Agilent s LC/MS Portfolio and Applications Examples Michael Scott LC/MS Product Specialist Agilent Technologies March 2013 For Research Use Only. Not for use in diagnostic procedures.
Agilent 6000 Series LCMS
Ultra High Definition LC/MS The Most Comprehensive Answer To Your Urgent Analytical Question Signal Response Sensitivity Dynamic Range Linearity Peak Resolving Power Peak Capacity Separation Speed Mass Spectrum Ionization Profile Mass Accuracy Isotopic Fidelity Mass Resolving Power Acquisition Rate
a few words about chromatography LC - MS
Agilent High Performance Infinity Series LC: Best in class Front End systems for MS Preparative LC Standard LC Ultra High Pressure LC Capillary & Nano LC High Sensitivity HPLC-Chip/MS The industry s most comprehensive LC portfolio to configure an integrated LC/MS system
Agilent LC-MS column configurations: Column Type Column I.D. Nano Capillary MicroBore 0.075, 0.10 mm 0.3, 0.5 mm 1.0 mm Column Lengths Particle Sizes 50, 150 mm 3.5 um 35, 150, 250 mm 30, 50, 150 mm NarrowBore 2.1 mm 15 250 mm 1.8, 3.5, 5 um Chip LC Nano LC Capillary LC Solvent Saver 3.0 mm 50 250 mm 3.5, 5 um Analytical 4.6 mm 15 250 mm 1.8, 3.5, 5um plus Prep columns 9.4, 21.2, 30, and 50 mm id Flow Rate Range 100 600 nl/min 3.5, 5 um 1 10 ul/min 3.5, 5 um 30 60 ul/min Analytical LC 0.1 0.3 ml/min 0.3 1 ml/min 1 1.5 ml/min Prep LC
Agilent s > 40 year heritage in mass spectrometry 1971, 5930A GC/MS 1971-2011 2005, 5975A 2008, 7000A ICP-MS 1994-2011 1994, 4500 Series 1976, 5992A 2011 7200A 2000, 7500 Series 2012 8800 QQQ LC/MS 1980-2011 Direct Liquid Injection 1980, 5985 SQ ESI on 5989 MS Engine 1994, 2000 2003 LC/QQQ Thermospray, 5987 Particle Beam, 5988 LC/MSD TOF 2006 LC/MSD, LC/MSD Ion Trap LC/QTOF Over 37,000 GC/MS, 4,500 ICP-MS and 6,500 LC/MS systems through June 2012 Agilent (and previously, as HP) has sold more quadrupole MS systems than any other mfr.
A big leap forward in 2006 Rough Pump Quad Mass Filter (Q1) Octopole 1 Turbo 1 Lens 1 and 2 Turbo 1 6100 Series SQ 10KV Detector Turbo 1 Collision cell incorporates axial acceleration for high speed MS/MS analysis 6500 Series Q-TOF 6400 Series QQQ Quad Mass Filter (Q1) Octopole 1 Quad Mass Filter (Q3) Lens 1 and 2 Collision Cell 10KV Detector Rough Over 15 Years in LC/MS Pump atmospheric sampling and patented orthogonal geometry - Result in industry leading sensitivity and robustness Turbo 1 Turbo 1 Rough Pump Turbo 1 Quad Mass Filter (Q1) Octopole 1 Turbo 1 Lens 1 and 2 Turbo 1 Collision Cell Turbo 1 Octopole 2 DC Quad Ion Pulser Turbo 2
Agilent offers a range of LC/MS systems Single quad Triple quad Ion trap TOF QTOF Page 11 Introduction to the Agilent LC/MS Portfolio Agilent Restricted 3/26/2013
Agilent 6000 Series LC/MS portfolio in 2013 1290 series UHPLC 6500 series Q-TOF 1 Model 3 Models 6200 series TOF Comprehensive Range of Software 3 Models 6400 series QQQ 4 Models 6100 series Quad
Agilent 6000 Series LC/MS portfolio High Resolution Qualitative Single Stage 6200 series TOF 6500 series Q-TOF Tandem Stage Quantitative 6100 series Quad Low Resolution 6400 series QQQ
Agilent MassHunter Workstation Software Instrument Control Real-time monitoring Method set-up Autotune Qualitative Analysis Chromatographic results Spectral results Find compounds Quantitative Analysis User filters Compound results Calibration curve
