Analytical & Bioanalytical Techniques Joshua Hicks Catalent Pharma Solutions September 2, 2015
NMR quantification vs. other methods NMR • For automated, internalstandard-free, absolute quantization… • NMR is accurate, precise and versatile • Superior to CLND • CLND • ELSD • Weighing ELSD Analytical Chemistry (2005), 77(14), 4354 -4365 2. 10. 11 1
Quantificaiton with Internal Standard (IS) • X mg of Compound • X mg of IS • Purity of IS Concentration of Compound? Purity of Compound? 2. 10. 11 2
Using PULCON NMR of Sample J Am Chem Soc. 2006 Mar 1; 128(8): 2571 -6. Reference Standard for PULCON & 10 m. M Calibrated to a sample of known concentration, known number of protons P 1 Calibration NS TE Integrals # Protons Concentration of Unknown 2. 10. 11 3
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) 2. 10. 11 4
A Tale of Two Probes Single sample, same instrument – does configuration matter? Error Automation (Acquisition and Analysis) Manual Analysis PULCON (region) LINEFIT Region LINEFIT 400 MHz BBO 8% 4 -5% 2 -3% 0. 7% 400 MHz QNP 2% <1% <0. 2% <0. 1% Reference Std ‘Unknown’ 2. 10. 11 5
Quantification of Compounds in Crude Extract * * 32. 8 m. M 2. 10. 11 6
Youden Ruggedness’ Trial 600 MHz vs. 400 MHz NMR Tube Quality Different concentration Different gravimetric balance Different pulse sequence Different days Mesh Size (Also Et. OH: Leaf) Chlorogenic Acid BBE Delta ~ 2% 600 400 7 2. 10. 11 7
Benefits of High Quality Data: L-Carvone Red = double presat Blue = Wetdc Water DMSO L-Carvone 2. 10. 11 100 Hz 8
QC/QA System Non-NMR user • Simple interface • Lockdown Automated • Suitability test • Acqu/Proc • Identification • Quantification • Report 2. 10. 11 9
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: — — 2. 10. 11 Or proton+carbon — • Proton only Or proton+hsqc Stop light evaluation of atom assignment 10
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