Page 71 - Application Handbook - Liquid Chromatography

Application No.L500
News

Aflatoxin standard solution was added to the complex Table 1 HPLC Analytical Conditions
crude drug Kakkonto prior to analysis. The pretreatment
procedure is shown in Fig. 2. This pretreatment was System : Nexera-i
Column
: Shim-pack FC-ODS
performed based on the proposed revision to the (150 mm L. × 4.6 mm I.D., 3 µm)
Japanese Pharmacopoeia 17th Edition. An AFLAKING Mobile Phase : A; Water/methanol/acetonitrile = 6/3/1 (v/v/v)
: B; Acetonitrile
immunoaffinity column (Horiba, Ltd.) was used in a Time Program : A Conc. /B Conc. = 100/0 (0.00 - 15.00 min) →
cartridge to remove impurities. Aflatoxin standard 10/90 (16.00 - 23.0 min) → 100/0 (24.00 - 34.00 min)
solution was added to the crude drug sample so each Flowrate : 0.80 mL/min
Column Temp. : 40 °C
aflatoxin was present at a concentration of 0.25 µg/kg Injection Volume : 20 µL
(total 1 µg/kg). This is equivalent to 1/10th the reference Detection : RF-20AXS, Ex. at 365 nm, Em. at 450 nm
concentration stipulated in the proposed revision to the Cell Temp. : 25 °C
Japanese Pharmacopoeia 17th Edition.
The example analysis of Kakkonto is shown in Fig. 3,
and the analytical conditions are shown in Table 1. An
example analysis of the sample with no added aflatoxin
standard solution is also shown for comparison. Since
an impurity peak was found after aflatoxin B2, which is
the last eluted aflatoxin, a column cleaning process was
added to the procedure. See Application News L428 for
an example analysis of the standard solution.

uV
Sample 1.0 g 5000 ■Peaks
4500 1. Aflatoxin G2a (G1)
(Aflatoxin standard solution) 2. Aflatoxin B2a (B1)
4000
3. Aflatoxin G2
4 mL acetonitrile/water/methanol = 6/4/1 (v/v/v) 3500 4. Aflatoxin B2
3000
Shake for 30 min 4
2500
3
2000 2
Centrifuge 1500 1
1000
(2 mL) : up to 50 mL with 500
4 % polysorbate 20 in phosphate buffered saline 0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min
(50 mL)
Fig. 3 Chromatogram of Kakkonto After TFA Derivatization
—HPLC Analysis
Clean-up with immunoaffinity column “AFLAKING” (Upper: With Standard Solution, Lower: Without
Standard Solution)
Elution by acetonitrile (3 mL)

Evaporation by N2 gas
0.1 mL TFA
Standing in dark at room temperature

for 15 min.

0.4 mL
water/acetonitrile = 9/1 (v/v)
HPLC

Fig. 2 Pretreatment Procedure