lc/qqq, tof and q-tof for the analysis of pesticide
TRANSCRIPT
1
LC/QqQ, TOF and Q-TOF forthe Analysis of Pesticide
Residues in Food and WaterE. Michael Thurman1 and Imma Ferrer1
1Center for Environmental Mass Spectrometry (CEMS), University of
Colorado, Boulder, CO, USA
The 43rd Western Canadian Trace Organic Workshop, Saskatoon, April
27-30, 2008
OutlineOOutlineutline
1. Introduction: our research deals with environmental mass spectrometry and the identification of pesticides in food and water samples.
2. Examples of identification: pesticides in food(vegetable samples and olive oil...) and waterusing a variety of instruments:
- LC/TOF-MS- LC/QqQ-MS - LC/Q-TOF-MS
3. Highlight advantages and disadvantages forevery instrument.
2
Southern Spain (Almería):Vegetable Garden of Europe: Our Food Story
Southern Spain (AlmerSouthern Spain (Almeríía)a):Vegetable Garden :Vegetable Garden of Europe: Our Food Storyof Europe: Our Food Story
Greenhouses and Drip Irrigation
30,000 Hectares
Environmental BackgroundEnvironmental Background
• Occurrence of pesticides in food is an important environmental issue. Use of agrochemicals at various stages of cultivation has an important impact in food protection and quality preservation.
• European Food Regulations (91/414/EEC) require MRL’s of <0.01 mg/kg (or 10 ppb) for non regulated pesticides in food.
Available Instruments, which one do we use? Advantages:
- LC/TOF-MS: provides elemental formula composition, specificity and selectivity (high resolving power).
- LC/QqQ-MS: routine analysis for target compounds, high sensitivity.
- LC/Q-TOF-MS: fragmentation information, screening and non-target analysis.
3
TargetsTargets
Types of analytes identified by LC-MS
PesticidesPesticides wewe are are lookinglooking forfor......
NonNon--targetstargets
PesticidesPesticides oror metabolitesmetabolites thatthat may show may show up in up in thethe samplesample......
UnknownsUnknowns
SomethingSomething wewe are are notnot expectingexpecting......
Identification by LC-MSR
outin
ean
alys
esN
ovel
Ana
lyse
s
LC/TOF-MS or LC/QqQ-MS
LC/TOF-MS or LC/Q-TOF-MS
LC/Q-TOF-MS or LC/TOF-MS
LC Conditions:• Column
Zorbax Eclipse XDB C8, 4.6 x 150 mm, 5 µm particles • Mobile phase
Acetonitrile and water with 0.1 % formic acid• Gradient
10 % organic isocratic for 5 min, then to 100 % in 25 min.Flow = 0.6 mL/min
Analytical MethodologyAnalytical MethodologyAnalytical Methodology
MS Conditions:
• Dual sprayer providing constant low flow ofreference solution for continuous auto-calibration.
• Positive ESI+, Capillary 4000V, Fragmentor 190V.
Extraction procedure:• Ethyl acetate method• QuEChERS method (S.J. Lehotay, Journal of AOAC International, 2005)
4
LC/TOF-MS (TOFFY I)Almeria, Spain: Food
2004
LC/TOF-MS (TOFFY II)Boulder, CO, USA: Water
