Agilent s New 7000B Quadrupole GC/MS/MS:

Agilent s New 7000B Quadrupole GC/MS/MS: Evolving technology for higher performance tools Terry L. Sheehan, Ph.D. GC/MS/MS Product Manager Agilent Technologies Santa Clara, CA Page 1

Triple Quadrupole MS/MS (also called tandem quadrupole) is changing the way scientists think about GC/MS Page 2

Transmission Quadrupole MS & MS/MS Sensitivity and Selectivity Scale Least Sensitive Most Sensitive Fast 12,500 u/s (5975C) Fast 6,250 u/s (7000) Targets & Non-targets < < Typical < 2,000 u/s Targets & Non-targets Dwell 20-50 ms Dwell 1-50 ms Targets only *Multiple Reaction Monitoring Also called SRM for Selected Reaction Monitoring (similar to SIM Selected Ion Monitoring) Page 3

Transmission Quadrupole MS & MS/MS Sensitivity and Selectivity Scale Least Sensitive Fast 12,500 u/s (5975C) Fast 6,250 u/s (7000) Targets & Non-targets Least Selective < < Typical < 2,000 u/s Targets & Non-targets Most Sensitive Dwell 20-50 ms Dwell 1-50 ms Targets only Most Selective Unit mass resolution < < Unit mass + AMDIS deconvolution Targets only per DBL Page 4

Summary of Relative Performance Factors GC/MSD Scan GC/MSD SIM GC/MSD Scan-DRS GC/TQ MS/MS S from analyte + +++ + +++ S from matrix -- -- - 0 N from neutrals = = = Ultra low N from matrix --- --- - 0 N from "bleed" Low Low Lower 0 MDL (clean) Very Low Lowest Lower Lowest MDL (very dirty) Low Low Lower Lowest Quant Error Low Low Lower Lowest MDL = S analyte N neutral + N matrix + N GC bleed Quant Error = S analyte S matrix + S matrix But scan is essential for non-targets (unknowns)! Page 5

Fundamental Benefits of MS/MS for GC Higher Selectivity to Identify Target Analytes in Complex Matrices Consistently High Sensitivity for Target Analytes in Complex Matrices Page 6

MS/MS Eliminates Scan and SIM Interferences Single Quad MS selectivity proportional to spectral resolution no selectivity against ions with same m/z interference analyte unit mass resolution Page 7

MS/MS Eliminates Scan and SIM Interferences Single Quad MS selectivity proportional to spectral resolution no selectivity against ions with same m/z interference analyte Product 2 unit mass resolution Product 3 Product 1 analyte Product Ion Spectrum Page 8

MS/MS Eliminates Scan and SIM Interferences Single Quad MS selectivity proportional to spectral resolution no selectivity against ions with same m/z interference analyte Triple Quad MS High probability that one or more of the product ions will be a unique dissociation product of the precursor only Product 2 unit mass resolution Product 3 Product 1 interference analyte Product Ion Spectrum Page 9

MS/MS Ensures Lowest Detection Limits EI: spectrum of analyte can also include ions from matrix, column bleed, gases, etc. Q1 SIM isolate precursor before CID chemical noise from these other ions is eliminated Page 10

MS/MS Ensures Lowest Detection Limits EI: spectrum of analyte can also include ions from matrix, column bleed, gases, etc. Product 2 Q1 SIM isolate precursor before CID Product 3 Product 1 chemical noise from these other ions is eliminated CID + Q2 SIM Lower m/z Product Ions measured against zero chemical noise for consistently lower LOQ and LODs Page 11

SIM Limited by Unit Mass Resolution in diesel fuel GC/MS Single Quad SIM Interfering matrix peaks = chemical noise 100 57 C 20 Eicosane 100 Hexachlorobenzene 284 43 Cl Cl Cl 71 50 85 50 142 Cl Cl Cl 29 99 113 127 141 155 169 183 197 211 225 238 253 282 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 (mainlib) Eicosane 107 71 177 118 95 24 35 83 130 4753 0 153 165 190 237 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 (mainlib) Benzene, hexachloro- 214 249 Page 12

SIM Limited by Unit Mass Resolution in diesel fuel GC/MS Single Quad SIM Interfering matrix peaks = chemical noise 100 57 100 284 43 Cl Cl Cl 71 50 85 50 142 Cl Cl Cl 29 99 113 127 141 155 169 183 197 211 225 238 253 282 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 (mainlib) Eicosane 107 71 177 118 95 24 35 83 130 4753 0 153 165 190 237 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 (mainlib) Benzene, hexachloro- 214 249 Page 13

