Pesticide Residue Analysis Webinar Series PART ONE: Sample Prep Tips and Tricks Using QuEChERS and Accelerated Solvent Extraction

Richard Fussell Vertical Marketing Manager, Food and Beverage

Mike Oliver Sample Preparation Product Manager

Aaron Kettle Dionex ASE system, Dionex AutoTrace SPE and Rocket Evaporator Systems Product Manager PP71597-EN 0315S

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Webinar Series Objectives

• To provide pesticide residue analysts with valuable information on development and optimization of method workflows for the analysis of pesticide residues in food.

• To understand the challenges, limitations and advantages of multi-residue workflows for pesticides residues in food

• To review the latest developments in: •  sample preparation •  targeted and screening techniques using GC-MS/MS and LC-MS/MS • software advances to help with data interpretation and reporting.

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1. Sample Prep: March 24th 2. LC-MS Analysis: April 29th

3. GC-MS Analysis: June 17th 4. Data Processing/Analysis: July 15th

Typical Pesticides Workflow Register at www.chromatographyonline.com/LCGCwebseminars

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Sample Homogenisation Before Extraction

•  Unavoidable steps in the analysis

•  Prerequisite to obtain representative sub-samples

Analytical Sample Laboratory Sample Test portion

•  Pesticide residues can be lost during processing

•  Critically important but receive relatively little attention

•  Not usually included in validation or estimates of measurement uncertainty

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Even Before Extraction…..

• Losses of a number of pesticides during sample processing at ambient temperatures may occur:

bitertanol, dichlofluanid, captan, chlorothalonil

Fussell, et al. Food Addit. Contam. 2007, 24:1247-1256.

• Add CO2

• Homogenise • Frozen sample

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Cryogenic Milling Effect on the Stability of Pesticides

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100 ca

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Ambient Cryogenic

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Cryogenic Milling Effect on Homogeneity

Fussell et al. J. Agric. Food Chem. 2007, 55, 1062-1070.

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Tips for Improved Stability and Homogeneity

• Cryogenic milling improves analyte stability and homogeneity (improved precision for small-scale methods)

• Smaller particle size allows better penetration of solvent and better extraction efficiency for incured residues

• If processing at ambient tempeartures work as quickly as possible and mix continuously during withdrawing of test portions

• If it is not possible to homogenise samples under cryogenic conditions, the analyst should at least recognise the potential losses and report results accordingly

• Do not forget potential losses of pesticides during storage of sample and avoid freeze/thaw cycles.

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Sample Extraction

• Objective; high recovery of analyte and low recovery of matrix components

• Analyte compatibility solubility (like dissolves like)

• Analyte selectivity minimise interfering matrix components

• Extraction efficiency incurred residues more realistic than laboratory spiked

samples (measure of analytical loses)

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Examples of Multi-Analyte Methods

Pesticide Residue Webinar: Sample Preparation Tips and Tricks Using QuEChERS

Mike Oliver Sample Preparation Product Manager Thermo Fisher Scientific

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Overview

• Analytical Challenges • Meeting the Challenge • How to choose the appropriate Sample Preparation Technique

• What is QuEChERS? • Quick (> 10 samples /hour) • Easy (simple process with few steps • Cheap (minimal solvent reagents and equipment • Effective (wide scope, accurate, precise, compatibility with GC-MS & LC-MS • Rugged (variations of method applicable to diverse sample types • Safe (non chlorinated solvents contained in capped tube)

• Trouble shooting • Applications

Weigh sample Extract dSPE Freeze? Filter?

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• Diverse and complex food matrices •  Increasingly large number of target compounds • Low limits of detection • High throughput (Efficient) • Cost effective

Analytical Challenges

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Reproducible accurate quantification and identification results are achieved by:

•  Appropriate sample preparation •  Removal of matrix co-extractives to minimise matrix effects •  Adjustment/buffering of pH: solubility and stability of analytes

•  Chromatographic performance • Separation and shape of peak • Speed in analysis

•  Use of high sensitivity instruments • Dilution (reduced matrix concentration) • Reduced maintenance, decreased instrument down-time

•  Good detector selectivity •  Reproducible products

How Do We Overcome the Challenges?

