Ultra-High Sensitivity in Triple Quadrupole LC/MS/MS Performance

Transcription

1 Ultra-High Sensitivity in Triple Quadrupole LC/MS/MS Performance John Hughes Sr. Applications Consultant Agilent Technologies with contributions from Linda Côté, John Fjelsted, Frank Kuhlman, Ning Tang Page 1

2 The Agilent 6460: Cutting Edge QQQ Performance With Agilent Jet Stream Technology Unmatched sensitivity Workflow improvements Faster method development The industry s most sensitive QQQ 100fg reserpine sensitivity with less than 10% RSD!!! Fast Pos/Neg switching Faster MRMs and more MRMs per time segment New Optimizer software enables faster MS/MS method development Page 2

3 Breaking the fg barrier with the new 6460A Triple Quad LC/MS/MS Performance of Agilent QQQ models High Sensitivity 6410 Femtogram barrier Femtograms Page 3

4 Agilent 6460 QQQ Performance Shattering the Femtogram Barrier 500 Attograms Verapamil, 500 attograms 1.8 μm C18, 2.1 x 50mm 0.4 ml/min Breakthrough Sensitivity 6460 triple quadrupole with Agilent Jet Stream technology breaks the femtogram barrier, shown here with 500 attograms of verapamil injected on-column, using unit resolution for both Q1 and Q3. Page 4

5 Agilent 6460 QQQ Performance 500 attograms verapamil and extended dynamic linear range Verapamil Five Decades of Linearity 6460 triple quad with Agilent Jet Stream technology exhibits outstanding performance with 5 decades of linearity from sub-femtogram to 100 picograms of verapamil injected on-column. Page 5

6 Why focus on improving the efficiency of ESI? Ionization and Transmission Efficiency in an Electrospray Ionization Mass Spectrometry Interface Jason S. Pagea, Ryan T. Kellya, Keqi Tanga and Richard D. Smith, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA Journal of the American Society for Mass Spectrometry Volume 18, Issue 9, September 2007, pages Ion transmission efficiency, also defined as the fraction of ES current that enters the mass analyzer, has traditionally been limited by losses at the mass spectrometer inlet and at the skimmer [[7] and [21]]. It has been estimated that only about one out of every analyte ions generated by ESI at atmospheric pressure is actually detected using present instrument designs [[7], [10] and [22]]. Page 6

7 Agilent Jet Stream* Ion Generation Gas Dynamics View Enhanced efficiency nebulizer Nebulizing gas Super-heated sheath gas 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 7

8 Agilent Jet Stream Ion Generation Thermal Dynamics View This plot is a simulation showing the thermal profile of the Agilent Jet Stream technology Thermal energy is focused to the nebulizer spray Thermal focusing produces the most efficient desolvation and ion generation possible! Super-heated N 2 sheath gas Nebulizer N 2 gas (near sonic velocity) Patent Pending Page 8

9 Agilent Jet Stream In Action Observing Thermal Gradient Focusing Temperature 25ºC at t = 0 min to 400ºC at t = 8 min Page 9

10 Narrower spray beam, higher concentrations Less light scattering shows increased droplet desolvation and high ion production. 2R Stop Temperature C at t = 8 min Page 10

11 Agilent Jet Stream animation Enter slide show mode to see the animation on this page. Page 11 11

12 Agilent Jet Stream available on 6460 QQQ and 6530 QTOF What else is new for these systems? Octopole 1 Quad Mass Filter (Q1) 6460 QQQ Quad Mass Filter (Q3) 6530 QTOF Lens 1 and 2 Collision Cell 10KV Detector Rough Pump Agilent Jet Stream* Turbo 1 Turbo 1 Turbo 1 Resistive Capillary* Turbo 2 2 nd Turbo Increased turbo stage 1 pumping conductance Quad Mass Filter (Q1) Octopole 2 Octopole 1 Lens 1 and 2 Collision Cell DC Quad Ion Pulser *Patent Pending Rough Pump Turbo 1 Turbo 1 Turbo 1 Turbo 2 Page 12

13 Reserpine sensitivity comparisons 6460 QQQ: > >> Signal/Noise vs QQQ for 500 femtogram injections on column Noise = 3x RMS noise 6410 QQQ New 6460 QQQ SNR = 36:1 SNR = 354:1 10x improvement 6 x x 10 4 Page 13

