Use of Ultra-High Performance LC-MS for Challenging Assays David Browne, Laboratory Manager (LC-MS-MS), Covance Laboratories, Harrogate
Department of Bioanalytical Services Determination of drug substance and metabolites in biofluids to support toxicokinetic and pharmacokinetic studies.
Theory - The Van Deemter Equation HETP = 2λd p + 2γD m + ωd 2 p v v D Lateral Diffusion m Longitudinal Diffusion Mass transfer HPLC H E T P P re s u re LINEAR VELOCITY
Theory - The Van Deemter Equation HETP = 2λd p + 2γD m + ωd 2 p v v D Lateral Diffusion m Longitudinal Diffusion Mass transfer UPLC H E T P P re s u re LINEAR VELOCITY
Theory - The Van Deemter Equation HETP = 2λd p + 2γD m + ωd 2 p v v D Lateral Diffusion m Longitudinal Diffusion Mass transfer 5 μm spherical particles
Theory - The Van Deemter Equation HETP = 2λd p + 2γD m + ωd 2 p v v D Lateral Diffusion m Longitudinal Diffusion Mass transfer 3 μm spherical particles
Theory - The Van Deemter Equation HETP = 2λd p + 2γD m + ωd 2 p v v D Lateral Diffusion m Longitudinal Diffusion Mass transfer 1.7 μm spherical particles
Efficiency gains with smaller particles System Efficiency and Velocity Smaller particle sizes: maximum resolution at higher linear velocity running at higher than optimal velocity gives minimal loss in resolution Therefore: higher flow rates are possible faster analysis shorter columns same resolution sensitivity is increased as the peak is sharper
Efficiency & Backpressure at constant velocity 500,000 System Efficiency and Backpressure 1.0 μm System Efficiency (Plates/m) 400,000 300,000 200,000 1.4 μm 1.7 μm 3 μm New equipment needed to take advantage of efficiency gains at higher pressure 100,000 5 μm 10 μm 0 0 20,000 40,000 60,000 80,000 100,000 120,000 140,000 Backpressure (psi)
Examples A robust bioanalytical method comprises 3 components Extraction Chromatography Detection Rough rule of thumb: at least 2 of these components must be highly selective. Which of the 3 components can we manipulate to obtain the biggest gains?
First example Endogenous compound Sensitivity required (10pg/mL) Does not give selective MS/MS transition Extraction: protein precipitation followed by selective SPE. Chromatography difficult due to co-extracting isoforms. Poor peak integrity presence of several peaks at the RT of the analyte Only 1 component is highly selective (extraction)
Vitamin Analogue R C H 3 CH 3 OH MS/MS Transition precursor ion (M + NH4)+ product ion loss of (NH3+H2O) non-selective ms/ms transition! HO OH
HPLC YMC ODS 3 µm, 2.1 x 100 mm, 0.3 ml/min
UPLC Waters ACQUITY BEH C18, 1.7 µm, 2.1 x 100 mm, 0.5 ml/min
HPLC vs. UPLC HPLC UPLC
Example 1 (UPLC vs. HPLC) UPLC Relatively high chromatographic resolution High peak purity/integrity Cycle time 7 minutes Same robustness as HPLC Increase in sensitivity x2 Lower injection volume HPLC Relatively low chromatographic resolution Low peak purity/integrity Cycle time 13 minutes Robust
Second example Steroid and prodrug Sensitivity required (100 pg/ml) Does not give selective MS/MS transition Extraction: C18 SPE. Chromatography very long due to isobaric interferences (25 minute cycle time) Only 1 component is highly selective (chromatography)
Steroid and Pro-drug Steroid: [M + Acetate] - OH Pro-drug: [M - H] - R O Isobaric interferences O R R
HPLC Phenomenex Luna C18, 3 µm, 2.0 x 100 mm, 0.2 ml/min
UPLC Waters ACQUITY BEH C18, 1.7 µm, 2.1 x 100 mm, 0.4 ml/min
HPLC vs. UPLC 100 pg/ml spiked extract injected on the two different systems * HPLC * UPLC
Example 2 (UPLC vs. HPLC) UPLC High chromatographic resolution Cycle time 6.5 minutes Same robustness as HPLC Increase in sensitivity x 4 HPLC High chromatographic resolution cycle time 25 minutes Robust
Third example Peptide - MW ~1850 Sensitivity required (10ng/mL) Does not give selective MS/MS transition Extraction: protein precipitation Chromatography difficult due to similar co-extracting analogues. Poor peak integrity presence of several peaks at the RT of the analyte No component is selective
Peptide MS/MS Transition precursor ion [M + 3H] 3+ product ion loss of (H 2 O) non-selective ms/ms transition!
