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TRANSCRIPT
©2011 Waters Corporation | COMPANY CONFIDENTIAL
Food Safety and Environmental
Analysis Solutions
Sample Preparation Strategies
Sevilla, 3 de Noviembre de 2011
©2011 Waters Corporation | COMPANY CONFIDENTIAL 2
Outline
Introduction — Challenges & Tools
Sample Preparation — Why Sample prep?
— Sample Prep techniques
— SPE: strategies and sorbents
Applications — Multiresidue methods
— Family-specific methods
©2011 Waters Corporation | COMPANY CONFIDENTIAL 3
Challenges Facing Food Safety and Environmental Laboratories
Staff Skills
Improve Productivity / Faster Analysis
Lower Cost per Analysis
Sample Preparation
Compliance Information Management
©2011 Waters Corporation | COMPANY CONFIDENTIAL 4
Waters Technology Focus
©2011 Waters Corporation | COMPANY CONFIDENTIAL 5
Chemistry Solutions for Sample prep
Sep-Pak® SPE Products - first silica-based SPE cartridge - great variety of sorbents: normal phase, reversed- phase and ion exchange - thousands of references in literature - Certified Sep-Pak
Oasis® SPE Products - polymeric sorbent (water-wettable, won’t dry out) - HLB, MCX, MAX, WAX, WCX - corticosteroids, beta-agonists, sulfonamides, pesticides, pharmaceuticals ...
Dispersive SPE: DisQuE - pre-packaged ―QuEChERS‖ sample preparation kit - designed for multi-pesticide residue analysis
©2011 Waters Corporation | COMPANY CONFIDENTIAL 6
Outline
Introduction — Challenges & Tools
Sample Preparation — Why Sample prep?
— Sample Prep techniques
— SPE: strategies and sorbents
Applications — Multiresidue methods — Family-specific methods
©2011 Waters Corporation | COMPANY CONFIDENTIAL 7
Why Sample Prep?
60% of the work activity and operating cost is spent on sample
preparation for introduction into the analytical system.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 8
Sample Preparation Non-Chromatographic Techniques
Techniques Advantages Disadvantages
Dilution
• Simple
• Cheap
• High throughput
• No cleanup
• No enrichment
Filtration
• Simple
• Fast
• No enrichment
• Potential analyte binding
Centrifugation
• Simple cleanup
• High throughput
• No enrichment
• Cumbersome
Liquid-Liquid
Extraction
• Best non-chromatographic
cleanup
• Enrichment
• Cumbersome
• Expensive
• Lots of solvent usage
©2011 Waters Corporation | COMPANY CONFIDENTIAL 9
Sample Preparation by SPE A Chromatographic Cleanup
Techniques Advantages Disadvantages
Solid Phase
Extraction
(SPE)
• Best cleanup
• Enrichment
• Fast
• Easy to automate
• Many sorbents available
for optimum cleanup
• May need multiple steps
• Not well understood
©2011 Waters Corporation | COMPANY CONFIDENTIAL 10
Why Use SPE in Food Safety and Environmental Analysis ?
Sample Enrichment
— To achieve sub ppb detection limits, usually the sample must
be concentrated 10-1000 times
o using liquid extraction (LE), up to 100 mL of solvent must
be evaporated after cumbersome manual extraction
o using SPE, only a few mL of solvent must be evaporated
after more user friendly procedure
Sample Cleanup
— Significant cleanup can often be accomplished using SPE
— Multiple SPE cartridges can often be used for optimum cleanup
For high sensitivity analyses, such as those employing LC/MS/MS, proper
sample preparation can be critical for minimizing matrix effects and
concentrating analytes of interest.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 11
SPE Strategies
1. Strategy #1
Dispersion Cleanup (DisQuE™ for QuEChERS)
acceptable cleanup for multiresidue screening
2. Strategy #2
Pass-through cleanup
more effective cleanup for multiresidue analysis
3. Strategy #3
Retention, cleanup, elution
most effective cleanup and enrichment for individual compound
class analysis
©2011 Waters Corporation | COMPANY CONFIDENTIAL 12
SPE Sorbents for Environmental and Food Safety Analysis
Normal-Phase Sorbents
— Silica, Alumina, Florisil®, Aminopropyl silica, PSA, Diol silica,
Reversed-Phase Sorbents
— Oasis® HLB
— C18, C8 etc (alkyl silica's)
— Graphitized carbon and activated carbon
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)
Dispersive SPE
— DisQuE
©2011 Waters Corporation | COMPANY CONFIDENTIAL 13
Outline
Introduction — Challenges & Tools
Sample Preparation — Why Sample prep?
— Sample Prep techniques
— SPE: strategies and sorbents
Applications — Multiresidue methods
— Family-specific methods
©2011 Waters Corporation | COMPANY CONFIDENTIAL 14
Applications
Multiresidue methods
— Pesticides in fruits and vegetables
— Veterinary residues in meat and milk
— Pesticides in water
— Pharmaceuticals in water
Family-specific methods
— PFC’s in water
— Nitrofurans in honey
— PAH’s in seafoods
— Quats in water
— Tetracyclines in liver
— Malachite green in fish
— others
©2011 Waters Corporation | COMPANY CONFIDENTIAL 15
Multi-residue Test Methods
Why are they attractive?
—More information per analytical run
—Streamlined (fewer) workflows
—Better asset utilisation
What are the technical challenges?
—―Universal‖ sample extraction (analytes and
matrices)
—Performance demands on separation
and detection
©2011 Waters Corporation | COMPANY CONFIDENTIAL 16
1) Pesticide multiresidue in fruits and vegetables
Dispersive Sample Preparation - “QuEChERS”
―Quick, Easy, Cheap, Effective, Rugged, Safe‖
Popular approach to performing sample cleanup
— Low material costs
— Method gaining world-wide acceptance
High throughput sample preparation and screening analysis
Combination of salting-out LLE and matrix dispersion
extraction
©2011 Waters Corporation | COMPANY CONFIDENTIAL 17
Typical Laboratory Workflow
Most ―QuEChERS‖ and dispersive SPE applications require
the user to source the individual components including vials,
sorbents and buffers.
