Preparative SFC QDa Applications in Natural Products Research Judith M. Rollinger Ulrike Grienke ÖSTERREICHISCHES TECHNOLOGIE SYMPOSIUM 2016, October 25, 2016
Pharmacognosy Group, UNIVIE Plants, fungi, microorganisms, marine organisms, are still the major source of new chemical entities for drug discovery! 64% = derived/inspired by nature 34% = natural cpds /derivatives Sources of drug substances (small chemical entities;1981 2011, n = 1078): Newman & Cragg (2012) J. Nat. Prod., 75, 311-335
Research Groups http://pharmakognosie.univie.ac.at/ Dirsch Lab: Molecular Targets Research focus: to understand the molecular interaction of natural products with proteins/signaling molecules within cells and to provide cellular models for the identification of bioactive natural products. Rollinger Lab: Phytochemistry & Biodiscovery We use interdisciplinary approaches (e.g. in silico guided, ethnopharmacology-driven) to identify, analyze, and purify multi-component mixtures from plant, fungal, or marine sources aiming at the characterization, the targeted isolation and discovery of bioactive secondary metabolites. Zotchev Lab: Pharmaceutical Biotechnology Discovery of natural product from microorganisms, supported by cutting edge biotechnologyrelated approaches, including genome-based bioprospecting, metabolic engineering and synthetic biology.
Aims Pharmaceutical lead and target identification from natural resources Quality control of herbal medicinal products Sustainable production of natural products by optimized extract procedures, chromatographic workflows and biotechnological approaches Educational work and young researchers training
... state-of-the-art equipment! Analytic lab for metabolic profiling, dereplication UPLC systems with different detectors (UV/Vis, DAD, QD, ELSD, CAD) UPLC - triple quadrupole MS with ESI and APCI; UPLC - ion trap mass spectrometer with ESI and APCI ion source GC-MS with EI- and CI-ionization; GC with flame ionization detectors (FID) Different HPLC systems with UV/Vis, DAD and/or ELSD for routine analysis High-performance TLC system with application unit, TLC visualizer and evaluation unit Extraction, fractionation, isolation Prep. supercritical fluid chromatograph (SFC) with parallel detection units (PDA, ELSD, QDa), fraction collector Prep. HPLC systems with autosampler, DAD and fraction collector; High performance counter-current chromatograph (HPCCC) Ultra-performance flash chromatograph combined with PDA and ELSD Chem. characterization of natural compounds/metabolites QTOF-MS with ESI and APCI ion source (with UPLC system; shared with Dep. Pharm. Chem.) NMR spectrometers (400 MHz & 500 MHz) with autosampler and cryoprobe (shared with Dep. Pharm. Chem) UV/VIS spectrophotometers Attenuated total reflection (ATR) - Fourier transform infrared (FTIR) spectrophotometer Biological characterization of natural compounds/multicomponent mixtures Caenorhabditis elegans Screening Plattform (in vivo Model) Different wild type strains (e.g., GFP marked) and genetically modified strains adapteto 96 well plates (for chemical genetics screens & therapeutic screen (neurodegen. diseases) In silico Screening Plattform: Expertise in computer-aided NP drug discovery; VS adapted NP databases (3Dmolecule-DB: >120.000 entries); access to modeling software (LigandScout, ROCs, ) See: http://pharmakognosie.univie.ac.at/research/phytochemistry-biodiscovery/equipment/
... our latest additions To complement our systems H-Class UPLC (with PDA, ELSD, QDa) including fraction manager SFC Prep-15 (with PDA, ELSD, QDa)
Instrumental set-up: Waters SFC Prep-15
Instrumental set-up: Waters SFC Prep-15
Instrumental set-up: Waters SFC Prep-15 CO 2 cylinders chiller N 2 generator
Pump for co-solvent and CO 2 pump with different channels for 6 co-solvents pump for CO 2 (cooled with ethylene glycol)
Oven, fraction collector, and autosampler column oven: automated column switching and temperature control of up to 10 columns (analytical & preparative) fraction collector & autosampler: accommodates both sample injection & fraction collection
Waters H-Class UPLC Our add-on detection configuration QDa MS-based triggering better collection precision by targeting only the mass of interest
further chemistries: Silica 2-EP Silica Columns for the SFC Prep-15
Columns available in our lab Dimension analytical preparative 4.6 x 250 mm 10 x 250 mm 4.6 x 250 mm 10 x 250 mm 4.6 x 250 mm 10 x 250 mm Silica 2-EP 4.6 x 250 mm 10 x 250 mm particle shape: spherical, particle size: 5 µm
Application example: POLYPORES strain 2 strain 1 one species strain 3 different strains different metabolite profile different bioactivity! first evaluation of metabolite profile many triterpenes difficult to detect with HPLC! Profiling with SFC! TLC System: DCM : MeOH : H 2 0 (10 : 1 : 0.25) finding the best source material for bioactive compounds VIS Vanillin/H 2 SO 4 Dresch, P. Aguanno MN, Rosam K, Grienke U, Rollinger JM, Peintner U. (2015): Fungal strain matters: Colony growth and bioactivity of the European medicinal polypores Fomes fomentarius, Fomitopsis pinicola and Piptoporus betulinus. AMB Express 5:4. Pictures: http://www.mycobank.org/
Comparison of metabolite profiles Strain 1 Strain 2 column Viridis BEH, 5 µm 4.6 x 250 mm flow rate make-up flow rate injection volume 5 µl 5 ml/min 3 ml/min Strain 3 sample conc. detection system back pressure 5 mg/ml ELSD 120 bar Strain 4 temp. 40 C Strain 5 Strain 6 Time (min) EtOH (%) 0 5 1 5 best bioactive starting material to 9.5 50 isolate the target compound 10.5 50 11 5 12 5
Application example: POLYPORES EtOH crude extract Step 1: liquid/liquid partition Step 2: Silica gel column DCM fraction active > 200 constituents 98% lanostane triterpenes highly similar molecular weight bad UV detection S 1 F 12 active
Purification of selected fractions of a polypore fungus HPLC-CAD S 1 F 12 Highly complex triterpene mixture! separated with SFC separated with Flash CC (RP18) SFC LC-parameter: stationary phase: Zorbax (4.6 x 150 mm, 3.5 µm); temperature: 35 C; mobile phase: A= water; B= CH 3 CN; flow rate: 1.0 ml/min; detection: CAD; injection volume: 10 µl; gradient: 0 min: 45% B; 42 min: 80% B; 43 min; 95% B.
