APPLICATIONS - Phenomenex

TN-013APPLICATIONSA Simple Approach to Fast and Practical Solid Phase Extraction (SPE)Method DevelopmentMichael Rummel, Terrell MathewsPhenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USASolid phase extraction is an effective technique for cleaning up andconcentrating samples. In the following application note we outlinea simple approach for solid phase extraction method developmentusing Strata ® and Strata -X SPE sorbents.Step 1. Sample Pre-treatmentReproducible, high efficiency solid phase extraction requires thatthe sample be made liquid prior to loading onto a SPE device. TheSPE sample should meet the following conditions:• Liquid of low viscosity (to pass through the cartridge).• Low solids or particulate contaminants (to prevent clogging).• Solvent composition that is suitable for retention (eachmechanism has different matrix solvent composition requirementsfor proper retention).Sample Pre-treatment RecommendationsBiological Samples (liquid)Urine, Wholeblood, Serum,Plasma, Bile, etc.Biological Samples (solid)Organ tissues,Feces, GIcontentsSample MatrixWater (waste,river, etc.)Soil, SludgeOintments,CreamsFruit, Vegetable,HerbsDilute sample 1:2 with appropriate buffer, precipitateproteins if proteinaceous (ZnSO 4, ACN), hydrolyzeurinary glucuronides, disruption of protein binding(sonication, enzymatic, acids/bases).Homogenize with organic or aqueous solventdepending upon analyte solubility. Settle, decant,centrifuge or filter supernatant. Perform direct MatrixSolid Phase Dispersion (MSPD) extraction on tissue.Buffer to appropriate pH and filter particulates fromsample.Homogenize with organic or aqueous solventdepending upon analyte solubility. Settle, decantand filter supernatant; perform Soxhlet extraction.Oil based:Dissolve in non-polar organic (hexane) and extractvia polar SPE.Water based:Dissolve in water or water miscible organic(methanol) and extract via non-polar SPE.Homogenize with organic or aqueous solventdepending upon analyte solubility and filtersupernatant. Use appropriate SPE mechanism forthe dissolution solvent (hexane = polar mechanism;aqueous = non-polar mechanism; methanol/ACN =either non-polar or polar after proper dilution).Step 2. Selecting Strata and Strata-X SorbentsIdentify the possible SPE retention mechanism: Reversed Phase(RP), Ion-Exchange (IEX) or Normal Phase (NP):The sample solvent composition will guide you towards anappropriate SPE mechanism.AqueousWater, Urine, Plasma,Blood, AqueousTissue Prep.SPEMechanismReversedPhaseNormalPhaseIon-Reversed PhaseStrata-X, XL, C18,PH, C8Ion-ExchangeStrata-X-C,X-CW, X-A, X-AW,XL-C, XL-CW,XL-A, XL-AW, SAX,SCX, WCX, NH 2, ABW,Screen-A, Screen-CPosExchangeNegAnalyteFunctional GroupRhydrocarbonaromaticR - OHhydroxylR - NH 2amino -Add H 2ONR 4+strongRNH 3+weakRSO 3- strongRCO 2- weakOrganicWater miscible, Methanol,Acetonitrile, AcetoneFor ionic species onlyIon-ExchangeStrata-X-C, X-CW, X-A,X-AW, XL-C, XL-CW,XL-A, XL-AW, SAX,SCX, WCX, NH 2, ABW,Screen-A, Screen-CSorbentFunctional GroupRhydrocarbonaromaticCNpolarOHpolar-O 2C—weak-O 3S—strong+H 3N—weak+R 3N—strongAdd HexaneOrganicNot water miscible,Hexane,DichloromethaneNormal PhaseStrata NH 2, CN, Si-1,FL-PR, EPH, AL-NOnce