Agilent Solutions for Ultra-High Performance LC/MS. Ken Miller Global Director of Marketing, LC/MS
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1 Agilent Solutions for Ultra-High Performance LC/MS Ken Miller Global Director of Marketing, LC/MS September 29, 2009
2 Agilent Solutions - Overview Serving The Diverse Needs of the Mass Spec Community 1290 Infinity UHPLC HPLC-Chip II Separations 7100 CE Technology 7890A GC MS Detector Technology Data Analysis $0.3 B s / Application Software 34% of total revenue Solutions 7000A GC Triple Quad 7700 Series ICP-MS 6430 Triple Quad LC/MS 6540 UHD Accurate Mass Q-TOF Mass Profiler Professional w/ Pathway Analysis Personal Compound AMRT Databases MassHunter Quant Analysis Enhancements Quick Start Solution Kits for LC, GC, LC/MS & GC/MS Petrochemical Food Safety Environmental Toxicology Metabolomics / Proteomics Standards & Workflows
3 Agilent 1290 Infinity LC The best liquid chromatography system for MS EVER! Lowest Delay Volume Pump (w/o) mixer: 10 µl Pump, Fixed Loop 20 µl Pump, Fixed Loop, JetWeaver 55 µl ALS precision for small volumes: Highest <1.5% from 0.5-1µLm, <0.7% from 1-2µL, Precision 2-20 µl (40 ul) ** Pump Active Damping: RT stability < 0.2 % (1.5 min runs)** Best Autosampler Performance <0.002% carry-over with Chlorhexidine Optional needle seat backflushing with FlexCube Fixed Loop or Variable Loop Injections Greatest Productivity p g 2mL/min for highest resolution per time Reduced Ion & Matrix Suppression HT-Solution for up to 2000 samples/day (ACR) Complete Integration and control from MassHunter Enables method conversion from/to any (U)HPLC ** preliminary Minimum Specs
4 1290 Infinity Binary Pump - A Peek Under The Hood! Degasser Solvent selection valve Jet Weaver High Resolution Pump drives Multi-layer Heat exchanger Silicon Carbide Pistons Channel A Purge Valve Channel B Page 4
5 1290 Infinity Compatible with any HPLC and UHPLC bar 1200 A new power range providing maximum performance, flexibility, compatibility and investment protection 1290 Infinity 1000 Acquity 800 Dionex RSLC Thermo Accela Agilent RRLC Shimadzu UFLCXR Standard LC ml/min
6 HPLC-Chip II Enhanced Performance & Applications Carbon Implantation Technology (patent pending) Optimized connections Improved precision 2X extended Chip life lower cost per analysis New PhosphoChip Integrated TiO 2 enrichment and analysis Improved sensitivity Much faster time-to-result t Powerful New Applications Combination with Agilent 6400-Series Triple Quads Pharma: ADME/DMPK microdosing Dried Blood Spots Targeted Protein/Peptide Quant
7 HPLC-Chip II Enhanced Performance & Applications Carbon Implantation Technology (patent pending) Optimized connections Improved precision 2X extended Chip life lower cost per analysis New PhosphoChip Integrated TiO 2 enrichment and analysis Improved sensitivity Much faster time-to-result t Powerful New Applications Combination with Agilent 6400-Series Triple Quads Pharma: ADME/DMPK microdosing Dried Blood Spots Targeted Protein/Peptide Quant
8 Relentless QQQ Innovation Fall B Winter ms Polarity Switching HotBox Option (2X) Agilent Jet Stream 5-10X Sensitivity 100 ms +/- Switching 2 ms MRMs 6430 Spring ms +/- Switching 1 ms MRMs Dynamic MRM Peptide Optimizer 6410 Fall fg Sensitivity Spec High Reliablity Very Affordable 6410A Spring ,000 m/z Mass Range Chip Cube Compatible Optimizer and DB Excel 2007 Reporting 2-5x Faster MassHunter MS Optimizer Compliance SW support for 21 CFR Part 11 MassHunter Quant DA Batch at a Glance Parameter-less Integrator Excel Reporting
