Overview. Introduction. NMR hyphenation modes HPLC-SPE-NMR. Advantages and limitations. Examples

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1 NMR hyphenation

2 verview Introduction NMR hyphenation modes HPLC-SPE-NMR Advantages and limitations Examples

3 Hyphenated techniques Two or more analytical techniques combined into one integrated and automated technique HPLC-DAD GC-MS HPLC-MS n HPLC-NMR

4 Direct NMR hyphenation

5 HPLC: ü Uses mixtures of various solvents ü ften additives (salts, buffers) ü Time scale: min ü Column loading µg scale NMR: ü Preferably pure deuterated solvents ü Preferably no other solutes than the analyte ü Time scale: many hours for 2D experiments ü Higher amounts of analytes preferred

6 Direct HPLC-NMR methods Continued-flow: 1D proton NMR spectra are acquired continuously during the chromatographic run. Stopped-flow: The flow is stopped when the peak of interest reaches the NMR flow cell and NMR spectrum acquired under static conditions. Time slice stopped-flow: Spectra are acquired in the stopped flow mode with small time intervals throughout the whole chromatogram or its part. Peak storage: The peaks of interest are transferred to the peak sampling unit where they are stored. They are then transferred to the flow cell after completion of the chromatography.

7 NMR flow cell

8 Continuous flow NMR

9 Continuous flow NMR

10 Disadvantages direct NMR hyphenation ü Problems with solvent peak suppression: D 2 in the mobile phase is a must. ü Stopped-flow often restricts the analysis to one peak per chromatogram (alleviated by loop storage device). ü nly a small fraction of the material eluted from HPLC contributes to NMR, broad peaks catastrophic. ü Sensitivity: analyte amount from a single HPLC injection available for NMR. ü Works well in favorable cases with major constituents but not with minor. ü Most published studies use pre-purified fractions. ü Unattractive cost/benefit ratio for direct HPLC-NMR?

11 Peak elution volume ml NMR cell Volume µl

12 Indirect NMR hyphenation: HPLC-SPE-NMR

13 GrAz2007, September 2-6 HPLC-SPE-NMR TRAPPING STAGE DRYING STAGE ELUTIN STAGE

14 Automated SPE unit (Spark Holland)

15 verview direct and indirect NMR hyphenation

16 SPE-history ü Use of pre-column SPE for analyte concentration popular since 1980 s ü Post column SPE in connection with FAB MS: Kokkonen et al., 1991 ü Griffith and Horton 1998: use of trapping column back-flushed with CD 3 CN (without drying) ü Nyberg et al., 2001, 2003: off-line HPLC-SPE-NMR ü Commercial automated SPE device: Spark Holland Prospekt device ü First paper, Exarchou et al., 2003

17 Major advantages Analyte focusing Analyte accumulation by multiple trapping NMR spectra acquired in deuterated solvents

18 Analyte focusing by HPLC-SPE-NMR Concentration SPE 2 x 10 mm Elution volume 30 µl NMR flow cell volume 60 µl Peak elution volume µl (4.6 mm i.d HPLC column) NMR cell sensitive volume µl Without focusing (direct methods) Peak elution volume ml

19 Analyte accumulation by HPLC-SPE-NMR (I) Isoflavonoids from Smirnovia iranica H 3 C H 3 C H 3 C H CH 3 Relative NMR signal intensity Trappings Lambert et al. J. Nat. Prod. 2005, 68,

20 Analyte accumulation by HPLC-SPE-NMR (II) Scopoletin from Croton membranaceus 6000 H 3 C H H NMR S/N ratio C18HD GP Cartridge loading, µg Lambert et al. Magn. Reson. Chem. 2005, 43,

21 Analyte accumulation by HPLC-SPE-NMR (III) Constituents of Harpagophytom procumbens H H H 3 C H H H H H 10 µl (5 mg/ml) H H 1 CH 3 H 3 C H 3 C H H 25 µl (40 mg/ml) H H 3 C CH 3 Clarkson et al. J. Nat. Prod. 2006, 69,

22 Cartridge elution solvent Constituents of Harpagophytom procumbens and Multiple Trappings eluting solvent b compound 1 c compound 11 d acetonitrile-d ( ( 2.9 methanol-d ( ( 6.1 chloroform-d ( 7.9 acetonitrile-d e acetonitrile-d 3 (2 trappings) e acetonitrile-d 3 (3 trappings) e acetonitrile-d 3 (5 trappings) n.d. acetonitrile-d 3 (8 trappings) n.d. acetonitrile-d 3 (10 trappings) n.d e µ H H H 3 C H H H H H 10 µl (5 mg/ml) H H 1 CH 3 H 3 C H 3 C H H 25 µl (40 mg/ml) H H 3 C CH 3 Clarkson et al. J. Nat. Prod. 2006, 69,

23 Replacement of HPLC solvent with deuterated solvent Perovskia atriplicifolia (Lamiaceae) ü Peak 3 trapped 6 times on C18 HD ü ns 128 ü No solvent suppression H Intens. [mau] 1 H CH H 3 C CH Time [min]

24 Replacement of HPLC solvent with deuterated solvent H H CH 3 H 3 C CH 3 HMBC

25 Threshold level trapping in HPLC-SPE-NMR experiment

26 Time-sliced HPLC-SPE-NMR experiment

H N 1 CH 2 N N H 3 C Stationary phases for HPLC-SPE-NMR Model natural products H H CH 3 N NH H 3 C H NCH H 3 H 3 C H 3 C 2 HCl CH 3 3 4 5 6 H H N HN CH 2 CH 3 H CH 3 H N N CH 3 N N CH 3 SPE