— DisQuE Dispersive Sample Preparation Kits eliminate this lab
outsourcing
o Improves consistency in extraction product
o Improves lab efficiency and workflow
o Single Vendor source improves quality
DisQuE offers:
o Cleaner devices with lower extractables
o Method and product support to enable customer success
o Different kits, wide offer
o Flexibility
©2011 Waters Corporation | COMPANY CONFIDENTIAL 18
DisQuE Procedure CEN Method
PN 176001903: DisQuE CEN Dispersive Sample Preparation Kit
Description
— DisQuE Tube 1 (Extraction):
o 50 mL centrifuge tube
o 4 g magnesium sulfate anhydrous
o 1 g sodium chloride
o 1 g trisodium citrate dihydrate
o 0.5 g disodium hydrogencitrate
— DisQuE Tube 2 (Clean-up):
o 2 mL centrifuge tube
o 150 mg anhydrous magnesium sulphate
o 25 mg of PSA
o 25 mg C18
©2011 Waters Corporation | COMPANY CONFIDENTIAL 19
DisQuE Procedure – Tube 1 CEN Method
Prepare Sample
Homogenize 10 g sample*
10 mL ACN** + IS
Liquid Fractionation Shake for 1 minute
Centrifuge 5 min>3000 g
Collection Remove acetonitrile for clean-up
DisQuE Extraction
Tube 1
*Depending on the matrices
**If highly acidic compounds add NaOH
Analytes
remain in
supernatant
Tube 1
Tube 1
©2011 Waters Corporation | COMPANY CONFIDENTIAL 20
Transfer
1 mL Extract Tube 1 to Tube 2
Shake vigorously for 1 minute Centrifuge >1500 x g
Transfer Collect to autosampler vial
Dilute if necessary
DisQuE Clean-Up
Tube 2
Tube 1
Tube 2
Analytes
remain
in
Supernatant
Transfer
Supernatant
to
Tube 2
Tube 2
DisQuE Procedure - Tube 2 CEN Method
Prepare Sample
Homogenize 10 g sample*
10 mL ACN** + IS
Liquid Fractionation Shake for 1 minute
Centrifuge 5 min>3000 g
Collection Remove acetonitrile
for clean-up
DisQuE Extraction
Tube 1
*Depending on the matrices
**If highly acidic compounds add NaOH
©2011 Waters Corporation | COMPANY CONFIDENTIAL 21
Recommended Extraction Conditions
Water can be added
during comminution step7.5 g5 gDried fruits
10 g5 gCereals
10 g2 gSpices
10 g5 gHoney
X = 10 g – water amount
in 10 g sampleX g10 g
Fruits & vegetables 25-
80% water content
-10 gFruits & vegetables >
80% water content
NoteWater
added
Sample
weightSample type
Water can be added
during comminution step7.5 g5 gDried fruits
10 g5 gCereals
10 g2 gSpices
10 g5 gHoney
X = 10 g – water amount
in 10 g sampleX g10 g
Fruits & vegetables 25-
80% water content
-10 gFruits & vegetables >
80% water content
NoteWater
added
Sample
weightSample type
©2011 Waters Corporation | COMPANY CONFIDENTIAL 22
Cleanup Tube 2
Provides additional cleanup
Sorbent choices
— PSA
— Graphitized Carbon Black (GCB)
— C18
Acetonitrile Layer (ANALYTES ARE HERE)
Sorbent (INTERFERENCES)
©2011 Waters Corporation | COMPANY CONFIDENTIAL 23
DisQuE Primary-Secondary Amine (PSA)
Primary-Secondary Amine (PSA)
A weak anion-exchanger with pKa 10.1 and 10.9
Stronger anion-exchanger than aminopropyl
Strong affinity and high capacity for fatty acids, organic acids
For QuEChERS, PSA removes
— Acidic interferences (ion-exchange mechanism)
— Carbohydrates and sugars (HILIC mechanism)
O
O H
S i
O
O
H
N
N H 2
720002707en
©2011 Waters Corporation | COMPANY CONFIDENTIAL 24
Optional sorbents in Tube 2
C18:
— will remove co-extracted fat and waxes often present in these matrices. Their present affects the ruggedness of the further analysis.
