1 SampliQ SPE and QuEChERS Advancements Agilent Technologies Month ##, 200X Page 1 Agenda • Agilent SPE Polymeric Phases o Addition of 60 μm resin size o Stron Ion Exchange Polymeric Resins (SCX,SAX) o New Weak Ion Exchange Polymeric Resin (WCX,WAX) • QuEChERS o Extensions of portfolio o Review of New Advancements in QuEChERS Applications Month ##, 200X Page 2
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Topic 1 Advances in Sample Prep ppt - Agilent Condition 1 mL Methanol Equilibrate 1 mL Water Wash 1 mL 25 mM Sodium Acetate, pH 4.5-5.0 Wash and Interference removal 1 mL Methanol
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SampliQ SPE and QuEChERS Advancements
Agilent Technologies
Month ##, 200XPage 1
Agenda
• Agilent SPE Polymeric Phases
o Addition of 60 µm resin sizeo Stron Ion Exchange Polymeric Resins (SCX,SAX)o New Weak Ion Exchange Polymeric Resin (WCX,WAX)
• QuEChERSo Extensions of portfolioo Review of New Advancements in QuEChERS Applications
Month ##, 200XPage 2
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What is the SampliQ Polymer?Features:• High retention, outstanding recovery, and
excellent reproducibility• High sorbent robustness: if cartridges
accidentally go dry during the SPE process, you will not risk losing analytes and/or reproducibility
• No leaking bonded phases or other leachables that can contaminate valuable extracts
• Compatibility with most organic solvents and aqueous solutions over a pH range of 0 to 14
• Spherical particles and narrow size distribution, which ensure reproducible flow characteristics
Month ##, 200XPage 3
Specifications:• spherical particles 25-35 µm• pore size 74-80Å using BET measure• pore size 1000Å using inverse size exclusion• surface area 475-505 m 2/g• particle quality controls
OPT • a novel polyamide polymer• has affinity for both hydrophobic and hydrophillic compounds
OPT
SCX• a sulfonic acid modified divinylbenzene polymer• has mixed-mode affinity for both basic and neutral compounds
SAX• a tertiary amine modified divinylbenzene polymer• has mixed-mode affinity for both acidic and neutral compounds
Polymer Performance is Robust
• Highly reproducible recoveries wet or dry
– Cartridges dried under vacuum for 10 minutes before the equilibration step • RSD’s of the recoveries for each of the compounds (n=5) very low • Compounds range from very polar, basic compounds to hydrophobic, neutral
*Method Development 96 Well Plate = 3 rows each of SAX, SCX, WAX, WCX; Method Development Cartridge Kit = 10 ea. of SAX, WAX, and OPT (Acids); and SAX, WCX, and OPT (Bases)
5982-3796
Polymer Method Development Products*
Polymer
60 um 30um
5
SampliQ WAX
•WAX (weak anion exchanger) resin is a neutral amine-modified divinylbenzene (DVB) polymer
•The polymeric resin exhibits retention for acidic/anionic, neutral and, more importantly, strong acids (e.g. sulfonates) that are irreversibly retained on strong anion exchange resins
•The amine functionality (pKa ~ 6) for the SampliQ WAX can be ionized or neutral (dependent on the pH of the buffer solution); unlike the strong anion exchange (SAX) resin (always ionized, pKa ~18) WAX offers a wider pH range
• The weak anion exchange resin exhibits both anion exchange and reversed phase behavior (mixed mechanism)
Month ##, 200XPage 9
SampliQ WAX
•The weak anion exchange resin is inert to a wide variety of solvents, stable in the pH range 0 to 14, and is water-wettable
•The weak anion exchange resin’s pKa ~6
Month ##, 200XPage 10
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Condition 1 mL Methanol
Equilibrate 1 mL Water
Wash 1 mL 25 mM Sodium Acetate, pH 4.5-5.0
Wash and Interference removal 1 mL Methanol
Dry 3 minutes
Dry 1 minute
Elute 2 x 1 mL 5% Ammonium Hydroxide/Methanol:ACN ( 20/80)
Load 1 mL sample (dilute 1:1 2% phosphoric acid in water pH 1.5-2.0)
Methanol fraction may be collected if basic and neutral compound capture is desired
Dry and reconstitute in mobile phase
Dry 1 minutes
SampliQ WAX ® Generic Method Process for Acidic/Anionic and Neutral Compounds
Month ##, 200XPage 11
Performance of the Weak Anion Exchange Resin for the Extraction of Acidic/Anionic and Neutral Compounds from Human Plasma
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0102030405060708090
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Exceptional Compound Recovery from SampliQ WAX
Acidic Compound Recovery from SampliQ WAX
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SampliQ WCX
•WCX (weak cation exchanger) is a neutral carboxylic acid-modified divinylbenzene (DVB) polymer
•The resin exhibits retention for basic/cationic, neutral and more importantly strong bases (quaternary amines) that are irreversibly retained on strong cation exchange resins
•The carboxyl functionality (pKa ~5) of the WCX resin can be ionized or neutral dependent on the pH of the buffer solution; unlike the strong cation exchange resin that is always ionized at all pH values, the WCX has a wider pH range.