27 H N 1 CH 2 N N H 3 C Stationary phases for HPLC-SPE-NMR Model natural products H H CH 3 N NH H 3 C H NCH H 3 H 3 C H 3 C 2 HCl CH H H N HN CH 2 CH 3 H CH 3 H N N CH 3 N N CH 3 SPE breakthrough curves H 3 CN H H 3 C H H N N H HCl H H H H H HC H H H H H H H H H H H 3 C H N CH 3 N H H CH CH 3 β-glc N + Cl œ CH 3 CH H CH H H H H H 3 C CH CH 3 H 3 C H CH CH H H H H H H H 3 C CH 3 H H H CH 3 CH Spark Holland Symbiosis instrument

28 Stationary phases for HPLC-SPE-NMR

29 Stationary phases for HPLC-SPE-NMR Effect of acetonitrile content in the loading solvent on retention of test compounds a Percent of compound retained on cartridge at defined acetonitrile content in mobile phase Compound 0% 3% 6% 9% 12% 15% Emetine hydrochloride (5) Dihydrorobinetin (11) Berberine chloride (15) Piperonylic acid (20) Flavone (24)

30 EXAMPLES

Harpagophytum procumbens (Devil's Claw) ü Indigenous to west-south Africa ü Used by local populations to treat fevers, blood diseases, digestive disorders, arthritis, rheumatism ü Introduced to

31 Harpagophytum procumbens (Devil's Claw) ü Indigenous to west-south Africa ü Used by local populations to treat fevers, blood diseases, digestive disorders, arthritis, rheumatism ü Introduced to Europe during the 20th century ü Gained international reputation as an antiinflammatory and anti-rheumatic agent ü Contradictory results of numerous in vivo and clinical studies Ethanol extract Petroleum ether extract Clarkson et al. J. Nat. Prod. 2006, 69, ; Clarkson et al. J. Nat. Prod. 2006, 69,

32 Constituents identified by HPLC-SPE-NMR Clarkson et al. J. Nat. Prod. 2006, 69, ; Clarkson et al. J. Nat. Prod. 2006, 69,

33 Detailed structural information from 2D spectra obtained in the HPLC-SPE-NMR mode Clarkson et al. J. Nat. Prod. 2006, 69, ; Clarkson et al. J. Nat. Prod. 2006, 69,

34 Sample concentration by HPLC-SPE-capNMR Total NMR cell volume 6 µl Active volume 1.5 µl Peak elution volume µl (4.6 mm i.d. HPLC column) SPE 1 x 10 mm Lambert et al. Anal. Chem. 2007, 79,

35 Combination of HPLC-SPE with capnmr for increased analyte focusing and mass sensitivity 2''''-8'''' 2'' C CH 7 H H 3 C 4' H 3 C 3 8 2'''-4''' 6 H C H 3 H 3' 7 CH 3 CH 3 15 CH 3 13 thapsigargin 4'''' CH 3 3'''' 2'' CH 14 3 H H 3 C 3 C 5'''' 4' H 3 C 3 8 2'''-4''' 6 H C H 3 H 3' 7 CH 3 CH 3 15 CH 3 13 thapsivillosin I 2'' 2''''-6'''' C CH 5 H H 3 C 4' H 3 C 3 8 2'''-4''' 6 H C H 3 H 3' 7 CH 3 CH 3 15 CH 3 13 thapsigargicin 2'' CH 14 3 H 3 C 4' H 3 C 3 8 2'''-4''' 6 H C H 3 H 3' 7 CH 3 CH 3 15 CH 3 13 nortrilobolide 4'''' 3'''' 2'' 5'''' CH 2'''' 14 3 H 3 C 4' H 3 C 3 8 2'''-4''' 6 H C H 3 H 3' 7 CH 3 CH 3 15 CH 3 13 thapsivillosin J H 3 C CH 3 3'' CH 3 H 3 C 4'' 4'' CH 3 3'' CH 3 5'' 5'' 2' 7' 2' 6' H CH 3 3''' CH 3 CH 3 3 7' 6' H 3 C 5''' 4''' 3'''' H CH 3 3''' CH 3 CH 3 3 5''' 4''' CH 3 C 5 H 11 H 3 C 4'' CH 3 3'' CH 3 H CH 3 3''' CH 3 CH 3 H 3 C 7' H 3 C 4'' 5'' 2' CH 3 3'' CH 3 5'' 7' 2' 6' ' CH H CH 3 3''' CH 3 CH 3 3 5''' 5''' 3'''' 4''' 4''' C 7 H 1 2''''-8'''' Lambert et al. Anal. Chem. 2007, 79,

36 Extended HPLC-PDA-MS-SPE-NMR-CD hyphenation for determination of structure and chirality Gossypol from Thespesia danis H H CH H HC H H H H 3 C CH 3 H 3 C CH 3 H 3 C CH 3 HPLC of crude extract UV and circular dichroism

37 Important areas of application of hyphenated NMR techniques ü Biomarker identification ü Toxicology GrAz2007, September 2-6 ü Assistance in metabolic profiling and fingerprinting ü Discovery of new pharmacologically active compounds ü Pharmaceutical analysis (drug impurities, metabolites)

38 Natural products-based drug discovery Extract Extract Isolation Structure elucidation High-resolution bioassays Structure elucidation Interesting Not interesting Interesting Not interesting Isolation

Dilution(*) Chromatography

WA20264 Poster # 184, HPLC 2002, Montreal, 4-5 June 2002 At-Column Column-Dilution for Preparative Dilution(*) Chromatography Cecilia Mazza, Jie Cavanaugh, Ziling Lu,Tom Sirard,Tom Wheat and Uwe Neue Waters