determination of anionic polar pesticides by lc-ms/ms · 2019. 8. 29. · fosetyl al phosphonic...

©2019 Waters Corporation 1

Determination of anionic polar pesticides by LC-MS/MS

Euan Ross

Principle Market Development Manager

Food and Environmental


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.


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


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


Challenges of Highly Polar Pesticides

Multi residue

approach

Retention

Reliability

Separation

Detection



Multi residue

approach

Retention

Reliability

Separation

Detection


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.


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


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



Multi residue

approach

Retention

Reliability

Separation

Detection


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


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


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


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)


Curve type 5

Curve type 2

Full Scanm/z 50 - 500

AMPA TIC

Understanding matrix complexity:Separating analytes with gradient curves


Analyte Separation

Gradient flexibility for separation in challenging commodities

AMPA

Glufosinate

N-acetyl

glufosinate

Glyphosate

MPPA

N-acetyl AMPA

Ethephon

Phosphonic acid

Fosetyl aluminum


Phosphoric Acid and Phosphonic AcidKey Separation from the QuPPe document (Hypercarb)

EURL-SRM/QuPPe v.10


Phosphoric Acid and Phosphonic Acid

Key Separation from the QuPPe document (DEA M1.7)

EURL-SRM/QuPPe v.10


Suspected phosphoric acid green grape97 > 6381 > 63


Key Separation using Waters DEA column

100 ng/mL phosphonic acid




Suspected phosphoric acid green grape97 > 6381 > 63




Suspected phosphoric acid pineapple97 > 6381 > 63

100 ng/mL phosphonic acid



Multi residue

approach

Retention

Reliability

Separation

Detection


Tandem Quadrupole Systems

Increasing Sensitivity

Refreshed XEVO TQ-S micro XEVO TQ-XS


Bringing Detection into the Routine


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


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





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


Compound name: Glyphosate





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


Calibration curve: 129.083 * x + -39.8463



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


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


Targetlynx: QuantitationData quality and peak flagging



Multi residue

approach

Retention

Reliability

Separation

Detection


Recovery of glyphosate across food commodities without IS correction

Method performance and repeatability:TrendPlot showing method trueness across food commodities


Reference retention time (TRef) TRef± 0.1

Tomato | Cucumber | Wheat flour

Retention time stability:Retention times you can rely on


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


Delivering a reliable and accessible method

Multi residue

approach

Retention

Reliability

Separation

Detection


Column flexibility and reliability:Achievable in the routine laboratory


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


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)


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



Multi residue

approach

Retention

Detection

Separation

Matrix complexity


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


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


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.


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

Blog | Read

chlorate and

perchlorate in milk

https://www.linkedin.com/pulse/example-critical-separations-anionic-polar-pesticides-euan-ross?trk=portfolio_article-card_title

http://www.waters.com/polarpesticides

https://www.linkedin.com/pulse/determination-chlorate-perchlorate-milk-using-torus-dea-euan-ross?trk=portfolio_article-card_title

determination of anionic polar pesticides by lc-ms/ms · 2019. 8. 29. · fosetyl al phosphonic...

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