purity by spe - kinesiskinesis.co.uk/media/wysiwyg/knowledebase/pdf/... · in 1996, waters...

Oasis Sample Extraction Products

Purity by SPE

2 [ ]3

Oasis Product Family Provides:

n Highest SPE Recovery

n Cleanest Extracts

n Lowest Matrix Effects

n Lowest Method Variability

THE MOST WIDELY USED SPE PRODUCTS IN BIOANALYTICAL LABORATORIES

* U.S. patents 5,882,521 (1996), 5,976,376 (1998), 6,106,721 (1999), 6,254,780 (2001), 6,322,695 (2001),

6,468,422 (2002), 6,726,842 (2004), 6,773,583 (2004), 6,723,236 (2004), additional patents pending.

2 [ ]3

The novel design of the Oasis® family of solid-phase extraction products

is intended to simplify and improve sample preparation. By combining the

appropriate sorbent, device format and methodology, bioanalytical scientists

are routinely able to achieve robust, reproducible and sensitive SPE methods.

Oasis SPE sorbents covered by nine US patents* are unique in their purity,

stability and retention characteristics. The innovative, patented Oasis µElution

plate, for the first time, enables elution volumes of clean extracts to be as low

as 25 µL while maintaining high analyte(s) SPE recovery.

[ ]4

WHY SOLID-PHASE ExTRACTION (SPE)?

Sample preparation is a key component of every analytical method (such as LC/MS/MS). By some estimates, 60-80% of the work activity and operating cost in the analytical laboratory is spent preparing samples for introduction into an instrument.

The importance of sample preparation, particularly SPE, stems from four major concerns—eliminating matrix effects including reducing ion suppression, lowering method variability, concentrating analyte(s) of interest, and improving analytical system performance.

SPE OffErS SOlut iOnS tO t hESE MajOr SaMPlE P rEParat iOn COnC ErnS

For high sensitivity analyses, such as those employing UPLC®/MS/MS, proper sample preparation can be critical for minimizing matrix effects and concentrating analytes of interest. Oasis sample preparation can be used with UPLC/MS/MS systems to provide the cleanest extracts.

The system shown here integrates the Xevo™ TQ MS, a highly advanced mass spectrometer, combining MassLynx™ informatics with innovative ScanWave™ and IntelliStart technologies, and the Waters ACQUITY UPLC® system.

Solid-phase extraction (SPE) has proven to be an effective tool for removing interferences, enabling sensitive, selective, and robust LC/MS/MS analysis.

EliMinat ing Mat rix EffECtS

Frequently, compounds of interest are present at levels too low for accurate and precise quantitation. SPE enables the enrichment of selected analytes without concentrating the interferences.

COnC Ent rat ing analyt E(S) Of int ErESt

Oasis SPE products successfully remove phospholipids and lyso-phospholipids, key contributors to ion suppression in LC/MS/MS analysis. Removal of these interferences results in lowering the method variability and increasing the MS response, hence reducing the LOQ.

rEduCing iOn SuP P rESSiOn

Advances in SPE technology, combined with robotic automation, make SPE not only a cost-effective, but also a time-efficient, sample-preparation technique. This improves analytical system performance by:

n Introducing the analyte(s) in an MS compatible solvent

n Extending analytical column lifetime, reducing system downtime/maintenance

n Minimizing ion suppression to improve signal response

iMP rOv ing analyt iCal SySt EM PErfOrManC E

CHEMISTRY

[ ]5

In 1996, Waters revolutionized SPE technology with the introduction of Oasis HLB, the first water-wettable—yet hydrophobic—polymeric sorbent, permanently changing SPE practice. The Oasis family includes five patented SPE chemistries for all your sample preparation needs:

Wat ErS innOvat iOnS in SOlid-PhaSE Ext raCt iOn

OASIS WCX

OASIS MCX

OASIS WAX

OASIS HLB

OASIS MAX

OASIS WCX

OASIS MCX

pKa <11 meq/g

pKa ~50.75 meq/g

OASIS WAX

pKa ~60.6 meq/g

Water-wettableStable from pH 0–14No silanol interactions

OASIS HLB

HydrophilicRetention of Polars

LipophilicRP Retention

OASIS MAX

pKa >180.25 meq/g

n OASIS MCx: Mixed-mode Cation-eXchange reversed-phase sorbent for bases

n OASIS MAx: Mixed-mode Anion-eXchange reversed-phase sorbent for acids

n OASIS WCx: Mixed-mode Weak Cation-eXchange reversed-phase sorbent for strong bases and quaternary amines

n OASIS WAx: Mixed-mode Weak Anion-eXchange reversed-phase sorbent for strong acids (e.g., sulfonates)

n OASIS HLB: Hydrophilic-Lipophilic-Balanced reversed-phase sorbent for acids, bases and neutrals

[ ]6 [ ]7

Unique Water-Wettable Oasis HLB Copolymer

N-VINYLPYRROLIDONE DIVINYLBENZENE

HYDROPHILIC-LIPOPHILIC BALANCE

Specific Surface Area: 810 m2/g, Average Pore Diameter: 80 ÅTotal Pore Volume: 1.3 cm3/g, Average Particle Diameter: 5 µm, 15 µm, 25 µm, 30 µm or 60 µm

Batch-to-Batch Reproducibility of Oasis HLB Sorbent — A Decade of Dramatic Consistency

Effect of Drying on Recovery

Oasis HLB Cartridge (30 mg) C18 Cartridge (100 mg)

% R

ecov

ery

Drying Time (minutes)

ProcainamideAcetaminophenRanitidinePropranololDoxepin

Drying Time (minutes)

% R

ecov

ery

0

20

40

60

80

100

0 4 80

20

40

60

80

100

0 5 10

No impact of sorbent drying on HLBHigh, consistent recovery

No Batch Reservations Needed: Consistently high recoveries over more than 10 years of production.

85

95

105

% R

ecov

ery

Procanamide – 1.90% RSD Ranitidine – 1.68% RSD Acetaminophen – 1.65% RSD

Recovery for:

Long-term, batch-to-batch reproducibility of traditional silica-based mixed-mode sorbents may be compromised by hydrolytic instability at pH extremes, relatively low ionic capacity, and difficulties in bonding. To assure performance consistency for large projects, careful analysts test for suitability, and then reserve successful, specific lots of sorbent.

Exceptional Batch-to-Batch reproducibility

Oasis HLB is a Hydrophilic-Lipophilic-Balanced, water-wettable, reversed-phase sorbent for all your SPE needs. It is made from a specific ratio of two monomers, hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene. It provides superior reversed-phase capacity with a neutral polar ‘hook’ for enhanced retention of polar analytes.

Waters has built a family of SPE sorbents which inherit some key features of this unique substrate: stability at pH extremes and in a wide range of solvents, extraordinary retention of polar compounds, and a relative hydrophobic retention capacity 3x higher than that of traditional silica-based SPE sorbents like C18.

Water-wettable Oasis sorbents exhibit excellent retention capacity for a wider polarity spectrum of analytes, even if the sorbent bed runs dry during conditioning or sample loading. This means that your SPE methods will be more rugged and robust, precluding the need for repeat sample preparation.

The advantage of having higher retention capacity [k] is that more analytes are retained with less breakthrough, improving the recovery and overall reproducibility of your SPE method.

Available in five particle sizes [60 µm, 30 µm, 25 µm, 15 µm, and 5 µm], Oasis HLB sorbent, in cartridge, plate, or column format, allows you to select the appropriate product based on the volume, viscosity, and turbidity of your sample.

universal Sorbent for acidic, neutral, and Basic Compounds

CHEMISTRY

Oasis sorbents have demonstrated excellent long-term, batch-to-batch reproducibility for over ten years. Our careful process design and stringent quality controls have set a new standard in batch-to-batch and lot-to-lot reproducibility for SPE sorbents. Our entire Oasis family of sorbents and devices is manufactured in Waters ISO 9000

registered facilities in compliance with cGMP guidelines of the U.S. Food and Drug Administration for Class 1 medical devices.

Multiple batches of Oasis HLB, MCX, MAX, WCX, and WAX have each been used successfully on validated bioanalytical assays in a regulated laboratory environment.

OaSiS hlB ChEMiSt ry

[ ]6 [ ]7

Drug–Sorbent Interactions on Oasis MCX Sorbent

Drug–Sorbent Interactions on Oasis MAX Sorbent

SO–3

SO–3

NO

MCX sorbentMixed-mode Cation eXchange

Reversed-phase interaction

Strong cation-exchange mode

Best retention at least 2 pH units below pKa

of the analyte

+CH3

CH 3

O

OH

NHH

Propranolol(basic drug)

Sulfonic-acid-cation-exchange capacity:

1.0 meq/g

Highly selective retention enables much stronger washes,resulting in very clean extracts.

