searching and finding unusual fatty acids and compounds of ... · anteiso-fatty acids acids •...

Walter Vetter

Universität Hohenheim Institut für Lebensmittelchemie

D-70593 Stuttgart

Searching and finding

unusual fatty acids and compounds

of the unsaponifiable matter

AK Vetter

stable isotope mass spectrometry

(IRMS)

halogenatednatural producs

polyhalogenated compounds

lipid analysis

enantiomer separations

methyl-branched fatty acids

food authenticy

countercurrent chromatography

GC/MS

halogenated flame

retardants

furan fatty acids

unsaponifi-able matter

standard compounds

[min] 20 25 30 35

16:0 18:0

18:1n-9 18:2n-6

18:3n-3 Irel

sesame oil

16:0 18:0

18:1n-9 18:2n-6

Irel

[min] 20 25 30 35

18:3n-3

14:0

18:0

18

:1n-

9

18:4

n-3

DHA

18:3

isom

ers

16:4

18

:1n-

7

20:3

n-3

18 22 26 30 [min]

20:3

n-6

Irel EPA 16:0 fish oil

15:0

12:0 10:0

16:0

a15:0

14:0

18:0

18:1n-9 16:1n-7 a17:0

16 18 20 22 24 26 28 30 [min]

Irel

milk fat

16:1

n-7

linseed oil

Gas chromatograms of fatty acids (as methyl esters) in food samples

anteiso-fatty acids acids

• methyl substitutent at (n-2) carbon ⇒ anteiso-fatty acid

• typical chain length: C14 and C16, odd carbon number (due to methyl group)

• usually found in ruminants and fish (barely in plants)

• usually occurring together with iso-fatty acids

(methyl-branch on second last carbon)

13-methyl tetradecanoic acid (i15:0) COOH

COOH 12-methyl tetradecanoic acid (a15:0)

Bacterial lipids

• anteiso- and iso-fatty acids dominate (in gram-positive bacteria)

⇒ up to 80% contribution to the total fatty acids

• occurrence of anteiso-fatty acids in food is mostly linked with the presence of bacteria

⇒ in gnotobiologic (germ-free) rats only present at traces [1]

[1] Y. Demarne, E. Sacquet, M. J. Lecourtier, J. Flanzy. Am. J. Clin. Nutr. 32 (1979) 2027-2032

GC/MS analysis of the fatty acids of Streptomyces avermitilis (Thurnhofer & Vetter)

16 17 18 19 20 21 [min]

i15:0 a15:0

i16:0

16:0 i17:0

a17:0

i14:0 14:0 15:0 17:0 18:0

Irel

Stable isotope analysis: GC-IRMS method

• eluate from the GC column led to combustion unit • organic carbon is transferred into CO2 • exact determination of the 13C/12C ratio is difficult

⇒ instead, measurement relative to reference standard

[13C/12C]sample - [13C/12C]standard δ13C [‰] = ——————————————— x 1000

[13C/12C]standard

• typical δ13C values in the range -10 to -40‰

Ref.: W. G. Mook WG (ed) (2000) Environmental isotopes in the hydrological cycle, principles and applications, Vol I: Introduction: theory, methods, review. UNESCO/IAEA, Vienna/Paris

δ13C values [‰] of fatty acids in suet

fatty acid* δ13C value [‰]

12:0 -26.6

14:0 -27.2

i15:0 -36.7

a15:0 -35.9

15:0 -30.5

i16:0 -37.6

16:0 -27.1

i17:0 -36.2

a17:0 -35.5

δ13C values verify:

• methyl-branched fatty acids

are depleted in 13C

compared to

straight-chain fatty acids

⇒ different sources!

