On-Line SPE with the Flexcube/6460 QQQ Dr. Sheher Bano Mohsin Mass Spectrometry Applications June 11, 2013 This overview of on-line Solid Phase Extraction (SPE) coupled with LC/MS/MS was created by Joe Weitzel in November 2012. This presentation provides 4 applications sections with data from Peter Abrahamsson (LC/MS PS EMEA), Thomas Glauner (EMEA Food team LC/MS chemist), Paul Stephens (EMEA Environmental marketing manager), Sheher Mohsin (AFO LC/MS AE) and customers: Tarun Anumol –University of Arizona, Sarah Gledhill – South East Water (UK). 11/15/2018

Introduction – Why Online SPE? Introducing the Flexcube for Online SPE Sensitivity of the Mass Spectrometers Application Examples - Water Samples - Samples with dirty Matrices 11/15/2018

Solid Phase Extraction (SPE) Wouldn’t it be great if you could automate SPE while reducing sample and solvent volumes? Most environmental customers find that manual SPE is a bottleneck in the lab. It is time consuming and lab intensive! Volume of water sample required can vary from 200ml – 1,000 ml. This also requires solvent for rinsing, eluting, and creating final volumes. 11/15/2018

Automated on-line SPE Load 500ul-1800 µl water sample on the enrichment cartridge. Valve switch, ‘backflush‘ desorb using HPLC gradient. Separate on the analytical column and collect MRM tranistions on the QQQ. Recondition enrichment cartridge during analysis. Ready for next sample after ~ 20 minutes. Basics of the Agilent on-line SPE LC/MS. Everything controlled by one software package –MassHunter! Customers have found that they can get 100-200 analyses per SPE cartridge depending upon the cleanliness of the water sample. SPE system can be used with LC/QQQ or LC/TOF, or LC/QTOF. 11/15/2018 4

High sensitivity 6460 Triple Quadrupole Sub-femtogram, on-column sensitivity (1pg reserpine; s/n 2000:1) Five Orders of Linearity Fast Pos/Neg switching, 30 millisec “Dynamic Multiple Reaction Monitoring” for MRMs scheduled by peak retention times instead of time segments Triggered MRM for confirmation Agilent Jet Stream Online SPE or enrichment can be used with either the LC/QQQ , LC/TOF, or LC/QTOF. MassHunter acquisition controls entire system. Most online SPE users utilize the 6460 LC/QQQ as the detector. Agilent Jet Stream thermal gradient focusing technology was developed to signifi cantly enhance sensitivity in ESI-MS by improving the desolvation and spatial focusing of ions. Superheated nitrogen sheath gas confi nes the nebulizer spray to more effectively dry ions and concentrate them in a thermal confi nement zone (Figures 1-3). Desolvation reduces noise. Full confi nement of the spray by the superheated nitrogen gas eliminates sample recirculation and reduces peak tailing 11/15/2018 5

Agilent MassHunter Software Instrument Control Real-time monitoring Method set-up Autotune Qualitative Analysis Chromatographic results Spectral results Find compounds Quantitative Analysis User filters Compound results Calibration curve Reporting Easily Customizable Based on Excel and XML technology MassHunter Software is available for LC/QQQ, LC/TOF, LC,QTOF, GC/MSD (data analysis only), GC/QQQ, GC/QTOF, and ICP-MS systems Entire on-line SPE LC/QQQ controlled by MassHunter SW 11/15/2018

Agilent Online SPE System Analytical column for separation Switching valve with TEC MS/MS Quaternary pump Autosampler Binary pump for gradient elution 11/15/2018

What’s new with Online SPE? Flexcube with Built in Valves and a Pump - all Controlled through Mass Hunter 11/15/2018

Agilent Online SPE System Switching valve with TEC Analytical column for separation Replace with Flexcube MS/MS Quaternary pump Autosampler Binary pump for gradient elution 11/15/2018

Agilent 1200 Infinity Series Online SPE solution Valves mounted in 1290 Infinity Flexible Cube Solvent selection valve for up to three solvents Low level detection 3 position Solvent Selection Valve helps with conditioning and loading SPE cartriges. Condition with up to 3 solvents in paralell condition with the other cartrige in analytical flow path Re-useable SPE cartriges. Flexible Advanced Valve Solutions. Valve heads easy to mount SPE Starter set Direct injection High Volume injection Flask Sampling Multi SPE Cartrges 2 position 10 port valve in the flex cube with 2 SPE cartrige, 1.8 ml of sample, Software: MassHunter MH B.06.00, driver release A.02.06 or higher to controll all features of 1290 Infinity Flexible Cube (G4227A) List price of 1290 Infinity Flexible Cube including 2 valve drives ca. 9 kUSD Reciprocating single-piston pump for flows up to 4 ml/min (60 bar) up to two Quick-Change valves, according to application

