Ultra-High Sensitivity in Triple Quadrupole LC/MS/MS 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 Our measure is your success. QQQ talk LCMS US Tour Winter 2009 Frank Kuhlman, Ning Tang
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Ultra-High Sensitivity in Triple Quadrupole LC/MS/MSTriple Quadrupole LC/MS/MS
Performance
John HughesSr. Applications ConsultantAgilent Technologies
with contributions fromLinda Côté, John Fjelsted, Frank Kuhlman Ning Tang
Page 1 Our measure is your success. QQQ talk LCMS US TourWinter 2009
Frank Kuhlman, Ning Tang
The Agilent 6460: Cutting Edge QQQ Performance
With…
Agilent Jet Stream Technology
Unmatched sensitivity
Workflow improvementsThe industry’s most
100fg reserpine sensitivity with less than 10% RSD !
Faster method development The industry’s mostsensitive QQQ
g p y
Fast Pos/Neg switching
Faster MRMs and more MRMs per time segment
New Optimizer software enables faster MS/MS method development
Page 2 Our measure is your success. QQQ talk LCMS US TourWinter 2009
Breaking the “fg barrier” with the new 6460A Triple Quad LC/MS/MSnew 6460A Triple Quad LC/MS/MS
Performance of Agilent QQQ models
Femtogram barrier
Performance of Agilent QQQ models
6410-High
6460
6410
6410 High Sensitivity
10-1 100 101 102 103
Femtograms
Page 3 Our measure is your success. QQQ talk LCMS US TourWinter 2009
Femtograms
Agilent 6460 QQQ PerformanceShattering the Femtogram Barrier – 500 Attogramsg g g
Verapamil 500 attogramsVerapamil, 500 attograms
1.8 μm C18, 2.1 x 50mm0.4 mL/min
Breakthrough Sensitivity6460 triple quadrupole with Agilent Jet Stream technology breaks the femtogram barrier shown here with
Page 4 Our measure is your success. QQQ talk LCMS US TourWinter 2009
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.
Agilent 6460 QQQ Performance –500 attograms verapamil and extended dynamic linear g p yrange
VerapamilVerapamil
Five Decades of Linearity6460 triple quad with Agilent Jet Stream technology exhibits outstanding performance with 5 decades of
Page 5 Our measure is your success. QQQ talk LCMS US TourWinter 2009
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.
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 RichlandBiological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA
Journal of the American Society for Mass SpectrometryVolume 18, Issue 9, September 2007, pages 1582-1590
“Ion transmission efficiency, also defined as the fraction of ES current that t th l h t diti ll b li it d b l t thenters 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 103–105 analyte ions generated by ESI at atmospheric pressure is actually detected usinggenerated by ESI at atmospheric pressure is actually detected using present instrument designs [[7], [10] and [22]].”
Page 6 Our measure is your success. QQQ talk LCMS US TourWinter 2009
Agilent Jet Stream* Ion Generation Gas Dynamics Viewy
N b li iNebulizing gas
“S h d” h h
Enhanced efficiency nebulizer
Nozzle voltage
“Super-heated” sheath gas
The super-heated sheath gas
g
Heated drying gas
e supe eated s eat gascollimates the nebulizer spray and creates a dramatically “brighter” source Resistive sampling
capillary*
Page 7 Our measure is your success. QQQ talk LCMS US TourWinter 2009
*Patent Pending
Agilent Jet Stream animation
Enter slide show mode to see the animation on this page.
Page 9 Our measure is your success. QQQ talk LCMS US TourWinter 20099
Herbicides in Spinach Matrix Herbicides in Spinach Matrix --1uL injection, 100ppt level1uL injection, 100ppt level
Page 23 Our measure is your success. QQQ talk LCMS US TourWinter 2009
(100 fg each on(100 fg each on--column)column)
Agilent MassHunter Workstation
More Innovations in M S t tWorkstation Mass Spectrometry Software
Page 24 Our measure is your success. QQQ talk LCMS US TourWinter 2009
MassHunter OptimizerAutomated MRM Method Development SoftwareAutomated MRM Method Development Software
T diti l MS/MS th d d l tTraditional 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
> O f DAYS f tt d d k!=> One or a few DAYS of unattended work!
Compound-specific optimization for MRM experiments• Selection and optimization of precursor and product ions• 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 Our measure is your success. QQQ talk LCMS US TourWinter 2009
MassHunter OptimizerAdvantages over previous solutionsAdvantages over previous solutions
Utmost flexibility via support of all common optimization modes:M l i f i ( i )• 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 includes analysis WITH column using fast LC.
