Samplicity™ System Delivers High-Performance Multi-sample Filtration While Streamlining Preparation of Chromatography Samples

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The Samplicity™ filtration system has helped researchers streamline chromatographic sample preparation in order to match the speed and throughput requirements of their analytical separations such as high/ultrahigh performance liquid chromatography (HPLC /UHPLC). An easy-to-use alternative to syringe filters, the Samplicity™ system enables simultaneous vacuum filtration of up to eight samples. Even difficult-to-filter samples with high viscosity or particulates can be processed in seconds. Our data show that the Samplicity™ system facilitates preparation of chromatography samples with high recovery, low extractables and very low passage of particulate impurities.

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  Data Sheet Abstract The Samplicity™ ltration system has helped researchers streamline chromatographic sample preparation in order tomatch the speed and throughput requirements o their analytical separations such as high/ultrahigh perormanceliquid chromatography (HPLC /UHPLC). An easy-to-use alternative to syringe lters, the Samplicity™ system enablessimultaneous vacuum ltration o up to eight samples. Even dicult-to-lter samples with high viscosity orparticulates can be processed in seconds. Our data show that the Samplicity™ system acilitates preparation o chromatography samples with high recovery, low extractables and very low passage o particulate impurities. Introduction Sample preparation prior to analysis helps to bring asample to a ormat that is compatible with the analyticaltechnique, reduces sample complexity, removesinterering impurities rom the matrix and therebyconcentrates the analyte prior to analysis. A typicalsample or HPLC / UHPLC needs to be particle-ree andcompletely soluble in the solvent compatible with thechromatography system.Due to its simplicity and eectiveness, membraneltration is a common sample preparation technique;however, it is one o the most neglected steps insample prep optimization. Not enough attention is paidto ltration and the choice o ltration devices andmaterials, leading to inconsistency and even ailure o the downstream analysis.Sample ltration prior to chromatography is mostcommonly perormed using syringe ltration. This isa serial, manual process, involving ltration o onesample at a time. Syringe ltration can also be very laborintensive depending on the sample type; samples whichare dicult to lter (such as particle laden or viscoussamples) require even higher pressures to lter, whichcan lead to atigue. Syringe ltering one or two samplesa day may present a mere inconvenience, but lteringlarge numbers o samples at a time can lead to severeatigue, musculoskeletal pain or repetitive stress injuries.Simpliying sample preparation, the Samplicity™system enables ltration o up to 8 samples directlyinto standard HPLC vials (12 x 32 mm) using vacuum-driven ltration. This avoids multiple transers that are Application Note The Samplicity ™ System DeliversHigh-Perormance Multi-sampleFiltration While StreamliningPreparation o ChromatographySamples EMD Millipore is a division o Merck KGaA, Darmstadt, Germany  2 Extractable analysis using HPLC-UV  Three solvents (Milli-Q® water, methanol (LC-MS grade),and acetonitrile (LC-MS grade) were ltered through0.20 µm and 0.45 µm Millex Samplicity™ lters or eachsolvent. 1 mL o solvent was applied to each lter andltrates were collected in vials. These vials were thenreplaced with new vials, and an additional 1 mL o eachsolvent was applied to the same lters and ltered intothe second set o vials. Samples and starting solventswere analyzed by reversed phase HPLC using C18 columnrun under gradient condition (0 – 100 % acetonitrile)ollowed by UV detection (214 and 254 nm) . Particle retention analysis Polystyrene latex particles (0.5 µm, Sigma Aldrich Cat.No. LB5 or 0.3 µm, Sigma Aldrich Catalogue No. LB3)were diluted to a 0.005% suspension in Triton® X-100and absorbance measured at 272 nm. 1.5 mL o eachparticle suspension was added to 0.20 µm or 0.45 µmMillex Samplicity™ lters and vacuum applied. Theabsorbance o each ltrate (as well as a Triton® X-100blank) at 272 nm was measured by UV spectroscopy andparticle retention calculated. Sample recovery fltering volatile solvents 1 mL o each solvent (acetonitrile, tetrahydrouran andacetone) was ltered through Millex Samplicity™ lters(0.45 µm) into preweighed vials. Immediately aterltration (Time 0), vials were weighed and placed backinto the system. Ater 10 minutes o additional vacuum(Time 10), the vials were weighed