“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.
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