Taking Full Advantage of UHPLC with Agilent 6460A Triple Quadrupole MS. Outline

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1 Taking Full Advantage of UHPLC with Agilent 0A Triple Quadrupole MS Dr. Anabel Fandino R&D Application Scientist LC/MS Division Agilent Technologies Santa Clara, CA utline Ultra High Definition LC/MS using the Agilent 90 Infinity UHPLC System coupled to the 0A Triple Quadrupole Mass Spectrometer Application example: High Throughput Metabolic Stability Study using cassette analysis and polarity switching Page LCMS - ASMS 009

2 Agilent UHPLC/MS/MS System - Relevance for High Throughput ADME assays Agilent 90 Infinity + RRHD columns 00 bar at ml/min Separation Speed Peak Resolving Power Agilent 90 Infinity LC and Agilent 0 Triple Quad MS Agilent 0 A Triple Quadrupole MS Dwell time - ms Polarity switching time 0 ms Agilent Jet Stream Technology High efficiency ion optics and detection Acquisition rate Sensitivity Dynamic Range Linearity, Precision, Accuracy Page LCMS - ASMS 009 Agilent 0A Triple Quadrupole MS Sample Liquid Quad Mass Filter (Q) Quad Mass Filter (Q) ebulizing gas ctopole Lens and Collision Cell 0KV Detector Superheated sheath gas Rough Pump Agilent Jet Stream* Turbo Turbo Turbo Turbo Enhanced Heated drying gas desolvation Resistive sampling capillary inlet to MS* *Patent Pending Page LCMS - ASMS 009

3 UHPLC/MS/MS Relevance for targeted Quantitation in Drug Discovery: in-vitro ADME need for: Speed (highest possible throughput): Short chromatographic run times / fast MS acquisition rate Resolution (to avoid overestimation in quantitation due to in-source CID conversion of a metabolite into its parent drug, to reduce ion suppression) Precise and accurate quantitation: An adequate number of data-points across the chromatographic peak is needed (at least 9-0 data points across the peak) Page LCMS - ASMS 009 Metabolic Stability study using cassette analysis Sample preparation Rat liver S9 preparation ADPH-regenerating system Phosphat -Buffer Substrate ( μm): Buspirone Verapamil Dextromethorphan Pre-incubate minutes at 7 C Take a μl aliquot from each incubate and stop by addition of AC at different time points Calculation of % parent remaining 0min min min 0min 0min min min min 0min min Work-up Cassette samples UHPLC/MS/MS Analysis Page LCMS - ASMS 009

4 UHPLC/MS/MS Polarity switching Substrates and internal standards: Buspirone, Dextromethorphan, Verapamil,, -d (ISTD), Dextrorphan-d (ISTD) UHPLC: Injection volume eedle wash Mobile phase Column Flow rate Gradient Stop time μl flushport (methanol:water 7:, 0.% formic acid), 0 seconds A = water + 0.% HCH B = acetonitrile + 0.% HCH RRHD Zorbax SB-C8,. x 0 mm, sub- μm.0 ml/min or. ml/min % B to 80% B.8 min or. min Speed + chromatographic resolution MS/MS: Drying Gas temperature 0 ºC MS/MS Resolution Unit Drying gas flow (nitrogen) 0 L/min Delta EMV 00 ebulizer gas (nitrogen) psig Time filtering off Sheath gas temperature 00 ºC MRM Transitions (+/-) or (non-switched) Sheath gas flow L/min ozzle voltage 0 V (+), 000 V (-) Dwell time ms (+/-) or ms (non-switched) Capillary voltage 000V (+/-) Pos/eg switching 0 ms Cycle Time ms (+/-) or ms (non-switched) Speed Page 7 LCMS - ASMS 009 Speed and data quality using polarity switching Flow rate. ml/min, 070 bar at %B, column: 0 C Flow rate.0 ml/min, 80 bar at %B, column: 0 C x0 8 + MRM (. ->.) Verapamil, min Avg W / = 0. sec points across W Rel. Area RSD [%] =.0 x0 + MRM (. ->.) Verapamil, 0.7 min Avg W / = 0.7 sec points across W Rel. Area RSD [%] =.9 x0 + MRM (8.0 ->. Buspirone,, 0. min 0. min Avg W / = 0.70 sec 0 points across W Rel. Area RSD [%] =. x0 + MRM (8.0 ->.. Buspirone, 0.0 min 0.0 min Avg W / =. sec points across W Rel. Area RSD [%] =.8 x0 + MRM (7. ->.) Dextromethorphan,, 0.0 min 0.0 min x0 + MRM (7. ->.) Dextromethorphan, min min. Avg W / = 0.98 sec 8 points across W Rel. Area RSD [%] =. Avg W / =. sec points across W Rel. Area RSD [%] =.7 x0 - MRM (9.0 -> 0.0, 0.7 min 0.7 min Avg W / = 0.7 sec 9 points across W Rel. Area RSD [%] = 7.8 Analysis time < 0.8 min x0 - MRM (9.0 -> MRM (9.0 -> 0.0 Analysis time <. min,.0 min.0 min Avg W / = sec points across W Rel. Area RSD [%] = Page 8 LCMS - ASMS 009

