Sample Preparation Strategies for Water Analysis - …

1 2007 Waters CorporationSample Preparation Strategies Sample Preparation Strategies for Water Analysisfor Water AnalysisHannah WhiteHannah WhiteWaters Business Development ManagerWaters Business Development Manager 2007 Waters Corporation 2 OutlineOutline Introduction Why Sample Prep ConsiderationsoChoices of tools Why SPEoPre-Treatment Strategies Traditional approaches Modern approaches oMixed ModeoReverse phase Summary Appendix 2007 Waters Corporation 3 For high sensitivity analyses, such as those employing LC/MS/MS, proper Sample preparationcan be critical for minimizing matrix effects and concentrating analytes of Sample Prep?Why Sample Prep?60% of the work activity and operating cost is spent on Sample Preparation for introduction into the analytical systemThree Purposes: Removes interferences from Sample matrix Concentrating analytes of interest Improving analytical system performances 2007 Waters Corporation 4 Sample Preparation TechniquesSample Preparation Techniques Sample Preparation - The simplification of Sample matrix and enrichment of target analyte(s) Types of Sample Prep include.

2 Dilution Centrifugation Filtration Liquid/Liquid Extraction Solid Phase Extraction 2007 Waters Corporation 5 Some ConsiderationsSome Considerations Solid samples usually start with organic or aqueous extract of tissue or soil initial extract is adjusted for optimal SPE enrichment and/or cleanupopH adjustmentosolvent adjustment acetone/acetonitrile/IPA suitable for aqueous dilution, Reversed-Phase and Mixed-Mode SPE ethyl acetate/DCM/MTBE can be exchanged to hexane for normal-phase SPE Aqueous samples ( Water , beverage, plasma/urine) pretreatment may be appropriateopH adjustmentofiltration/centrifugationopro tein precipitation Usually, aqueous samples can be analyzed using Oasis Reversed-Phase or Mixed-Mode SPE 2007 Waters Corporation 6 PrePre--Treatment Prior to SPET reatment Prior to SPEPre treatment: Solid samples (soil, tissue, etc.) shake, sonicate or soxhletoextract with polar organic solvent (methanol, acetonitrile); polarsoextract with organic solvent + drying agent (DCM, acetone).

3 Non-polars, multi-residue Non aqueous Liquidoif Water soluble, dilute with Water for reversed-phase (or mixed-mode) SPEoif hexane soluble, dilute with or exchange to hexane for NP-SPE Wastewater filter or centrifuge as necessaryofiltered solids and filter may require Analysis as solids 2007 Waters Corporation 7 Why Solid Phase ExtractionWhy Solid Phase Extraction Isolation of the analyte(s) of interest from the matrix Sample Cleanup removal of matrix interference Increased sensitivityoIncreased system uptimeoLonger column lifetime Enrichment of analyte(s) of interest oIncreased sensitivity Exchange to LC or GC compatible solventSPE is also faster and more suitable for automation compared with liquid-liquid extraction 2007 Waters Corporation 8 Short List of Sorbent Types Short List of Sorbent Types for SPEfor SPE Normal-Phase Sorbents (polar sorbents) Silica, Alumina, Florisil , Aminopropyl silica, Diol silica, GCB Reversed-Phase Sorbents (non-polar sorbents) Oasis HLB C18, C8 etc (alkyl silica's) Carbon based sorbents Ion Exchange Accell Plus CM, QMA Mixed Mode (ion-exchange/reversed phase) Oasis MAX, Oasis WAX (strong and weak anion-exchange) Oasis MCX, Oasis WCX (strong and weak cation-exchange) 2007 Waters Corporation 9 OutlineOutline Introduction Why Sample Prep ConsiderationsoChoices of tools Why SPEoPre-Treatment Strategies Traditional approaches Modern approaches oMixed ModeoReverse phase Summary Appendix 2007 Waters Corporation 10 SPE StrategiesSPE #1 Retention, cleanup, # #3 Dispersion 2007 Waters Corporation 111.

4 Load2. wash3. is loaded onto SPE sorbent Analyte(s) of interest are retained on interferences are washed off are eluted from sorbentSPE Strategy 1 SPE Strategy 1 RetentionRetention--CleanupCleanup--Elut ionElution 2007 Waters Corporation 12 SPE Strategy 2 SPE Strategy 2 PassPass--Through CleanupThrough is passed through sorbent and collected no Sample interferences are retained on sorbentpass through 2007 Waters Corporation 13 SPE Strategy 3 SPE Strategy 3 Dispersion CleanupDispersion Cleanup Bulk sorbent is added to Sample with agitation Sample is filtered or centrifuged Supernatant is collected for analysisThis is similar to pass-through cleanup, but less effective- Dispersion SPE is a one stage (one theoretical plate) cleanup- Pass-through SPE is a multi-stage cleanup 2007 Waters Corporation 14 OutlineOutline Introduction Why Sample Prep ConsiderationsoChoices of tools Why SPEoPre-Treatment Strategies Traditional approaches Modern approaches oMixed ModeoReverse phase Summary Appendix 2007 Waters Corporation 15 IonIon--Exchange and MixedExchange and Mixed--ModeMode Many compounds of environmental interest are weak acids ( dinoseb) or weak bases (i.)

