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LC-MS+NMRLC-MS+NMR
Building High Throughput Building High Throughput Natural Products LibrariesNatural Products Libraries
Bob AlbrechtProtasis Corporation
Copyright (c) 2008 by Protasis CorCopyright (c) 2008 by Protasis Corporation, All Rights Reservedporation, All Rights Reserved
22
LC / Mass Spectrometry AloneLC / Mass Spectrometry Alone
First choice for compound First choice for compound identificationidentification
Very sensitive and fast, but…Very sensitive and fast, but… Not enough information to elucidate Not enough information to elucidate
novel compoundsnovel compounds Often ambiguous Often ambiguous Ionization problems complicate Ionization problems complicate
identificationidentification
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33
NMR on LC-MS SamplesNMR on LC-MS Samples(No Fraction Collection)(No Fraction Collection)
LC/MSLC/MS Sample ReturnedSample Returned
Interesting?
NMRNMR
Easy for very-high-purity compoundsEasy for very-high-purity compounds
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44
Isolation, De-Replication, ElucidationIsolation, De-Replication, Elucidation
1.1. Extract / Prepare SampleExtract / Prepare Sample2.2. Analytical LC/MS With Fraction CollectionAnalytical LC/MS With Fraction Collection3.3. De-replicate with MSDe-replicate with MS4.4. Exchange solvent, concentrateExchange solvent, concentrate5.5. Run 1D NMR on unidentified or Run 1D NMR on unidentified or
ambiguous fractionsambiguous fractions6.6. De-replicate with 1D NMRDe-replicate with 1D NMR7.7. NMR Structure Elucidation Suite (HMBC, NMR Structure Elucidation Suite (HMBC,
13C, 15N, …) 13C, 15N, …) 8.8. ACD/Labs Structure Elucidator HelpsACD/Labs Structure Elucidator Helps
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55
CapNMR From Purified FractionsCapNMR From Purified Fractions SimpleSimple
– No tricky SPE methodNo tricky SPE method– No stop-flowNo stop-flow– No HPLC solvent suppression No HPLC solvent suppression – No re-shimmingNo re-shimming
InexpensiveInexpensive No interferencesNo interferences = easier elucidation = easier elucidation Complete NMR elucidationComplete NMR elucidation enabled by enabled by
having more timehaving more time Better match for de-replicationBetter match for de-replication libraries libraries
withwith NMR in deuterated solventsNMR in deuterated solvents Best NMR SensitivityBest NMR Sensitivity vs. online LC-NMR or vs. online LC-NMR or
LC-SPE-NMRLC-SPE-NMR Largest fraction capacityLargest fraction capacity vs. LC-SPE-NMR vs. LC-SPE-NMR
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66
Auto-sampler
HPLC Column
Splitter
MS
FractionCollectorUV
CapNMR
H11D, HMBC, 13C. 15N, …
LC-MS+NMRLC-MS+NMR
ACD/LabsStructure Elucidator
HPLC
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77
Micro-plateMicro-plateFraction CollectionFraction Collection
CapNMR is designed foranalytical-scale separations.CapNMR is designed foranalytical-scale separations.