9 6400 Triple Quadrupole Product Line Excellent Value and Performance 6410 Triple Quadrupole LC/MS Robust, Easy-to-Use. Overall lowest cost of ownership. Axial Acceleration Collision Cell Polarity switching MRM analysis Solid performance, good sensitivity 6430 Triple Quadrupole LC/MS Fast, robust, sensitive quantitation. Ideal for HPLC-Chip 6460 Triple Quadrupole LC/MS The world s most sensitive Triple Quadrupole LC/MS Fast optimization with Optimizer Fast polarity switching Dynamic MRM methods Complex multi-analyte analyses Targeted Protein Quantitation Agilent Jet Stream extreme, sub-fg sensitivity! Perfect for the most demanding applications Fast polarity switching Dynamic MRM methods
10 6540 UHD Accurate Mass Q-TOF LC/MS The Highest Performing Bench-top Q-TOF - By Far Exceptional Ultra High Definition Performance With No Trade-Offs Up to 40, resolution Excellent isotopic fidelity Mass measurement error down to 500 ppb 5 orders of linear dynamic range Femtogram-level sensitivity with Agilent Jet Stream FAST acquisition for UHPLC up to 20 spectra/second Made Possible by Continuing Technology Breakthroughs Ion Beam Compression (IBC) cools & focuses ion beam Extended Flight Tube with Enhanced Mirror Technology (EMT) New Photonis Fast Bipolar Detector The Ultimate Qualitative Analysis System Proteomics/Metabolomics Non-targeted food/environmental screening Impurity analysisa s Metabolite ID
11 MassHunter Workstation One software for all Agilent MS Systems Minimize learning (and maximize productivity) Minimize learning (and maximize productivity) with a common software for all of your mass spec systems
12 Driving Applications Forward New MassHunter Software Tools Enable Huge Productivity Gains High Throughput Quantitation MH Optimizer quickly and easily optimizes MS/MS signal Dynamic MRM methods deliver robust assays faster Easy export to WATSON LIMS Fast flexible Custom Reporting is 10X faster High Throughput Targeted Screening Personal Compound Databases (PCD) use accurate mass and RT Available for: Metabolomics 23,000 compounds Pesticides id 1,600 Toxicology/Forensics 7,000 New MS/MS Library Searching Capability Proteomics / Metabolomics & Non-targeted Screening New Mass Profiler Professional Intuitive workflows Powerful statistical tools rendered easy-to-apply Pathway Architect for direct biochemical pathway interrogation
13 Ultra High Definition Optimizing all Analytical Dimensions Sensitivity Dynamic Range Signal Response Linearity Peak Resolving Power Peak Capacity Separation Speed Mass Spectrum Ionization Profile Mass Accuracy Isotopic Fidelity Data Mining Mass Resolving Power Differential Profiling Acquisition Rate Annotation
14 High Definition Mass Spec for Fast LC Practical Considerations As peaks get narrower, the MS detector must scan faster. MS cycle times are reduced. x Excellent 4 Data Quality: Avg W W 1/2 = 0.63 sec 1/2 = 0.63 sec 3 Avg. W = 1.8 sec 5 points across W 1/2 13 points across W 2 Area RSD [%] = 4.7 SNR (3RMS) = W = 1.8 sec Counts vs. Acquisition Time (min) MS Cycle time = ms During each cycle, the MS system must: Triple Quad: analyze many MRM transitions and switch polarity TOF/Q-TOF: TOF: acquire as many accurate mass MS and MS/MS scans as possible
15 Agilent s New 6430 and 6460 Triple Quads The ideal Triple Quads for Agilent s new 1290 Infinity UHPLC p g y High Pressure 2mL/min fast chromatography high resolution columns High Performance. Reduce run times by > 50% Analyze > 1,000 samples per day Increase sensitivity with sharp peaks 1290/6460 Triple Quad 1290/6430 Triple Quad 6460 Triple Quad LC/MS 6430 Triple Quad LC/MS
16 Comparative sensitivity 6430 vs 6460 The 6460 is about 5 times more sensitive than 6430 for reserpine 6460 Typical S/N > 2,100:1 1 pg reserpine where noise = 1 X RMS 6430 Typical S/N > 400:1 1 pg reserpine where noise = 1 X RMS
17 Jet Stream Technology on 6460 Triple Quad: delivers more sensitivity with tighter spray plume Super heated gas confines spray plume to increase analyte concentrations near the orifice with gas dynamics
18 Sub-femotgram sensitivity with Attograms verapamil on-column
