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“MS Produktneuigkeiten
Agilent 6490 QQQ“
Volker Gnau
Agilent Technologies
Waldbronn
New 6490 LC Triple with iFunnel Technology
1290 Infinity LC / 6490 Triple: Clearly Better Together
6490 – The New High Sensitivity Triple Quadrupole
•
Ionization and Ion Transfer Technology
– Agilent Jet Stream Ion Generation
– Hexabore capillary
– Dual ion funnel (DIF) technology
• Two stages for ion focusing and gas removal
• Improvements for wide m/z range transmission
• Low capacitance
•
Collision Cell
– Hexapole field axial focusing curved collision cell
• Tapered cell structure for increased ion acceptance at entrance
• Reduced noise
•
Improved Quad Drive Electronics
– Improved Quad DC frequency response
– Higher RF power capability
– Quad drive frequency increased to 1.4 MHz
NiFunnel
E W I F UTechnology
N N E L T E C Hcaptures
N O L O G Y 10X more ions
Agilent Jet Stream
Hexabore Capillary
Dual Ion Funnel
• Thermal confinement
of ESI plume
• 6 capillary inlets
• Removes the gas but
captures the ions
• Efficient desolvation to
create gas phase ions
• Samples 10X more ion
rich gas from the source
• Captures the majority of
• Creates an ion rich zone
the gas from the source
region
• Removes neutral noise
• Extends turbo pump life
Hexabore Atmospheric Sampling….
•SIX bores
•HALF as long
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?
Compare:
Standard
Capillary is
180mm
Hexabore Sampling Capillary
• Hexabore sampling capillary increases
ion sampling efficiency up to 10X
compared to single bore
• Capillary length is reduced by 50%
• minimize high mobility ion loss
• pulls in more ion rich gas
Ion sampling gains are most
dramatic with the combination of
both Agilent Jet Stream technology
with the hexabore sampling
capillary
Agilent’s Two Stage Ion Funnel Can Handle the Gas Load
High
Pressure
Stage 1
Low
Pressure
Stage 2
Existing
RF Ion
Guide
Stage 1
8-12 Torr
Stage 2
1-3 Torr
Stage 1 offset breaks up the high pressure gas exiting the Hexabore Capillary
How Does an Ion Funnel Work?
RF Voltage
focuses the ions
to the center.
RF Drive
Unfocused
Ions and Gas
Enter
DC Voltage
accelerates the
ions to the exit.
Focused
Ions Out
DC Drive
Triple Quadrupole Instrument Configurations
6460 LC/MS (Skimmer Configuration)
Ion Detector
Cap
RF Ion
Guide
Q1
Coll
Cell
Q3
Ion Detector
Q3
6490 LC/MS (iFunnel Configuration)
Curved Collision cell
HB Cap
Dual Ion
Funnels
RF ion
guide
Q1
Novel Curved and Tapered Collision Cell Design
Efficient Ion Transmission over wide mass range
Reduces transmission of ionizer generated noise
RF & DC
Voltages Set
Confinement and
Axial Acceleration
Smaller
(3.5mm)
Exit
Aperture
Focuses
Ions into
MS2
Larger
(5 mm)
Entran
ce
Aperture
Accepts more
Ions
Improved Ion Sampling Efficiencies
Agilent Jet Stream
positive ion
Agilent Jet Stream
negative ion
11x
7x
7x
7x
6490
5x
14x
10x
7x
5x
10x
622
118
6460
922
322
302
1034
1222
113
200
400
600 800 1000 1200 1400
m/z
200 400 600 800 1000 1200 1400
m/z
The Value of Enhanced Resolution on 6490
Fluticasone propionate (inhalant) in human plasma 2.5 fg on-column)
1
x10
Enhanced Resolution gives increased selectivity for
analytes in complex matrices
Q1 Unit (0.7 amu)
Q2 Unit (0.7 amu)
7
Q1 Enhanced (0.4 amu)
Q2 Unit (0.7 amu)
6
5
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Acquisition Time (min)
2
2.2
2.4
5 pg/mL Fluticasone in plasma with new 6490
2.5 Femtogram on-column
Area RSD = 8.7% (n=5)
Mean accuracy = 104
Fluticasone propionate
x10
1
5.3
x10
5.3
5.2
5.2
5.1
5.1
5
5
4.9
4.9
4.8
4.8
4.7
4.7
4.6
4.6
4.5
4.5
4.4
4.4
4.3
4.3
4.2
4.2
1.765 min.
F
1
HO
S
O
CH3
O
O
CH3
CH3
F
CH3
MRM enhanced / unit resolution
(0.4 amu / 0.7 amu)
H
O
F
0.8 0.9
1
1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9
2
2.1 2.2 2.3 2.4 2.5
Acquisition Time [min]
Bisphenol A and Benzophenon – Comparison Agilent QQQ 6460 vs. 6490
LOD estimation
Bisphenol A
Quant ion
S/N = 6.4
Peak to peak
Based on height
250 fg o.c.