Mass Hunter Extensive application support BioConfirm Confirm identities and identify variants of proteins Metabolite ID Identify metabolites Mass Profiler METLIN Personal Metabolite Database Mass Profiler Professional Used for expression profiling Most comprehensive database made available on PC Chemometrics software to visualize large sample sets Analytical Studio Reviewer Fast analysis of large amounts of LC/MS data Easy Access Walk-up operation for instruments Spectrum Mill Identify proteins and peptides by fast database searches
Full integration: the lab, business applications, OpenLAB, the company network The Lab Business Applications GC/MS GC MS Office Legacy Data UV-vis LC PDF ELN Images ICP-MS Lab-on-a Chip CE Masshunter Workstation LIMS Research Drawings Agilent OpenLAB (Laboratory Informatics) Intranet Internet
LC/MS Ion sources Wide range of sampling conditions Sources are interchangeable between mass spectrometer platforms Multimode HPLC-Chip Multimode source Flow rates up to 2 ml/min Simultaneous operation APCI ESI NanoESI HPLC-Chip/MS Nanoelectrospray without the fuss Reproducible response and retention times On-chip enrichment for speed and sensitivity APPI AP-MALDI
6100 Series Single Quad
Quadrupole Operation Principles Each m/z value has a unique DC and RF potential that will allow that ion to be passed through the quadrupole analyzer
6100 Series Single Quadrupole LCMS Easiest Flexible Performance 6120 6130 6150
6100 Series Single Quadrupole LCMS
Agilent Jet Stream Technology Page 22
Agilent Jet Stream Enter slide show mode to see the animation on this page. Page 23 Agilent Restricted 23 June 2008, Denver
Agilent Jet Stream Ion Generation Gas Dynamics View Nebulizing gas Enhanced efficiency nebulizer Super-heated sheath gas Agilent Jet Stream Parameters Sheath Gas Flow Sheath Gas Temperature Nozzle Voltage Nebulizing Pressure Chamber Voltage Nozzle voltage Heated drying gas The super-heated sheath gas collimates the nebulizer spray and creates a dramatically brighter source Patent Pending Resistive sampling capillary Page 24
Heating the Sheath Gas in Jet Stream, time lapse Start Temperature 25 o C at t = 0 min Page 25
Narrower Spray Beam 2R Stop Temperature 400 o C at t = 8 min Page 26
Herbicides in Water Anglian Water Laboratories, Huntingdon, UK ppb For clean matrix, LC/MSD (single quad) is a very useful and inexpensive tool for screening and quantitation)
Phthalates in Children s Toys
6400 Series Triple quadrupole
Why a Triple Quad? Ancient proverb: On a dark night you can see more stars 31
Why a Triple Quad?
Multiple reaction monitoring (MRM) Quad Mass Filter (Q1) Quad Mass Filter (Q3) Collision Cell Spectrum with background ions (from ESI) 210 222 Q1 lets only target ion 210 pass through 210 Collision cell breaks ion 210 apart Q3 monitors only characteristic fragments from ion 210 for quant and qual. 165 268 280 158 191 210 158 191 170 210 250 290 190 210 150 170 190 210 160 190 no chemical background
Eight min Dynamic MRM Analysis 6460 QQQ - 250 Pesticide Screen. 500 ppt, dynamic MRM Retention Time Window = 12 sec Peak widths ~ 1 second Zorbax Eclipse Plus C18 2.1 x 100 mm (1.8 μm)
ASMS 2010 Agilent Introduces ifunnel Technology
N EW I F UNNEL TECHNOLOGY ifunnel Technology captures 6x more ions Agilent Jet Stream Thermal confinement of ESI plume Efficient desolvation to create gas phase ions Creates an ion rich zone Hexabore Capillary 6 capillary inlets Samples 6X more ion rich gas from the source Captures the majority of the gas from the source region Dual Ion Funnel Removes the gas but captures the ions Removes neutral noise Extends turbo pump life
