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cenapt.pharm.uic.edu UIC
CENAPT Workshop
Application of NMR beyond
Structure Elucidation
Hands-on Practice
qHNMR for
Purit...
cenapt.pharm.uic.edu UIC
Applications of NMR....
The ultimate use of NMR data
• ...to elucidate structures
– What is it? 1...
cenapt.pharm.uic.edu UIC
The HSCQ Insight Continuum
Structure
pre
Composition Quantity
M&Ms
No More
Multiplets!
1H Based D...
cenapt.pharm.uic.edu UIC
Why HSCQ to Go Beyond Structure?
• Residual Complexity is inherent to NPs
• Hydrogen is universal...
cenapt.pharm.uic.edu UIC
Goals of Workshop
• Q&A: Why go beyond structure?
– Provide rationales
– Perspectives of q-expert...
cenapt.pharm.uic.edu UIC
Quantitative NMR Conditions
• Structure & Quantity Concurrently
– Simultaneous
– All-in-one data ...
cenapt.pharm.uic.edu UIC
The qNMR Workflow
• Working towards the q
– Be aware of opportunity
– Adjust routine workflow
– A...
cenapt.pharm.uic.edu UIC
Sample Preparation
Quality and accuracy of
any result start HERE
Sample
preparation
qNMR
acquisit...
cenapt.pharm.uic.edu UIC
Sample Preparation:
Choice of qHNMR Method
Precious sample
~~ weight ?
~~ volume ?
Situation 1
1 ...
cenapt.pharm.uic.edu UIC
Purity Determination
Decision Flow Chart: What Kind of Method?
Can the sample be accurately
weigh...
cenapt.pharm.uic.edu UIC
Sample Preparation:
Choice of qHNMR Method
Precious sample
~~ weight ?
~~ volume ?
100% Method
Re...
cenapt.pharm.uic.edu UIC
Calibrant prepared according to the specifications
Sample Preparation: Overview
Final concentrati...
cenapt.pharm.uic.edu UIC
Sample Preparation: Calibrant
13
cenapt.pharm.uic.edu UIC
Sample Preparation: Calibrant
Choice based on
• Chemical shifts
• Chemical properties
(inerte/sta...
cenapt.pharm.uic.edu UIC
Sample Preparation with IC
(Example 1 = Several Precious Samples)
Sample
Calibrant (IC)
STEP 5STE...
cenapt.pharm.uic.edu UIC
Sample Preparation with IC
(Example 2 = Non-precious Powder Sample)
Sample
STEP 3STEP 1 STEP 2
ID...
cenapt.pharm.uic.edu UIC
Sample Preparation with EC
Sample
STEP 1 STEP 2
ID, MW
ID, MW, Purity, [EC]
[EC] ∝ [Sample]
In mM...
cenapt.pharm.uic.edu UIC
Sample Preparation:
Sealing your NMR Tube
Similar to the process of flame sealing an ampoule
Seal...
cenapt.pharm.uic.edu UIC 19
cenapt.pharm.uic.edu UIC
qHNMR acquisition
Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014)
2No Magic: Quant...
cenapt.pharm.uic.edu UIC
Key Parameters for Acquisition
Parameters Value Remarks
Relaxation delay
(D1)
Theory: 5 X T1 of c...
cenapt.pharm.uic.edu UIC
Other Parameters for Acquisition
Parameters Value Remarks
Number of scans
(ns)
64 ( 300-700 MHz, ...
cenapt.pharm.uic.edu UIC
Example: Adjustment of NS
An 1H NMR spectrum was acquired with
• NS = 4 ( co-addition of 4 FIDs),...
cenapt.pharm.uic.edu UIC
Example of Bruker Pulse Program
AQ
24
cenapt.pharm.uic.edu UIC
Bruker/Topspin Acquisition Parameters
Sample: Naringenin (69.42 mM) Magnetic Field Strength = 900...
cenapt.pharm.uic.edu UIC
Example of JEOL Pulse Program
26
cenapt.pharm.uic.edu UIC
JEOL/Delta Acquisition Parameters
✓
Changing the sweep
affects the acq_time
Automatic calculation...
cenapt.pharm.uic.edu UIC
Good Practice/Advise : Documentation
In the “comment” / title section
Add all information related...
cenapt.pharm.uic.edu UIC
Data Processing
Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014)
3 Enhancing the Qu...
cenapt.pharm.uic.edu UIC
Data Processing Checklist
Window function WDW
(Lorentzien-Gaussian)
GM Notes
Line broadening LB (...
