Agilent 6470 Triple QuadrupoleLC/MS System and EMR-Lipid

–The Combination for Confident

Pesticide Quantitation

Christoph Mueller

Agilent Technologies

LCMS Application Scientist

EMEAI Food Market Program Manager

Agenda

• 6470 QQQ innovations and quantitation performance

• Experimental• Sample Preparation• LC-QQQ method

• Evaluation of Matrix Removal

• Evaluation of Pesticide Recovery

6470 Innovations

Agilent 1290 Infinity II

High Speed Pump

Multisampler

Multicolumn Thermostat

Upgradeable to 6495 QQQ with iFunnel Technology

Experimental

• Samples• Avocado (high lipid content)• Spinach (low lipid content)

• Pesticides• Test mix from Agilent Pesticides tMRM Application Kit (G1733BA) with 250

pesticides• + some herbizides for negative ion performance evaluation• Spiked to samples before extraction to asses recovery 10 and 100 ng/g• Spiked to extracts after extraction 10 and 100 ng/g

• Extraction• EN QuEChERS extraction salt + PSA/C18 dSPE (for high lipid content)• EN QuEChERS extraction salt + EMR lipid dSPE + EMR lipid polish salts

EMR vs QuEChERS workflow

400 µL extract + 600 µL water in autosampler vial. (Spinach: 200 µL extract + 800 µL water)

Vortex 1 min, centrifuge at 5,000 rpm for 5 min.

Transfer 6 mL to Bond Elut EN dispersive SPE 15 mL tube (PSA/C18)

Centrifuge (5000 rpm, 5 min)

Add Bond Elut EN extraction salts, shake 1 min

5g Avocado (10 g spinach) + STD, 5 mL water, 10 mL ACN (1% acetic acid) in 50 mL centrifuge tube, shake 1 min

200 µL extract + 800 µL water in autosampler vial.

Vortex 1 min, centrifuge at 5,000 rpm for 5 min.

Transfer 5 mL of supernatant to a EMR—Lipid polish tube.

Vortex 1 min, centrifuge at 5,000 rpm for 5 min.

Add 5 mL water, then 5 mL of ACN extract to EMR-Lipid dSPE 15 mL tube

Centrifuge (5000 rpm, 5 min)

Add Bond Elut EN extraction salts, shake for 2 min

10 g Avocado (10 g spinach) + STD, 10 mL ACN (1% acetic acid) in 50 mL centrifuge tube.

PSA/C18 EMR

UHPLC and QQQ ParametersUHPLC column Agilent Zorbax Eclipse Plus C18 RRHD

2.1 x 150 mm, 1.8 µm

Mobile Phase A: water + 5 mM ammonium acetate + 0.1% acetic acidB: methanol + 5 mM ammonium acetate + 0.1% acetic acid

Gradient program

Min.031720

%B5

30100100

Flow rate 0.4 mL/min

Dynamic MRM acquisition with fast polarity switching

(for matrix evaluation: precursor ion scanand MS scan)

AVOCADO MATRIX

Evaluation of Matrix Removal

6x10

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

1.8

1.9

2

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 CID@20.0 (** -> **) Avocado EN acid 0.d

Counts 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

Precursor Ion Scan for Product Ion m/z 184 – Phosphatidylcholin- Phospholipids = Group of compounds known for causing ion supression- Q1 Scan – Collision C. – Q2 fixed at m/z 184

6x10

0.2

0.225

0.25

0.275

0.3

0.325

0.35

0.375

0.4

0.425

0.45

0.475

0.5

0.525

0.55

0.575

0.6

0.625

0.65

0.675

0.7

0.725

0.75

0.775

0.8

0.825

0.85

0.875

0.9

0.925

0.95

0.975

+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 CID@20.0 (** -> **) Avocado EN acid 0.d

Counts vs. Acquisition Time (min)15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8 17 17.2 17.4 17.6 17.8 18 18.2 18.4 18.6 18.8 19 19.2 19.4 19.6 19.8 20 20.2 20.4 20.6 20.8 21 21.2 21.4 21.6 21.8

5x10

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

3.8

4

4.2

4.4

4.6

4.8

+ESI EIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 CID@20.0 (778.6000, 780.0000, 782.8000, 784.8000 ... -> 184.0000) Avocado EN acid 0.d

Counts 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

Comparison of PC-(18:n/18:n) between EMR-Lipid and PSA/C18Increasing level of saturation of fatty acid Increasing level of removal

5x10

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

3.8

4

4.2

4.4

4.6

4.8

+ESI EIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 CID@20.0 (778.6000, 780.0000, 782.8000, 784.8000 ... -> 184.0000) Avocado EN acid 0.d

Counts vs. Acquisition Time (min)18.3 18.4 18.5 18.6 18.7 18.8 18.9 19 19.1 19.2 19.3 19.4 19.5 19.6 19.7 19.8 19.9 20 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9 21 21.1 21.2 21.3 21.4 21.5 21.6 21.7 21.8 21.9

