The Secrets of Solid Phase Extraction (SPE)for Sample Preparation

Agilent AccuBONDII, AccuBONDII ENV and EVIDEXII SPE

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What Is Solid-Phase Extraction (SPE)?

• Sample preparation technique with principles similar to those ofHPLC for selective adsorption of analytes or interferences from complex matrices

• Used for sample cleanup and analyte concentration preceding LC, GC, ion chromatography and other techniques

• Cost-effective alternative to/replacement for liquid-liquid extraction (productivity, solvent, waste)

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Advantages of SPE vs. Liquid-Liquid Extraction

• Improved throughput (parallel vs. serial processing)

• Decreased organic solvent usage and waste generation

• Higher and more reproducible recoveries

• Cleaner extracts (contamination, solvent impurities)

• No emulsions

• Tunable selectivity ( SPE phase choices, solvent mixtures)

• Readily automated

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Typical Applications of SPE

• Sample Cleanup– Combinatorial reaction cleanup before LC-MS or LC– Pharmacokinetic studies, dissolution testing – Isolate analytes from complex matrices – urine, plasma– Remove “column killers” or major interferences – Eliminate late-eluters to allow isocratic analysis

• Trace Enrichment– Environmental analysis– Pharmaceutical and Agrochemical applications

• Desalting• Solvent Exchange• Sample Preservation and Storage

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Application and Importance of SPE is Growing …Rapidly

• Applied in cartridge format for 20+ years —Critically important for sample cleanup and trace enrichment

(esp. environmental and pharmaceutical labs) • SPE usage is accelerating

—Combinatorial synthesis and high-throughput LC-MS analysis—Overall LC-MS usage increases and greater instrument uptime —Smaller samples requiring smaller bed masses (tubes and plates)—Minimization of organic solvents and waste—Ease of automation (cost, reproducibility, throughput)

• Liquid handling and SPE workstations• 96-well plate autosamplers

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Typical SPE Formats

Schematic Diagram of a 96-Well SPE Extraction Plate System*

* 3M Corp.Manifold System

Single ExtractionDisk

SPE Cartridge

Schematic Diagram of a 96-Well Plate Extraction System

(LC-GC, S9, May, 1998)

R. E. Majors, LC-GC, 15(4), 318 (1997)

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SPE Modes—”Digital Chromatography”Matrix Adsorption• Analyte(s) unretained (k ~ 0)• Matrix retained

(k >> 1)

• No preconcentration advantage • Eluates may not be as clean• Sample loading often gravity fed• Used less often than analyte

adsorption

Analyte Adsorption• Analyte(s) retained (k >> 1)• Matrix unretained

(k ~ 0)and/or strongly retained (k >> 1)

• Preconcentration factor• Cleaner extracts • Load at 1-3 drops/sec (recovery ∝

1/flow)• Capacity issues may be more

important

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Fundamental Steps for SPE Adsorption Mode

Prewash*

Remove contaminants that could elute with analyte

Precondition Load Wash Elute

Prepare cartridge to accept sample

Load sample and rinse reservoir(s)

Wash with solvent that won’t elute analyte

Elute analyte in smallest volume possible

If elution solventwill be stronger

than precond. solvent

1. MeOH or ACN

2. Weak solvent(water, buffer)

Weakly retained matrix compds elute

Analyte and othermatrix compds

retained

Elute analyteleaving highly

retained compds

1 2

*Not necessary

for Agilent SPE

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General SPE Method Development StrategySPE Adsorption Mode: Background

• Research the Problem —Previous SPE and analysis conditions for the analyte and matrix?

• Characterize the Analyte—Structure, pKa, polarity, functional groups —Solvent solubility and stability

• Any restrictions on final solvent and concentration (technique or instrument)?