cenapt.pharm.uic.edu UIC
Snapshot Processing in TopSpin
256 KAdjusted to
Apodization
1
2
31
cenapt.pharm.uic.edu UIC
Snapshot Processing in TopSpin
1
32
cenapt.pharm.uic.edu UIC
Snapshot Processing in Mnova:
Apodization and Zero Filling
33
cenapt.pharm.uic.edu UIC
Snapshot Processing in Mnova :
Window Function, Apodization
1
34
cenapt.pharm.uic.edu UIC
Snapshot Processing in Mnova:
Zero-filling to 256 K
2
35
cenapt.pharm.uic.edu UIC
Snapshot Processing in Mnova:
Baseline Correction
36
cenapt.pharm.uic.edu UIC
Phasing and Baseline Correction for Accurate
Integration
37
cenapt.pharm.uic.edu UIC
Example: Baseline Correction for Accurate
Integration (1/2)
38
cenapt.pharm.uic.edu UIC
Example: Baseline Correction and Integration
(2/2)
39
cenapt.pharm.uic.edu UIC
Data Analysis
4Turn NMR Spectra into
Quantity Numbers
40
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Identification of Impurities
1 qNMR experiment
• ID compound
• ID impurities
• Residual solvents
...
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Identification of residual solvents
42
cenapt.pharm.uic.edu UIC
Identification of Residual Solvents
43
cenapt.pharm.uic.edu UIC
Residual Solvent Calculator
http://www.commonorganicchemistry.com/Handouts/NMR
%20Residual%20Solv...
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Calculation of Purity
Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014)
Concentratio...
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Calculation of Purity:
100% Method
Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014)...
cenapt.pharm.uic.edu UIC
Sample = 100%
= % cpd +% impurities
Integration of all signals:
target compounds +
impurities
Cal...
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100% Method: Liquiritigenin Example
• 1H NMR processing
• 1H resonance assignment:
• Liquiritigen...
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Assignment and Signal Integration
49
cenapt.pharm.uic.edu UIC
Identification of Solvents
50
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100% Method: Liquiritigenin Example
• Integration of all possible 1H resonances
• Normalization o...
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Normalizing the Integrals to 100%
52
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Exporting the Integrals to a Spreadsheet
53
cenapt.pharm.uic.edu UIC
100% Method: Check List
• qHNMR acquisition & processing
• 1H resonance assignment:
• Target comp...
cenapt.pharm.uic.edu UIC
Pasting the Data into the Calculation
Spreadsheet
55
cenapt.pharm.uic.edu UIC
Attributing each Integral to Compounds with
Known ID (MW)
• Enter the parameters in the yellow
se...
cenapt.pharm.uic.edu UIC
Results with the residual solvent calculator
57
Liquiritigenin EtOACEtOH Acetone
With the 100% Ca...
cenapt.pharm.uic.edu UIC
Calculation of Purity:
with Internal Calibrant (IC)
Analyte Concentration = PIC
Standardized Inte...
cenapt.pharm.uic.edu UIC
Purity Determination with IC: Overview
Sample: Liquiritigenin
MW : 256.25
Qty: 0.80 mg
Calibrant ...
cenapt.pharm.uic.edu UIC
Calculation of Purity with Spreadsheet
Enter the parameters in the yellow sections
Automatic calc...
cenapt.pharm.uic.edu UIC
Calculation of Purity with Spreadsheet
Enter the parameters in the yellow sections
Automatic calc...
cenapt.pharm.uic.edu UIC
Calculation of Purity:
Mass Balance Approach
98.21% w/w 96.30% w/w
Ethyl Acetate
Ethanol
Acetone
...
cenapt.pharm.uic.edu UIC
Software-based Purity Determination
qNMR plugin (MestRenova ) Potency (CMC-assist, Topspin )
Tops...
cenapt.pharm.uic.edu UIC
Educative Blogs and Website
http://u-of-o-nmr-facility.blogspot.ca
http://cbc.arizona.edu/rss/nmr...
cenapt.pharm.uic.edu UIC
CENAPT Workshop
Application of NMR beyond
Structure Elucidation
Hands-on Practice
qHNMR for
Purit...
cenapt.pharm.uic.edu UIC
The HSCQ Insight Continuum
Structure
pre
Composition Quantity
M&Ms
No More
Multiplets!
1H Based D...
cenapt.pharm.uic.edu UIC
Why HSCQ to Go Beyond Structure?
• Residual Complexity is inherent to NPs
• Hydrogen is universal...
cenapt.pharm.uic.edu UIC
Residual Complexity: Inescapable?
68
Screen
Bioassay			PAR
Guidance
Biological
Verification
SAR,	...
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RC vs. Orthogonality
• Analysis & Purification of Bioactive Natural Products
• Meta analysis of t...
cenapt.pharm.uic.edu UIC
Why HSCQ to Go Beyond Structure?
• Residual Complexity is inherent to NPs
• Hydrogen is universal...
cenapt.pharm.uic.edu UIC
Hydrogen: Universal & Sensitive
• Relative sensitivity:
– 1H 1.000, 19F 0.845 (limited for NPs)
–...
cenapt.pharm.uic.edu UIC
Why HSCQ to Go Beyond Structure?
