GC/MS/MS Analysis of Thiabendazole in Grapefruit Extracts Varian Application Note Number 30 Steve Schachterle, Varian Chromatography Systems Uri Golner and Irina Tannenzapf, Ministry of Agriculture, Israel Key Words: Saturn 4D, MS/MS, Pesticide, Wave~Board Introduction The analysis of grapefruit for thiabendazole (TBZ) is an economically important analysis. The fungicide is applied as a postharvest treatment in the citrus packing houses. Generally the maximum regulatory levels are high (up to 10 ppm), but certain legislations consider TBZ a food additive when it comes to processed fruit (e.g. juices) and as such the residue level must not exceed 10 ppb. The analysis necessary to assure this is indeed a challenging one due to the complexity of the grapefruit background matrix. Matrix TBZ Discussion Conventional GC/MS does not always provide unambiguous results because the mass spectrum of a coeluting matrix component may have ions which coincide with those from TBZ at a concentration far above the desired detection limit for TBZ. Figure 1 shows the GC/MS chromatogram for TBZ in a grapefruit extract at the 500 ppb level. The inset spectra are for the matrix component and for TBZ. Note that the matrix component has ions at m/z 201 and 174, the two main ions for TBZ. Due to the partial chromatographic separation, it is not possible to measure very low levels of TBZ in the presence of this interfering component. Figure 2 shows the GC/MS/MS chromatogram for TBZ in the grapefruit extract at the 10 ppb level. The background from the matrix peaks does not appear in the mass chromatogram for m/z 174. Figure 3 compares the GC/MS total ion chromatogram with the GC/MS/MS total ion chromatogram. Note that the GC/MS/MS total ion chromatogram still contains several peaks; although it should be noted the level is 20 times lower than the GC/MS total ion chromatogram. Any ion at m/z 201 that dissociates under the excitation conditions will produce ions in the MS/MS chromatogram. Figure 1: Mass Chromatogram showing 500 ppb of TBZ in Grapefruit by GC/MS. Figure 2: 10 ppb of TBZ in Grapefruit by GC/MS/MS NOTICE: Varian, Inc. was acquired by Agilent Technologies in May 2010. This document is provided as a courtesy but is no longer kept current and thus will contain historical references to Varian. For more information, go to www.agilent.com/chem. This includes both molecular ions and fragment ions produced in the EI ionization process. The real advantage of MS/MS is that now the m/z 174 mass chromatogram shows only those m/z 174 ions which were formed by CID from m/z 201 and not those formed by electron ionization as seen in the GC/MS chromatogram. Figure 4 shows an external standard calibration curve prepared by spiking the grapefruit extract. The linearity is excellent with a wide dynamic range, even in the presence of the complex matrix. Instrument Conditions Gas Chromatograph Column: DB5-ms, 30m x 0.25 mm ID x 1.0 µm film thickness Flow Rate: 0.8 ml/min measured at 200°C Oven Program: 50°C for 1 minute. Program at 10°C/min to 275°C. Hold 13.5 minutes. Injector: SPI with high performance insert. 25°C no hold. Program at 200°C/min to 230°C. Hold until the end of the run. Injection Volume: 1 µl Injection Speed: 1µl/sec Mass Spectrometer Experimental The grapefruit concentrate was prepared for analysis through a modified Luke procedure (FDA-PAM 232.4) by a robotic sample preparation system. The extract contained 1 gram of soluble grapefruit in a final volume of 1 ml. After filtration, the acetone extract was evaporated with helium and reconstituted with methylene chloride. Mass Range Scan/sec Mult. delay Target Threshold Filament RF level Ion Trap Temperature Excitation voltage Excitation time MS 50-210 1 180 sec 25,000 1 20 µA 300 dacs 270°C - MS/MS 50-210 1 180 sec 5,000 1 80 µA 450 dacs 270°C 61 volts 40 ms Conclusion GC coupled with tandem mass spectrometry (MS/MS) using an ion trap can be used to identify and quantitate TBZ at low ppb levels, even in the presence of interfering components. Compared with standard GC/MS, the selectivity provided by the additional MS step effectively eliminates the response from the interfering component and lowers the detection limit dramatically. Figure 3: Total ion chromatograms of 500 ppb spike by GC/MS (top) and GC/MS/MS (bottom). Figure 4: GC/MS/MS External Standard Calibration Curve Prepared by Spiking the Grapefruit Extract. These data represent typical results. For further information, contact your local Varian Sales Office. GC/MS30:0694
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