Analysis of Polar Pesticides Using a Mixed-Phase Mode HPLC Column

Narong Chamkasem, Southeast Food and Feed Laboratory

(SFFL), U.S. Food and Drug Administration, Atlanta, GA

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Disclaimer

• The findings and conclusions in communicated in this presentation are those of the presenter . Any views/opinion expressed do not necessarily represent the views or official position of U.S. Food and Drug Administration (FDA) or the U.S. Department of Health and Human Services (DHHS).

• The names of vendors or manufacturers are provided as examples of available product sources; it does not imply endorsement of vendors, manufacturers, or products by FDA or DHHS.

3 www.fda.gov http://www.crl-pesticides.eu/library/docs/srm/meth_QuPPe.pdf

Existing LC/MS Methods Available for Highly Polar Pesticides

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There are different types of mixed-phase mode columns.

Credit: Thermo Scientific

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There are three types of Acclaim column

Acclaim Trinity Q1

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Salt, pH, % organic solvent are important parameters

Credit: Thermo Scientific

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Obelisc R 98% A (5 mM NH4-Formate pH 3) for 3 min then 100% B (30% acetonitrile 50 mM NH4-formate pH 3)

www.fda.gov

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Polar analyte by LC/MS in a positive mode (Acclaim Q1) 7:3 acetonitrile:water with 50 mM NH4-acetate pH 4.7

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Polar Analyte by LC/MS in a Negative Mode 50 mM NH4-formate pH 2.9

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First Method: Glyphosate in Milk 1 Pipette 1 g milk into a 15-mL tube 2 Add 3 mL Extracting solvent (50 mM acetic acid/10 mM Na2EDTA) 3 Shake 10 min (2000 stroke/min) on a Geno grinder and centrifuge at 3000 x g for 5 min 4 Passing 2 mL of the top layer through a 60 mg/3 mL Oasis HLB (no conditioned needed) 5 Mix the sample with IS and inject to LC/MS HPLC condition: Column - Trinity Acclaim Q1 (100 x 3 mm, 3 um), with a C18 SecurityGuard (4 x 3 mm), 10 uL Mobile phase: 50 mM ammonium formate (pH 2.9) at 0.5 mL/min, 35 ◦C, 6 min, MS condition: AB Sciex 5500 (Q-Trap), Ion-spray neg-mode, source temp 350 C,

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Standard in Solvent with Internal Standard (no need for matrix-matched standard)

Fortification Matrix Matrix Solvent Solventlevel with IS without IS with IS without IS

Glyphosate 25 ng/mL Recovery (%) 81 85 85 123RSD (%) 8.36 9.94 7.62 7.3

100 ng/mL Recovery (%) 107 110 111 106RSD (%) 2.78 2.76 2.19 3.25

500 ng/mL Recovery (%) 93 99 100 98RSD (%) 2.04 2.41 2.05 2.37

2000 ng/mL Recovery (%) 91 97 99 97RSD (%) 3.36 3.15 4.54 3.16

Glufosinate 25 ng/mL Recovery (%) 86 89 90 123RSD (%) 5.43 6.83 7.66 8

100 ng/mL Recovery (%) 106 106 105 96RSD (%) 3.62 3.62 4 4.94

500 ng/mL Recovery (%) 94 101 96 93RSD (%) 6.01 1.64 6.16 1.52

2000 ng/mL Recovery (%) 94 99 97 92RSD (%) 7.07 1.31 7.04 1.38

AMPA 25 ng/mL Recovery (%) 83 89 84 43RSD (%) 2.73 5.12 3.86 2.35

100 ng/mL Recovery (%) 104 115 106 28RSD (%) 6.65 2.98 7.06 3.28

500 ng/mL Recovery (%) 90 113 97 17RSD (%) 4.35 2.53 5.44 2.67

2000 ng/mL Recovery (%) 93 113 100 15RSD (%) 5.17 2.26 6.06 2.62

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Sample Extraction Procedure (Soybean/Corn)