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88
MS isn’t enough for MS isn’t enough for de novode novo structure structure
determination, but…determination, but… CapNMR Has The PowerCapNMR Has The Power
MS + 1D Proton NMR in deuterated solvent MS + 1D Proton NMR in deuterated solvent is often enough for de-replicationis often enough for de-replication
CapNMR enables full structure elucidation CapNMR enables full structure elucidation of unknowns of unknowns – 13C, 15N, HSQC, HMBC, HMQC, COSY, TOCSY, 13C, 15N, HSQC, HMBC, HMQC, COSY, TOCSY,
ROESY, NOESY without changing probes!ROESY, NOESY without changing probes! One-Minute NMR keeps the system One-Minute NMR keeps the system
running non-stoprunning non-stop
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99
Chromatographic Purification Chromatographic Purification StrategiesStrategies
Time / VolumeTime / Volume UVUV MSMS MS + UVMS + UV
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1010
Time / Volume Directed PurificationTime / Volume Directed Purification
1.0 2.0 3.0 4.0Time (min)
• Fractions can contain 2+ compounds• Less mass of purified material• Many fractions• Collects everything
9.0 9.2 9.4 9.6 9.8 10.0 10.2Time (min)
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1111
UV-Directed PurificationUV-Directed Purification
0.70 0.80 0.90 1.00 1.10Time (min)
• More pure compound• Few impure fractions• Too many worthless fractions• No chromaphore, no fraction
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1212
MS-Directed PurificationMS-Directed Purification
0.70 0.80 0.90 1.00 1.10Time (min)• Pure fractions
• MS picks valuable fractions• “Worthless” fractions may actually have value• Poor ionization can waste valuable fractions• Unknown mass range a problem
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1313
MS + UV PurificationMS + UV Purification
0.70 0.80 0.90 1.00 1.10Time (min)
• Poor ionization, fractions still collected• No chromaphore, fractions still colleted• Mass range can screen-out unlikely hits• NMR on fractions with interesting MS spectra
• Poor ionization, fractions still collected• No chromaphore, fractions still colleted• Mass range can screen-out unlikely hits• NMR on fractions with interesting MS spectra
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C18 HPLC of extract frommarine-derived Cephalosporium sp.
DAD detection
Bioactivity profile
Workflow Example: Natural Products DereplicationActive Peak(s)
14
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500 MHz8 g in 6 L CD3ODPRESAT 1.5 min
Recognisable features: 5 CH3 groups, of which 1 methoxyl,3 singlet CH3-C, 1 doublet CH3-C; 1 aldehyde
15
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Purification ScalePurification Scale UPLC™UPLC™ (2.1x150mm 1.7 (2.1x150mm 1.7µµ) )
– < ~2 mg sample, plenty for MS (.1< ~2 mg sample, plenty for MS (.1µµg) + CapNMR (50g) + CapNMR (50µµg)g)– Amazing chromatographic resolutionAmazing chromatographic resolution– highest purity fractionshighest purity fractions– CapNMR on CapNMR on 2.5%2.5% of total fraction mass of total fraction mass
AnalyticalAnalytical – (4.6x150mm 3-5 – (4.6x150mm 3-5µµ))– < ~7mg sample< ~7mg sample– Good chromatographic resolutionGood chromatographic resolution– CapNMR on CapNMR on 0.7%0.7% of total fraction mass of total fraction mass
Semi-PrepSemi-Prep– 50mg to grams of sample50mg to grams of sample– Longer runsLonger runs– Less resolution, more impure fractionsLess resolution, more impure fractions– Typical for compound librariesTypical for compound libraries– CapNMR on CapNMR on 0.1%0.1% of total fraction mass of total fraction mass
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Why Scale DownWhy Scale DownPurification + NMR?Purification + NMR?
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Is This Your NextIs This Your NextNatural Products Expedition?Natural Products Expedition?
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Use One-Minute NMR to Use One-Minute NMR to Do more with less sampleDo more with less sample
Solve Solve structures structures with with minimum minimum massmass
Most Most samples samples are not hitsare not hits
Only scale- Only scale- up when up when neededneeded
Fixed-Budget Throughput vs. Sample Mass
UPLC
Analyt
ical
Semi-P
rep
Sample MassCost / SampleThroughput
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2020
Chromatographic Resolution Chromatographic Resolution MattersMatters
3.70 3.80 3.90 4.00 4.10 4.20 4.30Time (min)
There are three compounds under this peak.The MS sees them, but you will not getthe purity you need for NMR elucidation.
There are three compounds under this peak.The MS sees them, but you will not getthe purity you need for NMR elucidation.
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Why notWhy not scale-down purification? scale-down purification?It Is a Trade-OffIt Is a Trade-Off
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2Time (min)
Next Blockbuster Drug (50ug)
You still need to scale-down NMR toElucidate the 0.1% fraction.You still need to scale-down NMR toElucidate the 0.1% fraction.
