1

MultiresidueMultiresidue Screening of Agricultural Screening of Agricultural Chemicals in a Food Matrix using Varian Chemicals in a Food Matrix using Varian SPE and Triple Quadrupole LCSPE and Triple Quadrupole LC--MS/MS MS/MS

Eugene Chang, Anita DerMartirosian, Guang Li, Nigel Simpson, and Ritu Arora

Varian, Inc.Lake Forest, CA

Marcus Kim

Varian Canada

•• According to Japanese Ministry of Agriculture, Forestry and FiAccording to Japanese Ministry of Agriculture, Forestry and Fishery shery Government data, Japan's domestic production supplies 40% of fooGovernment data, Japan's domestic production supplies 40% of foods ds consumed, based on calorie supply. 60% of foods consumed is impconsumed, based on calorie supply. 60% of foods consumed is imported. orted. Japan can no longer support its people's diet without imported fJapan can no longer support its people's diet without imported foodood

•• On May 29, 2006, JapanOn May 29, 2006, Japan’’s Ministry of Health, Labour, and Welfare (MHLW), s Ministry of Health, Labour, and Welfare (MHLW), equivalent to the FDA, introduced the equivalent to the FDA, introduced the ““positives listpositives list”” for detection of for detection of agricultural chemicals in foods. The legislation prohibited distagricultural chemicals in foods. The legislation prohibited distribution of ribution of foods that contained these chemicals above a certain level of mafoods that contained these chemicals above a certain level of maximum ximum residue limits (residue limits (MRLsMRLs).).

•• The agricultural chemicals include pesticides, feed additives, The agricultural chemicals include pesticides, feed additives, and and veterinary drugs. The regulations apply to all domestically prodveterinary drugs. The regulations apply to all domestically produced uced and imported foodstuffs and comprise a listing of almost 800 cheand imported foodstuffs and comprise a listing of almost 800 chemicalsmicals

2

•• A uniform level of 0.01 A uniform level of 0.01 ppmppm was established as the level having no was established as the level having no potential to cause damage to human healthpotential to cause damage to human health

•• The regulation requires that analysts apply a set of testing prThe regulation requires that analysts apply a set of testing protocols otocols that use classic cartridgethat use classic cartridge--based SPE and LC/MS or GC/MS techniquesbased SPE and LC/MS or GC/MS techniques

This talk will present data on a mix of 67 This talk will present data on a mix of 67 neutral, basic, and acidic chemicals analyzed neutral, basic, and acidic chemicals analyzed

by two different methods, both employing by two different methods, both employing SPE and LC/MS/MSSPE and LC/MS/MS

The previous system allowed for a very limited list of compounds for use as pesticides and veterinarydrugs, only 283 substances, and set a “zero tolerance” for compounds not specified by the government of Japan

3

The uniform limit has been set at 0.01 ppm so that the

estimated intake of agricultural chemicals does not exceed 1.5 µg/day when calculated based

on the food consumption of Japanese population. In

January 2005 the European Union (EU), established their

uniform level at 0.01 ppm

i.Agricultural chemicals and their decomposition products which are determined not to pose adverse health effects

ii.Specified agricultural chemicals for which registration withholding limits are not established

iii.Agricultural chemicals which are determined not to require any MRL in foreign countries and whose uses are not restricted.

4

• For fruits, vegetables and herbs, weigh out 20.0 g of the sample. For tea and hops, weigh out 5.00 g of the sample and let stand in 20 mL of water for 15 minutes

• Add 50 mL of acetonitrile and homogenize the sample. Filter the sample by suction. Add 20 mL of acetonitrile to the residue on the filter paper. Perform homogenization and suction filtration. Mix both filtrates. Add acetonitrile to the filtrate to make a 100 mL solution

ExperimentalStep 1: Liquid-liquid extraction for Methods I & II

5

• Add 50 mL of acetonitrile and homogenize the sample. Filter the sample by suction. Add 20 mL of acetonitrile to the residue on the filter paper. Perform homogenization and suction filtration. Mix both filtrates. Add acetonitrile to the filtrate to make a 100 mLsolution

Method I• Measure out 20 mL of the extracted solution. Add 10 g of sodium chloride and 20 mL of 0.5 mol/L phosphate buffer (pH 7.0) and shake. Let stand until the solution is clearly separated into layers. Discard the aqueous layer. Dry the acetonitrile layer over sodium sulfate (anhydrous) and filter

Method II• Measure 20 mL of the extracted solution. Add 10 g of sodium chloride and 20 mL of 0.01 mol/L hydrogen chloride and shake. Let stand until the solution is clearly separated into layers. Discard the aqueous layer. Dry the acetonitrile layer over sodium sulfate(anhydrous) and filter

• Concentrate the filtrate to dryness at 40°C orlower. Dissolve the residue in 2 mL of acetonitriletoluene (3:1)

• Concentrate the filtrate to dryness at 40°C or lower. Dissolve the residue in 2 mL of acetone/triethylamine/n-hexane (20:0.5:80)

• Collect the entire volume of effluent. Concentrate the effluent to 1 ml or less at 40°C or lower. Add 10 ml of acetone and concentrate to 1 ml or less at 40°C or lower. Add 5 ml of acetone to the concentratedsolution and concentrate to dryness

Elute the column with 10 ml ofacetone/triethylamine/n-hexane (20:0.5:80), and discard the effluent

Elute the column with 20 ml ofacetone/methanol (1:1). Collect the entire volume of effluent

• Dissolve the residue in methanol to make a 4 ml solution. Use this as the test solution

Concentrate the effluent to dryness at 40°C or lower. Dissolve the residue in methanol to make a 4 ml solution. Use this as the test solution.

