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7/28/2019 Agilent QQQ Data
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LC/MS Food SafetyApplications
J uly, 2006
Agilent TechnologiesAgilent Technologies
6000 Series LC/MS6000 Series LC/MS
for Food Safetyfor Food Safety
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Agilent LC/MS SolutionsAgilent LC/MS Solutions
• Single provider of:
– Single Quad
– Triple Quad– Ion Trap
– TOF
– Q-TOF
• Major solutions for Food
– High performance/low cost Agilent 6410 Triple Quad LC/MS
– High performance 6210 Agilent TOF LC/MS
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Nitrofuran MetabolitesNitrofuran Metabolites -- Agilent 6410 LC/MS TripleAgilent 6410 LC/MS Triple
QuadrupoleQuadrupole
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The four drugs furazoidone, furaltadone, nitrofurazone
and introfurantoin are veterinary drugs that belong to
family of nitrofuran antibiotics
Widely used for the treatment of gastrointestinal
infections in cattle, pigs and poultry
The European Union (EU) banned the use of nitrofuran
antibiotics in food-producing animals by listing them in
AnnexⅣ of Council Regulation 2377/90
The EU set a Minimum Required Performance Limit(MRPL) of 1 ug/kg that laboratories should at least detect
and confirm
BackgroundBackground
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EU RegulationsEU Regulations-- Minimum Required PerformanceMinimum Required Performance
Limits (MRPL)Limits (MRPL)
COMMISSION DECISIONof 12 August 2002
implementing Council Directive 96/23/EC concerning the performance of analytical
methods and
the interpretation of results
(2002/657/EC)
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2002/657/EC2002/657/EC
•Set’s criteria for screening (< 5 % β error)
•Set’s criteria for confirmation
–For LC/MS
•MS Points for confirmation
•MS ratio of at least two ions•Retention time tolerance
•Does not set level for nitrofurans
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EU RegulationsEU Regulations --NitrofuransNitrofurans
COMMISSION DECISION
of 13 March 2003
amending Decision 2002/657/EC as regards the setting of minimum required performance limits
(MRPLs) for certain residues in food of animal origin
(2003/181/EC)
Nitrofuran metabolites:
— furazolidone
— furaltadone— nitrofurantoin
— nitrofurazone’
Poultry meat
Aquaculture products 1 µg/kg for all
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Structures of Structures of NitrofuransNitrofurans andand MetaboliteMetabolitess
O
NN NH
O
O
N
O
O
H2NN NH
O
O
N
O O
NN
NH
O
O
Nitrofurantoin AHD NPAHD
O
NNH NH2
O
N
O
O
H2NNH NH2
O
Nitrofuranzone SEM NPSEM
NO O
NNH
O
NH2
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O
N NO
O
N
O
O
H2N N O
O
NO O
N N
OO
Furazolidone AOZ NPAOZ
O
NN
ON
O
O
O
N
O
Furaltadone AMOZ NPAMOZ
H2NN
O
O
N
O
N
O O
NN
OO
N
O
Structures of Structures of NitrofuransNitrofurans andandmetabolitemetabolitess
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HPLCHPLC ParametersParameters◆ HPLC system : Agilent 1100 series
◆ Column : C18, 2.1×150, 5 µm
◆ Injection Volumn : 50 µL◆ Flow rate : 0.3 mL/min
◆ TempTemp : 40: 40℃
◆ Mobile phase : A-0.1% Formic acid; B-Acetonitrile
◆ Gradient
109017.01109022
9551795512.0160401250501010900BATime
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◆ MS system : Agilent 6410 LC/MS/MS◆ Ion source : ESI
◆ Polarity : Positive
◆ Nebulizer gas : Nitrogen
◆ Ion spray voltage : 4000V
◆ Source temperature : 350℃
◆ Resolution : Q1 (unit) Q3 (unit)
◆ Scan mode : Multiple Reaction Monitoring (MRM)
◆ Conditions of MRM
MS ParametersMS Parameters
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Mass spectra of SC at different collision energiesMass spectra of SC at different collision energies
