一次性筷子中抗菌剂检测方案(液相色谱仪)

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Technical ReportAS/LC-050 High-performance Liquid Chromatograph Simultaneous Analysis of Fungicides (OPP， TBZ， DP) in Disposable Chopsticks Ortho-phenyl phenol (OPP)， thiabendazole (TBZ)， and diphenyl (DP) are classified as foodadditives (fungicides) for citrus fruits， etc.， and their residual amounts in food products areregulated. In 1994， OPP was detected from disposable chopsticks. As a result， the Ministry ofHealth Labor and Welfare (MHLW) specified that the amounts of OPP， TBZ， and DP indisposable chopsticks must be“not detectable”in Notice No. 1113002 of the Inspection andSafety Division， Department of Food Safety， and No. 1113002 of the Standard and EvaluationDivision， Department of Food Safety (November 13，2007). This time， based on the above issued notices， OPP， TBZ， and DP in disposable chopstickswere simultaneously analyzed. By using the HPLC system with the pressure resistance of60 MPa， the general-purpose analysis in accordance with the test method and the high-speedanalysis with a UHPLC column were performed， and the results were introduced here. High-performanceLiquid ChromatographChromaster@ General-purpose Analysis of OPP， TBZ， and DP Standard Solutions √/The above notices state that the limit for OPP， TBZ， and DP for a pair of disposable chopsticks is “not detectable”.The quantitation limit is 0.05 pg/mL (0.002 mg per pair) for OPP and TBZ and 0.5 pg/mL (0.02 mg per pair) for DP. The standard stock solutions are prepared by weighing 10 mg each of OPP， TBZ， and DP and making up the volume to100 mL with methanol (100 pg/mL each). To 10 mL each of these standard stock solutions， the mobile phase is addedto make up the volume to 100 mL (10 pg/mL each). The standard solutions for the calibration curve are prepared bydiluting this mixed solution appropriately with the mobile phase. Concentration (ug/mL) Figure 1 Chromatogram of Standard Solution (0.5 pg/mL) Figure 2 Calibration Curve Table 1 Analytical Conditions of General-purpose Analysis Column LaChrom II C18(5 pm) 4.6 mmI.D.×250 mm Mobile phase CH3CN/CH3OH/H20=5/60/35 (v/v) addition of 10 mmol/L SDSpH 2.7 (adjust with phosphoric acid) Flow rate 1.0 mL/min Column temperature 40°C Detector FL Excitation wavelength： 285 nm Fluorescence wavelength： 325 nmResponse Time： 1s Sampling Period： 200 ms Injection vol. 20pL √/The calibration curves for OPP and TBZ were prepared for the concentration range of 0.005 -10 pg/mLwhile the curve for DP was prepared for the range of 0.05-10 pg/mL. The correlation coefficient (R2)was 1.0000 for all of them， indicating a good linearity (Figure 2). General-purpose Analysis of OPP， TBZ， and DP in Disposable Chopsticks √/By following the procedure described in the notices (Figure 3)， OPP， TBZ，and DP were extracted from disposablebamboo and wood chopsticks， and the sample solutions were prepared. √/The formula to calculate the concentrations of OPP， TBZ， and DP for a pair of disposablechopsticks is shown. Dissolution amount per pair (mg) =Concentration of OPP， TBZ， DP in sample solution (ug/mL) x Solution volume (20 mL)x2/ 1000 Figure 3 Extraction Method for OPP， TBZ， and DP in Disposable Chopsticks Figure 4-1 Chromatogram ofDisposable Bamboo Chopstick Sample Solution Figure 4-2 Chromatogram of Disposable Wood Chopstick Sample Solution Table 2 Quantitative Analysis Results Sample Solution with Standard Addition Addition concentration(ug/mL(mg)) Sample