Composition of Antarctic krill oil and methods for its...

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part of Aker

Newcastle Australia November 2013

Composition of Antarctic krill oil and methods for its harvesting, production and qualitative and quantitative analysis.

Dr. Nils Hoem, Chief Scientist Aker BioMarine

In this presentation material from Spectral Service , Cologne has been used for illustrative purposes

© Aker BioMarine™


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Agenda

• Introduction - a few words about krill• What does krill contain?• How do we analyze krill and krill lipids?

- and some cumulative data.• Final words



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Nomenclature and abbreviations

FA fatty acidEPA eicosapentaenoic acid 20:5 n-3DHA docosahexaenoic acid 22:6 n-3DPA docosapentaenoic acid 22:5 n-3ASX AstaxanthinNMR nuclear magnetic resonanceGC Gass chromatographyLC Liquid chromatographyHPLC High pressure licuid chromatographyMS Mass spectrometryFID Flame ionization detection



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Antarctic Krill - Euphausia Superba

Zoo plankton (crustacean)

Antarctic food chain

Worlds largest biomass

Harvesting challenges

Catch is regulated by

CCAMLR



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Harvesting area and ice conditions



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Agenda


- and some cumulative data• Final words



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• Chitin (poly-b-1,4-D-N-acetylglucosamine)

• Proteins

• Lipids – high content of phospholipids

• Astaxanthin esters

What does krill contain?

Krill meat

Shell

Red color

http://upload.wikimedia.org/wikipedia/commons/f/fb/Chitosan_Haworth.gif



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Differences in structure and function of triglycerides and phospholipids

Triglyceride (fish oils) Phospholipids (krill oil)

MembraneFat cell

• Non-polar

• Hydrophobic – does NOT mix with water

• Polar

• Hydrophilic - mixes with water

Function

Charac-teristics

• Main structural component in all cell membranes

• Energy storage

Structural form G

l

y

c

e

r

o

l

Fatty acid

Phosphate

Fatty acid

G

l

y

c

e

r

o

l

Fatty acid

Fatty acid

Fatty acid X

X: choline; ethanolamine; serine; inositol; glycerol



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Main components in various krill material

Raw Krill (%) Krill oil (%) Krill powder (%)

5 93 60

2 45 28

3,7 55 32

0,4 25 13

0,035 3 1,3

12 n.a. 30

80 2 1

Total lipids

Polar lipids

Neutral lipid

Omega-3

Omega-6

Proteins

Water

.



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Lipid classes g/100g oil

Triacylglycerol 34

Diacylglycerol 0,7

Monoacylglycerol <1

Free fatty acids 7,9

Cholesterol 1,2

Cholesterol esters <0,5

Phosphatidylethanolamine 2,6

Phosphatidylinositol <1

Phosphatidylserine <1

Phosphatidylcholine 38

Lysophosphatidylcholine 3,4

Total polar lipids 44

Total neutral lipids 44,7

Total sum lipids 88,7

Lipid structure


Lipid classes

2,87,9

Free fatty acids

Phospholipids

Other

34,0

44,0

Triacylglycerol

g/100g of krill oil

Details



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Lipid classes g/100g oil

w-3 (usually < 30g/100g)

Eicosapentanoic acid (EPA) 13,2

Docosahexanoic acid (DHA) 7,7

a-Linolenic acid 1.2

Stearidonic acid 3

w-6

g-Linoleic acid 0,2

Arachidonic 0,3

w-9

Oleic acid 10,7

Other

Palmitic acid 13,5

Palmitoleic acid 2,5

Stearic acid 0,6

Fatty acid breakdown Details


Fatty acid profile

7,7

13,2 13,5

10,7

Other

Omega 3s

Omega 9s

Omega 6s

EPA

DHAOleic acid

Palmitic acid

g/100g of krill oil



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Agenda





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Analytical methods used on in the overall production of krill products 1/4

Parameter MethodFill weight Ph.Eur

Disintegration Ph.Eur

Chemistry

Protein ISO 5983-2

/NS-EN-ISO 16634-1

Water ISO 6496

Ash ISO 5984

Fat (B&D) Bligh&Dyer

Salt AOAC 937.09

Fluorine DIN 38405-4

Water in oil (Karl Fischer) AOCS Ca 2e-84

aW AOAC 978.18

Dry matter ISO 6496

Total volatile Nitrogen AOAC 920.03

TMAO Conway, E. I., and A. Byrne.