1 2 g sample in a 50-mL tube + 10 mL extracting solvent 2 Shake for 10 min and centrifuge for 5 min 3 Pass 2 mL to a 60 mg/3 mL Oasis HLB SPE 4 Mix the extract with IS and inject to LC/MS

N. Chamkasem and T. Harmon (2016) “Direct determination of glyphosate, glufosinate, and AMPA in soybean and corn by liquid chromatography/tandem mass spectrometry” Anal Bioanal Chem 408:4995-5004 LIB 4604 : Direct determination of glyphosate, glufosinate, and AMPA in egg by liquid chromatography/tandem mass spectrometry

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Soybean Recovery Data soybean (n = 7)

Analyte Fortification Matrix Matrix Solvent Solventlevel (µg/g) with IS no IS with IS no IS

Glyphosate 0.1 Recovery (%) 103 101 102 97RSD (%) 4.26 4.72 3.34 4.66

0.5 Recovery (%) 102 100 101 96RSD (%) 3.98 2.96 3.51 3

2 Recovery (%) 102 103 100 98RSD (%) 2.43 3.07 2.36 3

Glufosinate 0.1 Recovery (%) 102 95 101 76RSD (%) 4.28 5.2 4.13 4.86

0.5 Recovery (%) 102 100 98 75RSD (%) 3.95 1.6 3.83 1.69

2 Recovery (%) 98 104 94 76RSD (%) 2.99 3.85 3.07 3.75

AMPA 0.1 Recovery (%) 101 57 106 NARSD (%) 6.3 28.83 4.53 NA

0.5 Recovery (%) 108 78 107 NARSD (%) 6.35 5.76 4.36 NA

2 Recovery (%) 105 80 108 2RSD (%) 7.59 11.21 5.85 63.53

Calibration method

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Isocratic vs. Gradient Elution for Honey Sample

1 = AMPA 2 = glufosinate 3 = glyphosate 4 = matrix (sugar)

www.fda.gov Laboratory Information Bulletin (2016) no 4613

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Analysis of 2,4-D in Soybean 1 5 g sample + 15 mL water + 300 uL 5 N NaOH and shake 30 min and neutralize with 300 uL 5 N

HCl 2 Shake with 10 mL 1% formic in acetonitrile for 2 min, add QuEChERS salt to separate the layer,

centrifuge, 3 Mix 250 uL acetonitrile with 250 uL water in a syringeless filter vial and inject to LC/MS HPLC: Acclaim Q1 with 3:7 50 mM NH4-formate:acetonitrile at 0.5 mL/min

Std 1 ng/mL

Soybean blank

Soybean blank With 10 ng/g

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Recovery Results (no internal standard)

Note: the reported value was obtained from the sources, not the certified value.

Sample Found (ppm) Reported value (ppm) Source

by this methodSoybean (14A)` 8.2 10.1 USDASoybean (14B) 7.7 10.4 USDACorn Forage 7.4 9.4 Dow AgroSciencesCorn Stover 7.5 8.1 Dow AgroSciences

Soybean forage 10.5 11.2 Dow AgroSciencesSoybean hay 16.3 23.1 Dow AgroSciences

Matrix Fortification Recovery RSD (%)level (ng/g) (%)

Soybean 10 85.6 5.45100 90.5 2.93500 95.4 3.19

Corn 10 100.2 2.32100 98.3 2.7500 107.5 9.66

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Paraquat/Diquat in Potatoes

- 5 g sample in a 50-mL tube, add IS and 15 mL of 1:1 MeOH:0.1% HCl

- Shake 10 min on Geno grinder, heat for 15 min at 80 ◦C then shake for 2 min

- Centrifuge at 3,000 x g for 5 min - Pass thru an Oasis HLB (60 mg) - Inject 10 uL to LC/MS Column: Acclaim Q1 with 7:3 acetonitrile:50mM NH4-acetate(pH 4.7) at 0.5 mL/min for a total run time of 10 min.

