LC -MS+NMR
Transcript of LC -MS+NMR
LCLC--MS+NMRMS+NMR
Building High Throughput Building High Throughput
Natural Products LibrariesNatural Products Libraries
Bob AlbrechtProtasis Corporation
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LC / Mass Spectrometry AloneLC / Mass Spectrometry Alone
�� First choice for compound First choice for compound
identificationidentification
�� Very sensitive and fast, butVery 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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NMR on LCNMR on LC--MS SamplesMS Samples(No Fraction Collection)(No Fraction Collection)
LC/MSLC/MS Sample Returned
Sample Returned
Interesting?
NMRNMR
Easy for very-high-purity compoundsEasy for very-high-purity compounds
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Isolation, DeIsolation, De--Replication, ElucidationReplication, Elucidation
1.1. Extract / Prepare SampleExtract / Prepare Sample
2.2. Analytical LC/MS With Fraction CollectionAnalytical LC/MS With Fraction Collection
3.3. DeDe--replicate with MSreplicate with MS
4.4. Exchange solvent, concentrateExchange solvent, concentrate
5.5. Run 1D NMR on unidentified or Run 1D NMR on unidentified or ambiguous fractionsambiguous fractions
6.6. DeDe--replicate with 1D NMRreplicate with 1D NMR
7.7. 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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CapNMR From Purified FractionsCapNMR From Purified Fractions
�� SimpleSimple–– No tricky SPE methodNo tricky SPE method
–– No stopNo stop--flowflow
–– No HPLC solvent suppression No HPLC solvent suppression
–– No reNo re--shimmingshimming
�� 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 deBetter match for de--replicationreplication libraries libraries withwith NMR in deuterated solventsNMR in deuterated solvents
�� Best NMR SensitivityBest NMR Sensitivity vs. online LCvs. online LC--NMR or NMR or LCLC--SPESPE--NMRNMR
�� Largest fraction capacityLargest fraction capacity vs. LCvs. LC--SPESPE--NMRNMR
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Auto-sampler
HPLC Column
Splitter
MS
FractionCollector
UV
CapNMR
H11D, HMBC, 13C. 15N, …
LCLC--MS+NMRMS+NMR
ACD/LabsStructure Elucidator
HPLC
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MicroMicro--plateplate
Fraction CollectionFraction Collection
CapNMR is designed foranalytical-scale separations.
CapNMR is designed foranalytical-scale separations.
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MS isnMS isn’’t enough for t enough for de novode novo structure structure
determination, butdetermination, but……
CapNMR Has The PowerCapNMR Has The Power
�� MS + 1D Proton NMR in deuterated MS + 1D Proton NMR in deuterated
solvent is often enough for desolvent is often enough for de--replicationreplication
�� 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!
�� OneOne--Minute NMR keeps the system Minute NMR keeps the system
running nonrunning non--stopstop
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Chromatographic Purification Chromatographic Purification
StrategiesStrategies
�� Time / VolumeTime / Volume
�� UVUV
�� MSMS
�� MS + UVMS + UV
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Time / Volume Directed PurificationTime / Volume Directed Purification
1.0 2.0 3.0 4.0
Time (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 10.2
Time (min)
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UVUV--Directed PurificationDirected Purification
0.70 0.80 0.90 1.00 1.10
Time (min)
• More pure compound• Few impure fractions• Too many worthless fractions• No chromaphore, no fraction
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MSMS--Directed PurificationDirected Purification
0.70 0.80 0.90 1.00 1.10
Time (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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MS + UV PurificationMS + UV Purification
0.70 0.80 0.90 1.00 1.10
Time (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 from
marine-derived Cephalosporium sp.
DAD detection
Bioactivity profile
Workflow Example: Natural Products DereplicationActive Peak(s)
14
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500 MHz8 µµµµg in 6 µµµµL 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
�� UPLCUPLC™™ (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 massof total fraction mass
�� AnalyticalAnalytical –– (4.6x150mm 3(4.6x150mm 3--55µµ))–– < ~7mg sample< ~7mg sample
–– Good chromatographic resolutionGood chromatographic resolution
–– CapNMR on CapNMR on 0.7%0.7% of total fraction massof total fraction mass
�� SemiSemi--PrepPrep–– 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 massof total fraction mass
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Why Scale DownWhy Scale Down
Purification + NMR?Purification + NMR?
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Is This Your NextIs This Your Next
Natural Products Expedition?Natural Products Expedition?
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Use OneUse One--Minute NMR to 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 scaleOnly scale--up when up when neededneeded
Fixed-Budget Throughput vs.
