Targeted screening and quantitation of pesticide residues in green tea using a Quadrupole Time-of-Flight Mass Spectrometer Huan Lin 1 , Toshiya Matsubara 1 , Natsuyo Asano 1 , Mikie Shima 2 1 SHIMADZU Corporation, Kyoto, Japan 2 AISTI SCIENCE, Wakayama, Japan 2. Introduction With population growth and the development of agriculture and food industries, food safety is becoming a greater concern in recent years. The increasingly stricter regulations of residual food contaminants requires faster and more accurate analytic techniques. For this purpose, targeted screening by simultaneous MRM measurements using a triple quadrupole mass spectrometer is the most common strategy. Data acquisition using a high-resolution and accurate mass spectrometer (HRMS), such as a Time-of-Flight, is also carried out coupled with MRM-based analysis for the purpose of guaranteeing the robustness and reliability of screening. In addition, HRMS also provides a different advantage from MRM measurement because a compound which was not included as a target when acquiring data can be processed later without reacquiring data. Compounds in green tea samples were extracted with QuEChERS extraction after grinding with cryogenic milling (Figure 2). The resulted acetonitrile layers were collected and purified by a fully automated solid phase extraction system (ST-400; Aisti Science, Wakayama, Japan). Pesticide mixtures or green tea extracts were analyzed using a Quadrupole Time-of-Flight Mass Spectrometer (LCMS-9030; Shimadzu Corporation, Kyoto, Japan) coupled with conventional flow liquid chromatography (Nexera X2; SHIMADZU). LC separation was performed using a Raptor Biphenyl (2.1 mm × 100 mm, RESTEK) with binary gradient of 2 mmol/L ammonium formate + 0.002% formic acid in water and 2 mmol/L ammonium formate + 0.002% formic acid in methanol. Assignments with MS/MS spectra and predicted fragments from a molecular structure are performed using a third party’s software, ACD/MS Workbook Suite (ACD/Labs, Toronto, Canada). 3. Methods TP 239 Figure 1 LCMS-9030 quadrupole time-of-flight mass spectrometer High-Resolution and Accurate Mass Spectrometer Resolution power: > 30,000 FWHM at m/z 1,972 / 1,626 Mass accuracy: 1 ppm Maximum acquisition rate: 100 Hz Minimum polarity switching rate: 1 second 1. Overview The purpose of this study is to screen and identify pesticide chemicals in green tea. Compounds from green tea were extracted with QuEChERS method and analyzed with MS/MS measurement using a LC/Q-TOF-MS. 4. Results 4-1. Analysis conditions for pesticides We prepared a mixed standard sample of 157 pesticide compounds frequently used in local tea agriculture. The theoretical m/z of each chemical was calculated and listed up. Mass chromatogram was confirmed by a mass error tolerance of ± 10ppm. The retention time of each chemical was recorded by injection of standard sample by using the following instrument parameters. UHPLC(Nexera X2) Analytical Column: Restek Raptor™ Biphenyl (2.1 mmI.D. x 100 mmL., 2.7 µm) Mobile Phase A: 2 mmol/L Ammonium formate + 0.002% Formic acid – Water Mobile Phase B: 2 mmol/L Ammonium formate + 0.002% Formic acid – Methanol Gradient Program: 3%B (0 min) – 10%B (1 min) – 55%B (3 min) – 100%B (10.5 – 12 min) – 3%B (12.01 -15 min) Flow Rate: 0.4 mL/min Column Temperature: 35 ºC Injection Volume: 10 μL MS (LCMS-9030) Ionization: ESI positive Acquisition mode MS Nebulizing Gas Flow: 3 L/min Drying Gas Flow: 10 L/min Heating Gas Flow: 10 L/min Interface Temperature: 350 ºC DL Temperature: 150 ºC HB Temperature: 300 ºC TOF range: m/z 120 - 1000 Event Time: 0.4 s (Pulser inj.: 794) ID off Figure 3 shows MRM chromatograms of 157 pesticide chemicals. It took 12 minutes per LC-MS/MS analysis. Excellent separation and high sensitivity detection were obtained. Figure 3 Trace amounts of 157 pesticides (10 ppb) were quantified in one run of LCMS analysis with their fragmental MS/MS information. All peaks were extracted by theoretical m/z (± 10 ppm) from one full scan. 4-3. Identification of pesticides by structural information from MS/MS fragments. 4-2. Calibration curve for quantitative analysis Calibration curves for quantitative analysis are generated using the green tea extracts spiked with pesticide mixture. Among 157 pesticides, flufenoxuron and boscalid, were detected from the green tea extracts, estimated concentrations as 0.142 ppb and 0.231 ppb, respectively. As a result of formula prediction, the composition of corresponding peak on the MS spectrum completely agree with that of flufenoxuron and boscalid with the top score. Figure 6 Quantitative result of pesticide residues in green tea Flufenoxuron Boscalid In order to confirm the validity of the identification results, we further performed structural analysis by MS/MS acquisition of the corresponding peak. All of the major MS/MS fragments were assigned to structural fragments of flufenoxuron and boscalid with accuracies of 1 mDa or less. Figure 7 Accurate MS/MS fragments help identify flufenoxuron and boscalid; MS/MS spectra (left), fragmentation patterns and mass error (right) 5. Conclusions According to the official guideline of CODEX, and Japanese Ministry of Health, residual concentrations quantified here is far below the criteria defined for them. Thus, our results indicate that LCMS-9030 system successfully achieves robust and reliable residual screening in foods by coexistence with both high-sensitivity quantitation and qualification with high mass accuracy. Water 0.5 mL Smart-SPE C18-50 mg+PSA-30 mg: Purification eluate Fractionation 0.5 mL Smart-SPE C18-30 mg: Purification Elution MeCN – Water (8/2) 0.5 mL eluate Constant volume (2 mL, Water) Homogenization (13000 rpm, 1min.) NaCl 1 g MgSO 4 (anhydrous) 4 g TSCD* 1 g Shaking (1 min.) Centrifugation (3500 rpm, 5 min.) DHS** 0.5 g Green tea 2 g MeCN layer MeCN 10 mL Add 2ppm of mixed STDs 50 μL Precooling cryogenic grinding with dry ice Elution 2% FA in MeCN 0.5mL Extraction QuEChERS STQ - LC Purification Automatic SPE device ST-L400 Automatic SPE device ST-L400 *TSCD: trisodium citrate dihydrate **DHS: disodium hydrogen citrate sesquihydrate Figure 2. Sample preparation of pesticides in green tea Flufenoxuron Δ 0.18 ppm 475.0 480.0 485.0 490.0 495.0 500.0 505.0 m/z 0 500 1000 1500 2000 2500 Inten. 489.0435 MS spectrum Blank Green tea 8.0 9.0 10.0 11.0 0 25 0 50 0 75 0 100 0 125 0 150 0 175 0 XIC: 489.0435(+) 0 250 500 750 1000 1250 1500 1750 8.0 9.0 10.0 11.0 XIC: 489.0435(+) Boscalid Blank Green tea 6.0 7.0 8.0 9.0 0 250 500 750 1000 1250 1500 1750 2000 2250 XIC: 343.0399(+) 6.0 7.0 8.0 9.0 0 250 500 750 1000 1250 1500 1750 2000 2250 XIC: 343.0399(+) 335.0 337.5 340.0 342.5 345.0 347.5 350.0 352.5m/z 0 500 1000 1500 2000 2500 3000 3500 4000 Inten. 343.0397 Δ 0.74 ppm Screening software: LabSolutions Insight (Quantitation view) Residual compounds 1. Summary table (Compound quantitation results) 3. Calibration 2. Chromatogram Figure 4 Screening result of pesticide residues in green tea 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 min 0 300000 600000 900000 1200000 1500000 1800000 2100000 2400000 TIC (x 0.25) XIC C21H11N2O3F6Cl Flufenoxuron O NH O NH O Cl F F F F F F Δ 0.1778 ppm, Score 98.77 Measured spectrum Theoretical spectrum Error Margin = 1 ppm Figure 5 Example of formula prediction (LabSolutions Insight Explore) Element table Name Min Max Carbon 0 150 Hydrogen 0 300 Nitrogen 0 12 Oxygen 0 12 Fluorine 0 12 Sulphur 0 12 Chlorine 0 12 # of repetition Area RT (min) Conc. (ppb) 1 7,958 9.051 0.151 2 7,632 9.049 0.143 3 7,188 9.050 0.133 4 7,569 9.051 0.142 Average 7,587 9.050 0.142 CV (%) 3.605 0.009 4.484 Quantitative results # of repetition Area RT (min) Conc. (ppb) 1 9,948 7.274 0.233 2 9,854 7.235 0.231 3 9,886 7.237 0.231 4 9,764 7.235 0.229 Average 9,863 7.245 0.231 CV (%) 0.673 0.229 0.612 N C + O NH Cl N C + O NH Cl N Cl C + N N C + Cl N Cl C + O N H + C O NH NH C + F F C + F F NH 3 + O F F C + O F F Cl O F F NH 3 + F F Δ1.0 mDa O N NH Cl Cl Δ-0.9 mDa Δ-0.9 mDa Δ-0.7 mDa Δ-0.8 mDa Δ0.7 mDa Δ0.6 mDa Δ0.6 mDa Δ-0.8 mDa O NH O NH O Cl F F F F F F Δ-0.7 mDa Δ-0.8 mDa Δ-0.5 mDa Δ-0.5 mDa Δ-0.7 mDa Summary of assigned MS/MS fragments ( flufenoxuron ) Flufenoxuron Summary of assigned MS/MS fragments ( boscalid ) Boscalid