SIM Limited by Unit Mass Resolution in diesel fuel GC/MS Single Quad SIM Interfering matrix peaks = chemical noise At unit mass resolution SIM, HCB is buried by the more intense peaks from C 20 to C 22 hydrocarbons Page 14

MS/MS Succeeds Where MS Fails in diesel fuel GC/MS Single Quad SIM Interfering matrix peaks = chemical noise GC/MS Triple Quad SRM A chromatographer s dream: single peak on flat baseline Page 15

Pesticide Analysis: Comparing SIM to MS/MS SIM EIC (246) Apple Cabbage Ginseng X Orange X Spinach Page 16

Pesticide Analysis: Comparing SIM to MS/MS SIM EIC (246) MRM (2 transitions) Apple Cabbage Ginseng 246.0 -> 176.1, 246.0 -> 175.1 x10 4 Ratio=23.5 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0-0.1 11.8 11.9 12 12.1 12.2 12.3 A c q u isitio n T im e (m in ) 246.0 -> 176.1, 246.0 -> 175.1 x10 3 Ratio=22.9 8 7 6 5 4 3 2 1 0 11.8 11.9 12 12.1 12.2 12.3 A c q u is itio n T im e (m in ) 246.0 -> 176.1, 246.0 -> 175.1 x10 4 Ratio=23.3 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Counts Counts Counts 0 S/N = 448 S/N = 241 S/N = 446 X Orange 11.8 11.9 12 12.1 12.2 12.3 Acquisition Time (min) 246.0 -> 176.1, 246.0 -> 175.1 x10 3 Ratio=23.6 8 7 6 5 4 3 2 1 Counts S/N = 456 0 X Spinach 11.8 11.9 12 12.1 12.2 12.3 A c q uisitio n T ime (min ) 246.0 -> 176.1, 246.0 -> 175.1 x10 4 Ratio=24.0 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Counts 0-0.1 11.8 11.9 12 12.1 12.2 12.3 A c q u is itio n T im e (m in ) S/N = 260 Page 17

7000B MS/MS: High Performance Components The best technologies from the industry leading 5975C Series GC/MSD Heated monolithic gold plated quartz quadrupole Reliable, inert, high performance ion source High Signal/Low Noise Triple-Axis Detector PCI/NCI ion source and the 6400 Series LC/MS/MS Linear acceleration enhanced hexapole collision cell MassHunter software New inert, high temp, extraction EI source Split Flow Turbo Gold plated quartz, hyperbolic quadrupoles New Ramped Iris Triple-Axis Detector High pressure hexapole collision cell with Patent Pending Helium Quench Gas Hinged access to source and detector Page 18

7000B MS/MS: Hot (200ºC) Quartz Analyzers High temperature analyzers stay clean even with highly concentrated, complex matrices Gold plated quartz, hyperbolic quadrupoles Page 19

Proprietary Heated Quartz Quadrupole High temperature (200ºC) operation results in greater reliability with dirty samples MS/MS methods typically have more dirty samples and benefit even more from the higher temp quad analyzer Virtually eliminates the need to clean quads as a maintenance issue More stable tunes and methods over a longer period of time in real world sample environments Page 20

Hyperbolic, Gold-coated, Quartz - Proprietary! Why quartz? Low coefficient of linear expansion (expansivity; α) dl = α L 0 dt [t = ºC; dl = change in length (m); L 0 = length at 0 ºC] α varies greatly for different materials (m/m K x 10-6 ) Stainless steel 304 = 17.3; Stainless steel 316 = 16.0 Inconel (Ni alloy) = 12.6; Monel (Ni/Cu alloy) = 13.5 Glass pyrex = 4.0; Quartz, fused = 0.59 For change from 25 to 150 ºC, which material expands the LEAST? But what is the analytical value? Low thermal expansion means dimensions/alignments do not change After maintenance, the quartz quad can be baked at 200 ºC Under operation, the quartz quad can be run at 150, 180 or even 200 ºC Heat is the friend of GC/MS methods true for the source and analyzer! Page 21

More Information on the Agilent Website Detailed Video Page 22

7000B MS/MS: Collision Cell with Helium Quench Highly efficient dissociation of precursor ions Maximum transitions per second without product ion carryover (cross-talk) High pressure hexapole collision cell with Patent Pending Helium Quench Gas Reduction of metastable helium from EI source Page 23