•The WCX exhibits both cation exchange and reversed phase behavior (mixed mechanism)
Month ##, 200XPage 13
SampliQ WCX
•The weak cation exchange resin is inert to a wide range of solvents, stable in the pH range 0 to 14, and is water-wettable
Month ##, 200XPage 14
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SampliQ-WCX ® Generic Method Process for Strongly Basic/Cationic and Neutral Compounds
Month ##, 200XPage 15
Condition 3 mL methanol
Equilibrate 3 mL water
Wash 1 mL 25 mM phosphate buffer (pH 7)
Interference removal: 2 mL methanol
Dry 1 minutes
Dry <2 minute
Elute with 5% formic acid in methanol:ACN (60/40)
Recommended flow through cartridge: not faster than 1mL per minute
Load 1 mL prepared sample in 2% phosphoric acidspiked with internal standard
Methanol fraction may be collected if neutral compound capture is desired
Dry and reconstitute in mobile phase
Dry 3 minutes
Performance of the Weak Cation Exchange Resin for the Extraction of Strongly Basic/Cationic and Neutral Compounds
Month ##, 200XPage 16
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Atenolol Protryptyline Chlorpromazine
Basic Compounds Eluted by WCX
Basic Compounds
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Load:
Wash:
Elute 1:
Elute 2:
Equilibrate:
Condition:
Strong AcidspKa <1
Weak BasespKa 2-10
Weak AcidspKa 2-8
Strong BasespKa >10
Agilent SampliQ Polymeric A/N/B MethodNeutral
CompoundspKa 3-10
Weak Bases
Strong Acids
Strong Bases
Weak Acids
0.1% Formic Acid in Methanol
2% Formic Acid in Water
Prepared sample
2% Formic Acid
Methanol
Water
Prepared sample
5% NH4OH
2% Formic Acid in Methanol
Analyte:
Methanol
Water
Prepared sample
5% - 10% Methanol in Water
Methanol or 0.1% Formic Acid in
Methanol
Use: SampliQ OPT
Use: SampliQ SCX
Use: SampliQ WAX
Use: SampliQ SAX
Use: SampliQ WCX
Prepare Sample for SPE
Log P < 1.5 Log P < 1.5Log P > 1.3
Neutrals
5% NH4OH in Methanol
Weak Acids
100% Methanol
Weak Bases
100% Methanol
Month ##, 200XPage 17
Method Development: Tips and Tricks
•Identify the compounds you need to extract
• pKa, Log P and matrix: biological, food, environmental
• Analysis by HPLC will offer insight into possible SPE modes
• The more details available will facilitate quick method development
QuEChERS Second Step – Dispersive SPEin less than 20 min
Month ##, 200XPage 28
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Four Variations of the QuEChERS Method
�The original QuEChERS method was introduced in 2003, uses sodium chloride to enhance extraction
�AOAC 2007.01 uses sodium acetate as a buffer replacing sodium chloride and 1% acetic acid in acetonitrile
�The dual phase variation introduces the use of PSA and GCB cartridge to remove high levels of chlorophyll and plant sterols in the final extract without the loss of planar pesticides (polar aromatics) using an acetone:toluene solvent blend (3:1)
�European version is similar to the AOAC method, except the extraction uses sodium chloride, sodium citrate dihydrate and disodium citrate sesquihydrate instead of sodium acetate, and acid is added at the very end.
Month ##, 200XPage 29
Modifications to the Original Schenck Method- Buffered Extraction
�Ensure efficient extraction of pH dependent compounds and minimize degradation of base and acid labile pesticides
�Citrate Buffering for the µ-extraction at a compromise pH of 5-5.5, most acid / base compounds in the sample extracts, small amount of formic acid is added to the final extract after cleanup
�Acidic pesticides are directly analyzed from raw extract before PSA cleanup since they would be absorbed and not released by the sorbent
�Graphitized carbon black (GCB) PSA cartridge is used to remove plant pigments without the loss of planar compounds
Month ##, 200XPage 30
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Agenda
Traditional Sample Preparation Methods
What is QuEChERS?