R+CH 2

CH3

CH3

C H4 9N

NO

MAX sorbentMixed-mode Anion eXchange

Reversed-phase retention

Suprofen(acidic drug)

Quaternary-amine-anion-exchange capacity:

0.25 meq/g

S

CH 3O

COO–

Strong anion-exchange mode

Best retention at least 2 pH units above pKa

of the analyte

+


high Selectivity and Sensitivity for Basic CompoundsOasis MCX is designed to overcome the limitations of traditional silica-based mixed-mode SPE sorbents. It is a strong mixed-mode cation exchange, water-wettable, polymeric sorbent made repro-ducibly by a novel, Waters-patented process. Oasis MCX provides dual modes of retention—ion exchange, and reversed phase—on a single, clean, stable, high-surface-area, organic co-polymer that is stable from pH 0 to 14.

In addition to the pH stability typical of DVB polymers, Oasis MCX has far greater binding capacity than that of silica-based mixed-mode SPE sorbents. The ability to fully manipulate pH [0–14] during the development, optimization, and use of SPE methods on mixed-mode sorbents enables not only fast, straightforward, method development, but also ensures very rugged and robust procedures. There is no analyte breakthrough or loss of recovery due to dissolving silica particles at high pH or cleaving bonded phase at low pH.

Effective use of mixed-mode SPE sorbents for extraction of basic compounds from biological matrices (such as plasma, urine, bile, and tissue homogenates) requires high capacity (both reversed-phase for the retention of interferences and ion-exchange for selective retention of analytes). Typical silica-based mixed-mode sorbents are synthesized in a complex process that is hard to

Mixed-Mode Cation-eXchange and reversed-Phase Sorbent

high Selectivity and Sensitivity for acidic CompoundsOasis MAX is designed to overcome the limitations of traditional silica-based mixed-mode SPE sorbents. It is a strong mixed-mode anion-exchange, water-wettable, polymeric sorbent stable from pH 0 to 14. Now you can employ reliable SPE in methods to detect, confirm, or quantify acidic compounds and/or acidic metabolites in biological fluids. SPE procedures using the selectivity and rugged-ness of Oasis MAX enable separation of analytes from complex samples into two fractions: acidic compounds and basic/neutral compounds. Fractionated extracts can be analyzed by multiple analysis methods or even multiple techniques [LC/MS and GC/MS].

The novel, well-controlled Waters production process reproducibly delivers Oasis MAX with 0.25 meq/g of quaternary-amine-ion-ex-change capacity. Two particle sizes [60 µm and 30 µm] are available.

Mixed-Mode Anion-eXchange and reversed-Phase Sorbent

CHEMISTRY

OaSiS MCx ChEMiSt ry

OaSiS Max ChEMiSt ry

control, and they have relatively low exchange capacities, ranging from 0.06–0.2 meq/g. The novel, well-controlled Waters production process reproducibly delivers Oasis MCX with 1.0 meq/g of sulfonic-acid-ion-exchange capacity. Two particle sizes [60 µm and 30 µm] are available.

[ ]8

Drug-Sorbent Interactions on Oasis WCX Sorbent

Drug-Sorbent Interactions on Oasis WAX Sorbent


Weak cation-exchangeinteraction

WCX sorbentWeak Cation eXchange

COO -

N

N+

O

OCH3

CH3

CH3H C3

CH O3


Carboxylic-acid-cation-exchange capacity: 0.75 meq/g

+NH

NO

+NH


Weak anion-exchangeinteraction

WAX sorbentWeak Anion eXchange

SO–3

2


Piperazine-anion-exchangecapacity: 0.6 meq/g

high Selectivity and Sensitivity for Strongly Basic CompoundsOasis WCX is designed to provide superior sample preparation for strong bases and quaternary amines. It is a weak-cation-exchange, mixed-mode, water-wettable, polymeric sorbent, stable from pH 0 to 14. Oasis WCX has all the advantages of Oasis HLB. Rugged and highly selective SPE methods using Oasis WCX detect, confirm and quantify strongly basic compounds and quaternary amines in biological fluids.

The novel, well-controlled Waters production process reproducibly delivers Oasis WCX with 0.75 meq/g of carboxylic-acid-ion-exchange capacity. Three particle sizes [60 µm, 30 µm, and 5 µm] are available.

Mixed-Mode Weak Cation-eXchange and reversed-Phase Sorbent

high Selectivity and Sensitivity for Strongly acidic CompoundsOasis WAX is designed to provide superior sample preparation for strong acids. It is a weak-anion-exchange, mixed-mode, water-wettable, polymeric sorbent, stable from pH 0 to 14. Oasis WAX has all the advantages of Oasis HLB. Rugged and highly selective SPE methods using Oasis WAX detect, confirm, and quantify strongly acidic compounds in biological fluids.

The novel, well-controlled Waters production process reproducibly delivers Oasis WAX with 0.6 meq/g of piperazine-ion-exchange capacity. Three particle sizes [60 µm, 30 µm, and 5 µm] are available.

Mixed-Mode Weak Anion-eXchange and reversed-Phase Sorbent

OaSiS WCx ChEMiSt ry

OaSiS Wax ChEMiSt ry

CHEMISTRY

FORMAT

[ ]9

The entire Oasis family of sorbents and devices is manufactured in Waters ISO 9000 registered facilities in compliance with cGMP guidelines of the U.S. Food and Drug Administration for Class 1 medical devices.

OaSiS faMily Of SPE P rOduCtS

n OASIS µELUTION PLATES

n OASIS 96-WELL ExTRACTION PLATES

n OASIS SYRINgE-BARREL CARTRIDgES

n OASIS gLASS CARTRIDgES

n OASIS ON-LINE COLUMNS

[ ]10 [ ]11

FORMAT

OaSiS μElut iOn Plat ES

n Patented µElution plate design*

n Enabling technology facilitates elution volumes as low as 25 µL

n No evaporation and reconstitution necessary; just elute and shoot

n Ideal for small sample volumes

n Up to a 15x increase in concentration

n Compatible with most liquid-handling robotic systems for automated, reliable high-throughput SPE (HT-SPE)

OaSiS 96-W Ell Ext raCt iOn Plat ES

n Innovative two-stage well design (1999 R&D 100 Award winner)

n High throughput and high recovery

n Available in 5 mg, 10 mg, 30 mg, and 60 mg per well

n Compatible with most liquid-handling robotic systems for automated, reliable high-throughput SPE (HT-SPE)

OaSiS faMily Of SPE PrOduCtS

* U.S. patent 6,723,236

[ ]10 [ ]11

FORMAT

OaSiS SyringE-BarrEl Cart ridgES

n Ultra-clean syringe barrel and frits

n Available in cartridges ranging from 1 cc to 35 cc

n Available from 10 mg to 5 g of sorbent per cartridge

n Flangeless syringe-barrel cartridges available: 1 cc, 3 cc, 6 cc

n Also available: Plus cartridges with Luer inlet hub and outlet tip, 225 mg

OaSiS glaSS Cart ridgES

n Ultra-clean glass syringe with Teflon® frit

n Endocrine disruptors analysis at part-per-trillion levels

n Available in 5 cc (200 mg) configuration

OaSiS On-linE COluMnS

n Rugged, reproducible, ultra-fast, on-line analysis

n Compatible with all on-line analysis systems

n Wide choice of configurations, particle sizes, and sorbent chemistries

[ ]13[ ]12

Disk Technology

Ratio Bed H/D = 0.13

Wide and ShortSorbent Bed

Vacuum

�Elution Technology

Narrow and TallSorbent Bed

Vacuum

Ratio Bed H/D = 1.15

Elution Volume

Oasis µElution Technology Disk Technology

>85% recovery in 25 µL Minimum of 100 µL needed for best result

% R

ecov

ery

0

20

40

60

80

10025 µL

100 µL Spiked Saline

acetaminophen

practolol

N-acetylprocainamide

betamethasone

caffeine

naproxen

amitriptyline

propranolol

Oasis HLBµElution plate

Brand E C18 Brand S C18Brand E UR

75 µL 75 µL 75 µL

The innovative features of the Oasis µElution plates enable sensitive,robust, reproducible results without evaporation and reconstitution.

Comparison of µElution vs. Disk Technology

Recovery Comparison of µElution vs. Disk Technology

There are two predominant low-elution SPE technologies in the marketplace today: SPE Disk and Oasis µElution. These technologies differ greatly in performance primarily because of three features: aspect ratio, holdup volume and elution volume.

There is a correlation between a sorbent bed’s aspect ratio [height to diameter, H/D] and its performance in an SPE device. An H/D ratio less than 1.0 often compromises extraction efficiency. In SPE disk design (e.g. membrane/glass-fiber disk), a small mass of sorbent is embedded in a thin support structure having good flow properties but a small aspect ratio, H/D=0.13. In contrast, the Oasis µElution technology uses an internally tapered well, packed with high capacity Oasis sorbent, having an aspect ratio, H/D=1.15, nearly 9x that of SPE disk technology. An Oasis µElution plate well functions more like a chromatography column, minimizing loss of analyte during critical steps in the SPE procedure and enhancing overall extraction efficiency.