• Residual Complexity is inherent to NPs
• Hydrogen is universal...
cenapt.pharm.uic.edu UIC
No More Multiplets
• Multiplicity makes first order assumption
– d t q dd dt ddt dq ddq ddd dddd....
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Why Bother?
• Why 1H NMR?
– Protons are ubiquitous
– Gold Standard Experiment
• Why use NMR as an...
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TOCSY Multiplets by GARP {13C}1H
• HupA: Neurotopic agent (AD, PD)
• ABCD(E)(MN)(OP)X3Y3 spin sys...
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Why HSCQ to Go Beyond Structure?
• Residual Complexity is inherent to NPs
• Hydrogen is universal...
cenapt.pharm.uic.edu UIC
Think It Over!
• HS:1H-detected NMR experiments are the
modern workhorses of structural analysis
...
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Why HSCQ to Go Beyond Structure?
• Residual Complexity is inherent to NPs
• Hydrogen is universal...
cenapt.pharm.uic.edu UIC
Universal qHNMR
The Calibration Q: 100% - EC - IC - ECIC
• Four principal qHNMR methods
– Interna...
cenapt.pharm.uic.edu UIC
Why HSCQ to Go Beyond Structure?
• Residual Complexity is inherent to NPs
• Hydrogen is universal...
cenapt.pharm.uic.edu UIC
Free as in Beer!
• Price of quantitative conditions
in 1D 1H NMR (qHNMR): $0.00
– p90, D1, TD, et...
cenapt.pharm.uic.edu UIC
Purity Activity-Relationship (PAR)
• Is ursolic acid (MIC 32-128 µg/mL) a viable anti-TB lead?
– ...
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HSCQ Applications in NP Research
• Purity determination enhances research
integrity and significa...
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qHNMR for purity determination

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You will find here all the elements presented by the CENAPT team ( Drs. Guido Pauli and Charlotte Simmler) and pertaining to the NMR workshop at the American Society of Pharmacognosy (ASP 2017, Portland Oregon).

These slides summarize the different steps related to the implementation of quantitative NMR for purity analysis.

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qHNMR for purity determination

  1. 1. cenapt.pharm.uic.edu UIC CENAPT Workshop Application of NMR beyond Structure Elucidation Hands-on Practice qHNMR for Purity Determination asp 2017 portland 1
  2. 2. cenapt.pharm.uic.edu UIC Applications of NMR.... The ultimate use of NMR data • ...to elucidate structures – What is it? 1D/2D/nD • ...to determine composition – How complex is it? One vs. many • ...to quantify – How much of what is there? – Relative composition – Absolute content 2
  3. 3. cenapt.pharm.uic.edu UIC The HSCQ Insight Continuum Structure pre Composition Quantity M&Ms No More Multiplets! 1H Based Detection of 3
  4. 4. cenapt.pharm.uic.edu UIC Why HSCQ to Go Beyond Structure? • Residual Complexity is inherent to NPs • Hydrogen is universal atom in NPs • Hydrogen is most sensitive NMR nucleus • No More Multiplets! enhances rigor and reproducibility • CQ enforces thinking beyond HS - innovate • No weighing error in 100% qHNMR • Q is Free - as in beer! 4
  5. 5. cenapt.pharm.uic.edu UIC Goals of Workshop • Q&A: Why go beyond structure? – Provide rationales – Perspectives of q-experts – Recognize innovation • Q&A: How to go beyond structure? – The q-workflow • Q&A: When to go beyond structure? – Enable: go q-today! 5
  6. 6. cenapt.pharm.uic.edu UIC Quantitative NMR Conditions • Structure & Quantity Concurrently – Simultaneous – All-in-one data sets • No magic but routine! – Go quantitative – No excuses • Focus in 1D qNMR – 2DqNMR feasible • Q is Free - as in beer! 6
  7. 7. cenapt.pharm.uic.edu UIC The qNMR Workflow • Working towards the q – Be aware of opportunity – Adjust routine workflow – Acquire new skills Sample Preparation Acquisition Processing Assignment & Integration Calculation 1 2 3 4 7
