Sample Prep Options for GC users Tina Chambers Technical Specialist Sample Preparation tina.chambers@agilent.com 949-842-0493 Why Bother? • Removal of interferences which would otherwise affect detection of analyte •Removal of interferences which would otherwise affect instrumentation and/or columns • Concentration of an analyte to a detectable levels • Solvent switching into an analytically more compatible solvent Matching SPP to Analytical Method All GC based systems require highest degree of cleanliness, regardless of detector!! Sample Preparation Options •Dilute and shoot •Protein precipitation •Liquid-liquid extraction/solid supported liquid extraction •Solid phase extraction •QuEChERS Considerations for LLE/SLE - Polarity of the analyte – soluble in non-miscible organic? - Nature of interfering compounds – presence of lipids? - LODs/LOQs – is concentration necessary? TOXI-TUBES A & B ® • Contain solvents and salts ideally proportioned for Clinical/Forensic drug analysis • Ultimate in ease-of-use: just add sample, mix and centrifuge • Can be used with challenging matrices: post-mortem blood, gastric content, liver tissue etc. • Absolutely no method development required! Sample Preparation – Supported LLE (SLE) Hydromatrix™ - diatomaceous earth sorbent • • • • Composed of fossilized diatoms Purified at high temperatures High surface area for water adsorption Very polar surface Chem Elut™ - pre-assembled cartridges with Hydromatrix Combilut ™- 96-well plate filled with Hydromatrix The SLE Process Before Extraction Dry sorbent Apply Sample Extract with Organic Solvent Aqueous layer Organic layer The Chem Elut Method Aqueous sample being applied Solid support adsorbs water onto high surface area particles Organic extraction solvent Multi-Residue Confirmation of Pesticides in Honey using SLE App note SI-01002 Recovery comparison of pesticides between solid supported liquid-liquid extraction (SLE) on Chem Elut and classical liquid-liquid extraction (LLE). LLE/SLE summary - Polarity of compound and nature of interferences dictate whether this is suitable - pH adjustments and/or addition of salts can be helpful - LLE/SLE solvents are often a good choice for GC analysis Considerations for Solid Phase Extraction (SPE) - High degree of versatility (over 40 different chemistries available) - Concentrates dilute samples - Method development often necessary Solid Phase Extraction Triangle Often, both the the analyte and the matrix are known, it is sorbent choice which is the critical component Four Steps of SPE • Conditioning: Preparation of the sorbent prior to sample addition • Retention: Analytes of interest and other interferences adsorb onto the surface of the sorbent during sample addition • Washing: Elimination of undesired interferences • Elution: Selective desorption and collection of desired analytes from the sorbent The Four Steps of SPE – Selective Elution Green = Blue and Yellow Blue is more non polar than yellow Blue is retained Sample loading Retention Rinsing Elution Silica VS. • “True” polar/ion exchange possible • Wide range of chemistries • Wide range of established methods • Can be more selective • Conditioning is crucial Polymer • Inherent hydrophobicity (conditioning) • Higher capacity (sorbent mass/flow) • Polarity gradient in Plexa Polymers and Conditioning 450 ng/mL, LCUV 120% 100% % Recovery 80% Methanol Aqueous 60% Non-Conditioned 40% 20% 0% Atenolol Ranitidine Pseudoephedrine Quinidine Brompheniramine Mianserin Fluoxetine Silica and Conditioning Benefits by Design: Sorbent Quality Bond Elut Plexa Bond Elut Plexa PCX See Plexa Advantage Note, SI-1935 Oasis HLB Oasis MCX Confidentiality Label Size Distribution Confidentiality Label May 30, 2013 Non-Polar Method Development