TMA Biochem. J. 27:419-429, 1933.

Astaxanthin Roche. Method A 23

Esterified astaxanthin Roche. Method A 23



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Parameter Method

Ethoxyquin AOAC 996.13

Naturox

Free Fatty acids (FFA) AOAC Ca 5A-40

Peroxide value AOCS Cd 8b-90

Anisidin Value AOCS Cd 18-90

Acid value ISO 660

Viscosity Brookfield 021, 1.5 rpm

Saponification value AOAC 920.160

Iodine value AOCS Ja 14-91

Ethanol, flash point GC

Cadaverine Internal NOFIMA A55

Histamine Internal NOFIMA A55

Vitamin A, mg/100g J. Sci. Agri. Vol 29 p 697-702

alpha- Tocopherol, mg/100g

Fat classes, Total phospholipids 31 P-NMR or N A881



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Parameter Method

Fat classes, total lipids as fatty acids AOCS Ce 1b-89

Fatty acid profile

AOCS Ce 1b-89

Omega-3 Fatty acids

EPA (C20:5, n-3)

DHA (C22:6, n-3)

DPA (C22:5, n-3)

Omega-6 Fatty acids

Linoleic acid (C18:2, n-6)

Omega-9 Fatty acids

Oleic acid (C18:1, n-9)

Saturated fatty acids

Monounsaturated fatty acids

Polyunsaturated fatty acids

Total trans-fat



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Parameter Method

Metals and non-metals

Phosphorous ISO 6491/Internal NOFIMA method (A37)

Calcium ISO/CD 6869 / Internal NOFIMA method (A29)

Magnesium Internal NOFIMA method (A30)

Iron NMKL161 mod;ICP-MS/Internal NOFIMA method (A27)

Zink NMKL161 mod;ICP-MS

Arcenic (in-organic) Extraction/digestion, HPLC-ICP-MS

Arcenic (organic) Microwave assisted digestion, ICP-MS

Cadmium NMKL161 mod;ICP-MS / EN 15763:2009

Mercury ALC 208:112 / EN 15763:2009

Lead NMKL161 mod;ICP-MS / EN 15763:2009

Copper NMKL161 mod;ICP-MS / EN ISO 11885, mod 05

Tin EN ISO 11885, mod 05

Antimony EN 15763:2009

PCB's EN 1948 modified, HRGC/HRMS

Dioxins EN 1948 modified, HRGC/HRMS

Microbiology

TPC NF EN ISO 4833 / NordVal Ref.no.012

E.coli Petrifilm Select EC / NordVal Ref.no.014

Coliforms Petrifilm Select EC / NordVal Ref.no.014

Anaerobic Sulphitr. Clostridium NMKL 56

Bacillus Cereus Pres. NMKL 67

Staphylococcus Aureus NorVal. Ref. no. 019

Salmonella AES 10/4-025/04 / NordVal Ref.no.023

Enterobacter ISO 7402

Yeast and molds NorVal. Ref. no. 016



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Batch analyses performed on Superba krill oil



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Compositional analysis of 184 batches of krill oil 1/4


Polar lipids Neutral Lipids

Mean 44,361957Std Dev 3,3557175Std Err Mean 0,2473868Upper 95% Mean 44,850054Lower 95% Mean 43,873859Skewness -0,029825Kurtosis 1,2234362

Mean 41,078804Std Dev 4,3263125Std Err Mean 0,31894Upper 95% Mean 41,708077Lower 95% Mean 40,449532Skewness 0,0547301Kurtosis -0,695264