again. Recoveries werecalculated based on the initial weight o solvent. Thetypical holdup volume o the lter varied between70-100 µL; thereore, losses o 7-10% were expected. Results The holdup volume o a lter can aect the volume o sample recovered in the ltrate. We showed that MillexSamplicity™ lters had average holdup volumes o less than 70 µL, indicating that, or most applicationsinvolving ltration o 1-2 mL volumes, the volume o sample lost to holdup is not a signicant loss (Table 1).sometimes necessary with syringe ltration, simpliesworkfow and reduces time and atigue associated withsyringe ltration.The Samplicity™ system involves ltration o samplesthrough Millex Samplicity™ lter units. Here, we showperormance data or these lter units, demonstratingltration with low hold up volume, high samplevolume recovery, high analyte recovery (by mass), andlow extractables. Furthermore, we show that MillexSamplicity™ lter units provide accurate particleretention with respect to reported pore size and highsample volume recovery or volatile solvents. Methods   For all experiments, samples were ltered eitherthrough hydrophilic polytetrafuoroethylene (PTFE)Millex® syringe lters (EMD Millipore Catalogue No.SLCR025NK) with a 10 mL syringe, or through 0.20µm or 0.45 µm hydrophilic PTFE Millex Samplicity™lters (EMD Millipore Catalogue Nos. SAMPLG001 orSAMPLCR01) using the Samplicity™ ltration system(EMD Millipore Catalogue Nos. SAMPSYSGRor SAMPSYSBL). Determination o holdup volume and samplevolume recovery HPLC vials and Millex Samplicity™ lters (0.20 µmlters and 0.45 µm lters) were preweighed using ananalytical balance. Four Samplicity™ base units wereused or ltration. Milli-Q® water (2 mL) was applied toall lters and vacuum was applied until visual inspectionrevealed that ltration was complete. Vials werereweighed. The bottom o each lter was wiped using alaboratory wipe and the lters were weighed. Determination o analyte recovery Four drug tablets (ranitidine, loratadine, ibuproenand acetaminophen) were dissolved in their respectivedissolution media (900 mL) or 24 hours withconstant stirring at room temperature. 1.5 mL o theresulting drug suspensions were pipetted into 1.5 mLmicrocentriuge tubes and centriuged at high speed ina microcentriuge. Samples claried by centriugationwere analyzed by UV spectroscopy, and absorbancerecorded. These samples were considered to represent100% recovery. All samples were then ltered usingthe Samplicity™ ltration system using either 0.20 µmor 0.45 µm Millex Samplicity™ lters. Filtrates wereanalyzed by UV spectroscopy, and analyte concentrationsdetermined and % recovery determined in comparison tocentriuged sample. Pore sizeHoldupvolume,µL%CV  Volumefltered invial, µL%CV  0.45 µm68.75913.0821905.7310.7300.20 µm62.79012.6631907.7711.174 Table 1. Millex Samplicity™ flters eature holdup volumesless than 70 µL (N = 32). Experimental error is reported aspercent coefcient o variation (%CV).  3 Next, we tested the percent recovery (by mass)or our dierent analyte types, to simulate thepreparation o samples during dissolution testing,a common procedure during the development o pharmaceutical products. We ound that, or all ouranalytes tested, Millex Samplicity™ lters providedrecovery between 96-100% (Table 2).The Samplicity™ system saves signicant amounts o time by enabling users to process multiple samples atonce. Further, the system provides ergonomic benetsby enabling steps to be completed in batch ormat(assembly-line style) rather than repeated serially. Asa result, the Samplicity™ system requires only 7 stepsor ltration o 4 samples, while syringe ltrationrequires 24 steps or the same 4 samples (Table 3). Pore sizeAnalyteAverage percentdrug recoveryater fltration%CV  0.45 µmAcetaminophen 99.52 %1.22Loratadine95.88 %0.91Ranitidine 96.5 %0.76Ibuproen100.12 %0.130.20 µmAcetaminophen 99.9 %1.64Loratadine 96.16 %0.74Ranitidine 99.17 %0.52Ibuproen 99.99 %0.12 Table 2. High recovery (96-100%) o drug analytes aterfltration using the Samplicity™ system. Experimental error isreported as %CV. N = 16. Filtration System UsedNumber o steps orfltration o 4 samples Syringe Filter + Syringe24Samplicity™ System7 Table 3. The Samplicity™ fltration system requires ewersteps to flter 4 samples than syringe fltration. It is important that sample preparation methods donot introduce additional impurities into the sample,particularly or sensitive downstream analyses.Analytical results can be conounded by extractableimpurities in the sample, which can come rom alter, lter housing, or rom the lter manuacturingprocess. Chemical compatibility between the liquidbeing ltered and the lter system is important tominimize the extractables