Sample Mass
UPL
C
Ana
lytic
al
Sem
i-Pre
p
Sample Mass
Cost / Sample
Throughput
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Chromatographic Resolution Chromatographic Resolution
MattersMatters
3.70 3.80 3.90 4.00 4.10 4.20 4.30
Time (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 scalescale--down purification?down purification?It Is a TradeIt Is a Trade--OffOff
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Time (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
OneOne--Minute NMR Automation Minute NMR Automation
Integrates:Integrates:
–– NMR with CapNMR ProbeNMR with CapNMR Probe
–– LCLC--MSMS
–– ACD/Labs Structure Elucidation and ACD/Labs Structure Elucidation and
DatabasingDatabasing
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OneOne--Minute NMRMinute NMR
Manages Samplesfrom
Plates 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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OneOne--Minute NMR SoftwareMinute 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:Excellent NMR Results from:Excellent NMR Results from:Excellent NMR Results from:
• 20 20 20 20 –––– 200200200200µµµµg samples g samples g samples g samples
• 0.1% fraction for semi0.1% fraction for semi0.1% fraction for semi0.1% fraction for semi----prepprepprepprep
• 0.7% fraction for analytical scale0.7% fraction for analytical scale0.7% fraction for analytical scale0.7% fraction for analytical scale
• 2.5% fraction for UPLC2.5% fraction for UPLC2.5% fraction for UPLC2.5% fraction for UPLC
Excellent NMR Results from:Excellent NMR Results from:Excellent NMR Results from:Excellent NMR Results from:
• 20 20 20 20 –––– 200200200200µµµµg samples g samples g samples g samples
• 0.1% fraction for semi0.1% fraction for semi0.1% fraction for semi0.1% fraction for semi----prepprepprepprep
• 0.7% fraction for analytical scale0.7% fraction for analytical scale0.7% fraction for analytical scale0.7% fraction for analytical scale
• 2.5% fraction for UPLC2.5% fraction for UPLC2.5% fraction for UPLC2.5% 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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DualDual--flowflow--cell CapNMRcell CapNMR
1.1. Quickly deQuickly de--replicate with replicate with
1H1D1H1D
2.2. Elucidate unknown Elucidate unknown
structures with 13C, structures with 13C,
NOESY, NOESY, gHMBCgHMBC, , gHSQCgHSQC
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1200 Series Fraction Collector AS >
6100 Series Quadrupole LC/MS System
Agilent
AS = Analytical Scale
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Waters AutoPurification LC(/MS)
Semi-PrepSemi-Prep
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SFCSFC�� Normal Phase Chromatography with CO2 Normal Phase Chromatography with CO2
solventsolvent
�� Fractions autoFractions auto--concentrate concentrate
�� SemiSemi--prep scale availableprep scale available
�� TharSFCTharSFC has a high quality systemhas a high quality system
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Fraction ConcentrationFraction Concentration
Vacuum Centrifuge
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DonDon’’t Forget Quantitationt 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 havehave
�� CapNMR of purified fractions is ideal for CapNMR of purified fractions is ideal for quantitationquantitation
�� OneOne--Minute NMR Is the Ultimate System 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 nonERETIC signal for a non--interfering internal 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 5 ugug of 5 componentsof 5 components
�� Isolated Isolated proparacainproparacain
�� CapNMR at 600 MHzCapNMR at 600 MHz
�� 16 Scans (1 minute)16 Scans (1 minute)
�� 5 5 ugug of 1 component on columnof 1 component on column
Data Courtesy Novatia, LLCData Courtesy Novatia, LLC
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Online LCOnline LC--NMR vs. NMR vs. FractionationFractionation + CapNMR+ 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
OH
H3C
MW 740 -> agl 286
Data Courtesy of
Jean-Luc Wolfender
MetabolomicsUniversity of Geneva, Switzerland
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LC/UV/MSLC/UV/MS
of the of the chromatogramchromatogram
usedused for microfor micro--fractionationfractionation
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
Intensity
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
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
31,3325,6319,05
37,72
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 45,7241,7347,3410,78 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, 23,61-25,02NL: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 1000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
[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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CapCap--NMRNMR gCOSYgCOSY spectrumspectrum of fraction C47of fraction C47
O
OH
O
O
OH
O
OO
OH
OHHO
CH3
OH
OHHO
O
O
HO
OH
OH
H3C
NT=1 NI=512 Time=12 min
Data Courtesy of
Jean-Luc Wolfender
MetabolomicsUniversity of Geneva, Switzerland
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LCLC--MS+CapNMRMS+CapNMR for Natural 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 defractionation, and de--replicationreplication
�� Maximum performance CapNMR to Maximum performance CapNMR to
dede--replicate and elucidate replicate and elucidate de novode novo
structures structures
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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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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