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Polar lipids Neutral Lipids

Mean 44,361957Std Dev 3,3557175Std Err Mean 0,2473868Upper 95% M 44,850054Lower 95% M 43,873859Skewness -0,029825Kurtosis 1,2234362

Mean 41,078804Std Dev 4,3263125Std Err Mean 0,31894Upper 95% M 41,708077Lower 95% M 40,449532Skewness 0,0547301Kurtosis -0,695264



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Phosphatidyl choline Phosphatidyl ethanolamine

Mean 39,186413Std Dev 3,1427739Std Err Mean 0,2316884Upper 95% M 39,643537Lower 95% M 38,729289Skewness 0,2540793Kurtosis 2,1289064

Mean 2,0581522Std Dev 0,6258012Std Err Mean 0,0461347Upper 95% M 2,1491764Lower 95% M 1,9671279Skewness 0,5652078Kurtosis 0,3084509



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EPA 20:5 n-3 DHA 22:6 n-3

Mean 13,279348Std Dev 1,0746318Std Err Mean 0,0792229Upper 95% M 13,435656Lower 95% M 13,12304Skewness -0,041249Kurtosis -0,351436

Mean 5,9107065Std Dev 0,426184Std Err Mean 0,0314187Upper 95% M 5,972696Lower 95% M 5,848717Skewness 0,0461241Kurtosis -1,045097



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Analysis on separate lipid fractions


Krill

Krill lipids

Polar lipids Neutral lipids Free fatty acids

Separation by fractionation in protic solvent(s) and/or flash chromatography on silica



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Fatty acid profile in Krill oil lipid sub fractions absolute amounts at 60% PL

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

PL

TG

FFA



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Fatty acid profile in Kril oil lipid sub fractions relative amounts

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

PL

TG

FFA



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Krill oil dilution in Soft gel capsules



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Volume

=X0

Krill oil(TG+PL mix)

Triglyceride oil(fish oil)

Fresh capsuleVolume=X0

H2O

H2OH2OH2O

H2O

H2OH2O H2O

StorageH2O

H2OH2OH2O

H2O

H2OH2O H2O

H2O

H2O

H2OH2O

H2O

H2O

H2OH2O

VolumeX1=X0

Volume

X1> X0

KO (hydrophilic) gain weight and volume because of uptake of water

Found in experiments to be ≈ 10%

=

≠

Krill oil dilution in softgel capsules



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Omega-3 Phospholipid species analysis by LC-MS/MS

Winther, B., N. Hoem, et al. (2011). Lipids 46(1): 25-36.

PC general structure



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Omega-3 Phospholipid species

FA

EPA

PCh

FA

DHA

PCh

EPA

DHA

PCh

DHA

DHA

PCh

EPA

EPA

PCh

69 choline-containing PL’s detected

• 60 PL• 9 PLC

7 with ώ-3 FA in both SN-1 and SN2 position

7 PC-species with an alkyl-acyl composition

Phospholipids

Structure

•Polar

•Mixes withwater

Krill oil

A B

C ED

FA

FA

PCh

Winther, B., N. Hoem, et al. (2011). Lipids 46(1): 25-36.

Minor omega-3 species

Major omega-3 species



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Fatty acid composition in KO species analysis by LC-MS/MS 1/4

KO

KO-TG fraction

Spectral Service AG Study Report AKB33483

Emil-Hoffmann-Straße 33 D-50996 Köln (Germany) page 38 of 92

O:\Daten\Triglyceride\AKB334837 28/02/2012 16:48:21 AKB33483-7 NKO OmegaGen Cardio, 009012(V.2.0) Säule 39; APCI pos. (triglyapci2)V2.0 DEV G