Graphitized Carbon Black
— is used if the samples has a high content of carotinoides (e.g. red pepper, carrots) or chlorophyll (e.g. spinach, rucola salad, vine leaves
©2011 Waters Corporation | COMPANY CONFIDENTIAL 25
2) Pesticide Multiresidue in fruits and vegetables Pass-through SPE: Sep-Pak Carbon/Aminopropyl
Devices contain 500mg Graphitized Carbon Black
with 500mg Aminopropyl(NH2) or PSA Silica
Sep-Pak Vac 6cc Carbon/NH2
Graphitized Carbon 500mg
Aminopropyl silica 500mg
Frits
6cc Vac cartridge
Graphitized Carbon 500mg
PSA silica 500mg
Frits
6cc Vac cartridge
Graphitized Carbon 500mg
Aminopropyl silica 500mg
Frits
6cc Vac cartridge
Graphitized Carbon 500mg
PSA silica 500mg
Frits
6cc Vac cartridge
Sep-Pak Vac 6cc Carbon/PSA
©2011 Waters Corporation | COMPANY CONFIDENTIAL 26
Multi-Residue Screening GC/MS and LC/MS/MS Method
Condition
10 mL 25% toluene in acetonitrile
Sample Pre-Preparation *extract 20g of sample
Load
*prepared extract
Elute 20 mL
25% toluene in acetonitrile
Evaporate
Reconstitute Methanol for LC/MS/MS
1:1 ACN/Hexane for GC/MS
Sep-Pak®
Carbon/NH2
*Extraction
20 g sample to 50 mL of acetonitrile
Filter
adjust to 100mL using acetonitrile
Liquid Fractionation
20 mL extract/20 mL 0.5 M phosphate buffer (pH 7)/10 g NaCl
Collection
collect acetonitrile layer, dry with sodium sulfate
Evaporation
Reconstitution 2mL 25% toluene in acetonitrile
©2011 Waters Corporation | COMPANY CONFIDENTIAL 27
Multi-Residue Screening Sample: Spinach Extract
Before clean-up After clean-up
©2011 Waters Corporation | COMPANY CONFIDENTIAL 28
Multi-Residue Screening Recovery at 0.1 ppm
Pesticide %Rec Pesticide %Rec Pesticide %Rec
Abamectin 102.0 Dimethirimol 101.0 Naproanalide 115.9
Anilofos 111.7 Fenoxcarb 108.7 Oryzalin 103.8
Azinphos-methyl 107.6 Fermizon 112.6 Axycarboxin 85.1
Benzofenap 139.5 Formetanate HCl 86.7 Oxydemetone-methyl 108.0
Butafenacil 104.5 Furathiocarb 100.5 Phenmedipharm 102.2
Chloridazon 106.0 Imidacloprid 111.8 Pyrazolynate 72.7
Chromafenozide 108.2 Indoxacarb 121.2 Quizalofop-p-terfural 145.3
Clomeprop 104.4 Isoxaflutole 99.5 Simiconazole 106.0
Cloquintocet-mexyl 108.7 Lactofen 108.8 Thiacloprid 109.2
Clothianidin 101.5 Methoxyfenozide 103.3 Thiamethoxam 108.3
Cyazofamid 108.3 Milbemectin A3 114.5 Tridemorph 94.6
Cyflufenamide 110.1 Milbemectin A4 101.2 Triconazole 113.3
©2011 Waters Corporation | COMPANY CONFIDENTIAL 29
3) Veterinary drugs multiresidue - food
Still relatively scarce:
— Lack of volatility of drugs not compatible with GC/MS;
LC/MS/MS
— Wide range of polarities difficult to find a common extraction
and clean-up procedure
— Complex matrices (proteins, carbohydrates, fat ...)
— Low levels of detection and confirmation (i.e. 1ug/Kg or lower)
SPE can be a very valuable option
Veterinary drugs in tissue: Oasis HLB key to diminish ion supression, eliminating the main components of
the matrix (above all proteins) and analyte concentration
Kaufmann et al, Journal of Chromatography A, 1194 (2008) 66-79
Veterinary drugs in milk: Sep-Pak C18
©2011 Waters Corporation | COMPANY CONFIDENTIAL 30
4) Multiresidue analysis of veterinary drugs in milk
Sample pre-treatment:
Add 2mL acetonitrile to 2mL of milk. Vortex and centrifuge for 4 minutes at 8.000 x g. Take 2mL supernatant and add 3mL 0,2% HCOOH in acetonitrile. Centrifuge Analytes: Representative compounds from major classes of veterinary drugs: tetracyclines, sulfonamides, fluoroquinolones, macrolides, NSAID’s, steroids, beta-lactams and beta-andrenergids
*Method developed by Waters in the USA. Ref 720004089EN.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 31
5) Multiresidue analysis of 165 pesticides by HPLC/MS/MS
Situation: need of multiresidue methods with low LOD’s for common pesticides in water (lake, tap and mineral water)
Analytical challenges: analysis of pesticides and metabolites of very different chemical families and physico-chemical properties in one single determination step.
Waters Solution:
— Sample prep: Oasis HLB
— System: Quattro Micro
— Column: Atlantis dC18
D.Ortelli et al, Service de Protection de la Consommation, Geneve
©2011 Waters Corporation | COMPANY CONFIDENTIAL 32
Experimental conditions
LC-MS/MS:
System : HP 1100 (Agilent) + Quattro Micro
Column : Atlantis dC18 3μm 2.1x100mm plus a guard column
Mobile phase : Water (A) and MeOH (B) with 0.1 % HCOOH
Gradient : 0min:100/0 A/B, 3min: 40/60, 11min:10/90 until 15min
Flow rate : 300 μl/min
Cycle time : 23 min
Capillary: 3,5kV
Source T: 120ºC
Desolvation Gas: 600L/h at 350ºC
Cone gas 50L/h
Cone and Collision voltage optimised for each compound
The selected reaction monitoring (SRM) mode was used with the most abundant MS/MS transition for quantitation and a second one for confirmation of substance identity. two different runs needed. The first one for detection of 90 fungicides and insecticides and a second one for analysis of 75 herbicides.
Condition/Equilibrate 3 mL methanol, 3 mL water
Load Sample 500mL sample
Elute 4 mL MeOH
Post-treatment Evaporate and reconstitute in
100µL MeOH
Oasis® HLB
6cc 200mg
©2011 Waters Corporation | COMPANY CONFIDENTIAL 33
Results
LOD’s between 1ng/L and 100ng/L
Recoveries, RSD and accuracy values fulfill the criteria of
validation commonly admitted
©2011 Waters Corporation | COMPANY CONFIDENTIAL 34
6) Multiresidue analysis of Pharmaceuticals in Drinking Water - HLB
41 million Americans drink publicly
supplied water containing trace amounts
of pharmaceuticals
70% of Americans are ―very‖ concerned
about these drugs in drinking water
$6 billion spent by homeowners to further
treat drinking water (filtration systems,
filters, etc.)