A small holdup volume is necessary to minimize elution volume. SPE disks have holdup volumes in the 35–65 µL range, compared to that for an Oasis µElution bed plus frits which measures as little as 15 µL. Having 2–4x less holdup volume minimizes elution volume and reduces analyte loss during elution, improving both recovery and precision.

Oasis µElution technology vs. disk technology

n Elute in as little as 25 µL

n No evaporation and reconstitution

n Ideal for small sample volumes

n Up to a 15x increase in concentration

Now you can confidently perform SPE cleanup and analyte enrich-ment of very small sample volumes (10-25 µL) up to a maximum of 375 µL. The Waters Oasis µElution plate combines patented plate design, proven Oasis chemistries, and straightforward protocols that deliver high analyte recovery and clean extracts in elution volumes as low as 25 μL.

Wat ErS lat ESt innOvat iOn in SPE t EChnOlOgy

OaSiS �Elut iOn Plat ES

FORMAT

Minimizing SPE elution volume is critical for SPE performance, recovery, and precision. Normal elution volume range for an Oasis µElution plate is only 25–50 µL, far lower than the 75–300 µL typical for SPE disk technology.

Reproducible elution in only 25 µL enhances and streamlines SPE methods. SPE can be performed on very small sample volumes. Sample enrichment increases as much as 15-fold. A time-consuming step for eluate concentration by evaporation is no longer needed—neither is evaporation/reconstitution if the eluting solvent is properly chosen to be compatible with the LC/MS mobile phase. The innovative features of Oasis µElution plates enable sensitive, robust, repro-ducible results without evaporation and reconstitution. Analytical performance, in terms of sensitivity, selectivity, and ruggedness, is superior.

[ ]13

% R

ecov

ery

SPE Recovery for 200 ng/mL Imipramine and 200 ng/mL Atenolol on Oasis MCX µElution Plate

SPE Recovery for Polar andNon-Polar Analytes in Plasma Example

SPE Recovery for Polar andNon-Polar Analytes in Urine Example

Matrix Effects for Polar andNon-Polar Analytes in Plasma

0

20

40

60

80

100

50 100 150 200 250 300 350 µL

Loaded Plasma Volumes (Undiluted) Loaded Urine Volumes (Undiluted) Loaded Plasma Volumes (Undiluted)

-40

-30

-20

-10

0

10

20

30

40

50 100 150 200 250 300 350 µL

Atenolol (polar)

Imipramine(non-polar)

n = 8

% R

ecov

ery

Ion

Enha

ncem

ent

Ion

Supp

ress

ion

0

20

40

60

80

100

50 100 150 200 250 300 350 µL

SPE device capacity is defined as the total mass of analytes and

endogenous sample components retained by the sorbent bed under

loading conditions. Breakthrough will occur when the capacity of

the sorbent bed is exceeded. The physicochemical properties of

Oasis sorbents are designed to provide exceptionally high loading

capacity, even though each well in a Waters Oasis µElution plate

contains only 2 mg of Oasis sorbent.

To determine the Oasis μElution plate capacity, increasing volumes

of plasma and urine samples (from 50 µL to 350 µL in 50 µL

increments) were spiked with 200 ng/mL imipramine (non polar

base) and 200 ng/mL atenolol (polar base). The plasma aliquots

were diluted 1:1 with 4% aqueous H3PO4 and the urine aliquots

were diluted 1:1 with H2O and then loaded onto the µElution plate.

SPE recovery was calculated and plotted for each loading level.

µElution Plate loading Capacity

FORMAT

Waters award-winning plate design, with five chemistry and four sorbent-mass options, provides flexible high-throughput SPE in a single device. The Oasis 96-well plates are designed to be used on many manifold configurations and most robotic liquid handling systems. Oasis sorbents’ unique balance of hydrophobicity and water-

wettability means you will never have to worry about poor results if individual wells of the 96-well plate dry out. As always, you can expect Oasis SPE products to perform reliably, delivering high and reproducible recoveries for a wide range of analytes, including polar and basic compounds, with RSDs less than 5% [n=96].

v ErSat ilE, high-t hrOughPut OaSiS 96-W Ell Ext raCt iOn Plat ES

OaSiS 96-W Ell Plat ES

Waters 96-Well Plate Design

By varying frit size and/or placement, the same plate may be filled with various quantities of sorbent per well. Our design permits optimal recoveries, even with low sorbent weights for smaller elution volumes.

5 mg 10 mg 30 mg 60 mg

1999 R&D100 Award Two-Stage Well-Design

Condition:200 µL CH3OH

Equilibrate:200 µL H2O

Load:Various volumes of:

- Plasma diluted 1:1 with 4% H3PO4 in H2O - Urine diluted 1:1 with H2O

Wash 2:200 µL CH3OH

Wash 1:200 µL 2% CHOOH in H2O

Elute:2 x 25 µL 5% NH4OH in 60:40

CH3CN:CH3OH

Dilute:50 µL H2O

Inject:5 µL

SPE Protocol for Oasis MCX µElution 96-Well Plate

[ ]14

On-linE SPE COluMnS fOr lC /MS/MS

OaSiS On-linE COluMnS

FORMAT

All of these formats are available with the five Oasis patented sorbents (HLB, MCX, MAX, WCX, WAX) in a wide choice of particle sizes and dimensions. The Oasis On-Line columns make it possible to analyze a specific analyte in a sample matrix when combined with appropriate Waters narrow-bore analytical columns (such as XBridge™, SunFire™, Atlantis®, XTerra®, or Symmetry® columns).

1

2

3

On-Line System Configuration

IsocraticHPLC

GradientHPLC

10 Port Valve2 positions

Waste

OasisOn-Line Column

Narrow BoreAnalytical Column

MassSpectrometer

MassSpectrometer

Alliance HT

515System

LOADPOSITION

Waste

AnalyticalColumn

AnalyticalColumn

543

2

110

9

8

76

Oas

is

MassSpectrometer

Alliance HT

515System

INJECTIONPOSITION

Waste

543

2

110

9

8

76

Oas

is

There are three HPLC Oasis On-Line column configurations designed to fit all your on-line-analysis needs.

n The Oasis Cartridge Column fits into a Sentry™ holder that features a finger-tight fitting for fast, convenient replacement [1]

n The Oasis Direct-Connect Column can be screwed directly into a switching valve or connected to fittings like those for a conventional HPLC column [2].

n The Oasis Column features traditional HPLC column fittings and hardware [3].

[ ]14

METHODOLOgY

[ ]15

n OASIS 2x4 METHOD

n OASIS 2x4 OPTIMIzED METHODOLOgY

n OASIS 2x4 METHOD OPTIMIzED

FOR µELUTION PLATE

n OASIS HLB gENERIC METHOD

n OASIS HLB OPTIMIzED METHODOLOgY

n OASIS ADvANCED METHODOLOgY

OaSiS MEt hOdOlOgy

SPE method development starts with knowledge of the sample matrix—the nature and relative concentration of the analytes of interest as well as of potentially interfering endogenous compounds. Then, judicious choices of sorbent and solvents for load, wash, and elute steps create rugged, reliable, selective protocols.

Waters application chemists have devised several approaches to streamline this process.

[ ]16 [ ]17

METHODOLOgY

3a: OaSiS 2x4 MEthOd

The Oasis 2x4 Method is a simple, logical approach to the selection of an SPE sorbent and protocol. Two protocols and four sorbents provide the flexibility to extract acids, bases, and neutrals with high SPE recoveries while removing matrix components that may interfere with analysis.

Follow the simple steps outlined in this flow chart to achieve high recoveries and the cleanest extracts:

n Characterize your analyte [Neutral, Acid or Base, pKa].

n Select one of the four Oasis sorbents.

n Apply the indicated Protocol [1 or 2].

n Determine SPE recoveries by LC analysis. Neutrals WeakerBases

WeakerAcids

For Bases pKa 2-10:

Use Oasis MCX

For Acids pKa 2-8:

Use Oasis MAX

For Strong Bases pKa >10:

Use Oasis WCX

For Strong Acids pKa <1:

Use Oasis WAX

Apply Protocol 2

Prepare Sample

Condition/EquilibrateLoad Sample

5% NH4OH

Elute 1:100% CH3OH

Elute 2:2% HCOOH in CH3OH

Oasis 2x4 MethodOnly 2 protocols and 4 sorbents to

analyze all types of compounds:acids, bases, and neutrals

Prepare Sample


Wash: Wash:2% HCOOH

Elute 1:100% CH3OH

Elute 2:5% NH4OH in CH3OH

Base Strong Acid Acid Strong Base

Apply Protocol 1

The Oasis Sorbent Selection Plates and Cartridge Kit enable rapid development of SPE methods for LC/MS analysis. Having all four Oasis ion-exchange sorbents [MCX, MAX, WAX and WCX] in a single plate is convenient for scouting the best ways to accomplish efficient isolation of unknown, zwitterionic compounds, or mixtures of analytes with different retention/elution properties.