2008
1 GHz to 4 GHz DetectorAnalyst to Mass Hunter Software1100 to 1200 HPLCImprovements in accurate mass
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min
0.0
1.0e6
2.0e6
3.0e6
4.0e6
5.0e6
6.0e6
7.0e6
8.0e6
9.0e6
1.0e7
1.1e7
1.2e7
1.3e7
1.4e7
1.5e7
1.6e7
1.7e7
1.8e7
1.9e73.5
20.9
2.1
16.4
2.3
12.2 20.619.315.7 17.6
15.414.4 17.9 26.913.4
5.7 24.022.511.53.2 9.4
4.4 24.227.725.6
24.921.9 23.2
LC/TOF-MS Multi-residue Analysis LC/TOFLC/TOF--MS MultiMS Multi--residue Analysis residue Analysis
Pepper matrix, spiking level: 0.05 mg/Kg
3.56.1 7.5
11.7
15.4
16.2
17.7 21.0
21.7
22.1
22.523.9
25.9
27.2
27.6
28.6
29.2
CY
RO
MA
ZIN
E
CA
RB
EN
DA
ZIM
TH
IAB
EN
DA
ZO
LE
ME
TH
OM
YL
IMID
AC
LO
PRID
AC
ET
AM
IPR
ID
TH
IAC
LO
PRID
SPIN
OSA
D A
SPIN
OSA
D D
DIM
ET
HO
MO
RPH
AZ
OX
YST
RO
BIN
TR
IFL
UM
IZO
L HEXAFLUMURON
LUFENURON
TEFLUBENZURON
FLU
FEN
OX
UR
ON
Inte
nsity
Time (min)
5
0.20.1167.1040167.1040[M + H]+C6H10N6Cyromazine
0.5
-0.3
0.1
-0.4
-0.3
0.2
-0.1
-0.6
-1.3
-0.1
-0.3
0.1
-0.1
-0.3
-0.1
mDaError
1.0489.0435489.0440[M + H]+C21H11N2O3F6ClFlufenoxuron
-0.6510.9857510.9854[M + H]+C17H8N2O3F8Cl2Lufenuron
0.2380.9815380.9816[M + H]+C14H6N2O2F4Cl2Teflubenzuron
-0.8460.9889460.9885[M + H]+C16H8N2O3F6Cl2Hexaflumuron
-1.0346.0928346.0925[M + H]+C15H15N3OF3ClTriflumizol
0.5404.1242404.1243[M + H]+C22H17N3O5Azoxystrobin
-0.1388.1310388.1310[M + H]+C21H22NO4ClDimethomorph
-0.8746.4838746.4832[M + H]+C42H67NO10Spinosyn D*
-1.8732.4681732.4668[M + H]+C41H65NO10Spinosyn A*
-0.5253.0309253.0308[M + H]+C10H9N4ClSThiacloprid
-1.3223.0745223.0742[M + H]+C10H11N4ClAcetamiprid
0.5256.0596256.0597[M + H]+C9H10N5O2ClImidacloprid
-0.1185.0355185.0355[M + Na]+C5H10N2O2SMethomyl
-1.7202.0433202.0430[M + H]+C10H7N3SThiabendazole
-0.3192.0767192.0767[M + H]+C9H9N3 O2Carbendazim
ppmm/z calculatedm/z experimentalSelected ionFormulaCompound
LC/TOF-MS Accurate Mass MeasurementsLC/TOFLC/TOF--MS Accurate Mass MeasurementsMS Accurate Mass Measurements
Max. 2.7e5 cps.
16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5Time, min
0.01.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e51.6e51.7e51.8e51.9e52.0e52.1e52.2e52.3e52.4e52.5e52.6e52.7e5
Intensity, cps
20.9
(a) mass window: 0.2 Dam/z 233.0-233.2
Olive Oil, 10 ppb diuronXIC m/z 233
diuron
Unknown m/z 233.1533
TIC of +TOF MS: from Sample 3 (Aceite MSPD 10 ppb) of Aceite MSPD calibration.wiff Max. 5.9e6 cps.
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min
0.02.0e54.0e56.0e58.0e51.0e61.2e61.4e61.6e61.8e62.0e62.2e62.4e62.6e62.8e63.0e63.2e63.4e63.6e63.8e64.0e64.2e64.4e64.6e64.8e65.0e65.2e65.4e65.6e65.8e65.9e6
Intensity, cps
21.0
19.425.1 28.5
22.8 29.114.527.918.1 23.5 27.72.2 16.9 24.621.516.1 26.52.4 25.717.6
19.013.7 20.115.83.5 11.64.14.4 12.6
TIC
Max. 1.4e4 cps.
