John Edwards ACS NERM 2013 - Fish Oil Analysis -10-22-13

PLS Regression Model Comparison of 60 and 300 MHz 1H qNMR of EPA and DHA Omega-3 Fatty Acids Obtained at Different Points in a Fish Oil Nutritional Supplement

Manufacturing Process

John C. Edwards and Paul J. GiammatteoProcess NMR Associates, LLC, 87A Sand Pit Rd, Danbury, CT 06810 USA

Presented at the ACS North-East Regional Meeting October 23, 2013New Haven, CT

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

http://www.process-nmr.com/

http://www.nmrblog.com/

60 MHz NMR3-10 mm SamplesLab or On-Line

aspectimaging60 MHz NMR3-10 mm SamplesLab or On-Line

60 MHz NMR3-5 mm SamplesLab Only

42 MHz NMR3-5 mm SamplesLab Only

60 MHz NMR3-5 mm SamplesLab Only 60 MHz NMR

3-5 mm SamplesLab Only

45 MHz and 82 MHz NMR300 micron capillary injectionLab Only

85+ MHz NMR3-5 mm SamplesLab Only

Picospin 45 and 80

NMReady 60

Pulsar

Fourier 60

MobiLab™ SPECTRA 2T

Cryogen FreePermanent MagnetHigh ResolutionBenchtop NMR

60 and 90 MHz5 mm SamplesLab Only

60 MHz Reaction Monitoring

On-Line Process NMR 45 Systems Installed Since 1996

Predominantly in Petroleum and Petrochemical

2 x Varian Mercury 300 Liquids1 x Varian UnityPlus-200 SolidsMultiple 10/20/40 mm TD-NMR 20-25 MHzNMR MOUSE2 x Aspect-AI 60 MHz HiRes-NMR3 x Qualion 60 MHz HiRes NMR

MicroESRShimazu GCMNova(Verify, qNMR, DB), ACDLabsChemometrics: GramsIQ, Eigenvector Solo, Pirouette


b

b

a

g



50 sample initial data set from all points in manufacturing process.

First round of PLS regression analysis revealed concentration outliers that were linked to limitations in the GC method. This study was performed on improved GC data values.

Final 80 Sample models were utilized to validate the calibrations on a 24 sample validation set

Experimental Varian Mercury - 300 MHz NMR Supercon Spectrometer4 pulse on pure sample in 5 mm tube, Run UnlockedIn MNova 8.1.2 SPC Files Imported, Stacked, Binned at 3 Hz interval, Area Normalized to 100, Saved as Transposed Ascii MatrixPLS Regression Performed Thermo Grams IQ and Eigenvector Solo

300 MHz 1H NMR

DHA

EPA

Olefins

=C-CH2-C=

In chain

CH2

EtOOC-CH2-R

DHA

EPA

CH3-CH2-O-OC-CH2-R

CH3

EthylCH3

R-CH2-C=CH3

w6/ w9

w3

Comparison of High EPA and High DHA PUFA-Ester after Separation

DHA

EPA

EPA

EPA

EPA

300 MHz 1H NMR Spectra of Samples at Diferent Points in the Process

-200 -150 -100 -50 0 50 100 150 200-20

-15

-10

-5

0

5

10

15

Scores on LV 1 (98.04%)

Score

s o

n L

V 2

(0.6

4%

)

Samples/Scores Plot of SPC Files in MNova.xlsx

First Esterification

Clathration

Molecular Distillate

Pollock Oil

Separator Product

PUFA Ester

Clathration Raffinate

MUFA Ester

Salmon Oil

95% Confidence Level




300 MHz 1H NMR Data

Stacked and integrated.

Integrals Utilized in PLS Regression Against EPA/DHA content by GC

FTIR-ATR Spectra

NMR Spectra

Fusion of 1H NMR and FTIR-ATR Spectra – absolute intensity scaling




PLS Regression DataEPA Content (Wt%)

R2, SECV (Wt%)DHA Content (Wt%)

R2, SECV (Wt%)

60 MHz NMR 0.988, 1.68 0.991, 1.09

300 MHz NMR 0.993, 2.13 0.992, 1.13

Peak Integral Data 0.981, 2.71 0.964, 2.24

Fused - NMR-FTIR 0.993, 1.62 0.992, 1.08

Regression Results for Various NMR Approaches




Acknowledgements

Daniel EdwardsGeneric Customer

Aspect AI – NMR SystemsCosa-Xentaur

Think About Attending the 2nd AnnualPractical Applications of NMR in Industry Conference

February 3-5, 2014Charlotte, NC

Agenda and Registration Information can be found at: http://www.panicnmr.com/


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John Edwards ACS NERM 2013 - Fish Oil Analysis -10-22-13

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