Note that neutral analytes can be isolated from any of the four sorbents in the Elute 1 step of either protocol. Choose the particu-lar ion-exchange sorbent that is best at removing specific matrix interferences. A good example of this is shown below.

Oasis Sorbent Selection Plates and Cartridge Kit

Oasis MCXProtocol 1 – Elute 2

Oasis WAXProtocol 1 – Elute 1

Oasis MAXProtocol 2 – Elute 2

Oasis WCXProtocol 2 – Elute 1

0 50 100%

SPE Recovery

Cephalexin

[Cephalosporin antibiotic]

A difficult amphoteric analyte

pKa = 2.6

pKa = 7.3

Aliquots of prepared sample processed using Oasis 2x4 Method protocol designated for each

of 4 sorbents. Eluates from Elute 1 and Elute 2 steps analyzed by LC/MS/MS.

Clearly, Oasis MCX is the sorbent of choice.

S

NNH

O

HH

NH 2

O

HO ORecovery is high on all four Oasis sorbents,but removal of phospholipids, a primary causeof matrix ion suppression, is best on Oasis MCX.Protein precipitation is a poor choice for samplepreparation prior to LC/MS/MS analysis.

Cleanliness of 100% MeOH Elute 1 Monitoring 5 PhospholipidMRM Transitions

2% Phospholipids

%

100 1: MRM of 5 Channels ES+TIC

2.55e72.50

Oasis MCX — Protocol 1

1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 Min

%

0

1001: MRM of 5 Channels ES+

TIC2.55e7

2.522.60

Oasis WCX — Protocol 2

%


TIC2.55e72.522.60

Oasis MAX — Protocol 2 9% Phospholipids

11% Phospholipids

12% Phospholipids

%

100 1: MRM of 5 Channels ES+TIC

2.55e72.50

2.60

Oasis WAX — Protocol 1

%


TIC2.55e7

3.262.52 3.16

2.60

3.08

ProteinPrecipitation 100% Phospholipids

0 50 100%

SPE Recovery

O

OH

H H

O

OH

OH

Prednisone

Evaluating Oasis 2x4 Method for Cephalexin

Choosing Optimum Sorbent and Protocol for Neutral Compounds Example: Prednisone in Plasma

[ ]16 [ ]17

METHODOLOgY

Oasis 2x4 Method—Proof of Concept

To demonstrate the logic, simplicity, and effectiveness of the Oasis 2x4 Method, five rat plasma samples were prepared, each containing one of these characterized test analytes:

n Imipramine, a base – pKa of conjugate acid = 9.4

n Ibuprofen, an acid – pKa = 5.2

n Decanesulfonic acid, a strong acid – pKa < 0.5

n Valethamate, a quaternary amine [strong base] – pKa of conjugate acid > 12

n Prednisone, a neutral compound

Each plasma sample was diluted [1/1, v/v] and acidified with phos-phoric acid [4% in water]. Respective aliquots were then processed using the protocol and the Oasis ion-mixed-mode sorbent designated by the Oasis 2x4 Method for the corresponding analyte type. LC/MS/MS analysis was used to determine SPE recoveries.

The neutral analyte was processed on all four sorbents, as shown on the previous page. Of the four method options, Oasis MCX with Protocol 1 proved superior at removing nearly all the phospholip-ids, eliminating this major source of matrix effects, a known cause of ion suppression, loss of sensitivity, and inaccurate quantitation in LC/MS analysis.

Essentially quantitative recovery and excellent cleanup efficiency were achieved for each of the ionic or ionizable test analytes when the recommended Oasis 2x4 Method sorbent/protocol combination was used. These results are shown in the four figures below.

MRM ES+281.2 > 85.95

4.16e6

Oasis MCX

SPE Recovery: 108%n=4, 2–18% RSD

Imipramine [pKa = 9.4]

pKa <1

1 3 5 Min0

3.19

For BasespKa 2–10:

Use Oasis MCX

Protocol 1

Imipramine

N

N

Oasis WAX

1 3 5 min0

2.71

MRM ES+221.1 > 79.8

2.85e3


Decanesulfonic acid [pKa <0.5]

pKa ~6

For Strong AcidspKa < 1:

Use Oasis WAX

Protocol 1

Decanesulfonicacid

SO

O O– Na+

Oasis MAX


pKa ~18

Ibuprofen [pKa = 5.2]

MRM ES+205 > 161

1.34e4

1 3 5 min0

2.63

For AcidspKa 2–8:

Use Oasis MAX

Protocol 2

Ibuprofen

OH

O

Oasis WCX

MRM ES+306.2 > 163

2.10e6

1 3 5 min0

2.71

pKa ~5SPE Recovery: 106%

n=4, 9–13% RSD

Valethamate [pKa >12]

For Strong BasespKa > 10:

Use Oasis WCX

Protocol 2

Valethamate

ON+

O Br–

Oasis 2x4 Method Test on MCX: Base Isolation Oasis 2x4 Method Test on WAX: Strong Acid Isolation

Oasis 2x4 Method Test on MAX: Acid Isolation Oasis 2x4 Method Test on WCX: Strong Base Isolation

[ ]18 [ ]19

METHODOLOgY

Removes interferingacids [unionized] and

neutral compounds andacidic interferences

Locks ionized drug onstrong cation-exchangesites; removes proteins

and salts

Weak base removestertiary and

aromatic amines

Wash 3:1.5 mL 2.5% TEA in CH3OH

Wash 2:2 mL 100% CH3OH

Elute: 2 mL 5% NH4OH in CH3OH

Wash 1:2 mL 5% CH3OH in 0.1 N HCI

Load:10 mL spiked urine

(acidified with 100 µL 5 N HCI)

3B: OaSiS 2x4 OPt iMizEd MEthOdOlOgy

Optimization of the Oasis 2x4 Method on mixed-mode sorbents [MCX, MAX, WCX, WAX] takes full advantage of two different chromatographic separation mechanisms: reversed phase and ion exchange. Changes in pH are used to manipulate the respective ionization states of the exchange sites on the sorbent and of the acidic or basic moieties in analytes and interferences. In this way, retention can be directed selectively to occur through either hydrophobic or ionic interactions. Simultaneously, organic solvent concentration is modified to fine-tune elution based upon rela-

tive hydrophobicity. The result is cleaner extracts that reduce matrix effects, enhance the sensitivity, and lower the viability of the analytical method.

In the examples that follow, we will demonstrate how straightforward wash-elute studies, in which the percentage of organic solvent is systematically varied, are used to determine the optimal compo-sition of SPE mobile phases for selective wash and elution steps. Similar experiments may be designed for another variable such as pH or ionic strength.

A second example demonstrates the dramatic benefits of an optimized Oasis MCX method for determining methamphetamine in urine. A wash-elute study was used to find the best concentration of trieth-ylamine [TEA] for Wash 3 solution [below left]. In this way, tertiary

and aromatic amine interferences were removed selectively. As shown in the LC/UV data [below right], impressive RSDs were achieved for quantitation of the primary and secondary amine analytes.

3B–1: Oasis MCx 2x4 Method — Optimized for Bases

When the greatest selectivity and sensitivity for basic compounds [pKa 2–10] is required to achieve the lowest limits of detection [LOD] and quantitation [LOQ], optimization of Protocol 1 is recom-mended. As an example, a wash-elute study was performed to

determine the optimal retention and elution parameters for imip-ramine on Oasis MCX [below left]. These experimental data were then used to optimize a selective Oasis MCX SPE method [below right] to remove neutral, acidic, and basic interferences.

