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1 Click on Table of Contents. Then type desired page number & click OK. 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 NEW Oasis µelution Plates...28 Oasis On-Line Columns...31 Prospekt Cartridges...33 Oasis Glass Cartridges...33 Oasis Products Selection Guides...34 Ordering Information...36 SAMPLE PREPARATION 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 The Waters Extraction Manifold

2 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. 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 analytical 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 different 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. SAMPLE PREPARATION Minimizing ion suppression while improving signal response. There are many techniques used in sample preparation. Common methods include liquid-liquid extraction, centrifugation, filtration, precipitation, 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 versatile, methods of sample preparation. Utilizing low cost, pre-packed, disposable 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. Strategies for Solid-Phase Extraction There are two simple solid-phase extraction strategies for sample preparation. You can choose a cartridge sorbent, sample solvent and elution solvent(s) to cause: Strategy 1. Compound(s) of interest are unretained, while matrix interferences are adsorbed (captured) Load Sample Matrix Waters Sep-Pak Vac Elute Compound(s) of Interest Capture (retain) Interferences then Dispose of Cartridge 15

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. Load Sample Matrix Elute Compound(s) of Interest with Stronger Solvent 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, NH 2, 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. Waters Sep-Pak Vac Capture (retain) Compound(s) of Interest Waters Sep-Pak Vac 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. Wash out Interferences (Dispose) Collect Compound(s) of Interest as they Elute from Cartridge 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. 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 Generic Elution Protocol for Normal Phase Chromatography on Sep-Pak Cartridges (Silica, Alumina, Florisil, Diol, NH 2 ) ion-exchange The basic characteristics of each chromatographic mode are described below. There are many different types of sorbents for each mode and the Load Sample Using progressively more polar solvents to elute first and second compounds of interest 2 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. You may condition cartridge with nonpolar solvent Elute unwanted compounds (wash) Compound 1 Compound 2 Discard the used cartridge 16

4 Reversed-Phase Chromatography (non-polar sorbents) with Oasis HLB and Sep-Pak Cartridges Ion-Exchange Chromatography (charged sorbent particle surface) Sep-Pak Cartridges To perform reversed-phased chromatography with Sep-Pak cartridges, use a gradient of strongly to weakly polar solvents with nonpolar C 18, tc 18, C 8, tc 2, Diol, NH 2 or 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. 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 NH 2 as 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). SAMPLE PREPARATION Generic Elution Protocol for Reversed Phase Chromatography on Sep-Pak Cartridges (C 18, tc 18, C 8, tc 2, Diol, NH 2, CN) Generic Elution Protocol for Ion-Exchange Chromatography on Sep-Pak Cartridges (NH 2, Accell Plus QMA, Accell Plus CM) 2 Load Sample Using progressively stronger solvents to elute first and second compounds of interest Load Sample Using progressively stronger buffers (changing ph or ionic strength, or both) 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 Condition cartridge with water or buffer Elute unwanted compound(s) with Compound 1 Compound 2 more water or buffer Discard the used cartridge For Oasis HLB recommended protocols are provided to simplify methods development even more, see page 20. For Oasis MCX and Oasis MAX sorbents ion-exchange chromatography recommended protocols are provided to simplify methods development. See pages 24 and

5 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 reversedphase 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. Unique Water-Wettable Oasis HLB Copolymer O N 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 C 18 sorbents 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. N O N O Pore Dewetting Mechanism of Sorbent Pores (Silica Based C 18 ) N-Vinylpyrrolidone Hydrophilic Lipophilic Balance Optimal Properties for Reversed-Phase SPE Specific Surface Area: 810 m 2 /g Average Pore Diameter: 80 Å Total Pore Volume: 1.3 cm 3 /g Average Particle Diameter: 5 µm, 15 µm, 25 µm, 30 µm or 60 µm* * Depending on configuration Divinylbenzene Properly Wetted Pore Analytes properly retained in pore Analytes not retained Dewetted Pore If air passes through traditional C 18 cartridge the pores dry out. When sample is loaded, the analytes are not retained resulting in poor recoveries. 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 18

