[ product solution ] Waters Oasis µ Elution PlateS. Patented Innovation. Elution volume as low as 25 μl. No evaporation and reconstitution
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1 [ product solution ] Waters µ Elution PlateS Elution volume as low as 25 μl No evaporation and reconstitution Ideal for small sample volumes Up to a 15x increase in concentration Patented Innovation Now you can confidently perform SPE cleanup and analyte enrichment of very small sample volumes (1-25 µl) up to a maximum of 375 µl. The Waters µelution plate combines patented * plate design, proven chemistries **, and straightforward protocols that deliver high analyte recovery and clean extracts in elution volumes as low as 25 μl. Using the μelution plate achieves superior results compared to other conventional SPE formats in less time. This plate format produces concentrated extracts that can be directly injected into your LC/MS/MS, eliminating the need for the time-consuming evaporation step. Eluting in 25 µl without evaporation provides up to a 15 fold increase in analyte concentration, enabling sensitive, robust, and reproducible SPE results. Scientists in both pharmaceutical drug discovery and drug development can prepare the cleanest biological sample extracts for more sensitive LC/MS/MS analysis. *U.S. Patent 6,723,236 ** U.S. patents 5,882,521 (1996), 5,976,376 (1998), 6,16,721 (1999), 6,254,78 (21), 6,322,695 (21), 6,468,422 (22), 6,726,842 (24), 6,773,583 (24), 6,723,236 (24), additional patents pending.
2 Rapid and Sensitive Method for the Determination of Ropinirole in Human Plasma Ropinirole is a potent drug marketed under the name Requip in the U.S. and Adartrel in Europe. It is prescribed for both restless leg syndrome and Parkinson s disease. A bioanalytical method was developed and validated using MCX μelution plates for sample preparation and concentration, and an ACQUITY UPLC /MS/MS for highest sensitivity. The required LLQ (lower limit of quantitation) of.5 ng/ml (5 pg/ml) for ropinirole in human plasma was easily achieved. The response at.5 ng/ml is more than 1x the level found in the blank sample, surpassing the FDA guidelines for determining the limit of quantitation in bioanalytical methods. This example dramatically demonstrates both the powerful extraction capability and capacity of MCX mixed-mode cation-exchange sorbent in the μelution plate format and the high sensitivity, speed, and specificity capabilities of the combination of Waters ACQUITY UPLC and Quattro Premier XE MS systems. MCX μelution 96-well Plate Procedure 2 µl CH 3 2 µl H 2 6 L diluted human plasma (3 µl plasma diluted 1:1 with 4 H 3 P 4 in H 2 ) 2 µl 2 CH in H 2 2 µl CH 3 1 x 25 µl 5 NH 4 in CH 3 ACQUITY UPLC Conditions Column: ACQUITY UPLC BEH C 18, 2.1 x 5 mm, 1.7 μm Column part number: Mobile phase A: 1 mm NH 4 C, ph 9 Mobile phase B: CH 3 Flow rate:.5 ml/min Gradient: Time Profile Curve (min) A () B () Injection volume: 8 μl Column temperature: 45 C Sample temperature: 15 C Sample diluent: CH NH 4 Strong needle wash: 6:4 CH 3 CN:IPA +.5 HC (12 μl) Weak needle wash: 95:5 H 2 :CH 3 (5 μl) 96-well collection plate part number: UPLC /MS/MS Chromatogram of the XIC for a Sample of Ropinirole in Human Plasma at LLQ of.5 ng/ml > e ng/ml ropinirole S/N 125 >1X level in blank Blank Plasma > e Ropinirole Standard Curve and Linearity Data Residual ng/ml 2. Response ng/ml Ropinirole standard linear curve fit with 1/x 2 weighting, from.5 ng/ml to 1 ng/ml over 3.5 orders of magnitude, Correlation Coefficient: r =.99954, r 2 =.99818, and Calibration Curve Equation: * x