• Characterize the Sample Matrix—Possible interferences — similar functional groups, pKa, etc. —pH, ionic strength —Solvent solubility and stability—Qualitative and quantitative variability

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General SPE Method Development StrategySPE Adsorption Mode: Experimental

• Develop or apply effective HPLC or GC conditions to monitor progress— Assess recovery and eluate cleanliness

• Select and test sorbents—Determine which sorbents provide maximum analyte retention—Determine which eluent solvents yield highest recoveries

• Identify optimum wash solvent—Assess eluate cleanliness under conditions of maximum analyte

retention—Determine strongest wash solvent that will not elute analyte

• Test blank and fortified matrix—Assess eluate cleanliness and recovery using optimum wash and eluent

solvents• Test real samples and fortified samples

—Assess eluate cleanliness and recovery for different unfortified and fortified samples

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SPE Method ValidationVariables to Consider

• Validate SPE procedure—Sorbent (sorbent weight, different cartridges, different lots)—Preconditioning (strong solvent, weak solvent)—Loading solvent (% organic, pH, ionic strength, volume)—Wash solvent (% organic, pH, ionic strength, volume)

• Eluent (volume, % organic …)—Flow rates (loading, wash, elution)—Linearity and range (different analyte concs. and matrix loadings)—Analyte stability (loading solvent, eluent)—Matrix stability (loading solvent)

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Design Characteristics for SPE Family

• Provide a variety of popular chemistries to meet most application needs

• Provide silica and PS-DVB base materials• Provide most popular sizes of cartridges and bulk material for

“self-packers”• Pre-clean packing, tubes and frits – low organic extractables for

high sensitivity work• Economical for routine applications• Excellent quality assurance program to ensure consistent

results from batch-to-batch and year-to-year• Specialty phase for drugs of abuse testing

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Break Time

For Questions and AnswersPress *1 on Your Phone toAsk a Question

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SPE Products from AgilentAccuBONDII and EVIDEXII

• Improved AccuBONDII SPE Products—Solid Phase Extraction Cartridges—Bulk SPE Adsorbents and Accessories—Method Development Kits—Applications

• EVIDEXII SPE Specialty Cartridges—Drugs of Abuse—General Pharmaceutical Extractions—Applications

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AccuBONDII SPE Phases

Sulfonic acidStrong Cation Exchange (SCX)

Quaternary amineStrong Anion Exchange (SAX)

Silica, -CN, Diol, Amino, Alumina (acidic, basic, and

neutral), FlorisilNormal Phase (polar)

C2, C8, C18, Phenyl, ENV PS-DVB*Reversed Phase

Phases AvailableSPE Mode

* Newest phase

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AccuBONDII SPE Cartridges

• Silica and new PS-DVB cartridges available• Wide variety of cartridge sizes

—100, 200, 500 and 1000 mg—1, 3, and 6 mL tubes—96 well plates in final development—Tabless cartridges for automation (i.e. Gilson)

• Excellent quality assurance program—Random testing of cartridges

• surface characteristics• packing parameters

—Performance certificate in each box• Low extractables

—Pre-washed packings, frits, and tubes• Bulk packings available – 25 g bottles

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AccuBONDII SPE – Low Levels of ExtractablesTube and Frit

min0 4 8 12 16

min0 4 8 12 16

Agilent SPE AccuBONDII

Pre-washed Tube/Frit Only

Waters’ Sep-Pak C18 Tube/Frit Only

Programmed TemperatureGC-FID AnalysisSplitless Injection

Internal Standard

Total = 50 ppm

Total = 5 ppm

Internal Standard

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AccuBONDII SPE Method Development Kits

• SPE Method Development Kits – groups of phases that separate using the same mechanism

—Kit A (Reversed Phase)• C2, CN, Phenyl, C8 and C18

—Kit B (Normal Phase)• Si, Amino, Diol and CN

—Kit C (Ion Exchange)• SCX, SAX and Amino

• Multiple kits provide a wide variety of options for determining the best mechanism and phase type for your analyte and matrix

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AccuBONDII SPE Application Notes• Application Notes for AccuBONDII

—Environmental• Extraction of Chlorinated Pesticides in Water (SE-1)• Extraction of Polynuclear Aromatic Hydrocarbons in Water (SE-2)• Extraction of PCB’s in Water (SE-3)• Extraction of Triazines Using SPE Cartridges

—Pharmaceutical• Anabolic Steroids in Urine and Serum (SC-1)• Benzodiazepines in Serum (SC-2)• Caffeine and Metabolites in Serum (SC-3)• Barbiturates in Serum (SC-4)

—Available online:www.agilent.com/chem

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AccuBONDII Example ApplicationsTriazines in Different Matrices

• Characterize the Analyte• Structure, pKa, polarity,

functional groups • Solvent solubility and

stability• Any restrictions on final

solvent and concentration due to technique or instrument?

