Agilent s LC/MS Portfolio and Applications Examples
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1 Agilent s LC/MS Portfolio and Applications Examples Sean Orlowicz LC/MS Product Specialist Agilent Technologies 31August
2 Agilent s > 40 year heritage in mass spectrometry 1971, 5930A GC/MS , 5975A 2008, 7000A ICP-MS , 4500 Series 1976, 5992A A 2000, 7500 Series QQQ LC/MS Direct Liquid Injection 1980, 5985 SQ ESI on 5989 MS Engine 1994, 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.
3 Most Common Separation Techniques Gas Chromatography vs. Liquid Chromatography Approx 80% of known compounds can be separated by LC
4 Analyte Characteristics Required for LC vs. GC Volatility GC Yes ; Derivatization Time Consuming LC No; Sample must be soluble in mobile phase, LC/MS is API Atmospheric Pressure Ionization, all LC/MS is based on Chemical ionization Polarity GC Nonpolar to moderate polarity LC polar, non polar, ionic Thermal Lability GC Analyte must be able to survive high temperatures LC Analysis at room temperature Molecular Weight GC - < 1000 amu LC No theoretical upper limit e.g. large proteins
5 Comparison of HPLC and GC
6
7 Why would a lab choose LC-MS? Analytes which are difficult or impossible by GC/MS: Thermally-labile or non-volatile Poor chromatography or sensitivity Not derivatizable to solve the above problems [e.g., benzodiazepines] Existing GC/MS methods which are problematic or timeconsuming [e.g., THC in blood] Existing methods lack necessary sensitivity or specificity Improved productivity by: No derivatization, simpler sample prep Shorter runs (especially with modern LC columns)
8 Ultra high definition LC/MS Optimizing all analytical dimensions Signal Response Sensitivity Dynamic Range Linearity Separation Speed Peak Resolving Power Peak Capacity Mass Spectrum Mass Accuracy Isotopic Fidelity Mass Resolving Power Acquisition Rate
9 Agilent 6000 Series LC/MS portfolio 1290 series UHPLC 6500 series Q-TOF 6200 series TOF Comprehensive Range of Software 6400 series QQQ 6100 series Quad
10 Agilent 6000 Series LC/MS portfolio Single Stage High Resolution Qualitative 6200 series TOF 6500 series Q-TOF Tandem Stage Quantitative 6100 series Quad Low Resolution 6400 series QQQ
11 A big leap forward in 2006 Rough Pump Octopole 1 Turbo 1 Quad Mass Filter (Q1) Lens 1 and 2 Turbo Series SQ 10KV Detector Turbo 1 Collision cell incorporates axial acceleration for high speed MS/MS analysis 6500 Series Q-TOF 6400 Series QQQ Octopole 1 Quad Mass Filter (Q1) 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 Octopole 1 Turbo 1 Turbo 1 Quad Mass Filter (Q1) Lens 1 and 2 Turbo 1 Collision Cell Turbo 1 Octopole 2 DC Quad Ion Pulser Turbo 2
12 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
13
14 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 mm 1.8, 3.5, 5 um Chip LC Nano LC Capillary LC Solvent Saver 3.0 mm mm 3.5, 5 um Analytical 4.6 mm mm 1.8, 3.5, 5um plus Prep columns 9.4, 21.2, 30, and 50 mm id Flow Rate Range nl/min 3.5, 5 um 1 10 ul/min 3.5, 5 um ul/min Analytical LC Prep LC ml/min ml/min ml/min
15 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
16 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 Mille Identify proteins and peptides by fast database searches
17 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
18 6100 Series Single Quad
19 Conceptual Model of a Single Quadrupole Mass Spectrometer External Ionization Source Quadrupole Mass Filter Detector
20 Validation Results Anglian Water Laboratories, Huntingdon, UK ppb For clean matrix, LC/MSD (single quad) is a very useful and inexpensive tool for screening and quantitation)
21 6400 Series Triple quadrupole
22 Multiple reaction monitoring (MRM) Quad Mass Filter (Q1) Quad Mass Filter (Q3) Collision Cell Spectrum with background ions (from ESI) 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 no chemical background