Ref.: W. Vetter, S. Gaul, S. Thurnhofer, K. Mayer, Anal. Bioanal. Chem. 389 (2007) 597-604

* measured as methyl ester

Properties of methyl- branched fatty acids

• inertness towards oxidation

• low melting point

• changing physiological properties of lipids

⇒ increase of the membrane fluidity

• positive effect on the penetration

of other compounds into the skin

COOH

COOH

1,2-dimyristoyl-sn-glycero- 3-phosphatidylcholine (DMPC)

DMPC i15:0

pure

DMPC DMPC with

3 mol% i15:0

5 mol% i15:0

Ref.: F. Lindström, S. Thurnhofer, W. Vetter, G. Gröbner, Phys. Chem. Chem. Phys. 8 (2006) 4792-4797

Differential scanning calorimetry (DSC): DSC thermograms of lipids

A thought during a student lecture

• amino acids – building blocks of proteins – are chiral

(typically L-form)

• sugars – building blocks of carbohydrates – are chiral

(typically D-form)

• fatty acids – building blocks of lipids – are non-chiral

⇒ one notable exception: anteiso-fatty acids

Chirality of anteiso-fatty acids

• 1950ies: one-time investigation of suet and shark liver oils [1,2]

⇒ ~ 10 kg fat/oil converted into methyl esters

⇒ fractionation via distillation

⇒ repeated crystallization

⇒ x-ray analysis, optical rotation

result:

• all samples exclusively featured the (+)-enantiomer

Ref.: [1] R. P. Hansen, F. B. Shorland, N. J. Cooke, Biochem. J. 52 (1952) 203-207 [2] I. M. Morice, F. B. Shorland, Biochem. J. 64 (1956) 461-464

S-a15:0

R-a15:0

COOH

COOH

enantiopure standards not commercially available

GC enantiomer separation of anteiso-fatty acids

• not reported when we started

• only of α-, β- and γ-methyl-

substituted acids resolved [1][2]

• the more remote the methyl group

from the head group, the more difficult the enantiomer separation is [1][2]

[1] V. Karl, J. Gutser, A. Dietrich, B. Maas, A. Mosandl, Chirality 6 (1994) 427-434 [2] R. Stritzel, B. Dobner, F. Bringezu, P. Nuhn, J. High Res. Chrom. 19 (1996) 121-123

HOOC

HOOC

HOOC

HOOC

α-methyl

β-methyl

γ-methyl

10 carbons

Small problems had to be solved…

• synthesis of enantiopure standards [1]

⇒ Wittig reaction (olefination of chiral aldehydes via ylides)

⇒ hydrogenation of the resulting double bond

• searching for a chiral stationary GC phase [2]

⇒ testing of ~20 chiral stationary phases

⇒ improvement of the only promising one

• development of sensitive and selective method [3]

⇒ development of a GC/MS-SIM method

⇒ enrichment/isolation of anteiso-fatty acids by hydrogenation,

urea complexation and/or (Ag+) HPLC fractionation

1 year work

1 year work

1 year work

Ref.: [1] S. Thurnhofer, W. Vetter, Tetrahedron 63 (2007) 1140-1145

[2] S. Thurnhofer, G. Hottinger, W. Vetter, Anal. Chem. 79 (2007) 4696-4701

[3] S. Thurnhofer, W. Vetter, J. Agric. Food Chem. 53 (2005) 8896-8903

Fatty acid analysis

• important routine task in food science (and life sciences)

• classic method using GC/FID after formation of fatty acid methyl esters (FAME)

⇒ peak abundance correlates with amount

⇒ determination of relative contributions (“100% method“)

disadvantages of GC/FID • low selectivity

• co-elutions may be overlooked

• problems with low abundant fatty acids GC: gas chromatography

FID: flame ionisation detector

Coelution of monoenoic and anteiso-fatty acids

• co-elutions can hardly be omitted on 50 m columns

*

19.6 20.0 20.4

I rel

a17:0

[min]

17:0

i17:0 16:1n-7

17:0-ME: M+ = m/ z 284

16:1-ME: M+ = m/ z 268

(GC column: 50 m x 0.25 mm i.d. x 0.2 µm 100% cyanopropyl polysiloxane)

GC/MS chromatogram of a milk fat sample

Why GC/MS in SIM mode?