RT: 0.00 - 30.00 SM: 15B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

10011.15

9.80

12.698.60

6.9329.3514.46 27.6726.1624.3222.675.63 19.5418.002.841.77

7.70 8.606.204.08

11.14

2.9912.28 12.74 19.41 20.1814.462.14 21.33 23.13 25.07 26.95

4.74

10.138.76 10.738.522.13 4.25 15.66 18.41 19.5414.50 21.56 23.94 25.39 27.980.33

4.83

5.60 9.166.24 9.89 17.572.18 10.964.43 19.7616.80 21.64 23.14 25.63 28.060.34

3.573.89

6.23

6.662.842.18 7.97 10.40 19.5411.62 22.2519.16 23.9212.45 26.6616.70 29.46

6.27

5.804.532.82 7.57 11.9610.07 19.4613.36 20.5018.06 24.1216.60 25.65 28.120.95

NL:7.95E5

Base PeakMSAKB334837

NL:6.72E5

m/z=800.00-1100.00 MSAKB334837

NL:4.34E3

m/z=962.00-963.00 MSAKB334837

NL:4.02E3

m/z=988.00-989.00 MSAKB334837

NL:3.23E3

m/z=1016.00-1017.00 MSAKB334837

NL:8.14E3

m/z=968.00-969.00 MSAKB334837

Fig. 22 LC-MS analysis (+APCI) of AKB33483-7 with characteristic Fish oil triglycerides


RT: 0.00 - 30.00 SM: 15B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

10011.15

9.80

12.698.60

6.9329.3514.46 27.6726.1624.3222.675.63 19.5418.002.841.77

7.70 8.606.204.08

11.14

2.9912.28 12.74 19.41 20.1814.462.14 21.33 23.13 25.07 26.95

7.58

6.80 9.534.552.97 19.3712.23 20.48 22.3914.272.18 24.1217.98 25.69 27.41

9.55

5.528.50 11.142.98 19.446.07 21.07 22.202.06 18.23 23.9216.5814.38 25.74 27.58

10.77

6.089.362.82 5.54 6.59 14.19 24.0219.542.10 25.0415.17 20.4518.27 26.63 28.36

11.18

9.546.032.97 5.53 14.207.76 19.552.05 18.67 20.5315.18 23.48 24.46 26.15 27.85

NL:7.95E5


NL:6.72E5

m/z=800.00-1100.00 MSAKB334837

NL:3.35E4

m/z=842.00-843.00 MSAKB334837

NL:4.45E4

m/z=820.00-821.00 MSAKB334837

NL:5.89E4

m/z=848.00-849.00 MSAKB334837

NL:1.13E5

m/z=822.00-823.00 MSAKB334837

Fig. 23 LC-MS analysis (+APCI) of AKB33483-7 with characteristic Krill oil triglycerides

EPA-16:0-14:0

16:0-DPA-14:0

16:1-16:1-14:0

14:0-18:1-16:0



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KO?

