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SAMPLE

PREP

ARA

TION

SAMPLE PREPARATION

Solid-Phase Extraction

Introduction...15

Oasis

®

SPE Products

A Breakthrough in SPE...18

Oasis

®

HLB Cartridges, Plates, and Columns

for Reversed-Phase SPE ...20

Oasis

®

MCX Cartridges, Plates, and Columns

for Cation Exchange...24

Oasis

®

MAX Cartridges, Plates, and Columns

for Anion Exchange...25

Automation for Oasis

®

SPE Products ...27

High Throughput 96-well Extraction Plates ...28

N

EW

Oasis

®

µElution Plates ...28

Oasis

®

On-Line Columns ...31

Prospekt

Cartridges...33

Oasis

®

Glass Cartridges ...33

Oasis

®

Products Selection Guides ...34

Ordering Information...36

Sep-Pak

®

Cartridges and Accessories

NEW

Sep-Pak

®

96-well Plates ...39

Which Cartridge is Best for

your Application ...40

Which Sorbent is Best for

Sep-Pak

®

Cartridge Selection Guide ...42

your Application ...44

Sep-Pak

®

Cartridges...47

Sep-Pak

®

Vac Cartridges ...48

Sep-Pak

®

DNPH Cartridges ...51

Waters XpoSure

Cartridges ...52

Porapak

®

Rdx Cartridges ...53

(2)

SAMPLE

PREP

ARA

TION

Why SPE?

Sample preparation is a key component of every analytical analysis (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 the system.

The importance of sample preparation, in particular SPE, stems from three major concerns—removing interferences from sample matrix, concentrating analyte(s) of interest, and improving analytical system performance. SPE offers solutions to these major concerns of sample preparation: Removing interferences from sample matrix

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

Concentrating Analyte(s) of Interest

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

Improving analytical system performance

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

• Introducing the analytes in a MS compatible solvent. • Extending analytical column lifetime, reducing system

downtime/maintenance.

• Minimizing ion suppression while improving signal response. There are many techniques used in sample preparation. Common methods include liquid-liquid extraction, centrifugation, filtration, precipi-tation, and now more importantly, solid-phase extraction (SPE). Traditionally, the benefits, resulting from the chromatographic technology used in solid phase extraction, were not experienced by many analysts. This was due to several factors: a lack of awareness of how to be successful with the technique, simple methods development protocols and products that did not perform reproducibly.

Solid-Phase Extraction (SPE)

Today, advances in SPE product technology, as well as robotic equipment make this technique capable of very cost effective and time efficient sample preparation. SPE can be one of the simplest, yet most effective and versa-tile, methods of sample preparation. Utilizing low cost, pre-packed, dispos-able cartridges containing a chromatographic packing (sorbent), a sample analyte of interest is separated from other species in the sample matrix by loading the sample onto the device and selectively eluting the desired compound(s) using different solvents.

Convenient Technology for Improved Laboratory Operations

Convenience and ease-of-use are primary benefits of solid-phase extraction cartridges. This can allow you to maximize the productivity of your analyti-cal operation while saving you time and money. When compared to other sample preparation techniques, SPE cartridges also offer:

Faster sample prep —average time reduced by 2/3 Lower cost —less solvent and reagent consumption and less hazardous waste for disposal

Greater recoveries —minimal sample transfer Greater accuracy —no cross contamination

Powerful trace concentration (enrichment) of analytes Less sample handling —no emulsion problems

Reduced harm to labile samples —minimal evaporation Improved safety —due to reduced solvent/sample exposure and glassware

Easy automation —simultaneous batch processing of multiple samples

To satisfy all your methods development needs, Waters offers a complete range of different SPE cartridge and device designs. In addition, two differ-ent product lines are available: SEP-Pak® products containing traditional silica or alumina based sorbents, and Oasis®SPE products containing state of the art polymeric based sorbents.

Strategies for Solid-Phase Extraction

There are two simple solid-phase extraction strategies for sample prepara-tion. You can choose a cartridge sorbent, sample solvent and elution solvent(s) to cause:

Strategy 1. Compound(s) of interest are unretained, while matrix interfer-ences are adsorbed (captured)

Waters

Sep-Pak Vac

®

Load Sample Matrix

Elute Compound(s) of Interest

Capture (retain) Interferences – then Dispose of Cartridge

(3)

Strategy 2.Compound(s) of interest are retained, while matrix interferences are washed from the cartridge. The compound(s) of interest are then eluted with a stronger solvent.

The first strategy is usually chosen when the desired sample compound is present in high concentration. When compounds of interest are present at low levels, or multiple compounds of widely differing polarities need to be isolated, the second strategy is generally employed.

The second strategy may also be used for trace enrichment of extremely low level compounds and concentration of dilute samples. A complex matrix may be treated by both elution strategies to isolate different target analytes.

Modes of Chromatography

With either strategy, there are three different chromatographic modes to choose from. You can select :

• normal phase • reversed-phase • ion-exchange

The basic characteristics of each chromatographic mode are described below. There are many different types of sorbents for each mode and the selection of strategy, mode, sorbent, and elution solvents will depend upon the specific sample mixture and goal of the separation. This is a general guide for the steps you would use in a method.

Waters

Sep-Pak Vac

®

Load Sample Matrix

Wash out Interferences (Dispose)

Capture (retain) Compound(s)

of Interest

Elute Compound(s) of Interest with Stronger Solvent

Collect Compound(s) of Interest as they Elute

from Cartridge

Waters

Sep-Pak Vac

®

Normal Phase Chromatography (polar

sorbents) with Sep-Pak

®

Cartridges

To perform normal phase chromatography with Sep-Pak®cartridges, use a gradient of nonpolar solvents with polar Silica, Florisil®, NH2, Diol, CN, Alumina A, B, or N as a sorbent*.

1. You may condition the cartridge with six to ten hold-up volumes of nonpolar solvent, usually the sample solvent. 2. Load the sample into the cartridge.

3. Wash unwanted compounds with a nonpolar solvent. 4. Elute the first compound of interest with a polar solvent. 5. Elute remaining compounds of interest with progressively

more polar solvents.

6. When you recover all of your compounds, discard the used cartridge in an appropriate manner.

* Depending upon your chromatographic conditions, you may also use CN as a packing material for normal phase chromatography.

Generic Elution Protocol for Normal Phase Chromatography on Sep-Pak®Cartridges (Silica, Alumina, Florisil, Diol, NH2)

2

Load Sample Using progressively more polar solvents to elute first and second

compounds of interest You may condition cartridge with nonpolar solvent Elute unwanted compounds (wash) Compound 1 Compound 2 Discard the used cartridge

(4)

Generic Elution Protocol for Reversed Phase Chromatography on Sep-Pak®Cartridges (C18, tC18, C8, tC2, Diol, NH2, CN)

For Oasis®HLB recommended protocols are provided to simplify methods development even more, see page 20.

Load Sample Using progressively stronger solvents to elute first and second

compounds of interest

Solvate the bonded phase with methanol, then flush with water and buffer

Elute unwanted compounds (wash) Compound 1 Compound 2 Discard the used cartridge

Generic Elution Protocol for Ion-Exchange Chromatography on Sep-Pak®Cartridges (NH2, Accell Plus QMA, Accell Plus CM)

For Oasis®MCX and Oasis®MAX sorbents ion-exchange chromatography recommended protocols are provided to simplify methods development. See pages 24 and 26.