Oasis MCX Urine Application: Amphetamine and Methamphetamine

Optimized Oasis MCX Method for Imipramine

Basic drug is loaded inionized [or neutral] formif sample was pretreated

with acid [or base]

Removes interferingacids [unionized] and

neutral compounds andacidic interferences

Basic analyte iseluted; hydrophobicbasic interferences

are retained

Neutralizes bases;removes more polarbasic interferences

Condition/Equilibrate:1 mL CH3OH/H2O

Load sample:1 mL plasma or urine

Wash 1:1 mL 2% HCOOH

Wash 3:1 mL 5% NH4OH in CH3OH/H2O 55/45


Elute: 1 mL 5% NH4OH in CH3OH/H2O 95/5

Locks ionized drug onstrong cation-exchangesites; removes proteins

and salts

N

N

Imipramine

Maximum % Organic Wash

Indicates Peak Areaof Analyte in LC Analysisof Eluate

20% 30% 40% 50% 60% 70% 80% 90% 100%10%

[% CH3OH in H2O] : [conc. NH4OH] 95:5 v/v

Experiment for Oasis MCX 96-Well Plate 30 mg

10

0

20 30 40 50 60 70 80 90 100

Minimum % Organic Elute

% CH3OH in H2O

4 µg/mL n=6 Recovery RSD1. Amphetamine 96.8% 1.35%2. Methamphetamine 90.4% 4.01%3. Phentermine (I.S.)* — —* Internal Standard

0.00

0.01

AU

0 4 62

1 23

Blank withoutTEA Wash

Blank with2.5% TEA Wash

Spiked Samplewith 2.5% TEA Wash

Amphetamine Methamphetamine Phentermine

NH 2HN NH 2

Wash-Elute Study

Optimized Oasis MCX Method for Quantitation of Methamphetamine in Urine

[ ]18 [ ]19

METHODOLOgY

Experiment for Oasis WAX 96-Well Plate 30 mg

10 20 30 40 50 60 70 80 90 100

% CH3OH in H2O



20% 30% 40% 50% 60% 70% 80% 90% 100%10%

[% CH3OH in H2O] : [conc. NH4OH] 95/5 v/v

0


10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Experiment for Oasis MAX 96-Well Plate 30 mg

10 20 30 40 50 60 70 80 90 100


[% CH3OH in H2O] : [conc. HCOOH] 98/2 v/v

0



% CH3OH in H20

Wash-Elute Study

Wash-Elute Study

Optimized Oasis WAX Method for Decanesulfonic Acid

3B–3: Oasis Max 2x4 Method — Optimized for acids

When the greatest selectivity and sensitivity for acidic compounds [pKa 2–8] is required to achieve the lowest limits of detection [LOD] and quantitation [LOQ], optimization of Protocol 2 is recommended. As an example, a wash-elute study was performed to determine the

optimal retention and elution parameters for ibuprofen on Oasis MAX [below left]. These experimental data were then used to optimize a selective Oasis MAX SPE method [below right] to remove neutral, basic, and acidic interferences.

Locks ionized analyteon strong anion-exchange

sites; removesproteins and salts

Neutralizes acids;removes more polaracidic interferences

Acidic drug is loaded inionized [or neutral] formif sample was pretreated

with base [or acid]

Removes interferingbases [unionized] and

neutral compounds

Acidic analyte is eluted;more hydrophobic acidic

interferences are retained



Wash 1:1 mL 5% NH4OH

Wash 3:1 mL 2% HCOOH in CH3OH/H2O 45/55


Elute: 1 mL 2% HCOOH in CH3OH/H2O 90/10

Ionizes weak anion-exchange sites to

lock on analytes; removesproteins and salts

Neutralizes anion-exchange sites; removes

more polar stronglyacidic interferences

Removes interferingbases [ionized] and neutral

compounds [includingunionized acids]

Strongly acidic analyte iseluted; more hydrophobic

acidic interferencesare retained



Wash 1:1 mL 2% HCOOH

Wash 3:1 mL 5% NH4OH in CH3OH/H2O 25/75


Elute: 1 mL 5% NH4OH in CH3OH/H2O 80/20

3B–2: Oasis Wax 2x4 Method — Optimized for Strong acids

When the greatest selectivity and sensitivity for strong acids [pKa <1] is required to achieve the lowest limits of detection [LOD] and quantitation [LOQ], optimization of Protocol 1 is recommended. As an example, a wash-elute study was performed to determine the

optimal retention and elution parameters for decanesulfonic acid on Oasis WAX [below left]. These experimental data were then used to optimize a selective Oasis WAX method [below right] to remove neutral, basic, and acidic interferences.

Optimized Oasis MAX Method for Ibuprofen

S O

O O – Na +

Decanesulfonic Acid

OH

O

Ibuprofen

[ ]20 [ ]21

METHODOLOgY

Optimized Oasis WCX Method for Valethamate

3B–4: Oasis WCx 2x4 Method—Optimized for Strong Bases

When the greatest selectivity and sensitivity for strong bases such as quaternary amines [pKa >10] is required to achieve the lowest limits of detection [LOD] and quantitation [LOQ], optimization of Protocol 2 is recommended. As an example, a wash-elute study was

performed to determine the optimal retention and elution parame-ters for valethamate on Oasis WCX [below left]. These experimental data were then used to optimize a selective Oasis WCX SPE method [below right] to remove neutral, acidic, and basic interferences.

Ionizes weak cation-exchange sites to lock on

analytes; removesproteins and salts

Neutralizes cation-exchange sites; removes

more polar stronglybasic interferences

Removes interferingacids [ionized] and neutral

compounds [includingunionized bases]

Strongly basic analyte iseluted; more hydrophobic

basic interferencesare retained



Wash 1:1 mL 5% NH4OH

Wash 3:1 mL 2% HCOOH in CH3OH/H2O 25/75


Elute: 1 mL 2% HCOOH in CH3OH/H2O 75/25

O N +

O Br –

Valethamate

Experiment for Oasis WCX 96-Well Plate 30 mg

% CH3OH in H2O

10 20 30 40 50 60 70 80 90 100

10% 20% 30% 40% 50% 60% 70% 80% 90% 100%


[% CH3OH in H2O] : [conc. HCOOH] 98/2 v/v

0



Wash-Elute Study

The proven Oasis 2x4 method elution solvent is optimized to acc-ommodate the elutropic requirement of the small elution volume. Methanol is good as a generic elution solvent, but is often not strong enough for 25 µL elution volumes. The elution solvent recommended to be used with the μElution plate must possess a high enough elutropic strength to fully elute analytes in small volumes, and be appropriate for a diverse set of analytes.

The recommended elution solvent for the Oasis 2x4 method optimized for the µElution plate format is 60% CH3CN:40% CH3OH with a modifier. This was chosen as a starting point as it meets all of the above criteria.

Follow the simple steps outlined in this flow chart to achieve high recoveries and the cleanest extracts:

n Characterize your analyte [Neutral, Acid or Base, pKa].

n Select one of the four Oasis sorbents.

n Apply the indicated Protocol [1 or 2].

n Determine SPE recoveries by LC analysis.

3C: OaSiS 2x4 MEthOd OPt iMizEd fOr µElut iOn Plat E

Neutrals

For Bases pKa 2-10:

Use Oasis MCX

For Acids pKa 2-8:

Use Oasis MAX

For Strong Bases pKa >10:

Use Oasis WCX

For Strong Acids pKa <1:

Use Oasis WAX

Apply Protocol 2

Prepare Sample


5% NH4OH

Elute 1:100% CH3OH

Elute 2: 2% HCOOH in 60:40 CH3CN:CH3OH

Prepare Sample


Wash: Wash:2% HCOOH

Elute 1:100% CH3OH

Elute 2: 5% NH4OH in 60:40 CH3CN:CH3OH

Base Strong Acid Acid Strong Base

Apply Protocol 1

[ ]20 [ ]21

METHODOLOgY

Oasis 2x4 Method Proof of concept:Recovery Study:

To demonstrate the logic, simplicity, and effectiveness of the Oasis 2x4 method, five samples of rat plasma were prepared, each spiked with one of the previously characterized test analytes shown below:

— Imipramine: pKa = 9.4 (Base)

— Ibuprofen: pKa = 5.2 (Acid)

— Valethamate: pKa >12 (Quaternary Amine)

— Nonafluoropentanoic Acid: pKa <0.5 (Strong Acid)

— Prednisone: Neutral

Each plasma sample was diluted [1:1, v:v] and acidified with phos-phoric acid [4% in water]. Respective aliquots were then processed using the protocol and the Oasis ion-mixed-mode sorbent designat-ed by the Oasis 2x4 Method for the corresponding sample type. LC/MS/MS analysis was used to determine SPE recoveries. The neutral analyte was processed on all four sorbents used.

Analytes Spiked into Rat Plasma

% S

PE R

ecov

ery

Prednisone (n)Imipramine (b)

Prednisone (n)Nonafluoropentanoic Acid (sa)Prednisone (n)Ibuprofen (a)

Prednisone (n)Valethamate (sb)

Elute 1: CH3OH

Elute 2: 60:40 CH3CN/CH3OH + modifier

0

20

40

60

80

100

Oasis MCX Oasis MAX Oasis WCX Oasis WAX

Protocol 1 Protocol 2

Nonafluoropentanoic Acid(SA) pKa <0.5, 200 ng/mL

Ibuprofen (A)pKa = 5.2, 200 ng/mL

Imipramine (B)pKa = 9.4, 2 ng/mL

N

N

HO

O

FFFF

F F FFF

OH

O

Valethamate (QA)pKa >12, 2 ng/mL

ON+

O Br–

Prednisone (N)10 µg/mL

O

OH

H H

O

OH

OH

Oasis HLB Generic Method for Acids/Neutrals/Bases

[e.g., acidifyto disrupt drug–protein binding]

[Optional; e.g.,often not required inµElution plate format]

Prepare Sample(typically acidify)

Condition/Equilibrate:CH3OH/H2O

Load sample solution

Wash:CH3OH/H2O 5/95

Elute:CH3OH

Evaporate and Reconstitute(optional)

3d: OaSiS hlB gEnEriC MEthOd

In 1996 Waters introduced Oasis HLB, the first hydrophilic-lipophilic-balanced polymeric SPE sorbent. By careful design of its chemical structure and particle morphology [see page 6], we dramatically elevated the performance of SPE.