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Pesticide Analysis

• Many pesticide multiresidue methods (MRMs) are multi-facetted:

QuEChERS methods meets these demands

Traditional MRM QuEChERS Blending Shaking Filtration Centrifugation Large solvent volumes Small solvent volumes Multiple partitioning steps Single partitioning Transfers of entire extract Take aliquots (use I.S.) Lot of glassware Single vessel Evaporation/Concentration Large volume injection Classical SPE Dispersive SPE

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Extraction & Clean-up in a Tube

2) Dispersive SPE acetonitrile supernatant containing extracted residues Sample

matrix

Salts/water

centrifuge

What is QuEChERS?

Note: Add sample to the tube, then solvent, then sorbent then mix, to avoid agglomeration

centrifuge

1) Extraction

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QuEChERS Method Variations

Method Description

Original QuEChERS Method – introduced in 2003

Uses Sodium Chloride to enhance extraction

Dispersive AOAC 2007.01 Method Uses Sodium Acetate as a buffer replacing Sodium Chloride

Dual Phase Variation Uses PSA & GCB to remove high levels of chlorophyll and plant sterols

European Version Similar to AOAC method – uses sodium chloride, sodium citrate dihydrate and disodium citrate sesquihydrate

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Original QuEChERS Method:

1.  Weigh 15 g of homogenised (hydrated at least 80%) sample in a 50 mL centrifuge tube

2.  Add 15 mL acetonitrile (or 1:1 acetone/hexane, ethyl acetate) and IS 3.  Shake briefly 4.  Add 4 g anhydrous magnesium sulfate and 1g sodium chloride 5.  Shake by hand for 1 minute 6.  Centrifuge at 5,000 rpm for 5 min 7.  Transfer a portion of supernatent to a QuEChERS clean up tube 8.  Shake for 30 sec 9.  Centrifuge for 1 min at 6,000 rpm 10. Transfer 0.5 mL aliquot for analysis

Stage 1: Selecting the Method

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AOAC 2007.01 Method:

1.  Weigh 15 g of homogenised (hydrated at least 80%) sample in a 50 mL centrifuge tube

2.  Add 15 mL 1% acetic acid in acetonitrile (or 1:1 acetone/hexane, ethyl acetate) and IS

3.  Shake briefly 4.  Add 6 g anhydrous magnesium sulfate and 1.5 g anhydrous sodium acetate 5.  Shake by hand for 1 min 6.  Centrifuge at 3,700 rpm for 5 min 7.  Transfer a portion of supernatent to a QuEChERS clean up tube 8.  Shake for 30 sec 9.  Centrifuge for 1 min at 3,700 rpm 10. Transfer 0.5 mL aliquot for analysis and add TPP and either formic acid or

toluene / magnesium sulfate

Stage 1: Selecting the Method

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Advantages of AOAC 2007.01 Over Original Method: • Using sodium acetate as a buffer protects base sensitive compounds

•  Folpet • Dichlofluanid • Chlorthanonil

• The presence of acetic acid • PSA will absorb acetic acid •  Less sample clean-up • Higher baseline

• Only use this method if looking at specific compounds

• Dicofol • Captan •  Tolyfluanid

AOAC Method –Disadvantages

Stage 1: Selecting the Method

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Schenck Method – for Polar Aromatic Compounds: Using dual layer PSA/GCB cartridge

•  Extraction phase as AOAC method •  Pre-rinse the SPE cartridge with 5 mL of toluene •  Add an aliquot of the supernatent to the cartridge •  Start collection •  Elute with 6-12 mL of 3:1 acetone:toluene •  Concentrate for GCMS analysis –or- •  Concentrate to dryness and reconstitute in mobile phase for LC

analysis

Stage 1: Selecting the Method

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European EN15662 Method: 15 g modified version 1.  Weigh 15 g of homogenised (hydrated at least 80%) sample in a 50 mL

centrifuge tube 2.  Add 15 mL acetonitrile (or 1:1 acetone/hexane, ethyl acetate) and IS 3.  Shake briefly 4.  Add 6 g anhydrous magnesium sulfate, 1.5 g sodium chloride, 1.5 g sodium

citrate tribasic dihydrate, 0.75 g sodium citrate dibasic 5.  Shake by hand for 1 min 6.  Centrifuge at 5,000 rpm for 5 min 7.  Transfer a portion of supernatent to a QuEChERS clean up tube 8.  Shake for 30 sec 9.  Centrifuge for 1 min at 6,000 rpm 10. Transfer 0.5 mL aliquot for analysis

