TP 239
Targeted screening and quantitation of pesticide residues in green tea using a Quadrupole Time-of-Flight Mass Spectrometer
Huan Lin1, Toshiya Matsubara1, Natsuyo Asano1, Mikie Shima2
1 SHIMADZU Corporation, Kyoto, Japan 2 AISTI SCIENCE, Wakayama, Japan
Precooling cryogenic grinding with dry ice

STQ-LC

Green tea 2 g

1. Overview

Add 2ppm of mixed STDs 50 µL
MeCN 10 mL
Homogenization (13000 rpm, 1min.)
NaCl 1 g

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 withMS/MS measurement using a LC/Q-TOF-MS.

TSCD* 1 g
DHS** 0.5 g

Extraction
QuEChERS

4-2. Calibration curve for quantitative analysis

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.

Calibration curves for quantitative analysis are generated using the green tea extracts spiked
with pesticide mixture. Among 157 pesticides, flufenoxuron and boscalid, were detected fromthe green tea extracts, estimated concentrations as 0.142 ppb and 0.231 ppb, respectively. As aresult of formula prediction, the composition of corresponding peak on the MS spectrum
completely agree with that of flufenoxuron and boscalid with the top score.

2400000*TSCD: trisodium citrate dihydrate**DHS: disodium hydrogen citrate sesquihydrate

2100000

MgSO4 (anhydrous) 4 g

TIC (x 0.25)

1800000

Shaking (1 min.)2. Introduction

Centrifugation (3500 rpm, 5 min.)

1500000

MeCN layerWith population growth and the development of agriculture and food industries, foodsafety is becoming a greater concern in recent years. The increasingly stricter regulationsof 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 ahigh-resolution and accurate mass spectrometer (HRMS), such as a Time-of-Flight, isalso carried out coupled with MRM-based analysis for the purpose of guaranteeing therobustness and reliability of screening. In addition, HRMS also provides a different
advantage from MRM measurement because a compound which was not included as atarget when acquiring data can be processed later without reacquiring data.

1200000

Smart-SPE C18-50 mg＋PSA-30 mg: Purification
Elution 2% FA in MeCN 0.5mL

Smart-SPE C18-30 mg: Purification
Elution MeCN – Water (8/2) 0.5 mL

XIC

900000Automatic SPE device ST-L400

eluateWater 0.5 mL

Compounds in green tea samples were extracted with QuEChERS extraction after
grinding with cryogenic milling (Figure 2). The resulted acetonitrile layers were collectedand 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 inwater and 2 mmol/L ammonium formate + 0.002% formic acid in methanol. Assignmentswith 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).

Quantitative results

600000Purification# of repetition

Area

RT
(min)

Conc. (ppb)# of repetition

Area

RT
(min)

Conc. (ppb)

1

7,958

9.051

0.151

1

9,948

7.274

0.233

2

7,632

9.049

0.143

2

9,854

7.235

0.231

3

7,188

9.050

0.133

3

9,886

7.237

0.231

4

7,569

9.051

0.142

4

9,764

7.235

0.229

Average

7,587

9.050

0.142

Average

9,863

7.245

0.231

CV (%)

3.605

0.009

4.484CV (%)

0.673

0.229

0.612

Automatic SPE device

ST-L400

300000

eluate
0
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 minConstant volume (2 mL, Water)

Figure 2. Sample preparation of pesticides in green tea

4. Results

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.

4-1. Analysis conditions for pesticides

3. Methods

Flufenoxuron

Residual compounds

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 ofeach chemical was recorded by injection of standard sample by using the following
instrument parameters.

1. Summary table(Compound quantitation results)

175
0

XIC: 489.0435(+)

1750

15001500

12501250

10001000

750750500500250250Analytical 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)

8.0

2. Chromatogram

XIC: 489.0435(+)

9.0

10.0

2000
1500

2250 XIC: 343.0399(+)

2000

2000

1750

1750

1500

1500

1250

1250

1000

1000

750

750

500

500

250

250

0

11.0

8.0

9.010.011.0MS spectrum
489.0435

Δ 0.18 ppm

Summary of assigned MS/MS fragments (flufenoxuron)F

Δ-0.7 mDa
Δ-0.5 mDa
Δ-0.5 mDa F

O
C06.0

7.0

8.0

9.0

6.0

7.08.0F

9.0

3500
3000
2500

343.0397

Δ 0.74 ppm

FO++

NH

NH3

C
F

Δ-0.7 mDaF

Flufenoxuron

OF

F
Cl
F

500
475.0 480.0 485.0 490.0 495.0 500.0 505.0 m/z

F+
3NH

F

F+

C

10000O

F

1500

500

F

NHO

F

F

0335.0 337.5 340.0 342.5 345.0 347.5 350.0 352.5m/zFigure 4 Screening result of pesticide residues in green tea

Summary of assigned MS/MS fragments (boscalid)

FNFOCF

Δ-0.9 mDa
Δ-0.8 mDa

O
NH

10 μL

O

Cl

0.4 mL/min

Column Temperature: 35 ºC
Injection Volume:Δ-0.8 mDa

NH

2000
1000

Screening software: LabSolutions Insight(Quantitation view)

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 wereassigned to structural fragments of flufenoxuron and boscalid with accuracies of 1 mDa or less.

Inten.
4000

Inten.
2500

3. Calibration

2250 XIC: 343.0399(+)+00Green tea

Blank

Figure 6 Quantitative result of pesticide residues in green tea

4-3. Identification of pesticides by structural
information from MS/MS fragments.

Boscalid

Green tea

Blank

UHPLC（Nexera X2）

Flow Rate:

Boscalid

Flufenoxuron

Fractionation 0.5 mL

Δ-0.9 mDa+

Δ-0.8 mDaOOClO

NH

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

Figure 1LCMS-9030 quadrupole time-of-flight mass spectrometer

Measured spectrum

Ionization:

ESI positiveO

Acquisition modeMS

Cl

3 L/min

Drying Gas Flow:

10 L/minHeating Gas Flow:

10 L/minInterface Temperature:

350 ºCDL Temperature:

150 ºCHB Temperature:

300 ºCTOF range:

m/z 120 - 1000

Event Time:

0.4 s (Pulser inj.: 794)

ID

off

Δ 0.1778 ppm, Score 98.77

NC

C+

N+H+

N

ClCl

Cl

BoscalidCClCN

NHNH+ON

NC+N

ClΔ-0.7 mDa
Δ1.0 mDa

Δ0.6 mDaF
F

Nebulizing Gas Flow:

ONH

FFMS (LCMS-9030)

NH

Δ0.7 mDa
Δ0.6 mDa

F

Theoretical spectrum

FlufenoxuronCl

C21H11N2O3F6Cl

Figure 7 Accurate MS/MS fragments help identify flufenoxuron and boscalid;
MS/MS spectra (left), fragmentation patterns and mass error (right)

Element table
Name

Min

Max

Carbon0150

Hydrogen

0300

Nitrogen

012

Oxygen012

Fluorine

012

Sulphur

012

Chlorine

012Error Margin = 1 ppm

Figure 5 Example of formula prediction (LabSolutions Insight Explore)

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.