New Perspectives in Pesticide Analysis

Tim Anderson GC Product Manager Phenomenex

EPA Region 6 QA Conference October 2015 Overview Background & Objectives Challenges We’ve Heard Sample Preparation Techniques Developments in GC/LC Column Technology Background Pesticide analysis has historically driven by regulatory requirements Traditional approaches have been sufficient, but technological and intellectual advances offer the opportunity to improve upon older methods

Today’s Goal • Present an overview of options in sample prep, GC, and LC analysis that may provide significant increases in productivity and/or improvements in data quality Common Challenges

Variety of Wide Range of Wide Range of Compounds Chemical Properties Sample Matrices

Acidic

Neutral Basic SAMPLE PREP ANALYTICAL LC SPE

QuEChERS

GC LLE Analytical Options Analytical Sample Preparation Techniques Sample Preparation Goals

Start End

Extract Clean Up Concentrate Solvent Switch The Importance of Selectivity The ability to target specific analytes from any given sample matrix for extraction, concentration and clean up More selective sample preparation can lead to better analytical methods

After Liquid / Liquid After SPE Selectivity In Sample Preparation Higher analytical performance can be attained with more selective, intricate sample prep techniques

Solid Phase Extraction (SPE) QuEChERS Liquid / Liquid Extraction (LLE) Filtration Centrifugation Settling and Decanting Selectivity Homogenization Dilution Liquid-Liquid Extraction (LLE)

Advantages Disadvantages • Cheap • Non-specific and non- • Relatively quick (may still selective require blow-down) ― Co-extraction of interferences of similar chemistry (log P) • Simple • Hazardous solvents • No special equipment • Large volumes of waste needed Improvements Over LLE There has been an increase adoption of alternative sample prep techniques that addresses some of the drawbacks or deficiencies of LLE This includes QuEChERS and SPE

Increasing Selectivity

LLE QuEChERS SPE We All Know QuEChERS! The primary goal is to reduce matrix interferences in a sample • Typically is qualitative screening rather than quantitation Most applicable to multi-class screening • Analytes with widely varying chemistry; thus more selective choices like SPE are not as appropriate • Goal is to remove as much matrix interference as possible QuEChERS Technique Quick Easy Cheap Effective Rugged Safe For matrix removal and extraction of pesticides

Pros Cons • Screening for wide range of • Not the most selective sample pesticides prep technique • Applicable to many sample • Kits needed (logistical headache matrices if not available) • Reduce loss of key analytes • Method development is still • Quick and simple procedure required • Qualitative vs. quantitative The QuEChERS Process Step 1: Liquid Extraction Step 2: Dispersive SPE • Sample is homogenized • Supernatant is combined with • Add organic solvent + salts loose sorbent to extract target analytes • Interferences adhere to the • Centrifuge to pellet the sorbent homogenate • Spin to pellet sorbent, decant and analyze supernatant Pesticides in Spinach by LC/MS/MS

Liquid nitrogen / dry ice homogenization

Liquid Extraction / dSPE Steps Pesticides in Spinach by LC/MS/MS After Liquid Extraction MRM chromatogram of spinach extract spiked at 200 ng/g

After dSPE

16 QuEChERS vs. Solid Phase Extraction

QuEChERS Technique • Target analytes in solution are extracted with ACN and salts • Organic layer is transferred to a separate tube containing loose SPE sorbent • Matrix interferences bind to the sorbent, rather than the analytes • Supernatant containing the analytes is removed and analyzed

Solid Phase Extraction (SPE) • Liquid sample is applied to cartridge containing chemically- modified sorbent • Target analytes bind to sorbent through chemical interaction • Matrix interferences are washed away using organic or salts • Clean analytes are eluted and analyzed 17 Quick Review: Solid Phase Extraction In SPE, a support particle is modified with different functional groups • Silica or polymeric particles • Wide range of functional groups (RP, IEX, NP) Target analytes bind to the media • Matrix interferences are washed away using different washing protocols The key distinction is that you optimize your method to target & recover your analytes • More selective than QuEChERS Traditional Silica-Based SPE Media

Strata • C18 Advantages • Inexpensive • Available for normal phase Strata • C8 Disadvantages • Limited pH Strata stability (2-7) • Screen-C • Deconditioning! Polymeric SPE Media Strata-X • Reversed-phase Advantages • Resists deconditioning! • Larger capacity Strata-X-C • pH range 1-14 • Strong Cation Exchange Disadvantages • More costly than Si-based Strata-X-A • Strong Anion Exchange Why SPE?

