Multi-Residue Pesticide Analysis in Ginseng Powder: Optimized Cleanup After QuEChERS Extraction for UPLC-MS/MS And GC-MS/MS Analysis

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1 Multi-Residue Pesticide Analysis in Ginseng Powder: Optimized Cleanup After QuEChERS Extraction for UPLC-MS/MS And GC-MS/MS Analysis Michael S. Young, Kim Van Tran, and Jeremy C. Shia Waters Corporation, Milford, MA, USA APPLICATION BENEFITS Rapid QuEChERS extraction of WATERS SOLUTIONS ACQUITY UPLC I-Class System XEVO TQ-S Mass Spectrometer APGC ginseng powder. Simple, fast dspe cleanup prior to UPLC -MS/MS analysis; cleaner extracts mean less time spent on routine instrument maintenance. Straightforward SPE cleanup for GC-MS/MS analysis for longer column life and less injection port maintenance. Atmospheric pressure ionization for GC-MS/MS (APGC). Excellent performance for both UPLC-MS and APGC-MS using the Xevo TQ-S Mass Spectrometer only 3 minutes to convert from LC to GC operation with no source venting required. ACQUITY UPLC BEH C 18 Column DisQuE products for QuEChERS extraction and dspe cleanup Sep-Pak PSA/Carbon cartridge cleanup for GC-MS LCMS Certified Vials MassLynx V4.1 data system with Quanpedia data base INTRODUCTION The QuEChERS methods have simplified and streamlined sample preparation for pesticide analysis. Although effective for fruits, vegetables and many other types of samples there are challenges when this technique is applied to dried commodities. This is the second in a series of application notes that illustrate sample preparation for dried commodities. The first application note discussed the analysis of dried tea, a highly resinous leaf material. 1 This application note presents QuEChERS extraction and SPE-based cleanup strategies for multi-residue pesticide analysis of American ginseng powder, a highly resinous root material commonly used as an herbal supplement. For the analysis of dried ginseng powder, the sample is first equilibrated with water before extraction using the DisQuE pouch for CEN QuEChERS. Aliquots are then taken for cleanup and chromatographic analysis; one aliquot is subjected to a dspe cleanup specific for determination of base/neutral pesticides by UPLC-MS/MS. A second aliquot is taken and subjected to an SPE cartridge cleanup optimized for determination of base/neutral pesticides by GC-MS/MS analysis. Recovery data for target pesticides extracted from ginseng powder will be presented using these cleanup protocols. KEY WORDS QuEChERS, pesticides, SPE, ginseng, UPLC-MS/MS, GC-MS/MS, APGC 1

2 EXPERIMENTAL UPLC conditions System: ACQUITY UPLC I-Class GC conditions Instrument: Agilent 789 Column: ACQUITY UPLC BEH C 18, Injection Volume: 5 µl 2.1 x mm, 1.7 µm Column: Injection volume: J&W DB MS 3 m x.25 mm x.25 µm 2 µl splitless Temperature: 45 C Mobile Phase A: 1 mm ammonium acetate in water (ph 5.) Mobile Phase B: 1 mm ammonium acetate in methanol Flowrate:.45 ml/min Gradient: 1 B initial and hold to.25 minutes, linear gradient to 99 B at minutes, hold to 13. minutes, back to 1 B at 13.1 minutes, hold and re-equilibrate until 17 minutes MS conditions for UPLC Instrument: Xevo TQ-S Mode: Electrospray positive and negative (ES+, ES-) Capillary: 3. kv Extractor: 3. V Source temp.: 15 C Cone gas: 15 L/hr Flowrate: 2. ml/min helium (constant flow) Temperature program: 8 C initial, hold for.5 min, 12 C/min to 3 C and hold for 1 min MS conditions for APGC Instrument: Xevo TQ-S Mode: API positive Corona: 2.2 µa Source temperature: 15 C Probe temperature: 45 C Cone gas: 17 L/hr Aux gas: 25 L/hr Nebulizer gas: 4. Bar Collision gas (Argon):.18 ml/min LC-MS/MS cone and collision parameters, as well as MRM transitions used for this study are presented in Table 1. GC-MS/MS cone and collision parameters and MRM transitions used for this study are presented in Table 2. Desolvation temp.: 5 C Desolvation gas: L/hr Collision gas (Argon):.18 ml/min 2