2

Load Sample Using progressively stronger buffers (changing pH or ionic strength, or both) to elute first and second

compounds of interest

Condition cartridge with

water or

buffer Elute unwanted

compound(s) with more water or buffer

Compound 1 Compound 2 Discard the used cartridge

SAMPLE

PREP

ARA

TION

Reversed-Phase Chromatography (non-polar sorbents)

with Oasis

®

HLB and Sep-Pak

®

Cartridges

To perform reversed-phased chromatography with Sep-Pak® cartridges, use a gradient of strongly to weakly polar solvents with nonpolar C18, tC18, C8, tC2, Diol, NH2or CN as a sorbent. For improved performance, use the nonpolar, polymeric Oasis®HLB sorbent. Oasis®products provide simple, recommended methods to save time. In addition, they provide two significant benefits: increased sorbent capacity for smaller cartridge sizes, yielding higher sensitivity methods, as well as the ability to remain wetted, therefore avoiding the drying out effect of traditional sorbents that causes poor recoveries (see page 18).

1. Condition (wet/solvate) the sorbent with six to ten cartridge hold-up volumes of methanol or acetonitrile.

Equilibrate the cartridge with six to ten hold-up volumes of water or buffer. (For silica based sorbents -- Do not allow the cartridge to dry out (dewet). This is not a problem with Oasis®HLB devices). 2. Load the sample dissolved in strongly polar solvent. 3. Wash unwanted compounds with a strongly polar solvent. 4. Elute weakly held compounds of interest with a less polar solvent. 5. Elute more tightly bound compounds with progressively

more nonpolar solvents.

6. When you recover all of your compounds, discard the used cartridge in an appropriate manner.

Ion-Exchange Chromatography (charged sorbent

particle surface) Sep-Pak

®

Cartridges

To perform ion-exchange chromatography with Sep-Pak®cartridges, use a step gradient of pH or ionic strength with Accell™ Plus CM, Accell Plus QMA or NH2as a sorbent.

1. Condition the cartridge with six to ten hold-up volumes of deionized water or weak buffer.

2. Load the sample dissolved in a solution of deionized water or buffer.

3. Elute unwanted weakly bound compounds with a weak buffer. 4. Elute the first compound of interest with a stronger buffer

(change the pH or ionic strength).

5. Elute other compounds of interest with progressively stronger buffers.

6. When you recover all of your compounds, discard the used cartridge in an appropriate manner.

Ion-Exchange Chromatography (charged sorbent particle

surface) with Oasis

®

MCX ( Mixed Cation eXchange) or

MAX (Mixed Anion eXchange) sorbents.

These products provide a higher level of performance and come with simple, recommended methods to save time (see pages 24 and 26).

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Unique Water-Wettable Oasis®HLB Copolymer

Oasis

®

SPE Products

A Breakthrough in SPE

Introduction

In October 1977, Waters designed the first miniature cartridge columns (Sep-Pak®cartridges) containing silica-based adsorbents for SPE. New demands for sample preparation led to the development of a new, specially designed polymeric sorbent which performs optimally for reversed-phase SPE. The Oasis®HLB copolymer with unique Hydrophilic-Lipophilic Balance is unlike traditional SPE sorbents.

Today's goals for modern solid-phase extraction (SPE) are faster throughput, higher recovery and reproducibility, stronger retention and selectivity. Now SPE can outpace high throughput techniques such as LC/MS/MS.

The Oasis® HLB sorbent is a macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone. It provides reversed-phase capability with a special “polar-hook” for enhanced capture of polar analytes and excellent wettability.

High and Consistent Recoveries

Oasis® sorbents are water-wettable maintaining high retention and capacity for a wide spectrum of analytes, especially when the SPE column runs dry. When the sorbent pores dry-out, the chromatographic retention (capture) of the analytes is reduced, resulting in poor recovery. Traditional, silica based C18sorbents can easily dry-out, especially on a vacuum manifold if a particular cartridge flows quickly and allows air to be drawn in. Oasis®sorbents maintain proper wetting for more consistent performance (especially important for 96 well plate devices). Even if air passes through, the Oasis®pores do not dry out.

Optimal Properties for Reversed-Phase SPE Specific Surface Area: 810 m2/g

Average Pore Diameter: 80 Å Total Pore Volume: 1.3 cm3/g

Average Particle Diameter: 5 µm, 15 µm, 25 µm, 30 µm or 60 µm* * Depending on configuration

Hydrophilic – Lipophilic Balance

N O

N-Vinylpyrrolidone Divinylbenzene

N O N O

Pore Dewetting Mechanism of Sorbent Pores (Silica Based C18)

Analytes properly retained in pore

Properly Wetted Pore Dewetted Pore

If air passes through traditional C18cartridge the pores dry out.

When sample is loaded, the analytes are not retained resulting in poor recoveries.

Analytes not retained

Current Oasis®Patents:

Patent No. 5,882,521, Patent No. 6,254,780, Patent No. 5,976,376, Patent No. 6,322,695, Patent No. 6,106,721, Patent No. 6,468,422, Additional Patents Pending

(6)

Start with Oasis®HLB Recommended 1-D Method

Start with Oasis®MCX Recommended Method

Start with Oasis®MAX Recommended Method

Higher Retention Means Greater Capacity, No Breakthrough

Oasis®HLB 1 cc/30 mg and 1 cc/100 mg C

18cartridges were conditioned on a Waters vacuum

manifold. When the methanol reached the top of the upper frit in each cartridge, vacuum was maintained for different times to vary the cartridge drying time. The SPE protocol was then continued. The data shown are the average of three replicate extractions.

0 20 40 60 80 100 0 4 8 0 20 40 60 80 100 0 5 10 Oasis®HLB Cartridge (30 mg) (Excellent Recoveries) C18Cartridge (100 mg) (Variable Recoveries) Procainamide Acetaminophen Ranitidine Propranolol Doxepin

Drying Time (minutes) Drying Time (minutes)

% Recover

y

Effect of Drying on Recovery - Oasis®HLB Versus C

18Sorbents. No Stopcocks Required With Oasis®Since Air Does Not Dry

Out The Sorbent!

The variable recoveries seen with the C18sorbents, due to the drying out effect, are often the cause for “retests”, reducing laboratory productivity. In some laboratories 10% of samples are retests—this can be reduced using Oasis®sorbents.

Also, Oasis®sorbents retain polar compounds far better than bonded silica SPE sorbents. Note the poor recovery of the polar analyte Acetaminophen for C18. Oasis®sorbents work especially well when you need to capture metabolites (see figure above).

High Capacity — Use Less Sorbent

When transferring methods from a C18bonded phase to Oasis®products, keep in mind the greater capacity of the Oasis® sorbent. The Oasis® sorbent has 2-3x more surface area and shows a dramatic increase in k values compared to silica-based C18, This reduces breakthrough potential. In addition, you may be able to use 23less sorbent than you would with

C18(30 mg Oasis®HLB gives equivalent capacity to 100 mg C18).

Exceptional Batch-to-Batch Reproducibility

Because of poor stability at pH extremes and relatively low ionic capacity traditional silica based mixed-mode sorbents don’t have long-term batch-to-batch reproducibility and therefore require reservations of specific lots of sorbent for large projects. Oasis® sorbents have demonstrated excellent long-term batch-to-batch reproducibility for over 6 years. As a result of careful process design and stringent quality controls, a new standard has been set in batch-to-batch and lot-to-lot reproducibility for SPE sorbents. The Oasis®family of sorbents and devices are manufactured in a Waters ISO 9002 registered facility in compliance with cGMP guidelines of the U.S. Food and Drug Administration for class 1 medical devices.

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

Overall Guideline for Sorbent Selection

There are three available Oasis® sorbent chemistries which are designed to solve just about all of your Methods Development problems. They are all built upon this unique co-polymer and provide exceptional results. The sulfonated and Quaternary amine derivatives Oasis®MCX and MAX are designed for selective retention of basic, and acidic compounds respectively.