Bottled water not tested for
pharmaceuticals
EPA does not include pharmaceuticals
in its drinking water standards
EPA Method 1694: ―Pharmaceuticals and Personal Care Products in Water,
Soil, Sediment and Biosolids by HPLC/MS/MS‖ – December 2007
©2011 Waters Corporation | COMPANY CONFIDENTIAL 35
Pharmaceuticals in Drinking Water EPA Method 1694
Sample Preparation
• Oasis HLB, 20cc/1g LP, 60μm
• Acidic and basic fractions (typically 500 mL to 1 L)
Acidic Fraction (Adjust to pH 2.0)
Basic Fraction (Adjust to pH 10.0)
Condition
20 mL methanol
6 mL water
Load Sample
Wash
10 mL reagent water
Dry
Under vacuum, 5 min
Elute
12 mL methanol
Concentrate to final volume
Exchange to methanol
Condition
20 mL methanol
6 mL water
Load Sample
Wash
No Wash
Dry
Under vacuum, 5 min
Elute
6 mL methanol
9 mL 2% formic acid solution (v/v in methanol)
Concentrate to final volume
Exchange to methanol
©2011 Waters Corporation | COMPANY CONFIDENTIAL 36
Pharmaceuticals in Drinking Water EPA Method 1694
Results:
Average Recovery for Each Analyte(5 Replicates / Sample)
0
20
40
60
80
100
120
140
Reco
very
(%
)
Group 3
Group 1
Group 4
Group 2
Data by U.S. Environmental Protection Agency
©2011 Waters Corporation | COMPANY CONFIDENTIAL 37
Hydrophilic-Lipophilic Balanced
Copolymer
Oasis sorbents: HLB
•Water wettable
•Polar retention
•Stable across pH 1-14
•No silanol interactions
•High specific surface area (810 m2/g)
•High recoveries for acids, bases and neutrals
Hydrophilic monomer
Lipophilic
monomer
N O
Reversed-phase Retention Retention of Polars
©2011 Waters Corporation | COMPANY CONFIDENTIAL 38
Reversed Phase Retention SPE Starting Protocol
Condition cartridge — strongest solvent first (DCM, MTBE,
ethyl acetate)
— intermediate solvent next (methanol)
— weak solvent last (water)
Load Sample — dissolve in or exchange to weak
solvent (water or water/methanol)
Wash Cartridge — use strongest possible solvent
without eluting analyte (methanol/water)
Elute Cartridge with strong solvent* — methanol, IPA,MTBE, DCM
*Oasis HLB – elute solvent should have at
least 5 % methanol or IPA as polar modifier
Prepare Sample
Condition/Equilibrate 1 mL methanol, 1 mL water
Load Sample
Wash 1 mL 5% methanol/water
Elute 2 mL methanol
Evaporate, Reconstitute
Oasis® HLB
SPE Protocol
Conditions for 3 cc 60 mg cartridges
©2011 Waters Corporation | COMPANY CONFIDENTIAL 39
Applications: Family-specific methods
Tetracyclines in meat
PFC’s in water
Acidic herbicides in wine and grape
Bisphenol A and endocrine disruptors in water
Nitrofurans in honey
Dioxane in water
Triazine herbicides and metabolites in water
Sudan dyes in chili oils
Aromatic hydrocarbons in soil
PAH’s in seafoods
Quats in water
PFC’s in liver
Tetracyclines in liver
Sulfonamides in whole milk
Melamine in milk
Malachite green in fish
Beta-agonists in urine
Tilmicosin in serum
Acrylamide in potato products
Fluoroquinolones in liver
…
©2011 Waters Corporation | COMPANY CONFIDENTIAL 40
Tetracyclines in meat HLB
0 5 10 Minutes
Ab
so
rba
nce
@3
65
nm
0.0
04
AU
FS
1
nonspiked sample
2
3
spiked pork HPLC Method Column: Nova-Pak® C8
Mobile Phase: 20% acetonitrile in
50 mM oxalic acid/water
Flow Rate: 0.8 mL/min
Detection: UV @ 365 nm
Injection: 60 µL
Sample: 5 g homogenized meat,
extracted with 2 x 20 mL
of McIlvaine buffer (mixed
citrate/phosphate, pH 4.1 with
EDTA). The combined extracts
were filtered before SPE.
SPE: 6 cc, 200 mg Oasis® HLB
1. Oxytetracycline (96%)
2. Tetracycline (91%)
3. Chlortetracycline (81%)
HO O HO O
OH
CONH2
HN(CH
3)2
O
H
CH3
HO
©2011 Waters Corporation | COMPANY CONFIDENTIAL 41
PFC’s in water HLB
PFCs are retained and
concentrated on Oasis HLB
Cartridge (225mg)
The retained PFCs are eluted with
methanol
The eluent is evaporated to 500 µL
— PFCs are stabilized prior to analysis
by addition of 200 µL 2 mM
ammonium acetate
The eluent is diluted to mobile
strength with water
Sample is ready for UPLC analysis
Condition 5 mL methanol/10 mL water
Load 500 mL
Wash 2 mL water
Dry with N2 for 20 min
Elute 2 mL methanol
Evaporate to 500 µL Dilute to mobile phase conc.
Oasis® HLB PFC Method
PFC’s are Persistent Organic Pollutants of high current
interest worldwide.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 42
Groundwater 1 ng/L (ppt) Spiked Sample
Time2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
%
0
100
1
2 3
4
5
7 6
8
9
10 7a
Results (n = 6) Compound % Recovery 1 ppt
1. PF-butanesulfonate 105 (6) 2. PF-hexanoic acid 112 (11) 3. PF-heptanoic acid 124 (15) 4. PF-hexanesulfonate 103 (5) 5. PF-octanoic acid (PFOA) 122 (17) 6. PF-nonanoic acid 107 (9) 7. PF-octanesulfonate (PFOS) 104 (6) 8. PF-decanoic acid 109 (8) 9. PF-undecanoic acid 100 (9) 10. PF-dodecanoic acid 105 (12)
peak7a. PFOS isomers
LOQ < 0.5 ng/L (500 ppq)
The full combination of UPLC plus SPE enrichment plus LC/MS Certified Vials allows us to break the ppt barrier!
No contamination issues !!!
