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©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


Challenges Facing Food Safety and Environmental Laboratories

Staff Skills

Improve Productivity / Faster Analysis

Lower Cost per Analysis

Sample Preparation

Compliance Information Management


Waters Technology Focus


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


Outline





Applications — Multiresidue methods — Family-specific methods


Why Sample Prep?

60% of the work activity and operating cost is spent on sample

preparation for introduction into the analytical system.

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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


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


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.


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


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


Outline





Applications — Multiresidue methods

— Family-specific methods


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


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


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


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


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


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


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


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


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


Cleanup Tube 2

Provides additional cleanup

Sorbent choices

— PSA

— Graphitized Carbon Black (GCB)

— C18

Acetonitrile Layer (ANALYTES ARE HERE)

Sorbent (INTERFERENCES)


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


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


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


PSA silica 500mg

Frits

6cc Vac cartridge


Aminopropyl silica 500mg

Frits

6cc Vac cartridge


PSA silica 500mg

Frits

6cc Vac cartridge

Sep-Pak Vac 6cc Carbon/PSA


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


Multi-Residue Screening Sample: Spinach Extract

Before clean-up After clean-up


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


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


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.


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


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


Results

LOD’s between 1ng/L and 100ng/L

Recoveries, RSD and accuracy values fulfill the criteria of

validation commonly admitted


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


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


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


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


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


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


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

…


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


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.


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 !!!


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


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


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


For basic analytes

– Use basic wash, maximum methanol content

• Basic wash keeps analyte in neutral form, enhances retention


Optimization of Wash Steps


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


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


UPLC for Bisphenol A


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


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.


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


Honey Preparation Step 1

Retains: Polyphenolic constituents, waxes, and organic contaminants (removed from pass-though sample)

Does not-retain: Underivitized nitrofuran metabolites and sugars


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.


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


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)


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


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


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


Reversed-phase SPE limitations

Retention of polar analytes

Need to obtain a cleaner sample – more complex matrices

Better starting protocols


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


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


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


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)


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


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


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


Optimized SPE Protocol Paraquat/Diquat

Oasis® WCX SPE Method

Paraquat/Diquat

50:1 sample enrichment

Prepare Sample adjust to pH 7



Wash 1 mL pH 7 buffer/1mL methanol

Elute 1.5 mL ACN/water/TFA 84:14:2


Conditions for 3 cc cartridges

0.5 mL mobile phase

Prepare Sample



Wash 1 mL pH 7 buffer/1mL methanol

Elute 1.5 mL ACN/water/TFA 84:14:2


Conditions for 3 cc cartridges

0.5 mL mobile phase


LC-MS Conditions paraquat/diquat

→

→

→

→

→

→

1 2 3 4 5 6 7 8 1

100 1

100


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


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


PAH’s in seafood QuEChERS

Prepare Sample

Homogenize 15 g sample 15 mL ACN


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


PAH’s in seafoods

Acquity H Class with FLR and PDA detectors

Waters PAH 4,6x50mm 3µm column


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


Oasis Family of Mixed-Mode Sorbents: Reversed-Phase Retention and Ion Exchange


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


Load Sample

Wash:

5% NH4OH

Elute 1:

100% MeOH

Elute 2:

2% Formic Acid in MeOH

Protocol 2 Prepare Sample


Load Sample

Wash:

2% Formic acid

Elute 1:

100% MeOH

Elute 2:

5% NH4OH in MeOH

Protocol 1

Bases Strong

Acids

Strong

Bases Acids


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


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


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.


UPLC-MS-MS (MRM) analysis 5ng/ml spiked whole milk


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.


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.


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


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


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)


Fluoroquinolones in liver MAX; MAX+MCX

Reproducible method with recoveries higher than 70%

LOQ < 10ug/kg


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