How to choose the Right Kits?
Advantages of QuEChERS over Standard SPE
Applications
Month ##, 200XPage 31
SampliQ QuEChERS Kits Making it Easy to Select the Right Kit
Choose your Extraction Kit based on the QuEChERS method you’re using, and the pesticides you’re screening for
– All salts and sorbents are pre-measured according to the specific method you are using.
Choose the Dispersive SPE Kit based on the type of food you’re analyzing and the method you are following
– A look-up reference of foods and kits can be found at www.agilent.com/chem/quechers
Month ##, 200XPage 32
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SampliQ QuEChERS Selection Guide
Month ##, 200XPage 33
Extraction SPE kits
Month ##, 200XPage 34
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Extraction SPE kits
Month ##, 200XPage 35
Month ##, 200XPage 36
Dispersive SPE kits
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Month ##, 200XPage 37
Dispersive SPE kits( contd)
New !!
SampliQ QuEChERS Kits Making it Easy to Select the Right Kit Provides food testing labs with a simplified approa ch to preparing samples for pesticide analysis
•No guesswork, no measuring: Pre-packed Extraction Kits and Dispersive SPE Kits are assembled to suit specific food types and screening protocols
•Extraction salts are pre-weighed and provided in anhydrous packets, so you can conveniently add them at the correct time and improve results by reducing analyte degradation
•Sorbents and salts for the dispersive SPE step are supplied in 2 mL or 15 mL centrifuge tubes, for 1 mL, 6 mL or 8 mL aliquot volumes, as specified by the various QuEChERS methodologies in use today
•Agilent’s tight quality control processes ensure that all QuEChERS salts, sorbents and supplies are high quality, and free of contaminants, so results are accurate and reproducible
Month ##, 200XPage 38
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What Makes SampliQ QuEChERS Kits Unique?
• SampliQ QuEChERS kits have strong product advantages:
– Ease of selection: outline how to choose products based on analysis, regulatory method or sample size
– Pre-measured packets: provided in anhydrous packs ensure procedure is correctly followed, resulting in more accurate data
– Agilent provide ceramic homogeneizers to improve the recoveries
– Agilent provide standards
– Agilent focus on education: providing video
– Recognized brand, quality and WW technical support
Month ##, 200XPage 39
The value of the kits•Individually packaged extraction salts improves acc uracy of results
– Vacuum-sealed anhydrous foil packs ensure salt integrity
– Allows the user to add the extraction salts as specified in the approved methods to avoid exothermic reactions
– Exothermic reactions occur in competitor’s kits when the sample is added directly to the salts already in tubes degrading analytes and decreasing recoveries
•No need for additional centrifuge tubes
– Less total cost
– Less waste (green solution)
•Agilent design innovations
– Convenient finger-tip access to all tubes
– Minimizes handling of tubes and promotes GLP
– Effortlessly determine remaining quantity at a glance
– Stores easily on shelf or bench
• No amorphous, plastic bags to shove in a drawer
Month ##, 200XPage 40
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Cost Savings with Pre-made QuEChERS Kits
Month ##, 200XPage 41
Cost / 100 samples
weigh in the SPE material needed incl. salt etc. for 100 samples 30 min x 60 €/h
• Extraction step requires the shaking of extraction salts with comminuted fruit/vegetable
• Consistency in shaking can be an issue, effecting recoveries and pesticide analysis• Limited Consistency in QuEChERS applications
Agilent’s Response: SampliQ’s CH: Ceramic Homogenizers• Offers more consistent extraction of the sample with the salts• Breaks up salt agglomerates • Facilitates homogenation by angle cut• Increase recovery of pesticides from sample• Also available in dispersive sizes too, 2 mL and 15 mL
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Ceramic Homogenizers improve recoveries
Month ##, 200XPage 43
• Are made from a mixture of ceramic and alumina oxide• Are inert, showing no loss in pesticide recovery
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Reco
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Comparison of QuEChERS Method AOAC and EN With and Without Ceramic Homogenizers
AOAC Method with Ceramic HomogenizersEN Method with Ceramic HomogenizersAOAC Method without Ceramic HomogenizersEN Method without ceramic Homogenizers
Ceramic Homogenizers decrease shaking time
Month ##, 200XPage 44
Average recoveries of several pesticide types with Ceramic Homogenizers
20 seconds of shaking with the ceramic homogenizers give equivalent or better recoveries than 1 minute shaking without them.