When isolating a range of acidic, basic, and neutral compounds, a generic method using Oasis HLB is recommended to remove unretained matrix constituents [e.g., salts, sugars, polar lipids, proteins) while reproducibly retaining, and subsequently eluting, a broad chromatographic polarity range of acidic, basic, and neutral analytes.

When transferring methods from C18-bonded-silica phases, consider these three unique advantages of Oasis HLB:

n Its higher capacity [2–3x more surface area] means correspondingly less sorbent weight is required. Bed and elution volumes can be reduced. Smaller-scale- device formats are more effective.

n Its higher retention power [3–5x] reduces breakthrough, increas-es enrichment factors, and permits finer tuning of gradient steps for more selective washing and elution sequences. Especially hydrophobic analytes may require stronger elution solvents. Polar compounds like phenols [> 9x higher k] that interact via hydrogen bonding with the amide moieties in the HLB backbone may require a protic solvent [e.g., methanol] rather than an aprotic solvent [e.g., acetonitrile] for efficient elution.

n Its excellent stability over a pH range of 0–14 and compat-ibility with a broad range of organic solvents facilitates development of rugged, reliable methods.

[ ]22 [ ]23

METHODOLOgY

1

3

2

SampleBlank

SampleBlank

SampleBlank

1

3

20.004 AU 0.004 AU0.004 AU

12

3

10 min8642010 min8642010 min86420

Peak Identification:Peak 1: NorverapamilPeak 2: VerapamilPeak 3: Methoxyverapamil [I.S.]

Oasis HLB Generic Method

Prepare sample solution[acidified to disrupt protein binding]



Load sample:1 mL plasma

Elute:1 mL 100% CH3OH

Modified Oasis HLB Generic Method


1 mL CH3OH/H2O



Wash 2:1 mL 5% CH3OH with 2% NH4OH

Elute:1 mL 65% CH3OH with 2% CH3COOH

Optimized Oasis HLB Method


1 mL CH3OH/H2O

Wash 1:1 mL 5% CH3OH with 2% CH3COOH


Wash 3: 1 mL 65% CH3OH with 2% NH4OH

Wash 2:1 mL 5% CH3OH with 2% NH4OH

Elute:1 mL 65% CH3OH with 2% CH3COOH

Condition/Equilibrate: Condition/Equilibrate:

Effect of Method Development Optimization on Chromatography: Verapamil in Plasma

0

40

0 2 4 6 8 10 12 14

0

pH

Recommended pH range for silica

Recommended pH range for Oasis® Sorbents

32

8

16

24 Neutrals

Base [BH+]Acid [A–]

Base [B]

Acid Retained Base Retained

Low Retention for Base Low Retention for Acid

pKa = 4.8

pKa = 9.0

Acid [HA]

Rete

ntio

n Fa

ctor

k

Theory: Retention Factor [k] vs. pH for Acids, Bases, and Neutrals

2% CH3COOH Modified Wash 2% NH4OH Modified Wash

% MeOH

Maximum% Methanol Wash

Maximum% Methanol Wash

Minimum% Methanol Elution

0

0 20 40 60 80 100% MeOH

0

0 20 40 60 80 100

1

2

3

1

2

3

1. Norverapamil 2. Verapamil

3. Methoxyverapamil

indicatespeak area of analytein LC analysis of eluate

H 3CO

H 3CO

CN HN OCH 3

OCH 3

H 3CO

H 3CO

CNN OCH 3

OCH 3

H 3CO

H 3CO

CNN OCH 3

OCH 3

OCH 3

Wash-Elute Study for Verapamil and Metabolites in Plasma

3E: OaSiS hlB OPt iMizEd MEthOdOlOgyChemical and chromatographic principles may be applied to opti-mize methods on Oasis HLB. Selectivity is dramatically enhanced by tuning pH, as well as the ratio of organic solvent to water, in the mobile phase to manipulate retention.

If analytes or interferences are ionizable, then, as highly polar entities in their charged states, they may be eluted in weak mobile phases. If, by changing pH, they are converted to neutral form, they are retained primarily by the strength of their hydrophobic interaction with the sorbent surface. Stronger mobile phases, with higher organic solvent concentrations, will then be required for successful elution.

Published work by Waters chemists clearly demonstrates the benefits of such a 2-D [two-dimensional] process on Oasis HLB. The theory of retention, wash-elute studies for verapamil and two of its metabo-lites, and successive selectivity improvements made by refining the Oasis HLB method are summarized in the figures below.

[ ]22 [ ]23

METHODOLOgY

3f: OaSiS advanCEd MEthOdOlOgy

Waters Oasis product family of unique, patented sorbents in flexible device formats and our array of method development strategies simplify and streamline traditional SPE applications. They also

enable you to meet new sample preparation challenges, no matter whether they have been recently ripped from the headlines or previously relegated to the unsolvable SPE problem files.

For very complex matrices, the ultimate way to optimize cleanup and isolation is to use orthogonal separation modes [e.g., reversed phase and ion exchange] in tandem. Individual cartridges can be connected with an adapter. By careful planning of mobile phase compatibility and retention mode sequences, the final eluate from the first cartridge can be loaded directly into the second cartridge. Then, after discarding the first cartridge, the second can be washed and eluted with appropriately scaled volumes of solvent. A larger first cartridge removes the bulk of matrix interferences. A smaller second cartridge further concentrates the analyte(s) and maximizes the enrichment factor.

Animal tissue homogenates are complex matrices containing a host of endogenous interferences that may overload the ion-exchange and reversed-phase capacities of an SPE sorbent. Difficulty is further compounded when analytes, such as fluoroquinolone antibiotics, are amphoteric. Compared to a single-cartridge cleanup, the ability of a well-designed tandem Oasis MAX–MCX method to conquer such a challenge successfully is dramatic. Chromatograms at right demonstrate the selective isolation and confident determination of ciprofloxacin and enrofloxacin at sub-ppb levels.

Orthogonal SPE using two Cartridges—Most Powerful Methodology

HCl convertsamphoteric analytesto cations.They elute from MAXinto lower MCX bed wherethey are retained. Acidic interferencesare neutralized by HCl and washedout of both cartridges by CH3OH.

After NaOH treatment,MAX retains amphotericanalytes as anions.Wash steps remove basicand neutral matrix interferences.

Load:5 mL of prepared sample

Condition/Equilibrate:1 mL CH3OH/1 mL 5 N NaOH;

1 mL H2O

Wash 1:1 mL 5% NH4OH in H2O

Wash 2:1 mL CH3OH

Stage 1:Condition, Load, and Wash

Oasis MAX6 cc 150 mg Cartridge

Condition:1 mL CH3OH

Stage 2:ConditionOasis MCX

1 cc 30 mg Cartridge

Elute & Wash:2 mL 0.2 N HCl in CH3OH

Stage 3:Attach MCX Cartridge

to outlet of MAX Cartridge;Elute from MAX into MCX

500µL of 10% NH4OH in CH3OH

Wash:2 mL of CH3OH

Elute:

Neutralize eluate with HCOOHand bring to 1 mL withbuffered mobile phase.

Stage 4:Discard MAX Cartridge;

Wash & EluteMCX Cartridge

Smaller bed in 1-cc MCXCartridge enables elutionin a smaller volume,increasing enrichment factor.

MCX

Car

trid

ge

MCX

Car

trid

ge

MA

X Ca

rtri

dge

MCX

Car

trid

geM

AX

Cart

ridg

e

Ciprofloxacin

CiprofloxacinpKa of acid ~ 5pKa of base ~ 8–9

Enrofloxacin

Enrofloxacin

0 8 16 24 min

0 8 16 24 min

Chromatogram 2: Result from Tandem Oasis MAX + MCX

Chromatogram 1: Result from a single Oasis MAX Cartridge

N

F

HNN

O

OH

O

N

F

NN

O

OH

O

Tandem Oasis MAX–MCX Method for Amphoteric Fluoroquinoline Antibiotics

Tandem Oasis MAX – MCX Results for Fluoroquinolone Antibiotics in Beef Kidney – spiked at 1 µg/kg

[ ]24 [ ]25

Oasis hlB Sample Extraction Products Oasis MCx Sample Extraction Products (Cation Exchange)

DescriptionParticle

SizeQty. Part No.