Hexabore atmospheric sampling. 6 Bore Capillary Six bores, Half the restriction means 6 times the amount of atmospheric gas sampled AND 5 10X the number of ions sampled over wide mass range. But how do we handle all the extra gas molecules? Monolith With 6 Bores
Ion Funnel Operation RF Voltage focuses the ions to the center. RF Drive Unfocused Ions and Gas Enter Focused Ions Out DC Voltage accelerates the ions to the exit. DC Drive 38
Two-stage Ion Funnel can handle the gas load Stage 1 offset deflects the high quantity of gas exiting the hexabore capillary Line of Sight Stage 1 8-12 Torr Stage 2 1-3 Torr
New 6490 advances in sensitivity 50 fold in 4 years 10000 6490 SNR 1 pg reserpine 9000 8000 7000 6000 5000 4000 3000 2000 1000 6410 6460 0 2006 2007 2008 2009 2010
6490/iFunnel: Zeptomolar detection limits 100 attograms Verapamil on-column (200 zeptomoles) + MRM (455.3 -> 164.9) X10 1 Response 4.5 4.4 Blank 4.3 4.2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Acquisition Time (min)
Unprecedented six logs of linearity with 6490 100 attogram to 100 picogram Verapmil on-column 10000000 1000000 Y = 65283 x 0.390412 R 2 = 0.9976 100000 Area Response 10000 1000 100 10 1 0.1 1 10 100 1000 10000 100000 Femtograms
Estradiol in Human Plasma
6400 Series Triple Quad 6490 6460 6430 6420
6200 Series TOF
Formulas for TOF performance Resolving power = (m/z)/w 1/2 1000/0.025 = 40,000 922/0.022 = 41,909 Mass accuracy ( rel. mass error) actual - measured Mass accuracy 10 actual (1000.000-1000.001)/1000)x10 6 =1 ppm Therefore: 1 ppm @ 1000 m/z is 0.001Da (1 mda) 922.0092 W 1/2 = 0.022 923.0121 921.5 922.0 922.5 923.0 923.5 924.0 924.5 m/z, amu 6 In te n sity, co u n ts +TOF MS: 5.474 to 5.547 min from steroid2tofap42.wiff Agilent, subtracted (4.696 to... 1.16e5 1.10e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e4 0.00 Epitestosterone Resolving power: 6890 actual 289.2162 meas 289.2159-1.06 ppm 271.2051 269.1893 289.2159 287.1997 Max. 1.2e5 counts. 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z, amu
NPAMOZ at unit mass resolution 335.1352, -0.3 + 0.7 XIC of +TOF MS: 334.4 to 335.4 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff 6.8e4 Max. 2.0e5 cps. 123.06 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 In te n sity, cp s 4.0e4 3.5e4 3.0e4 123.05 2.5e4 2.0e4 1.5e4 1.0e4 5000.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min
NPAMOZ 335.1352 +/- 0.1 XIC of +TOF MS: 335.0 to 335.2 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff 3.4e4 Max. 1.7e5 cps. 123.06 3.2e4 3.0e4 2.8e4 2.6e4 S/N = 70:1 2.4e4 2.2e4 In te n sity, cp s 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min
NPAMOZ 335.1352 +/- 100 ppm (335.09939-335.16641) XIC of +TOF MS: 335.1 to 335.2 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 9.3e4 cps. 9000 8500 8000 7500 7000 S/N = 103:1 6500 6000 In te n sity, cp s 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min
NPAMOZ 335.1352 +/- 50 ppm (335.11614-335.14966) XIC of +TOF MS: 335.1 to 335.1 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 4.2e4 cps. 7500 123.05 7000 6500 6000 5500 S/N = 156:1 5000 In te n sity, cp s 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min