16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5Time, min
0.0500.0
1000.01500.02000.02500.03000.03500.04000.04500.05000.05500.06000.06500.07000.07500.08000.08500.09000.09500.0
1.0e41.1e41.1e41.2e41.2e41.3e41.3e41.4e41.4e41.4e4
Intensity, cps
21.3
(b) mass window: 0.05 Dam/z 233.00-233.05
diuron
Exp. mass m/z 233.0245
SelectivitySelectivity by LC/TOFby LC/TOF--MSMS
Diuron
Cl
NH
N
ClO
Theoretical mass: 233.0243
6
Example: Unknowns in Cucumbers
2 14 18 22 26 30Time, min
1.00e6
3.00e6
5.00e6
7.00e6
9.00e6
1.10e7
Intensity, cps
2.1
2.4
0.73.5
22.5
27.818.6 30.528.627.526.021.1 24.9
4.0
6 10
100 140 180 220 260 300 340 380m/z, amu
2.00e4
4.00e4
6.00e4
8.00e4
1.00e5
Intensity, counts
331.0435
285.0016
99.0080
127.0389353.0256
257.0066
-21.9821(M+Na)+
MH+
Isotope distribution tool for matching peak intensities
Database search for the empirical formula of C10H19O6PS2
100 140 180 220 260 300 340 380
m/z, amu
2.00e4
4.00e4
6.00e4
8.00e4
1.00e5
Intensity, counts
331.0435
285.0016
99.0080
127.0389353.0256
257.0066
Fragment ions
-21.9821
(M+Na)+
MH+
OH
O
O
3.3 ppm
O
O
HO
0.2 ppm
OH
O
S
PS
H3CO
H3CO
0.1 ppm
O
O
HO
S
PS
H3CO
H3CO
0.8 ppmS
PS
H3COH3CO
O CH3
O
O
OH
CH3
7
294.0 295.0 296.0 297.0 298.0 299.0 300.0 301.0 302.0 303.0 304.0 305.0m/z, amu
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
1.05e5
1.10e5
1.15e5
1.20e5
1.25e51.28e5 297.0558
299.0530
298.0587
301.0503300.0560
m/z = 297.0558
Chlorine isotopicpattern
2 Cl2 Cl
LCLC//TOFTOF--MS MS unknown unknown accurateaccurate mass mass sspectrpectrum from Strawberry sampleum from Strawberry sample
m/z = 299.0530
DatabaseDatabase ScreeningScreening SearchSearch
8
O
NN
H2C
Cl
Cl
C14H15Cl2N2O+
Exact Mass: 297.0556
Imazalil
Comparison for Target AnalysisLC/QqQ-MS vs LC/TOF-MS
9
5x10
0.20.40.60.8
11.21.41.61.8
Abundance vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3
+ TIC Product Ion (** -> **) Linuron.d
0.255 0.806 1.9171.3581 1 2 2 3 3 4 4
Linuron, CE voltage optimization
QqQQqQ: : OptimizationOptimization forfor MRM MRM transitionstransitions
5 V 10 V 15 V 20 V
4x10
0
0.5
1
1.5
Abundance vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
+ Product Ion (0.214-0.304 min, 12 scans) (249.0000 -> **) Linuron.d
248.8970
181.8980159.798362.0998 87.8994 217.5974132.9987
3x10
0
2
4
6
+ Product Ion (0.765-0.848 min, 11 scans) (249.0000 -> **) Linuron.d
248.8970159.7983 181.8980
61.9998 87.8994 217.8974132.9987 173.9981152.6984
3x10
02
46
+ Product Ion (1.325-1.407 min, 11 scans) (249.0000 -> **) Linuron.d
159.8983 181.8980
62.0998 248.9970217.0975132.698787.9994 152.7984 174.8981
3x10
0246
+ Product Ion (1.876-1.958 min, 11 scans) (249.0000 -> **) Linuron.d
159.7983 181.8980
132.798759.8998
216.997587.5994
5 V
10 V
15 V
20 V
...MRM ...MRM transitionstransitionsCompound Fragmentor Precursor Ion Product ions Collision V
3,4,5-Trimethacarb 80 194 137 5122 25
3-Hydroxycarbofuran 80 238 163 10220 5
Acephate 80 184 143 5125 10
Acetamiprid 80 223 126 1556 15
Acetochlor 120 270 148 10224 10
Acibenzolar-S-methyl 120 211 136 2091 25
Aclonifen 120 265 248 15193 15
Alachlor 80 270 162 15238 10
Aldicarb 80 116 89 570 5
Aldicarb Sulfone 80 223 148 576 5
Aldicarb Sulfoxide 80 207 89 5132 5
Aldoxycarb 80 223 149 15177 5
Ametryn 120 228 186 2096 25
Aminocarb 120 209 137 20152 10
Column: Agilent SB C18 4.6 x 150 mm (1.8 µm)Column temp: 25 CMobile phase: 10%ACN / 90%H2O (0.1% HCOOH)Flow-rate: 0.6 mL/minGradient: t0 = 10% ACN, t30 = 100% ACNInjection volume: 10 µL
10
5x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1
