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Matrix Effects in GC and LC. Michelangelo Anastassiades. GC-Injection - Split/Splitless. GC-Injection – A Critical Step. By Hans Mol. GC-Injection – A Critical Step. By Hans Mol. Overload. Liner volume. - PowerPoint PPT Presentation
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Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
1 EU Reference Laboratoryfor Pesticide Residues
requiring Single Residue Methods
Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
Michelangelo Anastassiades
Matrix Effects in GC and LC
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
2 EU Reference Laboratoryfor Pesticide Residues
requiring Single Residue Methods
Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
GC-Injection - Split/Splitless
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
3 EU Reference Laboratoryfor Pesticide Residues
requiring Single Residue Methods
Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
By Hans Mol
GC-Injection – A Critical Step
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
4 EU Reference Laboratoryfor Pesticide Residues
requiring Single Residue Methods
Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
GC-Injection – A Critical Step
By Hans Mol
Overload
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
5 EU Reference Laboratoryfor Pesticide Residues
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Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
The gas volume of the sample injected should be smaller than the internal volume of the liner.
If not, the liner is overloaded and backflash of the sample into various parts of the injector may occur. • Poor injection profiles• Bad peak-shapes• Adsorption onto various parts of inlet and possible carry over.
Typical liners for split/splitless injection are ~80 x 4mm = ~1ml volume
NOTE: Effective vol. available is lower as part of the liner will be filled with carrier gas.
Liner volume
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
6 EU Reference Laboratoryfor Pesticide Residues
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boiling point densityC g/ml μl vapor/μl injection
water 100 1 1491acetonitrile 82 0.78 511acetone 56 0.79 366ethyl acetate 77 0.9 275toluene 111 0.87 254hexane 69 0.66 206isooctane 99 0.69 162GC: 30 m x 0.25 mm, 70C, 60kPa
Expansion-Volumes of Solvents
GC-Injection
MW
184158889286114
Community Reference Laboratoryfor Pesticide Residues
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CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
7 EU Reference Laboratoryfor Pesticide Residues
requiring Single Residue Methods
Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
Large Volume Injections
By Hans Mol
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
8 EU Reference Laboratoryfor Pesticide Residues
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PTV for Large Volume Injections
By Hans Mol
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
9 EU Reference Laboratoryfor Pesticide Residues
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Requirements for robust GC analysis
• Avoid water- activates liner, retention gap, column- damages filament- has large expansion volume (upon evaporation)
Note: Water can be conveniently evaporated using PTV Azeotrop: MeCN:H2O = ~84:16- if H2O content is lower it will concentrate to 16% and then evaporate together w. MeCN; - if content is higher water will remain in the system
• Avoid non-volatile co-extractants (incl. Fat=triglycerides)- contaminate liner/retain less volatile analytes
• Avoid too high concentrations of volatile co-extractants- interfere in the chromatograms
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
10 EU Reference Laboratoryfor Pesticide Residues
requiring Single Residue Methods
Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
High Selectivity in Instrumental Analysis gives more flexibility in sample preparathion
GC analysis
MIPs/Immunoaffinity
Column chromatography Solid Phase Extraction Dispersive SPESelective extraction solventGeneric extraction solvent
GC-FID
GC-ECD GC-NPD GC-FPD GC-MS (quad/IT/TOF) GCxGC-MS, GC-MS/MS, GC-hrMSGCxGC-hrMS, GC-QTOF
Combination should be fit-for-purpose
Sample preparation
Interdependence of analytical steps
By Hans Mol
Incr
easi
ng s
elec
tivity
In
crea
sing
sel
ectiv
ity
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
11 EU Reference Laboratoryfor Pesticide Residues
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Questions:
What are the target analytes?
What is the matrix?
What is the desired LOQ?
What instrumentation is available in your laboratory?
LC-MS/MS available?
Analytes amenable to LC-MS/MS? yes no
use LC-MS/MS(+ GC for confirmatory purposes if possible)
yes no
GC analysis
Decisions Regarding Instrumental Analysis
By Hans Mol
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
12 EU Reference Laboratoryfor Pesticide Residues
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Dealing with Matrix-Effects in GC
with the help of Analyte Protectants (APs)
Community Reference Laboratoryfor Pesticide Residues
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WHAT ARE MATRIX-
EFFECTS ???
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
14 EU Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
15 EU Reference Laboratoryfor Pesticide Residues
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GC-Injector schematically
Syringe
GC-capillary
GC-linerHeater z.B.
250°C
Pneumatics
Sample injection
GC-Injection
Community Reference Laboratoryfor Pesticide Residues
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Ratios:• Peak-Areas: ~ 1,5:1• Peak-Heights: ~ 4:1• Peak-Width (at half height): ~ 1:3
8.70 8.75 8.80 8.85 8.90 8.95 9.00 9.05 9.10 9.15 9.20 9.25 9.30 9.35 9.40 9.450
5000
10000
15000
20000
25000
30000
RT= 8,80 min
RT= 8,92 min
WITHOUT Matrix co-extractives(in pure solvent)
e.g. Calibration standard
WITH Matrix co-extractives(Strawberry-Extract)
Analyte: Atrazine ; Matrix: Strawberry
Stronger Tailing
Apex-Shift towards longer RTs!