Stage 1: Selecting the Method

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Matrix Type Examples Sorbent Requirements for Clean-Up

General Matrices •  Apples •  Cucumber •  Melon

MgSO4, PSA Removal of excess water organic acids, fatty acids, sugars

Fatty Matrices •  Milk •  Cereals •  Fish

MgSO4, PSA, C18 Additional removal of lipids & sterols

Pigmented Matrices •  Lettuce •  Carrot •  Wine

MgSO4, PSA, C18, GCB Additional removal of pigments & sterols

High Pigmented Matrices •  Spinach •  Red Peppers

MgSO4, PSA, C18, GCB, Chlorofiltr™ Additional removal of chlorophyll

Stage 2: Select the Right Product

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Stage 2: Select the Right Product

Material Purpose

Magnesium Sulphate Removal of excess water

PSA (Primary / Secondary Amine)

Removal of organic acids, fatty acids, sugars

C18 Removal of lipids & sterols

GCB (Graphatized Carbon Black)

Removal of pigments & sterols

Chlorofiltr™ Removal of chlorophyll

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Thermo Fisher Scientific Products

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Troubleshooting QuEChERS

• Loss of planar Pesticides through GCB use • Use a product with less GCB • Use the Schenk method (add toluene to improve recovery)

Pesticides often lost when using GCB:

Carbendazim Mepanipyrm

Chlorothalonil Pentachloroaniline

Cyprodinil Phenmedipham

Demedipham Phosalone

Diflubenzuron Pymetozine

Flucycloxuron Pyrimethanil

Hexachlrobenzen Quinoxyfen

Hexaflumuron Thibendazole

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Troubleshooting QuEChERS

• Loss of acidic compounds from starting sample through PSA use • Decreased recovery of acephate, acrinathrin, carbaryl, chlorothalonil,

diclorvos, dimethoate, mevinphos, phosmet plus others e.g. • Pymetrozine

•  Imine (Schiff base) • Hydorlysis in acidic conditions

• Use a product without PSA e.g. MgSO4 & C18

• Loss of base sensitive pesticides • Addition of 0.1% acetic acid can improve stability

Base Sensitive Pesticides: Captan Dichlofluanid

Folpet Tolylfluanid

Dicofol Chlorothalonil

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Troubleshooting QuEChERS

• Poor recovery of pesticides • Verify sample hydration to at least 80% • Verify pesticide type and that correct products are being used • Effect of pH

• Decreased recovery of lipid soluble pesticides (hexachlorobenzene) • Use of C18 removes lipids •  Freeze out fat

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Troubleshooting QuEChERS

• Poor extraction efficiency/recovery •  Temperature of extraction

•  frozen samples require longer extraction time (thaw in solvent) • Shaking time

• depends on matrix (5-15 minutes using mechanical shaker recommended)

• Verify pesticide type and that correct products are being used • Effect of pH •  losses of lipid soluble pesticides (hexachlorobenzene) due removal of lipids

by C18 sorbent

•  Low moisture content commodities (e.g. Cereals, tea, etc) • rehydrate with water (10-20 min prior to addition of solvent) • do not exceed 20 min for cereals as activation of enzymes can degrade

OP pesticides •  freeze milled sample overnight to reduce chemical interferences •  freeze supernatant to remove lipid and also to increase ease of filtering

(if required)

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Application – Pesticides in Cucumber

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Application – Pesticides in Grapes

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Application – Pesticides in Lettuce

33 The world leader in serving science

Aaron Kettle Product Manager, Thermo Fisher Scientific March 24, 2015

Accelerated Solvent Extraction Technique: Overview and Uses for Pesticide Extraction

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The Challenge for Analysis

1.5 mL GC / LC Vial

How do we get pesticide residues out of these samples?

