targeted screening and quantitation of pesticide residues in … · 2020. 9. 2. · 4-2....
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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