Oasis®HLB (Hydrophilic-Lipophilic Balance) reversed-phase sorbent.

Oasis®MCX (Mixed-mode: Cation-eXchange and reversed-phase) sorbent. Oasis®MAX (Mixed-mode: Anion-eXchange and reversed-phase) sorbent.

A fast approach to successful methods development with Oasis®sorbents is as follows;

First, choose the best sorbent for your analyte(s), as diagramed below;

Data shown were obtained with two 3.9 mm x 150 mm columns, each packed with one of the sorbents, operated under the same conditions: mobile phase: 20 mM potassium phosphate, pH 7.0/methanol (95/5 v/v); temperature: 30 °C; flow rate: 1.0 mL/min; detection: UV @ 254 n

0 50 100 HO O H O OH N H3C O H HO HO N N N N O H CH3 CH3 O 3x 2x 6x 15x

C18Cartridge Oasis®HLB Cartridge

Retention Factor (k) Comparison

Salicylic acid

Acetaminophen Theobromine

Catechol

If a mix of Acids, Bases and Neutrals, or Unknowns

Reversed-Phase

If all are Bases

Cation Exchange

If all are Acids

Anion Exchange

See page 20 See page 24 See page 26

Determine Types of Analyte(s) in Sample Matrix (Acids, Bases, Neutrals)

SAMPLE

PREP

ARA

TION

Start with the Recommended Method for that sorbent. Batch-to-Batch Reproducibility of Oasis®HLB Sorbent

Recover

y (%)

Batch Number

No Batch Reservations Needed

65 75 85 95 105

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Recommended Generic Oasis®HLB SPE Method (1-D)*

Applying one or more of the following steps before loading your sample may improve

your results:

• Dilute sample 1:1 with buffer to improve flow during loading

**Add 20 µL (H2PO4) to disrupt

drug-protein interaction/ binding • Dilute 1:1 or greater with

0.1 N HCl or other acids • Filter through 0.45 µm membrane

• Centrifuge @ ≥3000 rpm

Sample Pretreatment Suggestion

1: Load (sample in acid— disrupt binding)** 2: Wash 5% MeOH 3: Elute 100% MeOH Condition 1 mL methanol Equilibrate 1 mL water Load 1 mL spiked sample** Wash (Mild Wash) 1 mL 5% methanol in water

Elute (Strong Elution) 1 mL methanol

* Volumes are given for the Oasis®

HLB cartridge 1 cc/30 mg

Evaporate & Reconstitute 40 ˚C/under nitrogen stream

200 µL mobile phase

One Simple Procedure: Many Applications on a Universal Sorbent—Reduced Methods Development Time

20 40 60 80 100 0 1-D Method

Seventeen drugs with a wide range of polarities were spiked in porcine serum, then extracted with 1 cc/30 mg Oasis®HLB cartridges, using the same

SPE method

(20 µL/mL H2PO4was used to disrupt drug-protein binding). % Recovery Acids Neutrals Bases n=6 RSDs < 3.5% IbuprofenNaproxen

Salicylic AcidSulfadiazineSulfamerazineAcetaminophen TheobromineParazanthineTheophylline Caffeine

ProcainamideRanitidineOxycodonePropranololNaltrexoneSalbutamol Doxepin

With silica-based sorbents (C18) you would need

several different chemistries to achieve the results

shown here

Oasis®HLB sorbent makes it fast and easy to develop sample preparation methods that deliver high, reproducible recoveries especially suited to LC/MS/MS analysis by providing the required selectivity and sensitivity.

A Simple and Fast Generic SPE Reversed-Phase Protocol

for Rapid Method Development for a Wide Range of

Compounds (1-D)

Limitations of traditional silica-based sorbents make you evaluate several different bonded phases and brands to obtain acceptable results. With Oasis® HLB, acidic, basic, and neutral compounds, whether polar or nonpolar, can be isolated reproducibly (RSDs <5%) with high recovery (>85%), using the same simple SPE protocol (see below).

This generic, 1-D method (1-Dimensional – only the organic strength is changed) has proven useful for a wide variety of compound types may be the only protocol required, reducing method development time.

Oasis

®

HLB Plates, Cartridges and Columns

5% 100% 1 3 2 pH High Low

Low Organic High Concentration

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Sorbent Amount and Solvent Selection

for the Generic Method

The suggested amount of sorbent in a cartridge or a plate required for your application, is given in the table below. Remember, because of the increased capacity of the Oasis®Sorbents, you can use less sorbent than you would normally need if you used a silica based packing. The solvent used for the elution step should be selected based on the polarity of the analyte. The second table below gives a selection of elution solvents and each solvent gives you different selectivity and elution strength.

The generic method (1-D) is an excellent starting protocol for methods development. If you were not able to meet all of your SPE goals, then the advanced 2-D protocol will provide a rapid way to chromatographically determine an even cleaner, more selective and sensitive result.

Sorbent Maximum Typical Elution

per device Mass Capacity Sample Volumes Volume

µElution Plate* 60 to 400 µg 10 to 375 µL 25 µL**

5 mg* 0.15 to 1 mg 10 to 100 µL ≤150 µL

10 mg 0.35 to 2 mg 50 to 200 µL 250 µL

30 mg 1 to 5 mg 100 µL to 1mL ≥400 µL

60 mg 2 to 10 mg 200 µL to 2 mL 800 µL

* Available only in 96-well plates ** µElution Plate requires no evaporation step

When converting from C18silica-based to Oasis®SPE sorbents, use approximately 2⁄3 less Oasis®

sorbent (100 mg C18sorbent = 30 mg Oasis®sorbent).

Capacity and Elution Volume of Oasis®

96-Well Plates and Cartridges

Solvent Solvent Type Relative Comments

Elution Strength**

Methanol proton donor 1.0 disrupts H-bonding Acetonitrile dipole-dipole 3.1 medium polarity drugs Tetrahydrofuran dipole-dipole 3.7 medium polarity drugs Acetone dipole-dipole 8.8 medium polarity drugs Ethyl Acetate dipole-dipole high nonpolar drugs and

GC compatible Methylene Chloride dipole-dipole high nonpolar drugs and

GC compatible * When using solvents other than methanol, add 10-30%(of proton donor solvent like methanol)

to disrupt H-bonding on the Oasis®HLB sorbent.

** High-Purity Solvent Guide. Burdick & Jackson Laboratories, Inc. Solvent Properties of Common Liquids, L.R. Snyder, J. Chromatogr., 92, 223 (1974); J. Chromatogr. Sci. 16, 223 (1978) Tips for Selecting Elution Solvents for the Generic SPE Method (1-D)* The elution solvent is selected based on polarity of analyte.

Note: Larger capacity cartridges are also available.

SAMPLE

PREP

ARA

TION

Better Results with a 2-Dimensional Method

Column: SymmetryShield™RP

8,

5 µm, 3.9 x 150 mm Temperature: 30 °C

Mobile Phase: 50 mM Phosphate pH 7: acetonitrile:methanol (41:37:22); Alliance®2690 Detection: UV at 230 nm Flow Rate: 1.0 mL/min.

Injection Volume: 40 µL (after evaporation and reconstitution in 200 µL water)

Peak Identification: Peak 1: Norverapamil Peak 2: Verapamil Peak 3: Methoxyverapamil (I.S.)