Online SPE with the Flex Cube Column Compartment Flexcube 1290 Autosampler Autosampler 6460 QQQ Autosampler with 900uL loop and extension seat capillary 1290 Binary Pump

Flex Cube and Autosampler 6 port valve 10 port valve Single Piston Pump Autosampler

Mass Hunter B 06.00 with Flexcube Control

System with alternating SPE loading: Load sample to SPE1 waste SPE starter set Flex cube pump Column Std. ALS 900 µL head SPE2 1260 Bin Pump SPE1 Load System with alternating SPE loading: Load sample to SPE1 waste

System with alternating SPE loading: Flex cube Column Mass Spec Load Std. ALS 900 µL head SPE2 1260 Bin Pump SPE1 Elute waste System with alternating SPE loading: Elute sample from SPE1 to column / Load sample 2 on SPE2

Application: Quantitative LC/MS/MS analysis of 24 trace pharmaceuticals and consumer products in water using on-line SPE enrichment. Tarun Anumol and Shane Snyder University of Arizona Sheher Mohsin – Agilent Applications Scientist Tarun Anumol and Shane Snyder are collaborators at the Un. of Arizona in Tucson. Sheher is located in Chicago, Il. This data will be published as an Agilent application note by February 2013. 11/15/2018

Compounds Analyzed Compound Class Atrazine Herbicide PFBS Fluoro-surfactant Bisphenol A Plasticizer PFOA Caffeine Stimulant PFOS Carbamazepine Anti-seizure Primidone Anticovulsant DEET Insect-repellant Simazine Estrone Hormone Sulfamethoxazole Antibiotic Fluoxetine Anti-depressant Atenolol Β-blocker Gemfibrozil Anti-cholesterol TCPP Flame-retardant Ibuprofen Analgesic Testosterone Meprobamate Anti-anxiety Triclocarban Anti-microbial Naproxen Pain-reliever Triclosan Diltiazem Anti-histamine Trimethoprim 24 compounds consisting of common Pharmaceutical and Consumer Products found in Western US wastewaters, perfluorinated compounds, herbicides, and a flame retardant. 11/15/2018

Summary Experimental Methods Collect 5 mL sample and store at 4C for preservation. Filter sample through 0.2 µm filters. Combine 1683 µL of sample (weight using analytical balance for accuracy) with 17 µL of a 10 ppb surrogate standard mix. Vortex for 30 sec. Analyze by UHPLC/MS/MS with the Agilent 1290 LC coupled to an Agilent 6460 Mass Spectrometer LC/MS/MS. Large volumes of sample are not required for this method. A 1.5 ml aliquot is taking by the autosampler from the 2 ml water sample in the autosampler vial. 11/15/2018

Chromatography Simultaneous analysis of 24 PPCPs in both ESI+ & ESI- mode. Injection Volume: 1.5 mL Cycle time (Extraction + Analysis): 18.5 min Trace Enrichment Cartridge: PLRP-S, 15 µm Analytical Column: Poroshell 120 EC, 2.1x50 mm The 18.5 minute cycle time include the on-line SPE and chromatography! PLRP-s polymeric reverse phase/polystyrene divinyl benzene/EC is endcapped 11/15/2018

Method Reporting Limits (ng/l) Analyte MRL Atenolol 15 Meprobamate 0.5 Atrazine 5 Naproxen 10 Bisphenol A PFBS Caffeine PFOA Carbamazepine 2.5 PFOS DEET 0.1 Primidone Estrone 20 Simazine 1.5 Fluoxetine Sulfamethoxazole Gemfibrozil TCPP Ibuprofen Triclocarban 1 Trimethoprim Triclosan S/N> 10 (by height) for 3 successive injections 11/15/2018

Evaluation of On-line Solid Phase Extraction and Direct Injection-LC/MS/MS for the Analysis of Chemicals on U.S. EPA’s Contaminant Candidate List 3 Sheher Bano Mohsin Confidential April 2010

Chemicals for LC/MS/MS Evaluation* 4,4’-methylenedianiline 3-hydroxycarbofuran bensulide clethodim fenamiphos fenamiphos sulfone fenamiphos sulfoxide methomyl quinoline tebuconazole tebufenozide thiodicarb 9 CCL3 chemicals 3 CCL3 degradates *Slide Taken From: Evaluation of On-line Solid Phase Extraction-LC/MS/MS for the Analysis of Chemicals on U.S. EPA’s Contaminant Candidate List 3 - Jody A. Shoemaker and Daniel R. Tettenhorst

Online SPE Instrumentation in Schaumburg Agilent 1200 Infinity Series Online-SPE solution system comprising: G1312B Binary Pump with modular degasser G1329B Standard Autosampler G4227A Flexible Cube with 2-position/10-port valves, G1316C Thermostatted Column Compartment. 6460 Triple Quadrupole LC/MS with Jet Stream Technology Online SPE Instrumentation in Schaumburg Confidential April 2010

Configuration showing the plumbing for direct on-column injection and on-line SPE.