HPLC Penomenex Jupiter C18 5 µm, 2.1 x 50 mm, 0.3 ml/min
UPLC Waters ACQUITY BEH C18, 1.7 µm, 2.1 x 50 mm, 0.5 ml/min
HPLC vs. UPLC HPLC UPLC
Example 3 (UPLC vs. HPLC) UPLC Relatively high chromatographic resolution High peak purity/integrity Cycle time 5 minutes Same robustness as HPLC Increase in sensitivity x5 Lower injection volume HPLC Relatively low chromatographic resolution Low peak purity/integrity Cycle time 5.5 minutes Robust
General Conclusions: UPLC vs. HPLC Sensitivity? Selectivity? Analysis time? Robustness? Ease of use? Cost? Increase Large increase Reduced Comparable Simple Conventional HPLC +~20% Is this the way forward in LC/MS-MS? Yes!
UPLC & API 4000 in combination UPLC Relatively high flow rates to maximise the gains of UHPLC For a 50 x 2.1 mm column, optimum flow is 0.5 to 1 ml/min API 4000 Works with relatively high flow rates, typically to 1.5 ml/min No real advantage in running higher flow rates Higher solvent usage UPLC/MS-MS A real advantage in running at higher flow rates, facilitated by the solvent capacity of the API 4000, and... No increase in solvent consumption due to shorter run times
Robustness Vitamin biomarker assay: ran 84 batches (approx. 100 extracted samples each) blocked columns - fixed by using a frit chromatography deteriorating (3 batches/column) PEEK needle bending - metal available shortly gradient from 50-98% organic Rt. stable
Disadvantages 50 µl maximum loop size 65 C upper limit on column oven PEEK injection needle Control through MassLynx or Empower only PEEK Needle
Jasco X-LC System capable of up to 15,000 psi Columns available down to 1.5 µm (although can t be used at these pressures!) Same Rheodyne valve as Waters Can also be used to retrofit CTC autosamplers
Agilent 1200 System capable of up to 8700 psi Removed > 350 µl dead volume Rapid Resolution HT Columns C18 and C8 (1.8µm) 4.6, 3.0 or 2.1 x 30 or 50 mm Systems available from Feb 2006
Columns - Alltech, Phenomenex and Thermo Alltech (1.5µm) HILIC, C18 and C8 2.1 or 4.6 x 10 or 20 mm 7 x 33 or 53 mm Hypersil Gold (1.9µm) C18 only 2.1 x 20, 30 or 50 mm Phenomenex (2.0µm) Synergy columns: 2.0, 4.0 and 4.6 x 10, 20, 30 or 50 mm
Towards a Different Future We may never know what motivated our fish ancestors to come out of the water. David Attenborough - Naturalist from Why Men Don t Listen and Women Can t Read Maps Allan and Barbara Pease 1999
Summary UPLC gives you: Improved selectivity Shorter runtime Greater sensitivity You just need the equipment to run it!
Acknowledgements Mohammed Abrar Sunetha Diaram Julian Potter Phill Turpin David Bakes
Questions?