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Recovery Data

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Chromatograms and Recovery Data

Matrix Spike levelng/g Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)

Russet Potato 10 101 3.9 81 5.5 98 5.7 92 6.525 89 9.2 86 10.4 93 4.1 93 4.3100 87 6.2 92 6.3 98 5.4 102 5.2500 91 8.3 98 8.0 94 3.1 98 3.1

Yellow Potato 10 97 11.9 96 11.7 106 7.5 121 6.425 97 10.6 96 10.6 98 6.7 102 6.3100 103 7.2 102 7.1 100 4.0 99 3.8500 101 7.1 100 7.0 100 4.3 98 4.0

Paraquat Diquat

std in matrix std in solvent std in matrix std in solvent

Paraquat Diquat

Laboratory Information Bulletin # 4618 (2016), tolerance paraquat (500 ng/g), diquat (100 ng/g)

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Method for Glyphosate (0.2), Maleic Hydrazide (0.2), Fosetyl al (10), and Ethephon(2) in Grape

- 5 g sample in a 50-mL tube + 16 mL of acetic-EDTA solution with 25% MeOH (20 mL final volume)

- Shake 10 min at 2000 stroke/min, centrifuge 3000 x g, pass thru an Oasis HLB (60 mg)

- Add IS and analysis by LC/MS - HPLC condition: 30 sec of water at 0.5 mL/min and step to 100% 50

mM ammonium formate (pH 2.9) for 13 min to remove matrix interfere that may coelute with Fosetyl Al in the next run, then equilibrate to water at 0.7 mL/min for 4 min

- The retention times of MH, glyphosate, fosetyl-Al, and ethephon are 2.7, 3.0, 4.3, and 4.4 min, respectively.

- MH has four prominent precursor/product ion transitions in the MS/MS mode at 111/82, 111/83, 111/55, and 111/42

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Sample Analytes solvent matrix % ME

Purple grape Maleic Hydrazide 210 59 28Glyphosate 239 214 89Fosetyl-Al 3972 296 7Ethephon 888 103 12

Green grape Maleic Hydrazide 195 62 32Glyphosate 254 252 99Fosetyl-Al 3041 387 13Ethephon 888 103 12

Slope

Matrix effect (%ME) evaluation Chromatograms of grapes blank spiked at 100 ng/g with signal/noise ratio

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Recovery Data at 100, 500, 2000 ng/g

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Chlormequat, Mepiquat, Daminozide, Cyromazine, ETU, PTU, Perchlorate,

Chlorate and Amitrole

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Additional Applications (more pesticides and plant growth regulator in fruits/vegetables)

A combination of HILIC-like separation + ion-exchange

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Recovery Data

Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)

Amitrol Sugar Snap beans 96 8.8 105 8.5 85 3.5Carrots 79 16.2 99 3.3 88 19.3Apples 82 11.9 93 5 82 2.2

Cyromazine Sugar Snap beans 80 10.7 103 3.4 88 2.1Carrots 85 5.2 94 1.8 88 22.9Apples 73 4.2 90 8.3 81 2

Mepiquat Sugar Snap beans 136 6.2 121 10.5 119 8.2Carrots 109 9.1 103 10.1 106 5.9Apples 127 8.5 112 6.1 107 8.8

Chlormequat Sugar Snap beans 100 9.7 101 4.5 104 5.7Carrots 82 13.4 93 9.5 94 6.4Apples 84 16.7 103 8.7 90 5.6

ETU Sugar Snap beans 86 11.4 97 6.8 82 5.9Carrots 82 1.3 100 2.8 88 20.8Apples 76 4.6 94 11.3 82 1.9

PTU Sugar Snap beans 94 5.9 111 2.1 92 4.8Carrots 98 4.2 117 1.7 107 20.1Apples 74 5 94 8.6 88 5.0

Perchlorate Sugar Snap beans 92 6.7 113 2.1 88 2.3Carrots 70 28.6 95 9.4 90 22.4Apples 74 3.5 94 7.9 85 1.6

Daminozide Sugar Snap beans 88 3.8 104 4.7 83 3.2Carrots 101 11.5 108 20.2 98 19.8Apples 74 4.9 89 11 76 3

20 ng/g 40 ng/g 250 ng/g

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More to come