PLS Regression Model Comparison of 60 and 300 MHz qNMR of EPA and DHA 1H

Omega-3 Fatty Acids Obtained at Different Points in a Fish Oil Nutritional

Supplement Manufacturing Process
John C. Edwards and Paul J. Giammatteo
Abstract
Process NMR Associates, LLC, 87A Sand Pit Rd, Danbury, CT 06810 USA
1H NMR of a series of samples taken from different points in the manufacturing process of a fish oil nutritional supplement were obtained on a 300 MHz superconducting NMR system and a cryogen-free bench-top 60 MHz NMR system.
The resulting spectra were utilized to develop single wide-range partial least-squares regression models of the EPA and DHA omega-3 fatty acid content of the fish oils at the various sampling points on the process. The process involves
initial concentration of the fatty acids by solvent precipitation, molecular distillation, ethyl ester esterification, clathration, and centrifugation. For each of the fatty acids a single full range PLS model was obtained utilizing the entire
integral binned/normalized spectrum or utilizing a series of normalized peak integrations rather than the entire spectrum. It was demonstrated that 60 MHz NMR spectra yielded identical model performance to the higher resolution 300
MHz spectra. The 60 MHz system is compact enough that it can be placed in the manufacturing plant environment for at-line utilization. Alternatively it can be packaged to provide on-line/in-line process control.
Sample ID NMR ID EPA (Area %) NMR ID DHA (Area %)
12JUN2424 FO3h002 21.55 FO3h002 13.34 Sample ID NMR ID EPA (Area %) NMR ID DHA (Area %)
12NOV9380 FO3h003 62.97 FO3h003 15.66 12JUL4800 FO3h061 0.07 FO3h061 0.00
11D12277 FO3h004 29.43 FO3h004 18.16 13JAN2039 FO3h062 1.67 FO3h062 0.03
In chain 12MAY2296 FO3h007 15.21 FO3h007 10.51 13FEB3550 FO3h063 0.11 FO3h063 0.00
Eicosaoentaenoic Acid (EPA) 20:5(n-3) CH2 12OCT8644
12OCT8354
FO3h008
FO3h009
7.18
16.95
FO3h008
FO3h009
0.23
10.04
12JUL4805
13MAY8786
FO3h064
FO3h065
0.03
0.09
FO3h064
FO3h065
0.00
0.00
13APR7031 FO3h010 36.35 FO3h010 16.47
1H NMR – 60 MHz 11K12672 FO3h011 61.09 FO3h011 21.26
12NOV8923 FO3h066 2.13 FO3h066 0.41
12JUN2380 FO3h067 0.83 FO3h067 0.45
13JAN2175 FO3h013 71.78 FO3h013 7.43
12MAR8096 FO3h014 41.40 FO3h014 25.91 12MAY2249 FO3h068 0.95 FO3h068 0.55
12NOV9549 FO3h015 1.19 FO3h015 0.06 12JUN2386 FO3h069 0.89 FO3h069 0.67