When selectivity for target analytes is Phenylurea Compounds from preferred mAU Rainwater 1000 • Most selective sample prep technique Higher recoveries of target analytes 800 No SPE Spike vs. Concentration of key analytes 600 SPE Extracted Can be automated 400 Spike Elimination of emulsions 200

0 0 2 4 6 8 10 12 14 min General SPE Screening Method Example for reversed phase procedure

(Volumes shown are for 30 mg sorbent mass) Paraquat/Diquat Extraction From Water • Condition with Methanol • Load Water • 1st Wash: Water • Elute with 5% Formic Sample Slowly Acid in Methanol • Equilibrate with Water • 2nd Wash: Methanol

• Dry Cartridge On Vacuum

Diquat

Ionic Interaction

Strata-X-CW Hydrophobic & Aromatic Interaction

Paraquat Sample Prep Summary Effective sample preparation is an essential component of most analytical methods Choice of sample prep technique is dependent on your analytical goals – what is most important? Advantages of QuEChERS and SPE include • Decreased down time • Better selectivity for specific pesticides • Cleaner samples / better analytical starting point GC Columns: Beyond the 5% Phenyl Traditionally Used GC Columns Choice commonly guided by government regulation (EPA, USDA, EN, etc.) Manufactured by every GC column producer

ZB-1 ZB-5 ZB-5ms ZB-35 ZB-50 ZB-1701 ZB-XLB

DB®-1 DB-5 DB-5ms DB-35 DB-17 DB-1701 DB-XLB

Rtx®-1 Rtx-5 Rtx-5ms Rtx-35 Rtx-17 Rtx-1701 Rtx-XLB

Rtx is a registered trademark of Restek Corporation. DB is a registered trademark of Agilent Technologies, Inc. Overcoming Common GC Productivity Thieves

Active compound breakdown Instrument downtime Coelutions of isomers or (Endrin, DDT, etc.) related to column lifetime structurally similar compounds Poor peak shapes

Potential remedy: contaminant removal

High temperature stable column High Temperature Science

Zebron™ Inferno™ ZB-1HT, ZB-5HT, ZB-35HT, Low Bleed Stationary Phase ZB-XLB-HT Deactivated Fused Silica

High Temperature Polyimide Resists pitting, brittleness, and breakage up to 430 °C High Temperature Benefits

Standard 35 Phase

Remove contaminants and see high boilers you may High Temp Stable 35 Phase be missing ZB-35HT Inferno™ Overcoming Common GC Productivity Thieves

Active compound breakdown Instrument downtime Coelutions of isomers or (Endrin, DDT, etc.) related to column lifetime structurally similar compounds Poor peak shapes

Optimized selectivity (resolve coelutions; resolve isomers; improve peak shapes)

Application-Specific Column Application-Specific GC Phases 1 5 % Phenyl Phase 35 % Phenyl Phase Sample 5 7

3 6 Deet 2 4 Terbacil Metribuzin

1 ZB-MultiResidue™-1 Bromacil ZB-CLPesticides™-1 7 Triadimefon (amiral) Better peak 5 3 shapes 2 4 6 Tricyclazole Better Hexazinone resolution

Comparative separations may not be representative of all applications. Overcoming Common GC Productivity Thieves

Active compound breakdown Instrument downtime Coelutions of isomers or (Endrin, DDT, etc.) related to column lifetime structurally similar compounds Poor peak shapes

