Chip-Based E-Tip SPE followed by Infusion MS Copyright by Jack Henion, 2015 Lecture 1, Page 30
Infusion -MS Analysis of Sample Mixtures HPLC Mass Spec Elution/spray solvent Pipette tip with micro SPE packing and isolated rapamycin Copyright by Jack Henion, 2015 Lecture 1, Page 31
Proposed Solid-Phase Extraction Format with Express-Tips Sample Loading Column Washing Column Drying with Air Compound Elution Directly to Mass Spec 5 step sample handling process can now be accomplished in 3 steps Copyright by Jack Henion, 2015 Lecture 1, Page 32
Work Flow XIC of +MRM (6 pairs): 809.8/756.8 amu from Sample 1 (DS_033009_6 channel_ IS Mix_Plasma - 1A-3-C) of DS_033009_6 channel_ I... Max. 3.5e4 cps. 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 4.0e5 In te n s ity, c p s 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 0.0 0.99 1.10 1.25 1.75 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Time, min Copyright by Jack Henion, 2015 Lecture 1, Page 33
Mass Spectrometry Experimental Mass Spectrometer: ABI/MDS/SCIEX 4000 QTRAP TriVersa NanoMate equipped with ESI chip Electrospray, Positive ion mode Direct chip-based infusion of elution from Express-Tip to MS 1.7kV Solvent A [10%] 20mM Ammonium Acetate in 100 % Water 0.1% FA ; Solvent B [80%] 100% MeOH 0.1% FA; Flow Rate 5ul/min Transitions monitored were: Rapamycin m/z 931.7 > m/z 864.7 Tacrolimus m/z 821.6 > m/z 768.6 Everolimus m/z 975.7 > m/z 908.7 Optimized Method on ABI/MDS/SCIEX 4000 QTRAP: CUR 2; CAD Medium; IHT 125 Deg. C; DP 60V; Entrance potential 10 V; CE 25V; Cell exit potential 10 V Copyright by Jack Henion, 2015 Lecture 1, Page 34 34
Example ion current profile of Everolimus, Rapamycin and Tacrolimus with Ascomycin as Internal Standard in Human Plasma Copyright by Jack Henion, 2015 Lecture 1, Page 35
On-Line SPE Why Bother? Automation Higher sensitivity More selectivity Relatively short run times Less ion suppression Copyright by Jack Henion, 2015 Lecture 1, Page 36
Copyright by Jack Henion, 2015 Lecture 1, Page 37
Vinorelbine in rat plasma Ref. Charles River Laboratories, Montreal Copyright by Jack Henion, 2015 Lecture 1, Page 38
Ultrafiltration HOW DOES IT WORK? Membrane filters available from 3K to 1,000K MW cut-off. Mechanism: Solution of the sample is placed on the membrane. Compounds with MW s above the membrane cut-off cannot pass through. Low MW compounds pass through as filtrate. Resulting sample can be relatively clean. Process can be automated off-line followed by on-line analysis. Advantages Rapid Usually good enough for LC/MS Simple and can be automated continued... Copyright by Jack Henion, 2015 Lecture 1, Page 39
Ultrafiltration (Con t) Disadvantages Limited sample clean-up Capacity limitations for filters Does not concentrate the sample Copyright by Jack Henion, 2015 Lecture 1, Page 40
Affinity Techniques WHY BOTHER? Highly selective interaction between a bound ligand the targeted analyte. Molecular recognition provides unique selectivity Release ligand via ph, ionic strength, or solvent. Advantages Good sample cleanup Good concentration Automation Disadvantages Limited quantitative capability Limited availability of specific antiserum Deactivation of columns can be a problem Difficult to prepare, purify and bond Ab s for small molecules Copyright by Jack Henion, 2015 Lecture 1, Page 41
Affinity Coupled with ultrafiltration Binding Ab (or ER) + PBS + Steroids (or Urine) Wash/Centrifugation 2 x 150 ml PBS Centrifuge 12 tubes 10,000 g Decomplexation 50 L 0.2% HCOOH + 40% ACN LC/LC/MS Copyright by Jack Henion, 2015 Lecture 1, Page 42
Intensity Analytical Example with Steroids 30 20 10 200 100 A: Male Urine B: Non-Gravid Female Urine progesterone ( 9) 1.15e1 cps 2.32e2 cps 400 200 C: Gravid Female Urine unknown (m/z 303-285) 5 -pregnan-3,20 -diol (13) 5 -pregnan-3 -ol-20-one (12) progesterone (9) unknown (m/z 319-301) 6.45e2 cps 4000 2000 D: Progestins Std 9 10 11 12 13 14 3 4 5 6 7 8 9 10 11 12 Time, min 1.32e3 cps Copyright by Jack Henion, 2015 Lecture 1, Page 43 15
Magnetic Beads for Sample Preparation Copyright by Jack Henion, 2015 Lecture 1, Page 44
Structures of Magnetic Beads Copyright by Jack Henion, 2015 Lecture 1, Page 45
Representative Bead Specs Copyright by Jack Henion, 2015 Lecture 1, Page 46
Magnet Bead Sample Preparation Copyright by Jack Henion, 2015 Lecture 1, Page 47
Serum Peptide Profiling Copyright by Jack Henion, 2015 Lecture 1, Page 48
Conclusions: Bead-Based Sample Preparation Copyright by Jack Henion, 2015 Lecture 1, Page 49
Biologics & Biomarker LC/MS Combined techniques Quantitative determination of biotherapeutics, antibodies, proteins, peptides, lipids, and small molecule biomarkers GLP-biotherapeutic studies Quantitative analysis for PD/PK/TK Biomarker studies Immunogenicity determination Advanced techniques Hamilton Star automated liquid handling robotics Immunoprecipitation using capture antibodies (magnetic bead assays) Immunoaffinity columns using capture antibodies (made in-house) Dionex Ultimate 3000 for multi-dimensional (up to 4-D) LC/NanoLC/MS Advion TriVersa NanoMate with ESI Chip Technology Thermo TSQ Vantage triple quadrupole mass spectrometers Copyright by Jack Henion, 2015 Lecture 1, Page 50