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