the general mechanism is identified, it will be necessary toidentify the most specific Strata or Strata-X sorbent by matchingthe analyte functional groups to the sorbent functional group.Strata or Strata-XSorbentC18-E, C18-U, C8C18-T, X, XLPH, SDBL, X, XLCN, NH 2Si-1, CN, EPHWCX, X-CW, XL-CWScreen-C, SCX, X-C, XL-CNH 2, X-AW, XL-AWScreen-A, SAX,X-A, XL-AStep 3. Sorbent Mass SelectionTo select the proper sorbent mass, it is first necessary todetermine the volume of sample needed to be extractedin order to meet method detection limits (not including buffer).Two tables are included below: Polymer-based andFor additional technical notes, visit www.phenomenex.comPage 1 of 4

TN-013APPLICATIONSsilica-based. This is necessary because the large surface areaof polymeric sorbents such as Strata -X have a higher analytecapacity per gram than Strata ® silica-based sorbents.Suggested Loading CapacityTable 1.Polymer-Based SorbentsSample MatrixSorbent MassStrata-X, X-C,X-CW, X-A, X-AWStrata-XL, XL-C, XL-CW, XL-A, XL-AWBlood, serum, plasma 30 mg 250 µL 125 µLUrine 30 mg 1 mL 500 µLFiltered tissue homogenates 60 mg 100 mg 50 mgEnvironmental SamplesSorbent MassStrata-X, X-C,X-CW, X-A, X-AWStrata-XL, XL-C, XL-CW, XL-A, XL-AWWater (particulate-free) drinking 200 mg 100 - 400 mL 50 - 200 mLWater (particulate-laden) rivers,runoff, etc.500 mg 100 - 400 mL 50 - 200 mLSoil extracts 500 mg 100 g 50 gTable 2.Silica-Based Sorbents (Strata C18, C8, SCX, SAX, WCX, NH 2, etc.)Sample MatrixSorbent MassBlood, serum, plasma 50 mg sorbent per 250 µLUrine 50 mg sorbent per 500 µLFiltered tissue homogenatesEnvironmental SamplesWater (particulate-free) drinkingWater (particulate-laden) rivers, runoff, etc.Soil extracts100 mg sorbent per 100 mg tissueSorbent Mass500 mg/100 mL - 500 mL sample1 g/100 mL - 500 mL sample1 g/100 g of soil extractSorbentMassStep 4. Method and Sorbent Volume SelectionThe volume of solvent needed for SPE processing is directly relatedto the mass of sorbent in the SPE tube and more specifically the“bed volume” of the SPE device. Intuitively we know more sorbentrequires more solvent, less sorbent = less solvent. Typically 4 – 16bed volumes are used in SPE methods.Sorbent Wash and Elution Volumes*StrataSilica-BasedSorbentMassPracticalMinimumWashandElutionVolume4 bedvolumesRecommendedWash andElution Volume8 bed volumesStrata-XPolymer-BasedSorbentMass*PracticalMinimumWashandElutionVolume4 bedvolumes10 mg 60 µL 120 µL 10 mg 100 µL 200 µL— — — 30 mg 300 µL 600 µL50 mg 300 µL 600 µL — — —— — — 60 mg 600 µL 1.2 mL100 mg 600 µL 1.2 mL 100 mg 1 mL 2 mL150 mg 900 µL 1.8 mL 150 mg 1.5 mL 3 mL200 mg 1.2 mL 2.4 mL 200 mg 2 mL 4 mL500 mg 3 mL 6 mL 500 mg 5 mL 10 mL1 g 6 mL 12 mL 1 g 10 mL 20 mL2 g 12 mL 24 mL — — —5 g 30 mL 60 mL — — —10 g 60 mL 120 mL — — —RecommendedWash andElution Volume8 bed volumes*Strata-X polymeric resins have a larger surface area than Strata silicabasedmaterial, hence requiring slightly more solvent per gram forprocessing. The elution volumes are specific to the chemical nature ofthe analyte being extracted, its concentration in the sample, the chemicalnature of the eluting solvent and the bed mass used. The aboveis a guideline. An elution study should be conducted