present in the ltrate. Wetested several solvents (acetonitrile and water shownin Figure 1) or their potential to extract impuritiesrom Millex Samplicity™ lters. Because these ltermembranes are composed o hydrophilic PTFE, whichhas broad chemical compatibility, ew impurities wereleached into either solvent (Figure 1). Extractableswere analyzed by UV spectroscopy at 214 nm.Typically, the lower the wavelength o absorbancedetection used, the higher the chance that theextractable impurities will be visible. Figure 1. S olvents fltered through Millex Samplicity™ flters (HPLC traces labeled “sample”)contained ew extractables when compared to blank solvents (HPLC traces labeled “blank”). Neither acetonitrile (let) nor water (right) extracted signifcant impurities rom 0.45 µm (top)or 0.20 µm (bottom) flters. SampleBlankSampleBlank 0.100.080.060.040.020.0-.020 2 4 6 8 10Time (min) Acetonitrile Extractables    A   b   s   o   r   b   a   n   c   e    (   2   1   4   n   m    ) 12 14 16 18 200.100.080.060.040.020.0-.020 2 4 6 8 10Time (min) Water Extractables    A   b   s   o   r   b   a   n   c   e    (   2   1   4   n   m    ) 12 14 16 18 20  A. Extractables rom 0.45 µm fltersB. Extractables rom 0.20 µm flters SampleBlankSampleBlank  0.070.050.060.040.020.030.010.0-0.010 2 4 6 8 10Time (min) Acetonitrile Extractables    A   b   s   o   r   b   a   n   c   e    (   2   1   4   n   m    ) 12 14 16 18 200.100.080.060.040.020.0-.020 2 4 6 8 10Time (min) Water Extractables    A   b   s   o   r   b   a   n   c   e    (   2   1   4   n   m    ) 12 14 16 18 20  A measure o membrane ltration perormance isthe degree to which the lter prevents particles largerthan its pore size rom passing into the ltrate. Wechallenged 0.45 µm lters with 0.5 µm diameter particlesand 0.20 µm lters with 0.3 µm particles. As shown inTable 4, both membranes caused retention o almost allparticles – 95% retention or 0.20 µm Millex Samplicity™lters and 100 % retention or 0.45 µm lters. Pore sizeAverage % retention o particles %CV  0.45 µm101.82%1.900.20 µm94.46%4.67 Table 4. Millex Samplicity™ flters retained latexmicrospheres with high (95-100%) efciency. Experimental error is reported as %CV. Certain analytical methods require that the concentrationo analyte in the sample be maintained during sampleltration. Particularly or these cases, it is important tominimize evaporation o solvent. To examine the recoveryo volatile solvents ater ltration by the Samplicity™system, we measured % recovery o the solventimmediately ater ltration (Time 0) as well as ater10 minutes o exposure to vacuum ollowing ltration(Time 10). For all three volatile solvents tested, percentrecovery was not signicantly altered ater prolongedexposure to vacuum (Table 5). Conclusion By streamlining sample preparation while still providinghigh perormance ltration, the Samplicity™ ltrationsystem has the potential to increase productivity anddata quality generated by liquid chromatography.Samples ltered using the Samplicity™ ltrationsystem will benet rom low extractables and ecientparticulate retention, rendering this sample preparationmethod compatible with sensitive downstream analysis,including highly sensitive analytical separations andmass spectrometry. Furthermore, the high analyterecovery, low holdup volume and good recovery o volatile solvents aorded by the Samplicity™ systemtranslate into the preservation o particularly precioussamples, saving time and cost o obtaining the startingmaterials or chromatography. Solvent% Recovery%CV Time 0Time 10Time 0Time 10 Acetonitrile88891%1%Tetrahydrouran92943%4%Acetone99962%5% Table 5. Percent recoveries o three volatile solvents were unchanged ater exposure tovacuum or ten minutes. Experimental error is represented as %CV. EMD Millipore, the M logo, Samplicity, and Millex Samplicity are trademarks o Merck KGaA, Darmstadt, Germany.Millex and Milli-Q are registered trademarks o Merck KGaA, Darmstadt, Germany.All trademarks o third parties are the property o their respective owners.Lit No. AN4411EN00 LS-SBU-12-05940 3/2012 Printed in the USA.© 2012 EMD Millipore Corporation, Billerica, MA USA. All rights reserved. www.emdmillipore.com/oices To Place an Order or ReceiveTechnical Assistance In the U.S. and Canada, call toll-ree 1-800-645-5476 For other countries across Europe and the world,please visit:  www.emdmillipore.com/ofces For Technical Service, please visit: www.emdmillipore.com/techservice Get Connected! Join EMD Millipore Bioscience on your avorite socialmedia outlet or the latest updates, news, products,innovations, and contests! acebook.com/EMDMilliporeBiosciencetwitter.com/EMDMilliporeBio
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