Matrix-Induced Peak Enhancement OVERESTIMATION OF RESULTS!!
Community Reference Laboratoryfor Pesticide Residues
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Analytes (interact with Active Sites)• Unwanted Retention/Tailing• Quasi-catalysed degradation (susceptible compounds)
„Matrix-Induced Peak Enhancement Effect“
Active Sites (on Surface of GC-Liner & Column )(Siloxanes & deposited non-volatile matrix-co-extractives)
Matrix-Components (in Excess) • Bloc active sites and protect analytes
GC-Liner
GC-Capillary
Community Reference Laboratoryfor Pesticide Residues
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CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
18 EU Reference Laboratoryfor Pesticide Residues
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Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
Number and type of active sitesNumber and type of active sites in the inlet and GC column in the inlet and GC column
Chemical structure of the analytesChemical structure of the analytes: : H-bondingH-bonding abilityability, , thermolability, volatilitythermolability, volatility
Analyte concentrationAnalyte concentration most pronounced at trace level most pronounced at trace level
Injection temperatureInjection temperature
Interaction timeInteraction time function of: analyte volatility (function of: analyte volatility (), inlet temperature (), inlet temperature (), flow ), flow raterate, , gas gas pressurepressure, , injection volumeinjection volume, , solvent expansion solvent expansion volumevolume, , column diamensionscolumn diamensions
Matrix type and concentrationMatrix type and concentration
Factors influencing in matrix-effects
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
19 EU Reference Laboratoryfor Pesticide Residues
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Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
START
FINISH
100 % 100 %
95 % 10 %
Influence of Analyte
Concentration
Community Reference Laboratoryfor Pesticide Residues
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20 EU Reference Laboratoryfor Pesticide Residues
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An additional effect… „Response Diminishment”Response Diminishment”
Accumulation of non-volatile matrix compounds on the surface of liner and a first part of GC column
Formation of new active sitesnew active sites
Increasing number of injections of matrix-containing extracts
Gradual decrease in analyte responsesand/or
increase in peak-tailing
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
CRL-SRMSAUDIS TRAININGSTUTTGART, DEC. 2009
21 EU Reference Laboratoryfor Pesticide Residues
requiring Single Residue Methods
Training on Pesticide Residues Analysis Bangkok, 17-21 september 2012
Matrix EffectsMatrix Effects
Response Response enhancementenhancement
Response Response diminishmentdiminishment
Community Reference Laboratoryfor Pesticide Residues
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Elimination Elimination oror CCompensation for ompensation for MMatrix atrix EEffects?ffects?
Analyte protectantsAnalyte protectants
Completely remove active sites in GC system
Completely remove matrix compounds
Standard additionMatrix-matched standardsIsotopically labeled internal standards
PracticalPractical
ELIMINATIONELIMINATION
COMPENSACOMPENSATIONTION
ImpossiblImpossible in e in
practicepractice
Impractical Impractical for routinefor routine
Community Reference Laboratoryfor Pesticide Residues
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Compensation of matrix effects (1)
Method of standard additionsMethod of standard additionsextra effortextra effort possible inaccuracies due to :possible inaccuracies due to :
concentration dependence oconcentration dependence off matrix effects matrix effects non-linear response of detector non-linear response of detector deterioration of the system as the samples deterioration of the system as the samples
are injected are injected
Community Reference Laboratoryfor Pesticide Residues
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24 EU Reference Laboratoryfor Pesticide Residues
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Peak Ratio Analyte/ISTD
Added amount of analyte to the aliquotx
x: absolute amount of analyte in aliquot before spiking (y=0)
y-intercept= ׀x׀ Slope
Method of standard additions
Withdraw from extract multiple aliquots of same volume and spike them with increasing amounts of standard
No spiking here
Good linearity is paramount because of
extrapolation
In case of violations: Standardaddition
Community Reference Laboratoryfor Pesticide Residues
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25 EU Reference Laboratoryfor Pesticide Residues
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Compensation of matrix effects (2)
Use of matrix-matched standardisationUse of matrix-matched standardisation need for enough blank matrix (ideally exactly the same need for enough blank matrix (ideally exactly the same
as the samples) and its long-term storageas the samples) and its long-term storage
extra time, laboextra time, labouur, and expense for preparation of the r, and expense for preparation of the blank extracts for calibration standardsblank extracts for calibration standards
greater amount of matrix material injected onto the greater amount of matrix material injected onto the column in a sequence column in a sequence greater GC maintenance greater GC maintenance
potentially greater potential for analyte degradation in potentially greater potential for analyte degradation in the matrix solutionthe matrix solution
Community Reference Laboratoryfor Pesticide Residues
using Single Residue Methods
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26 EU Reference Laboratoryfor Pesticide Residues
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Compensation of matrix effects (3)
Isotopically labelled internal standardsIsotopically labelled internal standards
Generally not availableGenerally not available for all pesticides for all pesticides (or expensive if available) (or expensive if available)
Restriction in the use of MS techniquesRestriction in the use of MS techniques
Occupy Measurement time Occupy Measurement time (theoretical consideration) (theoretical consideration)
Obviate the need for Matrix-MatchingObviate the need for Matrix-Matching
Community Reference Laboratoryfor Pesticide Residues
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Use of isotopically labelled ISTD in MS
MW 122.6
Formula: C5H13ClN
Chlormequat Chlormequat D4
MW 126.6
Formula C5H9D4ClN
Different mass, but exactly same behaviour during extraction, cleanup and chromatography!