Triazines

• Three major species – simazine, atrazine and propazine – all structurally similar

• Mode of Action: Herbicides • Practically insoluble in water

• Soil – large number of charged species-adjust pH to retain triazines and do ion-exchange

• Muscle tissue – large amounts of non-polar lipids – retain these and elute triazines using C18

• Corn oil – non-polar glycerides and fatty acids –weakly retained on diol while triazines are strongly retained

• Characterize the Sample Matrix• Solvent solubility and stability• pH, ionic strength• Possible interferences—similar

functional groups, pKa, etc. • Qualitative and quantitative

variability

N N

N

Cl

NH NH

R1R2

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SPE Methods for Triazines in Complex Matrices

MethanolMethanolAcetonitrile/K2HPO4ELUTE

HexaneWaterAcetic acid,

acetonitrile, water, 0.1 M K2HPO4

WASH

Diluted with hexaneDiluted with waterDiluted with acetic acidLOAD

Methanol, hexaneMethanolAcetic acidPRE-TREAT

NoneHomogenized in methanol

Shaken in acetonitrileEXTRACTION

DiolODSSCXCARTRIDGE

Corn OilMuscle TissueSoil

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HPLC Analysis of Triazines Extracted from Different Matrices

Triazines from Corn Oil with Diol

Triazines from Muscle Tissuewith ODS

Triazines from Soil with SCX

Column: C18, 4.6 x 150 mm, 5 mm Mobile Phase: 50% methanol:50% 0.01M K2HPO4 Flow Rate: 2 mL/minDetection: UV 254 nm Sample: Triazines 1. Simazine 2. Atrazine 3. Propazine

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AccuBONDII Example ApplicationsBarbiturates in Serum

• Characterize the Analyte• Structure, pKa, polarity,

functional groups • Solvent solubility and

stability• Any restrictions on final

solvent and concentration due to technique or instrument?

Barbiturates

• Non-polar side groups make retention on C18 possible – but retention not strong

• Mid pH will eliminate charge and improve retention

• Soluble in water• Final solvent easy to evaporate or

compatible with HPLC

• Serum has many components competing for retention

• Serum will vary from person to person

• Characterize the Sample Matrix• Solvent solubility and stability• pH, ionic strength• Possible interferences—similar

functional groups, pKa, etc. • Qualitative and quantitative

variability

N

NH

O

O

O

R1

R2

R3

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Analysis of Barbiturates in Serum AccuBONDII SPE C18 Cartridge

Solid Phase Extraction Method

CARTRIDGE: AccuBONDII ODS (C18) 6 mL/500 mg (P/N 188-1356 30/box)

Sample Preparation:

� Add 1 mL 0.5M K2HPO4 to 4 mL of serum Precondition:� 5 mL Acetone� 5 mL Deionized Water

Load:� 5 mL Sample (see sample preparation

above)

Wash� 2.0 mL 95% water:5% acetonitrile

Elute:� 3.0 mL AcetoneEvaporate and Reconstitute:� Evaporate Acetone in a nitrogen stream at

<45°C� Add 200 µL of acetonitrile:water (20/80)

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Break Time

For Questions and AnswersPress *1 on Your Phone toAsk a Question

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HPLC Analysis of Barbiturates Extracted from Human Serum

Column: C18, 4.6 x 150 mm, 5 mmMobile Phase: Time ACN Water

0 20 805 20 80

15 40 6020 40 60

Flow Rate: 1 mL/minDetection: UV 235 nmSample: Barbiturates

1. Barbital2. Allobarbital3. Aprobarbital4. Phenobarbital5. Butabarbital6. Alphenal7. Hexobarbital8. Amobarbital9. Mephobarbital10. Secobarbital

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Recovery Data for Barbiturates in Serum

8954.59Secobarbital5934.36Mephobarbital3934.20Amobarbital51024.12Pentobarbital6924.09Hexobarbital8873.46Alphenal14883.45Butalbital6883.29Butethal7893.06Butabarbital8782.87Phenobarbital9922.47Aprobarbital11821.99Allobarbital11701.00Barbital

Std. Dev.MeanRRTCompound

Serum spiked at 1mg/mLn= 4

RRT = retention time relative to barbital

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New AccuBONDII ENV for Environmental Analytes