• Condition a graphite carbon/ aminopropyl-silanized silica gel layered mini column (500 mg/500mg) with 10 ml of acetonitrile/ toluene (3:1) (Bond Elut Carbon/NH2, 500 mg/500mg, 6 ml). Apply the solution obtained from the Extraction step to the column and elute the column with 20 ml of acetonitrile/ toluene (3:1)

Condition a silica gel mini column (500 mg) with 5 ml of methanol, 5 ml of acetone, and 10 ml of n-hexane in this order (Bond ElutSi, 500 mg, 10 ml). Apply the solution obtained from the Extraction step to the column

Method I Method II

Step 2: Clean-up with Bond Elut Carbon/NH2 (Method I) and Bond Elut Silica (Method II)

6

Method I: MS-MS transition details

7

Method I: MS-MS transition details

Method II: MS-MS transition details

8

LC/MS/MS Analysis of Multiresidue Methods I and II onPursuit XRs C18 and Pursuit C18 on 320 LCMS

Method I: Chromatograms of 42 compounds in Tomatoat 100 ppb

-ve

-ve

-ve

9

Method II: Chromatograms of 25 compounds in Lemonat 100 ppb

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

Method I: Compounds in Standard and SpikedMatrices (Tomato and Lemon), Quantitation

10

Method II: Compounds in Standard and SpikedMatrices (Tomato and Lemon), Quantitation

S/N Ratio and Ion Ratio Identification

• Two fragment ions were monitored for each analyte for identification / confirmation. • At 10 ppb levels, two fragment ions of Azinphos-methyl and Imazaquin show good signal to noise• The ion ratios of daughter ion1 and ion2 of these compounds were used to help identify these compounds

11

Modifications made from Positive List procedure

Method I: changed drying gas temperature from 400°C to 220°C

Method II: changed drying gas temperature from 400°C to 250°C

Column temperature: Method I switched from 40°C to ambient for tridimorph isomers as separation was better at ambient

Linearity

12

Average Recoveries of Method I Compounds inTomato spiked at 10 ppb and100 ppb levels (n=3)

Average Recoveries of Method I Compounds inTomato spiked at 10 ppb and100 ppb levels (n=3)

13

Average Recoveries of Method II Compounds inTomato spiked at 10 ppb and100 ppb levels (n=3)

• All 67 compounds included in Multiresidue Methods I and II can be analyzed by using cartridge-based SPE and LC/MS/MS. The products developed include the following:

Method I:•Bond Elut Carbon /NH2 layered cartridge offering hydrophobic and anion exchange properties•Pursuit XRs C18 columns developed on a high surface area, 100Å, ultra-pure silica support• 320 LC-MS/MS triple quadrupole

Method II:• Bond Elut Silica• Pursuit C-18 columns based on 100Å, ultra-pure silica support• 320 LC-MS/MS triple quadrupole

• Good linearity from 10 ppb to 400 ppb was observed for most analytes with r2 greater than 0.98.• Recoveries for most analytes in both methods were in the range of 60%-140%. These recoveries are within the EU and CDFA requirements • Slight modifications were made to the drying gas temperature and column temperature compared to the existing Positive List procedure in order to see stable daughter ions and isomers

Conclusions

14

Acknowledgements

•• Eugene ChangEugene Chang•• Anita Anita DerMartirosianDerMartirosian•• GuangGuang LiLi•• Nigel SimpsonNigel Simpson•• RituRitu AroraArora

Thank you for your attentionThank you for your attention

Japan Positive List - Analytical Methods used

Analytical methods for residual compositional substrates of

agricultural chemicals, food additives and veterinary drugs in food

• Multi-residue method for agricultural chemicals by GC/MS (agricultural products)

• Multi-residue method I for agricultural chemicals by LC/MS (agricultural products)

• Multi-residue method II for agricultural chemicals by LC/MS (agricultural products)

• Multi-residue method for agricultural chemicals by GC/MS (animal and fishery products)

• Multi-residue method I for Veterinary drugs, etc by HPLC (animal and fishery products)

• Multi-residue method II for Veterinary drugs, etc by HPLC (animal and fishery products)

ReturnReturn

15

16

ReturnReturn

ReturnReturn

17

Method I: Chromatograms of 42 compounds in Tomatoat 100 ppb

-ve

-ve

-ve

ReturnReturn

ReturnReturn