19 Power Range for 1290 Infinity LC most powerful UHPLC bar 2.1mm ID 3 4.6mm ID Power = Pressure x Flow Rate Agilent 1290 Infinity Vendor A This app falls in the Power Range 800 Vendor E Vendor C Agilent RRLC Vendor D Standard LC ml/min
20 1290 Infinity - Ultra High Performance Separations Challenges for Quant sufficient sampling across peak mau Typical Peak Width ~ 2 sec min Peptide Map of Tryptic Digest of BSA run on Agilent RRHT Zorbax SB-C18, 2.1x150mm, 1.8µ
21 Polarity Switching: is it fast enough for UHPLC? A. Switch power supply from positive to negative B. Stabilize ion optics C. Run negative MRM transitions D. Switch power supply from negative to positive E. Run positive MRM transitions Q1 Q3 a Collision Cell Detector Q1 Q3 a Collision Cell Detector
22 Metabolism study with negative/positive MRMs: 6460 Triple Quad with 30 millisecond polarity switching Flow rate 1.5 ml/min, 1,070 bar Flow rate 1.0 ml/min, 850 bar x MRM ( > 165.1) Verapamil Verapamil, 0.54 min Avg W 1/2 = 0.44 sec 11 points across W Rel. Area RSD [%] = 4.0 RSD [%] = 4.0 x10 5 +MRM ( > 165.1) Verapamil Verapamil, 0.75 min Avg W 1/2 = 0.71 sec 15 points across W Rel. Rel. Area Area RSD RSD RSD [%] RSD [%] = [%] = 2.9 [ 2.9 x10 5 +MRM ( > Buspirone, 0.22 min Avg W1/2= 0.70 sec 20 points across W Rel. Area RSD [%] = 4.2 RSD [%] = 4.2 x MRM ( > Buspirone Buspirone, 0.40 min Avg W 1/2 = 1.3 sec 35 points across W Rel. Rel. Area Area RSD RSD [%] [%] = [%] = 1.8= RSD [%] = 1.8 x10 4 +MRM ( > 215.2) Dextromethorphan, D 0.30 min t th h Dextromethorphan Avg W 1/2 = 0.98 sec 28 points across W Rel. Area RSD [%] = 2.2 RSD [%] = 2.2 x MRM ( > 215.2) Dextromethorphan th h Dextromethorphan, 0.53 min Avg W 1/2 = 1.3 sec 35 points across W Rel. Area RSD [%] = 2.7 RSD [%] = 2.7 x MRM ( > Diclofenac Diclofenac, 0.73 min Avg W 1/2= 0.37 sec 9 points across W Rel. Area RSD [%] = 7.8 RSD [%] = 7.8 Analysis time < 0.8 min Counts vs. Acquisition Time (min) x MRM ( > MRM ( > Analysis time < min Diclofenac, 1.04 min 1.04 min Avg W 1/2= 0.52 sec 11 points across W Rel. Area RSD [%] = 6.5 RSD [%] = Counts vs. Acquisition Time (min)
23 Polarity switching with UHPLC & 6460 Triple Quad 30 msec polarity switching for sub-2 second peaks 3 x MRM ( > Time (min) Diclofenac Average W 1/2 = 0.37 sec Counts vs. Acquisition points across 1.5 sec peak 9 points across W Rel. Area RSD [%] = 7.8% W = 1.5 sec
24 Polarity switching versus non-switched method Correlation of methods Non-sw witched ana alysis Buspirone r 2 = Selected example: % Verapamil remaining r 2 = Dextromethorphan t 5 r 2 = t 35 t 25 t 15 t 10 Diclofenac r 2 = Pos/Neg switching analysis
25 No Time for Time Segment Breaks with UHPLC pesticide analysis with 1290/6460 LC/MS System Counts versus Acquisition iti Time
26 Dynamic MRM solves the crowded peak problem monitors ion transitions only when compounds elute Const. Cycle Time: Concurrent MRMs Variable dwell Time: Dynamic MRM: fewer concurrent MRMs than with Time Segments import retention times add a uniform retention time window 0.5 min for example
27 300 Pesticides: 15 min Analysis with DMRM Mean area RSD = 3.2% with Dynamic MRM Cycle Time: 200 msec Page 27
28 Eight Minute Analysis of 250 Pesticides 1290 Infinity UHPLC and 6460 Triple Quad& with DMRM x ppt, dynamic MRM Peak widths ~ 1 second Cycle time: 100 msec Min dwell: 2.5 msec Counts vs. Acquisition Time (min) Zorbax Eclipse Plus C x 100mm (1.8μm)
29 Target Screening of PPCPs in Water Matrices Using UHPLC and LC/MS/MS QQQ Pharmaceuticals and Personal Care Products (PPCPs) refer, in general, to any product used by individuals for personal health or cosmetic reasons or used by agribusiness to enhance growth or health of livestock. PPCPs comprise a diverse collection of thousands of chemical substances, including prescription and over-the-counter therapeutic drugs, veterinary drugs, fragrances, and cosmetics.