CH3 N+
CH3
CH3
Cl
HH
H H
CH3 N+
CH3
CH3
Cl
DD
D D
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Use of isotopically labelled ISTD in MS
Different mass, but exactly same behaviour during extraction, cleanup and chromatography!
Co-elutionCo-elution
Co-elutionCo-elution
Community Reference Laboratoryfor Pesticide Residues
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N
O
O
SC
Cl
Cl
Cl
H
H
H H
N
O
O
SC
Cl
Cl
Cl
D
D
D D
Use of isotopically labelled ISTD in MS
MW 296.6
Formula C9H4Cl3NO2S
Folpet Folpet D4
MW 300.6
Formula C9D4Cl3NO2S
Different mass, but exactly same behaviour during extraction, cleanup and chromatography!
Community Reference Laboratoryfor Pesticide Residues
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Degradation at• High pH• High temperatures
Phthalimide
Dicofol, Captafol, Tolylfluanid, Dichlofluanid, Pyridate
CAPTAN
e.g.:
• During sample prep. • In final extract• In GC-inlet
Similar behaviour:
FOLPET
Tetrahydrophthalimide
Degradation of Folpet and Captan
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Alternative: Isotopically Labelled ISTDs
FLV Folpet D4
y = 12,78x + 0,228
R 2 = 0,9979
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0 0,005 0,01 0,015 0,02 0,025 0,03 0,035
FLV BS 138
y = 0,1576x + 0,003
R2 = 0,7960
0,002
0,004
0,006
0,008
0,01
0 0,005 0,01 0,015 0,02 0,025 0,03 0,035
Fläche pur
y = 3E+06x + 45408
R2 = 0,8318
0
20000
40000
60000
80000
100000
120000
140000
0 0,005 0,01 0,015 0,02 0,025 0,03 0,035
FLV C aptan D6
y = 71,164x + 2,312
R2 = 0,9979
0
1
2
3
4
5
6
7
0 0,01 0,02 0,03 0,04 0,05 0,06 0,07
FLV BS 138
y = 1,0722x + 0,0252
R2 = 0,9585
0
0,02
0,04
0,06
0,08
0,1
0 0,01 0,02 0,03 0,04 0,05 0,06 0,07
Fläche pur
y = 6E+06x + 152616
R2 = 0,9436
0
100000
200000
300000
400000
500000
600000
0 0,01 0,02 0,03 0,04 0,05 0,06 0,07
Only via Area
via ISTD (PCB 138)
via ISTD (Folpet D4)
R2 =0.9979
R2 =0.7960
R2 =0.8318
R2 =0.9979
R2 =0.9436
R2 =0.9585
Only via Area
via ISTD (PCB 138)
via ISTD (Captan D6)
Folpet in Papaya (MRL=0.01 mg/kg)
0.019 mg/kg
0.022 mg/kg
0.018 mg/kg
No
violation
0.030 mg/kg
0.033 mg/kg
0.030 mg/kg
Unacceptable R2
Should be at least 0.995
Captan in Blueberries (MRL=0.01 mg/kg)
Dicofol is the next to check using isotopically labelled ISTD
ISTDs added
to extract
Violation
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THE USE OF ANALYTE PROTECTANTS
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WHAT ARE ANALYTE PROTECTANTS?
?ANALYTE
PROTECTANT
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PROTECTANT LORD ANALYTE
?
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ANALYTE
PROTECTANT
DANGER
?
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ANALYTE
PROTECTANT
?
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PROTECTANT
ANALYTE
?
