determination of anionic polar pesticides by lc-ms/ms · 2019. 8. 29. · fosetyl al phosphonic...
TRANSCRIPT
©2019 Waters Corporation 1
Determination of anionic polar pesticides by LC-MS/MS
Euan Ross
Principle Market Development Manager
Food and Environmental
©2019 Waters Corporation 2
Why is glyphosate such a challenge to analyse?
Method Step Challenges
Sample Preparation
Glyphosate is very polar, water soluble and insoluble in organic
solvents, and will bind to metal ions, therefore making the
extraction possibilities limited. Derivatization can be very time
consuming.
SeparationInsufficient retention on column using reverse phase, requires
derivatization, HILIC or IEX stationary phase.
Analysis
Within the extract, other water soluble matrix compounds can
also be found (proteins, sugars, amino acids, salts, etc.) that
interfere with the determination of glyphosate. The use of
derivatization is not compound specific and can lead to selectivity
issues.
Glyphosate used as a desiccant on cereal
crops to aid harvest-results in increased
frequency of residues in products such as bread and breakfast cereals
and beer.
©2019 Waters Corporation 3
Sample PreparationApproaching multi residue extraction with QuPPe method
Weigh homogenized sample (5 - 10g) into centrifuge tube
(adjust for water content)
Add cold methanol (10 ml) containing 1 % formic acid
Vortex thoroughly for 1 minutes
(cereals, wheat flour freeze for 2 hrs)
Centrifuge at 5000 rpm for 5 minutes
(chilled if possible)
Filter supernatant (0.45 µm, PVDF, filter) into a plastic vial
spike commodity with labelled IS or
reference material
©2019 Waters Corporation 4
Sample Preparation
Approaching multi residue extraction with QuPPe method
Compound % Recovery
(Trueness)
% RSDr
AMPA 100.2 6.6
Glyphosate 99.2 4.5
Glufosinate 112.0 3.1
Ethephon 100.3 7.0
Fosetyl 101.2 5.6
MPPA 106.8 6.0
0.010 mg/kg spike level, green grape
©2019 Waters Corporation 5
Challenges of Highly Polar Pesticides
Multi residue
approach
Retention
Reliability
Separation
Detection
©2019 Waters Corporation 6
Challenges of Highly Polar Pesticides
Multi residue
approach
Retention
Reliability
Separation
Detection
©2019 Waters Corporation 7
Calculating Column Void Volume
Void Volume (v) is the volume in a column which is not taken up by stationary
phase.
V = 0.7 x π x (i.d/2)2 x L
V = 0.7 x π x (2.1/2)2 x 100
V = 0.242 mL
Column Length
Column I.D.
©2019 Waters Corporation 8
Calculating the retention time corresponding to the void volume
Time required for one volume of mobilephase to exit the column
= V
F
Where v= void volume and F= flow rate of the LC method
t0 = 0.484
2.1 mm x 100mm
V =0.242 ml
F =0.5 ml/min
t0= 0.484 min
2.44
©2019 Waters Corporation 9
RetentionRetaining analytes to maximise performance
Time1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50
%
0
100
t0 = 0.97 min
The minimum acceptable retention time for the analyte(s) should be at minimum twice the retention time corresponding to
the void volume of the column (SANTE/11813/2017):
All analytes are eluting
after 1.5 minutes.
All analytes are retained
SANTE requirements: 0.484 min x 2
Retained if eluting after = 0.968 minutes
©2019 Waters Corporation 10
Challenges of Highly Polar Pesticides
Multi residue
approach
Retention
Reliability
Separation
Detection
©2019 Waters Corporation 11
Critical Separations to avoid false detections:Isobaric interferences from targeted analytes
AMPA
fosetyl-al
phosphonic acid
fosetyl isobaric interference in
AMPA and phosphonic acid MRM
transitions.
110 < 63
109 < 63
81 < 63
©2019 Waters Corporation 12
AMPA
N-acetyl AMPA
N-acetyl AMPA isobaric
interference in AMPA MRM
transitions.