30 EPA 1624 Method for Group 1 Pharmaceuticals 46 pharmaceuticals 22 minutes 2.1 x 100 mm column 3.5 micron EPA Standard Method 1200 RRLC X
31 EPA 1624 Method: 1290 UHPLC/6460 Triple Quad 46 pharmaceuticals 11 minutes 2.1 x 100 mm column 1.8 micron 1290 Infinity UHPLC 0.6 ml/min at 750 Bar
32 Pesticide application kit for 6400 Series Triples G1733AA -- Data Base Kit Pesticide DMRM Includes: 600-compound database, a positive and negative ion test mix with their analysis methods. The methods contain compound names, optimal settings, and retention times for the Dynamic MRM. The Application Kit Quick Start Guide The Application Kit Quick Start Guide shows how to run the test mixes and create a Dynamic MRM method.
33 Pesticide standard in the application kit diazinon pyraclostrobin malathion molinate metazachlor metosulam atrazine carbofuran metoxuron imazalil dimethoate thiabenzazole imazapyr aminocarb
34 1290 Infinity LC and the 6460 or 6430Triple Quad The Ideal, high-speed Quant System! Sensitivity from femtogram to nanogram Fast polarity switching 30 millisec Fast analysis times - < 2 min with good RSDs 4,000 DMRMs for complex samples > 1,000 Analyses per day 6460 Triple Quad LC/MS 6430 Triple Quad LC/MS Page 34
35 Agilent s New 6540 Ultra High Definition QTOF Research Performance in a Benchtop Format 40,000 Resolving Power <1 ppm MS <3 ppm MS/MS Mass Accuracy 20 Spectra/s 2 pg 50:1 Reserpine S/N 5 Decades in Spectrum Dynamic Range Excellent Linearity and Isotopic Fidelity Supports Standard ESI, Agilent Jet Stream and HPLC-Chip Unsurpassed Analytical Capacity in a Benchtop
36 Enhanced Ion Flight Tube and Mirror Technology Stable, Sensitive, High Resolution 1ppm/ C Expansion Coefficient for Inner Flight Tube virtually eliminates calibration drift due to flight tube elongation. 2 nd Order Temporal Focusing Ion Mirror uses high transmission Harp Grid for maximum sensitivity Ion Mirror 6530 Q-TOF 6540 UHD Q-TOF Octopole 2 Detector DC Quad Ion Pulser Turb Making Research Grade Performance o possible in a Benchtop Format
37 Ion Beam Compression (IBC)* Technology Drives Higher Resolution Active Ion Beam Compression simultaneously maximizes ion transmission and reduces beam divergence Exit from collision cell Into slicer and time-of-flight pulser region Narrowed beam slits enables a mass resolving power of 40K Active Ion Beam Compression is achieved with Agilent s Axial Ion Acceleration Technology applied to a tapered Ion guide design. * Patent pending Making Research Grade Performance possible in a Benchtop Format
38 Next Generation Ultra High Speed Detector New Bipolar TOF Detector New ultra fast and high efficiency scintillator New ultra fast response PMT design continues the tradition of high dynamic range and detector lifetime Developed by Photonis with Agilent TOF Technology Specifically enhances Resolution in 2Ghz Ext. Dynamic Range Mode 2 nsec/div Single Ion Response ~800 psec FWHM Making Research Grade Performance possible in a Benchtop Format
39 Ultra High Speed Acquisition From Agilent s Leadership in GHz Speed Electronics 4 GHz Acquisition for Maximum Resolving Power and <1ppm Mass Accuracy 5 Decades of in-spectrum Dynamic Range from 2-Channel x 2 GHz Dual Gain Mode Dual Input Agilent pre-amplifiers Picture of 4GHz board Goes here 4GH GHz (8bit)A Analog-Digital-Converter it l t Adapted from Agilent s High Speed Oscilloscope Systems Ultra High Speed FPGAs process and store transients in real time FPGAs 4 GHz Agilent ADC Making Research Grade Performance possible in a Benchtop Format