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EquipmentEquipment
One-Minute NMR Automation One-Minute NMR Automation Integrates:Integrates:– NMR with CapNMR ProbeNMR with CapNMR Probe– LC-MSLC-MS– ACD/Labs Structure Elucidation and ACD/Labs Structure Elucidation and
DatabasingDatabasing
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One-Minute NMROne-Minute NMRManages Samples
fromPlates and Vials
Interfaces with:• People (Web, e-mail)• NMR • LC-MS• ACD/Labs• OneMoon• Custom
Software
Interfaces with:• People (Web, e-mail)• NMR • LC-MS• ACD/Labs• OneMoon• Custom
Software
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One-Minute NMR SoftwareOne-Minute NMR Software• Web / E-Mail enabled
• Import Fraction Lists
• Run NMR Experiments
• Maintain NMR Quality
• Archive and Process Data
• Customizable
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CapNMRCapNMRSpecifically Designed for NMR of LC PeaksSpecifically Designed for NMR of LC Peaks
Excellent NMR Results from:
• 20 – 200µg samples
• 0.1% fraction for semi-prep
• 0.7% fraction for analytical scale
• 2.5% fraction for UPLC
Excellent NMR Results from:
• 20 – 200µg samples
• 0.1% fraction for semi-prep
• 0.7% fraction for analytical scale
• 2.5% fraction for UPLCCapNMR TXI Runs all of your NMRexperiments for structure elucidationwithout changing the probe!
1H, 13C, 15N, 1D, 2D:APT, INEPT, DEPT, NOE, COSY, RELAY, DQFCOSY, TQCOSY, TOCSY, HOMJ,NOESY, ROESY, INADEQUATE, PFG, gCOSY, gDQFCOS, HETCOR, HMQC, HSQC,HMBC, gHMBC, gHMQC, gHSQC…
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Dual-flow-cell CapNMRDual-flow-cell CapNMR
1.1. Quickly de-replicate with Quickly de-replicate with 1H1D1H1D
2.2. Elucidate unknown Elucidate unknown structures with 13C, structures with 13C, NOESY, gHMBC, gHSQCNOESY, gHMBC, gHSQC
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2727
1200 Series Fraction Collector AS >
6100 Series Quadrupole LC/MS System
Agilent
AS = Analytical Scale
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2828
Waters AutoPurification LC(/MS)
Semi-PrepSemi-Prep
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SFCSFC Normal Phase Chromatography with CO2 Normal Phase Chromatography with CO2
solventsolvent Fractions auto-concentrate Fractions auto-concentrate Semi-prep scale availableSemi-prep scale available TharSFC has a high quality systemTharSFC has a high quality system
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Fraction ConcentrationFraction Concentration
Vacuum Centrifuge
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Don’t Forget QuantitationDon’t Forget Quantitation You can only know how active your You can only know how active your
compound is if you know how much you compound is if you know how much you reallyreally have have
CapNMR of purified fractions is ideal for CapNMR of purified fractions is ideal for quantitationquantitation
One-Minute NMR Is the Ultimate System One-Minute NMR Is the Ultimate System For QuantitationFor Quantitation– No tuning between tubes = no recalibration No tuning between tubes = no recalibration
with standardwith standard– Consistent robotic injectionConsistent robotic injection– ERETIC signal for a non-interfering internal ERETIC signal for a non-interfering internal
standardstandard
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Isolated Model Compound NMR Isolated Model Compound NMR
5 Model Compound Mixture5 Model Compound Mixture– CapNMR at 600 MHzCapNMR at 600 MHz– 64 Scans (4 minutes)64 Scans (4 minutes)– 5 ug of 5 components5 ug of 5 components
Isolated proparacainIsolated proparacain CapNMR at 600 MHzCapNMR at 600 MHz 16 Scans (1 minute)16 Scans (1 minute) 5 ug of 1 component on 5 ug of 1 component on