5 E. aniline). weak acids can be ionized at high pH weak bases can be ionized at low pH Some compounds are strong acids ( PFOA) or strong bases ( chlorhexidine) that are ionic except at extreme pH values A few of these compounds are quaternary amines ( paraquat), ionic at all pH Ionizable Compounds 2007 Waters Corporation 16 Why MixedWhy Mixed--Mode?Mode? Mixed-Mode SPE extends pH range for good retention of acids or bases Retention can be by reversed-phase, ion-exchange or both Chose retention mode by adjusting pH ion-exchange allows for good retention in strong solventoacids can be retained by anion-exchange while bases/neutrals are washed off with strong solventobases can be retained on cation-exchange while acids/neutrals are washed off with strong solvent For environmental Analysis , mixed-mode SPE allows simultaneous retention of acids and bases 2007 Waters Corporation 17 OasisOasis Family of MixedFamily of Mixed--Mode Mode Sorbents:Sorbents.

6 ReversedReversed--Phase Retention and Ion ExchangePhase Retention and Ion Exchange 2007 Waters Corporation 18 Oasis MixedOasis Mixed--Mode Sorbents Mode Sorbents PFOS, PFOA (perfluoroacids and related compounds) Oasis WAX Acidic Herbicides Oasis MAX Quats Oasis WCX Pharmaceuticals/pesticides (organic bases) Oasis MCXS trategies for Isolation and Enrichment of Individual Compounds or Compound ClassesOasis 2x4 method 2007 Waters Corporation 19 IntroductionIntroduction Perfluorinated compounds (PFCs) such as perfluorooctanesulfonate and perfluorooctanoic acid are persistent organic pollutants (POPs) PFCs have been identified in environmental samples worldwide PFOS can be detected at low PPT levels in most humans PFOS commonly found in arctic fauna There is need for reliable analytical methods for PFCs in food, drinking Water , tissue, plasma and blood In this presentation we will discuss Sample Preparation for UPLC-MS determination of PFCs in Water and tissue samples 2007 Waters Corporation 20 UPLCUPLC--MSMS--MS SystemMS System ACQUITY Ultra Performance LC Using m particles, and at elevated pressures up to 15.

7 000 psi Shorter Analysis Time Higher Resolution Broad selectivity options Quattro Premier XE Fast acquisition rates Sensitive detection Oasis sorbents Cleaner samples 2007 Waters Corporation 21 GoalsGoals Develop an Acquity UPLCTM separation based on a recently published method* Adapt or modify the SPE protocol for UPLC River Water Sample Chicken Liver tissue Sample Lower the quantification limits to under 1 ppb in Chicken Liver tissue, and Low ppt level in River Water Sample *S. Taniyasu et. Chrom. A., 1093 (2005) pp89-97 2007 Waters Corporation 22 Structures of PFOS and PFOAS tructures of PFOS and PFOAF3 COOHFFFFFFFFFFFF perfluorooctanoic acidPFOApKa ~ 1perfluorooctanesulfonatePFOSpKa<<1 FFFFFFFFFFFFSOOO-F3 CFFPFOA and PFOS are Persistent Organic Pollutantsof high interest worldwide. 2007 Waters Corporation 23 OasisOasis 2x4 Method 2x4 Method For Acids, Bases, and NeutralsFor Acids, Bases, and NeutralsNeutralsFor Bases:pKa 2-10 Use Oasis MCXFor Strong AcidspKa < Oasis WAXFor Strong BasespKa >10 Use Oasis WCXFor AcidspKa 2-8 Use Oasis MAXP repare SampleCondition/EquilibrateLoad SampleWash:5% NH4 OHElute 1:100% MeOHElute 2:2% Formic Acid in MeOHProtocol 2 Prepare SampleCondition/EquilibrateLoad SampleWash:2% Formic acidElute 1:100% MeOHElute 2:5% NH4OH in MeOHProtocol 1 BasesStrong AcidsStrong BasesAcids 2007 Waters Corporation 24 Optimized SPE ProtocolOptimized SPE Protocolfor River Waterfor River WaterConditions for Oasis WAX 3 cc 60mg cartridgesPrepare SamplepH 3 Condition2 mL methanol/2 mL waterLoad200 mLWash #11 mL 2% Formic acidElute 1 (Wash #2)2 mL methanolElute 22 mL 1% conc.

