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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation INTRODUCTION GC-MS and LC-MS/MS are the two common techniques for pesticide residue analysis. Due to the wide range of physiochemical properties of pesticides, two separate analyses by GC-MS and LC-MS/MS are often required to cover multiple pesticides of interest. In general, LC is favored for polar, less volatile compounds, while GC is preferred for volatile, thermally-stable ones, and also for pesticides that do not ionize well in atmospheric pressure ionization. On the other hand, GC needs derivitization and extensive sample clean-up, while LC-MS/MS is prone to matrix co-eluting issues, either ion suppression or isobaric interference from matrix. UltraPerformance Convergence Chromatography (UPC 2® ) provides an alternative approach for pesticde residue analysis. UPC 2 uses compressed carbon dioxide, in supercritical state or sub-supercritical state, as its primary mobile phase, sub-two micron particles as the support of stationary phases, and cutting-edge chromatography system as the separation platform. The non polar nature of the neat carbon dioxide can be altered with polar solvents, and achieves separation for both highly nonpolar and polar compounds with unique selectivity. In addition, with the use of different stationary chemistries, its selectivity can be altered dramatically. These render UPC 2 a potential powerful tool for the separation of multiple pesticides with wide range of physiochemical properties. Recently, both GC and LC amenable pesticides with wide range of polarity have been successfully analyzed on a single SFC-MS/MS system in a single injection (1). The aim of this work is to demonstrate the capability of UPC 2 in simultaneous analysis of multiple pesticides with wide range of properties, and to explore the unique benefits of UPC 2 in multiple pesticide residue analysis. ANALYSIS OF MULTIPLE PESTICIDES BY SFC ANALYSIS OF MULTIPLE PESTICIDES BY SFC - - MS/MS WITH SUB MS/MS WITH SUB - - 2 MICR 2 MICR ON PARTICLE COLUMNS ON PARTICLE COLUMNS - - A FEASIBILITY STUD A FEASIBILITY STUD Y Y Jinchuan Yang, Brian Tyler, Joseph P. Romano, Jennifer A. Burgess Waters corporation, 34 Maple St, Milford, MA 01757, U.S.A. References 1.M. Ishibashi, T. Ando, M. Sakai, A. Matsubara, T. Uchikata, E. Fukusaki, T. Bamba, J. Chromatogra. A, 1266 (2012) 143–148 RESULTS Optimization of the make-up solvent The effects of make-up solvent flow rate, water concentration, and acidicity or basicity of make-up solvent on the MS peak area of 18 pesticides were investigated using 100ppb pesticides mixture solvent solution. The results of those investigation are shown in Fig. 1 to Fig. 3. Linearity and LOD The linearity of the MS response was investigated using both the spinach matched pesticide standard solutions and the solvent standard solutions with pesticide concentrations from 0.001ppb to 100ppb. The LODs were also estimated during the linearity study. The results are presented in Table 3. Selectivity The selectivity in UPC 2 separation can be easily changed by using different stationary phases. Fig. 4 is a comparison of separation of 17 pesticides on two different columns, HSS C18 SB column and BEH 2-EP column. Table 3. LODs and calibration curve linearity of 18 pesticides in solvent and in spinach extract CONCLUSION UPC 2 can simultaneously analyze residues of pesticides of wide range of polarity and MW. UPC 2 offers a solution for a high-throughput pesticide residue analysis since both GC