Phospholipid Removal Solutions - Phenomenex

Phospholipid Removal SolutionsRemove PhospholipidsRemove ProteinNo Method Developmentwww.phenomenex.com/Phree

One Quick Method, Three Big Advantages12Remove Proteins that can clog HPLC columnsEliminate Phospholipids that cause ion suppressionand increase your column lifetime!3No Method Development; one method for acids,bases, and neutralsRemove ProteinsSolvent Shielding Technology preventsdripping of organic solvent, allowing forprotein precipitation within the PhreePhospholipid Removal Product.Eliminate PhospholipidsThe Phree sorbent selectively removesphospholipids from precipitated plasmasamples.ProteinsPhospholipidsTarget Analyte© 2013 Phenomenex, Inc. All rights reserved.2Phenomenex l WEB: www.phenomenex.com

Phospholipid Removal SolutionsBe Free of Phospholipids and Proteins“We have demonstrated that Phree PhospholipidRemoval Plates resulted in >98 % decrease in thepeak area of the six phospholipids we monitoredin rat plasma compared to traditional protein crash.We highly recommend the use of these plates forhigh throughput LC-MS/MS bioanalytical samplepreparation.”Nina KhoshabaWIL Research3 Steps Above Traditional Protein Precipitation.............................pp. 4-5An Improved Phospholipid Removal Solution.................................pp. 6-7Skip the Method Development.......................................................pp. 8-9Technical Support...............................................................................p. 10Ordering Information..........................................................................p. 11Phenomenex lWEB: www.phenomenex.com3

3 Steps Above Traditional Protein Precipitation1. Protein Precipitation Does Not Remove PhospholipidsEndogenous phospholipids are a primary source of ion suppression and resulting matrix effects inbioanalytical LC/MS work. Ion suppression caused by the presence of phospholipids can result in:• irreproducible results• quantitation issues• loss in method sensitivity• matrix to matrix biasTotal Phospholipid ProfileProtein Precipitation vs. Phree Phospholipid Removal ProductsIntensity, cps200114.5e64.0e63.5e63.0e62.5e62.0e6Cleanup: 100 µL plasma plus 300 µL acetonitrilewith 1 % formic acidColumn: Kinetex ® 2.6 µm C18 100 ÅDimensions: 50 x 2.1 mmPart No.: 00B-4462-ANMobile Phase: A: 0.1% Formic acid in WaterB: 0.1% Formic acid in MethanolGradient: Time (min)00.515.515.5119.5% B6095956060Flow Rate: 400 µL/minDetection: Mass Spectrometer (MS) @ 425 °C;184 amuTemperature: 22 °C1.5e61.0e65.0e5App ID 2001100 1 23 4 5 6 7 8 9 1011 min~ 50x Zoom20012Intensity, cps9.0e48.5e48.0e47.5e47.0e46.5e46.0e45.5e45.0e44.5e44.0e43.5e43.0e42.5e42.0e41.5e41.0e45000.00.0Selectively removephospholipids with PhreePhree extracted plasmaProtein precipitated plasma0 1 23 4 5 6 7 8 9 1011 minApp ID 20012Phospholipid profile monitored using m/z 184-1844Phenomenex lWEB: www.phenomenex.comPlasma

Phospholipid Removal Solutions2. Reduce Ion SuppressionThe presence of phospholipids in plasma samples produces zones of ion suppression that correlateexactly with the phospholipid elution profile when analyzed via mass spectrometer (MS).1.2e61.0e6Protein Precipitated Plasma20013Ion suppressionis observedPhree Extracted Plasma200141.2e61.0e6Intensity, cps8.0e56.0e5Intensity, cps8.0e56.0e5Suppression zoneis eliminated4.0e52.0e5App ID 200134.0e52.0e5App ID 200140.00.2 0.6 1.0 1.4 1.8 2.2 2.6 min00 0.2 0.6 1.0 1.4 1.8 2.2 2.6 minSuppression ZonePhospholipids m/z 184-184Amoxapine was infused post-column to establish an ion suppression/enhancement profile with both protein precipitatedplasma (left) and Phree extracted plasma (right), showing that Phree can successfully reduce ion suppression.Amoxapine m/z 314-2713. Maximize Sensitivity and Column LifetimePhospholipids reduce the sensitivity of the MS signal and shorten column lifetime whenthey build up over time.Column Sensitivity after 250 Injections300000250000~5,000 µL plasma injected withoutsignificant loss of sensitivity200000Peak Area Counts150000Reduced sensitivity isimmediately observedDiclofenac in PhreeExtracted PlasmaDiclofenac in ProteinPrecipitated Plasma100000Phospholipid build up results insignificant loss of sensitivity5000000 50 100 150 200 250Injection Number (20 µL of Plasma)To assess the effect of phospholipid build up, repetitive 20 µL injections of diclofenac in protein precipitated plasma versus diclofenac in Phree extracted plasma were made.Phenomenex l WEB: www.phenomenex.com5