More Selective and Sensitive Reversed-phase Methods

Development Using 2-Dimensional (2-D) Strategy for

Oasis

®

HLB Sorbent

Meeting even more difficult Selectivity and Sensitivity Goals

The generic 1-D method can be expanded to meet even more challeng-ing selectivity and sensitivity goals. Uschalleng-ing a chromatographically based approach, more powerful methods can be developed, very predictably, to remove plasma, urine or any other sample matrix interferences. A comparison of the 1-D and 2-D strategies (below) shows that you can tailor the cleanliness of the baseline depending on your needs. A cleaner extract is achieved by simply manipulating the organic concen-tration and the pH. This is a 2-Dimensional approach, which now includes the powerful selectivity of pH adjustment to create a more sensitive and selective method. The impact of pH for reversed-phase applications is shown on the accompanying Retention Map. This is the basis of how this method development strategy was created (see next page).

AV AV 1 3 2 0.004 AU 1 4 2 3 5% 65% 100% pH 1 2 3 0.004 AU 10 8 6 4 2 0 1 2 5 4 5% 65% 100% pH 3 1 3 2 0.004 AU 5% 100% 1 3 2 pH Blank Minutes Sample Sample Sample High Low

Low Organic High Concentration

High

Low

Low Organic High Concentration

High

Low

Low Organic High Concentration Blank

Blank

AV

2-D Method with 2 washes 1-D Method (Generic)

2-D Method with 3 washes

1:Load 2: Wash 5% MeOH 3:Elute 100% MeOH

1: Load 2: Wash 5% MeOH 3:Wash High pH 65% MeOH 4: Elute Low pH 65% MeOH

1: Load 2: Wash Low pH 5% MeOH 3: Wash High pH 5% MeOH

(9)

This is called a “Retention Map” for Reversed-phase chromatography. We have plotted the retention (k) of a neutral, acidic and basic compound as a function of the pH. For a basic compound, it indicates that when a basic compound is ionized at low pH (2 units below its pKa), it is poorly retained by SPE. At high pH (2 units above its pKa), it is un-ionized, and strongly retained by reversed-phase SPE, with a high kvalue. This means that if we want to capture a base on our reversed-phase chromatographic sorbent, we need to LOAD and Wash at High pH, and then we can ELUTE the base in a cleaner extract at Low pH.

In general, when the ionizable compound is in its unionized state you will obtain a high k, excellent capture. In its ionized state, there is very little k, therefore it will be easy to elute from the sorbent.

Acid (Unionized) Acid (Ionized) Base (Unionized) Base (Ionized) Neutral 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Retention (k)

Base Neutral Acid

Mobile Phase pH

Recommended pH range of Oasis® Recommended pH range of Silica

Retention Factor Versus pH for Acids, Bases and Neutrals— Retention Map, Reversed-Phase, Oasis®HLB

An acidic compound behaves the same way relative to its’ ionization. Note that the retention of a neutral compound is not changed by adjusting the pH, it is affected by the organic concentration only.

pH 12 Un-ionized (Captured) pH 2 N A-B BH+ Ionized

Impact of pH

Load and Wash at Elute at Low pH

High pH to capture Base, to release Base

release acids, neutrals

pH 2 pH 12 N BH+ HA A-Un-ionized (Captured) Ionized

Load and Wash at Elute at

Low pH to capture Acid, High pH

release bases, neutrals to release Acid

Using this chromatographically based approach, a Wash and Elution Study is performed to determine the optimal SPE method using organic concentration and pH. (Always test your analytes for stability and solubil-ity at different pH values.)

Investing approximately 4 hours of development time, using this strategy, yields extremely powerful, and predictable results. Overall methods devel-opment time will decrease, with the added benefit of very clean, selective extracts. (This approach has solved many methods development problems over the last several years. Contact your Waters Technical Support Team for more information.)

The 2-D experiment (following page) demonstrates this approach. Twenty samples of the analytes in a saline solution (PBS) are loaded onto twenty conditioned and equilibrated cartridges or wells of a 96-well plate. Each cartridge is eluted with a different bottle of solvent (10 bottles contain Low pH solutions-each with different concentrations of methanol, and 10 High pH solutions). The eluate is then analyzed and the area count for each analyte is plotted relative to its’ elution bottle of solvent on two area count plots (Low pH and High pH). The area counts will be zero if the analyte is still captured on the cartridge.

Reviewing the two Area Count Plots, note that the analytes begin to elute with lower organic strength (~35%) at Low pH. At High pH it takes over 70% organic. (Based on the Retention Map for Reversed-Phase, this indicates that these analytes were bases, since they were unionized at High pH, had a higher k, and required more organic for elution.)

From these plots, we can design a 2-D method which will greatly reduce matrix interferences in two ways relative to the generic 1-D method; first, an additional stronger, more aggressive Wash step to get rid of more interferences; and second, a weaker, less aggressive Elution step that will release our analytes, but leave other interferences captured in the cartridge. Since this is determined chromatographically, it is a very predictable approach.

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SAMPLE

PREP

ARA

TION

2-D Wash Elute Study: Experiment for Oasis®HLB 30 mg 96-well Plate or Cartridge

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

0% 0%10% 20% 30% 40% 50% 60% 70% 80% 90%

2-D Wash Elute Study Results (Area Counts) Base Modified Wash High pH Acid Modified Wash Low pH

Prepare analytes in saline

Condition/equilibrate 20 cartridges 1 mL methanol/1 mL water

Load 1 mL spiked saline solution

▼ ▼

Elute with 1 mL

2% NH4OH in MeOH/water 2% CH3COOH in MeOH/water

150000 100000 50000 0 0 20 40 60 80 100 200000 150000 100000 50000 0 0 20 40 60 80 100 Maximum % Methanol Wash % MeOH Minimum % Methanol Elution Area (µV*sec) % MeOH % MeOH Area (µV*sec) Peak Area (µVs) Base 2% Ammonium

Methoxy-Hydroxide Verapamil Norverapamil verapamil 0-70% No Response No Response No Response

80% 34,095 47,482 6,358

90% 63,158 85,704 178,178

Peak Area (µVs) Acid 2%

Acetic Acid

Methoxy-Verapamil Norverapamil verapamil 0-30% No Response No Response No Response

40% 73,146 32,269 86,789 50% 121,820 68,259 137,899 60% 109,145 61,830 123,229 Condition 1 mL methanol Equilibrate 1 mL water Load 1 mL spiked sample Wash 1 1 mL 5% MeOH in water

with 2% acetic acid

Wash 2 1 mL 5% MeOH in water with 2% ammonium hydroxide

Wash 3 1 mL 65% MeOH in water with 2% ammonium hydroxide

Elute 1 mL 65% MeOH in water

with 2% acetic acid Evaporate & Reconstitute 40 ˚C/under nitrogen stream

200 µL water/mobile phase

Removes neutral polar and basic polar interferences

Removes neutral non-polar and basic lipophilic interferences

but not analytes Elute the basic analyte compounds in ionized

form Removes acidic polar interferences 2-D SPE 3-Wash Method Developed from

Wash Elute Study—Oasis®HLB

To obtain complete elution, the elution solvent should be acidic (2% acetic acid) and the methanol concentration should be greater than 60%, but less than 70%. Remember, the elution solvent should be able to fully elute the analytes, but not be too strong and contain too much organic to release undesirable neutral interferences from the sorbent. For this experiment, low pH with no more than 70% methanol should be used. Notice, we did not choose high pH for an elution condition, since this made the analytes retain more, and thus would require almost 100% methanol to elute them. This high percentage of an organic concentration would also elute undesirable interferences along with the analytes.

The cleanest extract was achieved for verapamil and its metabolite using the 2-D, 3-wash procedure instead of the 1-D generic method. The more selective method enables higher sensitivity for analyses at ng/mL levels.