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Dispersive SPE –Removal of Co-extractives
2,9
1,31,0
pH 5.4
pH 8.4pH 8.9
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
No PSA PSA 25 mg/mL PSA 50 mg/mL
mg
/mL
0
1
2
3
4
5
6
7
8
9
10
pH
va
lue
Amount of Co-extractives in the extract (mg/kg)
pH of Acetonitril Extract
PSA Cleanup and effect on pH
Drawbacks: pH rises (degradation risk) Matrix-Induced Analyte
Protection reduced
Solutions:
Addition of Acids (see later)
Addition of Analyte Protectants
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Analyte Protectants Principle
PSA cleanup
Addition of „Analyte
Protectants“(AP)
„ Protection“
Raw Extract
Cleaned up Extract
Cleaned-up Extract + AP
Standard + AP
Standard in Pure Solvent
Analyte Protectants help to reduce analyte Interactions with Active Sitesand thus Errors related to Matrix-Induced Peak Enhancement in GC
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Extract acidification improves GC-behaviour of Folpet, Captan and Dicofol
But better results by addition of ANALYTE PROTECTANTS-MIX to extracts
Use of Analyte Protectants to improve GC-Analysis
Errors due to Matrix Effects with and without Analyte Protectants
010
2030
4050
6070
8090
100
Dic
hlo
rvos
Dic
hlo
benil
Acephate
o-P
henylp
henol
Phenm
edip
ham
Carb
ofu
ran
HC
H,
gam
ma-
Chlo
roth
alo
nil
Vin
clo
zolin
Para
thio
n-m
Carb
ary
l
Meth
iocarb
Dic
lofluanid
Para
thio
n-e
Chlo
zolin
ate
Toly
fluanid
Pro
cym
idone
Capta
n
Folp
et
Flu
benzim
ine
Ditalim
fos
Bupirim
at
Bin
apacry
l
Trifloxystr
obin
Tetr
am
eth
rin1
Dic
ofo
l
Acrinath
rin
Bitert
anol
Ipro
dio
ne
Chin
om
eth
ionat
TP
P
ME
AN
ALL
Ab
so
lute
Err
or
[%]
Against Solvent w/o APs
Against solvent with AP
(no peak in
solvent)
Overestimations if quantified against
solvent-based calibr. std
• Folpet, Captan work perfectly • Dicofol still not fully satisfactory
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Analyte Protectants-Reduction of Matrix Induced Enhancement Errors
Errors eliminated if: Response in Matrix/Response in Solvent ~ 1
0,00 0,50 1,00 1,50 2,00 2,50 3,00
methamidophos
mevinphos
acephate
omethoate
vinclozolin
metalaxyl
chlorpyriphos
carbaryl
dichlofluanid
fenthion
cyprodinil
endosulfan I
imazalil
azinphos-methyl
coumaphos
Without AP With AP no AP with AP
1,0
Response in Matrix
Response in Solvent
Overestimations when using
Standards in Solvent
AP was added to both :Sample Extract and Calibration Standard (in pure Solvent)
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Analyte Protectants – Examples
Various Compounds Tested for “Protective Potential”. Best Protection : Polyhydroxy-Compounds (sugars, ~derivatives)
O
OH OH
OOH
OHOH O
OH
OHOH
OH
OHOH
OH
Sorbitol
Ethylglycerol
δ-Gulonolactone
Examples:
APs typically give broadly eluting peaks For protection over a broad volatility range use AP-Mixtures as each covers a different volatility range
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D-Sorbitol
Degradation products of L-Gulonic acid γ-lactone
First dimension (DB5-ms)
Seco
nd d
imen
sion
(BPX
-50)
3-Ethoxy-1,2-propandiol(end of the tail)
Elution of pesticides
GCGCGCGC/TOF-MS/TOF-MS of Analyte Protectants of Analyte Protectants
By J. Hajslova
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0
100
200
300
400
500
Dichlor
vos
Mev
inpho
s
Omet
hoat
e
Pirimica
rb
Vinclo
zolin
Met
alaxy
l
Pirimip
hos-
met
hyl
Met
hioca
rb
Chlorp
yrifo
s
Tolyl
fluan
id
Procy
mido
ne
Thiab
enda
zole
Endos
ulfan
I
Imaz
alil
Parat
hion-
met
hyl
Brom
opro
pyla
te
Phosa
lone
Cyhal
othr
in-l
Deltam
ethr
in
Azoxy
strob
in
Re
lativ
e in
ten
sity
(%
)
Comparison of Generated Data
Accurate data over a broad analyte spectrum Accurate data over a broad analyte spectrum !!
3-ethoxy-1,2-propandiol (4 mg/mL)
L-gulonic acid γ-lactone (1 mg/mL)
D-glucitol (1 mg/mL)
Baby food0.015 mg/kg
Without analyte protectants With analyte protectants
1340%1340% 1450%1450%
By J. Hajslova
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Strong interactions with active sites (H-Bond activity)
Similar volatility to analytes to be protected (so that protection extents during entire run)
Soluble in sample extract (not in non-polar solvents)
Not accumulating in GC-system
Not reactive with analytes (not inducing their degradation)
Minimal interference with analyte detection (small m/z)
Not deteriorating GC-column separation performance
Cheap and not hazardous
Analyte Protectants- Desirable properties
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Active Site masked by AP
Active Sites
PestizidPestizidePesticides
Aktive StelleAP- Elution-Band
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Community Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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51 EU Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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52 EU Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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53 EU Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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67 EU Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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68 EU Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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Community Reference Laboratoryfor Pesticide Residues
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55 potential APs have been individually tested
2-Deoxy-D-ribose, 4,6-O-Ethylidene-a-D-glucose, 4-Hydroxybenzoic acid, Adipic acid, Allylacetic acid, Formic acid, Succinic acid, Pyruvic acid, Brij92 (Diethylene glycol oleyl ether), Butyric acid, Citric acid, Caffeine, D-(+)-Galactose, D-(+)-Gluconic acid-δ-lactone, D-(+)-Glucose, D-(+)-Ribonic acid-γ-lactone, D-(+)-Xylose, D-Fructose, Diethylene glycol, DL-Malic acid, D-Mannite, D-Sorbitol, Acetic acid, Ethylene glycerol, Fumaric acid, Polyethylene glycols (mixture of), Gallic acid, Glutaric acid, Glycolic acid, Glycyl-glycine, Hexaethylene glycol, Caffeic acid, L-(+)-Arabinose, L-(+), Gulonic acid-γ-lactone, Lactose, L-Sorbose, Maleic acid, meso-Erythitol, Lactic acid, myo-Inositol, Oxalic acid, Pentaethylene glycol, Pimelic acid, Propyl gallate, Adonitol, Sucrose, Shikimic acid, Sorbic acid, Tetraethylene glycol, Triethylene glycol, Triglycerol, Valeric acid, Vanillic acid, Tataric acid, Xylitol
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Ranking of potential APs
The ranking system used the ratio of peak height and peak area for all pesticides covered by GC methods.
