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Judith M. Rollinger
Ulrike Grienke
Preparative SFC QDa Applications in
Natural Products Research
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
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 biotechnology-related approaches, including genome-based bioprospecting, metabolic engineering and synthetic biology.
http://pharmakognosie.univie.ac.at/
• 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
Aims
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 (3D-molecule-DB: >120.000 entries); access to modeling software (LigandScout, ROCs,…)
See: http://pharmakognosie.univie.ac.at/research/phytochemistry-biodiscovery/equipment/
.... state-of-the-art equipment!
To complement our systems …
... our latest additions
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
N2 generator
chiller
CO2 cylinders
Pump for co-solvent and CO2
pump with different channels for 6 co-solvents
pump for CO2
(cooled with ethylene glycol)
fraction collector & autosampler: accommodates both
sample injection &
fraction collection
column oven: automated column switching and temperature control of up to 10 columns (analytical & preparative)
Oven, fraction collector, and autosampler
“Our” add-on detection configuration
QDa
Waters H-Class UPLC
MS-based triggering better collection precision by targeting only the mass of interest
Columns for the SFC Prep-15
further chemistries: Silica 2-EP Silica
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
4.6 x 250 mm
10 x 250 mm Silica 2-EP
particle shape: spherical, particle size: 5 µm
Dimension analytical preparative
Columns available in our lab
VIS
Vanillin/H2SO4
TLC System:
DCM : MeOH : H20
(10 : 1 : 0.25)
Application example: POLYPORES
one species
strain 1
strain 2 strain 3
Pictures: http://www.mycobank.org/
first evaluation of metabolite profile
many triterpenes difficult to detect with HPLC!
Profiling with SFC!
finding the best source material for bioactive compounds
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.
different strains different metabolite profile different bioactivity!
column Viridis BEH, 5 µm 4.6 x 250 mm
flow rate 5 mL/min
make-up flow rate 3 mL/min
injection volume 5 µL
sample conc. 5 mg/mL
detection ELSD
system back pressure 120 bar
temp. 40°C
Time (min) EtOH (%)
0 5
1 5
9.5 50
10.5 50
11 5
12 5
Comparison of metabolite profiles
Strain 1
Strain 2
Strain 3
Strain 4
Strain 5
Strain 6
best bioactive starting material to isolate the target compound
EtOH
crude extract
DCM
fraction active
S1F12
active
Step 1: liquid/liquid partition
Step 2: Silica gel column
> 200 constituents 98% lanostane triterpenes highly similar molecular weight bad UV detection
Application example: POLYPORES
Purification of selected fractions of a polypore fungus
S1F12
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= CH3CN; 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.
HPLC-CAD
Highly complex triterpene mixture !
SFC method development for S1F12
• 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
CH3CN
flow rate 5 mL/min
make-up 3 mL/min
injection volume
20 µL
sample conc. 3 mg/mL
detection PDA-ELSD
system back pressure
120 bar
temp. 40°C
Time (min) co-solvent (%)
0 5
1 5
6 50
7 50
7.5 5
9 5
S1F12 best separated with EtOH as co-solvent
BEH
CSH FP
Silica 2-EP
best separation with BEH column further optimisation
SFC method development for S1F12
Scal
e-u
p
column Viridis BEH, 5 µm 4.6 x 250 mm (analyt.) 10 x 250 mm (prep.)
flow rate 5 mL/min (analyt.) 15 mL/min (prep.)
make-up flow rate
3 mL/min (analyt.) 5 mL/min (prep.)
injection volume
20 µL (analyt.) 150 µL (prep.)
sample conc.
3 mg/mL
detection PDA-ELSD
system back pressure
120 bar
temp. 40°C Time (min) EtOH (%)
0 8
9.5 8
10 50
11 50
11.5 8
12 8
Scale-up: analytical to preparative mode
good separation of single peaks
FractionLynx Method:
Development of fractionation method
Collection of 10 fractions
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
S1F12 SFC separation
fractions 5, 7, 9 monitoring with RP-HPLC-CAD: good separation on Prep-15 system enriched fractions still contain a few peaks
Fraction monitoring with RP-HPLC-CAD
1
2
3
4
5 6
7 8
9 10
9
7
5
HPLC monitoring
LC-parameter: stationary phase: Zorbax (4.6 x 150 mm, 3.5 µm); temperature: 35° C; mobile phase: A= water; B= CH3CN; 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-fractionation of S1F12 with Flash chromatography
column PuriFlash Column 15 C18 HP 6.0 g
solvents gradient: H2O/CH3CN
flow rate 5 mL/min
amount separated
~ 500 mg
detection PDA-ELSD
HPLC monitoring
LC-parameter: stationary phase: Zorbax (4.6 x 150 mm, 3.5 µm); temperature: 35° C; mobile phase: A= water; B= CH3CN; 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
Fraction 6
Fraction 7
Fraction 8
Separation of two co-eluting cpds
SFC-parameter: stationary phase: Viridis BEH, 5 µm 10 x 250 mm; temperature: 40°C; system back pressure: 120 bar, mobile phase: A= CO2; 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.
A B
A
B
HPLC monitoring
Fraction 6
Separation of two co-eluting cpds
SFC-parameter: stationary phase: Viridis BEH, 5 µm 10 x 250 mm; temperature: 40°C; system back pressure: 120 bar, mobile phase: A= CO2; 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.
HPLC monitoring
C C
Fraction 7
Separation of further cpds
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= CO2; 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.
D
D
Fraction 8
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
Thank you for your attention!
Dr. Ulrike Grienke Natalie Prenner Julia Zwirchmayr
Department of Pharmacognosy, University of Vienna, AUSTRIA
Acknowledgement
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