©2011 Waters Corporation | COMPANY CONFIDENTIAL 43
Condition/Equilibrate 3 mL solvent*/3 mL methanol/3 mL water
Load up to 500 mL sample
Wash
Elute
Prepare Sample
Solvent strength and pH are the variables that can be manipulated
in the wash and elute steps to produce an optimized SPE method
More aggressive wash
steps to wash off
interferences
Alternative eluents to
selectively elute analytes
and retain interferences
Modifications for Reduction of Interferences
Optimized Oasis® HLB Methods
©2011 Waters Corporation | COMPANY CONFIDENTIAL 44
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pH
Re
ten
tio
n F
acto
r (k
’)
Neutrals
Bases (BH+)
Theory
Neue
A-
B
Acids (HA)
Recommended pH range of Silica
Recommended pH range of Oasis®
( pKa = 4.8 ) ( pKa = 9.0 )
Retention Factor (k’) as Function of pH Reversed-Phase
©2011 Waters Corporation | COMPANY CONFIDENTIAL 45
For any type of analyte
• Use the highest organic content possible (usually methanol)
• Experimentally determine the optimum methanol content
– try 10%, 20%, 30%, 40% etc.
For acidic analytes
– Use acidic wash, maximum methanol content
• Acidic wash keeps analyte in neutral form, enhances retention
• Experimentally determine the optimum methanol content
For basic analytes
– Use basic wash, maximum methanol content
• Basic wash keeps analyte in neutral form, enhances retention
• Experimentally determine the optimum methanol content
Optimization of Wash Steps
©2011 Waters Corporation | COMPANY CONFIDENTIAL 46
Common elution solvents
Organic — MeOH
— MeOH/ACN (20:80)
— ACN/IPA (40:60)
— MTBE/MeOH (90:10)
o Methyl tert-butyl ether
Additives (prepare daily) — Formic acid (2%)
o Improves recoveries for basic compounds in reversed-phase sorbents
— Ammonium hydroxide (5%)
o Improves recoveries for acidic compounds in reversed-phase sorbents
©2011 Waters Corporation | COMPANY CONFIDENTIAL 47
Bisphenol A and endocrine disruptors in water HLB
BPA and endocrine disruptors are
retained and concentrated on Oasis
5cc 200mg Glass cartridge
Glass cartridge: designed
specifically for low level estrogens
in water
Condition
3 mL MTBE / 3mL MeOH/ 3 mL water
Load 200 mL sample pH 3
Wash 1 3 mL 10% MeOH in water
Reequilibrate
3 mL water
Wash 2 3 mL 10% MeOH, 2% NH4OH
in water
Elute 2x3 mL 10% MeOH in MTBE
Evaporate and reconstitute in
10% MeOH in water
Oasis® HLB
©2011 Waters Corporation | COMPANY CONFIDENTIAL 48
UPLC for Bisphenol A
©2011 Waters Corporation | COMPANY CONFIDENTIAL 49
Corticosteroids in urine HLB
Sample pre-treatment: Enzymatic hydrolisis.
Add 1ml acetate buffer (0,2M
sodium acetate) pH 5,2 to 3ml
urine. Add 50 l b-glucuronidase/
arilsulfatase.
Incubate at 55ºC for hours.
Centrifuge for 15 minutas at 3000rpm
and take supernatant.
Analytes: triamcinolone, prednisolone,
m-prednisolone, prednisone,
betamethasone, dexamethasone,
fluomethasone, bechlormethasone,
budesonide, desonide, flunisolide,
3’-OH-estanozolol, tetrahidrogestrinone,
17α-trembolone, cortisone, hydrocortisone.
SPE Protocol for
Oasis® HLB
Prepare sample
Condition/Equilibrate 3 mL methanol, 3 mL water
Load 3mL urine
Wash 1 3 mL MeOH:0,02M NaOH (4:6)
Wash 2 3mL water
Elute 3 mL methanol
Conditions for 3 cc 60 mg cartridges
*Method from a workshop. Tested also for liver with
good results.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 50
Nitrofurans in honey HLB
antibiotics banned by EU
for use in food animals
parent antibiotic seldom
found in biological
matrixes
metabolites are bound to
matrix proteins in tissue
metabolites may be bound
to sugars in honey
Furaltadone
N
O
ONH2 AOZ
©2011 Waters Corporation | COMPANY CONFIDENTIAL 51
Honey Preparation Step 1
Retains: Polyphenolic constituents, waxes, and organic contaminants (removed from pass-though sample)
Does not-retain: Underivitized nitrofuran metabolites and sugars
Condition/Equilibrate 2 mL methanol, 2 mL water
Pass-Through* 2g prepared honey in 5 mL of 0.1 N HCl
Wash 1* 2 mL water
Collect Quantitatively*
* The pass through fraction and wash steps are quantitatively combined into a
single 15 mL capped sample tube
Oasis® HLB, 3 cc, 60 mg
Sample Pre-Preparation A 2 g sample of honey is diluted to 5 mL with 0.12 M HCl
and hydrolyzed at 37oC for 30 min.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 52
Nitrofuran metabolites derivatisation
N O
O
N H 2
N H 2 N N H 2
O
H
N
O
N H 2
O N
O
N H 2 N
N H
O
O
NO 2
OHC N
O
O
N
NO 2
NO 2
N
N N H 2
O
H
NO 2
N N
N H
O
O
NO 2
N O
N
O
N
O
0.1M HCl, 37o, 16 hr AOZ
Semicarbizide (SC)
AMOZ
1-Aminohydantion (AH)
2NB-AOZ
©2011 Waters Corporation | COMPANY CONFIDENTIAL 53
Nitrofuran Antibiotic Metabolites Honey Step 2
The eluent from step 1 is quantitatively collected and 300 µL of 50 mM 2-nitrobenzaldehyde in DMSO is added for derivatization. The sample is derivatized for 16 hrs at 37 C.
The sample is adjusted to pH 7 by addition of 6 mL of 0.1 M K2HPO4 prior to SPE Step 2.