NPAMOZ 335.1352 +/- 20 ppm (335.1262-335.1396) XIC of +TOF MS: 335.1 to 335.1 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 1.0e4 cps. In te n sity, cp s 3200 3100 3000 2900 2800 2700 2600 2500 2400 2300 2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 S/N = 214:1 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Time, min 123.05
LC/ESI-TOF MS, using accurate mass 2.4e5 250.0434 2.0e5 1.6e5 1.2e5 8.0e4 252.0406 4.0e4 251.0473 0.0 250.0 250.5 251.0 251.5 252.0 252.5 253.0 253.5 m/z, amu
Table of possible chemical formulas for m/z 250.0434 (with < 2 ppm error) Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl2 250.0434-0.0336-0.1346 4 C9 H13 N2 O F P Cl 250.0432 0.1410 0.5640 4 C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5 C5 H16 N3 O F P2 Cl 250.0435-0.1715-0.6858-0.5 C3 H7 N10 O2 Cl 250.0436-0.2487-0.9949 5 C4 H13 N3 O7 Cl 250.0436-0.2541-1.0165-0.5 C4 H12 N5 O F2 P Cl 250.043 0.3407 1.3629 0.5 C6 H16 N3 F P Cl2 250.0437-0.3461-1.3845-0.5 C10 H17 O P2 Cl 250.0437-0.3712-1.4848 3 C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5
Table of possible chemical formulas for m/z 250.0434 (with < 2 ppm error) Eliminate compounds with two Cl Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl2 250.0434-0.0336-0.1346 4 C9 H13 N2 O F P Cl 250.0432 0.1410 0.5640 4 C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5 C5 H16 N3 O F P2 Cl 250.0435-0.1715-0.6858-0.5 C3 H7 N10 O2 Cl 250.0436-0.2487-0.9949 5 C4 H13 N3 O7 Cl 250.0436-0.2541-1.0165-0.5 C4 H12 N5 O F2 P Cl 250.043 0.3407 1.3629 0.5 C6 H16 N3 F P Cl2 250.0437-0.3461-1.3845-0.5 C10 H17 O P2 Cl 250.0437-0.3712-1.4848 3 C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5
Table of possible chemical formulas for m/z 250.0434 (with < 2 ppm error) Eliminate compounds with low C count Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl2 250.0434-0.0336-0.1346 4 C9 H13 N2 O F P Cl 250.0432 0.1410 0.5640 4 C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5 C5 H16 N3 O F P2 Cl 250.0435-0.1715-0.6858-0.5 C3 H7 N10 O2 Cl 250.0436-0.2487-0.9949 5 C4 H13 N3 O7 Cl 250.0436-0.2541-1.0165-0.5 C4 H12 N5 O F2 P Cl 250.043 0.3407 1.3629 0.5 C6 H16 N3 F P Cl2 250.0437-0.3461-1.3845-0.5 C10 H17 O P2 Cl 250.0437-0.3712-1.4848 3 C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5
Table of possible chemical formulas for m/z 250.0434 (with < 2 ppm error) Apply the Nitrogen Rule Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl2 250.0434-0.0336-0.1346 4 C9 H13 N2 O F P Cl 250.0432 0.1410 0.5640 4 C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5 C5 H16 N3 O F P2 Cl 250.0435-0.1715-0.6858-0.5 C3 H7 N10 O2 Cl 250.0436-0.2487-0.9949 5 C4 H13 N3 O7 Cl 250.0436-0.2541-1.0165-0.5 C4 H12 N5 O F2 P Cl 250.043 0.3407 1.3629 0.5 C6 H16 N3 F P Cl2 250.0437-0.3461-1.3845-0.5 C10 H17 O P2 Cl 250.0437-0.3712-1.4848 3 C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5 10 -> 1 compound
Molecular Formula Generator
Molecular Formula Generator
Formula Generation three criteria Scoring based on 1. Monoisotopic Mass 2. Isotope Spacing monoisotopic mass Isotope distribution C 10 H 9 ClN 4 O 2 S 3. Isotope Distribution Isotope spacing Page 59
Agilent 6230 TOF Pesticide Screening 224 pesticides, 50 pg on column 17 min. 3 min. Thiabendazole Peak Width=0.8 sec 23 data points 1.5 min. -1.8 ppm Spectral Resolution & Isotopic Fidelity
6500 Series QTOF design
QTOF 6500 Series 6510 6520 MMA > 2ppm RP > 20K MS2 Speed 10 Hz
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.