6 Effect of Drying on Recovery - Oasis HLB Versus C 18 Sorbents. No Stopcocks Required With Oasis Since Air Does Not Dry Out The Sorbent! % Recovery Oasis HLB Cartridge (30 mg) (Excellent Recoveries) Procainamide Acetaminophen Ranitidine Propranolol Doxepin Drying Time (minutes) Oasis HLB 1 cc/30 mg and 1 cc/100 mg C 18 cartridges 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. Higher Retention Means Greater Capacity, No Breakthrough C 18 Cartridge (100 mg) (Variable Recoveries) Drying Time (minutes) The variable recoveries seen with the C 18 sorbents, 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 C 18. Oasis sorbents work especially well when you need to capture metabolites (see figure above). High Capacity Use Less Sorbent When transferring methods from a C 18 bonded 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 C 18, This reduces breakthrough potential. In addition, you may be able to use 2 3 less sorbent than you would with C 18 (30 mg Oasis HLB gives equivalent capacity to 100 mg C 18 ). H 3 C H O HO O H N O N H N O CH 3 O CH 3 N HO N Acetaminophen OH HO Salicylic acid Theobromine Catechol Retention Factor (k) Comparison C 18 Cartridge 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: 254 n 3x 2x 6x Oasis HLB Cartridge 15x 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-tobatch 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. Batch-to-Batch Reproducibility of Oasis HLB Sorbent Recovery (%) No Batch Reservations Needed 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 reversedphase) 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; Determine Types of Analyte(s) in Sample Matrix (Acids, Bases, Neutrals) If a mix of Acids, Bases and Neutrals, or Unknowns Reversed-Phase Start with Oasis HLB Recommended 1-D Method Procanamide (1.90% RSD) Ranitidine (1.68% RSD) Acetaminophen (1.65% RSD) Batch Number If all are Bases Cation Exchange Start with Oasis MCX Recommended Method Start with the Recommended Method for that sorbent. If all are Acids Anion Exchange Start with Oasis MAX Recommended Method See page 20 See page 24 See page 26 SAMPLE PREPARATION 19

7 Oasis HLB Plates, Cartridges and Columns 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. Recommended Generic Oasis HLB SPE Method (1-D)* 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). 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 Evaporate & Reconstitute 40 C/under nitrogen stream 200 µl mobile phase * Volumes are given for the Oasis HLB cartridge 1 cc/30 mg Sample Pretreatment Suggestion 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 High ph Low Low 2 5% 1 Organic Concentration 100% 1: Load (sample in acid disrupt binding)** 2: Wash 5% MeOH 3: Elute 100% MeOH **Add 20 µl (H 2 PO 4 ) 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 3000 rpm 3 High One Simple Procedure: Many Applications on a Universal Sorbent Reduced Methods Development Time 100 % Recovery Acids Neutrals With silica-based sorbents (C 18 ) you would need several different chemistries to achieve the results shown here Ibuprofen Naproxen Salicylic Acid Sulfadiazine Sulfamerazine Acetaminophen Theobromine Parazanthine Theophylline Caffeine Procainamide Ranitidine Oxycodone Propranolol Naltrexone Salbutamol Doxepin Bases n=6 RSDs < 3.5% 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 H 2 PO 4 was used to disrupt drug-protein binding). 20

