Page 91 - Application Notebook - Solution for Food Safety

Application No. AD-0096 N
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 Experimental  Results and Discussion
Instrumental and Analytical Conditions Development of Fast UHPLC Method
An UHPLC system - Nexera X2 (Shimadzu Corporation) For well separation of the thirteen bisphenols studied, a
equipped with a fluorescence detector (RF-20Axs) was reference HPLC method has a long running time of
employed in this work. Separation of bisphenol A (BPA), 95 mins. As shown in Fig. 3, the current UHPLC method
bisphenol F (BPF) and other 11 derivatives are performed was optimized to achieve fast elution for every compounds
using a Shim-pack HR-ODS column (250 × 3.0 mm, 3 μm) with sufficient separation resolution, especially for the
with an optimized gradient elution program. Pure water separation of BADGE-H 2O and BADGE-H 2O-HCl at 19.4
and acetonitrile (ACN) were used as UHPLC mobile phases and 19.9 mins. Due to the similarity in compound
without any additive. The detailed analytical conditions structure and chemical properties, separation of these
of the UHPLC method are shown in Table 1. two peaks was a main obstacle to achieve fast analysis
speed. The results obtained show clearly the advantages
of an UHPLC column with small particle size (3 μm) of the
Standards and Spiked Milk Samples C18 stationary phase.
A mixed standard stock solution of thirteen bisphenols The main targets BPA (18.1 min) and BPF (13.4 min),
(Refer to Table 2) containing BPA, BPF and other BADGE (34.3 min) are separated completely without
derivative compounds were prepared in ACN/H 2O (30:70). any inference. BFDGE has two positional isomers,
A serial of calibration standards of concentrations from which appeared as a pair at 28.8 min and 30.0 min,
5 μg/L to 2,000 μg/L were prepared from the stock to set respectively.
up multi-point calibration curves. Two blank milk matrix
spiked with known concentrations of standards (100 and Noted that, another pair of positional isomers BFDGE-
1,000 μg/L) obtained from a third party laboratory were 2HCl appeared just before the BFDGE peaks at 26.9 min
used for evaluation of the method performance. and 27.9 min, respectively.

Table 1 UHPLC Conditions of Bisphenols and Derivatives
Column : Shim-pack HR-ODS (250 × 3.0 mm, 3 μm)
Mobile phase : A: Water B: Acetonitrile
Elution program : 0.1 min, 30 % B; 13 min, 45 % B; 37 min, 70 %
B; 38 to 43 min, 85 % B; 43.1 min, 30 % B.
Flow rate : 0.40 mL/min
Detection : Ex 235 nm, Em 317 nm
Oven temp. : 30 ºC
Injection : 10 μL

UHPLC-RF Chromatogram of Mixed Standards of Thirteen
Bisphenols at Concentration of 100 μg/L Each

Table 2 Summary of UHPLC Method and Performance Evaluation Results for Analysis of Thirteen Bisphenols
Calibration range:
Ret Time RSD (%), n=6 Sensitivity **
ID # Name 5-2000 μg/L
(min)
R 2 Accuracy (%) * 5 μg/L 100 μg/L LOD (μg/L) LOQ (μg/L)
1 BFDGE-2H2O 7.9 0.9998 100.9 1.0 0.9 0.7 2.0
2 BADGE-2H2O 9.5 0.9991 98.7 0.4 0.4 0.4 1.2
3 BPF 13.4 0.9997 101.7 0.7 0.3 0.8 2.5
4 BPA 18.1 0.9997 101.2 0.8 0.3 1.0 3.1
5 BADGE-H2O-HCL 19.4 0.9998 101.5 0.5 0.3 0.5 1.5
6 BADGE-H2O 19.9 0.9997 103.2 0.3 0.3 0.5 1.5
7 BFDGE-2HCL-1 26.9 0.9996 98.8 0.6 0.1 1.5 4.6
8 BFDGE-2HCL-2 27.8 0.9997 100.8 2.3 0.2 1.2 3.6
9 BFDGE-1 28.9 0.9997 99.3 0.9 0.3 1.4 4.2
10 BFDGE-2 30.0 0.9997 101.3 1.2 0.4 1.6 4.7
11 BADGE-2HCL 31.8 0.9997 98.9 1.8 0.3 0.6 1.7
12 BADGE-HCL 33.0 0.9997 101.7 0.5 0.4 0.7 2.2
13 BADGE 34.3 0.9997 100.7 1.0 0.5 0.5 1.6
* : Average of 12 concentration levels 5-2000 μg/L
** : Estimated using 5 μg/L mixed stds data based on S/N=3 for LOD and S/N=10 for LOQ