LC/MS/MS Parameters Binary Pump: • Solvent A: Water + 0.1% formic acid. Solvent B: ACN + 0.1% formic acid. Analysis Flow rate: 0.4 mL/min. Column: ZORBAX EclipsePlus C18 3.0x50mm 1.8 μm, 50 °C. Flexible Cube: Valves: 2 of 2-position/10-port Quick-Change valves. Flow rate: 1.0 mL/min. Solvent: A1: 3% MeOH/Water, B1: ACN/IPA/MeOH/Water 1/1/1/1 v/v/v/v. Standard Autosampler fitted with 900 μL metering head Auto-SPE Injection volume: 900 μL using a draw and eject speed of 1,000 μL/min. Direct Injection volume: 90 μL Triple Quadruple MS method with Agilent Jet Stream thermal gradient focusing technology: • Gas temperature: 300 °C. Gas flow: 10 L/min. • Nebulizer: 45 psi • Sheath gas temperature: 350 °C. Sheath gas flow: 11 L • Capillary: 4,000 Volt. Nozzle: 0 Volt

Dynamic MRM Transitions Cmpd Name Prec Ion Prod Ion Frag (V) CE (V) Cell Acc (V) Ret Time (min) Ret Window Polarity 4-4-diaminodiphenylmethane 199.1 106 120 28 3 6.74 1.81 Positive Bensulide 398 356 100 1 2 11.06 1.11 314 6 158 26 Carbofuran-3-hydroxy 238.1 181 110 8 8.15 0.94 163 11 Clethodim 360.1 206 12 4 11.67 1.15 166 130 27 Fenamiphos 304.1 234 150 15 10.28 1.03 217 23 202 31 Fenamiphos-sulfone 336.1 308 9.13 0.91 266 18 188 24 Fenamiphos-sulfoxide 320.1 292 9 8.64 0.99 233 171 Methomyl 107 170 17 1.14 95 Quinoline 103 30 6.97 77 Tebuconazole 308.1 125 42 10.49 1.05 70 Tebufenozide 353.2 297 85 10.85 1.09 133 Thiodicarb 355 80 9.17 1.28 108 14 88 Dynamic MRM Transitions

Superimposed chromatograms Showing Reproducibility of Retention time as well as Area Counts for two runs Alternating Between two Different SPE Cartridges. The calibration standard used has a concentration of 10 ppt (ng/L) each for all 12 compounds with quantifier and qualifier ions. Each run alternates between two different SPE cartridges.

Mix at 1 ppt Tebufenozide Tebuconazole Carbofuran-3-Hydroxy Quinoline Fenomiphos Bensulide Fenomiphos Sulfoxide Fenomiphos Sulfone 4,4-diaminodiphenyl methane Thiodicarb Methomyl Clethodim

Mix at 0.1 ppt

1 ppt and 0.1 ppt 1 ppt 1ppt 1 ppt 0.1 ppt 0.1 ppt 1 ppt 0.1 ppt 1 ppt

Direct Injection and online-SPE Same amount on column

Fenomiphos Sulfoxide 1.0 ppt 0.5 ppt 0.1 ppt

Fenomiphos Sulfoxide 0.1 ppt to 100 ppt On-line SPE

Fenomiphos Sulfone 1 ppt 0.5 ppt 0.1 ppt

Fenomiphos Sulfone 0.1 ppt to 100 ppt Online -SPE

Tebufenozide 1 ppt 0.5 ppt 0.1 ppt

Tebufenozide Online SPE

Thiodicarb

Thiodicarb Online SPE

Clethodim

Clethodim 0.1 ppt to 100 ppt Online-SPE

Bensulide 1 ppt 0.5 ppt 0.1 ppt

Bensulide Online SPE

Tebuconazole 1 ppt 0.5 ppt 0.1 ppt

Tebuconazole 0.1 ppt to 100 ppt Online SPE

Fenomiphos 1 ppt 0.5 ppt 0.1 ppt

Fenomiphos Online SPE

Carbofuran-3-Hydroxy 1 ppt 0.5 ppt 0.1 ppt

Carbofuran-3-Hydroxy Online SPE

Quinoline 1 ppt 0.5 and 0.1 ppt

Quinoline 0.1 to 1000 ppt Online SPE

Water Samples Analysis Water samples from a lake-fed municipal water supply (tap), residential well (ground), a suburban collection basin (WestLake) and a flood control reservoir (Spring Creek) were analyzed. Notably, both WestLake and Spring Creek are in the drainage basin of nearby public and private golf courses. Samples were filtered with 0.45 um disk filters prior to analysis.