  8. 8. cenapt.pharm.uic.edu UIC Sample Preparation Quality and accuracy of any result start HERE Sample preparation qNMR acquisition Spectrum processing Assignment & integration Calculation of purity 1 8
  9. 9. cenapt.pharm.uic.edu UIC Sample Preparation: Choice of qHNMR Method Precious sample ~~ weight ? ~~ volume ? Situation 1 1 qHNMR acq./proces. Purity: 100% Method Xx mg Sample Accurate weight Precise volume Precious sample Accurate weight Precise volume Situation 2 Situation 3 Sample MW Calibrant MW, Purity 1 qHNMR acq./proces. Purity: Internal Calibrant Sample MW 2 qHNMR acq./proces. Purity: External Calibrant 9
  10. 10. cenapt.pharm.uic.edu UIC Purity Determination Decision Flow Chart: What Kind of Method? Can the sample be accurately weighted? Do you have enough for replicate analysis ? Purity evaluation 100% method MS (HRMS) & tandem MS Identification of impurities Purity value depends on the identified impurities Limited time/ access to spectrometer Internal Calibrant (IC) Known purity & quantity unlimited time to spectrometer necessity to avoid sample contamination External Calibrant (EC) Known purity & quantity Purity value depends on the accuracy of sample preparation YESNO Same NMR acquisition processing 10
  11. 11. cenapt.pharm.uic.edu UIC Sample Preparation: Choice of qHNMR Method Precious sample ~~ weight ? ~~ volume ? 100% Method Relative method Purity % relative to ID impurities Routine Experiment Xx mg Sample Accurate weight Precise volume Precious sample Accurate weight Precise volume Internal Calibration External Calibration Absolute method Repetitive Preparation of IC with sample to analyze 1qNMR acquisition Reduced time of Analysis Absolute method EC accurately prepared once No contamination of sample 2 qNMR acquisitions 11
  12. 12. cenapt.pharm.uic.edu UIC Calibrant prepared according to the specifications Sample Preparation: Overview Final concentration: molarity close to the sample of interest Accurate Volume 5 mm tubes 3 mm tubes Solvent volume 500 uL 170-200 uL Weight sample 4 -12 mg <1- 4 mg Accurate weight 10 mM optimal 12
  13. 13. cenapt.pharm.uic.edu UIC Sample Preparation: Calibrant 13
  14. 14. cenapt.pharm.uic.edu UIC Sample Preparation: Calibrant Choice based on • Chemical shifts • Chemical properties (inerte/stable, non volatile) • Solubility • Relaxation delay 14
  15. 15. cenapt.pharm.uic.edu UIC Sample Preparation with IC (Example 1 = Several Precious Samples) Sample Calibrant (IC) STEP 5STEP 1 Qty [IC] ≈ [Sample] stock solution (commercial) STEP 2 STEP 3 STEP 4 ID, MW ID, MW, Purity Qty Max 500 µL (5 mm) Max 200 µL (3 mm) Tared vial + dried cpd 15
  16. 16. cenapt.pharm.uic.edu UIC Sample Preparation with IC (Example 2 = Non-precious Powder Sample) Sample STEP 3STEP 1 STEP 2 ID, MW ID, MW, Purity, [IC] [IC] ≈ [Sample] In mM Commercial IC or in house stock solution Adjust the concentration Qty Max 500 µL (5 mm) Max 200 µL (3 mm) 16
  17. 17. cenapt.pharm.uic.edu UIC Sample Preparation with EC Sample STEP 1 STEP 2 ID, MW ID, MW, Purity, [EC] [EC] ∝ [Sample] In mM OR Commercial solution Calibrant Qty Max 500 µL (5 mm) Max 200 µL (3 mm) STEP 3 STEP 4 STEP 4 17
  18. 18. cenapt.pharm.uic.edu UIC Sample Preparation: Sealing your NMR Tube Similar to the process of flame sealing an ampoule Sealing is important: • To preserve your EC • To avoid solvent evaporation 18 Beware with organic solvent use liquid nitrogen to freeze your sample
  19. 19. cenapt.pharm.uic.edu UIC 19
  20. 20. cenapt.pharm.uic.edu UIC qHNMR acquisition Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014) 2No Magic: Quantitative Conditions 20