to determine theappropriate volume to use.Generic MethodEach SPE mechanism/phase has a general set of solvent conditionsunder which SPE may be performed. Use the solvents/pHconditions listed below, volumes as determined in Method andSorbent Volume Selection.Reversed Phase SPE Method Normal Phase SPE Method Strong Ion-Exchange SPE MethodSorbent SDB-L, C18, C8, PH, CN, X, XL Silica, Florisil ® , NH 2 , CNAnion exchange: Screen-A, SAX, X-A, X-AW,XL-A, XL-AWCation exchange: Screen-C, SCX,X-C, X-CW, XL-C, XL-CWAnalyte Properties Low to moderate polarity (or nonpolar)HydrophobicNeutralized/unchargedPharmaceuticalsPesticides, herbicidesModerate to high polarity compounds(neutralized/uncharged)Pesticides Ionized/charged compounds Anion exchange: Acidic analytesCation exchange: Basic drugsSample/Matrix Aqueous, diluted with buffer Biological fluidsWaterConditioning Step 1. Solvation –polar organic solvents2. Equilibration –aqueous, buffersWash Step Aqueous buffers with 5 to 50 %polar organic solventElution StepPolar or non-polar organic solvent(s)with or without water, buffer and/orstrong acid or base1. Methanol2. Water or bufferMethanol: Water (5:95)Methanol: Acetonitrile(50:50)Non-polar organic solvents ormoderately polar organic solvents1. Solvation –polar organic solvents (optional)2. Equilibration –sample/matrix solventNon-polar organic solvents with alow concentration (1 to 5 %) ofmoderate to low polarity organicsolventsNon-polar organic solvents containinghigher concentrations (5 to 50 %)of moderate to high polarity organicsolventsHexane, chloroform, petroleumether, toluene or methylenechloride1. Methanol (optional)2. Hexane or chloroformHexane with 1 % THF, ethylacetate, acetone, acetonitrileor IPAHexane with 10 % THF, ethylacetate, acetone, acetonitrileor IPAAqueous; Low ionicstrength buffers(

))))) ) ))) ) )) )) ) ))))))))) ))))))) ) ))))TN-013APPLICATIONSStrata -X Polymeric SPE Sorbents• Clean extracts from biological sample matrices• Streamlined method development and simple processingStrata ® Silica SPE Sorbents• Extremely reproducible from batch-to-batch• Formats for large and small volume samplesSorbentStrata-XStrata-X-CStrata-X-CWStrata-X-AStrata-X-AW)nFunctionalGroup Mode Analyte)n)n)n)n)n)n)n)n)nn)n)n)n)n)nOṄ .Ṅ ..OS O -Ṅ . H +S O OO - -OOSOOOO OOOHCH3 CI -N +OHO -OHOCH3 NHNH 2NHNH 2NHNH 2NHOOHNH 2Reversed PhaseReversed Phaseand Strong CationExchangeReversed Phaseand WeakCation ExchangeReversed Phaseand Strong AnionExchangeReversed Phaseand WeakAnion ExchangePolar andNon-PolarBasesBases(includingQuaternaryAmines)AcidsAcids(includingSulfonicacids)Reversed PhaseC18-E• Extraction of hydrophobic molecules from aqueous and biological samplesC18-U• Increased extraction efficiency and enhanced clean up of hydrophobiccompounds that contain hydroxy or amine functional groups from water orbiological fluidsC18-T (wide pore)• Extracting large hydrophobic molecules (up to 75 kD) from water or biologicalmatricesC8• Extracting hydrophobic compounds from water or biological fluids that areretained too strongly on Strata C18-E or Strata-XPhenyl• Extracting aromatic hydrophobic compoundsCN• Extracting non-polar compounds that are retained too strongly on StrataC18-E or C8SDB-L (styrene-divinylbenzene)• Extraction of non-polar and polar moleculesStrata-XLStrata-XL-CStrata-XL-CWStrata-XL-AStrata-XL-AW)n)n)n)n)n)n)n)n)n)n)n)n)n)nn)n)nṄ .