LLPCPC(18:2/18:2)

OLPCPC(18:1/18:2)

OOPCPC(18:1/18:1)

Lyso-PC-(C18:n/0:0)

Significantly smaller than PC, but FA saturation has the same effect on level of removal6x10

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

1.8

1.9

2

+ESI EIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 CID@20.0 (518.5000, 520.5000, 522.5000 -> 184.0000) Avocado EMR 0-r002.d

Counts vs. Acquisition Time (min)15.7 15.8 15.9 16 16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9 17 17.1 17.2 17.3

Lyso-PC-(C18:3/0:0)Lyso-PC-(C18:2/0:0)

Lyso-PC-(C18:1/0:0)

Avocado EMR 10 [Fenpyroximat]

16.25 16.3 16.35 16.4 16.45 16.5

4x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

422.1 -> 366.2 Area=83779

422.1 -> 135.1 Area=20859

Avocado EN acid 10 [Fenpyroximat]

16.25 16.3 16.35 16.4 16.45 16.5

4x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

422.1 -> 366.2 Area=94675

422.1 -> 135.1 Area=22753

Assessment of Total Extracted MatrixMS Scan, High Temperature and Extended Chromatography

7x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5

3.75

4

4.25

4.5

4.75

5

5.25

5.5

5.75

6

6.25

6.5

6.75

7

+ESI TIC Scan Frag=135.0V CF=0.000 DF=0.000 Avocado EN acid 0.d

Counts 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 34 35 36 37 38 39

Diacylglycerols(+ some MGDG)

Triacylglycerols???

SPINACH MATRIX

Evaluation of Matrix Removal

Precursor Ion Scan for Product Ion m/z 184 – Phosphatidylcholin- Group of compounds known for causing ion supression- Q1 Scan – Collision C. – Q2 fixed at m/z 184

6x10

0

0.5

1

1.5

2

2.5

+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 CID@20.0 (** -> **) Avocado EN acid 0.d

6x10

0

0.5

1

1.5

2

2.5

+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 CID@20.0 (** -> **) Spinach EN 0.d

6x10

0

0.5

1

1.5

2

2.5

+ESI TIC Precursor Ion Frag=150.0V CF=0.000 DF=0.000 CID@20.0 (** -> **) Spinach EMR 0.d

Counts vs. Acquisition Time (min)14.8 15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8 17 17.2 17.4 17.6 17.8 18 18.2 18.4 18.6 18.8 19 19.2 19.4 19.6 19.8 20 20.2 20.4 20.6 20.8 21 21.2 21.4 21.6 21.8

Avocado PSA/C18

Spinach PSA/C18

Spinach EMR-Lipid

Assessment of Total Extracted MatrixMS Scan, High Temperature and Extended Chromatography

7x10

0

1

2

3

4

5

6

+ESI TIC Scan Frag=135.0V CF=0.000 DF=0.000 Avocado EN acid 0.d

7x10

0

1

2

3

4

5

6

+ESI TIC Scan Frag=135.0V CF=0.000 DF=0.000 Spinach EN 0.d

7x10

0

1

2

3

4

5

6

+ESI TIC Scan Frag=135.0V CF=0.000 DF=0.000 Spinach EMR 0.d

Counts 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 34 35 36 37 38 39

Avocado PSA/C18

Spinach PSA/C18

Spinach EMR-Lipid

PESTICIDE RESULTS

Excellent performance at default MRL (10µg/kg)

Pirimicarb

Signal Qualityin region with large max. number of concurrent MRMs

Uniconazole-P Fipronil Diflubenzuron Flusilazole

Fenamiphos Picoxystrobin Bupirimate Fenoxycarb

Recovery of Pesticides from Avocado MatrixEMR Lipid vs QuEChERS• 256 pesticides spiked

• Reference for recovery is solvent calibration, which results in greater deviation from 70-120% range.

EMR-Lipid PSA/C18

Lower Recovery with EMR-Lipid vs. PSA/C18

Lower Recovery in PSA/C18 vs. EMR-Lipid

Better Recovery with PSA/C18

Avocado EMR 10 [Propamocarb]

3.2 3.25 3.3 3.35 3.4 3.45

4x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

189.1 -> 102.1 Area=112744

189.1 -> 144.0 Area=7224

Avocado EN acid 10 [Propamocarb]

3.2 3.25 3.3 3.35 3.4 3.45

4x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

189.1 -> 102.1 Area=203910

189.1 -> 144.0 Area=12887

Avocado EMR 10 [Oxamyl]

3.8 3.85 3.9 3.95 4 4.05 4.1

4x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

237.0 -> 72.1 Area=63456

237.0 -> 90.1 Area=12043

Avocado EN acid 10 [Oxamyl]