Important properties of APs: many hydroxy groups (e.g. sugars) proton donation (e.g. acids) to protect
base-sensitive compounds such as captan, folpet and chlorothalonil
vapor pressure corresponding to pesticides
Ranking Analyte Protectant Score
1 D-(+)-Gluconic acid--lactone 2.06
2 4,6-O-Ethylidene--D-glucose 1.77
3 L-(+)-Arabinose 1.76
4 Triglycerol 1.72 5 D-Fructose 1.72
6 D-Sorbitol 1.72
7 Brij ' 92 1.60 8 D-(+)-Glucose 1.59
9 2-Deoxy-D-ribose 1.57
10 Adonitol 1.56 11 Xylitol 1.56
12 Shikimic acid 1.55
13 D-(+)-Xylose 1.55
14 D-(+)-Ribonic acid--lactone 1.42
15 Polyethylene glycols (mix of) 1.37
16 AP Mix 1 1.36 17 L-Sorbose 1.36
18 L-(+)-Gulonic acid--lactone 1.25
19 D-Mannite 1.24 20 Ethyl glycerol. 1.21
21 meso-Erythitol 1.08
22 Vanillic acid 1.06 23 Citric acid 1.01
24 Gallic acid 0.96
25 Caffeic acid 0.83
40 DL-Malic acid 0.25
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Influence of amount of analyte protectants
Beside the type of an analyte protectant also the amount is important!
Example Malic Acid:
increasing protection the more malic acid added
Best at 0.35 mg / mL MeCN(in this experiment)
Carb
aryl
Ditalim
ph
os
Parath
ion
-m
Dico
fol
Cap
tan
Fo
lpet
Ch
loro
thalo
nil
Diclo
fluan
id
To
lyfluan
id
Clo
zolin
ate
TP
P
Pro
cymid
on
pH 4pH 4.5
pH 5pH 5.5
pH 60
1
relative respo
ns
Malic Acid
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Latest mixture of analyte protectants
(E)
OH
OH
OH
O
OH
OO
OH
OH
OH
HO
AP 2-Mix
d-Gluconolactone
Shikimic acid
OH OOH
OHOH
OH
OHOH
OH
AP 1-Mix
Sorbitol
Ethyl glycerol
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Reduction of matrix-effect related errors using the new AP-mixture
Use of APs to Reduce Matrix-Induced Effect Related Errors
0 10 20 30 40 50 60 70 80 90 100 110
TPP (ISTD)
Vinclozolin
Tebufenpyrad
Quinoxyfen
Pyriproxyfen
Pyrimethanil
Pyridaben
Profenofos
Procymidon
Pirimiphos-methyl
Pirimicarb
Phosmet
Pendimethalin
OPP
Myclobutanil
Mirex
Metalaxyl
Mepanipyrim
Malathion
Kresoxim-methyl
Iprodion
Fludioxonil
Fipronil
Fenarimol
Esfenvalerat
Diethofencarb
Dichloran
Cyprodinil
Chlorpyrifos
Biphenyl
Azoxystrobin
Relative Error [%](absolute deviation between signals from pure-solvent standard to signals from matrix-containing standard)
with APs without APs
Average Error- without APs= 22 %
- with APs= 7 %
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Analyte protectants
When using APs, the presence of
matrix is less important.
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Literature AP
AP Publications:M. Anastassiades, K. Maštovská, and S.J. Lehotay J. Chromatogr. A, 1015 (2003) 163-184
K. Maštovska, S.J. Lehotay and M. Anastassiades, Anal Chem 77 (2005) 8129
AP-Posters:M. Anastassiades, S. Lehotay, 'Reduction of Analyte Degradation and Peak Tailing During GC Injection by Addition of Protecting Agents' 4th European Pesticide Residue Workshop in Rome/Italy Awarded as best Poster
M. Anastassiades, E. Scherbaum, B. Tasdelen: ‘Investigations on the use of analyte protectants for multiresidue GC analysis‘6th European Pesticide Residue Workshop in Corfu/Greece
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LC-MSLC-MS
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Why do we need LC-MS if GC-MS is available?