3 Sample preparation QuEChERS extraction Place 2 g ginseng powder and 1 ml reagent water into a 5 ml centrifuge tube. Let soak and equilibrate for 3 minutes. Add 1 ml acetonitrile, cap and vortex for 1 seconds and then shake well for a 1 minute. Add contents of DisQuE pouch for CEN QuEChERS and shake well for 1 minute. Centrifuge the sample at 4 RPM (rcf 325 x g) for 5 minutes and collect the supernatant. Aliquots of the supernatant are used for SPE cleanups. dspe cleanup for pesticides analysis by LC-MS Place 1 ml of QuEChERS extract into 2 ml DisQuE dspe tube (15 mg MgSO 4 /25 mg PSA/25 mg C 18, p/n ). Add 7 mg graphitized carbon black (GCB), vortex for 1 seconds and shake for 1 minute. Centrifuge the sample at 12 RPM (rcf 134 x g) for 4 minutes and collect the supernatant. Transfer 2 µl of supernatant to a LC-MS cetified vial and dilute to 1. ml with mobile phase A for LC-MS. SPE cleanup for pesticides analysis by GC-MS Dilute 1 ml of QuEChERS extract with 1 ml 3:1 acetone/toluene. Install a Sep-Pak PSA/carbon SPE cartridge on vacuum manifold with collection vessel in place. Place 2 mg anhydrous MgSO 4 atop the cartridge frit. Pass all of the diluted extract through cartridge and collect. Rinse the cartridge with 2 ml 3:1 acetone/toluene and collect (combine with pass-through fraction above). Evaporate to just below.5 ml, add 2 ml toluene and evaporate to.5 ml. 1 ppb spiked gingseng ging528_ ging528_ : MRM of 2 Channels ES > (Triazophos) 4.33e5 16: MRM of 2 Channels ES > (Triazophos) 1.59e Time Figure 1. LC-MS/MS analysis of ginseng, 1 ppb triazophos (transition for quantitation on top). Ginseng 1 ppb spk1 APGC_Ginseng_81213_ : MRM of 6 Channels API > (Diazinon) 2.15e5 RESULTS AND DISCUSSION Figure 1 shows a typical UPLC-MS/MS chromatogram obtained from a 1 ppb spiked sample of ginseng; the selected compound is triazophos. Figure 2 shows a typical APGC-MS/MS chromatogram obtained from a 1 ppb spiked sample of ginseng; the selected compound is diazinon APGC_Ginseng_81213_ : MRM of 6 Channels API > (Diazinon) 7.92e5 Time Figure 2. APGC-MS/MS analysis of ginseng, 1 ppb diazinon (transition for quantitation on top). 3

4 Recovery data for this study are presented in Tables 1 and 2 and were determined by comparison of the peak areas for samples spiked into the sample matrix prior to sample preparation with the peak areas for samples spiked after all sample preparation steps. UPLC-MS/MS (dspe Cleanup) Pesticide MRL ppb (EU) RT min MRM m/z (Cone V, Collision ev) Recovery ppb ( RSD) Acephate > (8,18) > 143. (8,8) 118 (39) 11 (8) Acetamiprid > 126. (3,2) 223. > 56.1 (3,15) 122 (6) 124 (5) Azoxystrobin > (2,25) 44.1 > (2,1) 11 (9) 14 (6) Boscalid > 37. (3,2) > (3,2) 15 (2) 18 (7) Carbaryl > 145 (3,1) 22. > 127 (3,26) 113 (13) 146 (4) Carfentrazone-ethyl > 346. (3,24) 412. > 266. (3,18) 91 (8) 134 (3) Chlorantraniliprole > 453. (35,2) 484. > (35,1) 84 (25) 16 (6) Clothianidin > 169. (3,12) 25. > 132. (3,17) 18 (33) 18 (7) Cyprodinil > 93. (4,33) 226. > 18. (4,3) 91 (16) 115 (6) Diazinon > 169. (3,22) 35.1 > 96.9 (3,35) 18 (7) 17 (8) DimethomorphE 5* > 3.9 (3,2) > 165. (3,4) 99 (12) 16 (7) DimethomorphZ 5* > 3.9 (3,2) > 165. (3,4) 98 (19) 97 (8) Fluopicolide > 173. (15,2) 385. > (15,2) 97 (15) 11 (2) Fenhexamid > 97.2 (32,22) 32.1 > 55.3 (32,38) 16 (11) 17 (8) Fenamidone > (3,16) > 92. (3,25) 112 (11) 13 (5) Imidacloprid > (3,19) > 29.1 (3,16) 92 (19) 116 (6) Metalaxyl > 22.1 (3,13) 28.1 > (3,17) 19 (6) 112 (4) Novaluron > 158. (3,19) 493 > 141. (3,4) 79 (14) 16 (6) Pyrethrins > (2,15) > (2,15) 81 (29) 125 (1) Pyrimethanil > 17. (42,24) 2. > 82. (42,24) 19 (4) 12 (4) Pyriproxifen > 96. (3,14) > (3,14) 115 (6) 16 (6) Pyroclostrobin > 163. (3,25) > (3,12) 96 (11) 95 (7) Sethoxydim > (15,2) > (15,15) 11 (12) 84 (5) Spinosad A > 142. (4,5) > 98.1 (4,5) 15 (12) 11 (8) Spinosad D > 142. (4,5) > 98.1 (4,5) 88 (22) 91 (5) Thiamethoxam 2 (2US) > 181. (3,22) 292. > 211. (3,13) 114 (22) 11 (1) Triazophos > (3,18) > (3,35) 16 (6) 11 (5) Trifloxystrobin > 186. (3,16) 49. > 145. (3,4) 96 (4) 97 (4) * sum of isomers Table 1. UPLC-MS/MS recovery data 4