8 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. Capacity and Elution Volume of Oasis 96-Well Plates and Cartridges 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 C 18 silica-based to Oasis SPE sorbents, use approximately 2 3 less Oasis sorbent (100 mg C 18 sorbent = 30 mg Oasis sorbent). Note: Larger capacity cartridges are also available. Tips for Selecting Elution Solvents for the Generic SPE Method (1-D)* The elution solvent is selected based on polarity of analyte. 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) 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 challenging selectivity and sensitivity goals. Using 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 concentration 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). 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) AV AV 1-D Method (Generic) AU 2-D Method with 2 washes AU 2-D Method with 3 washes 1 1 Sample Blank Sample Peak Identification: Peak 1: Norverapamil Peak 2: Verapamil Peak 3: Methoxyverapamil (I.S.) High ph Low Low 1: Load 2: Wash 5% MeOH 3: Elute 100% MeOH Blank High ph Low Low 1 5% 2 2 Organic Concentration 3 65% 100% 5% 1 4 Organic Concentration 1: Load 2: Wash 5% MeOH 3: Wash High ph 65% MeOH 4: Elute Low ph 65% MeOH 100% 3 High High SAMPLE PREPARATION 1 2 High 3 4 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. AV AU 2 4 Sample Blank 6 8 Minutes % 100% ph 5% Low Low Organic High Concentration 1: Load 2: Wash Low ph 5% MeOH 3: Wash High ph 5% MeOH 4: Wash High ph 65% MeOH 5: Elute Low ph 65% MeOH 21

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 pk a ), it is poorly retained by SPE. At high ph (2 units above its pk a ), it is un-ionized, and strongly retained by reversed-phase SPE, with a high k value. 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. Retention Factor Versus ph for Acids, Bases and Neutrals Retention Map, Reversed-Phase, Oasis HLB 60 Acid (Unionized) Base (Unionized) 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. Un-ionized (Captured) ph 2 ph 12 HA N BH+ Load and Wash at Low ph to capture Acid, release bases, neutrals Ionized A- Elute at High ph to release Acid Retention (k) Base (Ionized) Neutral Acid (Ionized) Mobile Phase ph Recommended ph range of Silica 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 solubility at different ph values.) Investing approximately 4 hours of development time, using this strategy, yields extremely powerful, and predictable results. Overall methods development 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.) Impact of ph Recommended ph range of Oasis Base Neutral Acid ph 12 ph 2 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. Un-ionized (Captured) B 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.) N A- Load and Wash at High ph to capture Base, release acids, neutrals Ionized BH+ Elute at Low ph to release Base 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. 22

10 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% % MeOH 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 2-D Wash Elute Study Results (Area Counts) Base Modified Wash High ph Area (µv*sec) 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% NH 4 OH in MeOH/water 2% CH 3 COOH in MeOH/water Maximum % Methanol Wash Acid Modified Wash Low ph Area (µv*sec) Minimum % Methanol Elution 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 C 18 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. 2-D SPE 3-Wash Method Developed from Wash Elute Study Oasis HLB Condition 1 ml methanol SAMPLE PREPARATION % MeOH % MeOH Equilibrate 1 ml water Acid 2% Acetic Acid Peak Area (µvs) 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, ,178 Methoxy- Verapamil Norverapamil verapamil 0-30% No Response No Response No Response 40% 73,146 32,269 86,789 50% 121,820 68, ,899 60% 109,145 61, ,229 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. 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 High ph Low Low 5% Organic Concentration 65% 100% High Removes neutral polar and basic polar interferences Removes acidic polar interferences Removes neutral non-polar and basic lipophilic interferences but not analytes Elute the basic analyte compounds in ionized form 1: Load 2: Wash Low ph 5% MeOH 3: Wash High ph 5% MeOH 4: Wash High ph 65% MeOH 5: Elute Low ph 65% MeOH 23