8 Ammonia in 10:90 methanol/MTBEO asis WAXO ptimized Protocol 1 Oasis WAX sorbent was selected for these analytes Logic: PFOA pKa ~1 PFOS pKa < 1 Oasis WAXNONNNNHHH++Hmixed-mode weak anion-exchangepKa~6 samples were evaporated and reconstituted in mLmobile phase 2007 Waters Corporation 25 SPE Protocol SPE Protocol Prepare SamplepH 3 Condition2 mL methanol/2 mL waterLoad200 mLWash #11 mL 2% formic acidWash #22 mL methanolElute 22 mL 1% conc. ammonia in 10:90 methanol/MTBEO asis WAXO ptimized Protocol 1@ pH 3 Sorbent, and analytes are fully charged(assures mixed-mode retention)Maximum load for good recovery of C3, C4 and PFBSA ssures sorbent is chargedRemoves neutrals and bases retainedby reversed-phaseMTBE based eluentminimizes elution of any retained humicmaterial 2007 Waters Corporation 26200mL river Water 6cc WAX _200uL recon Sm (SG, 1x1)2: MRM of 3 Channels Sm (SG, 1x1)2: MRM of 3 Channels in River WaterPFBS/PFOS in River Water100 ng/L (ppt)100 ng/L (ppt)BlankSpiked River WaterPFBSPFOS 2007 Waters Corporation 27200mL river Water 6cc WAX _200uL recon Sm (SG, 1x1)1: MRM of 5 Channels Sm (SG, 1x1)3: MRM of 5 Channels River WaterC3C3--C7 in River WaterC7 in River Water100 ng/L (ppt)100 ng/L (ppt)C5C4C3C7C6 2007 Waters Corporation 28200mL river Water 6cc WAX _200uL recon : MRM of 5 Channels.

9 MRM of 5 Channels in River WaterC12 in River Water100 ng/L (ppt)100 ng/L (ppt)BlankSpiked River WaterC12C8C10C9C11 2007 Waters Corporation 29 River Water RecoveriesRiver Water RecoveriesSpike Level g/LPFBS PFOS C3C4 C5 C6 C7 C8 C9 C10 C11 108 119 97 154 107 83 121 101 101 132 105 110 119 126 137 118 107 93 118 100 78 103 126 119 106 98 130 100 88 100 110 117 9999 102 102 92 115 9984681010410098 101 100 87 8982 103 99 101 66 Spike Level g/LPFBS PFOS C3C4 C5 C6 C7 C8 C9 C10 C11 108 119 97107 83 121 101 101 132 105 110 119 126 137 118 107 93 118 100 78 103 126 119 106 98 130 100 88 100 110 117 9999 102 102 92 115 9984681010410098 101 100 87 8982 103 99 101 66 Spike Level g/LPFBSPFOSC C4 C5 108 119 97107 83 121 101 101 132 105 110 119 126 137 118 107

10 93 118 100 78 103 126 119 106 98 130 100 88 100 110 117 9999 102 102 92 115 9984681010410098 101 100 87 8982 103 99 101 66 2007 Waters Corporation 30 Observations/RecommendationsObservations /Recommendations Fluorocarbon parts, tubing, etc. are potential sources of interferences UPLC fluidic lines were conditioned with 2% TFA in propanolfollowed with 4% conc. ammonia in Water (4 hours each step) Polypropylene (PP) lab ware may be best for Sample prep Do not use Teflon!! (possible positive interference) Analytes may adsorb to glass (possible negative interference) C3-C5 analytes are highly volatile Evaporative losses are possible, much more so at very low pH samples in glass vials may show loss of some analytes with time Analyze within 24 hrs of Sample prep 2007 Waters Corporation 31 ConclusionsConclusions Oasis WAX SPE method is effective for isolation and enrichment of C4-C8 perfluorosulfonic acids and C3-C12 perfluorocarboxylic acids from Water and tissue Acquity UPLC provides significantly reduced Analysis time and improved chromatographic behavior for these compounds compared with traditional HPLC The Quattro Premier XE API mass spectrometer, operated in MRM mode, provides outstanding sensitivity and selectivity for these compounds 2007 Waters Corporation 32 Acidic HerbicidesAcidic HerbicidesClOCH2 COOHCl2,4-DThese herbicides, such as 2,4-D, are used in cultivated agriculture, in pasture and rangeland applications, forest management and home and garden.