GC requires evaporation of analytes at high temperatures!
Consequently, analytesmust be volatile at 220 – 300°Cshould not be thermo labile
GC MS is not applicable for approximately 25 – 30% of all pesticides!
Some chemical classes cause problems in GC:
• aryloxyalkanoic acids (e.g. 2,4-D)
• benzoylureas (e.g. diflubenzuron)
• carbamates (e.g.desmedipham)
• neonicotinoids (e.g. imidacloprid)
• sulfonylureas (e.g. amidosulfuron)
Slide by Lutz Alder
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Comparison: sensitivity of GC-MS and LC-ESI-MS/MS
0%
20%
40%
60%
80%
100%
organo-P Carbamate organo-Cl Sulfonylurea Triazole Triazine Urea Pyrethroide Alkanoicacid
Other all
GC-MS better LC-MS/MS better equal sensitivity
L. Alder et al., Mass Spectrometry Reviews 25, 838-65 (2006)
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APCI INTERFACEAPCI INTERFACE
N2
SHEATH GAS
NEBULIZER GAS
LCELUENT
HEATER500 ºC
CORONA DISCHARGENEEDLE4-6 KV N2
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Atmospheric pressure Chemical ionization (APCI)
– evaporation of neutral droplets– ionization of neutral molecules induced by hot corona needle– competition for charge in gas phase
Declustering
3. Solvent/Analyte inteaction
Spraygas
Vacuum
2. Ionisation of solvent
Atmospheric pressure
Curtaingas
1. Corona-discharge
Solventl
Analyte
(~120°C)
LC
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IONSPRAYIONSPRAY
ATMOSPHERE VACUUMNEBULIZER GAS
LIQUID SHEATH
STAINLESS STEEL CAPILLARY HOLDER 3-8 KV
STAINLESS STEELHEATED CAPILLARY
LC FLOW
TOANALYZER
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Electrospray Ionization (ESI)
– Formation of droplets containing ions– Shrinking of droplets as solvent evaporates with time (heating
gas)– Ion release to gas phase by coulomb explosion (at high ion
density) or spontaneous evaporation from droplet surface – Competition for charges with matrix in solution– Works best with polar analytes which are either charged in
solution or easily ionized coming out of solution
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LCZ
Off axis
Orthogonal
LC-MS – Spray Types
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XIC of +MRM (175 pairs): 334.0/145.0 amu from Sample 12 (15740 S Ingwer R160 Ho) of DataMonitoring_2008_A_Q.wiff (Turbo Spray) Max. 4.8e5 cps.
12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e5
4.4e5
4.6e5
4.8e5
In
te
ns
ity
, c
ps
15.30
QuEChERSextract of
organic ginger
MRMs of Tebufenpyrad(m/z)
334 > 145334 > 117
Limitations of LC-MS/MSBy M. Jezussek LGL-Erlangen
XIC of +MRM (175 pairs): 334.0/145.0 amu from Sample 4 (Monitoring_2008 0,10 ug/ml Matrix: Spinat) of DataMonitoring_2008_A_Q.wiff (Turbo ... Max. 9.2e4 cps.
12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
8.5e4
9.0e4
In
te
ns
ity
, c
ps
15.39
Tebufenpyrad standard sol.
0.1µg/mLMRMs (m/z)334 > 145334 > 117
XIC of +MRM (175 pairs): 334.0/145.0 amu from Sample 13 (15740 ZV S Ingwer + 1 mg/kg Tebufenpyrad R160 Ho) of DataMonitoring_2008_A_... Max. 4.4e5 cps.
12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e5
4.4e5
In
te
ns
ity
, c
ps
15.33
14.56 15.02
QuEChERSextract of
organic ginger fortified with
Tebufenpyrad
MRMs (m/z)334 > 145334 > 117
LC-MS/MS: “probably yes”
Is it Tebufenpyrad?
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By M. Jezussek LGL-Erlangen
organic ginger extract+ Tebufenpyrad
Reference MS-spectrum of Tebufenpyrad
QuEChERS extract of organic ginger
Background-substracted MS-spectrum
Limitations of LC-MS/MS
GC-MS : “No”
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Matrix Effects in LC-MS applicationsMatrix Effects in LC-MS applications
-
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Matrix effects in LC/MS - Example
0
50
100
150
200
250
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7
2 g/ml
1 g/ml
0,5 g/ml
0,25 g/ml
0,125 g/ml
ohne Matrix
TPP Konzentration [µg/ml]
Fläche
Matrix
FIA mode, no column used
1998
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Unsaturated Just saturated Over-saturated
Cross-sectional views of droplets with increasing analyte concentration
Effect of Analyte Concentration on Analyte Response in ESI
Signal saturation is due to crowding at the droplet surface. Some analyte ions (AH+) are unable to reach the surface.