11 Oasis MCX Plates, Cartridges and Columns 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, 60 Base (Ionized) 50 Retention (k) Cation Exchange Reversed Phase Base (Unionized) Mobile Phase ph 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 ionexchange capacity (1 meq/gram). 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. Drug Sorbent Interactions on Oasis MCX Sorbent Reversed-Phase Retention Cation-Exchange Retention Basic Analyte (Combined) Retention 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 mechanisms. At low ph, the analyte is charged, and experiences maximum retention 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. MCX sorbent Mixed Mode Cation exchanger O N 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- SO 3 SO 3 phase retention. We can elute with a combination of high ph and high organic concentration. Strong cation-exchange mode best retention at least 2 ph units below pka Reversed-phase interaction CH 3 O + N H H CH 3 Sulfonic Acid Cation OH Exchange Capacity: Propranolol Basic Drug 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). Generic Oasis MCX Method for Extraction of Basic Compounds Condition 1 ml MeOH Equilibrate 1 ml H 2 O Load 3 ml spiked and acidified urine 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 Removes polar and acidic interferences Removes neutrals Oasis MCX 3 cc/60 mg cartridge 24

12 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. Oasis MAX Plates, Cartridges and Columns Comparison of Oasis MCX Cartridges to other SPE Silica-Based Products Using the Generic Oasis MCX Method 100 % Recovery (% RSD) % 0.7% 0.5% Oasis MCX 60 mg/3 cc 2.4% 3.2% 3.2% Brand A 300 mg/ 3 cc 13% 12% 1.1% Brand B 300 mg/ 3 cc Methadone Methadone metabolite (EDDP) Propranolol 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. Selectively Separate Primary and Secondary Amines from Tertiary and Aromatic Amines Using an Oasis MCX Cartridge 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). SAMPLE PREPARATION % Recovery Elute 1 Tertiary and aromatic amines with 5% TEA Elute 2 Primary and secondary amines with 5% NH 4 OH in methanol Elution Volume (µl of 5% TEA in Methanol) Procainamide (3 ) Toluidine (Aromatic) Doxepin (3 ) Amitriptyline (3 ) Nortriptyline (2 ) N,N-dimethylbenzylamine (3 ) N-methylbenzylamine (2 ) Benzylamine (1 ) Amphetamine (1 ) Methamphetamine (2 ) 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. Drug Sorbent Interactions on Oasis MAX Sorbent Reversed-phase Retention R+ Quaternary Amine Anion Exchange Capacity: 0.3 meq/g S O O N MAX sorbent Mixed Mode Anion exchanger COO CH 3 CH 3 + CH 2 N C 4 H 9 CH 3 Strong anionexchange mode Best retention at least 2 ph units above pka Suprofen Acidic Drug 25

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 since the analyte is now un-ionized, we get the maximum of the reversedphase retention. We can elute with a combination of low ph and high organic concentration. 60 Acid (Ionized) Generic Oasis MAX Method for Extraction of Acidic Drugs Retention (k) Acid (Unionized) Anion Exchange Reversed Phase Mobile Phase ph Reversed-Phase Retention Anion-Exchange Retention Acidic Analyte (Combined) Retention 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 Removes/elutes basic and neutral interferences The Retention Map plots the total k or capacity (retention) of a acidic analyte relative to ph. Note that the total k, is the sum of the two retention mechanisms. At high ph, the analyte is charged, and experiences maximum retention 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 Elute 2 2 ml 100 mm phosphoric acid/acetonitrile 1:1 (v/v) Alternative LC/MS Elution System: 2% formic acid in methanol As shown below, acidic compounds can be separated from basic and neutral compounds on the same Oasis MAX cartridge following the recommended protocol. High Recovery of Acidic, Basic and Neutral Compounds Using the Generic Oasis MAX Method Elute 1 (basic and neutral drugs) Elute 2 (acidic drugs) 1 2 I.S. 3 4 I.S. AV AV Minutes 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: 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 Sample Blank with I.S. Minutes Sample Blank with I.S. Results (n=6) Drugs µg/ml Type of Drug % Recovery % RSD Nortriptyline 5 Basic (Elute 1) Secobarbital 10 Neutral (Elute 1) Ketoprofen 5 Acidic (Elute 2) Naproxen 2.5 Acidic (Elute 2) *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. 26