• New PS-DVB SPE material for environmental applications—High surface area – 600 m2/g – provides good retention—Large particle size for high flow – 75 – 150 mm—High capacity for good retention – Capacity of caffeine 320 mg/g

• Provides high recovery of phenols in water – wide range of polarities and solubilities

—Higher recovery than with silica (phenol> 70%, other phenols>90%)

—Optimized for the analysis of phenols—High sample delivery rate for short extraction time

• Follow with GC or GC/MS analysis

• Being evaluated for other environmental analytes - pesticides and PAHs

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SPE Procedure for Extraction of Phenols

Solid Phase Extraction Method

CARTRIDGE:AccuBONDII ENV PS-DVB, 1000 mg*, 6

mL (P/N 188-3060, 30/box)

*1000 mg is required for optimal recovery

Sample and Recovery Standard Preparation:

� 1L water � Deuterated phenol, 2,4-

dibromophenol, and 2,4,6-tribromophenol recovery standards added at 10 ppb level

� Sample and standards mixed and pH lowered to <2 with 5N HCl

Precondition:� 9 – 12 mL Dichloromethane� 9 – 12 mL Methanol� 9 – 12 mL 0 05N HCl

Load:� 1.0 L water sample at 20 – 25 mL/min (see

sample preparation above)

Elute:� 9.0 mL Dichloromethane (dried with anhydrous

sodium sulfate)Evaporate and Reconstitute:� Evaporate Dichloromethane in nitrogen stream

at room temperature and transferred to silanized amber vial

� Bring to 900 µL of Dichloromethane + 100 µL of a solution containing 2,5-dibromotolune and 2,2’,5,5’-tetrabromobiphenyl at 0.05 mg/mL in Dichloromethane as internal standards

Publication Number of phenol application: 5988–5255ENAvailable on the Agilent web site: www.agilent.com/chem

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GC Analysis of Extractable Phenols

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AccuBONDII SPE Product FeaturesCOA, Packaging and Labeling

• Certificate of analysis with each box —Mass consistency—Flow rate consistency—Trace metal analysis—Extract cleanliness—QC chromatogram

• Packaged in trilayer laminated bag—Excludes air and water—Ensures clean product

• Individually printed tubes and plates—Easy identification—Versatility for method development

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Certificate of Performance for AccuBONDII – page 1

Description: AccuBONDII C8 100 mg 1 mL cartridgeCatalog No: 188-0310Lot No.: AMC18-0XRun No.: XXXXXXXThis AccuBOND product and sorbent have been manufactured, tested and assembled under the control of an ISO 9001 registered quality system.This AccuBOND SPE product has been subjected to the following QC tests:

Base Silica Characteristics Bonded Silica TestsSurface Area: 546 m2/g Carbon Loading: 13.0%Average Pore Size: 60Å Surface Coverage: 2.5 µmoles/m2Surface pH: 7 Extraction Residue: PassMetal Analysis: Pass Exchange Capacity: N/A

Particle Size Data Packed Cartridge TestParticle Shape: Irregular Cartridge Flow Resistance: PassAverage Particle Size (µm): 56 Frit Purity Test (GC): PassPercentage of Particles <10 µm: 0.00 Material Weight Check: PassPercentage of Particles <25 µm: 1.36Percentage of Particles <90 µm: 4.95

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Certificate of Performance for AccuBONDII — page 2

T e s t C o n d i t io n a n d R e s u lt s

C o n d it io n s :C o lu m n : 4 .6 x 2 5 0 m mM o b ile P h a s e : 7 0 /3 0 M e O H /H 2 OF lo w R a te : 2 .0 m l /m in

S p e c if i c a t io n s :K ’ b ip h e n y l = 3 .5 to 6 .5K ’ to lu e n e = 1 .5 to 3 .5K ’ a c e to p h e n o n e = 0 .6 to 1 .4T f u r a c i l = 0 .3 to 1 .5

R e s u l t s f o r L o t A M C 8 X -X XK ’ to lu e n e = 4 .5 P e a k 1 = U r a c i lK ’ d i m e th y la n i l in e = 2 .1 P e a k 2 = A c e to p h e n o n eK ’ n it r o b e n z e n e = 1 .0 P e a k 3 = T o lu e n eT f d im e th y la n i l in e = 1 .4 P e a k 4 = B ip h e n y l

N o t ic e :

M a te r ia l S a fe t y D a ta S h e e ts (M S D S ) a r e a v a i la b le a t :

h t tp : / /w w w .c h e m .a g i le n t .c o m /s c r ip ts /c a g _ m s d s s e a r c h .a s p

T h is p ro d u c t h a s p a s s e d a l l A g i le n t T e c h n o lo g ie s , in c . q u a l i t y c o n tro l s p e c i f ic a t io n s .