40 6540 Ultra High Definition Q-TOF Resolving Power Across the Mass Range 5 x R= R= R= R=30218 R= R= R= R= R= Counts vs. Mass-to-Charge (m/z) Resolution is Scan Rate Independent
41 Enhancements in Resolving Power High Resolution and Ext. Dynamic Range Modes solving Po ower Re High Resolution 6540 Ext. Dynamic Range 6530 High Resolution m/z Ext. Dynamic Range
42 6540 MS Resolution Is Invariant With Acquisition Rate - Pesticides Example Resolution azoxystrobin 404 m/z cyproconazole 292 m/z cyprodinil 226 m/z diazinon 305 m/z linuron 249 m/z paclobutrazol 294 m/z propazine 229 m/z tetraconazole 372 m/z tricyclazole 190 m/z Acquisition Rate (scans/second) ASMS 2009 UHPLC 6540 QTOF lunch and learn Page 42 June 2009
43 6540 Ultra High Definition QTOF Mass Accuracy Repetitive Injections 40pg reserpine on-column, 10 injections +ESI EIC( ) Scan Frag=240.0V Reserpine_40pgms3.d Counts vs. Acquisition Time (min) x Error Run (ppm) Mean 0.25 Std. Dev Isotope Obs % Calc % Obs m/z Calc m/z Diff (ppm) Counts vs. Mass-to-Charge (m/z) 250 ppb mass accuracy calibration and very accurate isotopic ratios
44 6540 Ultra High Definition QTOF Sensitivity Full Scan MS Mode 4 x10 1pg reserpine on-column 3 x10 (M+H)+ 2 S/N = 319 RMS 2 Resolution ~ 33, Ratio m/z Diff. Theor. Expt. Theor. Expt. (ppm) Counts vs. Acquisition Time (min) Counts vs. Mass-to-Charge (m/z)
45 Coeluting Metabolites With Parent Drug: Need Wide Dynamic Range Five decades of response in a single scan verapamil dihydroxy y metabolite of verapamil x million (M+H)+ 2.6 counts desmethyl metabolite monohydroxy metabolite counts (M+H) Counts vs. Mass-to-Charge (m/z) 25 counts Page 45
46 Resolving Isobaric Co-eluting Species Isobaric Co-eluting Pair m/z ppm Difference Tricyclazole Propazine Methiocarb Diethofencarb fragment Diethofencarb fragment Cyprodinil Cyproconazole - 37 Cl Paclobutrazol Clofentezine- 37 Cl Diazinon X (1.3 min) Differentiate by RT No fragment m/z Paclobutrazol X X 0.44 ppm R=29, Cyproconazole- 37 Cl X X X ppm Cyprodinil (0.53 min) R=30, X Mass-to-Charge (m/z) X Chlorfenvinphos X Triflumuron Tetraconazole Azoxystrobin fragment X X X Resolved seven pairs of isobaric coeluting species (ICS) which have similar retention times and accurate mass values for their adduct ions or fragments IMSC 2009 Infinity LC 6540 QTOF lunch and learn Page 46 August 2009
47 Identify These Pesticides By Isotopic Pattern And Mass 3 cyproconazole 4 paclobutrazol IMSC 2009 Infinity LC 6540 QTOF lunch and learn Page 47 August 2009
48 Pesticides in Pepper Matrix, 10 ppb 3 Scans/sec Acquisition Rate (M+H)+ Methomyl Imidacloprid (M+H) ppm R =28, (M+H)+ Diethofencarb Triflumuron (M+H) ppm R =31,700 TIC Acquisition Time (min) ppm Flufenoxuron ppm R =29,100 (M+H) R =24,200 (M+H) ppm (M+H)+ (M+H)+ (M+H)+ (M+H)+ R =34, (M+H)+ (M+H)+ (M+H) (M+H) (M+H) (M+H)+ (M+H)+ (M+H) (M+H) (M+H) Acquisition Time (min) IMSC 2009 Infinity LC 6540 QTOF lunch and learn Page 48 August 2009
49 6530 vs. 6540: Comparison of Peptide Mass Spectra Q-TOF Q-TOF CCTKPESERMPCTEDYLSLILNR Q-TOF Q-TOF Q-TOF Q-TOF IMSC 2009 Infinity LC 6540 QTOF lunch and learn Page 49 August 2009