Benzophenon
Quant ion
S/N = 44.4
Peak to peak
Based on height
500 fg o.c.
Bisphenol A
Quant ion
S/N = 7.3
Peak to peak
Based on height
500 fg o.c.
Benzophenon
Quant ion
S/N = 37.2
Peak to peak
Based on height
2500 fg o.c.
Bisphenol A
Quant ion
S/N = 28.1
Peak to peak
Based on height
2500 fg o.c.
Benzophenon
Quant ion
S/N = 56.7
Peak to peak
Based on height
5000 fg o.c.
Bisphenol A
Quant ion
S/N = 55.1
Peak to peak
Based on height
5000 fg o.c.
250 fg o.c.
Benzophenon
Quant ion
S/N = 82.4
Peak to peak
Based on height
Bisphenol A and Benzophenon – Comparison Agilent QQQ 6460 vs. 6490
Sample 1.1 (100 ng/ml in muesli extrakt, 5 ul)
Agilent QQQ 6490
Agilent QQQ 6460
Triggered MRM
Beispiel - tMRM
Die besten Resultate werden erzielt, wenn die
korrespondierenden MRMs am Maximum des Peaks
getriggert werden.
Idealer Workflow:
• Erreicht der primäre MRM einen definierten
Schwellenwert, wird triggered
MRM aktiviert
• Ist Steigung ≤ 0 MRMs werden für eine vorgegebene
Anzahl von Wiederholungen getriggered
• Fällt der primäre MRM unter den definierten
Schwellenwert, wird tMRM deaktiviert
Robustness Test in Human Plasma - Sample Preparation
• Verapamil concentration of 309 pg/mL (1 nM) in human plasma
• 100 μL of human plasma spiked with verapamil precipitated with ACN
(300 μL) containing 1% of formic acid and the ISTD reserpine
• Centrifugation for 15 minutes at 13200 rpm
• Supernatant (200 μL) diluted with 3 parts of water (e.g. 600 μL)
• 1 μL injected into the LC/MS system, corresponding to approx. 20 fg
injected on column
• LC/MS Method using C18, water/acetonitrile.
• LC/MS Run Time = 3 min
Ion Funnel Construction
Old design with many metal plates….
… resulting in a large capacitive load and
relatively large RF power electronics.
•The 6490 uses technology with only a
small conductive rim resulting in a
reduced capacitance load.
•Fast +/- ion switching
Gold
Plated Rim
Agilent’s Dual Ion Funnel – Different by Design
• PNNL poster describes the problems of contamination in their ion
funnel.
• Contamination is a result of deposition of materials from the LC
onto the surfaces of the funnel.
• Build up of material can alter tuning characteristics and degrade
ion transmission.
• Use of the Agilent Jet Stream, gold plated electrodes and funnel
optics together with efficient pumping minimizes potential
contamination.
• The ion optics of the dual ion funnel are designed to eliminate
transmission of neutrals into the mass spectrometer
Robustness Test – Over 4 Days of Continuous Use
Absolute Area (2000 injections / ~4 days)
Area RSD = 5.3%
Constant response
Abundance
1000
500
verapamil in human plasma (20 fg on column)
Below the sensitivity of other triple quadrupoles
0
500
1,000
Number of Injections
1,500
2,000
Simple and Easy Funnel Cleaning Procedure
Ion Funnel Maintenance
• As with all LC/MS systems routine
cleaning is necessary periodically.
• The ion funnel should be cleaned
periodically after extended periods of
operation, e.g. several thousands of
samples in biological matrix.
• The dual ion funnel is easily removed
•
Cleaning is performed by sonicating the
ion funnel assembly in a bath of 100%
isopropanol for 15 minutes.
Zusammenfassung
Agilent LC/MS has achieved a new, higher level of performance
 Combined operation in MassHunter software
Agilent 1290 Infinity LC allows fast, hi resolution separations
 Huge peak capacity for complex separations
 Excellent injection and RT precision
 Low delay volume for very fast methods
 Very low AS carry-over
Agilent 6460 & 6490 QQQ systems can keep up!
 Fast pos/neg switching
 Fast MRM and Dynamic MRM methods for triple quad
 Dynamic MRM databases for pesticides and forensics
 Triggered
 Excellent sensitivity and linear dynamic range
6490 Robustness Summary
• Robustness has been demonstrated for the analysis of verapamil
in human plasma.
• Continuous high sensitivity performance for the analysis of more
than 2000 samples - over 4 days of sample analysis.
• Area RSDs from the analysis were <6% and well within assay
acceptance criteria of <20 % for over 4 days.
• No contamination effects were observed.
• iFunnel cleaning not required after the verapamil analysis.
• Additional studies underway for larger sample sets, increased
injection volumes and protein quantitation.