• more sensitive and selective than full scan

19.6 20.0 20.4 [min]

Irel

19.6 20.0 20.4 [min]

Irel

17:0

a17:0

i17:0

M+

m/ z 284 measured in SIM mode

17:0

a17:0

i17:0

m/ z 284 extracted from full scan

Ref.: S. Thurnhofer, W. Vetter, J. Agric. Food Chem. 53 (2005) 8896-8903

(selected ion monitoring (SIM))

GC column: 50 m x 0.25 mm i.d. x 0.2 µm 100% cyanopropyl polysiloxane

Determination of fatty acid methyl esters by GC/MS-SIM

• sensitivity and selectivity

Ref:. W. Vetter, S. Thurnhofer,, Lipid Technol. 19 (2007) 184-186

0

10

20

30

[%]

m/z 74 m/z 87 m/z 79 m/z 81

saturated fatty acids (n=20)

monoenoic fatty acids (n=10)

dienoic fatty acids (n=4)

tri- to hexaenoic fatty acids (n=6)

GC/MS-SIM

GC/FID

Quantitative determination of individual fatty acids

• quantification requires use of internal standards (IS) not present in the sample

Ref:. S. Thurnhofer, K. Lehnert, W. Vetter, Eur. Food Res. Technol. 226 (2008) 975-983

(1) IS for sample cleanup

(addition before/after the extraction)

(2) syringe standard

(addition to GC/MS solution)

COOH

Cl

Cl

DC-11:0 14:0-EE

C

O

O

House method

lipid extraction using accelerated solvent extraction (ASE) for dry samples

or

microwave-assisted extraction (MAE) of aqueous samples

transesterification

GC/MS-SIM analysis

IS, ethyl ester

IS, DC-11:0


Microwave-assisted extraction (focused-open vessel; FOV-MAE)

• connection tool with water trap and nitrogen inlet

extraction flask

water trap

N2-inlet

reflux cooler

no commercial system, self-constructed, base instrument for acid digestions

Concentrations [g/100 g fat] of methyl-branched fatty acids in food

FAME (n = 3)

mozzarella (cow)

[g/100 g]

feta (cow)

[g/100 g]

feta cheese

[g/100 g]

human milk

[g/100 g]

i14:0 0.08 ± 0.00 0.08 ± 0.00 0.08 ± 0.00 0.02 ± 0.00

i15:0 0.20 ± 0.01 0.29 ± 0.01 0.21 ± 0.01 0.06 ± 0.00

a15:0 0.30 ± 0.01 0.38 ± 0.01 0.34 ± 0.03 0.08 ± 0.00

i16:0 0.12 ± 0.00 0.09 ± 0.01 0.07 ± 0.01 0.03 ± 0.00

i17:0 0.47 ± 0.01 0.56 ± 0.02 0.37 ± 0.02 0.11 ± 0.02

a17:0 0.39 ± 0.01 0.42 ± 0.02 0.37 ± 0.01 0.14 ± 0.00

• only these fatty acids were quantified using the corresponding ethyl esters as IS


Birth goo (Vernix caseosa)

1.14

1.16

1.18

1.20

1.22

1.24

1.26

1.28

1.30

log tR

12 13 14 15 16 17 18

Longest carbon chain

• detection of ~60 methyl-branched fatty acids (although germ-free)

Ref.: S. Hauff, W. Vetter, J. Chromatogr. A 1217 (2010) 8270—8278

The AOCS Lipid Library is of exceptional help, it´s our first aid kit when there are problems

Direct GC enantiomer separation of anteiso-fatty acids

• application of modified cyclodextrins

β-cyclodextrin (7 glucose units)

γ-cyclodextrin (8 glucose units)

α-cyclodextrin (6 glucose units)

• O-derivatisation at C-2, C-3 and C-6 provides a range of modified cyclodextrins suitable as chiral stationary GC phases

6-O-tert.-butyldimethylsilyl-2,3-di-O-methyl-β-cyclodextrin (β-TBDM)