50 Peptide Mapping: Serotransferrin x10 3 Peak width 1.1 sec 6 27 data points ppm Counts vs. Acquisition Time (sec) IMSC 2009 Infinity LC 6540 QTOF lunch and learn Page 50 August 2009
51 Malaria Metabolomics RESULTS AND BIOLOGICAL SIGNIFICANCE Page 51
52 Malaria Infected Red Blood Cell Study
53 PCA analysis of all Red Blood Cell samples reveals separation based on ph of extraction solvent ph 2 7 9
54 ID Browser Identified Arginine to be statistically differential in malaria infected cells Page 54
55 Pathway analysis in MPP showing differential abundances for three compounds in the urea cycle L Arg (Arginine) Ornithine Citrulline Infected Blood cells Non infected
56 6540 Ultra High Definition Q-TOF Compatible with unmatched chromatographic peak capacity and resolving power of latest 1290 Infinity UHPLC technology Superb MS mass accuracy and resolution invariant with acquisition rate Sophisticated identification software and algorithms handle complex samples, overlapping isotopic patterns
57 Proteomics - From Discovery Mode to Validation Peroxidase in Human Plasma Page 57
58 Discovery phase to Validation : MRM Selector Biomarker validation workflow Run samples on Q- TOF for protein ID in data-dependent d d t MS/MS mode. Step-1 Q-TOF Step-2 Spectrum Mill Search QTOF data using Spectrum Mill Use Spectrum Mill MRM Selector to create a list of MRM transitions with RT Import the MRM list into QQQ Acquisition software Run samples on QQQ in Dynamic MRM mode Step-3 QQQ Step Mass Prof Integrate the MR chromatograms Import quantitatio results into MPP perform statistica analysis Page 58
59 Depleted Human Plasma Sample analysis Replicate LC/MS runs HPLC Chip / QTOF Spiked with 0,0.5 and 5fmol per 0.5ug plasma Data Dependent Protein IDs from Spectrum Mill Page 59
60 MRM Selector Generates MRM method from discovery QTOF data Page 60
61 Dynamic MRM Page 61
62 Dynamic MRM Overlaid 2000 MRM chromatograms acquired in a single run using Dynamic MRM Page 62
63 Mass Profiler Professional Statistical Processing of MRM Data Four peptides from peroxidase were highlighted in green. The mean of 443 MRM abundances is displayed (black) to show the peptides from plasma did not vary from sample to sample. B1 B2 B3 A1 A2 A3 M1 M2 All Samples Page 63
64 Principle Component Analysis Matrix and 2 different peroxidase levels Samples at different peroxidase concentrations were correctly grouped together. Page 64
65 Hierarchical Clustering comparing different peroxidase concns. A condition was generated with peroxidase concentration color-coded on the tree branches, along with the peptide features labeled on each row. The heat map is colored from blue to red, where blue is low abundance and red is high abundance. The full view of all the features is on the left. The zoom view is M1 M2 A1 A2 A3 B1 B2 B3 on the right. Page 65
66 Sensitivity : Peroxidase in plasma matrix (per 0.5ug) M A B matrix 500amole 5fmol # MRM RT Cycle Min. Max. # window time dwell concurrent (min) (ms) (ms) MRM %RSD Area RSD RT (min) Reproducibility of MS response and RT Page 66
67 The 1290 Infinity UHPLC + Agilent MS: The Best LC/MS Solution Ever! Triple Quad 6460/6430/6410 Sensitivity! Productivity! 1290 resolution/speed Dynamic MRM, 30ms pos/neg switching TOF / Q-TOF Speed! (narrow peaks, fast scanning MS, maximum data/unit time) Comprehensive analysis Ultra-High Resolution Productivity!
68 THANK YOU!
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