QA

QC

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EVIDEXII SPE Cartridges for Drugs of Abuse in Urine

• Proprietary bonding chemistry— Mixed RP and cation-exchange

bonded phase• Designed for NIDA-5 Drug Classes

— Amphetamine/Methamphetamine— PCP (“Angeldust”)— Benzoylecgonine (Cocaine

metabolite)— Codeine and Morphine— THC-COOH (Marijuana metabolite)

• Tested with actual drugs of abuse— Ensures lot-to-lot reproducibility— Ensures high recoveries— Produces clean extracts with

excellent S/N ratios in GC-MS• Good general purpose pharmaceutical

mixed mode phase

NH2

CH3

NHCH3

CH3

N

N

O

O

O

OH

CH3

O

N

OH O

CH3

CH3

H

O

N

OHOH

CH3

H

THC-COOH

Amphetamine Methamphetamine PCP

Benzoylecgonine Codeine Morphine

O

OH

CH3

CH3CH3

H

H

OHO

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EVIDEXII Drugs of Abuse (DOA) Methods

• EVIDEXII Methods for NIDA drug classes

—One SPE cartridge type for all analytes and methods

—Two cartridge configurations 200 mg/3mL and 400 mg/6 mL for different sample sizes

—Step-by-step instructions—Robust procedures

• minor changes in reagent volumes and concentration do not affect results

—GC-MS analysis using column specific for DOA (DB-5 MS EVDX GC)

—Accurate, reproducible results (<5% RSD)

• Bottom Line: Results are defensible in court

Effect of Varying Method Conditions on Benzoylecgonine SPE Recovery

EVIDEXII methods are robust!

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EVIDEXII SPE Cartridges for Drugs of AbuseOpiates in Urine

Solid Phase Extraction Method

CARTRIDGE: EVIDEXII 400 mg, 6 mL(P/N 188-2946, Box of 30)

Precondition:� 6 mL methanol� 6 mL 0.1 M potassium phosphate (pH 6.0)� Do not let the phase go dryLoad:� Add 3 mL 0.1 M potassium phosphate (pH

6.0) to the cartridge� Attach an 8 mL reservoir� Add the urine sample

Rinse:� Remove reservoir� 3 mL water� 3 mL 0.1 M sodium acetate (pH 4.5)� 3 mL methanol

Elute:� Place a collection tube beneath cartridge� 3 mL methylene chloride/isopropyl alcohol/

NH4OH (78/20/2)� Collect the eluate

Codeine30 ng

Morphine30 ng

Codeine

Column: DB-5MS EVDXCarrier: Helium at 40 cm/secOven: 65 degrees for 1 min, 65-325 degrees at 20 degrees/minInjector: Splitless, 250°CDetector: MSD, 300°C transfer line

Clean extracts froma very dirty matrix

Morphine

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Recovery Levels (x±%, n = 8)

Level in ng/mL 1000 1750 2500Amphetamine 76 ±4 74 ±2 96 ±3Methamphetamine 85 ±1 71 ±3 98 ±3

Level in ng/mL 50 87.5 125PCP 90 ±3 91 ±2 88 ±3

Level in ng/mL 300 525 750 Benzoylecgonine 98 ±8 93 ±4 97 ±3

Level in ng/mL 600 1050 1500Codeine 99 ±1 97 ±3 98 ±1Morphine 96 ±2 96 ±3 93 ±4

Level in ng/mL 30 52.5 75THC-COOH 92 ±4 96 ±8 94 ±3

DOA Recoveries are Reproducible Using EVIDEXII SPE Cartridges

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Summary

• Solid Phase Extraction—Probably the most important technique for sample cleanup

and concentration today—Increases productivity, column lifetime and instrument uptime

• AccuBONDII

—Available with both silica and PS-DVB base materials—Many bonded phases for every sample type —Low extractable levels—prewashed tubes, frits, and packing—High quality SPE products for any sample type—Low extractables from packing, tubes and frits are

compatible with sensitive detectors like MS• EVIDEXII

—Accurate, reproducible, robust methods for drugs of abuse and other pharmaceutical applications

Appendix

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• General—Keep cartridges in sealed bags until use.—Store in zipper-locked bags or in desiccator once opened.—When using empty reservoirs attached to cartridges, use long

disposable pipets to ensure proper flow from reservoir to cartridge.—Use stopcocks to adjust/control flow through individual cartridges.