1.05
1.1
1.15
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
1.65
Abundance vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
+ MRM (282.0 -> 212.0) mix100_100pg_5May.d
1 1 2 25x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1
1.05
1.1
1.15
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
1.65
Abundance vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
+ MRM (282.0 -> 212.0) mix100_100pg_5May.d
1 1 2 2
ExtractedExtracted Ion Ion chromatogramschromatograms100 100 CompoundsCompounds by by QqQQqQ
100 pg on column or 0.01 ppm (Baby food levels)
Ab
un
da
nce
Acquisition Time (min)
6x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
24.4 24.6 24.8 25 25.2 25.4
305 -> 169.0 , 153.0
Ratio=52.2
+ MRM (25.047-25.408 min, 18 scans) 300 pesticide
Ab
un
da
nce
Mass-to-Charge (m/z)
5x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
150 175 200 225 250 275 300 325
169.0
153.0
De Brabander et al., TRAC, 20 (8) 2001
Ion Ratios by Ion Ratios by QqQQqQ
N
NCH3
CH3
CH3
OP
S
OH3CO
H3C
m/z=153
m/z=169
Diazinon
11
Diazinon - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs
Re
spo
nse
s
Concentration (ng/ml)
7x10
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
-10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115
y = 208335.0516 * x - 152717.0728R^2 = 0.99956076
3-Hydroxycarbofuran - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs
Re
spo
nse
s
Concentration (ng/ml)
5x10
0
1
2
3
4
5
6
7
-5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
y = 8408.3900 * x - 1375.3510R^2 = 0.99945613
CalibrationCalibration Data by Data by QqQQqQDiazinon (concentration range: 0.1-100 ppb)
3-Hydroxycarbofuran (concentration range: 5-100 ppb)
Green pepper matrix
Tomato matrix
QuantitationQuantitation by by QqQQqQ
12
MS/MS Interesting Isobaric Interference and example of how LC/TOF-MS could help
Diuron 233->160233->72
Fluometuron 233->160233->72
Diuron and fluometuron have the same isobaric precursor ion and same qualiferwith nearly identical ion ratios and similar retention times.
HN
N
O Cl
Cl
Diuron
C9H11Cl2N2O+
Exact Mass: 233.0243
O
N NH
F
FF
C10H12F3N2O+
Exact Mass: 233.0896
Fluometuron
1
10
100
400
Number ofTransitionsper group
Dwell time per ion100 mSec200 mSec 10 mSec
Dwell Time versus Number of MRM Transitions:“Where MRM meets Scan is Y”
ScanY
13
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
Abundance vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
+ MRM (353.0000 -> 228.0000) 300 pesticides_Tomato matrix_1 ppb.d
28.557
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
LODLOD’’ss by by QqQQqQ
1 ppb in tomato matrixCompound
Precursor Ion
Product ions
Pepper LOD
LOD Tomato r2
ppb ppb 3,4,5-Trimethacarb 194 137 0.9 0.5 0.999 122 3-Hydroxycarbofuran 238 163 5.0 5.0 0.999 220 Acetamiprid 223 126 0.2 0.5 1.000 56 Aclonifen 265 248 20.0 10.0 0.979 193 Atrazine 216 174 0.8 0.6 1.000 132 Bendiocarb 224 109 1.0 1.0 0.996 167 Buprofezin 306 201 0.7 0.8 0.999 116 Carbaryl 202 145 5.0 5.0 0.999 117 Diazinon 305 169 0.5 0.2 1.000 153 Dimethoate 230 199 0.7 0.7 0.994 171 Fuberidazole 185 157 1.1 1.0 1.000 156 Imazalil 297 159 10.0 10.0 0.850 255 Irgarol 254 198 1.0 1.0 0.999 156 Malathion 331 99 0.8 2.0 0.998 127 Propazine 230 146 1.0 0.5 1.000 188 Terbuthylazine 230 174 0.1 0.5 0.999 132
Accurate mass at 100 picograms with extracted ions
NN
H2NNO2
NCl
Imidacloprid
C9H11ClN5O2+
Exact Mass: 256.0596
N
N
N
Cl
NH
NH
C8H15ClN5+
Exact Mass: 216.1010
Atrazine
In Buffer Atrazine LOD is 20 pg and Imidacloprid is 100 pg.