Sample enrichment factor 2 g 200 µL (ca. 10:1)
Condition/Equilibrate 1 mL methanol, 1 mL water
Load prepared honey sample pH 7
Wash 1 2 mL water
Wash 2 2 mL 30 % methanol in water
Evaporate and Reconstitute
Dry 20 minutes
Elute 3 mL 2% formic acid in 90:10 MTBE/methanol
Conditions for HLB 3cc 60mg cartridges
©2011 Waters Corporation | COMPANY CONFIDENTIAL 54
Semicarbazide (MRM 291→166)
0.25 µg/Kg
0.25 ppb dual SPE
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00Time80
100
%
80
100
%
NFM_021705-011 Sm (SG, 2x1) 2: MRM of 2 Channels ES+ 209.1 > 166.2
6.08e3
NFM_021705-005 Sm (SG, 2x1) 2: MRM of 2 Channels ES+ 209.1 > 166.2
6.08e3
Single SPE
Dual SPE
2NB-semicarbazide
©2011 Waters Corporation | COMPANY CONFIDENTIAL 55
Ion Suppression Semicarbazide
Post extraction spiked blank honey samples (5 µg/kg, LC-MS-MS)
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Time 2
100
%
2
100
%
Single SPE
Dual SPE
60% suppression
15% suppression
The other metabolite derivatives showed similar suppression
©2011 Waters Corporation | COMPANY CONFIDENTIAL 56
Reversed-phase SPE limitations
Retention of polar analytes
Need to obtain a cleaner sample – more complex matrices
Better starting protocols
©2011 Waters Corporation | COMPANY CONFIDENTIAL 57
1: Desisopropylatrazine 2: Hydroxyatrazine 3: Desethylatrazine 4: Simazine 5: Cyanazine
6: Atrazine
Triazine herbicides and metabolites HLB generic protocol
HPLC Method Column: SymmetryShield™ RP8 Mobile Phase: A: 15% Acetonitrile in pH 6.7 phosphate buffer (5 mM) B: Acetonitrile Gradient: 100% A for 2 min, then linear gradient to 70% B in 25 min Flow Rate: 1.0 mL/min Detection: UV @ 214 nm (0.02 AUFS) Sample: 75 mL Injection: 75 µL
Minutes 0 5 10 15
1
2
3 4
5 6
spiked well water (200 ng/L)
nonspiked sample
atrazine
SPE: 3 cc, 60 mg Oasis® HLB
©2011 Waters Corporation | COMPANY CONFIDENTIAL 58
Results are given as % recovery with % RSD in parenthesis
Tap Water Well Water Spike level 500 ng/L Spike level 200 ng/L Compound 5 replicates 7 replicates
Desisopropylatrazine 98.4 (5.0) 95.6 (5.8)
Hydroxyatrazine 132 (1.3) 109 (11)
Desethylatrazine 106 (5.1) 104 (4.0)
Simazine n.a.* 97.7 (3.9)
Cyanazine n.a.* 93.1 (3.7)
Atrazine 101 (5.0) 101 (4.4)
Hydroxydesisopropylatrazine
Desethyldesisopropylatrazine * Simazine and Cyanazine were not included in the tap water spike
experiment
Triazine herbicides and metabolites HLB, Results
not recovered
©2011 Waters Corporation | COMPANY CONFIDENTIAL 59
Triazine herbicides and metabolites
MCX
For Bases
pKa 2-10
Select
Oasis® MCX
Prepare Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Elute 1:
100% MeOH
Elute 2:
5% NH4OH in MeOH
Protocol 1
for GC, use 90:10 MTBE/methanolic ammonia for elute 2
To recover acids and neutrals, analyze Elute 1
©2011 Waters Corporation | COMPANY CONFIDENTIAL 60
Oasis® MCX Method
Prepare Sample Adjust pH to 1,5 with HCl
Condition 1mL methanol/ 1 mL water
Load 100 mL sample
Wash #1 2 mL 0.1 N HCl
Wash #2 2 mL methanol
Elute 3 mL methanol(4% NH4OH)
Evaporate and Reconstitute
This method allows for high retention of bases in acidified
samples.
Matrix: drinking water
Wash #1 locks basic analytes to the resin by cation exchange
Wash #2 (methanol) contains acids and neutrals retained by
reversed-phase interaction. This fraction may be analyzed for those compounds if desired.
Conditions for 6cc 150mg cartridges
©2011 Waters Corporation | COMPANY CONFIDENTIAL 61
Triazine herbicides and metabolites in water MCX
Column: SymmetryShield™ RP18 3.9 x 150 mm Mobile Phase: A: phosphate buffer (20 mM, pH 6.8) B: acetonitrile Gradient: 95% A for 2 min then linear to 25% A in 20 min Flow Rate: 0.8 mL/min Detection: PDA (215nm) Injection: 80 µL
1. hydroxydesisopropylatrazine
2. desethyldesisopropylatrazine
3. hydroxydesethylatrazine
4. desisopropylatrazine
5. hydroxyatrazine
6. desethylatrazine
7. atrazine
Minutes
1
2
3
4 5
6
7
0 20 10
PD
A (0
.08
AU
FS
)
200 ng/L
spiked sample
non-spiked sample
©2011 Waters Corporation | COMPANY CONFIDENTIAL 62
Paraquat/Diquat in water
For Quats
Select
Oasis® WCX
N N CH3CH3
++N N CH3CH3
+++
+N
N+
+N
N
The Oasis WCX cartridge was
chosen for these analytes
Logic: quats are
cationic at all pH
values
quats can be eluted
from Oasis WCX with
acidic solvent
©2011 Waters Corporation | COMPANY CONFIDENTIAL 63
Optimized SPE Protocol Paraquat/Diquat
Oasis® WCX SPE Method
Paraquat/Diquat
50:1 sample enrichment
Prepare Sample adjust to pH 7
Condition 1mL methanol/ 1 mL water
Load up to 25 mL sample
Wash 1 mL pH 7 buffer/1mL methanol
Elute 1.5 mL ACN/water/TFA 84:14:2
Evaporate and Reconstitute
Conditions for 3 cc cartridges
0.5 mL mobile phase
Prepare Sample
Condition 1mL methanol/ 1 mL water
Load up to 25 mL sample
Wash 1 mL pH 7 buffer/1mL methanol
Elute 1.5 mL ACN/water/TFA 84:14:2
Evaporate and Reconstitute
Conditions for 3 cc cartridges
0.5 mL mobile phase
©2011 Waters Corporation | COMPANY CONFIDENTIAL 64
LC-MS Conditions paraquat/diquat
→
→
→
→
→
→
1 2 3 4 5 6 7 8 1
100 1
100
©2011 Waters Corporation | COMPANY CONFIDENTIAL 65
Isolation of Aromatic Hydrocarbons from Soil
Prepare Sample
Condition/Equilibrate 4 mL DCM, 10 mL hexane
Load Sample 0.25 mL of pre-extract in hexane
Elute 1 3 mL hexane
Elute 2 2 mL DCM
Evaporate to Final Volume (usually to 1 mL – not to dryness)
Sep-Pak® Silica
SPE Protocol
Pre-extraction Soils are mixed with sodium sulfate and
extracted with DCM or DCM/acetone.