AH+
AH+
A
AH+AH+
AH+
AH+
A
AH+
AH+AH+
AH+
AH+
AH+
AH+
A
AH+
AH+
AH+ AH+A
AH+AH+
A
AH+AH+
AH+
AH+ AH+
A
AAH+
AH+
A
AH+ AH+AH+
AH+
AAA
AH+
A
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COMPETITION between analyte and electrolyte ions for conversion to gas-phase ions decreases analyte response.
1. Surface competition
2. Charge competition
OAc- + AH+ HOAc + A
Electrolyte concentration
Analyte response
Matrix Effects on Analyte Response in ESI
NH4+
NH4+
NH4+
NH4+OAc-
NH4+OAc-
AH+
AH+NH4+
AH+OAc-
NH4+
A
A
NH4+
NH4+NH4+
AH+
AH+
A
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Matrix effects in LC-API-MS(/MS)
Ionization efficiency in the API source is influenced usually suppression
Effect takes not place during injection or elution (unlike GC)
matrix components and analytes must co-elute
Differences in column retention or deviations in the elution profile may shift position of matrix peak relative to analyte and alter the effect
Differences from commodity to commodity
Spurious matrix effect when matrix from previous injections coelutes
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Compare the calibration curves 1-5-10-25-50-100-250-500 µg/l
If the Slope matrix/Slope solvent: >1 signal enhancement
=1 no matrix effect
<1 signal supression
Standard in SOLVENT; in TOMATO; in PEAR and in ORANGE matrix
Spiroxamine
y = 1085,3x + 2745,3R2 = 0,9984
y = 1270,6x + 1482,3R2 = 0,9999
0
100000
200000
300000
400000
500000
600000
700000
0 100 200 300 400 500 600
Concentration (ng/ml)
Inte
nsi
ty (
cps)
solvent
matrix
Matrix effect: - 15 %
Signal supression in pear
Fipronil
y = 9,5105x - 13,16R2 = 0,997
y = 5,9001x + 47,606R2 = 0,9915
0
1000
2000
3000
4000
5000
6000
0 100 200 300 400 500 600
Concentration (ng/ml)
Inte
nsi
ty (
cps)
matrix
solvent
Matrix effect: + 61 %
Signal enhancement in orange
Matrix Effects in LC/MS – How to detect?
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Metalaxyl
y = 255,98x + 1671,8
R2 = 0,9992
y = 256,1x + 1418,6
R2 = 0,9984
0
20000
40000
60000
80000
100000
120000
140000
0 100 200 300 400 500 600
Concentration (ng/ml)
Inte
ns
ity
(c
ps
)
solvent
matrix
Matrix effect: 0 %
No matrix effect in tomato
Hexythiazox
y = 22,47x - 114,88R2 = 0,9981
y = 89,566x + 764,82R2 = 0,9957
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
0 100 200 300 400 500 600
Concentration (ng/ml)In
ten
sity
(cp
s)
solvent
matrix
Strong signal supression in orange
Matrix effect: -75 %
Matrix Effects in LC/MS - Examples
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Agüera et al. J Chromatogr A 1045 (2004) 125-135
Determination of triflumizole by LC-ESI-MS (m/z 346):Extreme suppression in the case of pepper extracts
Matrix Effects in LC/MS - Examples
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Tomato Pear Orange
9
24
109
71
19
31
5047
3
22 24
5145
8
0
20
40
60
80
100
120
<-50% -50-(-20)% -20 - 0 % 0 - 20 % 20 - 50 % >50 %
Tomate Pera Naranja
fuerte moderado suave suave moderado fuerte
supresión de la señal aumento de la señal
Signal supression Signal enhancement
strong moderate soft strongmoderatesoft
Strong matrix effect
~ 13% of compounds
TomatoPear
Orange
Matrix Effects in LC/MS - Examples
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Intensity ratio matrix standards / standards in solvent
5 matrices x 7 levels x 100 pesticides (= 3500 data)
0%
10%
20%
30%
40%
50%
60%
70%
0-2
0%
20
-40
%
40
-60
%
60
-80
%
80
-10
0%
10
0-1
20
%
12
0-1
40
%
14
0-1
60
%
16
0-1
80
%
18
0-2
00
%
>2
00
%
ESI response in matrix / response in solvent
Perc
en
tag
e o
f to
tal
cases
Tomato
Lemon
Raisin
Wheat flour
Avocado
No problem for screeningMatrix matched standards needed for exact quantification
Matrix Effects in LC/MS - Examples
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Effects of salts on ionization efficiency
Carbamates typical MRM transitions:
[M+H]+ [M - CH3-N=C=O]+
In the presence of Na+ salts :[M+Na]+ [Na]+
Pyrethroides Significant ionization in the presence of NH4
+ salts only
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Dealing with Matrix Effects in LC
Use of isotopically labelled internal standardsExpensive but applicable for single residue methods (see SRM-presentation)
Standard additions ApproachMost used when MRL violation has to be checked
DilutionSimple way as long as instrument sensitivity is sufficientBe careful to avoid precipitations
e.g. non-polar compounds when diluting with water
Better Cleanupeffect only partly eliminated
Better Chromatographic SeparationCan be very helpful (e.g. UPLC) but not always achievable
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• Cleanup• Dilution• Lowering the injection volume …only reduce Matrix effects
Matrix-matched calibration is better but not always practicable!