Tebuconazole in Water Spring Creek 50 ppt WestLake 37 ppt Ground 0.9 ppt Tap 0.004 ppt

Lake Water and Ground water Results are based on the average of duplicate analyses, for the one quantifier transition (308.1 to 70.0) and one qualifier transition (308.1 to 125.0). Tebuconazole is used as a fungicide for turf control. Both WestLake and Spring Creek are in the drainage basin of nearby public and private golf courses. Lake Water Amount ~ 50 ppt Ground Water

Performance data for all compounds present in the study based on the calculated signal to noise at 0.1 ppt and assuming an LOQ at approximately 10: 1 signal-to-noise. The signal to noise calculation is based on peak area and noise is RMS x 3. Compound Name Level (ppt) Signal to Noise (S/N) Nominal LOQ @ 10:1 S/N 4-4-diaminodiphenylmethane 0.1 25 0.04 Bensulide 10.3 Carbofuran-3-hydroxy 34.4 0.03 Clethodim 7 0.15 Fenamiphos 301.2 0.005 Fenamiphos-sulfone 7.9 Fenamiphos-sulfoxide 8.2 Methomyl 8.3 Quinoline* 500 0.002 Tebuconazole 427 0.0025 Tebufenozide 9.4 Thiodicarb 9 * uncertainty due to unresolved carryover

Trace level analysis of herbicides in drinking and surface water by online-SPE LC/triple quadruploe MS to the lower ppt range Edgar Naegele, Agilent Technologies R&D and Marketing GmbH & Co. KG, Waldbronn, Germany Confidential April 2010

MRM and dynamic MRM MS method Confidential April 2010

Chromatograms of a calibration standard with a concentration of 100 ppt (ng/L) each for all 28 pesticides measured by the final SPE-LC dynamic MRM method with quantifier and qualifier ion. Confidential April 2010

Water samples from a tap, Rhine river, and a spring spiked with 25 ppt of all 28 pesticides. The retention time window from 14.5 minutes to 19.1 minutes is shown. The table shows the average measured concentrations of all pesticides (n=12) within the retention time window dependent on the sources of water together with precision RSD and accuracy. Confidential April 2010

Conclusions We have demonstrated an integrated, cost effective scheme to do on-line SPE coupled to LC/MS/MS. The technical barriers and potential technology transfer problems associated with traditional online SPE system have been avoided. The direct coupling of the SPE cartridges (PLRP-S, 2.1x12.5mm, 15-20um) to the LC/MS/MS system was done through the integrated valves. The use of Dynamic MRM enables complex mixtures to be efficiently analyzed. We have demonstrated excellent performance with challenging contaminants on the contaminant list. LOQ for all components was nominally 1 ppt or lower. Results for real samples, including ground water, lake and flood control reservoir, the latter two obviously containing visible swimming organisms, produced measurable tebuconazole results.

Expand Your Versatility Agilent 1200 Infinity Series Online SPE solution Expand Your Versatility Boost Performance enrichment of analytes and matrix removal for utmost sensitivity Save Time individual configurations for broad automation flexibility Reduce Costs 1290 Infinity Flexible Cube contains a built-in pump For online sample preparation, upgrade your system with Agilent 1290 Infinity Flexible Cube featuring built-in pump to condition and flush re-usable cartridges solvent selection valve Reduce costs: the 1290 Infinity Flexible Cube comes with a built-in piston pump, so that for many applications no second LC pump is needed, i.e. save second LC pump in your set-up

Journal of Chromatography A, 1286 (2013) 166– 174 Quantitative analysis of naphthenic acids in water by liquid chromatography–accurate mass time-of- flight mass spectrometry Ralph Hindle, Matthew Noesthedena, Kerry Perub, John Headley Vogon Laboratory Services Ltd., Unit 4, 34 Griffin Industrial Pt., Cochrane, Alberta T4C 0A3, Canada Water Science & Technology Directorate, Environment Canada, 11 Innovation Blvd., Saskatoon, SK S7N 3H5, Canada Confidential April 2010