Critical Separations to avoid false detections:Isobaric interferences from targeted analytes
81 < 63 m/z
©2019 Waters Corporation 13
Chromatographically resolving matrix interferences:RADAR as a method development tool to under matrix complexity
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
5
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
m/z 133
(precursor)
m/z 115
(fragment)
Suspected malic acid
134.0874 g/mol
RADAR
TIC
RADAR
XIC
RADAR
XIC
©2019 Waters Corporation 14
Understanding matrix complexity:Separating analytes from matrix interferences to minimise matrix effects
AMPA in tomato extract
Glufosinate in tomato extract
malic acid standard
(prepared in QuPPe extraction solution)
suspected malic acid peak in tomato extract
(confirmed by ion ratios of the standard)
©2019 Waters Corporation 15
Curve type 5
Curve type 2
Full Scanm/z 50 - 500
AMPA TIC
Understanding matrix complexity:Separating analytes with gradient curves
©2019 Waters Corporation 16
Analyte Separation
Gradient flexibility for separation in challenging commodities
AMPA
Glufosinate
N-acetyl
glufosinate
Glyphosate
MPPA
N-acetyl AMPA
Ethephon
Phosphonic acid
Fosetyl aluminum
©2019 Waters Corporation 17
Phosphoric Acid and Phosphonic AcidKey Separation from the QuPPe document (Hypercarb)
EURL-SRM/QuPPe v.10
©2019 Waters Corporation 18
Phosphoric Acid and Phosphonic Acid
Key Separation from the QuPPe document (DEA M1.7)
EURL-SRM/QuPPe v.10
©2019 Waters Corporation 19
Suspected phosphoric acid green grape97 > 6381 > 63
Phosphoric Acid and Phosphonic Acid
Key Separation using Waters DEA column
100 ng/mL phosphonic acid
©2019 Waters Corporation 20
Phosphoric Acid and Phosphonic Acid
Key Separation using Waters DEA column
Suspected phosphoric acid green grape97 > 6381 > 63
©2019 Waters Corporation 21
Phosphoric Acid and Phosphonic Acid
Key Separation using Waters DEA column
Suspected phosphoric acid pineapple97 > 6381 > 63
100 ng/mL phosphonic acid
©2019 Waters Corporation 22
Challenges of Highly Polar Pesticides
Multi residue
approach
Retention
Reliability
Separation
Detection
©2019 Waters Corporation 23
Tandem Quadrupole Systems
Increasing Sensitivity
Refreshed XEVO TQ-S micro XEVO TQ-XS
©2019 Waters Corporation 24
Bringing Detection into the Routine
©2019 Waters Corporation 25
0
5
10
15
20
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8
RSD
r (%
)
Tru
enes
s (%
)
Series1 Series2 Series3 Series4 Series5 Series6SANTE defined tolerances
Method performance and repeatability:Accuracy and precision
©2019 Waters Corporation 26
Detection to quantitation:Accurately quantifying residues, well below current MRLs
Compound name: Ampa
Correlation coefficient: r = 0.999334, r^2 = 0.998668
Calibration curve: 40.9902 * x + 5.22407
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc-0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Response
-0
1000
2000
3000
4000
Conc
Resid
ual
-4.0
-2.0
0.0
2.0
4.0
6.0
8.0
Compound name: Glyphosate
Correlation coefficient: r = 0.997185, r^2 = 0.995378
Calibration curve: 653.409 * x + 31.5536
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc-0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Response
-0
10000
20000
30000
40000
50000
60000
Conc
Resid
ual
-10.0
-5.0
0.0
5.0
10.0
Calibration series representing 0.005 to 0.2 mg/kg in matrix
AMPA Glyphosate
©2019 Waters Corporation 27
Compound name: Glyphosate
Correlation coefficient: r = 0.999119, r^2 = 0.998238
Calibration curve: 530.208 * x + 72.8129
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Standard Addition Concentration : 0.137329
Conc-0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Res
pons
e
-0
20000
40000
Conc
Res
idua
l
-5.0
0.0
5.0
10.0
min5.050 5.100 5.150 5.200 5.250 5.300 5.350 5.400 5.450 5.500 5.550 5.600 5.650 5.700 5.750
%
0
100
HPEU_021_220819_120
QuPPe Flour 2.5 ng/mLGlyphosate
5.35
1378
min
%
0
100
HPEU_021_220819_120
QuPPe Flour 2.5 ng/mLGlyphosate
5.35
1580
0.010 mg/kg
Compound name: Glufosinate
Correlation coefficient: r = 0.999725, r^2 = 0.999451
Calibration curve: 129.083 * x + -39.8463
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc-0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Res
pons
e
-0
2500
5000
7500
10000
12500
Conc
Res
idua
l
-5.0
-2.5
0.0
2.5
5.0
min2.850 2.900 2.950 3.000 3.050 3.100 3.150 3.200 3.250 3.300 3.350
%
0
100
HPEU_021_220819_120
QuPPe Flour 2.5 ng/mLGlufosinate
3.05
233
2.85
min
%
0
100
HPEU_021_220819_120
QuPPe Flour 2.5 ng/mLGlufosinate
3.05
307
0.010 mg/kg – 0.400 mg/kg wheat flour matrix matched 0.010 mg/kg
Detection to quantitation:Accurately quantifying residues, below current MRLs
0.010 mg/kg – 0.400 mg/kg wheat flour matrix matched
©2019 Waters Corporation 28
Red Grape SampleEU MRL Table Grape: 1 mg/kgMeasure Concn: 0.144 mg/kg
Green Grape matrix matched 0.020 – 0.200 mg/kg
Targetlynx: QuantificationIncurred residues and standard addition
©2019 Waters Corporation 29
Targetlynx: QuantitationData quality and peak flagging
©2019 Waters Corporation 30
Challenges of Highly Polar Pesticides
Multi residue
approach
Retention
Reliability
Separation
Detection
©2019 Waters Corporation 31
Recovery of glyphosate across food commodities without IS correction
Method performance and repeatability:TrendPlot showing method trueness across food commodities
©2019 Waters Corporation 32
Reference retention time (TRef) TRef± 0.1
Tomato | Cucumber | Wheat flour
Retention time stability:Retention times you can rely on
©2019 Waters Corporation 33
Green Grape, batch injection 38
Red Grape, batch injection 62
Pineapple, batch injection 86
Wheat Flour, batch injection 119
Wheat Flour, batch injection 145
100 ng/mL in vial matrix matched
standards of glyphosate
through a single batch of sample
analysis.