A good chromatographic separation remains mandatory
Important to know which analytes are significantly affected in which commodity (database can help)!
Dealing with Matrix Effects in LC
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Reduction of supression by cleanup
-1 1 3
-4
215
-1
-2
400
662 1
-14
-3
10
-4-4-5
1
-1
57 6 75
-1 -1 -2-9 -10 -9
-18 -17
-9 -8-9
-50
-40
-30
-20
-10
0
10
201 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 30 31 32 34 34 35 36 37 38 39 40 41 42
Extract of Raisins (1 g/ mL) - Raw
Deviation on average: 5 %
-50
-40
-30
-20
-10
0
10
201 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 30 31 32 34 34 35 36 37 38 39 40 41 42
-1 -2-6
1
-7-2-3
0
-11
1
-7-11
10 63
-6-6 -5
0 -1
-1 -1
-1
-17
-4
-1
711 63 23 202 3332
-4
-50
-40
-30
-20
-10
0
10
201 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 30 31 32 34 34 35 36 37 38 39 40 41 42
Extract of Raisins (1 g/ mL) - after D-SPE
Deviation on average: 4 %
Suppression in raisins sample is low, thus cleanup has minimal effect.
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Reduction of supression by cleanup
-11
7
-26
-49
-14
-67
-4
-61
-27
3
-16
-6
-17-21
-29 -34-27-22
-31-29
-4
15
-17-22
-6-10
-27
-36
-15-16
-21-25-28
-65 -44
11
-16
5
-43-44-50
-40
-30
-20
-10
0
10
201 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 30 31 32 34 34 35 36 37 38 39 40 41 42
Extract of Oranges (1 g/ mL) - Raw
Deviation on average: 24 %
0
-6
-15 -15
1 3
-2
-29
-5 -4-6
05 4 3 3 5 5
-3
4
-36
-10
1114 15
-64
-7-12
9
-17
-59
-21
-8-9
69 8
-11
-22-21
-50
-40
-30
-20
-10
0
10
201 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 20 22 23 24 25 26 27 28 29 30 31 32 34 34 35 36 37 38 39 40 41 42
Extract of Oranges (1 g/ mL) - after freeze-out / D-SPE
Deviation on average: 12 %
Citrus are notoriously problematic as regards matrix effects in LC-MS/MS- Effects are significantly reduced on average but still remain unacceptably high for various compounds! - Analysts should be aware of this! Dilution, Matrix-matching, Std-addition…
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The use of ECHO technique
Eluent AInjectorPump
Detector
HPLC-column
43
2
Waste
Aux. pump-
Eluent A
16
5
Sam
ple
Standard
Pre column
1st injection: standard
By Lutz Alder
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Eluent A
InjectorPumpDetector
HPLC-column
1
43
2
65
Eluent B
Sta
nd
ar
d
Sample
Waste
Pre column
Aux. pump-
Eluent A
The use of ECHO technique
2nd injection: sample (50 seconds later)
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Definition:
Significant matrix effects if signal difference (reduce or enhance) at least 30%
Occurrence of significant matrix effects with 58 standards in solvents
with external stds. with ECHO stds. cucumber 7 4 lemon 40 14 raisin 22 10 wheat flour 6 6
Summary: ECHO standards reduced effects only by 50%.
The use of ECHO technique - Results
Reason: suppression changes virtually from second to second
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Postcolumn infusion to study matrix effects
pump A
pump B
no injection
syringe pumpwith analyte
T-piece
ESI interface
Mass spectrometer
column
0
50000
100000
150000
200000
250000
300000
350000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
time [min]
inten
sity [
cps] Suppression area
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By H. Stahnke
Postcolumn infusion to study matrix effects
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Comparison of matrix effects in different matrixes
Str
on
g e
ffec
tsW
eake
r ef
fect
s
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Matrix-Effects – Comparison GC vs. LC-MS
GC applications LC-MS applications (ESI-mode)
Effects take place in inlet and column Effects take place in ion source (after the chromatogr.run)
Most commonly signal enhancement Most commonly signal suppression
Virtually not influenced by chromatographic resolution Greatly influenced by chromatographic resolution
May change as sequence is progressing due to surface contamination with low volatility
components (matrix induced diminishment effect)Typically not changing as sequence is progressing
Matrix (e.g. electrolyte composition) may influence MS-spectra and ion-yield
Different from analyte to analyte
Different from matrix to matrix
Can be overcome using matrix-matched calibration
Can be overcome using standard additions approach
Can be overcome using isotopically labeled ISTDs
Can be overcome using AP approachAnalyte Protectants (AP) approach does not work
Echo-peak approach does not work Echo-peak approach does work (with limitations)
Ion-yield and MS-spectra are constant
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THE HUMAN FACTOR!!
(Ensure: training possibilities, pleasant working environment)
of a successful lab is…
Don’t forget: The…
(SKILLED and MOTIVATED PEOPLE)
AΩ&
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Thank you very much for your Attention