14 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. Automation Request your free Oasis Applications Notebook (Literature Code EN) at /oasis or contact your local sales office. Automation of Oasis Sample Extraction Cartridges and 96-Well Plates SAMPLE PREPARATION 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 /Prospekt II 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 Premier Mass Spectrometer with the Waters 1525µ Binary Pump and the 2777 Sample Manager using MassLynx 4.0 Software. 27

15 High Throughput 96-Well Extraction Plates 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. Waters 96-Well Plate Design 1999 R&D 100 Award Two-Stage Well Design 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. 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 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 evaporation step with the new Oasis µelution plate. * Patent Pending Excellent Recovery in 25 µl Elution OASIS µelution Technology >85% Recovery in 25 µl 25 µl µl SPIKED SALINE acetaminophen practolol 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 reproducible SPE results. % Recovery Oasis HLB µelution plate N-acetyl procainamide betamethsone caffeine naproxen amitripyline propranolol The innovative features of the new Oasis µelution plates enable sensitive, robust, reproducible results without evaporation and reconstitution. 28

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 throughput 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 sensitivity 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. Protein Precipitation Generic Method Add 50 µl spiked plasma (1 to 1000 pg/µl), 50 µl IS (10 pg/µl) in H 2 O 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% NH 4 OH and 50 µl H 2 O Inject 20 µl Oasis HLB Generic Method Oasis HLB µelution Plate** Part Number Condition with 200 µl MeOH Equilibrate with: 200 µl H 2 O Load 50 µl spiked rat plasma, 50 µl IS (10 pg/µl) in H 2 O Wash with 200 µl 5% MeOH in water Elute with 25 µl ACN:IPA 40:60 + 2% FA Dilute with 50 µl H 2 O Inject 20 µl Oasis MCX Generic Method Oasis MCX µelution Plate** Part Number Condition with 200 µl MeOH Equilibrate with 200 µl H 2 O Load 50 µl spiked rat plasma, 50 µl IS (10 pg/µl) in H 2 O Wash 1: 200 µl Water + 2% FA Wash 2: 200 µl MeOH Elute with 25 µl ACN:IPA 40:60 + 2% NH 4 OH Dilute with 50 µl H 2 O Inject 20 µl SAMPLE PREPARATION Increase in Selectivity (More Selective Methods) ** Tomtec Quadra 96 used. Oasis HLB and MCX µelution Plate versus Protein Precipitation LC Conditions Column: XTerra MS C 18, 2.1 x 30 mm, 3.5 µm Mobile phase A: Water % NH 4 OH Mobile phase B: ACN % NH 4 OH 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 Propranolol m/z Amitriptyline m/z Nortriptyline m/z Propranolol, 1 ng/ml Amitriptyline, 0.1 ng/ml 100 % Protein Precipitation Intensity: 7.50 x % Protein Precipitation Intensity: 9.71 x % Oasis HLB Intensity: 3.03 x x sensitivity without evaporation 100 % Oasis HLB Intensity: 3.62 x x sensitivity without evaporation % Oasis MCX Intensity: 1.90 x x sensitivity without evaporation 100 % Oasis MCX Intensity: 8.91 x x sensitivity without evaporation Time (mins) Time (mins)

17 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 sensitivity gains and delivers high performance without the time consuming evaporation step. Oasis MCX µelution Plate versus Glass Fiber and Membrane 96-well Disk Plates 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 Terfenadine 0.5 ng/ml 100 % S/N = Intensity: S/N = 82 Intensity: Intensity: 5.75 x x10 6 S/N = x10 7 % 6x Sensitivity % Terfenadinealcohol 0.5 ng/ml 100 % 2.54 S/N = Intensity: S/N = 17 Intensity: Intensity: 3.03 x x10 S/N = x10 5 % 16x Sensitivity % Terfenadinecarboxylate 0.5 ng/ml 100 % 2.70 S/2 = Intensity: S/N = 40 Intensity: Intensity: 7.30 x x10 5 S/N = x10 5 % 18x Sensitivity % Time (mins) Time (mins) Time (mins) Final extract diluted 1:2 with H 2 O Extracted using manufacturers recommended protocol Final extract diluted 1:2 with H 2 O Extracted using manufacturers recommended protocol Final extract diluted 1:2 with H 2 O Extracted using manufacturers recommended protocol Validation Assay of Terfenadine and Its Metabolites Using The Oasis MCX µelution Plate Calibration range: 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% 30