solutionconcentration(ug/mL) Content(mg) Recoveryrate(%) Disposablebamboochopsticks OPPTBZ DP 0.05 (0.002)0.05 (0.002)0.50 (0.020) 0.050 0.050 0.478 0.0020.0020.019 100.0100.095.7 Disposablewood chopsticks OPPTBZDP 0.05 (0.002)0.05 (0.002)0.50 (0.020) 0.0490.049 0.471 0.0020.0020.019 98.098.094.1 v The fungicides were not detected from the disposable chopstick samples (Figure 4). High-speed Analysis of OPP， TBZ， and DP Standard Solutions √The 4.6 mm l.D. x250 mm column with 5 pm particle for HPLC was changed to the 3.0 mm I.D. x100 mmcolumn with 1.9 um particle for UHPLC and the high-speed analysis of OPP， TBZ， and DP was performed. √In high-speed analyses， the sample will diffuse if system volumes including those in the tube and detectorflow cell are large， resulting in the reduced resolution capability and poor peak shapes. This time， the lowdiffusion tube kit (P/N 892-0820) and the flow cell for UHPLC 6440 FL detector (P/N 889-0861) were used forthe analysis. The response time and sampling period， the set conditions for the detector， were also changed to thesettings suitable for the high-speed analysis. 1.0E+07 Figure 6 Calibration Curve Table 3 Analytical Conditions of High-speed Analysis Figure 5 Comparison of General-purpose Analysisand High-speed Analysis(0.5ug/mL Standard solution) Column LaChromUltra ll C18 (1.9 pm)3.0 mm l.D.×100 mm Mobilephase CH3CN/CH3OH/H20=5/60/35 (v/v)Addition of 10 mmol/L SDS pH 2.7 (adjust with phosphoric acid) Flow rate 0.4 mL/min Columntemperature 40°C Detector FL Excitation wavelength： 285 nm Fluorescence wavelength： 325 nm Flow cell for 6440 FL detector is usedResponse Time： 0.05 s Sampling Period： 50 ms 10 pL Injection vol. 10 pL √/By changing to the high-speed analysis from the general-purpose analysis， the analysis time for eachanalytical run was reduced to 1/2 and the mobile phase consumption was reduced to 1/5. As the systemvolume is also extremely small， a high number of theoretical of plates (N) was obtained even by thehigh-speed analysis (Figure 5). √/The correlation coefficient (R2) for the calibration curve of the each component was 1.0000 even for thehigh-speed analysis， showing the linearity equivalent to that of the general-purpose analysis (Figure 6). High-speed Analysis of OPP， TBZ， DP in Disposable Chopsticks Figure 7-1 Chromatogram of Disposable BambooChopstick Sample Solution Figure 7-2 Chromatogram of Disposable WoodenChopstick Sample Solution Table 4 Quantitative Analysis Results Sample Solution with Standard Addition Addition concentration (ug/mL(mg)) Sample solutionconcentration(ug/mL) Content(mg) Recovery rate (%) Disposablebamboochopsticks OPPTBZDP 0.05(0.002)0.05 (0.002)0.50 (0.020) 0.048 0.0020.002 96.694.3 93.9 0.046 0.461 0.180 Disposablewoodchopsticks OPPTBZ 0.05 (0.002) 0.0480.048 0.0020.0020.019 96.098.094.9 DP 0.05 (0.002)0.50(0.020) 0.466 √'In the high-speed analysis of OPP， TBZ， and DP in disposable chopsticks， it was possible to obtainthe quantitative analysis results equivalent to those by the general-purpose analysis (Figure 7， Table4).√The system with the pressure resistance of 60 MPa is compatible with the high-speed analysis usinga UHPLC column， and the shorter analysis time can also result in the reduced mobile phaseconsumption. Page. √('OPP，TBZ， and DP were added at the concentrations of the quantitation limits and the recovery rates weredetermined. Good results were obtained with the rates of and for bamboo chopsticksand wooden chopsticks， respectively (Figure ， Table ).Page.