  21. 21. cenapt.pharm.uic.edu UIC Key Parameters for Acquisition Parameters Value Remarks Relaxation delay (D1) Theory: 5 X T1 of compounds Practice: 60 sec (if T1 unknown) Accuracy of the integral values Pulse width (90 pulse: P1) to be determined using the 360 null Function of solvent nucleus, sample Acquisition time (AQ) 4 sec Optimization of digital resolution Spectral window (SW) transmitter offset (O1P) SW: 30 ppm – O1P:7.5 ppm SW: 20 ppm – O1P: 4.5 ppm For an optimal baseline correction (edge effects), and digital resolution FID size (TD) 32 K-64 K For better resolution AQ = (TD/[2x SW]) Number of Data Points (TD) = 2 x Spectral Width (SW in ppm X field strength) x Acquisition Time (AQ) 21
  22. 22. cenapt.pharm.uic.edu UIC Other Parameters for Acquisition Parameters Value Remarks Number of scans (ns) 64 ( 300-700 MHz, RT probe) 32 (> 700 MHz, RT probe) Optimization of the S/N for precise quant.>250:1 Receiver gain (rg) To be determined generally chosen not to high to avoid FID truncation Depends on sample concentration and field strength Temperature Should be constant Records and document Sample spinning NO • Sensitivity (S/N) ∝√ ns • ns can be adjusted according to: • Molar concentration • Magnetic field strength • Type of probe ( RT vs. Cryoprobe) 22
  23. 23. cenapt.pharm.uic.edu UIC Example: Adjustment of NS An 1H NMR spectrum was acquired with • NS = 4 ( co-addition of 4 FIDs), and • gave a S/N of 50 for the 1H resonance of interest Question how many scans (ns) should be considered for a S/N of 250? (S/N) ∝√ ns (250 / 50)2 = 52 = 25 (S/N)2 ∝ ns ns = 25 x 4 ≈ 100 Proportionality factor Number of scans for S/N 250 23
  24. 24. cenapt.pharm.uic.edu UIC Example of Bruker Pulse Program AQ 24
  25. 25. cenapt.pharm.uic.edu UIC Bruker/Topspin Acquisition Parameters Sample: Naringenin (69.42 mM) Magnetic Field Strength = 900 MHz D1 = 60 sec P1 = 10.5 µsec AQ = TD /( 2 x sw x field strength) AQ = 180176/ (2 x 25 x 900) 25
  26. 26. cenapt.pharm.uic.edu UIC Example of JEOL Pulse Program 26
  27. 27. cenapt.pharm.uic.edu UIC JEOL/Delta Acquisition Parameters ✓ Changing the sweep affects the acq_time Automatic calculation of 90 pulse width Angle of the Excitation Pulse = 90 Digital Resolution (DR) ≤ 0.25 Hz DR = (SW/ real data_points) Real data points AQ = Real Data_points /( 2x sw x field strength 27
  28. 28. cenapt.pharm.uic.edu UIC Good Practice/Advise : Documentation In the “comment” / title section Add all information related to your sample: • Compound ID = name, MW, qty in NMR tube • Solvent ID , volume • Calibrant ID, MW, qty in NMR tube • Pulse parameters (ns, AQ, d1, P1) • Date 28
  29. 29. cenapt.pharm.uic.edu UIC Data Processing Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014) 3 Enhancing the Quality of NMR Spectra 29
  30. 30. cenapt.pharm.uic.edu UIC Data Processing Checklist Window function WDW (Lorentzien-Gaussian) GM Notes Line broadening LB (Hz) -0.3 Improve signal shape and s/nGaussian max position GB 0.05 Zero filling 256 K real data point SI Size of the real spectrum = 262144 Baseline correction “abs” command 5thorder polynomial FT : gfp = Gaussian window multiplication + FT + phase correction (1D) Automatic and manual phasing adjustment Assignment : ID target compound (ID impurities) Signal integration ( ideally integral width = 5 X fwhh) 1 2 3 4 5 6 30
  31. 31. cenapt.pharm.uic.edu UIC Snapshot Processing in TopSpin 256 KAdjusted to Apodization 1 2 31
  32. 32. cenapt.pharm.uic.edu UIC Snapshot Processing in TopSpin 1 32
  33. 33. cenapt.pharm.uic.edu UIC Snapshot Processing in Mnova: Apodization and Zero Filling 33
  34. 34. cenapt.pharm.uic.edu UIC Snapshot Processing in Mnova : Window Function, Apodization 1 34
  35. 35. cenapt.pharm.uic.edu UIC Snapshot Processing in Mnova: Zero-filling to 256 K 2 35
  36. 36. cenapt.pharm.uic.edu UIC Snapshot Processing in Mnova: Baseline Correction 36
  37. 37. cenapt.pharm.uic.edu UIC Phasing and Baseline Correction for Accurate Integration 37
  38. 38. cenapt.pharm.uic.edu UIC Example: Baseline Correction for Accurate Integration (1/2) 38