Ṅ .OSOO SNHNHCH3 CI - 2CH3Ṅ .H +O -H +O OS O -OOHOOOHOOṄ .ON +OOOOHOHO -OS O -NHNH 2ONHNH 2NHNH 2Large ParticleReversed PhaseLarge ParticleReversed Phaseand Strong CationExchangeLarge ParticleReversed Phaseand WeakCation ExchangeLarge ParticleReversed Phaseand Strong AnionExchangeLarge ParticleReversed Phaseand WeakAnion ExchangePolar andNon-PolarBasesBases(includingQuaternaryAmines)AcidsAcids(includingSulfonicacids)Normal PhaseCN• Normal phase sorbent that can effectively extract polar compounds fromnon-polar solventsNH 2• Extraction of strong anions from aqueous samplesEPH (Extractable Petroleum Hydrocarbon)• Fractionation of aliphatic and aromatic extractable hydrocarbons from soiland water samplesSilica• Extraction of polar compounds that are similar in structureFlorisil ® (FL-PR)• Extraction of pesticides from environmental samplesAlumina-N• Extraction of polar compounds from food and environmental samplesCation ExchangeWCX (weak cation exchange)• Extraction of quaternary aminesSCX (strong cation exchange)• Extraction of 1˚, 2˚ and 3˚ amines from biological fluidsScreen-C (mixed-mode cation exchange)• Extraction of basic drugs from biological matrices such as blood, serumand urineAnion ExchangeWAX (weak anion exchange)• Extraction of strong acids from aqueous solventSAX (strong anion exchange)• Extraction of organic acidsScreen-A (mixed-mode anion exchange)• Extraction of acidic drugs from biological matrices such as blood, serumand urineABW (specialty phase)• Fractionation of neutral compounds such as amides from acidic and basicanalytesIf Strata or Strata-X SPE products do not perform as well or betterthan your current SPE product of similar phase, mass and size,return the product with comparative data within 45 days for a FULLREFUND.For additional technical notes, visit www.phenomenex.comPage 3 of 4

TN-013APPLICATIONSORDERING INFORMATIONTubes 1 mL (100/box) 3 mL (50/box) 6 mL (30/box)Strata ® Silica-based sorbentsPhase 50 mg 100 mg 100 mg 200 mg 500 mg 200 mg 500 mg 1 gC18-E 8B-S001-DAK 8B-S001-EAK 8B-S001-EBJ 8B-S001-FBJ 8B-S001-HBJ 8B-S001-FCH 8B-S001-HCH 8B-S001-JCHC18-U — 8B-S002-EAK — 8B-S002-FBJ 8B-S002-HBJ — 8B-S002-HCH 8B-S002-JCHC18-T — 8B-S004-EAK — 8B-S004-FBJ 8B-S004-HBJ — 8B-S004-HCH 8B-S004-JCHC8 — 8B-S005-EAK — 8B-S005-FBJ 8B-S005-HBJ — 8B-S005-HCH 8B-S005-JCHPhenyl — 8B-S006-EAK — 8B-S006-FBJ 8B-S006-HBJ — 8B-S006-HCH 8B-S006-JCHSCX — 8B-S010-EAK 8B-S010-EBJ 8B-S010-FBJ 8B-S010-HBJ — 8B-S010-HCH 8B-S010-JCHWCX — 8B-S027-EAK — 8B-S027-FBJ 8B-S027-HBJ — 8B-S027-HCH 8B-S027-JCHSAX — 8B-S008-EAK 8B-S008-EBJ 8B-S008-FBJ 8B-S008-HBJ — 8B-S008-HCH 8B-S008-JCHNH 2 — 8B-S009-EAK — 8B-S009-FBJ 8B-S009-HBJ — 8B-S009-HCH 8B-S009-JCHCN — 8B-S007-EAK — 8B-S007-FBJ 8B-S007-HBJ — 8B-S007-HCH 8B-S007-JCHSi-1 — 8B-S012-EAK — 8B-S012-FBJ 8B-S012-HBJ — 8B-S012-HCH 8B-S012-JCHFlorisil ® — — — — 8B-S013-HBJ — 8B-S013-HCH 8B-S013-JCHEPH — — — — 8B-S031-HBJ — — —AL-N — — — — 8B-S313-HBJ — — 8B-S313-JCHStrata Mixed-mode sorbents (for drugs of abuse)Phase — 100 mg 100 mg 150 mg 200 mg — 200 mg 500 mg —Screen-C — 8B-S016-EAK 8B-S016-EBJ 8B-S016-SBJ 8B-S016-FBJ — 8B-S016-FCH 8B-S016-HCH —Screen-A — 8B-S019-EAK — — 8B-S019-FBJ — 8B-S019-FCH 8B-S019-HCH —Strata Polymeric sorbentsPhase 50 mg 100 mg — 200 mg 500 mg 200 mg 500 mg 1 gSDB-L 8B-S014-DAK 8B-S014-EAK — 8B-S014-FBJ 8B-S014-HBJ 8B-S014-FCH 8B-S014-HCH 8B-S014-JCHStrata -X Polymeric sorbentsPhase 30 mg 60 mg 60 mg 200 mg 500 mg 100 mg 200 mg 500 mgStrata-X 8B-S100-TAK 8B-S100-UAK 8B-S100-UBJ 8B-S100-FBJ 8B-S100-HBJ 8B-S100-ECH 8B-S100-FCH 8B-S100-HCHStrata-X-C 8B-S029-TAK — 8B-S029-UBJ 8B-S029-FBJ 8B-S029-HBJ 8B-S029-ECH 8B-S029-FCH 8B-S029-HCHStrata-X-CW 8B-S035-TAK — 8B-S035-UBJ 8B-S035-FBJ 8B-S035-HBJ 8B-S035-ECH 8B-S035-FCH 8B-S035-HCHStrata-X-A 8B-S123-TAK — 8B-S123-UBJ 8B-S123-FBJ 8B-S123-HBJ 8B-S123-ECH 8B-S123-FCH 8B-S123-HCHStrata-X-AW 8B-S038-TAK — 8B-S038-UBJ 8B-S038-FBJ 8B-S038-HBJ 8B-S038-ECH 8B-S038-FCH 8B-S038-HCHStrata-XL 8B-S043-TAK — 8B-S043-UBJ 8B-S043-FBJ 8B-S043-HBJ 8B-S043-ECH 8B-S043-FCH 8B-S043-HCHStrata-XL-C 8B-S044-TAK — 8B-S044-UBJ 8B-S044-FBJ 8B-S044-HBJ 8B-S044-ECH 8B-S044-FCH 8B-S044-HCHStrata-XL-CW 8B-S052-TAK — 8B-S052-UBJ 8B-S052-FBJ 8B-S052-HBJ 8B-S052-ECH 8B-S052-FCH 8B-S052-HCHStrata-XL-A 8B-S053-TAK — 8B-S053-UBJ 8B-S053-FBJ 8B-S053-HBJ 8B-S053-ECH 8B-S053-FCH 8B-S053-HCHStrata-XL-AW 8B-S051-TAK — 8B-S051-UBJ 8B-S051-FBJ 8B-S051-HBJ 8B-S051-ECH 8B-S051-FCH 8B-S051-HCHTerms and ConditionsSubject to Phenomenex Standard Terms & Conditions, which may be viewed atwww.phenomenex.com/TermsAndConditions96-well plates and online cartridges also available. Visit www.phenomenex.comTrademarksStrata is a registered trademark of Phenomenex, Inc. Strata-X is a trademark of Phenomenex,Inc. Florisil is a registered trademark of U.S. Silica Co.Page 4 of 4Copying or re-use of this information is not allowed without written permission from Phenomenex.Contact Phenomenex for more information on product offerings and FREE samples.Strata-X is patented by Phenomenex, Inc. U.S. Patent No. 7,119,145© 2011 Phenomenex, Inc. All rights reserved.www.phenomenex.comPhenomenex products are available worldwide. Email us at international@phenomenex.comAustraliaAustriaBelgiumCanadaDenmarkt: 02-9428-644401-319-1301+31 (0)30-2418700(800) 543-36814824 8048f: 02-9428-644501-319-1300+31 (0)30-2383749(310) 328-77684810 6265auinfo@phenomenex.com anfrage@phenomenex.com beinfo@phenomenex.com info@phenomenex.comnordicinfo@phenomenex.comFrancet:01 30 09 21 10f:01 30 09 21 11franceinfo@phenomenex.comThe Netherlandst:030-2418700f:030-2383749nlinfo@phenomenex.comGermany06021-58830-006021-58830-11anfrage@phenomenex.comNew Zealand09-478095109-4780952nzinfo@phenomenex.comIreland01 247 5405+44 1625-501796eireinfo@phenomenex.comNorway810 02 005+45 4810 6265nordicinfo@phenomenex.comItaly051 6327511051 6327555italiainfo@phenomenex.comPuerto Rico800) 541-HPLC(310) 328-7768info@phenomenex.com.comLuxembourg+31 (0)30-2418700+31 (0)30-2383749nlinfo@phenomenex.comUnited Kingdom01625-50136701625-501796ukinfo@phenomenex.comFinland(09)4789 0063+45 4810 6265nordicinfo@phenomenex.comMexico001-800-844-5226001-310-328-7768tecnicomx@phenomenex.comAll other countries:Corporate Office USA(310) 212-0555(310) 328-7768info@phenomenex.comTN56000111_L