5 Where Does this Example Fall in the 90 Infinity Range bar.mm ID.mm ID Power = Pressure x Flow Rate Agilent 90 Infinity This app falls in the Power Range Vendor A Vendor E Vendor C Agilent RRLC Vendor D Standard LC ml/min Page 9 LCMS - ASMS 009 Speed and data quality using polarity switching x MRM (9.0 -> 0.0, narrowest peak Average W / = 0.7 sec 9 points across W Rel. Area RSD [%] = 7.8% W =. sec Cycle time = ms Page 0 LCMS - ASMS 009

6 Switched vs. non-switched analysis Area response Flow rate.0 ml/min, 870 bar at %B, column: C x0 + MRM (. ->.) Avg. Switched +/- to + only response = % Verapamil x0 + MRM (. ->.) x0 + MRM (8.0 ->.) Buspirone Avg. Switched +/- to + only response = 0% Buspirone x0 + MRM (8.0 ->.) x0 + MRM (7. ->.) Dextromethorphan Avg. Switched +/- to + only response = 09% Dextromethorphan x0 + MRM (7. ->.) x0 - MRM (9.0 -> 0.0 Avg. Switched +/- to - only response =70% x0 - MRM (9.0 -> Polarity Switching MRM transitions ( targets + internal standards) Dwell time = ms Cycle time = ms o Polarity Switching MRM transitions ( targets + internal standard) Dwell time = ms Cycle time = ms Page LCMS - ASMS 009 Switched vs. non-switched analysis Precision and correlation of methods Rel. Area RSD [%], n= Buspirone Verapamil Dextromethorphan Time [min] pos./neg. pos.only pos./neg. pos.only pos./neg. pos.only pos./neg. neg. only RSD [%] < 0 (switched) RSD [%] < 9 (non-switched) on-switched analysis Buspirone Selected example: Y = 0.99 x % Verapamil remaining r = t 80 t Dextromethorphan 70 0 y = 0.99 x Y = 0.99 x.88 0 r = r = t 0 t 0 Y =.7 x. t 0 r = Correlation of methods (switched vs. non-switched): R : Pos/eg switching analysis Page LCMS - ASMS 009

7 Chromatographic resolution x0 + MRM (0.0 ->... 7 min Buspirone -oxide (metabolite) C H (0?.) x0 + MRM ( (8.0 -> -. >.099) Buspirone (parent drug incubated + artifact) Buspirone 0. min Incubated parent drug. C H (8?.). 7 min In-source deoxygenation decomposition 0. min Excellent resolution between critical peaks Buspirone (In- source CID artifact build from Buspirone - oxide) Page LCMS - ASMS 009 Sensitivity Agilent Jet Stream vs. conventional ESI at mobile phase flow rate =.0 ml/min (t min sample) x0. + MRM ( >.099) Verapamil 0.79 x MRM ( >.099) t-ajs-column-r00.d Smooth () Buspirone.7 H C CH CH HCl.. CH CH CH H C Agilent Jet Stream Area RSD [%] =.9 Area: x Agilent Jet Stream Area RSD [%] =.7 Area: x. S/: x.7. S/: x. Conventional ESI Area RSD [%] = Conventional ESI Area RSD [%] = x MRM ( >.99) Dextromethorphan CH 0.0 x MRM ( > ) H CH Agilent Jet Stream Area RSD [%] =. Area: x S/: x Agilent Jet Stream Area RSD [%] =. Area: x S/: x H H Cl Cl Conventional ESI Area RSD [%] = Conventional ESI Area RSD [%] = Page LCMS - ASMS 009 7

8 Conclusions Agilent UHPLC/MS/MS for Drug Discovery assays Polarity Switching Flexibility for cassette design. ml/min at 070 bar High Throughput analysis (Elution time < 0.8 min) High speed MS/MS analysis with cycle time as low as ms to monitor transitions and switch polarity Relative area RSD [%] < 0%, enabled by collecting enough data points (at least 9 points) across the chromatographic peaks (as narrow as W / = 0.7 seconds) Comparable results (% parent drug remaining) when using polarity switching or nonswitched analysis Excellent chromatographic resolution to avoid overestimation in quantitation due to in-source CID conversion of a metabolite into its parent drug Excellent sensitivity Cassette analysis at high mobile phase flow rates Page LCMS - ASMS 009 8