Transmision Hexapole Cell: Better Transmission Characteristics Mass Range Transmission Quadrupole Hexapole Octapole 1.2 1 0.8 0.6 0.4 0.2 0 0 500 1000 1500 m/z GC/MS m/z=1050 Quadrupoles are the best mass filters (analyzers) Hexapoles and octapole are the best transmission devices Page 24

500 Transitions/sec: Why Is This Important? Narrow GC peaks (width <1-2 s) Sufficient data points (15) to perform acceptable/accurate quantification Many compounds to monitor in single run (multiresidue) when coeluting peaks are quite frequent Regulated environment where QC checks are required to ensure data validity Confirmatory transitions according to the 96/23/CE directive Page 25

Measured Collision Cell Clearing Time 1 msec No product ion signal = no cross-talk at 500 transitions per second Page 26

Collision Cell Process: Typical Description Collision Cell 1 ml/min N 2 Collision Gas Quad Analyzer Quad Analyzer Source Precursor Ions In collision induced dissociation Product Ions Out Detector This is a good description for LC/MS/MS, but it is not complete for GC/MS/MS Page 27

Collision Cell Process: Full Description for GC/MS Source He* + Quad Analyzer Collision Cell Precursor Ions In 1 ml/min N 2 Collision Gas collision induced dissociation Quad Analyzer Product Ions Out At the detector, metastable helium generates neutral noise. + He* Detector A high population of highly energetic helium metastables are produced in an EI source; since metastable helium is not charged, it can pass through mass analyzer field into the collision cell and through to the HED-EM In GC/MS, neutral noise is buried in much higher chemical noise. In GC/MS/MS, chemical noise is greatly reduced so neutral noise is a critical source of noise. Page 28

Collision Cell Process with Helium Quench Gas Collision Cell 1 ml/min N 2 Collision Gas Quad Analyzer Quad Analyzer Source He* + Precursor Ions In collision induced dissociation Product Ions Out + He* Detector He Buffer Gas He* + He 2 He + heat Transmission of metastable helium to the detector is greatly reduced; the Triple-Axis detector further reduces neutral noise for ultra-low neutral noise. Page 29

Benefit from Helium Quench tested with hexachlorobenzene +MRM(283.79999->213.90039) 1pgHCB_std_cond_UnkGain_2090EMV_He0pct_05.d x103 Noise(RMS) =20.08; SNR(6.470min) =341.0 Same Signal 7.5 7 6.5 6.470 Noise Decreased 6 5.5 5 Helium Quench Gas OFF 4.5 4 +M RM(283.79999->213.90039)1pgHCB_std_cond_UnkG ain_2090em V_He0pct_05.d S)=20.08;SNR(6.470m in)=341.0 x102 Noise(RM 2 1.9 1.8 3.5 1.7 3 1.6 2.5 1.5 S/N 341:1 2 1.5 1.4 1.3 1.2 1 1.1 0.5 4.628 1 6.301 0.9 0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 Countsvs. AcquisitionTime(min) 5.8 5.9 6 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 0.8 6.9 0.7 0.6 0.5 +MRM(283.79999->213.90039) 1pgHCB_std_cond_UnkGain_2090EMV_He20pct_04.d x103 Noise(RMS) =6.03; SNR(6.465min) =1294.0 4.7 4.75 4.8 4.85 4.9 4.95 5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4 5.45 5.5 5.55 5.6 5.65 5.7 5.75 5.8 5.85 5.9 5.95 6 6.05 6.1 6.15 6.2 Countsvs.AcquisitionTim e(m in) Same Signal 8 7.5 7 6.465 6.5 6 5.5 5 Helium Quench Gas ON 4.5 4 3.5 3 2.5 2 S/N 1294:1 1.5 1 0.5 0 4.1 4.2 Page 30 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 Countsvs. AcquisitionTime(min) 5.8 5.9 6 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7 In MS apps, neutral noise is not visible it is buried by more intense chemical noise. Since MS/MS eliminates chemical noise, neutral noise is visible and must be minimized.

7000B MS/MS: New Higher Sensitivity Source Increase signal for precursor ions using an extractor source design New inert, high temp, extraction EI source Page 31

Lab-Proven Inert Source Solid, inert materials, not a coating that can be damaged by cleaning Stay-Clean (350ºC) design extends maintenance intervals with dirty samples Source tune parameters in the Autotune file Dual filaments reduces maintenance intervals 1000 s of satisfied MSD users Partition between source and analyzer compartments support dual stage pumping Page 32

7000B Extraction Source External View - Assembled Like the 7000A source, the 7000B extraction source is more open than the MSD source looks similar to the CI source At first look, it does not look any different than the 7000A source Page 33

7000B Extraction Source Expanded View of Components Component #7 is a new insulator And there is new connection to the lens #11 Design uses combination of push (repeller) and pull (extractor lens) to increase ion intensity Page 34

7000B Increased Signal Extractor source cutaway Extractor Source w/ Ramped Iris Detector Push-Pull Principle: Repeller and Extractor Standard Source & Detector mode Page 35

Page 36 Why Is the New Extractor Source More Valuable for Triple Quad MS/MS than Single Quad MSD?