• tests of >20 chiral stationary phases only partly successful with β-TBDM ⇒ improvement of the initial phase (collaboration with G. Hottinger, BGB-Analytik)

10% β-TBDM 50% β-TBDM thin film standard film β-TBDM:

R1= R2 = methyl

• lowers elutions temperature

• resolution decreases

• better interaction

• better resolution

Ref: S. Thurnhofer, G. Hottinger, W. Vetter, Anal. Chem. 79 (2007) 4696-4701

Enantioselective determination of a17:0 on β-TBDM

racemates spiked with S-enantiomer

elution order: R < S

pure S-enantiomer

⇒ required GC run time 8–10 h elution time

460 480 500 520 540 560 580 [min]

TIC: C10355ST.D

Irel

460 480 500 520 540 560 580 [min]

TIC: C10353ST.D

Irel

460 480 500 520 540 560 580 [min]

TIC: C10351ST.D

Irel


racemates partly resolved

a17:0-ME a18:0-ME

R S

S

R S

S

Sample preparing for enantiomer separation: (1) urea complexation

(2) silver ion chromatography

urea complexation

⇒ separation of major saturated fatty acids

Ag+-HPLC

⇒ separation of unsaturated fatty acids

12:0

i14:0

14:0

a15:0

i15:0 i16:0 16:0

i17:0 a17:0

13:0

branched-chain fatty acids enriched from milk fat

15:0

15.0 16.0 17.0 18.0 19.0 [min]

Irel


Results: Chirality of anteiso-fatty acids

• anteiso-fatty acids are predominantly S-configurated in milk fat and fish oil

(S. Thurnhofer, G. Hottinger, W. Vetter, Anal. Chem. 79 (2007) 4696-4701)

• yet, up to 10% R-anteiso-fatty acids detected in milk fat and fish

• higher share of R-anteiso fatty acids in polar lipids

(S. Hauff, G. Hottinger, W. Vetter, Lipids (2010) 357-365

• R-anteiso-fatty acids cannot be synthesized by the classical

biosynthesis via isoleucine as the primer

⇒ a hitherto mostly unknown biosynthesis pathway must exist

(D. Eibler, H. Abdurahman, T, Ruoff, S. Kaffarnik, H. Steingass,

W. Vetter, PLOS One 12 (2017) e0170788)

Two dimensional HPLC/GC chromatogram of fish oil fatty acids (as methyl esters)

• excel-programmed 2D evaluation (T. Kapp, W. Vetter, J. Chromatogr. A 1216 (2009) 8391-8397) • similar approach as

GCxGC disadvantage • time consuming (~2 days per sample)

advantage • good orthogonality • higher amounts (post

analysis possible)

Ref.: S. Hauff, W. Vetter, Anal. Bioanal. Chem. 396 (2010) 2695-2707

Retention time GC/EI-MS [min]15 20 25 30 35

HPLC

frac

tion n

umbe

r

20

25

30

35

40

45

50

55

600.0 0.1 0.2 0.3 0.4 >0.5

18:1

18:0

20:1

20:5 21:5n-3

18:3

20:2

18:2

18:5

19:1

20:3

18:4 12:0

20:4

22:6

22:5

22:1

C16

C18 C20 C22

16:2 16:3

16:4

16:1

16:0

penta- tetra-

tri-

di-

mono- saturated

HPLC/GC - 2D Konturplot Ref.: S. H

auff, W. Vetter, Anal. Bioanal.

Chem. 396 (2010) 2695-2707

14:0

retention time GC/EI-MS [min]

PUFAs in the HPLC/GC plot of a fish oil

• many, many PUFAs 18

20

22

24

26

28

30

32

34

20 22 24 26 28 30 32

0.0 0.1 0.2 0.3 0.4 >0.5

16:4 isomers

16:3 isomers

16:2 isomers

16:1 isomers

18:4 isomers

18:3 isomers

18:2 isomers

18:5n-3

17:1n-87-Me-16:1n-10

20:5n-3

20:4 isomers

20:3 isomers

21:5n-3

22:6n-3

22:5 isomers

22:4 isomers

Ret

entio

n tim

e G

C/E

I-M

S [m

in]

