Page 72 - Application Handbook - Liquid Chromatography

Application No.L500
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n Analysis by Direct Detection
mV
Although aflatoxins B1 and G1 have a low fluorescence 150
intensity, using the RF-20A XS highly-sensitive 140 ■Peaks 1 3
fluorescence detector allows for direct detection 130 1. Aflatoxin G2
2. Aflatoxin G1
without derivatization. We performed direct detection 120 3. Aflatoxin B2
using the RF-20AXS, and also attempted to shorten the 110 4. Aflatoxin B1
analysis time by using the Shim-pack XR-ODS Ⅱ high- 100
90
performance column. Fig. 4 shows analysis of the 80
aflatoxin standard solution without TFA derivatization 70
(each aflatoxin at 20 µg/L), and Fig. 5 shows the same 60
analysis performed at low concentrations (each aflatoxin 50
at 0.1 µg/L). Analytical conditions are shown in Table 2. 40
30
The relative standard deviation (n=6) of the area 20 2 4
measured upon analysis of aflatoxin B1 at 0.1 µg/L was 10
2.6 %. This result shows that sufficient analytical 0
sensitivity can be obtained by using the RF-20AXS even 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min
without performing TFA derivatization. Using the RF-
20AXS also shortens the analysis time to approximately Fig. 4 Chromatogram of Aflatoxin Standard Solution by Direct
1/3rd of the analysis time with TFA derivatization. Fig. 6 Detection—UHPLC Analysis (each 20 µg/L, 10 µL)
shows the calibration curves for each aflatoxin in the
concentration range of 0.1 to 20 µg/L. Good linearity
2
was obtained with all four compounds, with an R of
0.9999 or above. mV
0.80 ■Peaks
0.75 1 3
0.70 1. Aflatoxin G2
0.65 2. Aflatoxin G1
0.60 3. Aflatoxin B2
Table 2 UHPLC Analytical Conditions 0.55 4. Aflatoxin B1
0.50
System : Nexera-i 0.45
Column : Shim-pack XR-ODS Ⅱ 0.40
(100 mm L. × 3.0 mm I.D., 2.2 µm) 0.35
Mobile Phase : A; Water 0.30
: B; Methanol 0.25
: C; Acetonitrile 0.20
Time Program : A Conc. /B Conc. /C Conc. = 65/30/5 (0.00 - 5.50 min) → 0.15 2 4
15/5/80 (5.51 - 7.0 min) → 20/80/0 (7.01 - 9.00 min) → 0.10
65/30/5 (9.01 - 12.00 min) 0.05
Flowrate : 1.00 mL/min 0.00
Column Temp. : 50 °C -0.05
-0.10
Injection Volume : 10 µL 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min
Detection : RF-20AXS, Ex. at 365 nm, Em. at 450 nm
Cell Temp. : 25 °C
Fig. 5 Chromatogram of Aflatoxin Standard Solution by Direct
Detection—UHPLC Analysis (each 0.1 µg/L, 10 µL)

Area (×1000) Area (×1000) Area (×1000) Area (×1000)
650 1000 140
600 50 Aflatoxin B2 130
550 Aflatoxin G2 45 Aflatoxin G1 900 R 2 :0.9999 120 Aflatoxin B1
R 2 :0.9999
R 2 :0.9999
R 2 :0.9999
500 40 800 110
450 700 100
400 35 600 90
350 30 80
300 25 500 70
60
250 20 400 50
200 15 300 40
150 30
100 10 200 20
50 5 100 10
0 0 0 0
0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
Conc. Conc. Conc. Conc.
Fig. 6 Aflatoxin Standard Solution Calibration Curves—Direct Detection (each 0.1 to 20 µg/L, 10 µL)