Docosahexaenoic Acid (DHA) 22:6(n-3) 12OCT8378

12DEC1218
FO3h016
FO3h017
11.73
43.38
FO3h016
FO3h017
12.23
19.30
13MAR5560
13MAY8227
FO3h070
FO3h076
0.92
1.50
FO3h070
FO3h076
0.37
0.01
Ethyl 12APR9931 FO3h018 6.07 FO3h018 2.78 13MAY7819 FO3h077 1.18 FO3h077 0.02
12OCT8371 FO3h019 9.77 FO3h019 0.72 13APR7778 FO3h078 1.35 FO3h078 0.02
CH3 11L12732 FO3h020 58.93 FO3h020 23.41 13MAR5331 FO3h079 42.67 FO3h079 0.00
13APR6723 FO3h022 43.91 FO3h022 21.52
EtOOC-CH2-R 13JAN1968 FO3h024 0.00 FO3h024 0.00
12OCT7628 FO3h080 26.45 FO3h080 0.00
13MAR5547 FO3h085 1.17 FO3h085 0.49
Olefins 12APR9371 FO3h025 26.97 FO3h025 12.82
13MAR5556 FO3h086 1.01 FO3h086 0.40
13APR7428 FO3h026 44.55 FO3h026 19.30
13JAN1885 FO3h087 6.28 FO3h087 0.00 600
11AUG2057 FO3h027 16.69 FO3h027 9.89
=C-CH2-C= 13APR6478 FO3h029 62.79 FO3h029 19.43 12SEP7590 FO3h088 13.82 FO3h088 0.02
Integration of Peaks to Produce Multivariate Spectra
12MAY1569 FO3h030 37.29 FO3h030 22.03 25368NT FO3h005 14.21 FO3h005 9.54
12OCT8363 FO3h031 9.71 FO3h031 0.38 26601NT FO3h021 10.62 FO3h021 5.18 500
CH2-C= 25950NT FO3h028 6.87 FO3h028 4.53
CH3-CH2-O-OC-CH2-R CH3 12APR9555
12MAY2332
FO3h032
FO3h033
32.00
38.79
FO3h032
FO3h033
15.10
26.75 25802NT FO3h040 8.77 FO3h040 5.75
DHA 11L12746 FO3h034 41.87 FO3h034 23.25 26314NT FO3h046 12.10 FO3h046 5.69
EPA 13MAR5577 FO3h035 35.49 FO3h035 43.99
400
25424NT FO3h056 9.56 FO3h056 6.02
12OCT8590 FO3h036 0.30 FO3h036 0.00 25788NT FO3h057 9.01 FO3h057 5.85
12NOV9183 FO3h037 34.09 FO3h037 8.09 26857NT FO3h058 11.34 FO3h058 5.70
12MAY1988 FO3h038 15.44 FO3h038 9.82 300
26408NT FO3h059 12.70 FO3h059 5.58
11AUG2066 FO3h039 60.08 FO3h039 24.52

Data
26910NT FO3h060 10.86 FO3h060 5.95
13APR6283 FO3h041 12.41 FO3h041 57.59
12MAY1596 FO3h042 36.36 FO3h042 21.49 25460NT FO3h071 10.01 FO3h071 6.85
200
13JAN1876 FO3h044 29.23 FO3h044 13.78 26199NT FO3h072 11.79 FO3h072 5.41
11A12125 FO3h045 45.99 FO3h045 33.51 26392NT FO3h074 12.25 FO3h074 5.36
13APR7558 FO3h047 24.57 FO3h047 14.32 26412NT FO3h075 13.13 FO3h075 5.46
12MAR8793 FO3h048 45.86 FO3h048 33.61 26457NT FO3h081 13.22 FO3h081 5.48 100
13APR7427 FO3h050 58.13 FO3h050 24.66 25458NT FO3h082 10.43 FO3h082 6.99
12JUN2398 FO3h051 0.75 FO3h051 0.40 25474NT FO3h083 14.18 FO3h083 7.27
12NOV9492 FO3h052 48.26 FO3h052 23.12 25473NT FO3h084 13.78 FO3h084 6.85 0
11H12519 FO3h053 48.35 FO3h053 23.76

300 MHz 1H NMR 12A12876 FO3h055 11.67 FO3h055 7.19

-100
Expansion of 1 2 3 4 5 6
Variables
7 8 9 10 11

NMR Processing for Multivariate Analysis Correlated Region

24 Sample
Validation

Linear regression model using

0.04
Variables/Loadings Plot for 1H NMR - 55 Fish Oil Samples - Processed - 8-9-13 - Transposed.xlsx Partial Least Squares calculated with the SIMPLS algorithm
Developed 18-Sep-2013 01:02:051.68
80 4 100 4 Author: John@JOHN-NEW-HP
0.03
X-block: 1H NMR - 55 Fish Oil Samples - Processed - 8-9-13 -
3 3
80 Transposed.xlsx 49 by 135
Q Residuals (0.10%)
Q Residuals (0.12%)

0.02
60 Included: [ 1-12 14-23 25-33 35-38 40 42-44 46-55 ] [ 1-135 ]
Y Stdnt Residual 1