5 APGC-MS/MS (PSA/Carbon Cartridge Cleanup) Pesticide MRL ppb (EU) RT min MRM m/z (Cone V, Collision ev) Recovery ppb ( RSD) Acephate > 94.8 (1,2) > (1,1) 66 (6) 62 (5) Bifenthrin > (2,1) > (2,1) 86 (2) 122 (15) Carfentrazone ethyl > (2,3) > 31.8 (2,3) 85 (8) 74 (7) Chlorpyrifos methyl > (35,2) > (35,1) 73 (4) 77 (7) Chlorfenapyr > 27.8 (2,2) 48.7 > (2,1) 74 (6) 82 (5) Diazinon > (2,2) 34.9 > (2,1) 88 (6) 81 (5) Deltamethrin > (2,2) 55.6 > 28.7 (2,1) LOQ 72 (9) Fenvalerate > (1,4) > (1,1) 61 (4) 75 (9) L-Cyhalothrin > (15,2) > (15,1) 77 (8) 77 (16) Propetamphos (US) > (1,2) > (1,1) 83 (13) 79 (9) Pyriproxyfen > 95.8 (1,2) > (1,2) 79 (11) 76 (8) Phenothrin > (2,4) 35.9 > (2,2) 77 (8) 88 (9) Resmethrin > 17.9 (25,1) > (25,1) 52 (9) 56 (1) Trifluralin > (3,1) > (3,2) 81 (6) 79 (7) Table 2. APGC-MS/MS recovery data A modified QuEChERS method was used for this study. The ginseng powder sample is first mixed with water and allowed to equilibrate before the addition of acetonitrile and QuEChERS salts. This procedure was shown to be effective for extraction of a wide range of pesticide residues from ginseng. An aliquot of the QuEChERS extract was cleaned up using dspe prior to LC-MS analysis. Figure 3 shows the cleanup obtained; most of the colored matrix compounds are removed and therefore will not be deposited on the cone. Another aliquot of the QuEChERS extract was cleaned up using an SPE cartridge (carbon/psa) prior to GC-MS analysis. Without the SPE cleanup, only a few sampled could be analyzed by GC-MS before injection port and column maintenance was required; after SPE cleanup hundreds of samples were analyzed without any routine maintenance. Figure 3. QuEChERS extracts obtained from powdered ginseng; without dspe cleanup (left) and with dspe cleanup (right) 5

6 CONCLUSIONS The modified QuEChERS approach was effective for recovery of a wide range of pesticides from ginseng powder. dspe provided cleanup suitable for UPLC-MS/MS analysis. Reference 1. Multi-Residue Pesticide Analysis in Tea: Optimized Cleanup After QuEChERS Extraction for UPLC-MS/MS and GC-MS/MS Analysis, Waters Application Note EN. A PSA/carbon cartridge based SPE cleanup was highly effective for GC-MS/MS analysis using APGC-MS. One Xevo TQ-S Mass Spectrometer can be shared to provide excellent performance for both UPLC-MS and APGC-MS. Only 3 minutes are required to convert from LC to GC interface. No venting of the mass spectrometer is required for the conversion. Waters, The Science of What s Possible, ACQUITY UPLC, ACQUITY, UPLC, Sep-Pak, MassLynx, and Xevo are registered trademarks of Waters Corporation. DisQue and Quanpedia are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. 214 Waters Corporation. Produced in the U.S.A. March EN AG-PDF Waters Corporation 34 Maple Street Milford, MA 1757 U.S.A. T: F: