Targeted screening and quantitation of pesticide residues in green tea using a Quadrupole Time-of-Flight Mass SpectrometerHuan Lin1, Toshiya Matsubara1, Natsuyo Asano1, Mikie Shima2

1 SHIMADZU Corporation, Kyoto, Japan 2 AISTI SCIENCE, Wakayama, Japan

2. IntroductionWith 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. OverviewThe 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 pesticidesWe 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. ConclusionsAccording 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

MgSO4 (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

PurificationAutomatic 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/z0

500

1000

1500

2000

2500Inten.

489.0435MS 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

0XIC: 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/z0

500

1000

1500

2000

2500

3000

3500

4000Inten.

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 min0

300000

600000

900000

1200000

1500000

1800000

2100000

2400000

TIC (x 0.25)

XIC

C21H11N2O3F6Cl

Flufenoxuron

O

NH

O

NH

O

ClF

F

F

F

F

F Δ 0.1778 ppm, Score 98.77Measured 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 AreaRT

(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 AreaRT

(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

NC+

O

NH

Cl

NC+

O

NH

Cl

N

Cl

C+

N

N

C+

ClN Cl

C+

ON

H

+C

O

NH

NH

C+

F

FC+

F

F

NH3+

OF

F

C+

OF

F

Cl

O

F

F

N H3

+

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

ClF

F

F

F

F

F

Δ-0.7 mDaΔ-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