4x10
0
0.2
0.4
0.6
0.8
11.2
1.4
1.6
1.8
Abundance vs. Mass-t o-Charge (m/z)100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
+ProductIon(9.738min)(209.2->**)nitrofuran_MS2_100_Frag_5CE_3.d
209.0166.1 192.1
134.2
107.9119.4 147.9
4x10
0
0.2
0.4
0.6
0.8
1
Abundance vs. Mass-t o-Charge (m/z)100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
+ProductIon(9.721min)(209.2->**)nitrofuran_MS2_100_Frag_10CE_4.d
192.2
149.1134.2 166.2
209.1
103.8119.0
CE 10VCE 10V
CE 5VCE 5V
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MRM Conditions of nitrofuran metabolitesMRM Conditions of nitrofuran metabolites
559292150150166.3166.3209.1209.1
559292150150134.2134.2249.1249.1AHAH9.959.95
559292150150104.2104.2249.1249.1
559292150150134.3134.3236.0236.0AOZAOZ10.810.8
559292150150104.3104.3236.0236.0
559292150150192.3192.3209.1209.1SCSC88
559292150150262.4262.4335.1335.1
559292150150291.4291.4335.1335.1AMOZAMOZ00
Collision
Engergy (V)
Fragmentor
(V)
Dwell
(ms)ProductPrecursor CompoundTime
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Low Outlier
High Outlier
Quant Ion Integration
Rel Ion Ratio
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Ion RatiosIon Ratios-- Instant Graphic ReviewInstant Graphic Review
R e l a t i v e A b u n d a n c e ( % )
AcquisitionTime(min)
2x10
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
2.4 2.6 2.8 3 3.2 3.4 3.6
335.1->291.1 ,262.1
Ratio=24.0
Relative Peak AbundanceQuant Ion
Relative Peak A... Qualifier
(outside limits-colored)
}20 %
Tolerance
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Calibration curve of AMOZCalibration curve of AMOZ
Linear range: 10 ppt ~ 10ppb
RR22=0.99985=0.99985
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MRM EIC of MRM EIC of Nitrofuran MetabolitesNitrofuran Metabolites (100(100 pptppt))
2x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
Abundance vs. Acqui si t i on Ti me (mi n)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
+MRMMRM(249.1->134.3)Quan_Qualify_100ppt_2.dSmooth(1)
1 1 2 2 3 3 4
AMOZAMOZ
SCSC
AHAH
AOZAOZ
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2x10
0.5
1
1.5
2
2.5
3
3.5
Abundance vs. Acqui si t i on Ti me (mi n)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 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
+MRMMRM(335.1->291.4)Quan_Qualify_100ppt_2.dSmooth(1)
4.5131 1
1x10
4.8
55.2
5.4
5.6
5.8
6
6.2
6.4
6.6
+MRMMRM(335.1->262.4)Quan_Qualify_100ppt_2.dSmooth(1)
4.481
4.592
1.997
1 1
AMOZ 100AMOZ 100
pptppt
331.3331.3→291.4 (Quant Ion)291.4 (Quant Ion)
AMOZ 100AMOZ 100 pptppt
335.1335.1→262.4 (Qualify Ion)262.4 (Qualify Ion)
EIC at TransitionsEIC at Transitions m/zm/z 229191 andand m/zm/z 262262
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2x10
1
2
3
4
5
6
Abundance vs. Acqui si t i on Ti me (mi n)11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 2
+MRMMRM(236.0->134.3)Quan_Qualify_100ppt_2.dSmooth(1)
11.194
12.323 13.15911.702
4 4
2x10
0.5
0.6
0.7
0.8
0.9
1
1.1
+MRMMRM(236.0->104.3)Quan_Qualify_100ppt_2.dSmooth(1)
11.197
11.767
4 4
EIC Transitions atEIC Transitions at m/zm/z 113434 andand m/zm/z 110404
AOZ 100AOZ 100 pptppt
236.0236.0→134.3 (Quant Ion)134.3 (Quant Ion)
AOZ 100AOZ 100 pptppt
236.0236.0→104.3 (Qualify Ion)104.3 (Qualify Ion)
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2x10
0.5
0.6
0.7
0.8
0.9
Abundance vs. Acqui si t i on Ti me (mi n)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2
+MRMMRM(335.1->291.4)10ppt_2.dSmooth(1) Noise(RMS)=8.54;SNR(4.481min)=6.5
4.481
3.175
7.999
1 1 2 2 3 3 4 4
1x10
5
5.5
6
6.5
7
7.5
+MRMMRM(209.1->192.3)10ppt_2.dSmooth(1) Noise(RMS)=14.69;SNR(9.838min)=4.0
9.838
9.480
1 1 2 2 3 3 4 4
