NMR-Based Test Methods - How to Change the Paradigm of f NMR - PowerPoint PPT Presentation

The Practical Non-Existance of f Official NMR-Based Test Methods - How to Change the Paradigm of f NMR Exclusion The Perspective of a Small NMR Service Company John Edwards, Process NMR Associates Validation Workshop – PANIC NMR Conference – February 19, 2016 – Houston Texas

ASTM NMR Test Methods E2977: Standard Practice for Measuring and Reporting Performance of Fourier Transform Nuclear Magnetic Resonance (FT-NMR) Spectrometers for Liquid Samples D7171-05: Standard Test Method for Hydrogen Content of Middle Distillate Petroleum Products by Low- Resolution Pulsed Nuclear Magnetic Resonance Spectroscopy D4273-05: Standard Test Methods for Polyurethane Raw Materials Determination of Primary Hydroxyl Content of Polyether Polyols D4875-05: Standard Test Methods of Polyurethane Raw Materials: Determination of the Polymerized Ethylene Oxide Content of Polyether Polyols D5017-96: Standard Test Method for Determination of Linear Low Density Polyethylene (LLDPE) Composition by Carbon-13 Nuclear Magnetic Resonance D5292-99: Standard Test Method for Aromatic Carbon Contents of Hydrocarbon Oils by High Resolution Nuclear Magnetic Resonance Spectroscopy F2259-03: Standard Test Method for Determining the Chemical Composition and Sequence in Alginate by Proton Nuclear Magnetic Resonance ( 1 H NMR) Spectroscopy F2260-03: Standard Test Method for Determining Degree of Deacetylation in Chitosan Salts by Proton Nuclear Magnetic Resonance ( 1 H NMR) Spectroscopy USP <2251> Adulteration of Dietary Supplements with Drugs and Drug Analogs. Poloxamer Testing USP <761> : General Chapter – Nuclear Magnetic Resonance Spectroscopy Krill Oil Monograph – 1 H and 31 P NMR, Beta Glucan Monograph – 1 H NMR, Heparin Sodium Monograph – 1 H NMR European Pharmacopoeia Salmon Oil, Farmed 01/2005:1910 13 C NMR for distribution of fatty acids.

ISO 10565:1998 Oil and Moisture in Seeds by Low Field NMR Spectroscopy 8292 Animal and vegetable fats and oils -- Determination of solid fat content -- Pulsed nuclear magnetic resonance method 21461:2006 Rubber - Determination of the aromaticity of oil in vulcanized rubber compounds 21561:2005 Styrene-butadiene rubber (SBR) - Determination of the microstructure of solution- polymerized SBR 11543:2000 Modified starch -- Determination of hydroxypropyl content -- Method using proton nuclear magnetic resonance (NMR) spectrometry 10632:2000 Oilseed residues -- Simultaneous determination of oil and water contents -- Method using pulsed nuclear magnetic resonance spectroscopy AOCS Cd 16b-93 : (revised in 2000): Direct Method - Determination of Solid Fat Content -- Pulsed Nuclear Magnetic Resonance Method Cd 16-81 : (revised in 2000): Indirect Method - Determination of Solid Fat Content -- Pulsed Nuclear Magnetic Resonance Method Ak5-01 (01) : Oil Content of Oilseed Residues by NMR Ak3-94 (00) : Oil Content in Rapeseed and other Oilseeds by NMR Ak4-95 (99): Simultaneous Determination of Oil and Moisture Contents of Oilseeds using Pulsed Nuclear Magnetic Resonance Spectroscopy American Herbal Pharmacopoeia Aloe Vera - 1 H NMR Method for Quality Inspection and Identification Canadian Crude Quality Technical Association Maxxam V1.04 11/05 : Determination of Olefin Content of Crude Oils, Condensates and Diluents by 1 H NMR

1 H qNMR is very well referenced in the Guide to NMR Method Development and Validation academic literature but is – Part I: Identification and Quantification still not an official standard method validated by a standards organization

Organic Aloe Vera Juice Hand Fillet with Pulp Batch OAVWP0116 Dry Matter Composition Product Composition Product Composition Component Wt% Wt% (mg/L) Acemannan Wt% 13.96 0.158 1549 16.00 Glucose Wt% 0.181 1776 27.89 Malic Acid Wt% 0.315 3096 Lactic Acid 0.00 0.000 0 Citric Acid 22.07 0.249 2449 0.16 Pyruvic Acid 0.002 18 0.00 Sorbate 0.000 0 Benzoate 0.00 0.000 0 0.00 Acetoin 0.000 0 0.00 2,3-butandiol 0.000 0 Whole Leaf Marker (WLM) Not Observed Not Observed Trace Observed Isocitrate (WLM-1) 0.000 0 0.00 Isocitrate Lactone (WLM-2) 0.00 0.000 0 0.00 Acetic Acid 0.002 17 Succinic Acid 0.00 0.000 0 Formic Acid 0.00 0.000 0 0.00 Fumaric Acid 0.000 0 Ethanol 0.00 0.000 0 Glycine 0.00 0.000 0 0.00 Propylene Glycol 0.000 0 0.00 Sucrose 0.000 0 Dry Matter Weight (%) 1.13

