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

Transcription

1 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

2 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. 2

3 Existing LC/MS Methods Available for Highly Polar Pesticides 3

4 There are different types of mixed-phase mode columns. Credit: Thermo Scientific 4

5 There are three types of Acclaim column Acclaim Trinity Q1 5

6 Salt, ph, % organic solvent are important parameters Credit: Thermo Scientific 6

7 Obelisc R 98% A (5 mm NH4-Formate ph 3) for 3 min then 100% B (30% acetonitrile 50 mm NH4-formate ph 3) 7

8 Polar analyte by LC/MS in a positive mode (Acclaim Q1) 7:3 acetonitrile:water with 50 mm NH4-acetate ph 4.7 8

9 Polar Analyte by LC/MS in a Negative Mode 50 mm NH4-formate ph 2.9 9

10 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 Na 2 EDTA) 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, J. of Regulatory Science 02(2015)

11 Standard in Solvent with Internal Standard (no need for matrix-matched standard) Fortification Matrix Matrix Solvent Solvent level with IS without IS with IS without IS Glyphosate 25 ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%) Glufosinate 25 ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%) AMPA 25 ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%) ng/ml Recovery (%) RSD (%)

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13 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: LIB 4604 : Direct determination of glyphosate, glufosinate, and AMPA in egg by liquid chromatography/tandem mass spectrometry 13

14 Soybean Recovery Data soybean (n = 7) Calibration method Analyte Fortification Matrix Matrix Solvent Solvent level (µg/g) with IS no IS with IS no IS Glyphosate 0.1 Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Glufosinate 0.1 Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) AMPA 0.1 Recovery (%) NA RSD (%) NA 0.5 Recovery (%) NA RSD (%) NA 2 Recovery (%) RSD (%)

15 Isocratic vs. Gradient Elution for Honey Sample 1 = AMPA 2 = glufosinate 3 = glyphosate 4 = matrix (sugar) Laboratory Information Bulletin (2016) no

16 Analysis of 2,4-D in Soybean 1 5 g sample + 15 ml water 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 J. Reg. Science 2(2016)

17 Recovery Results (no internal standard) Matrix Fortification Recovery RSD (%) level (ng/g) (%) Soybean Corn Sample Found (ppm) Reported value (ppm) Source by this method Soybean (14A)` USDA Soybean (14B) USDA Corn Forage Dow AgroSciences Corn Stover Dow AgroSciences Soybean forage Dow AgroSciences Soybean hay Dow AgroSciences Note: the reported value was obtained from the sources, not the certified value. 17

18 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. J. of Reg Science 05(2017)1-8 18

19 Recovery Data 19

20 Chromatograms and Recovery Data Paraquat Diquat Paraquat Diquat Matrix Spike level std in matrix std in solvent std in matrix std in solvent ng/g Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Russet Potato Yellow Potato Laboratory Information Bulletin # 4618 (2016), tolerance paraquat (500 ng/g), diquat (100 ng/g) 20

21 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 21

22 Matrix effect (%ME) evaluation Chromatograms of grapes blank spiked at 100 ng/g with signal/noise ratio Slope Sample Analytes solvent matrix % ME Purple grape Maleic Hydrazide Glyphosate Fosetyl-Al Ethephon Green grape Maleic Hydrazide Glyphosate Fosetyl-Al Ethephon

23 Recovery Data at 100, 500, 2000 ng/g 23

24 Chlormequat, Mepiquat, Daminozide, Cyromazine, ETU, PTU, Perchlorate, Chlorate and Amitrole 24

25 Additional Applications (more pesticides and plant growth regulator in fruits/vegetables) A combination of HILIC-like separation + ion-exchange 25

26 Recovery Data 20 ng/g 40 ng/g 250 ng/g Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Amitrol Sugar Snap beans Carrots Apples Cyromazine Sugar Snap beans Carrots Apples Mepiquat Sugar Snap beans Carrots Apples Chlormequat Sugar Snap beans Carrots Apples ETU Sugar Snap beans Carrots Apples PTU Sugar Snap beans Carrots Apples Perchlorate Sugar Snap beans Carrots Apples Daminozide Sugar Snap beans Carrots Apples

27 More to come 27

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