The extract is exchanged to hexane (weak
solvent). The extract must be water-free.
Aliphatic hydrocarbons are removed.
This fraction may be collected and
analyzed if desired.
Aromatic hydrocarbons are eluted with
strong solvent.
Conditions for 6 cc 500 mg silica cartridges
©2011 Waters Corporation | COMPANY CONFIDENTIAL 66
Aromatic Hydrocarbons Recovered from Diesel Contaminated Soil (GC-FID)
Sep-Pak® Silica SPE
Minutes 0 5 10 15 20 25 30 35
naphthalene
2-methylnaphthalene
1-methylnaphthalene
phenanthrene
No SPE FID
Gas Chromatography
Column: RTX-5, 30 m x 0.25 mm (ID) 0.25 µm film
1 mL/min Helium carrier gas 35o C for 1 min, then 8oC/min to 320
©2011 Waters Corporation | COMPANY CONFIDENTIAL 67
PAH’s in seafood QuEChERS
Prepare Sample
Homogenize 15 g sample 15 mL ACN
Liquid Fractionation Shake for 1 minute
Centrifuge >1500 x g
Collection Remove acetonitrile for clean-up
DisQuE Extraction
Tube 1
Transfer 1 mL Extract
Tube 1 to Tube 2
Shake vigously for 1 minute Centrifuge >1500 x g
Transfer Collect to autosampler vial
Dilute if necessary
DisQuE Clean-Up
Tube 2
©2011 Waters Corporation | COMPANY CONFIDENTIAL 68
PAH’s in seafoods
Acquity H Class with FLR and PDA detectors
Waters PAH 4,6x50mm 3µm column
©2011 Waters Corporation | COMPANY CONFIDENTIAL 69
Tetracyclines in liver MAX
Sample pre-treatment Extract 1g of liver with 10 mL McIlvaine
buffer (citrate/phosphate buffer pH 4,1 with
EDTA).
Sonicate. Centrifuge. Filter.
UPLC/MS/MS Analysis: Aquity UPLC + Quattro Premier
Column: Acquity BEH RP18 2,1x50mm 1,7μm
Recoveries: Higher than 80%
SPE Protocol for
Oasis® MAX
Prepare sample
Condition/Equilibrate 2ml methanol, 2ml water
Load 10ml pre-treated sample
Wash 1 2ml 5% NH4OH in water
Wash 2 1ml MeOH
Elute 2 x 750μl 2% HCOOH in MeOH
Evaporate and reconstitute
N2 (47ºC), 300μl water:MeOH (9:1)
Conditions for MAX 3cc 60mg cartridges * Method from a workshop
HO O HO O
OH
CONH2
HN(CH
3)2
O
H
CH3
HO
©2011 Waters Corporation | COMPANY CONFIDENTIAL 70
Oasis Family of Mixed-Mode Sorbents: Reversed-Phase Retention and Ion Exchange
©2011 Waters Corporation | COMPANY CONFIDENTIAL 71
Oasis® 2x4 Method:
Neutrals
For Bases:
pKa 2-10
Use Oasis® MCX
For Strong Acids
pKa <1.0
Use Oasis® WAX
For Strong Bases
pKa >10
Use Oasis® WCX
For Acids
pKa 2-8
Use Oasis® MAX
Prepare Sample
Condition/Equilibrate
Load Sample
Wash:
5% NH4OH
Elute 1:
100% MeOH
Elute 2:
2% Formic Acid in MeOH
Protocol 2 Prepare Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Elute 1:
100% MeOH
Elute 2:
5% NH4OH in MeOH
Protocol 1
Bases Strong
Acids
Strong
Bases Acids
©2011 Waters Corporation | COMPANY CONFIDENTIAL 72
Oasis® 2x4 Method
Select Sorbents
For Strong Acids
pKa <1.0
Select
Oasis® WAX
For Strong Bases*
pKa >10
Select
Oasis® WCX
For Acids
pKa 2-8
Select
Oasis® MAX
For Bases
pKa 2-10
Select
Oasis® MCX
Characterize your Analyte and Select the Sorbent
Neutrals
are Retained on
all Sorbents
*also quats
©2011 Waters Corporation | COMPANY CONFIDENTIAL 73
Retention Factor (k’) as Function of pH Mixed-Mode Cation-Exchange
Oasis® MCX
pH
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
k’
Acids (HA)
Neutral
Bases (BH+)
A-
B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
k’
Acids (HA)Neutral
Bases (BH+)
A-
B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 140 1 2 3 4 5 6 7 8 9 10 11 12 13 14
k’
Acids (HA)Neutral
Bases (BH+)
A-
B
Oasis® MAX
indicates pH range for best retention of both acids and bases
pH
©2011 Waters Corporation | COMPANY CONFIDENTIAL 74
Sulfonamides in whole milk MCX
Sample pre-treatment: None !!!!!
