LC-MS Plus NMR
description
Transcript of LC-MS Plus NMR
LC-MS Plus NMRLC-MS Plus NMR
Building And Confirming Building And Confirming Modern Molecular LibrariesModern Molecular Libraries
Bob AlbrechtProtasis Corporation
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 verificationOften ambiguous verification Ionization problems complicate Ionization problems complicate
identificationidentification
CapNMR From Purified FractionsCapNMR From Purified Fractions SimpleSimple
– No tricky SPE method or LC solventsNo tricky SPE method or LC solvents– 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 Best NMR SensitivityBest NMR Sensitivity vs. online LC- vs. online LC-
NMR or LC-SPE-NMRNMR or LC-SPE-NMR Largest fraction capacityLargest fraction capacity vs. LC- vs. LC-
SPE-NMR (384+)SPE-NMR (384+)
Auto-sampler
HPLC Column
Splitter
MS
FractionCollectorUV
CapNMR
H11D, HMBC, 13C. 15N, …
LC-MS+NMRLC-MS+NMR
ACD/LabsStructure Elucidator
HPLC
Micro-plateMicro-plateFraction CollectionFraction Collection
CapNMR is designed foranalytical-scale separations.
MS isn’t enough for MS isn’t enough for novelnovel structure determination, structure determination,
but…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 verification, but when is often enough for verification, but when it isn’t…it isn’t…
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
Chromatographic Purification Chromatographic Purification StrategiesStrategies
Time / VolumeTime / Volume UVUV MSMS MS + UVMS + UV
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)
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
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
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
C18 HPLC of extract frommarine-derived Cephalosporium sp.
DAD detection
Bioactivity profile
Workflow Example: Natural Products DereplicationActive Peak(s)
John W Blunt, Department of Chemistry, University of Canterbury, Christchurch NEW ZEALAND
12
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
13
John W Blunt, Department of Chemistry, University of Canterbury, Christchurch NEW ZEALAND
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
Purification Scale-Down Saves Purification Scale-Down Saves Time and MoneyTime and Money
t ≈ $
∆t + ∆$ = ∆productivity
Analytical ScaleAnalytical ScaleChromatographic Resolution MattersChromatographic Resolution Matters
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.
But… But… Trade-Off With SensitivityTrade-Off With Sensitivity
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2Time (min)
.0.1% Impurity
You still need to scale-down NMR toElucidate the 0.1% fraction. A secondanalytical run resolves a poorly resolvedsemi-prep fraction.
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
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
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 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…
Dual flow cell now available!
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
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
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
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
ThanksThanks John W Blunt, Department of Chemistry,
University of Canterbury, Christchurch NEW ZEALAND
Jean-Luc Wolfender, University of Geneva David Detlefsen, Novatia
Protasis Corporationhttp://protasis.com508-481-4163