Reduce activity contributed by the column

Highly Inert, Well-Deactivated Column What You Want Reduced tailing, resolution, good detection limits How You Get It – Manufacturing

High purity fused silica Every column is specially Low bleed 5% phenyl- glass is used – but even deactivated to reduce arylene phase is applied the purest has inherent activity No retention time shifts from silanol activity other 5 % phenyl-arylenes

Engineered Self Cross-Linking™ Bonding

Enviro-Inert™ Technology How You Get It – Quality Control 2 tests to ensure SVOC success

Traditional Test Mix EPA 8270D Test Mix 1 2 Efficiency, Polarity, Bleed, Activity • Is the DFTPP Tune Mix with pyridine addition • Better measure of activity

EPA ZB-SemiVolatiles Test Probe Measure Requirement Requirement Pyridine Peak Not Specified ≥ 0.6 Response Pentachlorophenol Peak Skew ≤ 2.0 ≤ 2.0 Peak Not Specified ≥ 0.3 Response Benzidine Peak Skew ≤ 2.0 ≤ 2.0 DDT Breakdown < 20 % < 20 % Why is Pyridine Important? Good gauge of column lifetime

Indicator of column activity

“Pyridine may perform poorly … Therefore, if pyridine is to be determined in addition to other target analytes, it may be necessary to perform separate analyses.” --US EPA 8270D

Rxi is a registered trademark of Restek Corporation. Phenomenex is in no way affiliated with Restek Corporation or Agilent Technologies, Inc. Conditions were the same for all columns tested. Comparative separations may not be representative of all applications. Reduced Activity On A 5ms

Pyridine ZB-SemiVolatiles RF = 1.06 PCP No RF = 0.37 Breakdown Not all 5ms columns are equal

® Pyridine HP -5ms Ultra Inert RF = 0.28 PCP DDT RF = 0.20 Breakdown

HP is a registered trademark of Agilent Corporation. Phenomenex is in no way affiliated with Agilent Technologies, Inc. Conditions were the same for all columns tested. Comparative separations may not be representative of all applications. Productivity Advantage Improved peak shapes and responses for active compounds out-of-the-box • Improved RSD values when calibrating instrument • Eliminates downtime caused by new columns failing Tune Mix requirements

Stands up to contamination better to give longer lifetime • Improved quantitation for active compounds across all concentrations • Low concentrations – stronger response • All concentrations – improved peak shape allows easier and more consistent integration

Overcomes SVOC productivity thieves to increase productivity GC Summary

Active compound breakdown Instrument downtime (Endrin, DDT, etc.) Coelutions of isomers or related to column lifetime structurally similar compounds Poor peak shapes

High temperature stable Highly Inert, Optimized Column Selectivity column Well-Deactivated Column Trends Multi-residue Complex matrices Updated older methods Lower limits Larger lists Improved instrumentation Developments in LC Column Technology The Evolution of LC Media The primary trend in LC media development has been the movement towards particles with increasingly higher efficiency

Kinetex®

10 µm 5 µm 3 µm 1.7 µm

1970 1980 1990 2000 2010

Irregular Silica Fully Porous Silica Kinetex Core-Shell Media Column Efficiency (N) The efficiency of a column is a function of the amount of band tR broadening • Columns that cause a lot of peak broadening have low efficiency • Columns that produce very narrow peaks have high efficiency W1/2 Narrower peaks = closely-eluting peaks are easier to separate!

W Kinetex® Core-Shell Technology Monodispersed silica particles consisting of an impermeable silica core surrounded by a layer of fully porous silica

Porous What is the core-shell advantage? Shell • Kinetex core-shell columns provide significantly greater efficiency than columns packed with fully porous media • Can deliver UHPLC-equivalent performance on conventional HPLC systems What are the benefits? • Improved resolution • Increased sensitivity • Increase productivity

