qHNMR for purity determination

cenapt.pharm.uic.edu UIC
CENAPT Workshop
Application of NMR beyond
Structure Elucidation
Hands-on Practice
qHNMR for
Purity Determination
asp 2017 portland 1

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

The HSCQ Insight Continuum
Structure
pre
Composition Quantity
M&Ms
No More
Multiplets!
1H Based Detection of
3

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

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

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
6

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

Sample Preparation
Quality and accuracy of
any result start HERE
Sample
preparation
qNMR
acquisition
Spectrum
processing
Assignment
&
integration
Calculation
of purity
1
8

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
Purity: Internal Calibrant
Sample
MW
Purity: External Calibrant
9

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

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

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

Sample Preparation: Calibrant
13

Sample Preparation: Calibrant
Choice based on
• Chemical shifts
• Chemical properties
(inerte/stable, non volatile)
• Solubility
• Relaxation delay
14

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

Sample Preparation with IC
(Example 2 = Non-precious Powder Sample)
Sample
STEP 4STEP 1 STEP 3
ID, MW
ID, MW, Purity, [IC]
[IC] ≈ [Sample]
In mM
OR
Commercial IC
stock solution
Adjust the concentration
STEP 2
Calibrant
Qty
Max 500 µL (5 mm)
Max 200 µL (3 mm)
16

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

Sample Preparation:
Flame 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

qHNMR acquisition
Pauli et al. Journal of Medicinal Chemistry 57, 9220–31 (2014)
2No Magic: Quantitative
Conditions
20

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

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

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

Example of Bruker Pulse Program
AQ
24

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

Example of JEOL Pulse Program
26

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

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

Data Processing
3 Enhancing the Quality
of NMR Spectra
29

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

Snapshot Processing in TopSpin
256 KAdjusted to
Apodization
1
2
31

Snapshot Processing in TopSpin
1
32

Snapshot Processing in Mnova:
Apodization and Zero Filling
33

Snapshot Processing in Mnova :
Window Function, Apodization
1
34

Zero-filling to 256 K
2
35

Baseline Correction
36

Phasing and Baseline Correction for Accurate
Integration
37

Example: Baseline Correction for Accurate
Integration (1/2)
38

Example: Baseline Correction and Integration
(2/2)
39

Data Analysis
4Turn NMR Spectra into
Quantity Numbers
40

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

Identification of residual solvents
42

Identification of Residual Solvents
43

Residual Solvent Calculator
http://www.commonorganicchemistry.com/Handouts/NMR
%20Residual%20Solvent(s)%20Calculator.xlsx 44

Calculation of Purity
Concentration (molarity) α [integral Area/ Number of nuclei]
1 qNMR experiment
• ID compound
• ID impurities
• Residual solvent
• Moisture analysis
• Purity
4b
45

Calculation of Purity:
100% Method
Sample
preparation
qNMR
acquisition
Spectrum
processing
Assignment
&
integration
Calculation
of purity
46

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
impurity
cpd
cpdNMR Baseline expansion

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

Assignment and Signal Integration
49

Identification of Solvents
50

100% Method: Liquiritigenin Example
• Integration of all possible 1H resonances
• Normalization of integrals
100% for 1H
Do not integrate –
Exchangeable 1H
Residual HDO
51

Normalizing the Integrals to 100%
52

Exporting the Integrals to a Spreadsheet
53

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

Pasting the Data into the Calculation
Spreadsheet
55

Attributing each Integral to Compounds with
Known ID (MW)
• Enter the parameters in the yellow
sections
• Automatic calculation in the blue
section
56

Results with the residual solvent calculator
57
Liquiritigenin EtOACEtOH Acetone
With the 100% Calc. spreadsheet:
98.21% 0.08% 0.38 % 0.09%

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

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??

Calculation of Purity with Spreadsheet
Enter the parameters in the yellow sections
Automatic calculation in the blue section
60

Calculation of Purity with Spreadsheet
Enter the parameters in the yellow sections
Automatic calculation in the blue section
61

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

Software-based Purity Determination
qNMR plugin (MestRenova ) Potency (CMC-assist, Topspin )
Topspin 3.5 pl7: free for Academia
63

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

CENAPT Workshop
Application of NMR beyond
Structure Elucidation
Hands-on Practice
qHNMR for
asp 2017 portland 65

The HSCQ Insight Continuum
Structure
pre
Composition Quantity
M&Ms
No More
Multiplets!
1H Based Detection of
66

reproducibility
67

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

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

reproducibility
70

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

reproducibility
72

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

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

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

reproducibility
76

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

reproducibility
78

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)

reproducibility
80

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

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)

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)

qHNMR for purity determination

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