KO-TG fraction

FO: EPA-EPA-EPA

FO: DHA-EPA-EPA

FO: DPA-DHA-EPA

FO: 18:1-DHA-EPA




RT: 0.00 - 30.00 SM: 15B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

10011.15

9.80

12.698.60

6.9329.3514.46 27.6726.1624.3222.675.63 19.5418.002.841.77

7.70 8.606.204.08

11.14

2.9912.28 12.74 19.41 20.1814.462.14 21.33 23.13 25.07 26.95

4.74

10.138.76 10.738.522.13 4.25 15.66 18.41 19.5414.50 21.56 23.94 25.39 27.980.33

4.83

5.60 9.166.24 9.89 17.572.18 10.964.43 19.7616.80 21.64 23.14 25.63 28.060.34

3.573.89

6.23

6.662.842.18 7.97 10.40 19.5411.62 22.2519.16 23.9212.45 26.6616.70 29.46

6.27

5.804.532.82 7.57 11.9610.07 19.4613.36 20.5018.06 24.1216.60 25.65 28.120.95

NL:7.95E5


NL:6.72E5

m/z=800.00-1100.00 MSAKB334837

NL:4.34E3

m/z=962.00-963.00 MSAKB334837

NL:4.02E3

m/z=988.00-989.00 MSAKB334837

NL:3.23E3

m/z=1016.00-1017.00 MSAKB334837

NL:8.14E3

m/z=968.00-969.00 MSAKB334837



RT: 0.00 - 30.00 SM: 15B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

10011.15

9.80

12.698.60

6.9329.3514.46 27.6726.1624.3222.675.63 19.5418.002.841.77

7.70 8.606.204.08

11.14

2.9912.28 12.74 19.41 20.1814.462.14 21.33 23.13 25.07 26.95

7.58

6.80 9.534.552.97 19.3712.23 20.48 22.3914.272.18 24.1217.98 25.69 27.41

9.55

5.528.50 11.142.98 19.446.07 21.07 22.202.06 18.23 23.9216.5814.38 25.74 27.58

10.77

6.089.362.82 5.54 6.59 14.19 24.0219.542.10 25.0415.17 20.4518.27 26.63 28.36

11.18

9.546.032.97 5.53 14.207.76 19.552.05 18.67 20.5315.18 23.48 24.46 26.15 27.85

NL:7.95E5


NL:6.72E5

m/z=800.00-1100.00 MSAKB334837

NL:3.35E4

m/z=842.00-843.00 MSAKB334837

NL:4.45E4

m/z=820.00-821.00 MSAKB334837

NL:5.89E4

m/z=848.00-849.00 MSAKB334837

NL:1.13E5

m/z=822.00-823.00 MSAKB334837




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KO?

KO-TG fraction

EPA-EPA-EPA

DHA-EPA-EPA

DPA-DHA-EPA

18:1-DHA-EPA



O:\Daten\Triglyceride\AKB334831 28/02/2012 12:15:22 AKB33483-1 Nature's Way Krill Oil(V.2.0) Säule 39; APCI pos. (triglyapci2)V2.0 DEV G

RT: 0.00 - 30.00 SM: 15B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

1004.87

3.50 9.83 11.226.26

8.62 29.8921.99 23.01 25.38 28.0821.0612.37 19.3516.943.09 15.911.50

TG fraction

4.87

6.26

7.55 8.549.56 10.832.88 12.822.14 19.7815.08 20.81 26.7316.87 22.43 24.31 29.58

TG 20:5/20:5/20:5

4.77

10.927.14 8.544.01 14.8712.392.87 15.81 18.43 26.8720.48 21.40 24.71 29.540.38

TG 22:6/20:5/20:5

4.85

11.086.30 8.542.11 12.532.89 15.09 17.55 26.8719.69 21.82 29.7123.49

TG 22:5/22:6/20:5

5.18

6.064.03 6.94 9.23 12.512.08 13.49 19.7115.20 17.44 26.7521.93 24.39 28.14

TG 18:1/22:6/20:5

6.31

5.804.79 8.93 12.019.862.89 13.50 14.89 26.8720.0419.16 21.54 24.85 29.730.65

NL:3.93E5


NL:3.69E6

m/z=800.00-1100.00 MSAKB334831

NL:1.79E5

m/z=962.00-963.00 MSAKB334831

NL:2.99E5

m/z=988.00-989.00 MSAKB334831

NL:1.05E5

m/z=1016.00-1017.00 MSAKB334831

NL:1.32E5

m/z=968.00-969.00 MSAKB334831



RT: 0.00 - 30.00 SM: 15B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

1004.87

3.50 9.83 11.226.26

8.62 29.8921.99 23.01 25.38 28.0821.0612.37 19.3516.943.09 15.911.50

TG fraction

4.87

6.26

7.55 8.549.56 10.832.88 12.822.14 19.7815.08 20.81 26.7316.87 22.43 24.31 29.58

TG 20:5/16:0/14:0

7.61

6.374.64 9.574.332.87 12.26 20.4219.29 21.5412.58 23.50 26.8515.49 28.01

TG 16:0/22:5/14:0

9.61

5.58 8.544.83 6.33 11.252.13 20.4012.24 23.8019.26 26.5521.4116.9615.41 29.790.33