Extractor Source Increases Signal for All Ions EI: spectrum of analyte can also include ions from matrix, column bleed, gases, etc. Single Quad MS Chemical Noise Limited Analyte Signal Increased Chemical Noise Increased S/N does not improve Page 37

Extractor Source Increases Signal for All Ions EI: spectrum of analyte can also include ions from matrix, column bleed, gases, etc. MS/MS still eliminates chemical noise Product 2 Q1 SIM isolate precursor before CID Product 3 Product 1 chemical noise from these other ions is eliminated CID + Q2 SIM Page 38

Why Is the New Extractor Source Valuable for MS/MS? Single Quad MS Chemical Noise Limited Analyte Signal Increased Chemical Noise Increased S/N does not improve Triple Quad MS/MS Product Ion Signal Limited Product Ion Signal Increased Chemical Noise Does NOT Increase S/N (and LOD) are improved Page 39

Unique Entrance Lens Eliminates Pre-filter Improved lens The last lens, which extends into the heated monolithic quadrupole, optimizes coupling of the source to the analyzer. Voltage on this lens is dynamically ramped synchronously with analyzer voltage to focus maximum ion signal for every m/z into the center of the quadrupole field. Page 40

7000B MS/MS: New Ramped-Iris Detector Ramped-iris improves transfer of ion from the exit of the second analyzer into the Triple-Axis detector New Ramped Iris Triple-Axis Detector Page 41

High Performance Triple-Axis Detector with New Dynamically Ramped Iris Maximum reduction of neutral noise The combination of helium quench gas and the Triple-Axis design delivers ultra low noise Long life and high linearity Triple channel electron multiplier Gain Normalization corrects tune file for detector aging Consistent long term method sensitivity Design proven in the 5975C MSD hyperbolic quartz transmission quadrupole analyzer Y ion beam steering rod shield for secondary particles X Offset Z high energy dynode triple channel electron multiplier Page 42

Ramped-Iris Triple-Axis Detector Improved high mass peak shape Improved mid to high mass sensitivity Mass Iris (V) Signal Increase 69-1 1.0X 131-8 1.1X 264-13 1.5X 414-20 2.5X 502-30 2.8X 614-35 4.0X Page 43

7000B GC/MS/MS Specifications Installation Checkout Specifications EI SRM > 500:1 for 100 fg OFN PCI SRM (CH4) > 50:1 for 100 fg BZP Typical Precision EI MRM Pk Area RSD < 8% for 100 fg OFN Typical Sensitivity EI Scan > 300:1 for 1 pg OFN NCI SIM (CH4) > 200:1 for 10 fg OFN OFN = octafluoronaphthalene BZP = benzophenone Page 44

Column Backflush: Many Wonderful Advantages for GC/MS/MS of Complex Matrices Page 45

What Real Value Does High Performance EPC Have If Matrix Carryover Destroys Rt Precision? Norm. 10 140 00 120 00 100 00 Runs without Backflushing Retention times shift ~4-5 sec during 10 runs 80 00 60 00 40 00 20 00 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2 m in Norm. 160 00 140 00 10 Runs with Backflushing 120 00 100 00 80 00 60 00 40 00 20 00 0 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 m in Page 46

Productivity: CFT Backflush More analyses per hour... Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance on column and ion source Page 47

Multiple Backflush Configurations Multiple Backflush Devices Backflush from the start of the column Backflush guard column only Backflush from the end of the column Backflush entire column Backflush from the middle of the column Backflush a percentage of the column Example: at tee between two 15 m columns New Pressure Controlled Tee Kit Purged Ultimate Union Page 48

Backflush Mid-Column (Backflush Mode) Split/Splitless Injection Port Pressure / Flow Controller 7890A GC Vent Decrease inlet pressure during backflush Pressure Controlled Tee 5975C MSD or 7000B MS/MS 15-m HP-5ms (0.25mm id x0.25um) 15-m HP-5ms (0.25mm id x0.25um) Page 49