amenable and LC amenable pesticides can be simultaneously analysis on a single system. Coupling of Xevo TQ-S MS with UPC 2 provides excellent detection sensitivity that meet stringent regulations. The selectivity in UPC 2 separation can be conveniently changed with different stationary phases using the same mobile phase, which provides a solution to co-eluting issues. Additional investigation is needed on the reproducibility and robustness of this technique. METHODS Pesticides standards: Eighteen pesticides having different basic chemical structures, wide range of polarities, and molecular weights were chosen in this study. Table 1 lists their formula, MWs, log P ow , and common analysis techniques. Spinach matrix extract: Waters DisQuE™ kit was used and AOAC QuEChERS protocol was followed in the preparation of spinach extract. Details of the procedure can be found in DisQuE Care and Use Manual (Waters Lit. 715001888). Standard solutions: Spinach matrix matched pesticide standard solutions were prepared by mixing 800μL acetone water (3/1 v/v) mixture, 100μL matrix extract, and 100μL pesticide standard stock solution at certain concentrations. The pesticide standard stock solutions were prepared by serial dilution of 1mg/ mL pesticide solutions with acetone water (3/1) mixture to certain concentrations. UPC 2 Conditions: Column: ACQUITY ® UPC 2 BEH-2EP, 3.0x100mm, 1.7μm Column Temp: 65 o C Sample Temp: 4 o C Injection vol.: 2μL Flow rate: 1.5mL/min Back pressure: 1500 psi Mobile phase A: CO 2 Mobile phase B: MeOH with 10mM ammonium formate Gradient: Initial hold at 100%CO 2 for 2 minutes, linear gradient from 0% to 10% modifier B in 5 minute, then a second gradient from 10% to 45% modifier B in 2 minute, and hold at 45% B for 1 minute before return to initial condition. Make-up solvent: MeOH + 1%H 2 O + 0.1% NH 4 OH Make-up flow rate: 0.2mL/min Xevo ® TQ-S MS Conditions: Capillary voltage, 3000 V; source temperature, 150 ◦C; desolvation temperature, 600 ◦C; cone gas flow rate, 150 L/h; desolvation gas flow rate, 1000 L/h; collision gas flow rate, 0.15 mL/min. The dwell time: auto (6 to 34 ms). MRM transitions were optimized by combining the infusion of pesticide standard solutions with the make-up solvent at 200μL/min. Table 2 shows the optimized MRM transition parameters for the 18 pesticides. Figure 2. Effects of addition of trace water in make-up solvent on peak area. Data shown are normalized to the peak areas obtained without any water added to make-up solvent (methanol). All other conditions are the same. Figure 1. Effect of make-up solvent flow rate on peak areas of 18 pesticide standards. All data are normalized to the peak areas obtained at 0.5mL/min make-up flow rate. All other conditions are the same. Figure 3. Effects of addition of acid (0.1% acetic acid) or base (0.1% ammo- nium hydroxide) in make-up solvent on peak area. Data shown are normal- ized to the peak areas obtained without any acid or base (methanol with 1% water). All other conditions are the same. No. Pesticide Formula MW Monoisotopic MW log P ow Analysis Technique 1 1 Diquat dibromide C12H12Br2N2 344 341.9367 -4.6 IC or IP-LC 2 Fosetyl C6H18O9P3Al 354 353.9979 -2.7 IC 3 Maleic hydrazide C4H4N2O2 112 112.0273 -1.96 LC 4 Methamidophos C2H8NO2PS 141 141.0013 -0.8 GC 5 Methomyl C5H10N2O2S 162 162.0463 0.09 LC 6 Acetamiprid C10H11ClN4 223 222.0672 0.8 LC, GC 7 Carbendazim C9H9N3O2 191 191.0695 1.51 LC 8 Azinphos-methyl C10H12N3O3PS2 317 317 2.75 GC 9 Phosmet C11H12NO4PS2 317 317 2.78 GC 10 Thifluzamide C13H6Br2F6N2O2S 528 525.8421 