  39. 39. cenapt.pharm.uic.edu UIC Example: Baseline Correction and Integration (2/2) 39
  40. 40. cenapt.pharm.uic.edu UIC Data Analysis 4Turn NMR Spectra into Quantity Numbers 40
  41. 41. cenapt.pharm.uic.edu UIC Identification of Impurities 1 qNMR experiment • ID compound • ID impurities • Residual solvents • Moisture analysis • Purity level Sample preparation qNMR acquisition Spectrum processing Assignment & integration Calculation of purity 4a 41
  42. 42. cenapt.pharm.uic.edu UIC Identification of residual solvents 42
  43. 43. cenapt.pharm.uic.edu UIC Identification of Residual Solvents 43
  44. 44. cenapt.pharm.uic.edu UIC Residual Solvent Calculator http://www.commonorganicchemistry.com/Handouts/NMR %20Residual%20Solvent(s)%20Calculator.xlsx 44
  45. 45. cenapt.pharm.uic.edu UIC Calculation of Purity Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014) Concentration (molarity) α [integral Area/ Number of nuclei] 1 qNMR experiment • ID compound • ID impurities • Residual solvent • Moisture analysis • Purity 4b 45
  46. 46. cenapt.pharm.uic.edu UIC Calculation of Purity: 100% Method Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014) Sample preparation qNMR acquisition Spectrum processing Assignment & integration Calculation of purity 46
  47. 47. cenapt.pharm.uic.edu UIC Sample = 100% = % cpd +% impurities Integration of all signals: target compounds + impurities Calculation of Purity % w/w Relative Determination of Purity: The 100% Method 47 100% method Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014) impurity cpd cpdNMR Baseline expansion
  48. 48. cenapt.pharm.uic.edu UIC 100% Method: Liquiritigenin Example • 1H NMR processing • 1H resonance assignment: • Liquiritigenin (MW= 256.25) • Impurities = – Ethanol (MW = 46.06) – Ethyl Acetate (MW= 88.11) – Acetone (MW = 58.09) @ @@ @ ! ! * * 48
  49. 49. cenapt.pharm.uic.edu UIC Assignment and Signal Integration 49
  50. 50. cenapt.pharm.uic.edu UIC Identification of Solvents 50
  51. 51. cenapt.pharm.uic.edu UIC 100% Method: Liquiritigenin Example • Integration of all possible 1H resonances • Normalization of integrals 100% for 1H Do not integrate – Exchangeable 1H Residual HDO 51
  52. 52. cenapt.pharm.uic.edu UIC Normalizing the Integrals to 100% 52
  53. 53. cenapt.pharm.uic.edu UIC Exporting the Integrals to a Spreadsheet 53
  54. 54. cenapt.pharm.uic.edu UIC 100% Method: Check List • qHNMR acquisition & processing • 1H resonance assignment: • Target compound • Impurities (solvent, congeners) • Integration of all possible 1H resonances • Normalization of integrals to 100% for 1H • for the target compound • Export all integrals to spreadsheet 54
  55. 55. cenapt.pharm.uic.edu UIC Pasting the Data into the Calculation Spreadsheet 55
  56. 56. cenapt.pharm.uic.edu UIC Attributing each Integral to Compounds with Known ID (MW) • Enter the parameters in the yellow sections • Automatic calculation in the blue section 56
  57. 57. cenapt.pharm.uic.edu UIC Results with the residual solvent calculator 57 Liquiritigenin EtOACEtOH Acetone With the 100% Calc. spreadsheet: 98.21% 0.08% 0.38 % 0.09%
  58. 58. cenapt.pharm.uic.edu UIC Calculation of Purity: with Internal Calibrant (IC) Analyte Concentration = PIC Standardized Integrals of the Analyte X Calibrant Concentration Standardized Integrals of the Calibrant Sample preparation qNMR acquisition Spectrum processing Assignment & integration Calculation of purity 4c 58
  59. 59. cenapt.pharm.uic.edu UIC Purity Determination with IC: Overview Sample: Liquiritigenin MW : 256.25 Qty: 0.80 mg Calibrant (IC) MW: 212.12 Qty: 0.77 mg Purity:99.54 IC : 1H target : 1H 𝑃 [%] = 𝑛𝐼𝐶 ∙ 𝐼𝑛𝑡𝑡 ∙ 𝑀𝑊𝑡 ∙ 𝑚𝐼𝐶 𝑛𝑡 ∙ 𝐼𝑛𝑡𝐼𝐶 ∙ 𝑀𝑊𝐼𝐶 ∙ 𝑚 𝑠 ∙ 𝑃𝐼𝐶 = 1 x 100 t = target compound = liquiritigenin 256.25 0.77 99.54 1 x 120.19 212.12 0.80x x x x = 96.30% w/w 59 Qty (mic) = n/MW → Intic → InttTrue Qty (mt) = n/MW??