2
Y Stdnt Residual 1

2
60
Preprocessing: Mean Center
1
Reg Vector for Y 1

1 0.01 Y-block: DHA GC Content.xlsx 49 by 1

40 0 Included: [ 1-12 14-23 25-33 35-38 40 42-44 46-55 ] [ 1 ]
0 40
0 Preprocessing: Mean Center
-1
-1 Num. LVs: 8
20 20
-2 -0.01
-2 Cross validation: venetian blinds w/ 7 splits
-3 RMSEC: 0.861604 RMSECV: 1.21912
-3 0 Bias: -1.77636e-015 CV Bias: 0.0288731
0 -0.02 0 5 10 15 20 25 30 0 0.2 0.4 0.6 0.8
0 5 10 15 20 25 30 0 0.2 0.4 0.6 0.8 Hotelling T^2 (99.90%) Leverage R^2 Cal: 0.992859 R^2 CV: 0.986012
Hotelling T^2 (99.88%) Leverage
-0.03
20 40 60 80 100 120
Variable
50 Variables/Loadings Plot for 1H NMR - 55 Fish Oil Samples - Processed - 8-9-13 - Transposed.xlsx
0.5
80 50 Linear regression model using 40
Partial Least Squares calculated with the SIMPLS algorithm
Scores on LV 2 (1.51%)

40 0.4
Scores on LV 2 (1.05%)

Developed 18-Sep-2013 00:44:009.58

Y CV Predicted 1

20
60 0.3
Y CV Predicted 1

Author: John@JOHN-NEW-HP 30
X-block: 1H NMR - 55 Fish Oil Samples - Processed - 8-9-13 - 0 0.2

40 0 Transposed.xlsx 49 by 135 20
Included: [ 1-12 14-23 25-33 35-38 40 42-44 46-55 ] [ 1-135 ] 0.1
Reg Vector for Y 1

-20
Preprocessing: Mean Center 10 0
20 Y-block: EPA GC Content.xlsx 49 by 1
-40
Included: [ 1-12 14-23 25-33 35-38 40 42-44 46-55 ] [ 1 ] -0.1
0
Preprocessing: Autoscale 0 10 20 30 40 50 -400 -200 0 200 400
0 -50 Num. LVs: 8 Y Measured 1 Scores on LV 1 (94.95%) -0.2
0 20 40 60 80 -400 -200 0 200 400
Cross validation: venetian blinds w/ 7 splits
Y Measured 1 Scores on LV 1 (94.95%) -0.3
RMSEC: 1.54907 RMSECV: 2.19429

EPA PLS Regression Calibration – 300 MHz Bias: 0 CV Bias: -0.128876

R^2 Cal: 0.993514 R^2 CV: 0.987032
DHA PLS Regression Calibration – 300 -0.4

MHz -0.5
20 40 60
Variable
80 100 120

Variables/Loadings Plot for SPC Files in MNova.xlsx Linear regression model using
1.5
15 4 Partial Least Squares calculated with the SIMPLS algorithm
30 4 Developed 18-Sep-2013 01:45:09.376
3
Author: John@JOHN-NEW-HP
Q Residuals (0.08%)

1 25
Y Stdnt Residual 1

2 X-block: SPC Files in MNova.xlsx 49 by 220

Q Residuals (0.09%)

10 2
Y Stdnt Residual 1

20
Included: [ 1-3 5-8 10-23 25-33 35-38 40-44 46-55 ] [ 1-220
1
]
0.5
Reg Vector for Y 1

0 Preprocessing: Mean Center

15 0
5 -1 Y-block: DHA GC Content.xlsx 49 by 1
10 Included: [ 1-3 5-8 10-23 25-33 35-38 40-44 46-55 ] [ 1 ]
0
-2 -2 Preprocessing: Mean Center Num. LVs: 8
5 Cross validation: venetian blinds w/ 7 splits
-3
0
0 5 10 15 20 25 30 0 0.2 0.4 0.6 0.8 RMSEC: 0.613656 RMSECV: 1.13119
-0.5 0 -4
Hotelling T^2 (99.92%) Leverage 0 5 10 15 20 25 30 0 0.2 0.4 0.6 0.8 Bias: 0 CV Bias: -0.118407
Hotelling T^2 (99.91%) Leverage R^2 Cal: 0.99739 R^2 CV: 0.99137
-1 Variables/Loadings Plot for SPC Files in MNova.xlsx
80 20 20 40 60 80 100 120 140 160 180 200 220 1.5
Variable
60 20
Scores on LV 2 (0.51%)

Linear regression model using 50

60 10 1
Scores on LV 2 (0.36%)
Y CV Predicted 1

Partial Least Squares calculated with the SIMPLS algorithm 10

Y CV Predicted 1

Developed 18-Sep-2013 01:26:000034 40

40 0 Author: John@JOHN-NEW-HP 0.5

X-block: SPC Files in MNova.xlsx 48 by 220 30 0

Reg Vector for Y 1

Included: [ 1-23 25-33 35-38 40 42-43 46-50 52-55 ] [ 1-220 0

20 -10 20
]
-10
Preprocessing: Mean Center
10
0 -20
Y-block: EPA GC Content.xlsx 48 by 1 -0.5