TG 18:1/16:1/14:0

10.86

6.16

4.87 9.522.88 7.622.10 19.9314.6912.03 21.0815.56 26.7822.7918.84 27.78

TG 14:0/18:1/16:0

11.24

9.616.054.872.88 7.742.15 12.58 19.8614.22 21.09 26.7515.52 24.80 27.7718.79

NL:3.93E5


NL:3.69E6

m/z=800.00-1100.00 MSAKB334831

NL:1.22E4

m/z=842.00-843.00 MSAKB334831

NL:2.16E4

m/z=820.00-821.00 MSAKB334831

NL:2.46E4

m/z=848.00-849.00 MSAKB334831

NL:4.23E4

m/z=822.00-823.00 MSAKB334831




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Fatty acid composition in KO species analysis by LC-MS/MS 4/4Spectral Service AG Study Report AKB33483



RT: 0.00 - 30.00 SM: 15B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

50

100

50

100

50

100

50

100

50

100

50

100

50

100

AKB33483-1

4.903.53 9.86 11.256.29

8.65 29.9222.02 23.04 25.41 28.1121.0912.40 19.3817.903.12 12.851.53

AKB33483-2

11.25

9.86

12.818.65

7.01 14.52 29.1128.1226.4524.4921.4420.605.71 18.1917.062.911.28

AKB33483-3

11.199.86

8.5912.25 12.726.973.51 29.5328.1525.7124.5722.9118.126.38 20.4816.842.87

AKB33483-4

9.8611.23

8.67 12.323.56 12.767.00 25.40 29.4827.3822.1621.1618.3017.012.88 4.461.07

AKB33483-5

11.229.86

8.68 12.33 12.773.55 7.00 29.5328.0826.4624.503.16 22.9120.9615.53 17.036.63

AKB33483-6

11.209.86

12.768.663.55 6.99 14.49 29.3328.253.15 15.28 26.0624.4922.7818.37 20.835.74

AKB33483-7

11.219.86

12.758.666.99 29.4114.52 27.7326.5824.3822.735.69 19.6018.062.902.43

NL:3.93E5


NL:1.18E6


NL:6.26E5


NL:6.34E5


NL:6.65E5


NL:8.25E5


NL:7.95E5


Fig. 24 LC-MS (+APCI, Base Peak): AKB33483-1 to -7


RT: 0.00 - 30.00 SM: 15B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

50

100

50

100

50

100

50

100

50

100

50

100

50

100

AKB33483-1

4.90

6.29

7.584.46 8.5711.202.91 12.35 14.53 19.81 20.8416.40 26.7622.46 24.34 29.611.08

AKB33483-2

8.597.694.21 6.172.92 11.22

12.33 19.6412.77 20.752.29 21.7514.49 23.66 25.29 27.08

AKB33483-3

19.05 19.2918.794.17 7.74 8.644.76 19.958.85 11.172.99 20.8214.13 14.3912.252.17 21.88

23.76 25.50 27.101.33

AKB33483-4

4.14 7.87 19.26 19.474.87 8.60 18.8011.173.04

20.5513.96 14.27 21.702.2223.89 25.11 26.930.54

AKB33483-5

8.647.706.264.15

11.183.0619.60 19.9718.7812.31 14.49 21.622.20 23.41 25.15 27.01

AKB33483-6

8.647.73

5.71 8.904.17 11.203.05 19.1912.27 18.9312.79 20.152.25 21.40 23.21 24.93 26.74

AKB33483-7

7.76 8.666.264.14

11.20

3.0512.34 19.47 20.2414.522.20 21.6518.07 23.19 25.13 26.92

NL:3.69E6

m/z=800.00-1100.00 MSAKB334831

NL:5.20E5

m/z=800.00-1100.00 MSAKB334832

NL:4.17E5

m/z=800.00-1100.00 MSAKB334833

NL:4.12E5

m/z=800.00-1100.00 MSAKB334834

NL:5.13E5

m/z=800.00-1100.00 MSAKB334835

NL:5.34E5

m/z=800.00-1100.00 MSAKB334836

NL:6.72E5

m/z=800.00-1100.00 MSAKB334837

Fig. 25 LC-MS (+APCI, TG fraction): AKB33483-1 to -7



part of Aker

Analysis of KO by NMR

• 31P-NMR: has become standard method for phospholipid classification and quantitation (AKBM)