  60. 60. cenapt.pharm.uic.edu UIC Calculation of Purity with Spreadsheet Enter the parameters in the yellow sections Automatic calculation in the blue section 60
  61. 61. cenapt.pharm.uic.edu UIC Calculation of Purity with Spreadsheet Enter the parameters in the yellow sections Automatic calculation in the blue section 61
  62. 62. cenapt.pharm.uic.edu UIC Calculation of Purity: Mass Balance Approach 98.21% w/w 96.30% w/w Ethyl Acetate Ethanol Acetone Congener (5-OH) HDO 0.38% w/w 0.08% w/w 0.09% w/w 1.25% w/w 0.38% w/w 0.08% w/w 0.09% w/w 1.24% w/w ~~1.91% w/w Internal Calibration100% Method 62
  63. 63. cenapt.pharm.uic.edu UIC Software-based Purity Determination qNMR plugin (MestRenova ) Potency (CMC-assist, Topspin ) Topspin 3.5 pl7: free for Academia 63
  64. 64. cenapt.pharm.uic.edu UIC Educative Blogs and Website http://u-of-o-nmr-facility.blogspot.ca http://cbc.arizona.edu/rss/nmr/pdfs/params.pdf http://nmr-analysis.blogspot.com Mnova related Blog Parameters of NMR acq. Ottawa University NMR blog http://www.nmr.ucdavis.edu/useful-links/ 90 pulse width calibration (Bruker) http://web.mit.edu/speclab/www/PDF/DCIF-90pulse-Bruker-j07.pdf http://www.asdlib.org/onlineArticles/ecourseware/Larive/qnmr4.htm Practical aspects of qNMR 64 http://nmr-analysis.blogspot.com/2009/11/basis-on-qnmr-integration-rudiments.html http://pubs.acs.org/paragonplus/submission/jmcmar/jmcmar_purity_instructions
  65. 65. cenapt.pharm.uic.edu UIC CENAPT Workshop Application of NMR beyond Structure Elucidation Hands-on Practice qHNMR for Purity Determination asp 2017 portland 65
  66. 66. cenapt.pharm.uic.edu UIC The HSCQ Insight Continuum Structure pre Composition Quantity M&Ms No More Multiplets! 1H Based Detection of 66
  67. 67. cenapt.pharm.uic.edu UIC Why HSCQ to Go Beyond Structure? • Residual Complexity is inherent to NPs • Hydrogen is universal atom in NPs • Hydrogen is most sensitive NMR nucleus • No More Multiplets! enhances rigor and reproducibility • CQ enforces thinking beyond HS - innovate • No weighing error in 100% qHNMR • Q is Free - as in beer! 67
  68. 68. cenapt.pharm.uic.edu UIC Residual Complexity: Inescapable? 68 Screen Bioassay PAR Guidance Biological Verification SAR, PAR Pharmacology BIO- ASSAY Metabo- lome Purification NP Purity Analysis Organism Preparative Scale n NATURAL PRODUCT cNP SCE Residual Complexity0lim n = ¥® COMPLEXITY Analytical Scale RC Pauli GF et al., J. Nat. Prod.75, 1243 (2012) http://go.uic.edu/residualcomplexity
  69. 69. cenapt.pharm.uic.edu UIC RC vs. Orthogonality • Analysis & Purification of Bioactive Natural Products • Meta analysis of the literature – 1998/9 – 2004/5 – 2009/10 – 13 journals – 80,000 pages screened – Nearly 2,000 publications – Prospective parameters • Asking 15 questions; exemplary answers: – The average # of isolation steps is 2.4 – Silica gel indispensable in 57–63–71% of studies – Purity determined (LC, qNMR) for <0.5% of NPs 69Pauli GF et al., J. Nat. Prod. 75, 1243 (2012) z BIOME Bioassay Metabolome SCE RC T
  70. 70. cenapt.pharm.uic.edu UIC Why HSCQ to Go Beyond Structure? • Residual Complexity is inherent to NPs • Hydrogen is universal atom in NPs • Hydrogen is most sensitive NMR nucleus • No More Multiplets! enhances rigor and reproducibility • CQ enforces thinking beyond HS - innovate • No weighing error in 100% qHNMR • Q is Free - as in beer! 70
  71. 71. cenapt.pharm.uic.edu UIC Hydrogen: Universal & Sensitive • Relative sensitivity: – 1H 1.000, 19F 0.845 (limited for NPs) – 31P 0.07, 13C 0.016, 15N 0.001 • Ubiquitous, but sometimes rare – Hydrogen deficient, low H/C-ratio cpds 71 hypericin b-amyrin C30 2 x CH 3 x CH3 C30 7 x CH 18 x CH2 8 x CH3
  72. 72. cenapt.pharm.uic.edu UIC Why HSCQ to Go Beyond Structure? • Residual Complexity is inherent to NPs • Hydrogen is universal atom in NPs • Hydrogen is most sensitive NMR nucleus • No More Multiplets! enhances rigor and reproducibility • CQ enforces thinking beyond HS - innovate • No weighing error in 100% qHNMR • Q is Free - as in beer! 72
  73. 73. cenapt.pharm.uic.edu UIC No More Multiplets • Multiplicity makes first order assumption – d t q dd dt ddt dq ddq ddd dddd......m! • What is a multiplet? – Signal with much structural information • Are all multiplets the same? – Type I: first order, but uninterpreted ØFull 1D/2D interpretation to understand! – Type II: second or higher order ØFull spin analysis to understand! • General trend: information loss/ignorance 73