An Improved Phospholipid Removal SolutionRemove All Classes of PhospholipidsLysophosphatidylcholines and phosphatidylcholines both contribute to matrix effects. Remove allclasses of phospholipids using Phree Phospholipid Removal Products.1.5e6Intensity, cpsIntensity, cps1.4e61.5e61.3e61.4e61.2e61.3e61.1e61.2e61.0e61.1e69.0e51.0e68.0e59.0e57.0e58.0e56.0e57.0e55.0e56.0e54.0e55.0e53.0e54.0e52.0e53.0e51.0e52.0e501.0e5acetonitrile with 1 % formic acidColumn: Kinetex ® 2.6 µm C18 100 ÅDimensions: 50 x 2.1 mmPart No.: 00B-4462-ANMobile Phase: A: 0.1% Formic acid in WaterB: 0.1% Formic acid in MethanolSupelco ® HybridSPE ® 60Plasma Cleanup: 100 µL plasma plus 300 µLPhenomenex ® Gradient: Time (min) % BPhree0 600.5 95Agilent ® Captiva NDLipids15.5 9515.51 60Flow Rate: 400 µL/minDetection: Mass Spectrometer (MS) @ 425 °C;184 amuTemperature: 22 °C0 12 3 4 5 6 7 8 9 10 min00 12 3 4 5 6 7 8 9 10 min~ 25x Zoom4.8e4Intensity, cpsIntensity, cps4.4e44.8e44.0e44.4e43.6e44.0e43.2e43.6e42.8e43.2e42.4e42.8e42.0e42.4e41.6e42.0e41.2e41.6e480001.2e44000800004000Phree is most effective atremoving all classes of phospholipids1 2 3 4 5 6 7 8 9 10 minApp ID 200150Phospholipid profile monitored1using m/z2184-1843 4 5 6 7 8 9 10 minAgilent is a registered trademark of Agilent Technologies Inc. Captiva is a trademark of Agilent Technologies, Inc.Supelco and HybridSPE are registered trademarks of Sigma-Aldrich Co.Comparative separations may not be representative of all applications.6Phenomenex lWEB: www.phenomenex.com

Phospholipid Removal SolutionsExtended Phospholipid CapacityThe Phree sorbent has an extended capacity for phospholipids, allowing you to load up to 400 µL ofplasma without significant breakthrough of phospholipids.Intensity, cps200464.6e54.4e54.2e54.0e53.8e53.6e53.4e53.2e53.0e52.8e52.6e52.4e52.2e52.0e51.8e51.6e51.4e51.2e51.0e58.0e46.0e44.0e42.0e41200 1 2 3 4 5 6 7 8 9 10 11 minPhospholipid profile monitored using m/z 184-184~ 40x ZoomPlasma Cleanup: 400 µL plasma plus 1.2 mLacetonitrile with 1 % formic acidColumn: Kinetex ® 2.6 µm C18 100 ÅDimensions: 50 x 2.1 mmPart No.: 00B-4462-ANMobile Phase: A: 0.1% Formic acid in WaterB: 0.1% Formic acid in MethanolGradient: Time (min)00.515.515.5119.5® Phree Waters ® Ostro % B6095956060Flow Rate: 400 µL/minDetection: Mass Spectrometer (MS) @ 425 °C;184 amuTemperature: 22 °CApp ID 20046Waters Corporation states that the true maximum recommended plasma volume that can be processed using the Ostro plate is 350 µL due to well volume limitations.Ostro is a trademark of Waters Corporation. Comparative separations may not be representative of all applications.Phenomenex lWEB: www.phenomenex.comPhenomenex7