Ultra High Definition QTOF 6540 UHD
6540 Ultra High Definition QTOF Mass Accuracy Repetitive Injections 40pg reserpine on-column, 10 injections +ESI EIC(609.28066) Scan Frag=240.0V Reserpine_40pgms3.d 0.511 0.0 0.25 0.5 0.75 1 Counts vs. Acquisition Time (min) Error Run (ppm) 1 0.96 2-0.17 3 0.25 4 0.02 5 0.39 6 0.13 7 0.01 8 0.52 9 0.04 10 0.30 Mean 0.25 Std. Dev 0.32 x10 1.2 1 0.8 0.6 0.4 0.2 0 5 Isotope 609.28081 Obs % Calc % Obs m/z Calc m/z Diff (ppm) 1 100 100 609.28081 609.28066 0.25 2 35.87 37.24 610.28425 610.28393 0.52 3 8.58 8.59 611.28650 611.28671-0.34 4 1.41 1.48 612.28987 612.28941 0.75 5 0.14 0.21 613.29210 613.29203 0.11 610.28425 611.28650 612.28987 613.29210 609 610 611 612 613 614 Counts vs. Mass-to-Charge (m/z) 250 ppb mass accuracy calibration and very accurate isotopic ratios
6540 Ultra High Definition QTOF Sensitivity Full Scan MS Mode- 1 picogram 4 x10 S/N = 319 RMS Excellent sensitivity, accuracy and resolution at 1 pg on column 3 x10 (M+H)+ 2 2 Resolution ~ 33,000 609.280347 Ratio m/z Diff. Theor. Expt. Theor. Expt. (ppm) 100.00 100.00 609.2807 609.2803-0.51 37.24 36.36 610.2839 610.2832-1.25 8.59 8.39 611.2867 611.2878 1.77 1.48 1.45 612.2894 612.2926 5.29 1 1 610.283168 611.287790 612.292649 0 0.0 0.5 1 1.5 Counts vs. Acquisition Time (min) 0 607 608 609 610 611 612 613 614 615 616 Counts vs. Mass-to-Charge (m/z)
6540 Ultra High Definition QTOF Wide Dynamic Range G2421A Tune Mix m/z 322 1 sec spectrum Dynamic Range: 5,000,000 100 Peak height 5,000,000 6 x10 4 3.5 3 2.5 2 1.5 322.05432 20535 = 50,000 5 x10 2.75 2.5 2.25 2 1.75 1.5 1.25 1 0.75 0.5 0.25 0 323.05854 20415 4 x10 4 323.33313 16656 3.5 3 2.5 2 1.5 1 0.5 0 324.05976 21517 324.32499 11265 324 324.5 325 325.5 326 326.5 Counts vs. Mass-to-Charge (m/z) 324.05976 21517 324.32499 11265 3 x10 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 325.06230 21835 325 325.5 326 326.5 Co 325.06230 21835 325.06230 21835 Peak Height 100 326.20440 17254 326.20440 17254 326.20440 17254 323 323.5 324 324.5 325 325.5 326 326.5 Counts vs. Mass-to-Charge (m/z) 1 0.5 0 323.05854 20415 324.05976 21517 325.06230 21835 326.20440 17254 322 322.5 323 323.5 324 324.5 325 325.5 326 Counts vs. Mass - to - Charge ( m/z ) +ESI Scan (#27) Frag =180.0V 2GHz_accF.d
Dynamic Range Page 68
ifunnel Technology Revolutionizes Ion Sampling Proven10X Sensitivity Breakthrough For The 6490 QQQ Agilent Jet Stream Hexabore Capillary Dual Stage Ion Funnel 10X Sensitivity Breakthrough for 6550 QTOF
New 6550 ifunnel QTOF 10X Sensitivity Gain Enables Applications Sensitivity Dramatically improved quantitative capabilities New Qual/Quan Workflows Superior metabolite and protein detection Non-targeted compound screening Comprehensive Performance Enhancements Mass Resolution >40,000 50 spectra /sec MS and 33 spectra/sec MS/MS 5 orders of linear dynamic range <1 ppm MS mass accuracy; <2 ppm MS/MS Unrivalled sensitivity 6550 ifunnel Q-TOF LC/MS System ASMS 2010
Agilent QTOF Design Incorporates Cumulative Hardware Innovations Flight tube Common with TOF Collision cell Common with QQQ Ion optics Common with Q & QQQ Ion Mirror Octopole 1 Quad Mass Filter (Q1) Octopole 2 Detector Lens 1 and 2 Collision Cell DC Quad Ion Pulser Rough Pump Turbo Turbo Turbo Turbo TOF and QTOF Fundamentals Page 71 March 2007
Targeted MS/MS mode for Caffeine Calculate chemical formula given accurate mass measurement and using elements C, H, N, and O C 6 H 8 N 3 O + - 0.09 ppm CH 3 N C 5 H 8 N 3 + - 3.91 ppm CH 3 138.0662 O C + N CH 3 N H C + N N N C 3 H 5 N 2 + - 23.15 ppm CH + N N 69.0463 CH 3 CH 3 110.0717 195.0871 C 8 H 11 N 4 O 2 + + 2.84 ppm H 3 C O + N N H CH 3 O N N CH 3 Proposed structures
MS-MS Data Resolved
ID Browser: Identification of Statistically Differential Compounds
6500 Series QTOF Portfolio 6520 Femtomole sens ppm MMA >20K RP 10Hz MSMS 6540 UHD Femtomole sens ppb MMA >40K RP 20Hz MSMS 6550 ifunnel Attomole sens ppb MMA >40K RP 33Hz MSMS
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