SFC method development for S 1 F 12 analytical column screening: Viridis SFC columns (particle size: 5 µm, dimension: 4.6 x 250 mm) BEH CSH Fluoro-Phenyl Silica 2-EP co-solvent screening: MeOH EtOH 2-propanol CH 3 CN Time (min) co-solvent (%) 0 5 1 5 6 50 7 50 7.5 5 9 5 flow rate make-up injection volume sample conc. detection system back pressure 5 ml/min 3 ml/min 20 µl 3 mg/ml PDA-ELSD 120 bar temp. 40 C
SFC method development for S 1 F 12 S 1 F 12 best separated with EtOH as co-solvent Silica 2-EP CSH FP BEH best separation with BEH column further optimisation
Scale-up Scale-up: analytical to preparative mode column Viridis BEH, 5 µm 4.6 x 250 mm (analyt.) 10 x 250 mm (prep.) flow rate make-up flow rate injection volume sample conc. detection 5 ml/min (analyt.) 15 ml/min (prep.) 3 ml/min (analyt.) 5 ml/min (prep.) 20 µl (analyt.) 150 µl (prep.) 3 mg/ml PDA-ELSD Time (min) EtOH (%) 0 8 9.5 8 10 50 11 50 11.5 8 12 8 system back pressure 120 bar temp. 40 C good separation of single peaks
Development of fractionation method S 1 F 12 SFC separation FractionLynx Method: 1 2 3 4 5 6 7 8 9 10 split/collector delay: 9 sec PDA/ELSD delay: 2 sec fractionation based on timed events Collection of 10 fractions
Fraction monitoring with RP-HPLC-CAD 5 1 2 3 4 5 6 7 8 9 10 7 HPLC monitoring 9 fractions 5, 7, 9 monitoring with RP-HPLC-CAD: LC-parameter: stationary phase: Zorbax (4.6 x 150 mm, 3.5 µm); temperature: 35 C; mobile phase: A= water; B= CH 3 CN; flow rate: 1.0 ml/min; detection: CAD; injection volume: 10 µl; gradient: 0 min: 45% B; 42 min: 80% B; 43 min; 95% B. good separation on Prep-15 system enriched fractions still contain a few peaks
Pre-fractionation of S 1 F 12 with Flash chromatography Fraction 6 Fraction 7 HPLC monitoring Fraction 8 column solvents flow rate amount separated detection PuriFlash Column 15 C18 HP 6.0 g gradient: H 2 O/CH 3 CN 5 ml/min ~ 500 mg PDA-ELSD LC-parameter: stationary phase: Zorbax (4.6 x 150 mm, 3.5 µm); temperature: 35 C; mobile phase: A= water; B= CH 3 CN; flow rate: 1.0 ml/min; detection: CAD; injection volume: 10 µl; gradient: 0 min: 45% B; 42 min: 80% B; 43 min; 95% B. Pre-purified fractions further separation via SFC
Separation of two co-eluting cpds Fraction 6 A A B B HPLC monitoring SFC-parameter: stationary phase: Viridis BEH, 5 µm 10 x 250 mm; temperature: 40 C; system back pressure: 120 bar, mobile phase: A= CO 2 ; B= EtOH; flow rate: 15.0 ml/min; detection: ELSD; injection volume: 250 µl; gradient: 0 min: 10% B; 8 min: 10% B; 9.5 min: 50% B; 11.5 min: 50% B; 12 min: 10% B; 13 min: 10% B.
Separation of two co-eluting cpds Fraction 7 C C HPLC monitoring SFC-parameter: stationary phase: Viridis BEH, 5 µm 10 x 250 mm; temperature: 40 C; system back pressure: 120 bar, mobile phase: A= CO 2 ; B= EtOH; flow rate: 15.0 ml/min; detection: ELSD; injection volume: 250 µl; gradient: 0 min: 8% B; 9.5 min: 8% B; 10 min: 50% B; 11 min: 50% B; 11.5 min: 8% B; 12 min: 8% B.
Separation of further cpds Fraction 8 D D HPLC monitoring SFC-parameter: stationary phase: Viridis BEH, 5 µm 10 x 250 mm; temperature: 40 C; system back pressure: 120 bar, mobile phase: A= CO 2 ; B= EtOH; flow rate: 15.0 ml/min; detection: ELSD; injection volume: 250 µl; gradient: 0 min: 8% B; 9.5 min: 8% B; 10 min: 50% B; 11 min: 50% B; 11.5 min: 8% B; 12 min: 8% B.
Integration of SFC in NP workflows + SFC is orthogonal to C18 + inexpensive and non-toxic carbon dioxide + easy handling via automated sample collection through FractionLynx Application Manager + beneficial alternative to purification and analytical applications
Acknowledgement Dr. Ulrike Grienke Natalie Prenner Julia Zwirchmayr Department of Pharmacognosy, University of Vienna, AUSTRIA Thank you for your attention!