• Validated method• Relatively large Round Robin Test (21 different labs with some variation in

instrumentation)

• 1H-NMR and 13C-NMR increasingly used for• Total omega-3• Total number of double bounds• EPA and DHA and their sn1, sn2 positioning• Ether-phospholipids• Other lipids• Other impurities (TMNO)



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Analysis of KO by NMR - phospholipids 1/2



Fig. 8731

P-NMR spectrum of test item AKB33483-6_A + TPP (1 whole capsule)

Fig. 8831

P-NMR spectrum of test item AKB33483-6_B + TPP (filling)



4 METHODS

4.1 NMR Spectroscopy

1H-NMR spectrum and

13C-NMR spectra were recorded to characterise the test items. Approx.

200 mg of each test item have been dissolved in 1 ml DEUTERATED SOLVENTS.

The actually used NMR parameters appear on the spectrum plot.

For quantification according to standard operation procedure SAA-MET001-02 appropriate amounts

of each test item and of internal standard have been exactly weighed (s. Table “Calculation of

content”, Chapter 5 - NMR Spectroscopy), dissolved and measured. Integrated signals of test items

and internal standard have been used for calculation.

To characterize the test item and to determine the content of the phospholipids "Nuclear Magnetic

Resonance Spectra“ of phosphorus (31

P-NMR spectra) were recorded. The quantifications were

carried out against a reference item according to the standard operation procedure SAA-MET002-02.

The procedure corresponds to „USP method <761> NUCLEAR MAGNETIC RESONANCE“.

For the quantification two measurements were performed for each sample: one with 1 whole capsule

and one with only the filling of 1 whole capsule. Each test item and 20 mg of the internal standard

(TPP) were exactly weighed (see tables “Calculation of content”, Chapter 6 - NMR Spectroscopy),

dissolved in 10 ml CDCl3 / MeOH / Cs-EDTA, aq. (2/4/4) and measured. The integrated signals of

test item and internal standard were used for calculation.

The ratio of integrals per atom corresponds to the molar ratio of the compared substances. For

calculation software Microsoft Excel 14.0 was used.



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Analysis of KO by NMR - phospholipids 2/2



Tab. 17 Calculation of phospholipid-content AKB33483-6_A (1 whole capsule)

TPP Integral Molecular weight mMol Initial weight [mg] Content [%] Number P

Internal Standard 21.64 326.29 0.0647 21.14 99.9 1

Test item 784.51 1

Excel 14.0 Integral Molecular weight mMol Content [mg] Weight-% Mol-%

PC 49.26 790.0 0.1473 116.3797 14.84 62.86

1-LPC 2.20 534.5 0.0066 3.5166 0.45 2.81

2-LPC 16.60 534.5 0.0496 26.5345 3.38 21.18

PI 0.00 907.0 0.0000 0.0000 ---*) ---*)

LPI 0.00 629.5 0.0000 0.0000 ---*) ---*)

PS 0.00 833.0 0.0000 0.0000 ---*) ---*)

LPS 0.00 555.5 0.0000 0.0000 ---*) ---*)

SPH 0.00 812.0 0.0000 0.0000 ---*) ---*)

PE 4.42 770.0 0.0132 10.1782 1.30 5.64

LPE 1.34 492.5 0.0040 1.9736 0.25 1.71

APE 3.14 1032.0 0.0094 9.6909 1.24 4.01

PG 0.00 820.0 0.0000 0.0000 ---*) ---*)