0 20 40 60 80 -200 -100 0 100 200 Included: [ 1-23 25-33 35-38 40 42-43 46-50 52-55 ] [ 1 ] 0 -20
Y Measured 1 Scores on LV 1 (97.98%) Preprocessing: Mean Center Num. LVs: 8 0 10 20 30 40 50 60 -200 -100 0 100 200
-1
Cross validation: venetian blinds w/ 7 splits Y Measured 1 Scores on LV 1 (97.83%)
RMSEC: 1.53792 RMSECV: 2.30361
EPA PLS Regression Calibration – 60 MHz Bias: 1.42109e-014 CV Bias: -0.0513138
DHA PLS Regression Calibration – 60 MHz -1.5
20 40 60 80 100 120 140 160 180 200 220
R^2 Cal: 0.994135 R^2 CV: 0.986901 Variable

80 0.8
Variables/Loadings Plot for aaa-1H-300MHz_IntelligentIntegrals.xlsx
0.6
Variables/Loadings Plot for aaa-1H-300MHz_IntelligentIntegrals.xlsx
EPA - SECV DHA - SECV
60 4
3
0.4
PLS Regression Data (Wt%) (Wt%)
0.6 3
Y Stdnt Residual 1 EPA

2 50
Q Residuals (0.04%)

60
Q Residuals (0.03%)

Y Stdnt Residual 1

2
1
0.4
40
0.2
60 MHz NMR 2.31 1.13
1
Reg Vector for Y 1
Reg Vector for Y 1 EPA

0.2
40 0 30 0
0
300 MHz NMR 2.19 1.22
-1 0
20 -1 -0.2
20
-2 -0.2
10 -2
-0.4
Peak Integral Data 2.98 2.24
-3 -0.4
-3
0
0 0 5 10 15 20 25 30 0 0.2 0.4 0.6 0.8
0 5 10 15 20 25 30 0 0.2 0.4 0.6 0.8
-0.6 Hotelling T^2 (99.97%) Leverage
-0.6 Fused - NMR-FTIR 1.89 1.08
Hotelling T^2 (99.96%) Leverage

EPA PLS Regression Calibration – Peak Integrals -0.8

1 2 3 4 5 6 7 8 9 10 11
DHA PLS Regression Calibration – Peak Integrals -0.8
1 2 3 4 5 6 7 8 9 10 11
Variable 60 100 Variable 140
80 100
Partial Least Squares calculated with the SIMPLS algorithm 50 Partial Least Squares calculated with the SIMPLS algorithm
Scores on LV 2 (1.20%)

Developed 18-Sep-2013 02:14:037.15 Developed 18-Sep-2013 02:34:051.61 120 Fused 1H NMR and FTIR-ATR
Scores on LV 2 (1.33%)

Y CV Predicted 1
Y CV Predicted 1 EPA

50
60
50 Author: John@JOHN-NEW-HP 40 Author: John@JOHN-NEW-HP
X-block: aaa-1H-300MHz_IntelligentIntegrals.xlsx 49 by 11 X-block: aaa-1H-300MHz_IntelligentIntegrals.xlsx 49 by 11
30 100
Included: [ 1-12 14-23 25-33 35-38 40 42-44 46-55 ] [ 1-11 ] Included: [ 1-12 14 16-23 25-33 35-42 44 46-55 ] [ 1-11 ]
40 0
Preprocessing: Mean Center 20 Preprocessing: Mean Center
0
Y-block: EPA GC Content.xlsx 49 by 1 Y-block: DHA GC Content.xlsx 49 by 1 80
10
20 Included: [ 1-12 14-23 25-33 35-38 40 42-44 46-55 ] [ 1 ] -50
Included: [ 1-12 14 16-23 25-33 35-42 44 46-55 ] [ 1 ]
Preprocessing: Mean Center Num. LVs: 5 Preprocessing: Mean Center Num. LVs: 5
Data