HPLC fraction number

A

Ref.: S. Hauff, W. Vetter, Anal. Bioanal. Chem. 396 (2010) 2695-2707

• non-aqueous RP-HPLC with three C18 columns

HPLC/GC plots of branched chain fatty acids

16 17 18 19 20 2125

30

35

40

450.00 0.05 0.10 0.15 >0.20

4,8,12-triMe-13:0

i15:0 a15:0

i16:0

pristanic acid

i17:0

i18:0

phytanic acid

a17:0

7-Me-16:1n-10

16 17 18 19 20 21

45

40

35

30

25

HP

LC f

ract

ion

num

ber

*

Irel

Retention time GC/EI-MS [min] 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0

15:0 i16:0

a16:0

16.0 20.5 21.0

• HPLC fractionation allowed to detect traces of a16:0

• most likely produced by α-oxidation of a17:0

parameter organic milk*

phytanic acid + PUFA (ALA, EPA) +

CLA +

furan fatty acids +

“+“ higher content in organic milk than in conventional milk due to green feed

Valuable minor fatty acids in milk

• structural feature: furan moiety in the carbon chain

• substituted with one (“M“) or two (“D“) methyl groups

• short terms: 9M5 9D5

• “D“-furan fatty acids are more widespread but much less stable

⇒ partly absent in processed or stored samples

• excellent antioxidants (very effective protectors of PUFAs)

• degradation products responsible for off flavor of soy (among other)

Features of furan fatty acids

Methylfuran Dimethylfuran

1 3 5 7 9

1 3 5 1 3 5

1 3 5 7 9

5 9

M

D

5 9

M

Ref.: G. Spiteller, Lipids 40 (2005) 755-771

0

2

4

6

8

10

12

14

16

18

winterconventional

milk fat

winterorganicmilk fat

summerconventional

milk fat

summerorganicmilk fat

fish,raw

olive oil rapeseed oil soybean oil,refined

fura

n ac

id a

cid

inta

ke p

er c

apita

& d

ay

[mg]

Calculated daily intake of furan fatty acids

per capita [mg], in Germany

Ref.: C. Wendlinger, W. Vetter, J. Agric. Food Chem. 62 (2014) 8740-8744

• minor fatty acids, low concentrations (typically <0.1% contribution to the total fatty acids

• but: widely spread in virtually all plants and animals

• discovered, studied in the 1970s (R.L. Glass, H. Schlenk, F.D. Gunstone)

• mostly forgotten, studied again ~1985-1995 (G. Spiteller, W. Grosch)

• almost forgotten since the 2000s

• no reference standards commercially available

⇒ research only possible with house-prepared standards

Furan fatty acids

Compound isolation using countercurrent chromatography (CCC)

• CCC is a well-established method in natural product isolation [1][2]

• all liquid based chromatographic method (no solid support) [3]

• allows injection and isolation of gram-amounts of analytes [3]

• barely used in field of lipid compounds [1]

Ref.: [1] J. B. Friesen, J. B. McAlpine, S.-N. Chen, G. F. Pauli, J. Nat. Prod. 78 (2015) 1765-1796 [2] I. A. Sutherland, D. Fisher, J. Chromatogr. A 1216 (2009) 740-753 [3] Y. Ito, J. Chromatogr. A 1065 (2005)145-168

https://www.scopus.com/authid/detail.uri?origin=resultslist&authorId=7004413652&zone=

Applications of countercurrent chromatography (CCC) according to

SciFinder (1981-2015)

• currently >300 CCC papers/year, number is increasing

• ~3% in the field of lipid compounds

CCC instrumentation • CCC systems same setup as HPLC

instruments except the column

⇒ instead: CCC centrifuge with

multilayer coils

mobile phase

fraction collector

pump injection valve

stationary phase

CCC centrifuge with multilayer

bobbins

detector

centrifuge

CCC method development

• CCC is different to liquid-liquid extractions as it aims to distribute the analyte evenly between both phases