MRM EIC SpeMRM EIC Specctrum of AMOZ and SC (10trum of AMOZ and SC (10 pptppt))
S/N=6S/N=6
S/N=4S/N=4
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2x1 0
0. 6
0. 8
1
1. 2
1. 4
1. 6
1. 8
2
2. 2
2. 4
2. 6
2. 8
3
3. 2
Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )1 2 3 4 5 6 7 8 9 1 0 11 1 2 1 3 1 4 1 5 16 1 7 1 8 1 9 20 21 2 2
+ MRM MR M ( 2 3 6 .0 -> 1 34 . 3 ) N F r e a l s a m p l e B l a n k _ 1 . d Sm o o t h ( 1 )
1 1 2 2 3 3 4 4
2x1 0
0. 6
0. 8
1
1. 2
1. 4
1. 6
1. 8
2
2. 2
2. 4
2. 6
2. 8
3
3. 2
3. 4
3. 6
3. 8
Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )1 2 3 4 5 6 7 8 9 1 0 11 1 2 1 3 14 15 16 17 1 8 1 9 20 2 1 2
+ M R M M RM (2 4 9 .1 - > 13 4 .2 ) N F _ M RM s p i k e 1 00 p p t _ 2.d S m o o t h ( 1)
1 0 . 0 9 3
1 1 2 2 3 3 4 4
Spike Nitrofuran Metabolites in TilapiaSpike Nitrofuran Metabolites in Tilapia (100(100 pptppt))
BlankBlank
SpikeSpike AMOZ AMOZ
SC SC
AOZ AOZ
AH AH
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The Reproducibility of Nitrofuran MetabolitesThe Reproducibility of Nitrofuran Metabolites
1.051.050.10.1AMOZAMOZ
2.012.010.10.1SCSC
1.581.580.10.1AOZAOZ
8.078.070.10.1AHAH
RSDRSD
(%)(%)
ConcentrationConcentration
((ng/mng/mLL))CompoundsCompounds
a Each value was the average of 8 replicates (n=8).
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Malachite Green and MetaboliteMalachite Green and Metabolite
Agilent 6410 Triple QuadrupoleAgilent 6410 Triple Quadrupole
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MalachiteMalachite GGreen (MG)reen (MG)
Among other veterinary drugs, theAmong other veterinary drugs, the triphenyltriphenyl--methanemethanedye malachite green (MG) is a popular substance, anddye malachite green (MG) is a popular substance, and
prevents fungal and parasitic infections in aquaculture,prevents fungal and parasitic infections in aquaculture,includingincluding saprolegniasissaprolegniasis, white spot disease, and, white spot disease, andciliates.ciliates.
MG is prevalently reduced intoMG is prevalently reduced into leucoleuco--malachite greenmalachite green(LMG) and deposited in fatty tissue of the fish.(LMG) and deposited in fatty tissue of the fish.
C
N
N
Cl
Malachite Green
C
N
N
H
Leucomalachite Green
reduction
(Green) (Colorless)
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EU Regulatory Limit for Malachite Green and itsEU Regulatory Limit for Malachite Green and its
MetaboliteMetabolite
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HPLC equipped with a postHPLC equipped with a post--column unit for oxidation of LMG andcolumn unit for oxidation of LMG and
with either an absorbance or MS detector for detection in troutwith either an absorbance or MS detector for detection in trout
(eggs, fry and muscle), catfish (muscle and plasma) and in trout(eggs, fry and muscle), catfish (muscle and plasma) and in trout
muscle. The reactorsmuscle. The reactors (5 cm(5 cm××4mm)4mm)used in these studies were filledused in these studies were filled
withwith 2525%% PbOPbO22
Other method LCOther method LC--APCIAPCI--MS in catfish and trout without the use of MS in catfish and trout without the use of
a reactor for the conversion of LMG. This study aimed at thea reactor for the conversion of LMG. This study aimed at the
development and validation of a procedure for sample processingdevelopment and validation of a procedure for sample processing
and of an HPLC analysis method for determination of residues of and of an HPLC analysis method for determination of residues of
MGMG
The use of LCThe use of LC – –ESIESI--MSMS – –MS analysis was explored for MS analysis was explored for
confirmation of detected residues of this drug in such matrices.confirmation of detected residues of this drug in such matrices.