  74. 74. cenapt.pharm.uic.edu UIC Why Bother? • Why 1H NMR? – Protons are ubiquitous – Gold Standard Experiment • Why use NMR as an expensive balance? – Ubiquitous 1H means universal detector – Identical calibrant not required! – Think twice about $$s • Why Multiplets - are you serious? – Yes! 74 The Multiplet Pond
  75. 75. cenapt.pharm.uic.edu UIC TOCSY Multiplets by GARP {13C}1H • HupA: Neurotopic agent (AD, PD) • ABCD(E)(MN)(OP)X3Y3 spin system – 15H/11 spins, 38 J-couplings, including 31(!) long-range (4-6J) +baseline corrected resolution enhanced original signal H-7 triplet? calculated residual ddddddqq ! is "TOCSY-like" Huperzine A H-7 H N H3C CH3 O H2N Niemitz et al. MRC 45, 878 (2007) Shao-Nong Chen
  76. 76. cenapt.pharm.uic.edu UIC Why HSCQ to Go Beyond Structure? • Residual Complexity is inherent to NPs • Hydrogen is universal atom in NPs • Hydrogen is most sensitive NMR nucleus • No More Multiplets! enhances rigor and reproducibility • CQ enforces thinking beyond HS - innovate • No weighing error in 100% qHNMR • Q is Free - as in beer! 76
  77. 77. cenapt.pharm.uic.edu UIC Think It Over! • HS:1H-detected NMR experiments are the modern workhorses of structural analysis – 1D, COSY, TOCSY, (ed-)HSQC, HMBC • Why not use all 1H information? – H,H couplings – H,C couplings (13C satellites anybody?) • Why not use all NMR information? – Composition, Analogues – Quantitative composition, purity 77
  78. 78. cenapt.pharm.uic.edu UIC Why HSCQ to Go Beyond Structure? • Residual Complexity is inherent to NPs • Hydrogen is universal atom in NPs • Hydrogen is most sensitive NMR nucleus • No More Multiplets! enhances rigor and reproducibility • CQ enforces thinking beyond HS - innovate • No weighing error in 100% qHNMR • Q is Free - as in beer! 78
  79. 79. cenapt.pharm.uic.edu UIC Universal qHNMR The Calibration Q: 100% - EC - IC - ECIC • Four principal qHNMR methods – Internal Calibration Absolute Method (IC Abs-qHNMR) – External Calibration Absolute Method (EC Abs-qHNMR) – Combined External & Internal Calibration Absolute Method (ECIC Abs-qHNMR) • Required instrument validation and calibration • qNMR beats (our) balances?! – Uncalibrated Relative (100%) Method (Rel-qHNMR) • No weighing involved, no weighing error • Requires detailed interpretation of NMR data (education) 79Pauli et al. J. Med. Chem. 57, 9220-9231 (2014)
  80. 80. cenapt.pharm.uic.edu UIC Why HSCQ to Go Beyond Structure? • Residual Complexity is inherent to NPs • Hydrogen is universal atom in NPs • Hydrogen is most sensitive NMR nucleus • No More Multiplets! enhances rigor and reproducibility • CQ enforces thinking beyond HS - innovate • No weighing error in 100% qHNMR • Q is Free - as in beer! 80
  81. 81. cenapt.pharm.uic.edu UIC Free as in Beer! • Price of quantitative conditions in 1D 1H NMR (qHNMR): $0.00 – p90, D1, TD, etc. are a matter of awareness, not cost • HNMR is essentially already quantitative ØAdjust parameters to run qHNMR routinely! • Dynamic range – Instrument time: 1H 5 min vs 2D/13C 5 hrs – For ~1% level, need to detect 13C satellites 81 Free Beer Tomorrow! 𝑆/𝑁 ≈ 𝑁𝑆 𝑆/𝑁 ≈ 𝑡
  82. 82. cenapt.pharm.uic.edu UIC Purity Activity-Relationship (PAR) • Is ursolic acid (MIC 32-128 µg/mL) a viable anti-TB lead? – Eight UA accessions: declared vs. found 1-16 % differences • qNMR Answer: Inverse correlation between purity and activity • qNMR Net Outcome Pure UA is essentially inactive 82 O OH CH3 CH3 CH3 CH3 CH3 HO H H H 1 4 6 9 10 12 19 14 20 17 24 23 25 26 27 28 29 30 CH3H3C Purity [%] 65 70 75 80 85 90 95 100 105 Anti-TBMIC[ug/ml] 0 50 100 150 200 250 300 SI=IC50/MIC[ug/ml] 0.0 0.1 0.2 0.3 0.4 0.5 IC50VERO 0 5 10 15 20 25 30 % purity vs MIC H37Rv % purity vs MIC H37gfp % purity vs SI % purity vs IC50 VERO Jaki et al. J. Nat. Prod. 71, 1742-8 (2008)
  83. 83. cenapt.pharm.uic.edu UIC HSCQ Applications in NP Research • Purity determination enhances research integrity and significance • Drug discovery – Bioactive impurities or degradation products – Purity Activity Relationships (PAR) & qPAR* – Isolation yield prediction • Product Quality Control – Botanical Standardization • Reaction control 83*Qiu et al., J. Nat. Prod. 76, 413-9 (2013)

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