• Use mass balance for all fractions to determine fate of analyte (adsorption to surfaces, loading effluent, washes, eluate, etc.) during method development.

—Residual water can be removed effectively by centrifugation (5000 rpm, 5 min.) compared to drying with vacuum or nitrogen.

—Cartridge capacity for analytes and matrix is typically about 1-3% of cartridge bed weight (ion-exchange not included).

—NEVER allow the cartridge to dry out until the elution step.

SPE Tips For Improving Recovery and Precision

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SPE Tips For Improving Recovery and Precision

• Prewash—Remove all strong prewash solvent for GC (e.g., dichloromethane,

hexane, ethyl acetate) before preconditioning and loading.• Precondition

—Make sure pH is correct for ion-suppression (acids) or minimal silanol interactions (bases).

—Leave ~1-2 mm of preconditioning solvent above sorbent bed to prevent bed from drying.

—Leave ~1/4 to 1/2 of tube volume above sorbent bed when using empty reservoir above cartridge.

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SPE Tips For Improving Recovery and Precision

• Load—Leave ~1/4 to 1/2 tube volume above sorbent bed when using tube

reservoir above cartridge.—Use drop wise solvent flow when time/throughput is not a major

concern.• Wash

—Wipe needles of manifold before elute step to minimize contamination of eluate.

• Elute—When choosing eluent, consider ease of evaporation if reconstitution is

needed.—Allow cartridge/plate to soak with eluent for 0.5 - 1 min. (➚ recovery).—Sometimes several smaller eluent aliquots can improve recovery.

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SPE Extraction and Analysis of Triazines in Several Matrices

• Triazine pesticides may need to be extracted from many different sample matrices

• One SPE procedure will not be right for all sample types

• A variety of SPE bonded phases and mechanisms will be needed for different matrices

—Reversed-phase—Normal phase—Ion-exchange

• AccuBONDII family provides the necessary variety of SPE cartridges for anymatrix

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Extraction and Separation of PAHs from Water AccuBONDII ODS

Solid Phase Extraction Method

CARTRIDGE: AccuBONDII , 500 mg, 6 mL (P/N 188-1356, 30/box)

Sample Preparation:� Add 2 mL Isopropanol to 20 mL Water Sample� Mix thoroughly

Precondition: � 5 mL Methylene Chloride � 5 mL Methanol� 5 mL Deionized Water � Add ~1 mL Deionized Water to top of bed

Load:� Attach 24-mL sample reservoir to cartridge� Add sample to reservoir. Ensure flow into cartridge with glass pipet.� Apply vacuum and keep flow rate <10 mL/min. Slower flow gives

better results.

Wash:� 3 mL 50:50 Acetonitrile/Deionized Water� Apply vacuum until 30 sec. after wash has passed through cartridge� Centrifuge cartridge at 1000-1500 rpm for 5 min. (removes excess

water)

Elute:� 3 mL Methylene Chloride and collect eluent.� Evaporate to 50-200 µL. Do not apply heat or smaller PAHs will be

volatilized.� Add Methylene Chloride to bring sample to final volume of 200 µL.

Inject 2 µL.

CompoundAvg.