Analytical Approaches for Determination of Analytical Approaches for Determination of
Residues of MGResidues of MG
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Tissue sample (5g)
Add 1mL 0.25 g/mL Hydroxylamine hydrochloride
Add 2mL 1mol/L p-toluene sulfonic acid
Add 2ml 0.1 mol/L NH4Ac buffer, pH4.5
Add 40ml acetonitrile
homogenized for 2 min
PPtSupernatant(acetonitrile phase)
Add 20ml acetonitrile
filter
Supernatant
35ml water, 30ml dichloromethane
Collect dichloromethane phase, add 20ml dichloromethane in upper layer
LC/MS/MS
Concentration
The analysis of residues of MG and LMG in TilapiaThe analysis of residues of MG and LMG in Tilapia
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HPLCHPLC ParametersParameters
◆ HPLC system : Agilent 1100 series
◆ Column : C18, 2.1×150, 5 µm
◆ Injection Volumn : 10 µL
◆ Flow rate : 0.3 mL/min
◆ Mobile phase : A-10mmol NH4Ac (pH=4.5); B-Acetonitrile◆ Gradient
30701330708.0195589552
5050130700BATime
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◆ MS system : Agilent 6410 LC/MS/MS◆ Ion source : ESI
◆ Polarity : Positive
◆ Nebulizer gas : Nitrogen
◆ Ion spray voltage : 4000V
◆ Source temperature : 350℃
◆ Resolution : Q1 (unit) Q3 (unit)
◆ Scan mode : Multiple Reaction Monitoring (MRM)◆ Conditions of MRM
MS ParametersMS Parameters
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TIC of MG and LMG withTIC of MG and LMG with
DifferentDifferent Fragmentor Fragmentor VoltagesVoltages
7x10
1
2
3
4
5
Abundance vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 1
+EIC(329.4,331.4m/z)ScanoptimizingFRG70_3.d
1 1
7x10
1
2
3
4
5
+EIC(329.4,331.4m/z)ScanoptimizingFRG90_4.d
1 1
7x10
1
2
3
4
5
6
+EIC(329.4,331.4m/z)ScanoptimizingFRG100_5.d
1 1
70V
90V
110V
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Mass spectra of MG at different collisionMass spectra of MG at different collision
energiesenergies5x10
0
0.5
1
1.5
2
2.5
3
3.5
4
Abundance vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 35
+ProductIon(5.549min)(329.3->**)optimizingMS2_FRG100_CE20_2.d
329.3
313.5208.3 284.3236.9193.1
268.4
5x10
0
0.2
0.4
0.6
0.81
1.2
1.4
1.6
1.8
2
+ProductIon(5.549min)(329.3->**)optimizingMS2_FRG100_CE30_3.d
329.3
313.4
208.2
285.3251.4237.2 270.3192.8164.6 298.9217.4134.3
5x10
0
0.2
0.4
0.6
0.8
1
1.2
+ProductIon(5.549min)(329.3->**)optimizingMS2_FRG100_CE40_4.d
313.3
208.2
329.4284.2165.3 241.4
270.3192.1 251.4221.0134.1 298.9
91.5117.9 202.0179.8
CE 20VCE 20V
CE 30VCE 30V
CE 40VCE 40V
C
N
N
[M+H]+ m/z 329
-CH4 -dimethylaniline
m/z 313 m/z 208
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MRM Conditions of MG and LMGMRM Conditions of MG and LMG
30301001004040239.2239.2331.3331.3
30301001004040316.3316.3331.3331.3LeucomalachLeucomalach
iteite greengreen77
40401001004040208.2208.2329.3329.3
40401001004040313.3313.3329.3329.3MalachiteMalachite
greengreen00
Collision
Engergy (V)
Fragmentor
(V)
Dwell
(ms)ProductPrecursor CompoundTime
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Calibration curve of malachite greenCalibration curve of malachite green
Linear range: 10 ppt ~ 10ppb
RR22=0.9995=0.9995
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Calibration curve of Calibration curve of LeucomalachiteLeucomalachite greengreen
Linear range: 10 ppt ~ 10ppb
RR22=0.9994=0.9994
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3x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