3 [ product solution ] Sample Enrichment: Up to a 15X Concentration Factor ng/ml risperidone 375 µl sample diluted with 375 µl 4 H3P4 loaded onto MCX plates Elution in 25 µl Recovery = 98 11X increase in S:N 17X increase in area counts Elution in 375 µl > e6 MCX µelution plate 15X concentration > e6 MCX 1 mg plate No concentration Straightforward Methodology The 2x4 method is a simple, logical approach to the selection of an SPE sorbent and protocol. Two protocols and four sorbents provide the flexibility to extract acids, bases, and neutrals with high SPE recoveries while removing matrix components that may interfere with the analysis. This flow chart outlines the simple steps required to achieve high recovery and the cleanest extracts: Characterize your analyte [neutral, acid or base; pka]. Select one of the four sorbents. Apply the indicated protocol [1 or 2]. Determine SPE recoveries by LC analysis. 2x4 Method ptimized for μelution Plate For Bases pka 2-1: Use MCX For Strong Acids pka <1: Use WAX For Acids pka 2-8: Use MAX For Strong Bases pka >1: Use WCX MCX µelution Plate Protocol 2 µl CH 3 2 µl H 2 75 µl diluted plasma (375 µl plasma diluted 1:1 with 4 H 3 P 4 in H 2 ) 2 µl 2 CH in H 2 2 µl CH 3 MCX 1-mg Plate Protocol 5 µl CH 3 5 µl H 2 75 µl diluted plasma (375 µl plasma diluted 1:1with 4 H 3 P 4 in H 2 ) 5 µl 2 CH in H 2 5 µl CH 3 Apply Protocol 1 Prepare Sample Condition/Equilibrate Load Sample Wash: 2 HC Elute 1: 1 CH 3 Elute 2: 5 NH 4 in 6:4 CH 3 CN:CH 3 Neutrals Apply Protocol 2 Prepare Sample Condition/Equilibrate Load Sample Wash: 5 NH 4 Elute 1: 1 CH 3 Elute 2: 2 HC in 6:4 CH 3 CN:CH 3 25 µl 5 NH 4 in 6:4 CH 3 CN:CH 3 Inject: 5 µl 375 µl (125 µl x 3) 5 NH 4 in 6:4 CH 3 CN:CH 3 Inject: 5 µl Base Strong Acid Acid Strong Base Those who have used the 2x4 method with cartridges will note that the elutropic strength of the final elution solvent in the above protocols has been increased to optimize recovery in minimal elution volumes. We recommend as a starting point, using a 6:4 ratio of acetonitrile to methanol, not 1 methanol; this provides the optimal elution strength, viscosity, and solubility properties appropriate for high analyte recovery of a diverse set of analytes in only 25 µl.
4 2x4 Method Proof of Concept Recovery Study To demonstrate the logic, simplicity, and effectiveness of the 2x4 method, five samples of rat plasma were prepared, each spiked with one of the previously characterized test analytes shown below: Proof of Concept: Analytes Spiked into Rat Plasma Matrix Effects Study Matrix effects* are an alteration in MS response caused by interfering components in a sample. They may cause a loss of signal, ion suppression or gain in signal, ion enhancement. Phospholipids or lysophopholipids have been identified and reported as a major contributor to matrix effects in plasma samples.** Matrix effects decrease analytical method robustness and reproducibility, raise limits of detection/quantitation, and may lead to spurious results. N N Imipramine (B) pka = 9.4, 2 ng/ml F F F F H F F F F F Nonafluoropentanoic Acid (SA) pka <.5, 2 ng/ml Br Valethamate (QA) pka >12, 2 ng/ml Ibuprofen (A) pka = 5.2, 2 ng/ml N + H H H Prednisone (N) 1 µg/ml SPE cleanup using 2x4 method with µelution plates effectively reduces plasma interferences and their corresponding matrix effects. Quantitative Assessment of Matrix Effects: Matrix Effects Data for the 2x4 Method Proof of Concept Blank Sample Matrix (No analyte(s)) Standard Solution (Analyte(s)) Protocol 1 Protocol 2 1 MCX WAX MAX WCX Response Post-Extracted Spiked Sample Matrix Effects = ( -1) x 1 Response Standard Solution SPE Recovery Spike Standards into Extracted Martix Both samples should be in the same composition solution Calculated matrix effects of <±15 are generally acceptable 2 Imipramine (b) Ibuprofen (a) Valethamate (sb) Post-Extracted SPIKED Sample Elute 1: CH 3 Elute 2: 6:4 CH 3 CN/CH 3 + modifier Nonafluoropentanoic Acid (sa) Each plasma sample was diluted [1:1, v:v] and acidified with phosphoric acid [4 in water]. Respective aliquots were then processed using the protocol and the mixed-mode ion-exchange sorbent designated by the 2x4 method for the corresponding sample type. SPE recovery was determined by LC/MS/MS analysis. The neutral analyte was processed on all four sorbents used. Ion Suppression Ion Enhancement MCX Imipramine WAX Nonafluoropentanoic Acid MAX Ibuprofen WCX Valethamate All matrix effects are less than our ±15 criteria for acceptance * Neue and McDonald, Waters Whitepaper ** Chambers et al., J. Chromatogr. B, 27, Jun 1;852 (1-2): 22-34
5 μelution Plat e Loading Capacit y SPE device capacity is defined as the total mass of analytes and endogenous sample components retained by the sorbent bed under loading conditions. Breakthrough will occur when the capacity of the sorbent bed is exceeded. The physicochemical properties of sorbents are designed to provide exceptionally high loading capacity, even though each well in a Waters µelution plate contains only 2 mg of sorbent. To determine the μelution plate capacity, increasing volumes of plasma and urine samples (from 5 µl to 35 µl in 5 µl increments) were spiked with 2 ng/ml imipramine (non polar base) and 2 ng/ml atenolol (polar base). The plasma aliquots were diluted 1:1 with 4 aqueous H 3 P 4 and the urine aliquots were diluted 1:1 with H 2 and then loaded onto the µelution plate. SPE recovery was calculated and plotted for each loading level. SPE Recovery for 2 ng/ml Imipramine and 2 ng/ml Atenolol on MCX µelution Plate Recovery SPE Recovery for Polar and Non-Polar Analytes in Plasma Example µl Loaded Plasma Volumes (Undiluted) SPE Recovery for Polar and Non-Polar Analytes in Urine Example SPE Protocol for MCX µelution 96-well Plate Recovery µl CH 3 2 µl H 2 Various volumes of: - Plasma diluted 1:1 with 4 H 3 P 4 in H 2 - Urine diluted 1:1 with H 2 2 µl 2 CH in H 2 2 µl CH 3 2 x 25 µl 5 NH 4 in 6:4 CH 3 CN:CH 3 Dilute: 5 µl H 2 Inject: 5 µl Ion Suppression Ion Enhancement µl Loaded Urine Volumes (Undiluted) Matrix Effects for Polar and Non-Polar Analytes in Plasma µl Loaded Plasma Volumes (Undiluted) Atenolol (polar) Imipramine (non-polar) n = 8
6 96-well µelution Plates (1/pkg) Description HLB 3 µm MCX 3 µm Part Number BA BA MAX 3 µm WCX 3 µm Elution Plate WAX 3 µm Sorbent Selection Plate, 3 rows each MCX, WAX, MAX and WCX Manifold for Extraction PlateS Description Qty Part Number Extraction plate manifold for 96-well plates 1/box Extraction plate manifold kit A (includes extraction plate manifold, reservoir tray, sealing cap and 35 µl sample collection plate) WAT97944 Disposable reservoir tray 25/box WAT58942 SPE Vacuum Pump (Includes two gauges and pressure regulator) Sample collection plate, 35 µl 5/box WAT58943 Sealing cap for 96-well collection plate 5/pkg WAT58959 SPE vacuum pump 115 V, 6 Hz Vacuum Box Gasket Kit SPE vacuum pump 24 V, 5 Hz Vacuum box gasket kit Kit includes: 2 foam top gaskets 2 orange -rings Austria and European Export (Central South Eastern Europe, CIS and Middle East) , Australia , Belgium , Brazil , Canada x225, China , CIS/Russia , Czech Republic Denmark , Finland , France , Germany , Hong Kong Hungary , India and India Subcontinent , Ireland , Italy , Japan Korea , Mexico , The Netherlands , Norway , Poland Puerto Rico , Singapore , Spain , Sweden , Switzerland Taiwan , United Kingdom All other countries: Waters Corporation U.S.A / Waters, The Science of What s Possible,, ACQUITY UPLC, UPLC and Quattro Premier are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. 28 Waters Corporation. Printed in the U.S.A. July EN IH-DS Waters Corporation 34 Maple Street Milford, MA 1757 U.S.A. T: F:
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