Oasis HLB cartridge 1 cc/10 mg 30 µm 100/box 186000383Oasis HLB cartridge 1 cc/30 mg 30 µm 100/box WAT094225NEW Oasis HLB cartridge 1 cc/30 mg 30 µm 1000/box 186003908Oasis HLB flangeless cartridge 1 cc/30 mg 30 µm 100/box 186001879Oasis HLB cartridge with Gilson ASPC adapter

1 cc/10 mg 30 µm 500/box 186000988

Oasis HLB cartridge with Gilson ASPC adapter

1 cc/30 mg 30 µm 500/box WAT058882

Oasis HLB cartridge 3 cc/60 mg 30 µm 100/box WAT094226Oasis HLB flangeless cartridge 3 cc/60 mg 30 µm 100/box 186001880Oasis HLB cartridge with Gilson ASPC adapter

3 cc/60 mg 30 µm 500/box WAT058883

Oasis HLB cartridge 6 cc/200 mg 30 µm 30/box WAT106202Oasis HLB 3 cc/400 mg 60 µm 100/box 186003849NEW Oasis HLB cartridge 3 cc/540 mg 60 µm 100/box 186004134Oasis HLB flangeless cartridge 3 cc/540 mg 60 µm 100/box 186003852Oasis HLB cartridge 6 cc/150 mg 30 µm 30/box 186003365Oasis HLB cartridge 6 cc/150 mg 60 µm 30/box 186003379Oasis HLB cartridge 6 cc/500 mg 60 µm 30/box 186000115Oasis HLB cartridge 12 cc/500 mg 60 µm 20/box 186000116Oasis HLB cartridge 20 cc/1 g 60 µm 20/box 186000117Oasis HLB cartridge 35 cc/6 g 60 µm 10/box 186000118Oasis HLB Plus cartridge 225 mg 60 µm 50/box 186000132Oasis HLB Vac RC cartridge 20 cc/30 mg 30 µm 50/box 186000382Oasis HLB Vac RC cartridge 20 cc/60 mg 30 µm 50/box 186000381Oasis HLB glass cartridge 5 cc/200 mg 60 µm 30/box 186000683Oasis HLB Prospekt 2/Symbiosis cartridge* 1.0 x 10 mm 30 µm 96/box 186001196Oasis HLB Prospekt 2/Symbiosis cartridge* 2.0 x 10 mm 30 µm 96/box 186003925Reservoir 30 cc for Oasis cartridges 48/box WAT011390Reservoir 60 cc for Oasis cartridges 12/box WAT024659Reservoir adapter for 1 cc, 3 cc, 6 cc cartridges 10/box WAT054260Reservoir adapter for 12 cc, 20 cc, 35 cc cartridges 10/box WAT048160Reservoir adapter for 5 cc cartridges, Teflon 10/pkg 405000934

Oasis HLB column 2.1 x 20 mm 5 µm 1/pkg 186002034Oasis HLB column 3.0 x 20 mm 5 µm 1/pkg 186002037Oasis HLB column 3.9 x 20 mm 5 µm 1/pkg 186002040Oasis HLB cartridge column 3.9 x 20 mm 5 µm 1/pkg 186001413Oasis HLB column 4.6 x 20 mm 5 µm 1/pkg 186002043Oasis HLB column 2.1 x 20 mm 15 µm 1/pkg 186002035Oasis HLB column 3.0 x 20 mm 15 µm 1/pkg 186002038Oasis HLB column 3.9 x 20 mm 15 µm 1/pkg 186002041Oasis HLB cartridge column 3.9 x 20 mm 15 µm 1/pkg 186001414Oasis HLB column 4.6 x 20 mm 15 µm 1/pkg 186002044Oasis HLB column 2.0 x 15 mm 25 µm 1/pkg 186001792Oasis HLB column 2.1 x 20 mm 25 µm 1/pkg 186002036Oasis HLB cartridge column 2.1 x 20 mm 25 µm 1/pkg 186000706Oasis HLB column 3.0 x 20 mm 25 µm 1/pkg 186002039Oasis HLB column 3.9 x 20 mm 25 µm 1/pkg 186002042Oasis HLB column 4.6 x 20 mm 25 µm 1/pkg 186002045Holder kit for 2.1 x 20 mm cartridge column 1/pkg 186000262Holder kit for 3.9 x 20 mm cartridge column 1/pkg WAT046910Extraction column connector 1/pkg WAT082745Inline precolumn filter kit 1/pkg WAT084560Replacement filters 5/pkg WAT005139Replacement steel gaskets 1/pkg WAT084567

Oasis HLB µElution plate 2 mg/96-well 30 µm 1/pkg 186001828BAOasis HLB plate 5 mg/96-well 30 µm 1/pkg 186000309Oasis HLB plate 10 mg/96-well 30 µm 1/pkg 186000128Oasis HLB plate 30 mg/96-well 30 µm 1/pkg WAT058951Oasis HLB plate 60 mg/96-well 60 µm 1/pkg 186000679

* For use with Spark Holland Prospekt 2 and Symbiosis systems

DescriptionParticle

SizeQty. Part No.

Oasis MCX cartridge 1 cc/30 mg 30 µm 100/box 186000252Oasis MCX flangeless cartridge 1 cc/30 mg 30 µm 100/box 186001881Oasis MCX cartridge 1 cc/60 mg 60 µm 100/box 186000782Oasis MCX cartridge 3 cc/60 mg 30 µm 100/box 186000254Oasis MCX flangeless cartridge 3 cc/60 mg 30 µm 100/box 186001882Oasis MCX cartridge 3 cc/60 mg 60 µm 100/box 186000253Oasis MCX cartridge 6 cc/150 mg 30 µm 30/box 186000256Oasis MCX cartridge 6 cc/150 mg 60 µm 30/box 186000255Oasis MCX cartridge 6 cc/500 mg 60 µm 30/box 186000776Oasis MCX cartridge 20 cc/1 g 60 µm 20/box 186000777Oasis MCX cartridge 35 cc/6 g 60 µm 10/box 186000778Oasis MCX Plus cartridge 225 mg 60 µm 50/box 186003516Oasis MCX Vac RC cartridge 20 cc/60 mg 30 µm 50/box 186000261Oasis MCX Vac RC cartridge 20 cc/60 mg 60 µm 50/box 186000380Oasis MCX Prospekt 2/Symbiosis cartridge* 10 x 1 mm 30 µm 96/box 186002098

Oasis MCX direct connect column 2.1 x 15 mm 30 µm 1/pkg 186002050Oasis MCX column 2.1 x 20 mm 30 µm 1/pkg 186002046Oasis MCX cartridge column 2.1 x 20 mm 30 µm 1/pkg 186002051Oasis MCX column 3.0 x 20 mm 30 µm 1/pkg 186002047Oasis MCX column 3.9 x 20 mm 30 µm 1/pkg 186002048Oasis MCX column 4.6 x 20 mm 30 µm 1/pkg 186002049

Oasis MCX µElution plate 96-well 30 µm 1/pkg 186001830BAOasis MCX plate 10 mg/96-well 30 µm 1/pkg 186000259Oasis MCX plate 30 mg/96-well 30 µm 1/pkg 186000248Oasis MCX plate 30 mg/96-well 60 µm 1/pkg 186000250Oasis MCX plate 60 mg/96-well 60 µm 1/pkg 186000678

ORDERINg INFORMATION

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Oasis Method development Kits

Description Particle Size Part No.

Oasis sorbent selection plate3 rows each:MCX, MAX, WCX, WAX

96-well 30 µm 186003249

NEW Oasis µElution Sorbent Selection Plate, 3 rows each:MCX, MAX, WCX, WAX

96-well 30 µm 186004475

Oasis sorbent selection cartridge kit, 10 each:MCX, MAX, WCX, WAX

1 cc/30 mg 30 µm 186003463

Oasis Max Sample Extraction Products (anion Exchange)

DescriptionParticle

SizeQty. Part No.