Abundance vs. Acqui si t i on Ti me (mi n)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
+MRMMRM(329.3,331.3->**)malachitegreen_200606127.d
8.409
5.440
8.118 9.660
1 1 2
MRM EIC of MG and LMG (100MRM EIC of MG and LMG (100 pptppt))
MGMG
LMGLMG
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EIC spectra of ions atEIC spectra of ions at m/zm/z 313 and313 and m/zm/z 208208
2x10
0
0.5
1
1.5
2
2.5
3
3.5
Abundance vs. Acqui si t i on Ti me (mi n)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
+MRM(329.3->313.3)malachitegreen_200606127.d
5.4481 1 2
2x10
00.2
0.4
0.6
0.8
1
1.2
1.4
1.6
+MRMMRM(329.3->208.2)malachitegreen_200606127.d
5.4361 1 2
Malachite Green 100Malachite Green 100 pptppt
329.3329.3→313.3 (Quant Ion)313.3 (Quant Ion)
Malachite Green 100Malachite Green 100 pptppt
329.3329.3→208.2 (208.2 (QualQual Ion)Ion)
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3x10
0
0.2
0.4
0.6
0.8
1
Abundance vs. Acqui si t i on Ti me (mi n)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
+MRMMRM(331.3->239.2)malachitegreen_200606127.d
8.409
8.118 9.660
1 1 2
1x10
0
1
2
3
4
5
6
+MRMMRM(331.3->316.3)malachitegreen_200606127.d
8.3951 1 2
EIC spectra of ions atEIC spectra of ions at m/zm/z 239 and239 and m/zm/z 316316
LeucomalachiteLeucomalachite Green 100Green 100pptppt
331.3331.3→239.2 (Quant Ion)239.2 (Quant Ion)
LeucomalachiteLeucomalachite Green 100Green 100
pptppt
331.3331.3→316.3 (Qualify Ion)316.3 (Qualify Ion)
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2x10
0
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
Abundance vs. Acqui si t i on Ti me (mi n)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
+MRMMRM(331.3->239.2)MG_10ppt_MRM_a_2.d
8.394
9.4528.725
1 1 2
MRM EIC SpeMRM EIC Specctrum of MG and LMG (10trum of MG and LMG (10 pptppt))
MGMG
LMGLMG
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1
1 .5
2
2 .5
3
3 .5
4
4 .5
5
5 .5
6
6 .5
7
7 .5
8
Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 1 1 .5 1
+ M R M M RM (3 3 1 .3 - > 23 9 .2 ) S p i k e _ B l a n k _ 1 .d
1 1 2 2
3x1 0
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
3
3 .2
3 .4
3 .6
Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 1 1 .5
+ M R M M RM (3 2 9 .3 - > 31 3 .3 ) S p i k e 1 p p b _ 1 . d
1 1 2 2
Spike MG and LMGSpike MG and LMG
in Tilapiain Tilapia
(1 ppb)(1 ppb)
BlankBlank
SpikeSpikeMGMG
LMGLMG
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1
1 .5
2
2 .5
3
3 .5
4
4 .5
5
5 .5
6
6 .5
7
7 .5
8
Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 1 1 .5 1
+ M R M M RM (3 3 1 .3 - > 23 9 .2 ) S p i k e _ B l a n k _ 1 .d
1 1 2 2
2x1 0
0
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
Ab u n d a n c e v s . Ac q u i s i t i o n T i me ( mi n )0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 1 0 1 0 .5 1 1 11 .5 1
+ MRM MRM (3 3 1 . 3 -> 23 9 . 2 ) S p i k e 1 0 0 p p t _ 1. d
1 1 2 2
Spike MG and LMGSpike MG and LMG
in Tilapiain Tilapia
(100(100
pptppt
))
BlankBlank
SpikeSpike
MGMG LMGLMG
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The Reproducibility of MG and LMGThe Reproducibility of MG and LMG
2.252.250.10.1LMG (LMG (m/zm/z 331331.3.3→239239.2.2))
3.3.52520.10.1MG (MG (m/zm/z 329329.3.3→313.3313.3))
RSDRSD
(%)(%)
ConcentrationConcentration
((ng/mng/mLL))
CompoundsCompounds
a Each value was the average of 8 replicates (n=8).