23 Eight min Dynamic MRM Analysis 6460 QQQ Pesticide Screen. 500 ppt, dynamic MRM Retention Time Window = 12 sec Peak widths ~ 1 second Zorbax Eclipse Plus C x 100 mm (1.8 μm)
24 N EW I F U N N E L T E C H N O L O G Y 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
25 Jet Stream ion generation - gas dynamics view Super-heated sheath gas The super-heated sheath gas collimates the nebulizer spray and creates a dramatically brighter source Heated drying gas Resistive sampling capillary
26 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
27 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 Torr Stage Torr
28 SNR 1 pg reserpine New 6490 advances in sensitivity 50 fold in 4 years
29 6490/iFunnel: Zeptomolar detection limits 100 attograms Verapamil on-column (200 zeptomoles) Response + MRM ( > 164.9) X Blank Acquisition Time (min)
30 Unprecedented six logs of linearity with 6490 Area Response 100 attogram to 100 picogram Verapmil on-column Y = x R 2 = Femtograms
31 6200 Series TOF
32 Formulas for TOF performance Resolving power = (m/z)/w 1/2 1000/0.025 = 40, /0.022 = 41,909 Mass accuracy ( rel. mass error) Massaccuracy actual - measured 10 actual ( )/1000)x10 6 =1 ppm Therefore: m/z is 0.001Da (1 mda) W 1/2 = m/z, amu 6 I n t e n s it y, c o u n t s +TOF MS: to min from steroid2tofap42.wiff Agilent, subtracted (4.696 to e5 1.10e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e Epitestosterone Resolving power: 6890 actual meas ppm Max. 1.2e5 counts m/z, amu
33 NPAMOZ at unit mass resolution , XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff 6.8e4 Max. 2.0e5 cps e4 6.0e4 5.5e4 5.0e4 4.5e4 I n t e n s it y, c p s 4.0e4 3.5e4 3.0e e4 2.0e4 1.5e4 1.0e Time, min
34 NPAMOZ /- 0.1 XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff 3.4e4 Max. 1.7e5 cps e4 3.0e4 2.8e4 2.6e4 S/N = 70:1 2.4e4 2.2e4 I n t e n s it y, c p s 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min
35 NPAMOZ /- 100 ppm ( ) XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 9.3e4 cps S/N = 103: I n t e n s it y, c p s Time, min
36 NPAMOZ /- 50 ppm ( ) XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 4.2e4 cps S/N = 156: I n t e n s it y, c p s Time, min
37 NPAMOZ /- 20 ppm ( ) XIC of +TOF MS: to amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 1.0e4 cps. I n t e n s it y, c p s S/N = 214: Time, min
38 LC/ESI-TOF MS, using accurate mass 2.4e e5 1.6e5 1.2e5 8.0e e m/z, amu
39 Table of possible chemical formulas for m/z (with < 2 ppm error) Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl
40 Table of possible chemical formulas for m/z (with < 2 ppm error) Eliminate compounds with two Cl Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl
41 Table of possible chemical formulas for m/z (with < 2 ppm error) Eliminate compounds with low C count Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl
42 Table of possible chemical formulas for m/z (with < 2 ppm error) Eliminate compounds with odd N Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl > 1 compound
43 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 ppm Spectral Resolution & Isotopic Fidelity
44 6500 Series QTOF design
45 Table of possible chemical formulas for m/z (with < 2 ppm error) Eliminate compounds with even N Formula Calculated m/z mda error error (ppm) DBE C10 H13 N2 F Cl C9 H13 N2 O F P Cl C5 H12 N5 F2 Cl C5 H16 N3 O F P2 Cl C3 H7 N10 O2 Cl C4 H13 N3 O7 Cl C4 H12 N5 O F2 P Cl C6 H16 N3 F P Cl C10 H17 O P2 Cl C13 H10 N O F Cl > 1 compound
46 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 ppm CH 3 N C 5 H 8 N ppm CH O C + N CH 3 N H C + N N N N C 3 H 5 N ppm N CH + CH CH C 8 H 11 N 4 O ppm H 3 C O + N N H CH 3 O N N CH 3 Proposed structures
47 Typical MS chromatogram? TIC BPC
48 Thank you!
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