3.8 3.85 3.9 3.95 4 4.05 4.1

4x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

237.0 -> 72.1 Area=157305

237.0 -> 90.1 Area=29811

Avocado EMR 10 [Bifenazate]

12.65 12.7 12.75 12.8 12.85 12.9 12.95 13

4x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

301.1 -> 198.2 Area=20500

301.1 -> 170.1 Area=10103

Avocado EN acid 10 [Bifenazate]

12.65 12.7 12.75 12.8 12.85 12.9 12.95 13

4x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

301.1 -> 198.2 Area=107094

301.1 -> 170.1 Area=54042

Propamocarb Oxamyl Bifenazate

EMR

PSA/C18

Better Recovery with PSA/C18

Avocado EMR 10 [Spirotetramat]

12.95 13 13.05 13.1 13.15 13.2

4x10

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

374.1 -> 330.3 Area=17378

374.1 -> 302.2 Area=14487

Avocado EN acid 10 [Spirotetramat]

12.95 13 13.05 13.1 13.15 13.2

4x10

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

374.1 -> 330.3 Area=55790

374.1 -> 302.2 Area=46358

Avocado EMR 10 [Tribenuron-methyl]

10.4 10.45 10.5 10.55 10.6 10.65

3x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

396.0 -> 155.1 Area=18365

396.0 -> 181.1 Area=10656

Avocado EN acid 10 [Tribenuron-methyl]

10.4 10.45 10.5 10.55 10.6 10.65

3x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

396.0 -> 155.1 Area=30147

396.0 -> 181.1 Area=18051

Tribenuron-methyl Spirotetramat

EMR

PSA/C18

Better Recovery with EMR-Lipid

Avocado EMR 10 [Quinmerac]

5.1 5.15 5.2 5.25 5.3 5.35

4x10

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

222.0 -> 204.0 Area=60598

222.0 -> 141.1 Area=17360

Avocado EN acid 10 [Quinmerac]

5.1 5.15 5.2 5.25 5.3 5.35

4x10

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

222.0 -> 204.0 Area=6033

222.0 -> 141.1 Area=1759

Avocado EMR 10 [Triclopyr]

9.35 9.4 9.45 9.5 9.55 9.6 9.65 9.7 9.75

2x10

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

253.9 -> 195.9 Area=938

255.9 -> 197.9 Area=957

Avocado EN acid 10 [Triclopyr]

9.35 9.4 9.45 9.5 9.55 9.6 9.65 9.7 9.75

2x10

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

253.9 -> 195.9 Area=267

255.9 -> 197.9 Area=158

Avocado EMR 10 [Dichlorprop]

9.9 9.95 10 10.05 10.1 10.15 10.2

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

233.0 -> 161.0 Area=3332

235.0 -> 163.0 Area=2090

Avocado EN acid 10 [Dichlorprop]

9.9 9.95 10 10.05 10.1 10.15 10.2

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

233.0 -> 161.0 Area=906

235.0 -> 163.0 Area=623

Quinmerac Triclopyr Dichlorprop

EMR

PSA/C18

Better Recovery with EMR-Lipid

Avocado EMR 10 [2,4,5-T]

10.2 10.25 10.3 10.35 10.4 10.45

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

252.9 -> 194.9 Area=2463

252.9 -> 158.9 Area=498

254.9 -> 196.9 Area=2423

Avocado EN acid 10 [2,4,5-T]

10.2 10.25 10.3 10.35 10.4 10.45

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

252.9 -> 194.9 Area=548

252.9 -> 158.9 Area=140

254.9 -> 196.9 Area=571

Avocado EMR 10 [Bispyribac]

11.3 11.35 11.4 11.45 11.5 11.55 11.6

3x10

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

431.0 -> 275.1 Area=37806

431.0 -> 413.1 Area=11233

Avocado EN acid 10 [Bispyribac]

11.3 11.35 11.4 11.45 11.5 11.55 11.6

3x10

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

431.0 -> 275.1 Area=14581

431.0 -> 413.1 Area=5362

Avocado EMR 10 [Moxidectin]

17.3 17.35 17.4 17.45 17.5 17.55

3x10

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

*640.4 -> 622.2 Area=1103

640.4 -> 528.2 Area=29

Avocado EN acid 10 [Moxidectin]

17.3 17.35 17.4 17.45 17.5 17.55

3x10

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

*640.4 -> 622.2 Area=880

640.4 -> 528.2 Area=35

2,4,5-T Bispyribac Moxidectin

EMR

PSA/C18

Summary

• EMR-lipid clean-up dramatically reduces lipid contentinjected onto colum, while maintaining analyte recovery aswith other dSPE sorbents.

• EMR-lipid clean-up improves robustness of the method andlongevity of column and mass spectrometer.

• Productivity gains through less maintenance downtime.

• Sensitive and robust 6470 QQQ is perfect match for theanalysis of diluted extracts after EMR-lipid clean-up.

• Thank You!