Sample post-treatment: Evaporate and reconstitute in 0,5ml
mobile phase
UPLC/MS/MS:
Acquity UPLC + Quattro Premier XE (ESI+)
Protocolo EFS para
Oasis® MCX
Condition/Equilibrate 2ml methanol, 2ml water
Load 5ml whole milk
Wash 1 2ml 5% MeOH in water
Wash 2 1ml 0,5M aqueous HCl
Wash 3 2ml 20% MeOH in water
Wash 4 1ml MeOH
Elute 2,5ml 90:10 MeOH:water (200mM NH4HCO3 pH 8,5)
Conditions for MCX cartridges 3cc 60mg * Method developed by Waters in the USA.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 75
UPLC-MS-MS (MRM) analysis 5ng/ml spiked whole milk
©2011 Waters Corporation | COMPANY CONFIDENTIAL 76
Malachite green in fish MCX
Sample pre-treatment Add 50 l TMPD solution (1mg/mL) to 1g sample
(derivatization for UV analysis).
Add 10mL McIlvaines buffer (pH 2.6) / methanol (50:50)
solution; homogenize for 45s.
Centrifuge at 5000rpm for 20min. Take supernatant.
Repeat extraction and centrifugation. Combine both
extracts and load into SPE cartridge.
Analysis by UPLC/MS/MS or UPLC/UV
SPE Protocol for
Oasis® MCX
Condition/Equilibrate 2mL methanol, 2mL water
2mL McIlvaines buffer (pH 2.6)
Load 20mL extract
Wash 1 2mL 0,1N HCl
Wash 2
2 x 2.5mL water
Wash 3 2mL 50% MeOH:water
Wash 4 3mL hexane, vacuum dry
Elute Ethyl acetate:MeOH:NH4OH 5% (50:45:5)
Dry and reconstitue in 100µL 50% acetonitrile
Conditions for MCX 3cc 60mg cartridges * Method developed by Waters.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 77
Beta-agonists in urine MCX
Sample pre-treatment Enzymatic hydrolisis.
Add 1ml sodium acetate buffer 0,2M pH 5,2 to 4ml
urine. Add 50 l b-glucuronidase/arilsulfatase.
Incubate for 4h at room temperature.
Centrifuge for 15 minutes at 3000rpm and take
supernatant. Adjust to pH 8,8 with NaOH.
Analytes:
cimaterol, salbutamol, zilpaterol, cimbuterol,
clenciclohexerol, clenbuterol, ractopamine,
clenproperol, mabuterol, hidroximetilclenbuterol,
mapenterol, brombuterol
SPE Protocol for
Oasis® MCX
Prepare sample
Condition/Equilibrate 4ml methanol, 4ml water,
Load 2,5ml pre-treated sample
Wash 1 4ml water
Wash 2 4ml acetate buffer 0,1M pH 4
Wash 3 4ml MeOH
Dry the cartridge for 3-5 minutes
Elute 4ml DCM:IPA (8:2) 5% NH4OH
Conditions for MCX 6cc 150mg cartridges * Method from a workshop. Also tested in liver.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 78
Tilmicosin in pork serum MCX
Sample pre-treatment
300μl serum + 300μl 4% H3PO4.
UPLC/MS/MS analysis:
Acquity UPLC + Quattro Premier (ESI+)
SPE Protocol for
Oasis® MCX
Prepare sample
Condition/Equilibrate 500μl methanol, 500μl water,
Load 600μl diluted serum
Wash 1 500μl 2% HCOOH in water
Wash 2 300μl MeOH
Elute 300μl 5% NH4OH in MeOH
Conditions for MCX 10mg 96-well plates * Method from a workshop
©2011 Waters Corporation | COMPANY CONFIDENTIAL 79
Fluoroquinolones in liver MAX; MAX+MCX
Sample pre-treatment
Extract 2g of liver with 30mL 50mM phosphate
buffer, pH 7,4.
Centrifuge.
Analytes: ciprofloxacin, enrofloxacin,
sarafloxacin
Analysis by HPLC/MS/MS:
Alliance 2695 + Quattro Micro
Column: Atlantis dC18 4,6x150mm 5μm
SPE Protocol
Oasis® MAX
Prepare sample
Condition/Equilibrate 1ml methanol, 1ml 5N NaOH, 1ml water,
Load sample 5mL pre-treated sample
Wash 1 1mL 5% NH4OH in water
Wash 2 1mL MeOH
Elute 3ml 4% HCOOH in MeOH
Evaporate and reconstitute
N2 at 47ºC, 400μl mobile phase
Conditions for MAX 150mg cartridges * Method developed by Waters.
N
N N
COOH F
C H 3
©2011 Waters Corporation | COMPANY CONFIDENTIAL 80
Fluoroquinolones in liver Optimized method
Condition/Equilibrate 1 mL methanol, 1 mL 5 N NaOH, 1 mL water
Load 5 mL de pre-treated sample
Wash #1 1 mL 5 % NH4OH in water
Wash #2 1 mL methnol
Elute 2mL 0,2N HCl in MeOH
SPE Oasis® MAX (150 mg, 6 cc)
Load Elution from MAX cartridge
Wash 2ml de Methanol
Elute 500ul 10% NH4OH in MeOH
Neutralize with formic acid and bring to 1mL with mobile phase
SPE Oasis® MCX (30 mg, 1cc)
©2011 Waters Corporation | COMPANY CONFIDENTIAL 81
Fluoroquinolones in liver MAX; MAX+MCX
Reproducible method with recoveries higher than 70%
LOQ < 10ug/kg
©2011 Waters Corporation | COMPANY CONFIDENTIAL 82
Waters Food Safety Solutions
Considering all of a laboratories needs in
developing complete, cost-effective and compliant food
safety solutions.
Innovative Technologies
Comprehensive Solutions
Customer Satisfaction
©2011 Waters Corporation | COMPANY CONFIDENTIAL 83
Questions?