LC 6 Pt. Calib. Curve Test Kit EN Method 5190-0495
GC 6 Pt. Calib. Curve Test Kit EN Method 5190-0496
Why QuEChERS Standards:
Month ##, 200XPage 46
• Ready to use for QuEChERS extractions-no dilution necessary
• Support GC and HPLC with MS, MS/MS, and selective detectors
• Takes all variability out of making standards and calibration curves, CRM (certified reference material), manufactured in accordance with ISO 9001, concentrations verified by ISO 17025 accredited laboratory, calibrated balances and glassware, weights traceable to NIST in compliance with ANSI/NCSL Z-540-1
• Convenient storage and usage in sealed amber ampoules, lot number and expiration date
24
Agenda
Traditional Sample Preparation Methods
What is QuEChERS?
How to choose the Right Kits?
Advantages of QuEChERS over Standard SPE
Applications
Month ##, 200XPage 47
Traditional Methods
Blending
Filtration
Large solvent volumes
Multiple partitioning steps
Transfers of entire extract
Lots of glassware
Evaporation Concentration
Classical SPE
Shaking
Centrifugation
Small solvent volumes
Single partitioning step
Take aliquots (use I.S.)
Single vessel
Large volume injection
Dispersive SPE
Advantages: QuEChERS vs. Standard Sample Prep
Month ##, 200XPage 48
QuEChERS
25
Advantages of Dispersive SPE Over Standard SPE
Month ##, 200XPage 49
Dispersive SPE =
No SPE Apparatus No Flow Control needed
No SPE Cartridges No Elution Solvent
No Vacuum No Dilution of Extract
No pretreatment No Solvent Evaporation
No Channeling Less Sorbent
No Drying Out Less Time
No Collection Less Cost
Advantages of Dispersive SPE Over Standard SPE
Month ##, 200XPage 50
Standard SPE method QuEChERS Method
Sample Processing: 120 min. 30 min. 25% of the time
Solvent usage: 60-90 ml Solvent usage: 10-15 ml
Chlorinated Solvents: 20-30 ml Chlorinated Solvents: None
Save 65%-92% of solvent Reduce Hazardous Waste by 56-87%Recoveries typically > 90% *
* A Comparison of Solid Phase A Comparison of Solid Phase Extraction Cleanups for use with the Extraction Cleanups for use with the QuEChERS Method QuEChERS MethodFRANK J. SCHENCK Southeast Regional Laboratory, FDA, Atlanta, GA Southeast Regional Laboratory, FDA, Atlanta, GAJon Wong & Michael Hennessy Jon Wong & Michael HennessyCFSAN, FDA, College Park, MD CFSAN, FDA, College Park, MD
26
QuEChERS Advantages
�� A batch of 6-12 extracts can be prepared in 30-40 m in by a single analyst with ≈$1-3 of disposable materials per sample and generate <12 mL solvent waste and only a single re-usable item (FEP tube) for cleaning.
� Consistently high recoveries (mostly 90-110% with R SDs < 5%) of a wide range of GC- and LC-amenable pesticide s are achieved from many matrices.
� A low cost, highly effective sample preparation tec hnique which can be used on most food matricies for a wide range of pesticide classes
Month ##, 200XPage 51
Agenda
Traditional Sample Preparation Methods
What is QuEChERS?
How to choose the Right Kits?
Advantages of QuEChERS over Standard SPE
Applications
Month ##, 200XPage 52
27
Month ##, 200XPage 53
Applications
Analysis of Pesticide Residues in Spinach Using Agi lentSampliQ QuEChERS AOAC Kits by GC/MS (Publication 5990-4305EN)
This application note describes the use of kits QuEChERS AOAC sample preparation approach for extraction and cleanup of 18 GC-amenable multiple pesticide class residues in spinach
In order to address the significant loss of planar pesticides caused by graphitized carbon black (GCB) in dispersive SPE, a modified method with addition of toluene was employed for the planar pesticides.
The target pesticides in the spinach extracts were then analyzed by gas chromatography/mass spectrometry (GC/MS) operating in selective ion monitoring (SIM) mode
Month ##, 200XPage 54
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GC/MS of a Spinach Extraction by QuEChERS
Month ##, 200XPage 55
GC/MS of a Spinach Extraction by QuEChERS
Month ##, 200XPage 56
50 ng/g Fortified Spinach
Blank
29
Results original method without toluene
Month ##, 200XPage 57
Results original method with toluene
Month ##, 200XPage 58
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Current Trends in QuEChERS Research
•In general the QuEChERS methodology is expanding out into others areas
• Drug Residue Analysis: Pharmaceuticals in Whole Blood
• Anthelmintics in Animal Tissue
• Veterinary Drugs in Animal Tissue*
Month ##, 200XPage 59
Expanding the QuEChERS ConceptVeterinary Drugs in Animal Tissue
A method for the determination of 11 Quinolone anti biotics in bovine liver has been established:
• Analytes were extracted and cleaned up from bovine liver with Agilent SampliQ QuEChERS kits.
