Agilent s LC/MS Portfolio and Applications Examples

Agilent s LC/MS Portfolio and Applications Examples Sean Orlowicz LC/MS Product Specialist Agilent Technologies 31August

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.

Most Common Separation Techniques Gas Chromatography vs. Liquid Chromatography Approx 80% of known compounds can be separated by LC

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

Comparison of HPLC and GC

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)

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

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

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

A big leap forward in 2006 Rough Pump Octopole 1 Turbo 1 Quad Mass Filter (Q1) 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 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

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 Prep LC 0.1 0.3 ml/min 0.3 1 ml/min 1 1.5 ml/min

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 Mille Identify proteins and peptides by fast database searches

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

Conceptual Model of a Single Quadrupole Mass Spectrometer External Ionization Source Quadrupole Mass Filter Detector

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)

6400 Series Triple quadrupole

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)

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

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

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

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

SNR 1 pg reserpine New 6490 advances in sensitivity 50 fold in 4 years 10000 6490 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) Response + MRM (455.3 -> 164.9) X10 1 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 Area Response 100 attogram to 100 picogram Verapmil on-column 10000000 1000000 Y = 65283 x 0.390412 R 2 = 0.9976 100000 10000 1000 100 10 1 0.1 1 10 100 1000 10000 100000 Femtograms

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) Massaccuracy actual - measured 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 I n t e n s it y, c o u n t s +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 I n t e n s it y, c p 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 I n t e n s it y, c p 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 I n t e n s it y, c p 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 I n t e n s it y, c p 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. I n t e n s it y, c p 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) Eliminate compounds with odd N 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

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

Table of possible chemical formulas for m/z 250.0434 (with < 2 ppm error) Eliminate compounds with even N 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

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 N C 3 H 5 N 2 + - 23.15 ppm N CH + CH 3 69.0463 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

Typical MS chromatogram? TIC BPC

Thank you!