18 Oasis On-Line Columns for LC/MS or LC/MS/MS Generally, drugs present in biological matrices should not be directly introduced 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 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 exceptional 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. 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. Unique Water-Wettable Oasis HLB Copolymer N O N-Vinylpyrrolidone O N Hydrophilic Lipophilic Balance Optimal Properties for Reversed-Phase SPE Specific Surface Area: 810 m 2 /g Average Pore Diameter: 80 Å Total Pore Volume: 1.3 cm 3 /g Average Particle Diameter: 5 µm, 15 µm, 25 µm, 30 µm or 60 µm* * Depending on configuration N O Divinylbenzene SAMPLE PREPARATION O N O N SO 3 SO 3 R+ CH 3 + CH 2 N C 4 H 9 CH 3 Oasis HLB Sorbent Oasis MCX Sorbent Oasis MAX Sorbent 31

19 Oasis On-Line Columns 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 Sentry holder 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 analytical columns (such as Atlantis, XTerra, or Symmetry columns). Ultra Fast On-Line System Configuration Oasis On-line Column LOAD POSITION 515 System INJECTION POSITION 515 System Isocratic HPLC Waste Port Valve 2 positions Waste Oasis Waste Oasis Gradient HPLC Narrow Bore Analytical Column (Optional) Mass Spectrometer Alliance HT Mass Spectrometer Analytical Column (optional) Alliance HT Mass Spectrometer Analytical Column (optional) 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 Column Lifetime 2.1 x 20 mm cartridge column 50 µl injection 0.0 Minutes 1.0 Overlay of Toluamide after 360 Plasma injections. 32

20 Oasis Prospekt Cartridges On-Line SPE with Oasis Sorbent in Prospekt Cartridges Waters Oasis sorbents are available in Prospekt 1 and Prospekt 2 cartridges for use with the Spark Holland Prospekt /Symbiosis systems. The narrow-bore PVDF cartridge is 1.0 mm x 10 mm contain approximately 2.5 mg of Oasis sorbent. The Oasis sorbents in Prospekt cartridges show the same performance advantage for pharmaceutical compounds over silica-based sorbents. Recovery Comparison of Oasis HLB vs. Bonded Silica Prospekt Cartridge Oasis HLB C 18 C 2 Clonidine % Recovery Terconazole * Data were provided by Dr. Francis Beaudry, a Principal Research Scientist of Phoenix International Life Sciences Inc. in Montreal, Quebec, Canada. SAMPLE PREPARATION Oasis Glass Cartridges for PPT Detection Levels Waters Oasis glass cartridges are available in 5 cc (200 mg) configuration 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 # EN). Endocrine Disruptors PPT Recovery of Estrogens from River Water, LC/MS, 5 ng/l Spike Level, n=4 Results Ions Monitored (m/z) Recovery % RSD 1. bisphenol A b-estradiol a-ethynylestradiol estrone diethylstilbestrol Recovery of Phthalates and Nonylphenol from River Water, GC/MS, 200 ng/l Spike Level, n=4 Results Recovery % RSD 1. dimethylphthalate dirthylphthalate n-nonylphenol dibutylphthalate benzylbutylphthalate bis(ethylhexyl)phthalate 60 8 ISTD. o-terphenyl (internal standard) 33