Reliable PerformanceGlyphosate multiple commodities in a single run
©2019 Waters Corporation 34
Delivering a reliable and accessible method
Multi residue
approach
Retention
Reliability
Separation
Detection
©2019 Waters Corporation 35
Column flexibility and reliability:Achievable in the routine laboratory
©2019 Waters Corporation 36
Methodology:LC-MS/MS
Solvent A 0.9% formic acid in LCMS water
Solvent B 0.9% formic acid in aectonitrile
Time (min) %A %B Curve
0 10 90 -
4.50 60 40 2
8.50 60 40 6
15.50 10 90 1
Time (min) %A %B Curve
0 10 90 -
4.00 85 15 5
13.00 85 15 6
18.50 10 90 1
Solvent A50 mM ammonium formate with 0.9% formic
Acid
Solvent B 0.9% Formic Acid in acetonitrile
AMPA
Glyphosate
Glufosinate
MPPA
N-Acetyl Glufosinate
N-Acetyl AMPA
N-Acetyl Glyphosate
Ethephon
HEPA
Fosetyl-Al
Phosphonic Acid
Chlorate
Perchlorate
Bromate
Method A: with buffer Method B: without buffer
Waters’ Anionic Polar
Pesticides column
©2019 Waters Corporation 37
Chromatogram at 0.1 mg/kg in Wheat flour
Phosphonic acid
Fosetyl Al
AMPA
Glyphosate
N-acetyl glyphosate
N-acetyl glufosinate
Ethepon
Glufosinate
MPPA
AMPA - 6.25%
Glyphosate – 2.79%
Glufosinate – 1.35%
All compound showed %RSD < 10% for 0.1 mg/kg (n=15) and 0.25 mg/kg (n=15)
©2019 Waters Corporation 38
Time0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
%
0
100
AMPA Method BGlufosinate Method A
AMPA Method A
Glyphosate Method A
Glufosinate Method B
Glyphosate Method B
Detection Improvements using formic acid mobile phase:0.010 mg/kg in tomato QuPPe extract
©2019 Waters Corporation 39
Challenges of Highly Polar Pesticides
Multi residue
approach
Retention
Detection
Separation
Matrix complexity
©2019 Waters Corporation 40
Chlorate and perchlorate in milk and brusselsprouts
1,9
2
2,1
2,2
2,3
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
Retention Time (min) Chlorate in Matrix RT in matrix
Lower Limit - 0.1 min
Upper Limit + 0.1 min
Time0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
1000.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
100
Compound name: Chlorate
Coefficient of Determination: R^2 = 0.997883
Calibration curve: -0.00109872 * x^2 + 4.82846 * x + 236.465
Response type: External Std, Area
Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x, Axis trans: None
Standard Addition Concentration : 48.9732
-0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
Response
-0
2000
Resid
ual
-5.0
0.0
5.0
QuPPe solvent, passed through Oasis
PRiME HLB + PVDF filter
Compound name: ChlorateResponse Factor: 4.77771RRF SD: 0.152779, % Relative SD: 3.19775Response type: External Std, AreaCurve type: RF
-0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Response
-0
200
400
600
Resid
ual
-5.0
0.0
perchlorate
chlorate
©2019 Waters Corporation 41
Summary
Method
solution
Retention
Reliability
Separation
Detection
Achieved on standard UPLC-MS/MS technology, without the need to derivatise.
Providing performance to exceed the current regulatory (MRLs) requirements in crude food extracts.
Delivering comprehensive solution for success and confidence with application support.
©2019 Waters Corporation 42
Interested in finding out more
Blogs on LinkedIn Application noteWebinar with slides and Q&A
Blog | Read
critical separations of
anionic polar pesticides
in food commodities
#polarpesticides
Visit: www.waters.com/polarpesticides
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chlorate and
perchlorate in milk