1. Analytical & Bioanalytical Techniques
Joshua Hicks
Catalent Pharma Solutions
September 2, 2015

2. NMR quantification vs. other methods
For automated, internal-standard-free, absolute quantization…
NMR is accurate, precise and versatile
Superior to
CLND
ELSD
Weighing
CLND
ELSD
Analytical Chemistry (2005), 77(14),

3. Quantificaiton with Internal Standard (IS)
X mg of Compound
X mg of IS
Purity of IS
Concentration of Compound?
Purity of Compound?

4. Using PULCON & 10 mM Concentration of Unknown
J Am Chem Soc Mar 1;128(8):
Reference Standard for PULCON
NMR of Sample
Calibrated to a sample of known concentration, known number of protons
10 mM
&
P1 Calibration NS TE
Integrals
# Protons
Concentration of Unknown

5. Accuracy of Analysis?
Considerations for Reducing Error in Quantification
Sample Prep
Parameter Set
Field Strength (less error at higher fields)
Probe
Instrument stability
Method of integration (Region or Linefit)
Room stability
SOPs
Calibrant
Region (pure)
Linefit (overlap)

6. Automation (Acquisition and Analysis)
A Tale of Two Probes
Single sample, same instrument – does configuration matter?
Error
Automation (Acquisition and Analysis)
Manual Analysis
Reference Std
PULCON
(region)
LINEFIT
‘Unknown’
Region
400MHz BBO 8%
4-5%
2-3%
0.7%
400MHz QNP 2%
<1%
<0.2%
<0.1%

7. Quantification of Compounds in Crude Extract* * * *
32.8mM

8. Youden Ruggedness’ Trial
600 MHz vs. 400 MHz
Different gravimetric balance
Different days
NMR Tube Quality
Different pulse sequence
Different concentration
Mesh Size
(Also EtOH:Leaf)
Chlorogenic Acid BBE
Delta ~ 2%
600
400 7

9. Benefits of High Quality Data: L-Carvone
Red = double presat
Blue = Wetdc
Water DMSO
L-Carvone
100 Hz

10. QC/QA System Non-NMR user Simple interface Lockdown Automated
Suitability test
Acqu/Proc
Identification
Quantification
Report

11. Towards Automatic Analysis
Quantification
Proton, fluorine, and phosphorus
Identification of residual solvents
Assist with identification of ‘unknown’ signals
System:
PDF report
Concentration verification (based on spectra and submitted compound)
Verification by either:
Proton only
Or proton+carbon
Or proton+hsqc
Stop light evaluation of atom assignment

12. discover more.
CATALENT PHARMA SOLUTIONS 14 SCHOOLHOUSE ROAD SOMERSET, NJ 08873