44 The Core-Shell Advantage More Efficiency  Increased Sensitivity More Efficiency  Improved Resolution

Fully VWD1 Porous A, Wavelength=254 nm 3µm (JL030414\JEFF Column 2014-03-03 14-44-29\SULFA0000001.D)

mAU 3.074

50

3.332

0.653 4.196 45 mAU 3.756

40 3.914

30 2.979

20

10 2.768

4.282 4.062 0

0 1 2 3 4 min ® Kinetex VWD1 A, Wavelength=254Core nm (JEFF- 2014-03-03Shell 11-38-35\SULFA0000001.D) 2.6µm Column mAU

70 Increased 3.018

3.317 0.567

60 4.176 Resolution 3.777 50 60 mAU 3.915 40 33% Increase in 2.932 30

20 Peak Height 3.085 2.754

10 4.243

0

0 1 2 3 4 min The Core-Shell Advantage More Efficiency  Increased Productivity Fully Porous 5µm 250 x 4.6mm C18 Column

Kinetex® 2.6µm 100 x 4.6mm C18 Column Equivalent Performance in ½ the Run Time Beyond A Standard C18 Only Multiple Kinetex® Biphenyl Hydrophobic Interactions • Unique stationary phase chemistry Interactions Mechanisms • High degree of selectivity for polar & basic analytes • Delivers the efficiency benefits of the Kinetex core- shell particle

The selectivity of Kinetex Biphenyl is very distinct (orthogonal) from the typical C18 phases • Use Kinetex Biphenyl when a standard C18 doesn’t have the right selectivity • Start your method development with Kinetex Biphenyl when you have polar, basic analytes that don’t have enough polar retention on a standard alkyl-bonded phase Biphenyl Selectivity

Stationary phase based upon: Aromatic pi-pi interactions – between aromatic rings and pi electrons of target molecules and the double aromatic rings of the Kinetex® Biphenyl ligand

Hydrophobic interactions – between carbon skeleton of ligand and target analytes • Hydrophobicity of Kinetex Biphenyl (i.e. retention of analytes based primarily on hydrophobic interactions) is less than C18

Weak ionic or dipole interactions with the phenyl rings • High electron density • Behaves almost as a weak cation exchanger, giving enhanced retention of many basic analytes Polar Retention of Kinetex® Biphenyl

XIC of +MRM (88 pairs): 215.000/91.900 Da ID: SULFAGUANIDINE-2 from Sample 9 (Vet mix_KTX C18_010) of 031214.wiff (Turbo Spr... Max. 9.0e4 cps.

1.3e6 1.3e6 1.2e6 1.2e6 Kinetex 2.6µm C18 1.1e6 1.1e6 1.0e6 9.5e5 9.0e5 8.5e5 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5

Intensity, cps 5.5e5 Poor retention on C18 5.0e5 4.5e5 4.0e5 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 0.54 5.0e4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min

XIC of +MRM (88 pairs): 215.000/91.900 Da ID: SULFAGUANIDINE-2 from Sample 7 (Vet mix_Biphenyl_007) of 031214.wiff (Turbo Spr... Max. 5.9e4 cps.

2.1e6

2.0e6

1.9e6 1.8e6 Kinetex 2.6µm Biphenyl 1.7e6 1.6e6 Enhanced 1.5e6

1.4e6

1.3e6 1.2e6 Retention of 1.1e6

1.0e6

Intensity, cps 9.0e5 8.0e5 Early-Eluters 7.0e5

6.0e5

5.0e5

4.0e5

3.0e5

2.0e5

1.0e5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min Pesticide Screening Better separation of early elutors Column: Kinetex® Biphenyl 5µm Dimensions: 100 x 2.1 mm Mobile Phase: A: 5 mM Ammonium Formate in H2O B: 5 mM Ammonium Formate in MeOH Gradient: Time %B 0.01 10 1.00 10 10.00 90 15.00 90 15.10 10 20.00 10

Flow Rate: 0.5 mL/min Column Temp: 35 °C 200+ pesticides with great sensitivity for ® Detector: AB SCIEX 4500 QTRAP polar pesticides in under 20 minutes Neonicotinoids by LC/MS/MS Nicotine-related neurotoxic insecticides Less toxic than organophosphate and carbamate pesticides May be related to honey bee die-offs