DPG 0.00 774.0 0.0000 0.0000 ---*) ---*)

PA 0.00 746.0 0.0000 0.0000 ---*) ---*)

LPA 0.00 468.5 0.0000 0.0000 ---*) ---*)

Rest 1.40 812.0 0.0042 3.3997 0.43 1.79

Sum 78.36 21.89 100.00

Phosphorus 0.93*) = not observed, no signal assignment Balance R180Comment: Initial weight corresponds to process accuracy of 0.5% with security factor 5. Sartorius R 180 D-*D1

Tab. 18 Calculation of phospholipid-content AKB33483-6_B (filling)

TPP Integral Molecular weight mMol Initial weight [mg] Content [%] Number P

Internal Standard 24.82 326.29 0.0623 20.34 99.9 1

Test item 442.66 1

Excel 14.0 Integral Molecular weight mMol Content [mg] Weight-% Mol-%

PC 47.46 790.0 0.1191 94.0616 21.25 63.13

1-LPC 2.00 534.5 0.0050 2.6819 0.61 2.66

2-LPC 15.98 534.5 0.0401 21.4280 4.84 21.26

PI 0.00 907.0 0.0000 0.0000 ---*) ---*)

LPI 0.00 629.5 0.0000 0.0000 ---*) ---*)

PS 0.00 833.0 0.0000 0.0000 ---*) ---*)

LPS 0.00 555.5 0.0000 0.0000 ---*) ---*)

SPH 0.00 812.0 0.0000 0.0000 ---*) ---*)

PE 4.07 770.0 0.0102 7.8622 1.78 5.41

LPE 1.26 492.5 0.0032 1.5568 0.35 1.68

APE 3.03 1032.0 0.0076 7.8448 1.77 4.03

PG 0.00 820.0 0.0000 0.0000 ---*) ---*)

DPG 0.00 774.0 0.0000 0.0000 ---*) ---*)

PA 0.00 746.0 0.0000 0.0000 ---*) ---*)

LPA 0.00 468.5 0.0000 0.0000 ---*) ---*)

Rest 1.38 812.0 0.0035 2.8112 0.64 1.84

Sum 75.18 31.23 100.00

Phosphorus 1.32

*) = not observed, no signal assignment Balance R180Comment: Initial weight corresponds to process accuracy of 0.5% with security factor 5. Sartorius R 180 D-*D1



part of Aker

Analysis of KO by NMR – proton NMR for total n-3 1/4



Fig. 821H-NMR spectrum of test item AKB33483-6 (CDCl3/MeOD)

Fig. 831H-NMR spectrum of test item AKB33483-6 (CDCl3/DMSO-d6)



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Analysis of KO by NMR – proton NMR for total n-3 2/4

Product

ABCDEFG



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Analysis of KO by NMR – carbon NMR for fatty acids inPL and TG fractions 3/4



Fig. 8413

C-NMR spectrum of test item AKB33483-6 (CDCl3/MeOD)

Fig. 8513

C-NMR spectrum of test item AKB33483-6 (CDCl3/DMSO-d6)



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Analysis of KO by NMR – carbon NMR for fatty acids inPL and TG fractions 4/4



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Analysis of KO by NMR ether PL 1/2



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Agenda





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Analysis of KO by NMR

• Krill oil is a complex mixture of lipids • Krill oil can not be directly compared with regular Triglyceride oils

• Analytical methods needs to be adapted specifically for the KO matrix

• Regular methods for determination of oxidative status of the lipid mixture is not sensitive nor specific enough to be more than marginally useful (e.g. Anisidine or TBARS methods)

• Krill oils will always have more free fatty acids as well as water content than TG, thus are more prone to hydrolysis than TG-oils

• Methods that can distinguish between TG from KO and FO is desirable• Methods for direct (and sensitive enough) measurement of molar amount of

double bounds could be useful to investigate oxidative status and development

• Krill oil standards, both PL and TG are needed

Composition of Antarctic krill oil and methods for its...

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