Important Note: Traditional, Silica based C18 sorbents should not be exposed to High pH because the silica particle substrate dissolves, which can contaminate your extracts with silicates. Oasis® HLB works well because it is a copolymer, which doesn’t dissolve at high pH.

From these results, the wash solvent(s) can be either acidic, up to 30% methanol containing 2% acetic acid, or basic, up to ~70% methanol containing 2% ammonium hydroxide, since no analyte is eluted under these conditions (your choice depending on the type of sample matrix interferences present – in some cases, you can add multiple wash steps for even more sensitive methods). If we just wanted one more wash, it should be aggressive as possible, therefore we should chose high pH

and 65% methanol. 1 2 5 4 5% 65% 100% pH 3 High Low

Low Organic High Concentration

1: Load 2: Wash Low pH 5% MeOH 3: Wash High pH 5% MeOH

(11)

High Selectivity and Sensitivity for Basic Compounds— Oasis®MCX Sorbent for Cation Exchange

Obtain selective retention of basic drugs with cation-exchange groups on the sorbent surface. The Oasis®MCX sorbent has a tightly controlled ion-exchange capacity (1 meq/gram). There are no silanol groups to compli-cate the retention mode or method development. This novel, water-wettable, polymeric sorbent is stable from pH 0 to 14, making method development simple and fast.

Oasis

®

MCX Plates, Cartridges and Columns

Drug–Sorbent Interactions on Oasis®MCX Sorbent

SO–3 SO–3

N O

MCX sorbent Mixed Mode Cation eXchanger

Reversed-phase interaction

Strong cation-exchange

mode

best retention at least 2 pH units below pKa

+ CH 3 CH 3 O OH N H H

Propranolol Basic Drug

Sulfonic Acid Cation Exchange Capacity: 1.0 meq/g

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

Since this ion exchange sorbent is synthesized from the reversed-phase Oasis® HLB copolymer, it features two retentions mechanisms (cation exchange and reversed-phase) which can be manipulated very predictably (please refer to the Oasis®MCX Retention Map).

This Retention Map plots the total k or capacity (retention) of a basic analyte relative to pH. Note that the total k, is the sum of the two retention mecha-nisms. At low pH, the analyte is charged, and experiences maximum reten-tion primarily from the ion-exchange mechanism, however, there is also a slight amount of reversed-phase contribution for the combined retention. If your goal is to capture basic analytes and then wash out interferences aggressively, the Load and Wash steps should be at low pH to obtain maximum capture.

At high pH, the ion exchange retention mechanism shuts-off because the analyte becomes un-ionized. Only reversed-phase retention is present, but since the analyte is now un-ionized, we get the maximum of the reversed-phase retention. We can elute with a combination of high pH and high organic concentration.

Oasis®MCX Retention Map for Basic Analytes

Impact on Retention Factor (k) for a Base by Changing Mobile Phase pH in Cation Exchange and Reversed-Phase Mode,

Base (Ionized) Base (Unionized) Cation Exchange Reversed Phase 0 10 20 30 Retention (k) 40 50 60 1 Reversed-Phase Retention Cation-Exchange Retention Basic Analyte (Combined) Retention 2 3 4 5 6 7 Mobile Phase pH 8 9 10 11 12 13 14 Load

3 mL spiked and acidified urine Condition 1 mL MeOH Equilibrate 1 mL H2O Wash 1 2 mL 0.1N HCI Wash 2 2 mL methanol Elute Bases 2 mL 5% ammonium hydroxide in methanol Evaporate and Reconstitute 300 µL 20% methanol in water Oasis®MCX 3 cc/60 mg cartridge

Removes polar and acidic interferences

Removes neutrals Generic Oasis®MCX Method for Extraction of Basic Compounds

(12)

SAMPLE

PREP

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TION

0 200 400 600 800 1000 0 25 50 75 100 85 15 0 20 40 60 80 100 0.7% 0.5% 2.4% 3.2% 3.2% 13% 12% 1.1% 0.3% Methadone Oasis®MCX 60 mg/3 cc 300 mg/Brand A 3 cc Brand B 300 mg/ 3 cc Methadone metabolite (EDDP) Propranolol % Recover y (% RSD) % Recover y Procainamide (3˚) Toluidine (Aromatic) Doxepin (3˚) Amitriptyline (3˚) Nortriptyline (2˚) N,N-dimethylbenzylamine (3˚) N-methylbenzylamine (2˚) Benzylamine (1˚) Amphetamine (1˚) Methamphetamine (2˚)

Elution Volume (µL of 5% TEA in Methanol)

Elute 1 Tertiary and aromatic

amines with 5% TEA Elute 2 Primary and secondary amines with 5% NH4OH in methanol

Comparison of Oasis®MCX Cartridges to other SPE Silica-Based

Products Using the Generic Oasis®MCX Method

Selectively Separate Primary and Secondary Amines from Tertiary and Aromatic Amines Using an Oasis®MCX Cartridge

As shown, one protocol with minimum wash steps gives extractions fast enough to keep pace with your analytical system.

In some cases, with urine samples, no conditioning or equilibration of cartridges is required to achieve excellent results for basic drugs.

The use of the 2-D approach can be very successful for the Oasis®MCX sorbent as well. A pH modified wash system allows optimization of the method and it produces a very selective protocol for basic compounds.

We can also fractionate the tertiary and aromatic amines in an Elute 1 step using 5% TEA (triethyl amine) in MeOH, with very little release of the primary and secondary amines. To elute the primary and secondary amines ammonium hydroxide is used.

Oasis®MAX plates, cartridges and columns High Selectivity and

Sensitivity for Acidic Compounds using Anion Exchange

The Oasis®MAX sorbent has a tightly controlled ion exchange capacity of 0.3 meq/gram ensuring reproducible SPE protocols for extraction of acidic compounds and metabolites from biological fluids. There are no silanol groups to complicate the retention mode or method development. This novel, water-wettable, polymeric sorbent is stable from pH 0 to 14, making method development simple and fast.

Since this ion exchange sorbent is synthesized from the reversed-phase Oasis® HLB copolymer, it features two retentions mechanisms (anion exchange and reversed-phase) which can be manipulated very predictably (please refer to the Oasis®MAX Retention Map, page 26).

Oasis

®

MAX Plates,

Cartridges and Columns

Drug–Sorbent Interactions on Oasis®MAX Sorbent

R+ CH 2 CH3 CH3 C H4 9 N N O MAX sorbent Mixed Mode Anion eXchanger

Reversed-phase Retention

Suprofen Acidic Drug

Quaternary Amine Anion Exchange Capacity:

0.3 meq/g S CH 3 O COO– Strong anion-exchange mode Best retention at least 2 pH units above pKa

(13)

Oasis®MAX Retention Map for Acidic Analytes

Impact on Retention Factor (k) for an Acid by Changing Mobile Phase pH in Anion-Exchange and Reversed Phase

0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Acid (Ionized) Acid (Unionized) Anion Exchange Reversed Phase Retention (k) Reversed-Phase Retention Anion-Exchange Retention Acidic Analyte (Combined) Retention Mobile Phase pH

The Retention Map plots the total kor capacity (retention) of a acidic analyte relative to pH. Note that the total k, is the sum of the two retention mecha-nisms. At high pH, the analyte is charged, and experiences maximum reten-tion primarily from the ion-exchange mechanism, however, there is also a slight amount of reversed-phase contribution for the combined retention. If your goal is to capture acidic analytes and then wash out interferences aggressively, the Load and Wash steps should be at high pH to obtain maximum capture.

At low pH, the ion exchange retention mechanism shuts-off because the analyte becomes un-ionized. Only reversed-phase retention is present, but

As shown below, acidic compounds can be separated from basic and neutral compounds on the same Oasis®MAX cartridge following the recom-mended protocol.