Recovery (%)

Std. Dev. (%)

Naphthalene 80 122-Chloronaphthalene 78 8

Acenaphthylene 81 9Fluorene 99 9

Phenanthrene 98 9Fluoranthene 91 8

Chrysene 97 12Benzo(b)-fluoranthene 60 8

Benzo(a)pyrene 55 8Indene(1,2,3-c,d)pyrene 60 10

Benzo(g,h,i)perylene 59 10

Water Spiked at 50 ppb

Cl

CI0126C 45

Sample Preparation:• Add 0.5 mL hydrochloric acid to 5 mL urine• Cap• Heat at 120°C for 20 min• Cool to room temperature• Add 0.75 mL 10 N NaOH• Adjust to pH 6.5-7.5 with ~2.5 mL 0.5 M phosphoric acid

Condition:• 6 mL methanol• 6 mL 0.1 M K2HPO4 (pH 6.0)• Do not let the phase go dry

Load:• Add 3 mL 0.1 M K2HPO4 to the cartridge• Attach an 8 mL reservoir• Add the urine sample

Rinse:• Remove reservoir• 3 mL water• 3 mL 0.1 M sodium acetate (pH 4.5)• 3 mL methanol

Elute:• Place a collection tube beneath cartridge• 3 mL methylene chloride/isopropyl alcohol/ NH4OH (78/20/2)• Collect the eluant

Analysis Preparation:• Concentrate the eluant to dryness• Do not over dry• 75 µL ethyl acetate• 25 µL BSTFA/1% TMCS• Cap• Heat at 60°C for 10 min• Inject 1-2 µL

• All flow rates should not exceed 5 mL/min

Reagents:• 10 N NaOH

40 g sodium hydroxide in 100 mL DI water.• 0.1 M K2HPO4 (pH 6.0)

1.74 g potassium phosphate, dibasic (anhydrous) in 100 mL DI water. Adjust to pH 6.0 ± 0.1 with phosphoric acid.

• 0.1 M sodium acetate (pH 4.5)0.82 g in 100 mL DI water. Adjust to pH 4.5 ± 0.1 with glacial acetic acid.

• Methylene chloride/isopropyl alcohol/NH4OH (78/20/2)78 mL methylene chloride, 20 mL isopropyl alcohol, 2 mL ammonium hydroxide. Make fresh daily.

• BSTFA/1% TMCS9 mL N,O-bis(trimethylsilyl)trifluoroacetamide, 0.1 mL trimethylchlorosilane, or can be purchased premixed.

Other reagentsMethanol

DI waterEthyl acetateHydrochloric acidPhosphoric acid

Typical SPE Extraction Protocol for Opiates

CI0126C 46

Certificate of Performance for AccuBONDII

Description: AccuBOND C8 100mg 1mL cartridgeCatalog No: 188-0310Lot No.: AMC18-0XRun No.: XXXXXXX

This AccuBOND product and sorbent have been manufactured, testedAnd assembled under the control of an ISO 9001 registered quality system.

This AccuBOND SPE product has been subjected to the following Q.C. tests:

Base Silica Characteristics Bonded Silica Tests

Surface Area: 546 m2/g Carbon Loading: 13.0 %Average Pore Size: 60 Å Surface Coverage: 2.5 µmoles/mSurface pH: 7 Extraction Residue: PassMetal Analysis: Pass Exchange Capacity: N/A

Particle Size Data

Particle Shape: IrregularAverage Particle Size (µm): 56Percentage of Particles <10µm: 0.00Percentage of Particles <25µm: 1.36

Packed Cartridge Test

Cartridge Flow Resistance: PassCartridge Purity Test (GC): PassFrit Purity Test (GC): PassMaterial Weight Check: Pass

Percentage of Particles >90µm: 4.95

Test Condition and Results

Conditions:Column: 4.6 x 250 mmMobile Phase: 70/30 MeOH/H2OFlow Rate: 2.0 ml/min

Specifications:K’ biphenyl = 3.5 to 6.5K’ toluene = 1.5 to 3.5K’ acetophenone = 0.6 to 1.4Tf uracil = 0.3 to 1.5

Results for Lot AMC8X-XXK’ toluene = 4.5 Peak 1 = UracilK’ dimethylaniline = 2.1 Peak 2 = AcetophenoneK’ nitrobenzene = 1.0 Peak 3 = TolueneTf dimethylaniline = 1.4 Peak 4 = Biphenyl

Notice:

Material Safety Data Sheets (MSDS) are available at:

http://www.chem.agilent.com/scripts/cag_msdssearch.asp

This product has passed all Agilent Technologies, inc. quality control specifications.

2

QA

QC

CI0126C 47

HPLC Column Technical Support

800-227-9770 (phone: US & Canada)*

302-993-5304 (phone)*

* Select option 4, then option 2.916-608-1964 (fax)www.agilent.com/chem