System: API 4500 MS + Shimadzu LC-30 UFLC Mobile Phase: Water with 0.1% formic acid ACN with 0.1% formic acid Gradient: 5-70%B in 3min Flow rate: 600 µL/min Temp: 40C Sample: 1. Dinotefuran 2. Nitenpyram Imidacloprid 3. Clothianidin 4. Acetamiprid 5. Thiacloprid 6. Imidacloprid 7. Thiamethoxam Enhance Selectivity: Solvent Choice Neonicotinoids using acetonitrile

XIC of +MRM (28 pairs): 224.900/127.900 Da ID: Acetamiprid-1 from Sample 6 (KNX 2.6u C18_neonico) of ... Max. 7.7e4 cps.

4.8e5 4.6e5 4.4e5 ® 4.2e5 4.0e5 Kinetex 2.6µm 3.8e5 3.6e5 3.4e5 3.2e5 C18 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5

Intensity,2.0e5 cps 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 1 18 35 52 70 87 104 121 138 155 172 189 207 224 241 258 Time, min

XIC of +MRM (28 pairs): 224.900/127.900 Da ID: Acetamiprid-1 from Sample 11 (KNX 2.6u Biphenyl_neonic... Max. 8.0e4 cps.

4.6e5 4.4e5 4.2e5 4.0e5 3.8e5 Kinetex 2.6 3.6e5 µm Slight/moderate 3.4e5 3.2e5 3.0e5 Biphenyl shifts in 2.8e5 2.6e5 2.4e5 2.2e5 retention and 2.0e5

Intensity, cps 1.8e5 1.6e5 1.4e5 selectivity 1.2e5 1.0e5 8.0e4 6.0e4 using 4.0e4 2.0e4 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 1 18 35 52 70 87 104 121 138 155 172 189 207 224 241 258 Time, min acetonitrile Methanol to Enhance Selectivity Neonicotinoids using methanol XIC of +MRM (28 pairs): 202.990/129.100 Da ID: Dinotefuran-1 from Sample 18 (KNX 2.6u C18_neonico in ... Max. 6.7e5 cps.

6.8e5 1.25 6.5e5 ® 6.0e5 Kinetex 2.6µm C18

5.5e5

5.0e5

4.5e5

4.0e5

3.5e5

3.0e5

Intensity, cps

2.5e5

2.0e5

1.5e5

1.0e5

5.0e4

0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 1 18 35 52 70 87 104 121 138 155 172 189 207 224 241 258 275 292 309 326 344 361 378 395 412 Time, min

XIC of +MRM (28 pairs): 202.990/129.100 Da ID: Dinotefuran-1 from Sample 17 (KNX 5u Biphenyl_neonico ... Max. 4.5e5 cps.

5.4e5

5.0e5

1.55 Kinetex 2.6µm Biphenyl 4.5e5 Dramatic 4.0e5 3.5e5 changes in 3.0e5

2.5e5 retention and

Intensity, cps

2.0e5

1.5e5 selectivity using 1.0e5 methanol 5.0e4

0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 1 18 35 52 70 87 104 121 138 155 172 189 207 224 241 258 275 292 309 326 344 361 378 395 412 Time, min Summary: LC Columns Columns packed with core-shell media are able to provide significantly greater efficiency than columns packed with fully porous media • Improved resolution • Improved sensitivity • Improved productivity Stationary phase selectivity is also crucial to the success of any method • Phases that complement standard C18 phases, such as the Biphenyl, can provide the necessary to resolve mixtures that are difficult to chromatograph on the go-to C18s • Every lab that has a Kinetex C18 should have a Kinetex Biphenyl Get the Environmental Edge www.phenomenex.com/Edge • SPE Method Development Tool • 1,000s of Application Notes • On-Site Lab Demos • Environmental Edge Newsletter • Technical Notes • Digital Learning Tutorials

Thank You! Questions?