Generic Oasis®MAX Method for Extraction of Acidic Drugs

Condition 1 mL methanol Equilibrate 1 mL water Load* 3 mL sample Wash 2 mL 50 mM sodium acetate pH 7/5% methanol Wash 2 (Elute 1) 2 mL methanol Elute 2 2 mL 100 mM phosphoric acid/acetonitrile 1:1 (v/v)

Alternative LC/MS Elution System: 2% formic acid in methanol

Removes/elutes basic and neutral interferences

1 0 2 4 6 8 10 0 2 4 6 8 10 2 I.S. 3 4 I.S. Results (n=6)

Drugs µg/mL Type of Drug % Recovery % RSD

Nortriptyline 5 Basic (Elute 1) 94.34 0.78 Secobarbital 10 Neutral (Elute 1) 88.53 1.03 Ketoprofen 5 Acidic (Elute 2) 91.64 2.35 Naproxen 2.5 Acidic (Elute 2) 103.43 3.04 Minutes Sample Blank with I.S. Sample Blank with I.S. AV AV Minutes

Elute 1 (basic and neutral drugs) Elute 2 (acidic drugs)

* 1 mL urine hydrolyzed (1M KOH 60 °C, 15 minutes) and adjusted to pH 2 with phosphoric acid; diluted 1:1 with 10 mM sodium acetate, pH 2. Column: SymmetryShield™3.5 µm, 4.6 mm x 100 mm

SymmetryShield™Guard column, 5 µm, 3.9 mm x 20 mm

Mobile Phase: 20 mM potassium phosphate, pH 2.7/methanol, 52:48 v/v Detector: UV @ 214 nm Temperature: 30 °C Flow Rate: 2 mL/min

Injection Volume: 10 µL of extract with internal standard

Peak Identification 1. Nortriptyline 2. Secobarbital 3. Ketoprofen 4. Naproxen I.S. Butylparaben

High Recovery of Acidic, Basic and Neutral Compounds Using the Generic Oasis®MAX Method

since the analyte is now un-ionized, we get the maximum of the reversed-phase retention. We can elute with a combination of low pH and high organic concentration.

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SAMPLE

PREP

ARA

TION

Oasis

®

SPE Products

Oasis®products come in a full range of device formats to meet your SPE requirements—the new µElution plates, on-line columns, 96-well plates, and single-use cartridges.

Try Oasis®and successfully meet your SPE challenges.

Request your free Oasis®Applications Notebook (Literature Code 720000609EN) at www.waters.com/oasis or contact your local sales office.

Automation of Oasis

®

Sample Extraction Cartridges and 96-Well Plates

Oasis®SPE products are compatible with the following liquid handling and/or SPE automation systems • Packard MultiProbe II®Robotic liquid

handling system

• Tomtec Quadra96®and Quadra96® SV Pipetting Station

• Hamilton Microlab®SPE Workstation • Beckman Biomek®2000 Laboratory

Automation Workstation • Zymark RapidTrace®Automated

SPE Workstation • Gilson ASPEC XL4 • Gilson ASPEC XL

• Gilson 215 SPE Liquid Handler • Tecan Genesis

• Prospekt™/ProspektII

Waters LC/MS/MS Systems for Bioanalysis Featuring the

Waters

®

Quattro Premier

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. Oasis® Sample Extraction Products can be used with Waters LC/MS/MS Systems including this system that integrates the Waters® Quattro PremierMass Spectrometer with the Waters®1525µ Binary Pump and the 2777 Sample Manager using MassLynx™4.0 Software.

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Excellent Recovery in 25 µL Elution

OASIS®µElution Technology >85% Recovery in 25 µL % Recover y 0 20 40 60 80 100 25 µL 100 µL SPIKED SALINE acetaminophen practolol N-acetyl procainamide betamethsone caffeine naproxen amitripyline propranolol

Oasis® HLB µElution plate Waters 96-Well Plate Design The Oasis®sorbents come in several different mass amounts, in a 96-well

plate configuration that can be used on many manifolds and automated instruments. Because of the unique sorbent properties (no drying-out effect), each well gives high and reproducible recoveries for a wide range of analytes including polar and basic drugs, with RSDs ≤5% (n=96). Waters offers the standard high-throughput 30 mg and 60 mg Oasis® 96-well plates as well as 5 mg and 10 mg 96-well low elution plates.

5 mg 10 mg 30 mg 60 mg

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.

1999 R&D 100 Award Two-Stage Well Design

High Throughput 96-Well Extraction Plates

Oasis

®

µElution Plates for Ultra Low Elution Volumes

Elute in as little as 25 µL with no evaporation/reconstitution Ideal for small sample volumes

Up to a 25x increase in sensitivity

Waters Newest Innovation in SPE Technology

The new Oasis®µElution plate combines patented* plate design, proven Oasis®chemistries, and recommended protocols enabling elution volumes as low as 25 µL. Now for the first time you can perform SPE clean-up and concentration of very small sample volumes.

Achieve superior results compared to protein precipitation in the same or less time using the Oasis® µElution Plate. This plate produces extracts that can be directly injected, eliminating the time-consuming evaporation step. Just elute and shoot. Eluting in 25 µL without evaporation provides up to 25x increase in sample concentration, enabling sensitive, robust, and repro-ducible SPE results.

The innovative features of the new Oasis®µElution plates enable sensitive, robust, reproducible results

without evaporation and reconstitution.

Scientists in both drug discovery and drug development can prepare biological samples for LC/MS/MS analysis using a more efficient, generic methodology and can successfully eliminate the time consuming evapora-tion step with the new Oasis®µElution plate.

(16)

Speed and Throughput

Comparison of Oasis®µElution Plate and Protein Precipitation —

Superior Results, Less Time and Effort

The Oasis®µElution plate optimizes the configuration of the highly efficient Oasis®sorbents (HLB, MCX, MAX), enabling elution volumes as low as 25 µL, providing fast clean up with improved performance over protein precipitation. Time consuming evaporation and reconstitution steps are eliminated, compressing preparation cycle time and increasing through-put capabilities.

Extraction Protocols

The protein precipitation generic method includes both a centrifugation and an evaporation step, which produces the cleanest sample extract possible for protein precipitation. The final sample volume (75 µL) is the same for all three generic methods. The Oasis® generic methods produce cleaner, extracts than protein precipitation, demonstrated by the improved sensi-tivity with the generic Oasis®HLB (4x) and the generic Oasis®MCX (9x to 25x) methods. The Oasis® methods enable improved sensitivity by reducing ion suppression. Achieve superior results compared to protein precipitation in less time using the Oasis®µElution Plate.

