Methanol = 50/50 (v/v)
Time Program : B Conc. 0 % (0 - 20 min) → 50 % (20.01 - 25 min) Fig. 2 Chromatogram of a Standard Mixture (0.1 ppm each)
→ 0 % (25.01 - 45 min)
Flowrate : 1.0 mL/min
Column Temp. : 40 ˚C
Injection Volume : 10 µL
Detection : UV 220 nm (Cell temp. 45 ˚C)

Table 2 Repeatability (0.1 ppm, n=6)
R.T.%RSD Area %RSD
Nivalenol 0.09 0.68
Deoxynivalenol 0.06 0.76