columncolumnData Courtesy Novatia, LLCData Courtesy Novatia, LLC
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3333
Online LC-NMR vs. Fractionation + CapNMROnline LC-NMR vs. Fractionation + CapNMR
On flow NMR
NT=512
CAP-NMR of fraction C47
NT=16
O
OH
O
O
OH
O
OO
OH
OHHO
CH3
OH
OHHO
O
O
HO
OH
OHH3C
MW 740 -> agl 286
Data Courtesy of
Jean-Luc Wolfender
MetabolomicsUniversity of Geneva, Switzerland
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3434
LC/UV/MSLC/UV/MSof the chromatogram of the chromatogram
used for micro-fractionationused for micro-fractionation
RT: 0,00 - 60,08 SM: 7B
0 5 10 15 20 25 30 35 40 45 50 55 60
Time (min)
0
1
2
3
4
5
6
7
8
9
uAU
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
Inte
nsity
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bun
dan
ce
22,97
33,32
29,3728,63 39,4435,25 40,6930,1822,56 42,88 46,775,23 49,61 53,3618,587,25 16,99 56,304,28 12,809,04 59,211,45
31,3325,6319,0537,72
26,95
22,60
6,4837,3216,82
8,81
21,89 38,5044,54
46,36
35,9615,50 49,6128,77 40,28
40,7957,7514,595,97 11,69 57,0451,973,401,85
32,2127,7922,908,5725,13
21,72
33,2534,61
16,6820,23 37,18
6,58
38,36
18,88 39,8815,77 45,7241,73 47,3410,78 15,10 51,805,84 53,16 58,972,79
NL:6,95E5
m/z= 739,5-740,5 F: - c ESI Full ms [ 140,01-1000,02] MS exfl10mg
NL:1,68E7
Base Peak F: - c ESI Full ms [ 140,01-1000,02] MS exfl10mg
NL:9,90
Channel A UV exfl10mg
exfl10mg #676-690 RT:22,87-23,34 AV:15 SB: 77 21,55-22,66, 23,61-25,02 NL:5,92E5F: - c ESI Full ms [ 140,01-1000,02]
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
[M-H]-
739,3
477,5
445,6
740,8
421,6216,2741,8
506,6446,7921,7313,3 775,8609,8
281,3 863,8593,7 922,9738,8253,3 567,7404,9387,6 776,8468,5364,9 957,7742,9670,4515,2 650,8 832,2 902,0 997,5956,1
nm200 250 300 350 400 450 500 550
mAU
-200
0
200
400
600
800
1000
1200
DAD1, 21.878 (1449 mAU, - ) of FL10MG.D
m/z 739
TIC
UV (254nm)
Data Courtesy of
Jean-Luc Wolfender
MetabolomicsUniversity of Geneva, Switzerland
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3535
Cap-NMR gCOSY spectrum of fraction C47Cap-NMR gCOSY spectrum of fraction C47
O
OH
O
O
OH
O
OO
OH
OHHO
CH3
OH
OHHO
O
O
HO
OH
OHH3C
NT=1 NI=512 Time=12 min
Data Courtesy of
Jean-Luc Wolfender
MetabolomicsUniversity of Geneva, Switzerland
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3636
LC-MS+CapNMR for Natural LC-MS+CapNMR for Natural Products is:Products is:
Higher performance analytical scale Higher performance analytical scale chromatographychromatography
Fast online MS detection, Fast online MS detection, fractionation, and de-replicationfractionation, and de-replication
Maximum performance CapNMR to Maximum performance CapNMR to de-replicate and elucidate de-replicate and elucidate de novode novo structures structures
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3737
J Biomol Tech. 2003 June; 14(2): 136–142. PMCID: Mass-Based Fraction Collection of Crude Synthetic Peptides in Analytical and Preparative Scale
The importance of hyphenated techniques in the discovery of new lead compounds from natureJean-Luc Wolfender, Emerson Ferreira Queiroz, Kurt HostettmannExpert Opinion on Drug Discovery, August 2006, Vol. 1, No. 3, Pages 237-260(doi: 10.1517/17460441.1.3.237
ASMS 2008"Evaluation of Column Retentivity with large injection volume for High Sensitive and High-Throuput LC-MS/MS Quantitative Analysis." J. Watanabe, et. al. Takara Bio – Japan
Further ReadingFurther Reading
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3838
ThanksThanks
Jean-Luc Wolfender, University of Geneva
David Detlefsen, Novatia
Get your Natural Products LC-MS+CapNMR system from Protasis Corporation.http://protasis.com508-481-4163