V i 2 l 2009 A t ti ti f LC/MS th d fVersion 2, early 2009: Automatic creation of LC/MS method for large numbers of compounds from Compound Database, using “Scheduled MRM”
Page 26 Our measure is your success. QQQ talk LCMS US TourWinter 2009
MassHunter OptimizerCompound Entry & Method Setupp y p
Page 28 Our measure is your success. QQQ talk LCMS US TourWinter 2009
MassHunter OptimizerStep 2 – Setup Precursor Ion SelectionStep 2 – Setup Precursor Ion Selection
• Define up to 4 adducts and/or charge states for each polarity• Define exclusion of ions based on m/z or intensity
Page 29 Our measure is your success. QQQ talk LCMS US TourWinter 2009
MassHunter OptimizerStep 3 – Setup Product Ion SelectionStep 3 – Setup Product Ion Selection
• Optimizer will find the 4 most abundant product ions• Define minimum product ion mass• Define exclusion of ions based on m/z intensity or neutral losses
Page 30 Our measure is your success. QQQ talk LCMS US TourWinter 2009
Define exclusion of ions based on m/z, intensity or neutral losses
Run 1: The precursor ion is selected based on user criteria (adduct p (ions, charge states) and Fragmentor voltage is optimized
(optional): fine adjustment of Fragmentor voltage Run 2: Coarse product ion scan finds the largest 4 product ions with p g p
corresponding Collision Energy (default CE 0, 10, 20, 30, 40).(optional) fine adjustment of Collision Energy
Post Run: Populates Compound Database with the 4 best transitionsw/ optimum Fragmentor Voltages and Collision Energies
Prints Optimization Report
Page 32 Our measure is your success. QQQ talk LCMS US TourWinter 2009
MassHunter OptimizerStep 6 –View Results for ProjectStep 6 –View Results for Project
• Results for the project can be immediately viewed after optimization
Page 33 Our measure is your success. QQQ talk LCMS US TourWinter 2009
MassHunter OptimizerStep 7 – Build LC/MS method from Cmpd DBStep 7 – Build LC/MS method from Cmpd DB
• Select compounds in database after filtering• Select transitions for each compound (all or individual)
Page 35 Our measure is your success. QQQ talk LCMS US TourWinter 2009
• Easily drop into template LC/MS method
MassHunter “Scheduled MRM”Increased Utility and PerformanceIncreased Utility and PerformanceNew applications require quantitation of 100 – 1000 compounds in one MRM method !co pou ds o e et od• 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 times• Tedious to set up; problematic if changes in retention times
WITH Scheduled MRM:A t ti t f l i ti t ith t i t ti• 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 Our measure is your success. QQQ talk LCMS US TourWinter 2009
y g p
Scheduled MRMIncreased Utility and PerformanceIncreased Utility and Performance
Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Time segments Time segments MRM
Compounds (x/block)
Cycle Time (sec)50 800.5 0.8 1 0.7
100 70
Scheduled MRM
2x shorter cycle times better for narrow chromatographic peaks, more
Max Coincident
Cycle Time (sec) 0.2 0.4 0.4 0.320 40 40 30
Page 37 Our measure is your success. QQQ talk LCMS US TourWinter 2009
2x shorter cycle times better for narrow chromatographic peaks, more analytes, longer dwell time per analyte.
Application example for new software features –Optimizer and Scheduled MRMOptimizer and Scheduled MRMPeptide quantitation from protein digests for protein biomarker studies
•SpectrumMill Peptide Selector to choose useful peptides
•QQQ Optimizer to create MRM methods for quantitation•QQQ Optimizer to create MRM methods for quantitation
•HPLC-Chip MS/MS for rapid and reliable quant with small sample quantitiessample quantities
•MassHunter Quantitative Analysis for efficient data processing
Page 38 Our measure is your success. QQQ talk LCMS US TourWinter 2009
Agilent HPLC-Chip/QQQ LCMS TechnologyNanospray chip configuration brings new era in high sensitivity quantitation
NanoLC system forl ti l h t h
HPLC Chip Cube system
analytical chromatography
CapLC pump for sampleloading on enrichment column
6410 QQQ LCMS
Sensitivity: down to low amolDynamic range: up to 105
Page 40 Our measure is your success. QQQ talk LCMS US TourWinter 2009
y g p
Chromatographic PerformanceProtein Digest Mixture g
3
6
x107
Intensity
EIC m/z 985 615.7 secs
Conventional nanospray LC/MS
Conventional nanospray LC/MS
1
2
2
4
EIC m/z 985.6
0
3
0
2.0x108
6 secsHPLC-Chip/MS HPLC-Chip/MS
1
2
0.5
1.0
1.5
32% increase
EIC m/z 985.6
Red ced MS comple it + red ced ioni ation competition = impro ed ID