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The Answer is Sample Preparation

• Extraction • Recovers analytes from the sample • Eliminates compounds that interfere with the analysis (Clean Up)

• Evaporation • Concentrates extracted analytes for analysis • Reduces volume of extract

• Depending on the matrix-analyte combination, sample preparation can be the source of substantial error

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Analysis Techniques!

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Sample Preparation…

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Franz von Soxhlet (1848 – 1926)

In the Beginning There Was Soxhlet...

de facto standard for solvent extraction

Slow, high solvent usage

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Now . . . Accelerated Solvent Extraction

•  Low end system •  Ideal for low-

throughput labs • Smaller footprint that

is economically priced

•  Fast & efficient extraction of a single sample

• High end system •  Ideal for high

throughput labs requiring automation

• Unattended extraction of up to 24 samples

• Mixing or selection of three different solvents for complex extractions

Thermo Scientific™ Dionex™ ASE™ 150 and 350 Accelerated Solvent Extractor

Dionex ASE 150 system Dionex ASE 350 system

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Key Parameters for Accelerated Solvent Extraction

• Temperature •  Increases analyte solubility •  Increases diffusion rate • Decreases solvent viscosity

• Solubility •  Increases as temperature increases • E.g. Anthracene solubility increases 13 fold in DCM (50 oC to 150 oC)

• Viscosity • Decreases as temperature increases • Allows solvents to migrate through the matrix easier

• Surface Tension •  Increased temperature decreases solvent surface tension • Lower surface tension allows the solvent to better cover the matrix

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1 min

15-20 min Total Time

1-2 min

0.5 min

3-5 min

5-9 min

static cycle

Cell loaded into oven

Fill, heat, equilibrate

Static extraction

Fresh solvent rinse

Nitrogen purge

Filtered extract

Overview of Accelerated Solvent Extraction

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Schematic of the Extraction Cell

Dionex ASE In-Line Clean-up

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What About Thermally Labile Compounds and Carryover?

TN 206: Investigation of Thermal Degradation TN 207: Investigation of Carryover

Accelerated Solvent Extraction Does Not Degrade Labile Compounds and is Exhaustive!

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TN 206: Evaluating Thermal Degradation

• Spike DDT and Endrin on sand at 5 ppb level • Measure recovery and monitor for the presence of DDD, DDE, Endrin Aldehyde

and Endrin Ketone • Monitor the recovery of other temperature sensitive compounds such as o-toluidine and dichlorobenzidine

Compound Ext. Temp. %Recovery %RSD

DDT 150 °C 103 3.9

Endrin 150 °C 110 2.4

O-Toluidine 150 °C 104 10.3

Dichlorobenzidine 150 °C 106 13.2

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TN 207: Investigation of Carryover with PAHs and PCBs

•  5 g of SRS sample (soil contaminated at 11%, N=3)

•  First extract = 106.1% average recovery of PAH by HPLC

•  MDQ was 0.2 mg/kg, Certified concentration of PAHs ranged from 31 – 1500 mg/kg

•  Second extract (solvent blank) = PAHs not present

•  8 g of sediment from NIST(SRM 1939, 35% extractable, N=3)

•  First Extract = 101.8% average recovery of PCB by GC-ECD

•  MDQ – 0.1 µg/kg, Certified concentration of PCBs ranged from 180 to 3700 µg/kg

•  Second extract (solvent blank) = PCBs not present

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Use of Adsorbents Improves Selectivity

TN 210: In-Line Removal of Interferences List of Adsorbents and Uses

In-Cell Adsorbents May Eliminate the Need for Offline Clean Up Procedures

Adsorbent and Uses

Carbon Removes organics and nonpolar compounds

Copper Removes sulfur

Ion-exchange Resins

Removes organics, ionic interferences for IC and IC/MS analysis

C8 - C18 Resin Removes organics, polar compounds, lipids, colors

Acid-impregnated Silica Gel

Removes lipids

Alumina Removes nonpolar lipids, colors

Florisil Removes nonpolar lipids

Silica Gel Removes nonpolar lipids

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Dionex ASE Prep MAP Moisture Absorbing Polymer