• Serving Size 8 fl oz. Alcohol and Tobacco Tax • Servings Per Container 2 and Trade Bureau (TTB) • Amount Per Serving • Calories 30 • Calories from Fat 0 • % Daily Value* • Total Fat 0g 0% • Cholesterol 0mg 0% • Sodium 10mg 1% • Total Carbohydrate 7g (2%) • Sugar 2g • Protein 0g • Probiotic Content • Bacillus coagulans GBI-30 6086: 1 billion organisms • S. Boulardii: 1 billion organisms • Antioxidants and Organic Acids • EGCG 100mg • Glucuronic Acid 10mg • L(+) Lactic Acid 25mg • Acetic Acid 30 mg

USP General Chapter 2251 Adulteration of Dietary Supplements with Drugs and Drug Analogs

Typical Herbal Extracts

300 MHz NMR – PDE5i Standards

Component Wt% = 100 x (Component Molar Integral / Std Molar Integral) x (Wt Std / Wt Sample) x (Component Mw / Std Mw)

Component Wt% = 100 x (Component Molar Integral / Std Molar Integral) x (Wt Std / Wt Sample) x (Component Mw / Std Mw)

ASTM E1655 - 05(2012) Standard Practices for Infrared Multivariate Quantitative Analysis ASTM D6122 - 15 Standard Practice for Validation of the Performance of Multivariate Online, At-Line, and Laboratory Infrared Spectrophotometer Based Analyzer Systems Correlative (Chemometric Methods) NMR Spectrometer Specification 42 MHz, 60 MHz, 80 MHz, 300 MHz, 400 MHz, 700 MHz?

Experimental In chain 300 MHz 1 H NMR Varian Mercury - 300 MHz NMR Supercon Spectrometer CH 2 4 pulse on pure sample in 5 mm tube, Run Unlocked In MNova 8.1.2 SPC Files Imported, Stacked, Binned at 3 Hz interval, Area Normalized to 100, Saved as Transposed Ascii Matrix Ethyl PLS Regression Performed Thermo Grams IQ and Eigenvector Solo CH 3 EPA- a EPA- b w3 DHA CH 3 Olefin Ole ins w6/ w9 CH3-CH CH3 CH 2 -O-OC OC-CH CH 2 -R EtO tOOC-CH CH 2 -R CH 3 =C =C-CH CH 2 -C= C= R-CH 2 -C= EPA DHA

www.process-nmr.com www.nmrblog.com Tel: 203-744-5905

www.process-nmr.com www.nmrblog.com Tel: 203-744-5905

3 Hz binning interval 0.5 Hz binning interval

www.process-nmr.com www.nmrblog.com Tel: 203-744-5905

www.process-nmr.com www.nmrblog.com Tel: 203-744-5905

Spinsolve 1H NMR data at 42.5 MHz of a series of fish oils and esterified omega-3 fatty acids

Model Omega-3 EPA Content (wt%) in Fish Oil and Linear regression model using Partial Least Squares calculated with the SIMPLS algorithm Pharmaceutical Grade Supplements Developed 27-Jul-2014 15:03:05.984 Author: John@JCEPNA X-block: Final Fish Oil Spectra - 43MHz.csv 55 by 830 Correlation between 40 second 1H NMR at 42.5 MHz (John@JCEPNA@20140727T140753.60670200 m:20140727140753.619) and primary analysis method – 50 minute GC analysis Included: [ 1-55 ] [ 1-830 ] 6 Preprocessing: Mean Center 3 Y-block: EPA-GC Data.xlsx 55 by 1 5 2 (John@JCEPNA@20140727T141758.52236711 Q Residuals (0.09%) Y Stdnt Residual 1 m:20140727141758.529) 4 1 Included: [ 1-55 ] [ 1 ] 3 0 Preprocessing: Mean Center Num. LVs: 8 -1 2 Cross validation: venetian blinds w/ 7 splits RMSEC: 1.40566 RMSECV: 2.20172 -2 1 Bias: 0 CV Bias: -0.00054578 -3 R^2 Cal: 0.995028 R^2 CV: 0.987838 0 0 10 20 30 40 0 0.2 0.4 0.6 0.8 Hotelling T^2 (99.91%) Leverage SSQ Table Percent Variance Captured by Regression Model 80 15 -----X-Block----- -----Y-Block----- 10 Scores on LV 2 (2.48%) Comp This Total This Total 60 Y CV Predicted 1 ---- ------- ------- ------- ------- 5 1 95.78 95.78 84.51 84.51 40 0 2 2.48 98.26 3.47 87.98 -5 3 0.29 98.55 9.21 97.19 20 4 0.76 99.31 0.59 97.78 -10 5 0.43 99.73 0.60 98.38 0 -15 6 0.07 99.80 0.77 99.15 7 0.06 99.86 0.17 99.33 -20 -20 0 20 40 60 80 -100 -50 0 50 8 0.04 99.91 0.18 99.50 Y Measured 1 Scores on LV 1 (95.78%)