Backflush with Purged Ultimate Union: Lipid Peroxidation Products in Blood 4-hydroxy-2,3-nonenal (HNE), an indicator of oxidative stress, and its metabolite, 1,4- dihydroxynonene (DHN) PCI with NH 3 App Note 5989-9359EN Late eluting peaks were quickly contaminating the source Page 50

Backflush with Purged Ultimate Union: Loss of Response without Backflush with backflush without backflush App Note 5989-9359EN Page 51

Backflush with Purged Ultimate Union: Loss of Response without Backflush with backflush These were the first backflush results; the system is getting clearer without backflush App Note 5989-9359EN Page 52

Break for Questions For questions, at break please dial *1 on your phone, or type onto the Q&A box at any time during the presentation. Page 53 Page 53 Pesticide Analysis with New Agilent Jet Stream Technology December 12, 2008

MassHunter Software Page 54

MassHunter Software Common Workflow for all Mass Spec. applications ChemStation Single Quad LC/MS ChemStation Single Quad GC/MS (MSD) LC/MS Acquisition Module Translator Translator Triple Quad LC/MS, LC/TOF, LC/QTOF MassHunter Quant and Qual modules GC/MS Acquisition Module Triple Quad GC/MS MassHunter format Page 55

MassHunter Data Analysis Applications One software for all your Agilent MS applications Quantitative Analysis Target Compound Analysis High throughput quantitation Qualitative Analysis Discovery & Method Development Find features Identify compounds Page 56

Quantitative Analysis View Results Information Page 57

Quantitative Analysis View Results Information Compound Information Page 58

Quantitative Analysis View Results Information Compound Information Calibration Information Page 59

Results Information: All Results for batch in one view Easy visualization for all results Customize columns for desired data Easy Navigation Compound and Calibration views change when new compound is selected Reduce data by filtering on sample type, compound, time segment, and outlier Page 60

Results Information: Detailed compound view Detailed compound information is easily seen Page 61

Results Information: Highlight outliers Compounds with results outside specified ranges are highlighted 30 metrics are available Custom Calculation is possible More to be added i.e. Peak metrics Page 62

Results Information: Highlight outliers Compounds with results outside specified ranges are highlighted 30 metrics are available Custom Calculation is possible More to be added i.e. Peak metrics Page 63

Results Information : Simple Metrics Plot Columns Ability to see trends in a batch Ability to diagnose quantitation problems easily Missing ISTD amount Retention time drift Page 64

Compound Information: All relevant information in one view Compound Integration Qualifier Information Spectrum Information ISTD Integration ISTD Qualifier Information ISTD Spectrum Information Page 65

Calibration Curve Views Can include ISTD, QC, and CC data points on curve Point information is available as pop-up QC point Page 66

Calibration Curve Assistant Display all possible Calibration Curve fit options Review R2, Standard error, or Max % Residual Can sort, filter, or browse options Page 67

Calibration Curve Assistant Filter possible calibration curves based on requirements Compare selected calibration fit (black) with proposed (blue). Linear All points Ignore origin Page 68

Reporting and Data Transfer Use of XML results format Easy data exchange LIMS data exchange Copy/Paste views from MassHunter Microsoft Excel reporting engine Powerful Features Excel Macros and formulas can be created for custom result manipulation Flexible Reports and Layouts Custom Layouts and graphics properties. Preconfigured report template examples Page 69

Productivity of GC/MS/MS Sample complexity forces the change from MS to MS/MS Could not meet required LOD/LOQ in real samples Could not avoid interferences from matrix ions (isobaric ions) Page 70

Productivity of GC/MS/MS Sample complexity forces the change from MS to MS/MS Could not meet required LOD/LOQ in real samples Could not avoid interferences from matrix ions (isobaric ions) Practical aspects of productivity MS/MS selectivity for less sample preparation MS/MS sensitivity for less injected on-column In turn, less maintenance MS/MS selectivity for faster analysis cycles MS/MS selectivity lessens the requirement for column selectivity Less bakeout or backflush may be needed Caution: selectivity is a type of blindness and can be misleading MS/MS selectivity for faster and easier peak integration What can be easier than a flat baseline and a single peak Page 71

Break for Questions For questions, at break please dial *1 on your phone, or type onto the Q&A box at any time during the presentation. Page 72 Page 72 Pesticide Analysis with New Agilent Jet Stream Technology December 12, 2008

Thank You for Attending Today s eseminar Terry L. Sheehan, Ph.D. GC/MS/MS Product Manager Agilent Technologies, Santa Clara, CA Page 73