Phospholipid Removal SolutionsHigh Recoveries of Target AnalytesThe Phree general protocol has been developed to produce high recoveries of acids, bases,and neutrals while simultaneously removing all classes of phospholipids.Absolute Recoveries of Acids, Bases, and Neutrals100%90%80%70%Absolute Recovery (%)60%50%40%30%20%10%DiclofenacNaproxenPrednisoneAcetaminophenMetoprololAmitriptylineAcidsNeutralsBases0%AnalyteRecovery data was obtained by calculating the average absolute recoveries of analytes extracted from 3 Phree plates.Phenomenex l WEB: www.phenomenex.com9

Sample Preparation Specialists areReady to Assist YouTwo Levels of Method Development SupportLevel 1Contact one of our dedicated sample preparationspecialists for immediate method developmentassistance.Level 2Send your sample to our analytical servicesgroup for custom method development. Visitwww.phenomenex.com/PhenoLogix formore information.Get StartedContact your Sample Preparation Specialist:By email: Support@phenomenex.com“Phenomenex’s prompt support isvery important for us to achieveour work. I thank them for theirpersistent support and innovativeproducts.”Allena JiWyeth10Phenomenex lWEB: www.phenomenex.com

Phospholipid Removal SolutionsOrder Phree Now!Ordering InformationPhree Phospholipid Removal ProductsPart No. Description Unit Price8B-S133-TAK Phree Phospholipid Removal 1 mL Tube 100/box8E-S133-TGB Phree Phospholipid Removal 96-Well Plates 2/boxAccessoriesCollection Plates (deep well, polypropylene)AH0-7192 Strata ® 96-Well Collection Plate 350 µL/well 50/pkAH0-7193 Strata 96-Well Collection Plate 1 mL/well 50/pkAH0-7194 Strata 96-Well Collection Plate 2 mL/well 50/pkAH0-8635 Strata 96-Well Collection Plate, 2 mL Square/Round-Conical 50/pkAH0-8636 Strata 96-Well Collection Plate, 2 mL Round/Round, 8 mm 50/pkAH0-7279 Strata 96-Well Collection Plate, 1 mL/well Round, 7 mm 50/pkSealing MatsAH0-8597 Sealing Mats, Pierceable, 96-Square Well, Silicone 50/pkAH0-8598 Sealing Mats, Pre-Slit, 96-Square Well, Silicone 50/pkAH0-8631 Sealing Mats, Pierceable, 96-Round Well 7 mm, Silicone 50/pkAH0-8632 Sealing Mats, Pre-Slit, 96-Round Well 7 mm, Silicone 50/pkAH0-8633 Sealing Mats, Pierceable, 96-Round Well 8 mm, Silicone 50/pkAH0-8634 Sealing Mats, Pre-Slit, 96-Round Well 8 mm, Silicone 50/pkAH0-7362 Sealing Tape Pad 10/pkVacuum ManifoldsAH0-6023* SPE 12-Position Vacuum Manifold Set, for tubes eaAH0-6024* SPE 24-Position Vacuum Manifold Set, for tubes eaAH0-8950 Strata 96-Well Plate Manifold, Universal with Vacuum Gauge ea*Manifolds include: Vacuum-tight glass chamber, vacuum gauge assembly, polypropylene lid with gasket, male andfemale luers and yellow end plugs, stopcock valves, collection rack assemblies, polypropylene needles, lid supportlegs. Waste container included with 12-positive manifold.If Phree Phospholipid Removal products do not perform as well or betterthan your current phospholipid removal product, return the product withyour comparative data within 45 days for a full refund.TipFor more information about Phenomenexsample preparation products, visitwww.phenomenex.com/sampleprepinfoTerms and ConditionsSubject to Phenomenex Standard Terms and Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions.TrademarksStrata and Kinetex are registered trademarks of Phenomenex. Phree and Solvent Shielding Technology are trademarks of Phenomenex.Agilent is a registered trademark, and Captiva is a trademark of Agilent Technologies, Inc. Supelco and HybridSPE are registered trademarks of Sigma-Aldrich Co. Waters is a registeredtrademark, and Ostro is a trademark of Waters Corporation.DisclaimerPhenomenex is in no way affiliated with Agilent Technologies, Sigma Aldrich, or Waters Corporation. Comparative separations may not be representative of all applications.The opinions stated herein are solely those of the speaker and not necessarily those of any company or organization.© 2013 Phenomenex, Inc. All rights reserved.Phenomenex lWEB: www.phenomenex.com11

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