⇒ determination of the partitioning factor

KU/L = ≈ 0.4 – 2.5

• even distribution: KU/L = 1; acceptable range: KU/L = 0.4 – 2.5

⇒ the goal challenge is to find a biphasic solvent system in which the analytes are ~ evenly distributed (and resolved)

[concentration in upper phase]

[concentration in lower phase]

⇒ see tutorial on CCC in the AOCS Lipid Library

Isolation of the valuable furan fatty acid 11D5

[min] 10.0 18.0 26.0

11D5-EE

Irel

10.0 18.0 26.0 [min]

Irel

26.6

Irel

11D5-EE

DPA-EE

DHA-EE

27.4 [min]

CCC injection: 1 g

yield: 19 mg 11D5

purity: 99%

solvent consumption: 100 mL

time per mg 11D5: 3.4 min

Ref: M. Müller, K. Wasmer, W. Vetter, J. Chromatogr. A (2018), in press (DOI: 10.1016/j.chroma.2018.04.069)

• excellent antioxidant

• no standards available

• limited research

11-(3,4-dimethyl-5-pentylfuran-2-yl)-undecanoic acid (11D5)

CCC isolation of fatty acid methyl esters

hexane/methanol/water (350/175/2) for fatty acid methyl esters (FAME) Irel

26 [min] 10 14 18 22

14:0

16:0

16:1n-7

18:0

18:1n-9

20:5n-3

22:6n-3

10 14 18 22

14:0

16:0

18:4n-3 *

35 [min] 10 15 20 25 30

methyl ester

of 16:4n-1

Irel

OMe

O

1 6 9 16 15 12

Ref.: D. Li, M. Schröder, W. Vetter, Chromatographia 75 (2012) 1-6

• equivalent chain length (ECL) rule:

⇒ 2 carbons ~ 1 DB

• no problem with 16:4 (ECL: 12:2 / 10:1 / 8:0)

fish oil (FAMEs)

terminal DB

CCC

Sample fractionation and analyte isolation via countercurrent chromatography (CCC)

1. isolation of lipid compounds for use as standards/in biotests

• examples: - isolation of uncommon fatty acids

- isolation of phytosterols, tocopherols, carotenes etc.

2. fractionation of lipids or lipid fractions by CCC

• detection of minor compounds usually “invisible“ without fractionation

• examples: - detection of 430 fatty acids in one butter sample

- discovery of aromatic fatty acids in milk fat

Detailed analysis of a butter sample

21 g butter

16.9 g butter oil

17.1 g FAME

CCC #1 41 fractions

à 5 mL

urea complexation

4.7 g residue 0.2 g filtrate

CCC #2 31 fractions

à 7 mL

0.2 g 5 g

• CCC #1 from FAME fraction (major fatty acids) • CCC #2 from filtrate of urea

complexation (rare trace fatty acids) • 430 different fatty acids • >100 PUFAs

• several rare fatty acids

Ref.: M. Schröder, W. Vetter, J. Am. Oil Chem. Soc. 90 (2013) 771-790

Detailed analysis of a butter sample

50 70 90 110 130 150 170 m/z

Irel

55

59

74

83

87

98 101

111

129 136

O

O

CH3O

185 [M-1]+

155 [M-31]+

158 [M-28]+

143 [M-43]+

10 15 20 25 30 35 [min]

8:0

9:0

10:0 10:1

12:1 12:2 9-oxo-9:0

Irel

O

H O

O

HO

H O O

O

HO

O

9 10

11

• potential formation from oleic acid (lipid oxidation)


Aromatic fatty acids in butter O

OCH3

60 80 100 120 140 160 180 200 220

91

105 183

119 135 220 151 164

Irel

M+

188

[M-32]+ 92

O

OCH3

60 80 100 120 140 160 180 200 220 240

91

74

216 248 87

104

92

Irel

M+ [M-32]+

O

OCH3

60 80 100 120 140 160 180 200 220 240 260 280 0

91

74 258

290 87

92

104 M+ [M-32]+

Irel

65

65

91

Ph-9:0

Ph-12:0

16 18 20 22 24 26 28 30

Ph-12:0

Ph-13:0

m/z 91

Ph-3:0

Ph-11:0

Ph-10:0 Ph-8:0

Ph-9:0

Irel

[min]