• Extraction was performed using SampliQ EN extraction kits and 5% FA in Acetonitrile.
• Clean up was performed using SampliQ dispersive SPE kits (25 mg C18 and 150 mg MgSO4), for drug residue in animal tissue (NEW)
• Extracted samples were then analyzed by LC/MS/MS
• Limits of Quantitation (LOQ) were 5.0 ng/g
• Calibration curves were linear over the range of 5.0 to 400 ng/g
• The sample pre-fortified recoveries were between 62.0% and 113.1% with RSD (n=6) values between 2.2% and 13.4%
Month ##, 200XPage 60
31
Weigh 2 g homogenized liver sample ( ±0.05g) in 50 mL centrifuge tube
Spike 100 µL of IS spike solution, 50 µL of QC spik e solution if necessary vortex 30 sec
Centrifuge @ 4000rpm for 5min
Transfer 1 mL of upper ACN layer to SampliQ QuEChER S dispersive-SPE 2 mL tube
Vortex 1min, centrifuge @ 13000rpm for 3 min with m icro-centrifuge
Add 10 mL of 5% FA in ACN, and shake vigorously for 30 sec
Add SampliQ EN QuEChERS extraction kit, and shake v igorously for 1 min
Transfer 800 µL extract to another tube, blow down @ 40°C with N 2
Sample are ready for LC/MS/MS analysis
QuEChERS Extraction Procedure for Bovine Liver
Month ##, 200XPage 61
Add 8 mL of 30 mM KH 2PO4, pH 7.0 buffer, vortex
Reconstitute into 800 µL 1:9 MeOH/H 2O w/ 0.1% FA, vortex and sonicate
Filter samples w/ 0.22 µm Cellulose Acetate Spin Fi lter
LC-MS/MS Beef Liver Blank and 200 ng/g Fortified L iver ExtractsA
B
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IS
3
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8 9
10 11
LC/MS/MS Chromatograms of A) liver blank extract, and B) 200ng/g fortified liver extract. Peaks identification: 1. Pipemidic acid,2. Ofloxacin, 3. Ciprofloxacin, 4. Danofloxacin, 5. Lomefloxacin, 6. Enrofloxacin, 7. Sarafloxacin, 8. Cinoxacin, 9., Oxolinic acid, 10. Nalidixic acid, 11. Flumequine, IS (internal standard). Norfloxacin.
Month ##, 200XPage 62
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LC-MS/MS Beef Liver
Month ##, 200XPage 63
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Pip
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Recovery and ReproducibilityR
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Instruments and Methods – LC/MS/MS
time (minutes) % B Flow Rate (mL/min)0 15 0.3
0.2 15 0.38.0 75 0.39.0 100 0.311.5 Stop
Month ##, 200XPage 64
Chromatography System: Agilent 1200 HPLC System
HPLC conditions:• Column: Agilent Eclipse Solvent Saver Plus Phenyl-hexyl Column, 150×3.0mm 3.5µm (PN: 959963-312). • Flow rate: 0.3ml/min Column Temperature: 30oC • Injection volume: 10µl• Mobile phase: A) 5mM Ammonium Acetate in H2O, pH3.0, B) 1:1 Methanol/Acetonitrile.
• Gradient: Post Time: 4min
Mass Spectrometry System: Agilent 6410 Triple Quad Mass Spectrometrywith electrospray ionization.
Conditions: Polarity: Positive Gas Temperature: 325ºC Gas Flow: 8L/minNebulizer: 50psi Capillary : 4000VSolvent cut: 5min
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QuEChERS Advancements
Month ##, 200XPage 65
� Data presented shows that the QuEChERS methodology can be used for other residues besides pesticides
� Very large scope of possibilities, with very simple sample preparation technique, “ just enough sample preparation ”
� Relatively green and very cost effective technique when compared to Liquid-Liquid Extraction and Solid Phase Extraction
QuEChERS – As a conclusion
Month ##, 200XPage 66
� A ready to use and fast solution for pesticides analysis in Fruits and Vegetables
� Very effective, improve cost and time savings : improve the laboratory productivity!
� SampliQ QuEChERS Kits easy to choose, extract up to 250 pesticides in a row
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Additional Resources - QuEChERS
Check www.agilent.com/chem/quechers
Description of Extraction and Dispersive Kits
Description of QuEChERS SOP
Suggested SampliQ QuEChERS Dispersive Kits by Food Type and Method