Oasis®HLB and MCX µElution Plate versus Protein Precipitation

0.50 1.00 1.50 2.00 2.50 3.00 3.79 3.83 3.83 3.50 4.00 4.50 5.00 2 100 % 7 100 % 16 100 % Propranolol, 1 ng/mL Amitriptyline, 0.1 ng/mL Time (mins) 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 4.75 5.18 4.67 4.48 5.00 5.50 6.00 2 100 % 4 100 % 17 100 % Time (mins) Protein Precipitation Oasis®MCX Oasis®HLB Intensity: 7.50 x103 Intensity: 3.03 x104 Intensity: 1.90 x105 Protein Precipitation Oasis®MCX Oasis®HLB Intensity: 9.71 x103 Intensity: 3.62 x104 Intensity: 8.91 x104 4x sensitivity without evapora-tion 4x sensitivity without evapora-tion 25x sensitivity without evapora-tion 9x sensitivity without evapora-tion LC Conditions Column: XTerra®MS C 18, 2.1 x 30 mm, 3.5 µm Mobile phase A: Water + 0.5 % NH4OH

Mobile phase B: ACN + 0.5 % NH4OH

Flow rate: 0.2 mL/min LC conditions: 5% - 95% B 1 min. Temperature: room temp. LC Instrument: Waters Alliance®2795

MS Conditions

MS Instrument: Micromass Quattro Triple Quadrupole Ion source: ESI (+) Source temperature: 150 °C Gas cell: 2.0 e-3 bar Argon Desolvation temperature: 350 °C Drying gas flow: 500 L/hr Cone gas flow: 50 L/hr

Cone voltage: 25 volts Collision energy: 20 Capillary voltage: 3.5 Kv

MRM transition: Metoclopramide (IS) m/z 299.8 →226.7 Propranolol m/z 259.9 → 154.9 Amitriptyline m/z 278.1 → 232.9 Nortriptyline m/z 263.9 → 190.8

Oasis®HLB Generic Method

Oasis®HLB µElution Plate**

Part Number186001828

Protein Precipitation Generic Method

Oasis®MCX Generic Method

Oasis®MCX µElution Plate**

Part Number 186001830

Increase in Selectivity (More Selective Methods) Add 50 µL spiked plasma (1 to

1000 pg/µL), 50 µL IS (10 pg/µL) in H2O

Condition with 200 µL MeOH Equilibrate with: 200 µL H2O Load 50 µL spiked rat plasma, 50 µL IS (10 pg/µL) in H2O Wash with 200 µL 5% MeOH

in water

Condition with 200 µL MeOH Equilibrate with 200 µL H2O Load 50 µL spiked rat plasma,

50 µL IS (10 pg/µL) in H2O Wash 1: 200 µL Water + 2% FA

Wash 2: 200 µL MeOH Elute with 25 µL ACN:IPA 40:60 + 2% NH4OH Dilute with 50 µL H2O

Inject 20 µL Elute with 25 µL ACN:IPA

40:60 + 2% FA Dilute with 50 µL H2O

Inject 20 µL Add 1 mL ACN to precipitate plasma

Centrifuge the plasma at 3600 rpm for 30 minutes*

Transfer the supernatant to another 2 mL 96-well container

Evaporate the solution to dryness*

Reconstitute with 25 µL ACN:IPA (40:60) + 2% NH4OH and 50 µL H2O

Inject 20 µL

** Tomtec Quadra 96®used.

SAMPLE

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Sensitivity and Precision

Comparison of Oasis®MCX µElution Plate and Glass Fiber and

Membrane 96-well Disk Plates — Up to 4x Increase in Sensitivity The Oasis®µElution plate shows superior performance when compared to other low elution disk plate products used according to recommended protocols. A generic Oasis® MCX method for terfenadine with 50 µL elution, dilution and direct injection shows better sensitivity than the

membrane and glass fiber plates, which require a 150 µL elution volume for complete analyte recovery. The Oasis® µElution plate enables sensi-tivity gains and delivers high performance without the time consuming evaporation step. 2.92 2.00 2.50 3.00 3.50 -10 100 % 2.00 2.50 3.00 3.50 -10 100 % 2.45 2.62 2.00 2.50 3.00 3.50 -10 100 % 2.92 -10 100 % -10 100 % 2.47 2.63 2.00 2.50 3.00 3.50 2.00 2.50 3.00 3.50 2.00 2.50 3.00 3.50 -10 100 % 3.02 -10 100 % -10 100 % 2.54 2.70 2.00 2.50 3.00 3.50 2.00 2.50 3.00 3.50 2.00 2.50 3.00 3.50 -10 100 % Intensity: 5.75 x106 Intensity: 7.44 x106 Intensity: 1.14 x107 Intensity: 3.03 x104 Intensity: 7.14 x104 Intensity: 3.11 x105 Intensity: 7.30 x104 Intensity: 1.15 x105 Intensity: 5.04 x105

Time (mins) Time (mins) Time (mins)

Glass Fiber SCX, 96-well Plate

250 µL plasma loaded 150 µL elution

Membrane MPC 96-well Plate

250 µL plasma loaded 150 µL elution

Oasis®MCX µElution Plate 250 µL plasma loaded

50 µL elution

Final extract diluted 1:2 with H2O

Extracted using manufacturers recommended protocol

Final extract diluted 1:2 with H2O

Extracted using manufacturers recommended protocol

Final extract diluted 1:2 with H2O

Extracted using manufacturers recommended protocol

Terfenadine 0.5 ng/mL Terfenadine-alcohol 0.5 ng/mL Terfenadine-carboxylate 0.5 ng/mL 6x Sensitivity 16x Sensitivity 18x Sensitivity

Validation Assay of Terfenadine and Its Metabolites Using The Oasis®MCX µElution Plate • Calibration range: 0.5-200 ng/mL, 7 levels

• 6 Calibration curves per day over four days • Analyte recoveries over calibration range: ≥95%

• Inter-day %RSD of back-calculated standard concentrations over four days (24 values) ≤5.6%

• Intra-day %RSD of back-calculated standard concentrations within one day (6 values) ≤8.0%

Oasis®MCX µElution Plate versus Glass Fiber and Membrane 96-well Disk Plates

S/N = 82 S/N = 61 S/N = 357 S/N = 11 S/2 = 51 S/N = 17 S/N = 40 S/N = 178 S/N = 927

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SAMPLE

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Oasis

®

On-Line Columns for LC/MS or LC/MS/MS

Generally, drugs present in biological matrices should not be directly intro-duced into the LC/MS/MS. If they are directly injected onto the system, column clogging and analyte ion suppression due to the presence of proteins, lipids, salts and other endogenous material are inevitable. Therefore a sample cleanup is a must before LC/MS/MS analysis. In addition to the off-line sample cleanup, on-line sample preparation also provides a fast and efficient way to cleanup the sample before LC/MS/MS analysis. In the Oasis® On-Line Column for LC/MS/MS application, a diluted plasma sample is injected onto the Oasis®On-Line column. The column is then purged of most protein and interferences. A fast gradient is run to elute the target analyte, a fraction of this flow is diverted to the mass spectrometer for detection (see next page).

Oasis®sorbents are water-wettable enabling high capacity and retention of a wide spectrum of analytes. This feature of the Oasis® sorbent is especially important when the Oasis®On-Line column is cycled from 100% aqueous to 100% organic mobile phase. Also the polymeric nature of Oasis®enables column use under pH extremes (pH 1-14). (Note: low and variable results are often obtained for silica-based columns under such conditions) Oasis®On-Line columns also provide exceptional batch-to-batch and column-to-column reproducibility.

Oasis

®

Patented Chemistry for Oasis

®

On-Line Columns

There are three available Oasis®sorbent chemistries which are designed to meet just about all of your on-line LC/MS/MS needs. They are all built upon unique water-wettable Oasis®HLB copolymer and provide excep-tional results. The sulfonated and Quantenary amine derivatives Oasis® MCX and MAX are designed for selective retention of basic and acidic compounds respectively:

Oasis®HLB

(Hydrophilic-Lipophilic Balance) reversed-phase sorbent. Oasis®MCX

(Mixed-mode: Cation-eXchange and reversed-phase) sorbent. Oasis®MAX

(Mixed-mode: Anion-eXchange and reversed-phase) sorbent.