14 Limits of Detection (Estrone 3- sulfate) in Negative Ion Mode 100 fg on-column LOD 25 femtogram 100 fg on-column LOD 0.9 femtogram! x MRM ( > ) ES AR-BE-01. Noise (RMS) = 3.15; SNR (0.330min) = x SNR (0.370min) > Sub-Femtogram LOD! Page 14

15 6460 Triple Quad compared to 6410 Triple Quad 6410 Triple Quad: Tramadol (LOD 23 fg) Propoxyphene (LOD 33 fg) Phencyclidine (LOD 54 fg) 6460 Triple Quad: Tramadol (LOD 3 fg) Propoxyphene (LOD 2 fg) Phencyclidine (LOD 3.5 fg) x more S/N x more S/N x more S/N Page 15

16 Five Order of Linearity with new 6460 Triple Quad Alprazolam 22 femtograms to 22 nanograms! 12 calibration levels Alprazolam-P - 12 Levels, 12 Levels Used, 42 Points, 42 Points Used, 0 QCs x10 2 y = * x E-005 R^2 Y = x = R 2 = Relative Responses Relative Concentration Page 16

17 Agilent Jet Stream Performance Ruggedness & Reproducibility 6460 QQQ 500 Injections of Alprazolam in Spiked Human Plasma Extract, ~ 10hrs. 5 pg injections of Alprazolam in human plasma extract Internal Standard = 1.45 % RSD for amount Area Response External Standard = 3.76% RSD for areas Relative Response Injection Number Page 17

18 6460 QQQ performance: some additional application examples Page 18

19 Agilent Jet Stream Performance - Ruggedness Minimal Effect of Matrix on Response Results show 4 therapeutics in analyzed in solvent and plasma Plasma Solvent Verapamil Relative Response: % Sertraline Paroxetine Fexofenadine Page 19

20 Cyprodinil: LOD 21 femtograms with 100 msec +/- switching and 5 msec dwell times 100 fg, S/N=14 Positive ion Blank Page 20

21 Chloramphenicol: LOD 50 femtograms with 100 msec +/- switching and 5 msec dwell times 250 fg, S/N=15.7 Negative ion x MRM ( > 152.0) 01_10MRM_Blank-r010.d Blank x MRM ( > 152.0) 04_10MRM_STD_50ppt-r001.d Noise (PeakToPeak) = 1.02; SNR (1.50min) = 15.7 * Counts vs. Acquisition Time (min) Page 21

22 Agilent 6460 QQQ Trace analysis of pesticides x TIC MRM (** -> **) 50ppt-r001.d Prometon Ametryn Simazine Terbutryn Atrazine Prometryn Promazine Jetstre am Result s Counts vs. Acquisition Time (min) Solvent Std, 1uL injection, 50ppt (50 fg each on-column) Page 22

23 Agilent 6460 QQQ Trace analysis of pesticides x x TIC MRM (** -> **) pinch_blank1-r001.d Blank Blank + TIC MRM (** -> **) pinch_100ppt1-r001.d Prometon Prometryn Promazine 3 Sample Ametryn Simazine Terbutryn Atrazine Counts vs. Acquisition Time (min) Herbicides in Spinach Matrix -1uL injection, 100ppt level (100 fg each on-column) Page 23

24 Agilent MassHunter Workstation More Innovations in Mass Spectrometry Software Page 24

25 MassHunter Optimizer Automated MRM Method Development Software Traditional MS/MS method development: Manual optimization of even three parameters, for dozens of compounds => MANY Days to WEEKS of tedious, interactive work WITH Optimizer: Optimization can be fully automated for multiple compounds => One or a few DAYS of unattended work! Compound-specific optimization for MRM experiments Selection and optimization of precursor and product ions Supports optimization with multiple methods (e.g. pos then neg) Creation of a compound database with optimized parameters for re-use Page 25

26 MassHunter Optimizer Advantages over previous solutions Utmost flexibility via support of all common optimization modes: Manual infusion (syringe pump) Automatic infusion (via loop injection at lower flows) Flow injection analysis without column (FIA) Analysis with column (multiple compounds per run) Optimization WITH column for highest success: Infusion or FIA can result in 20% of cmpds not optimizing Optimizer is designed for injection analysis WITH column using fast LC. Version 2, early 2009: Automatic creation of LC/MS method for hundreds of compounds from Compound Database, using Scheduled MRM Page 26

