lc -ms+nmr

LCLC--MS+NMRMS+NMR

Building High Throughput Building High Throughput

Natural Products LibrariesNatural Products Libraries

Bob AlbrechtProtasis Corporation

Copyright (c) 2008 by Protasis Copyright (c) 2008 by Protasis

Corporation, All Rights ReservedCorporation, All Rights Reserved 22

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



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



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



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



Auto-sampler

HPLC Column

Splitter

MS

FractionCollector

UV

CapNMR

H11D, HMBC, 13C. 15N, …

LCLC--MS+NMRMS+NMR

ACD/LabsStructure Elucidator

HPLC



MicroMicro--plateplate

Fraction CollectionFraction Collection

CapNMR is designed foranalytical-scale separations.

CapNMR is designed foranalytical-scale separations.



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



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 4.0

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)



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



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



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



C18 HPLC of extract from

marine-derived Cephalosporium sp.

DAD detection

Bioactivity profile

Workflow Example: Natural Products DereplicationActive Peak(s)

14



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



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


�� 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




Why Scale DownWhy Scale Down

Purification + NMR?Purification + NMR?



Is This Your NextIs This Your Next

Natural Products Expedition?Natural Products Expedition?



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



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.



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.



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



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



OneOne--Minute NMR SoftwareMinute NMR Software

• Web / E-Mail enabled

• Import Fraction Lists

• Run NMR Experiments

• Maintain NMR Quality

• Archive and Process Data

• Customizable



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…



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



1200 Series Fraction Collector AS >

6100 Series Quadrupole LC/MS System

Agilent

AS = Analytical Scale



Waters AutoPurification LC(/MS)

Semi-PrepSemi-Prep



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



Fraction ConcentrationFraction Concentration

Vacuum Centrifuge



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



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



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



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




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




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



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



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

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