• Designed to remove moisture from wet samples (in-cell, in-line) as well as from the extract (in-vial, off-line)

• The polymer is a free flowing white granular material that can be easily mixed with Dionex ASE Prep DE (diatomaceous earth) dispersant and used for the moisture removal

• Dionex ASE Prep MAP polymer replaces the use of sodium sulfate and does not clump in the extraction cell

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In-Cell Water Removal of Different Matrices

• Accelerated solvent extraction conditions: 100 ºC, 5 min heat, 5 min static, 3 cycles, flush 30%, purge 120 s, hexane

Temperature (ºC)

Sample Amount of water in the sample (g)

Dispersant, (polymer and DE)

Water Observed in Collection Bottle (g)

100 Soil 7.00 4 g each No

100 Apple 8.49 4 g each No

100 Carrot 8.79 4 g each No

100 Pear 8.49 4 g each No

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Key ASE Pesticide Applications Summary

Matrix Analytes Determinative Step Application Note

Oyster Tissue Organochlorine Pesticides GC-MS AB 152

Raw Potatoes Organochlorine Pesticides GC-ECD AN 332

Bananas Organochlorine Pesticides GC-ECD AN 332

Apple Puree Organophosphorous Pesticides GC-NPD AN 343

Carrot Puree Organophosphorous Pesticides GC-NPD AN 343

Animal Feed Organochlorine Pesticides GC-MS AN 349

Beef Kidney Atrazine GC-MS AN 359

Soil Toxaphene GC-MS AN 352

Soil, Clay, Sediment Organochlorine Pesticides GC-MS AN 320

Soil, Sediment Organophosphorous Pesticides GC-NPD AN 319

Tobacco Organochlorine Pesticides GC-ECD AN 1099 (Pending Publication)

Visit www.thermoscientific.com/samplepreparation

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Summary of U.S EPA Equivalency Study for Accelerated Solvent Extraction

Compound Class Comparison Technique Relative Recovery

Organochlorine pesticides (OCP)

Automated Soxhlet 97.3%

Organophosphorus pesticides (OPP)

Automated Soxhlet 99.2%

Semivolatiles (BNA) Soxhlet 98.6%

Chlorinated herbicides Shake method 112.9%

Polychlorinated biphenyls (PCB)

Various reference materials

98.2%

Polycyclic aromatic hydrocarbons (PAH)

Various reference materials

104.8%

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U.S. EPA Method Technique Solvent Used Per Sample

Extraction Time Per Sample

3545A Accelerated Solvent Extraction

15 – 45 mL 10-15 min

3540 Soxhlet 300 – 500 mL 18 hours

3541 Automated Soxhlet 50 mL 2 hours

8151 Shaker 300 mL 2 hours

3546 Microwave* 25 mL 15 min

*Requires additional cooling and filtering steps (~ 45 min/sample)

Comparison to Other Techniques (U.S. EPA Methods)

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Thermo Scientific Rocket Evaporator for Automated Evaporation • Samples are loaded into the

rotor and placed under vacuum

• Low pressure steam is used to heat the samples

• The steam condenses on the flasks/tubes, which are cold due to the solvent(s) boiling inside them

• Condensate is thrown off the spinning flask and recycled

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Flasks For Evaporating or Concentrating

•  400 mL flask that concentrates sample into GC autosampler vials

•  Vial is insulated so that only solvent in flask evaporates

•  450 mL Evaporation Flask

•  Used for evaporating to dryness

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Dionex ASE Puck and Flip Flop Vials

• Pucks accept 60 mL ASE vials • Each puck accepts 3 vials • Total capacity per system is 18 vials • Allows direct transfer of ASE vial to

evaporator

• GC Vial is inserted into the flip flop funnel

• ASE vial is inserted directly into the Rotor

• GC vial is placed directly into autosampler once complete

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Total Workflow with Fast Simplified Sample Prep

Integrated Workflow Solutions for Sample Preparation

Questions?