27 MassHunter Scheduled MRM Increased Utility and Performance New applications require quantitation of compounds in one MRM method! Food and environmental analysis (e.g. pesticides) Targeted quantitation of proteins via peptides (proteomics) WITHOUT Scheduled MRM: Need to manually set up multiple time segments to maximize dwell time Tedious to set up; problematic if changes in retention times WITH Scheduled MRM: Automatic setup of overlapping time segments without user intervention Fewer MRMs per unit time results in longer dwell time => incr sensitivity Unaffected by chromatographic time shifts Page 36

28 Scheduled MRM Increased Utility and Performance Time segments MRM Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4 Time (min) Compounds (x/block) Cycle Time (sec) Scheduled MRM Max Coincident Cycle Time (sec) x shorter cycle times better for narrow chromatographic peaks, more analytes, longer dwell time per analyte. Page 37

29 Application example for new software features Optimizer and Scheduled MRM Peptide quantitation from protein digests for protein biomarker studies SpectrumMill Peptide Selector to choose useful peptides QQQ Optimizer to create MRM methods for quantitation HPLC-Chip MS/MS for rapid and reliable quant with small sample quantities MassHunter Quantitative Analysis for efficient data processing Page 38

30 Agilent Proteomics Biomarker Workflow SAMPLE1 Extraction SAMPLE2 Candidate Biomarker Identification DATA Depletion 6520 QTOF Candidates Fractionation Proteolytic Digest Extraction Validation 6410 QQQ Identification Page 39

31 High Throughput : 2D-Gel Spot x BPC( [-1]) Scanec-gelspots-03.d min run time Countsvs. AcquisitionTime(min) Page 40

32 Using Spectrum Mill Peptide Selector for Optimizing MRM Transitions Perform in silico digest Filter digestion product peptides as desired by user (mass, composition) Predicts best product ions from most likely MS/MS cleavage for each peptide Can provide Uniqueness of the peptide sequence in the database: helps select peptides most likely to provide unique information Page 41

33 SM Peptide Selector Results for Catalase Page 42

34 MassHunter Optimizer Compound(s) setup Page 43

35 MassHunter Optimizer optimization results for peptides Page 44

36 Agilent HPLC-Chip/QQQ LCMS Technology Nanospray chip configuration brings new era in high sensitivity quantitation NanoLC system for analytical chromatography HPLC Chip Cube system CapLC pump for sample loading on enrichment column 6410 QQQ LCMS Sensitivity: down to low amol Dynamic range: up to 10 5 Page 45

37 HPLC-Chip/MS Interface: making nanoesi almost routine Stator Autosampler Waste Nanopump Side View Rotor inner rotor Rotor outer rotor Stator Microvalve HPLC-Chip Page 46

38 Chromatographic Performance Protein Digest Mixture 3 2 Conventional nanospray LC/MS Intensity x Conventional nanospray LC/MS EIC m/z secs HPLC-Chip/MS 32% increase 0 x secs HPLC-Chip/MS EIC m/z Time [min] Time [min] Reduced MS complexity + reduced ionization competition = improved ID Page 47

39 Limit of Quantitation in the Low Amol Range Peroxidase 10 amol to 10 fmol spiked into 1 µg human serum x x x x MRM ( > ) per10a-c2-1ug-r002.d + MRM ( > ) per100a-c2-1ug-r001.d + MRM ( > ) per1f-c2-1ug-r001.d + MRM ( > ) per10f-c2-1ug-r001.d 10 amol peroxidase in 1ug serum 100 amol peroxidase in 1ug serum 1 fmol peroxidase in 1ug serum 10 fmol peroxidase in 1ug serum Counts vs. Acquisition Time (min) Page 48

40 External Quantitation Curve of Peroxidase Peptide DTIVNELR From 10 amol to 10 fmol Spiked into Human Serum peptide1-4 Levels, 4 Levels Used, 12 Points, 12 Points Used, 0 QCs x10 4 y = * x R^2 = Responses peptide1-4 Levels, 4 Levels Used, 12 Points, 12 Points Used, 0 QCs Responses x y = * x R^2 = amol 100amol 1fmol Concentration (fmol/ ul) R 2 = Concentration (fmol/ ul) Page 49

41 Excellent Reproducibility of MS Response HSA Peptide LVNEVTEFAK from 10 amol to 1 pmol (n=6) RSD fmol R 2 = All RSDs are within 15% Page 50

42 New 6460 Triple Quad LC/MS Breaks the femtogram barrier for some compounds New Technology Agilent Jet Stream Higher Signal Strength, great RSDs even in low fg region New Acquisition Software - Scheduled MRMs New Method Development Tool MH Optimizer Agilent is committed to continuous innovation in: Quadrupole technology Brighter sources Innovative software Page 51

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