Method Development Considerations • What is the partition coefficient (log P) of the un-ionized compound? log Poctanol/water = log ([solute]octanol/[solute]water) • If log P > 1.5, good candidate for hydrophobic method • If log P unknown, “rule of thumb”: four covalently bound carbon atoms or benzene ring minimum requirement • If compound is ionizable, determine pKa • Load acids at pH<pKa by 2 units minimum • Load bases at pH>pKa by 2 units maximum Method Development Considerations Solubility characteristics of target compound? Method Development Considerations • Select suitable solvents • Prepare 0%-100% concentrations 120000 100000 80000 60000 40000 20000 0 Elution Profile 0% MeOH 10% MeOH 20% MeOH 30% MeOH 40% MeOH 50% MeOH 60% MeOH 70% MeOH 80% MeOH 90% MeOH 100% MeOH • Plot recoveries Elution Profile Method Development Consideration • Highest % organic with low recoveries for wash • Lowest % organic with high recoveries for elution 120000 100000 80000 60000 40000 20000 0 Elution Profile 0% MeOH 10% MeOH 20% MeOH 30% MeOH 40% MeOH 50% MeOH 60% MeOH 70% MeOH 80% MeOH 90% MeOH 100%… • Try acid/base modifiers Elution Profile Interactions on Ion Exchange Sorbents OH Silica base SO3- NH3+ CO2H +N CO2OCON(CH3)2 Electrostatic attraction Ion Exchange Nomenclature STRONG: Ionic group is always charged (+ or -) WEAK: Ionic group is variably charged (+ or -) CATIONS: (+) Found in basic compounds ANIONS: (-) Found in acidic compounds Extract weak ions with strong exchangers and strong ions with weak exchangers! Ion Exchange Sorbent Selection • All bases except quarternary amines can be processed on Plexa PCX or Bond Elut SCX • Only carboxylic acids can be processed on Plexa PAX or Bond Elut SAX • Phosphates, sulfates and other STRONG acids should be processed with weak anion exchangers (Bond Elut NH2, PSA) Interactions on Ion Exchange Sorbents OH 50% Silica base SO3- NH3+ CO2H 50% If the pKa=9 and the pH=9 NH2 CO2H Method Development Considerations What is the pKa of your compound? pKa= -log Ka and Ka = [A-][H+]/[HA] • If pH=pKa, 50% of the compound is ionized and 50% is neutral • To ensure full charge or full neutralization, employ the rule of 2 Interactions on Ion Exchange Sorbents OH 100% Silica base SO3- NH3+ CO2H 0% If the pKa=9 and the pH=7 NH2 CO2H Interactions on Ion Exchange Sorbents OH 0% Silica base SO3- NH3+ CO2H 100% If the pKa=9 and the pH=11 NH2 CO2H Important Consideration for Ion Exchange • Reduce ionic strength of “salty” matrices by dilution • Consider competitive binding when choosing bed mass • Remember that ALL polymeric exchangers are mixedmode, elute in organic solvent • Some organic should be present even with silica based ion exchangers because of carbon linkers QuEChERS (Pronounced “catchers”) • Quick, Easy, Cheap, Effective, Rugged, and Safe • Portmanteau: blend of 2 or more words • www.quechers.com • Introduced in 2003: M. Anastassiades, S.J. Lehotay, D. Stajnbaher, and F.J. Schenck, J. AOAC Int 86 (2003) 412 • Validated in 2005, with subsequent modification in 2007 • AOAC 2007.01 and European Method EN 15662 • Streamlined approach that makes it easier and less expensive to examine pesticide residues in food 5/30/2013 QuEChERS First step extraction 1) Weigh sample, add water if needed, spike 2) Add 10ml ACN 3) Vortex 4) Add salt packet 5) Shake 1 minute 6) Centrifuge at 4,000 rpm for 5 minutes Second Step – Dispersive SPE 7) Choose d-SPE kit based on matrix characteristics 8) Transfer 1-8ml alliquot, vortex 1 minute 9) Centrifuge 11) Analyze by GC/MS or LC/MS Page 37 QuEChERS Applications • Pesticides • PAHs • Hormones • Antibiotics • Acrylamides • Vet Drug Residues • Animal tissue • Fish and Shellfish • Fruits and Vegetables • Food oils • Soil • Baby Food Page 38 Questions?