3.56 GC 11 Tralomethrin C22H19Br4NO3 665 660.8098 5.05 GC 12 Amitraz C19H23N3 293 293.2 5.5 LC, GC 13 Emamectin benzoate(B1a) C49H77NO13 888 887.5395 5.76 LC 14 Chlorfluazuron C20H9Cl3F5N3O3 541 538.963 5.8 LC 15 Acequinocyl C24H32O4 385 384.2301 6.2 LC 16 Pyridaben C19H25ClN2OS 365 364.1376 6.37 GC 17 Cypermethrin C22H19Cl2NO3 416 415.0742 6.6 GC, LC 18 Etofenprox C25H28O3 377 376.2038 7.05 LC Note: 1. Analysis technique that is commonly used for pesticide is listed. Compound Ionization mode Parent ion [m/z] Cone Voltage [V] Daughter ion [m/z] Collision Energy [eV] Daughter ion [m/z] Collision Energy [eV] 1 Diquat Dibromide ESI+ 183 25 157 18 168.2 15 2 Fosetyl Alumium ESI- 109.1 20 81 10 63 15 3 Maleic Hydrazine ESI+ 113 20 85 10 67 15 4 Methamidaphos ESI+ 142 30 93.9 12 124.9 9 5 Methomyl ESI+ 185.1 20 64 8 128 6 6 Acetamiprid ESI+ 223 20 125.9 20 56 12 7 Carbendazim ESI+ 192 20 159.9 15 131.9 27 8 Azinphos-methyl ESI+ 339.9 15 132 14 159.9 10 9 Phosmet ESI+ 317.9 18 160 10 132.9 35 10 Thifluzamide ESI+ 528.7 20 488.7 25 167.9 24 11 Tralomethrin ESI+ 682.6 25 440.6 12 276.7 30 12 Amitraz ESI+ 294.1 15 163.1 13 253.1 13 13 Emamectin Benzoate ESI+ 886.3 50 158 30 82 80 14 Chlorfluazuron ESI+ 539.8 40 382.9 25 158 15 15 Acequinocyl ESI+ 357 20 329.1 15 203 15 16 Pyridaben ESI+ 365 20 309 8 147 20 17 Cypermethrin ESI+ 432.9 20 190.9 10 127 30 18 Etofenprox ESI+ 394.1 25 359 8 177 13 MRM transition 1 MRM transition 2 Pesticide RT (min) LOD (ppb) Linear Range (ppb) R 2 LOD (ppb) Linear Range (ppb) R 2 1 Diquat Dibromide 9.220 0.500 1 - 100 0.996 0.500 1 - 100 0.996 2 Fosetyl Alumium 8.540 2.500 2.5 - 100 0.989 5.000 10 - 100 0.976 3 Maleic Hydrazine 8.100 5.000 10 - 100 0.997 10.000 25 - 100 0.996 4 Methamidaphos 5.180 0.025 0.100 - 100 0.998 0.050 0.050 - 100 0.997 5 Methomyl 5.470 2.500 5 - 100 0.991 2.500 5 - 100 0.990 6 Acetamiprid 7.990 0.025 0.050 - 100 0.996 0.050 0.050 - 100 0.997 7 Carbendazim 5.430 0.100 0.250 - 100 0.998 0.100 0.250 - 100 0.999 8 Azinphos-methyl 5.700 0.050 0.100 - 100 0.998 0.050 0.100 - 100 0.997 9 Phosmet 5.420 0.100 0.100 - 100 0.998 0.100 0.100 - 100 0.998 10 Thifluzamide 6.930 0.005 0.010 - 100 0.997 0.025 0.025 - 100 0.998 11 Tralomethrin 6.55, 6.99, 7.44 0.500 1 - 100 0.995 0.500 1 - 100 0.997 12 Amitraz 1 4.250 - - - - - - 13 Emamectin Benzoate 8.490 0.050 0.100 - 100 0.993 0.050 0.100 - 100 0.991 14 Chlorfluazuron 7.860 0.025 0.050 - 100 0.995 0.025 0.050 - 100 0.998 15 Acequinocyl 6.260 0.010 0.100 - 100 0.995 0.050 0.050 - 100 0.998 16 Pyridaben 5.220 0.025 0.100 - 100 0.997 0.025 0.100 - 100 0.997 17 Cypermethrin 4.86,5.06 0.100 0.250 - 100 0.997 N/A 2 6.8 - 104 2 0.998 18 Etofenprox 4.870 0.050 0.100 - 100 0.998 0.050 0.100 - 100 0.998 1: Data not available due to experimental error 2: 43ppb residue cypermethrin was found in blank spinach extract Solvent Spinach Figure 4. Comparison of separation of pesticides in UPC 2 on different columns (HSS C18 SB column vs BEH 2-EP column). The same mobile phase elution gradient was used for both columns. Pesticide standard solvent solutions were prepared by mixing 100μL pesticide stock solution with 800μL acetone water (3/1) mixture and 100μL MeCN with 1% acetic acid. Table 1. List of pesticides in this study UPC 2 -MS/MS Analysis: Waters UPC 2 system was coupled with Xevo TQ-S MS. A splitter for MS was used to incorporate make-up solvent and split eluent to MS probe before the pressure regulator. MassLynx 4.1 was used for instrument control and for data process. Table 2. MRM Transition parameters for the 18 pesticides.