Oasis MAX cartridge 1 cc/30 mg 30 µm 100/box 186000366Oasis MAX flangeless cartridge 1 cc/30 mg 30 µm 100/box 186001883Oasis MAX cartridge 3 cc/60 mg 30 µm 100/box 186000367Oasis MAX cartridge 3 cc/60 mg 60 µm 100/box 186000368Oasis MAX flangeless cartridge 3 cc/60 mg 30 µm 100/box 186001884Oasis MAX cartridge 6 cc/150 mg 30 µm 30/box 186000369Oasis MAX cartridge 6 cc/150 mg 60 µm 30/box 186000370Oasis MAX cartridge 6 cc/500 mg 60 µm 30/box 186000865Oasis MAX Plus cartridge 225 mg 60 µm 50/box 186003517Oasis MAX Vac RC cartridge 20 cc/30 mg 30 µm 50/box 186000372Oasis MAX Vac RC cartridge 20 cc/60 mg 30 µm 50/box 186000371Oasis MAX Vac RC cartridge 20 cc/60 mg 60 µm 50/box 186000378Oasis MAX Prospekt 2/Symbiosis cartridge* 10 x 1 mm 30 µm 96/box 186002099

Oasis MAX direct connect column 2.1 x 15 mm 30 µm 1/pkg 186002056Oasis MAX column 2.1 x 20 mm 30 µm 1/pkg 186002052Oasis MAX cartridge column 2.1 x 20 mm 30 µm 1/pkg 186002057Oasis MAX column 3.0 x 20 mm 30 µm 1/pkg 186002053Oasis MAX column 3.9 x 20 mm 30 µm 1/pkg 186002054Oasis MAX column 4.6 x 20 mm 30 µm 1/pkg 186002055

Oasis MAX µElution plate 2 mg/96-well 1/pkg 186001829Oasis MAX plate 10 mg/96-well 30 µm 1/pkg 186000375Oasis MAX plate 30 mg/96-well 30 µm 1/pkg 186000373Oasis MAX plate 60 mg/96-well 30 µm 1/pkg 186001256Oasis MAX plate 60 mg/96-well 60 µm 1/pkg 186001205

* For use with Spark Holland Prospekt 2 and Symbiosis systems

Oasis WCx Sample Extraction Products (Weak Cation Exchange)

DescriptionParticle

SizeQty. Part No.

Oasis WCX 1 cc cartridge 30 mg 30 µm 100/box 186002494Oasis WCX 3 cc cartridge 60 mg 30 µm 100/box 186002495NEW Oasis WCX 6 cc cartridge 150 mg 30 µm 30/box 186002498Oasis WCX 1 cc cartridge 30 mg 60 µm 100/box 186002496Oasis WCX 3 cc cartridge 60 mg 60 µm 100/box 186002497Oasis WCX Plus cartridge 225 mg 60 µm 50/box 186003518Oasis WCX µElution plate 2 mg/96-well 30 µm 1/pkg 186002499Oasis WCX 96-well plate 10 mg/96-well 30 µm 1/pkg 186002501Oasis WCX 96-well plate 30 mg/96-well 30 µm 1/pkg 186002503Oasis WCX Prospekt 2/Symbiosis cartridge* 1.0 x 10 mm 30 µm 96/box 186002892

Oasis WCX 2.1 x 20 mm column 30 µm 186002505Oasis WCX 3.9 x 20 mm column 30 µm 186002507Oasis WCX 2.1 x 20 mm column 5 µm 186002510Oasis WCX 3.9 x 20 mm column 5 µm 186002512

Oasis Wax Sample Extraction Products (Weak anion Exchange)

DescriptionParticle

SizeQty. Part No.

Oasis WAX 1 cc cartridge 30 mg 30 µm 100/box 186002489Oasis WAX 3 cc cartridge 60 mg 30 µm 100/box 186002490Oasis WAX 6 cc cartridge 150 mg 30 µm 30/box 186002493Oasis WAX 1 cc cartridge 30 mg 60 µm 100/box 186002491Oasis WAX 3 cc cartridge 60 mg 60 µm 100/box 186002492Oasis WAX Plus cartridge 225 mg 60 µm 50/box 186003519Oasis WAX µElution plate 96-well 30 µm 1/pkg 186002500Oasis WAX 96-well plate 10 mg/96-well 30 µm 1/pkg 186002502Oasis WAX 96-well plate 30 mg/96-well 30 µm 1/pkg 186002504NEW Oasis WAX 96-well plate 60 mg 30 µm 1/pkg 186003915Oasis WAX Prospekt 2/Symbiosis cartridge* 1.0 x 10 mm 30 µm 96/box 186002893

Oasis WAX 2.1 x 20 mm column 30 µm 186002508Oasis WAX 3.9 x 20 mm column 30 µm 186002509Oasis WAX 2.1 x 20 mm column 5 µm 186002511Oasis WAX 3.9 x 20 mm column 5 µm 186002513

[ ]26


Description Qty Part No.

Extraction plate manifold for Oasis 96-well plates 1/box 186001831

Extraction plate manifold kit A(includes extraction plate manifold, reservoir tray,sealing cap and 350 µL sample collection plate)

WAT097944

Extraction plate manifold kit B(as kit A, with 1 mL sample collection plate)

WAT097945

Extraction plate manifold kit C(as kit A, with 2 mL sample collection plate)

WAT097946

Manifold for Extraction Plate Manifold for Extraction Cartridges

accessories for Extraction Plate Manifold accessories for Extraction Columns and Cartridges

Oasis 96-well µElution Plate (Requires Manifold Spacer)

Oasis 96-well Plate (No Spacer Required)

Collection Plate

Spacer for µElution Plate (Included

with Manifold Kit)Extraction Plate

Manifold186001831

Waters Extraction Manifold

Description Part No.

Waters extraction manifold, 20-position without rack (includes 20 needle tips, 25 plugs, and ejector tool)

WAT200677

Waters extraction manifold, 20-position (complete with rack for 13 x 75 mm tubes)

WAT200606


WAT200607


WAT200608


WAT200609


Disposable reservoir tray 25/box WAT058942Sample collection plate, 350 µL 50/box WAT058943Sample collection plate, 2 mL 50/box WAT058958Sealing cap for 96-well collection plate 50/pkg WAT058959SPE vacuum pump 115 V 60 Hz 725000417SPE vacuum pump 240 V 50 Hz 725000418

Vacuum box gasket kit Kit includes: 2 foam top gaskets 2 orange O-rings

186003522


Holder kit for 2.1 x 20 mm cartridge column 1/pkg 186000262Holder kit for 3.9 x 20 mm cartridge column 1/pkg WAT046910Extraction column connector 1/pkg WAT082745Inline precolumn filter kit 1/pkg WAT084560Replacement filters 5/pkg WAT005139Replacement steel gaskets 1/pkg WAT084567SPE vacuum pump 115 V 60 Hz 725000417SPE vacuum pump 240 V 50 Hz 725000418Reservoir, 30 cc (for Oasis Plus, Light, Vac & Classic cartridges) 48/pkg WAT011390Reservoir, 60 cc (for Oasis Plus, Light & Vac cartridges) 12/pkg WAT024659Adapter, male-male Luer (for Oasis Classic cartridges) 100/pkg WAT024310

Adapter (to attach reservoir to 1, 3 & 6 cc Oasis Vac cartridges)

12/pkg WAT054260

Adapter (to attach reservoir to 12, 20 & 35 cc Oasis Vac cartridges)

10/pkg WAT048160

SPE Vacuum Pump (Includes two gauges and pressure regulator)

Vacuum Box Gasket Kit

[ ]26 [ ]27

Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 07

Australia 61 2 9933 1777

Belgium 32 2 726 1000

Brazil 55 11 5094-3788

Canada 1 800 252 4752 x2205

China 86 21 6879 5888

CIS/Russia +7 495 3367000

Czech Republic 420 2 617 1 1384

Denmark 45 46 59 8080

Finland 09 5659 6288

France 33 1 30 48 72 00

Germany 49 6196 400600

Hong Kong 852 29 64 1800

Hungary 36 1 350 5086

India and India Subcontinent 91 80 2837 1900

Ireland 353 1 448 1500

Italy 02 265 0983

Japan 81 3 3471 7191

Korea 82 2 820 2700

Mexico 52 55 5200 1860

The Netherlands 31 76 508 7200

Norway 47 63 84 60 50

Poland 48 22 833 4400

Puerto Rico 1 787 747 8445

Singapore 65 6273 1221

Spain 34 93 600 9300

Sweden 46 8 555 11 500

Switzerland 41 56 676 70 00

Taiwan 886 2 2543 1898

United Kingdom 44 208 238 6100

All other countries: Waters Corporation U.S.A. 1 508 478 2000

1 800 252 4752

www.waters.com

Sales Offices

©2008 Waters Corporation. Waters, ACQUITY UPLC, Atlantis, IntelliStart, MassLynx, Oasis, Quattro Premier, ScanWave, Sentry, Sirocco, SunFire,

Symmetry, The Science of What’s Possible, UPLC, XBridge, Xevo and XTerra are trademarks of Waters Corporation. Teflon is a trademark of DuPont.

All trademarks used are acknowledged. All rights reserved.

July 2008, 720001692EN 2008 IH-WA

The quality management system of Waters’ manufacturing facilitiesin Taunton, Massachusetts and Wexford, Ireland complies with the International Standard ISO 9001:2000 Quality Management and Quality Assurance Standards. Waters’ quality management system is periodically audited by the registering body to ensure compliance.

purity by spe - kinesiskinesis.co.uk/media/wysiwyg/knowledebase/pdf/... · in 1996, waters...

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