Unique Water-Wettable Oasis®HLB Copolymer

Optimal Properties for Reversed-Phase SPE Specific Surface Area: 810 m2/g

Average Pore Diameter: 80 Å Total Pore Volume: 1.3 cm3/g

Average Particle Diameter: 5 µm, 15 µm, 25 µm, 30 µm or 60 µm* * Depending on configuration

Hydrophilic – Lipophilic Balance

N O N-Vinylpyrrolidone Divinylbenzene N O N O SO–3 SO–3 N O R+ CH 2 CH3 CH3 C H4 9 N N O +

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Ultra fast On-Line SPE for LC/MS/MS

Ultra fast on-line analysis can improve the speed and throughput of LC/MS analysis of small molecules from biologic sample matrices.There are three Oasis® On-Line column configurations designed to fit all your on-line analyses needs.

• The Oasis®cartridge column fits into a Sentryholder that features a finger-tighten fitting for fast and convenient replacement (1). • The direct connect column can be screwed directly into a switching

valve or connected to fittings like a conventional HPLC column (2). • The Oasis®column features traditional HPLC column fittings and

hardware (3).

All of these formats are available with the three Oasis®patented sorbents (HLB, MCX, and MAX), and in a wide choice of particle sizes as well as dimensions. The Oasis® On-Line columns make it possible to analyze a specific analyte or metabolite from a complex sample matrix (such as plasma or serum). No off-line sample preparation is needed; just dilute the sample and inject.

Ultra fast on-line analysis of multiple analytes can also be accomplished using the Oasis®On-Line column combined with Waters narrow-bore analyt-ical columns (such as Atlantis™, XTerra®, or Symmetry®columns).

Up to 360 injections were achieved on a single Oasis® HLB 2.1mm x 20 mm cartridge column without any visible deterioration of the peak shape.

Centrifugation, dilution and/or acidification of the sample prior to analysis may actually provide even longer column lifetimes.

Oasis

®

On-Line Columns

1

2

3

Ultra Fast On-Line System Configuration

Isocratic HPLC Gradient HPLC 10 Port Valve 2 positions Waste Oasis® On-line Column Narrow Bore Analytical Column (Optional) Mass Spectrometer Mass Spectrometer Alliance HT 515 System LOAD POSITION Waste Analytical Column (optional) 5 4 3 2 1 10 9 8 7 6 Oasis ® Mass Spectrometer Alliance HT 515 System INJECTION POSITION Waste Analytical Column (optional) 5 4 3 2 1 10 9 8 7 6 Oasis ®

Oasis®On-Line Column Lifetime

0.0 Minutes 1.0

Overlay of Toluamide after 360 Plasma injections. 2.1 x 20 mm

cartridge column 50 µL injection

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Recovery Comparison of Oasis®HLB vs.

Bonded Silica Prospekt™Cartridge

SAMPLE

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On-Line SPE with Oasis®Sorbent in ProspektCartridges

Waters Oasis® sorbents are available in Prospekt1 and Prospekt2 cartridges for use with the Spark Holland Prospekt™/Symbiosissystems. The narrow-bore PVDF cartridge is 1.0 mm x 10 mm contain approximately 2.5 mg of Oasis®sorbent.

The Oasis® sorbents in Prospektcartridges show the same performance advantage for pharmaceutical compounds over silica-based sorbents.

* Data were provided by Dr. Francis Beaudry, a Principal Research Scientist of Phoenix International Life Sciences Inc. in Montreal, Quebec, Canada.

Clonidine % Recovery Terconazole C2 Oasis®HLB C18

Oasis

®

Glass Cartridges for PPT Detection Levels

Endocrine Disruptors

PPT Recovery of Estrogens from River Water, LC/MS, 5 ng/L Spike Level, n=4

Recovery of Phthalates and Nonylphenol from River Water, GC/MS, 200 ng/L Spike Level, n=4

Results Ions Monitored (m/z) Recovery % RSD

1. bisphenol A 227 113 11 2. 17b-estradiol 271 93 15 3. 17a-ethynylestradiol 295 96 12 4. estrone 269 87 5 5. diethylstilbestrol 267 75 5 Results Recovery % RSD 1. dimethylphthalate 130 15 2. dirthylphthalate 86 12 3. n-nonylphenol 90 11 4. dibutylphthalate 110 11 5. benzylbutylphthalate 110 8 6. bis(ethylhexyl)phthalate 60 8 ISTD. o-terphenyl (internal standard)

Waters Oasis®glass cartridges are available in 5 cc (200 mg) configura-tion with Teflon®frits for trace analysis at parts per trillion level. Each lot is tested for the presence of bisphenol A and other phenols and phthalates, assuring that endocrine disruptors in water samples can be analyzed to part per trillion levels.

See the full application in the Oasis® Applications Notebook (Waters Literature Code #720000609EN).

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Sorbent

Box of 100 Box of 100 Box of 100 Box of 500 Box of 100 Box of 100 Box of 500 Box of 30 Oasis®HLB 30 µm 186000383 WAT094225 186001879 WAT058882 WAT094226 186001880 WAT058883 N/A

Oasis®HLB 60 µm N/A N/A N/A N/A N/A N/A N/A N/A

Oasis®MCX 30 µm N/A 186000252 186001881 186001888 186000254 186001882 N/A 186000256

Oasis®MCX 60 µm N/A 186000782 N/A N/A 186000253 N/A N/A 186000255

Oasis®MAX 30 µm N/A 186000366 186001883 N/A 186000367 186001884 N/A 186000369

Oasis®MAX 60 µm N/A N/A N/A N/A 186000368 N/A N/A 186000370

Description 5 mg/ 10 mg/ 30 mg/ 60 mg/ 96-well 96-well 96-well 96-well 1/Pkg 1/Pkg 1/Pkg 1/Pkg Oasis®HLB 30 µm 186000309 186000128 WAT058951 N/A

Oasis®HLB 60 µm N/A N/A N/A 186000679

Oasis®MCX 30 µm N/A 186000259 186000248 N/A

Oasis®MCX 60 µm N/A N/A 186000250 186000678

Oasis®MAX 30 µm N/A 186000375 186000373 186001256

Oasis®MAX 60 µm N/A N/A N/A 186001205

Oasis

®

Cartridge Selection Guide

Oasis

®

96-well Plates

3 cc/60 mg Gilson Adapter 6 cc/ 150 mg 3 cc/60 mg Flangeless 3 cc/ 60 mg 1 cc/30 mg Gilson Adapter 1 cc/30 mg Flangeless 1 cc/ 30 mg 1 cc/ 10 mg NEW NEW

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SAMPLE

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TION

Box of 30 Box of 100*/500** Box of 30 Box of 20 Box of 20 Box of 10 Box of 50 Box of 50 Box of 50 Box of 30

WAT106202 N/A N/A N/A N/A N/A N/A 186000382 186000381 N/A

N/A N/A 186000115 186000116 186000117 186000118 186000132 N/A N/A 186000683

N/A 186001216** N/A N/A N/A N/A N/A N/A 186000261 N/A

N/A N/A 186000776 N/A 186000777 186000778 N/A N/A 186000380 N/A

N/A 186001855* N/A N/A N/A N/A N/A 186000372 186000371 N/A

N/A N/A 186000865 N/A N/A N/A N/A N/A 186000378 N/A

6 cc/ 200 mg 6 cc/400 mg Flangeless 6 cc/ 500 mg 12 cc/ 500 mg 20 cc/ 1 g 35 cc/ 6 g Plus 225 mg Vac RC 30 mg Vac RC 60 mg Glass Cartridge 5 cc/200 mg

Waters Manifold for

Oasis

®

96-well Plates

NEW

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

Manifold 186001831

Description NEW µElution 96-well 1/pkg Oasis®HLB 30 µm 186001828BA Oasis®MCX 30 µm 186001830BA Oasis®MAX 30 µm 186001829

Oasis

®

96-well

µElution Plates

References

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