14
QQQ inScan
1
10
100
300
Instrument Detection Limit10 pg1 pg 100 pg
Screening Pesticides:TOF detection limit is X and the “Crossover Point is XY”
QQQ inMRM
TOF is Full Scan2 ppm Accuracy
QQQ inScan
Scan
Number ofTransitionsper group
Examples of Environmental Samples byLC/Q-TOF-MS
15
Q-TOF Targeted Analysis300 compounds in 25 minutes
N
N
N
Cl
NH
NH
C8H15ClN5+
Exact Mass: 216.1010
Atrazine
Atrazine Frag ions
Answering our “Puzzle” by LC/TOF-MS and LC/TOF-MS-MS
N N
N
HN CH3
H2N
C5H8N5+
Exact Mass: 138.0774
N N
NHNH3C
H2N CH3
Cl
CH3
N
NH2N
Cl
C2H3ClN3+
Exact Mass: 104.0010
N
NNH3C
C4H6N3+
Exact Mass: 96.0556
NH2
H2N Cl
CH4ClN2+
Exact Mass: 79.0058
HN N
NH2N
HN CH3
Cl
C5H9ClN5+
Exact Mass: 174.0541
EE
N N
NH2N
NH3
Cl
C3H5ClN5+
Exact Mass: 146.0228
N
NNHH3C
Cl
C4H7ClN3+
Exact Mass: 132.0323
C8H15ClN5+
Exact Mass: 216.1010
N
NHN
C2H2N3+
Exact Mass: 68.02432
N
HNH3C
C3H7N2+
Exact Mass: 71.0604
Atrazine Pathway
C3H4N5+
Exact Mass: 110.04612
N
HNN
N
H2N
Atrazine pathwayof fragmentationfrom standard andfrom the analysisof a water sampleLawrence, KS
16
TOF compared with QQQ...
Screening and ID of pesticide by QqQ is favorite method but there are limits to dwell times and number of ions.
LC/TOF-MS offers another approach, which takes full scan data and searches database by accurate mass or by formula. Example of 100+ compounds including fragment ions.
QqQ more sensitive by 10-100 times, if transitions are few (1-20).
Cross-Over Point is 100-150 transitions and 10 msec dwell per window or about 20-40 pg on column.
TOF compliments QqQ for food analysis, especially non-targets and large screenings (isotope clusters useful) .
Q-TOF offers additional structural information, specially for fragment ions (good structural elucidation capability).
Rapid Resolution is easily possible with Q-TOF in food matrices from 25 to 5 minutes for a large number of compounds.
Accuracy remains excellent even with 2 to 3 second peaks, something not possible by QqQ with so many compounds.
Either TOF or Q-TOF can be used for screening and confirmation in food matrices.
Q-TOF offers additional information…
17
Antibiotic in Water
Example of 4 GHz High Resolution for Antibiotic Analysis in Water
H2N
SNH2
O O
N
N
OCH3
OCH3C12H15N4O4S+
Exact Mass: 311.0809
H3N N
N
OCH3
OCH3
C6H10N3O2+
Exact Mass: 156.0768
H2N
S
O O
C6H6NO2S+
Exact Mass: 156.0114
4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8+ Scan (7.929-8.012 min, 8 scans) Sulfadimethoxine_4GHz_190V.d
156.0769
156.0115
Counts vs. Mass-to-Charge (m/z)155.7 155.8 155.9 156 156.1 156.2 156.3 156.4 156.5
H2N
SNH2
O O
N
N
OCH3
OCH3C12H15N4O4S+
Exact Mass: 311.0809
H3N N
N
OCH3
OCH3
C6H10N3O2+
Exact Mass: 156.0768
H2N
S
O O
C6H6NO2S+
Exact Mass: 156.0114
18
AcknowledgmentsAcknowledgmentsAcknowledgments
Funding from the Ministerio de Educacion y Ciencia(Project AGL-2004-04838)
Dr. Jerry Zweigenbaum, Agilent Technologies, Inc
Department of Civil, Environmental, andArchitectural Engineering, University of Colorado, Boulder, CO USA