-50 0 60
Cross validation: venetian blinds w/ 7 splits 0 10 20 30 40 50 60 -500 0 500 Cross validation: venetian blinds w/ 7 splits
0 Y Measured 1 Scores on LV 1 (98.13%)
0 20 40 60 80 -500 0 500 RMSEC: 2.36013 RMSECV: 2.97738 RMSEC: 1.8631 RMSECV: 2.24092
40
Y Measured 1 EPA Scores on LV 1 (97.98%) Bias: 0 CV Bias: -0.0524533 Bias: -1.24345e-014 CV Bias: 0.00464222
R^2 Cal: 0.984945 R^2 CV: 0.976055 R^2 Cal: 0.974603 R^2 CV: 0.963534
20

Partial Least Squares calculated with the SIMPLS algorithm

EPA Correlation with Combined and Scaled 1H NMR and FTIR-ATR Partial Least Squares calculated with the SIMPLS algorithm DHA Correlation with Combined and Scaled 1H NMR and FTIR-ATR Developed 18-Sep-2013 03:07:000039 0
300 4 X-block: NMR_FTIR Scaled.xlsx 49 by 1016
X-block: NMR_FTIR Scaled - Sample 24 Removed.xlsx 49 by 998
Included: [ 1-32 34-37 39 41-43 45-49 51-54 ] [ 1-139 447-682 1159-1799 ] 300 4
250 3 Included: [ 1-3 5-8 10-32 34-37 39-43 45-54 ] [ 1-163 468-665 1152-
Preprocessing: Mean Center
Y Stdnt Residual 1 EPA

-20
250 1788 ]
Q Residuals (0.15%)

200 400 600 800 1000 1200 1400 1600

Y Stdnt Residual 1 DHA

2 Y-block: EPA GC Content - Sample 24 Removed.xlsx 49 by 1

Q Residuals (0.13%)

2 Variables
200 Preprocessing: Mean Center
1 Included: [ 1-32 34-37 39 41-43 45-49 51-54 ] [ 1 ] 200
Y-block: DHA GC Content - Sample 24 Removed.xlsx 49 by 1
150 Preprocessing: Mean Center Num. LVs: 6
0 150 0 Included: [ 1-3 5-8 10-32 34-37 39-43 45-54 ] [ 1 ]
Cross validation: venetian blinds w/ 7 splits
100
Preprocessing: Mean Center Num. LVs: 7
-1 RMSEC: 1.45399 RMSECV: 1.8994 100
-2 Cross validation: venetian blinds w/ 7 splits
Bias: -7.10543e-015 CV Bias: -0.11802
50 -2 50 RMSEC: 0.677763 RMSECV: 1.08301
R^2 Cal: 0.994649 R^2 CV: 0.990961
Bias: -5.32907e-015 CV Bias: 0.010442
0
-3
0.25
Variables/Loadings Plot for NMR_FTIR Scaled - Sample 24 Removed.xlsx 0
0 10 20 30 40
-4
0 0.2 0.4 0.6 0.8 R^2 Cal: 0.996822 R^2 CV: 0.992286 Conclusion
0 5 10 15 20 25 30 0 0.2 0.4 0.6 0.8
Hotelling T^2 (99.85%) Leverage
0.2
Hotelling T^2 (99.87%) Leverage
0.3
Variables/Loadings Plot for NMR_FTIR Scaled - Sample 24 Removed.xlsx Wide range PLS correlation models can be readily built
0.25
based on 60 MHz NMR data. Various spectral ‘de-resolution’
0.15 60
80 200 100 0.2
techniques may make these models transferable between
0.1 50 NMR data sets obtained at varying magnetic field strengths.
Scores on LV 2 (4.42%)
Y CV Predicted 1 DHA

0.15
Scores on LV 2 (10.80%)

Reg Vector for Y 1 EPA

50
Y CV Predicted 1 EPA

At-line and in-line permanent magnet NMR systems can

Reg Vector for Y 1 DHA

0.05
60 100 40 0.1

0
30 0
0.05 yield the high quality correlations as data obtained on
40 0 -0.05
20
0
Much higher field superconducting NMR systems.
-50 -0.05
-0.1
10 -0.1
20 -100 -0.15 -100
-0.15
0
-0.2
0 10 20 30 40 50 60 -500 0 500
-0.2
Y Measured 1 DHA Scores on LV 1 (85.17%) 200 400 600 800 1000 1200 1400 1600
0 -200 Variable
0 20 40 60 80 -500 0 500 -0.25
200 400 600 800 1000 1200 1400 1600
Y Measured 1 EPA Scores on LV 1 (85.65%) Variable