• previously not known to occur in milk (and other food)

• potential formation from phenylalanine as the primer


Cyclic fatty acids

60 80 100 120 140 160 180 200 220 240 260 280 300 320 m/z

74 87

143 199 310

83

M+

Irel

60 80 100 120 140 160 180 200 220 240 260 m/z

57

74 87

94 143

268 83 M+

Irel

O

O

83 74

87

H+

O

O 83 74

87

H+

60 80 100 120 140 160 180 200 220 240 260 m/z

55 74

87

97 111 129

143

157 199

239 282

83 M+ [M-43]+

Irel O

O

83 74

87

H+

• previously known to occur in milk

• potential formation with aromatic fatty acids by hydrogenation?

M. Schröder, W. Vetter, J. Am. Oil Chem. Soc. 90 (2013) 771-790

Very nonpolar lipid compounds

squalane tricaprylin

tripalmitin (PPP)

cholesteryl stearate (18:0-CE)

sitosterol

• except sitosterol no polar groups

(-OH)

log KOW~21

log KOW~10

log KOW~15

log KOW~9.7

log KOW~14.6

• log KOW 7 -20

• main interest in this study: log KOW >15

Introduction of benzotrifluoride as modifier in solvent systems

• bridging solvents between the biphasic system [1]

Ref.: [1] M. Englert, S. Hammann, W. Vetter, J. Chromatogr. A 1388 (2015) 119-125

100

100

10

20

30

40

50

60

70

80

90

80

70

60

50

40

30

20

10

0 0 10 20 30 40 50 60 70 80 90 100

0

90

benzotrifluoride

tie line

ternary phase diagram

32.5

17.5

50

• well suited composition and properties:

• Hex/ACN/BTF: 10 / 6.5 / 3.5

• settling time: < 20 sec

Isolation of carotenoids from carrot juice using hexane / acetonitrile / benzotrifluoride (BTF)

0 20 40 60 80 100 120 140 160 180 200

abso

rban

ce (

450

nm)

time [min]

β-carotene

lutein α-carotene

tail-to-head (140 min) head-to-tail (60 min)

O H

O H

Irel

α-carotene

32 mg

purity ≥95%

lutein

4 mg

purity ≥97%

4 8 12 16 20 24 28 32 [min] 0

β-carotene

51 mg

purity ≥95%

CCC/vis-chromatogram

Irel

Irel

4 8 12 16 20 24 28 32 [min] 0

HPLC/UV chromatograms (450 nm)

β-carotene

α-carotene

lutein

dual mode

CCC cut: 95-105 min

CCC cut: 115-130 min

CCC cut: 160-172 min

Ref.: M. Englert, S. Hammann, W. Vetter, J. Chromatogr. A 1388 (2015) 119-125

100 mg injected

Difference between “Hex / ACN“ and the “Hex / ACN / BTF“ solvent system

Phase composition of solvent systems determined by GC/FID [1]

solvent system lower upper

hexane / ACN

[1]

1.2 / 98.8

99.5 / 0.5

hexane / ACN / BTF

[2] 29.0 / 56.7 / 14.3

76.0 / 12.5 / 11.6

Ref.: [1] M. Englert, W. Vetter, J. Chromatogr. A 1342 (2014) 54–62 [2] M. Englert, S. Hammann, W. Vetter, J. Chromatogr. A 1388 (2015) 119-125

equal distribution partitions stronger into lower phase

• >1/4th hexane partitions into lower phase • difference in polarity decreased

KU/L of lipid compounds in the BTF system (Hex/ACN/BTF, 10:6.5:3.5)

Ref.: M. Englert, W. Vetter, J. Chromatogr. A 1342 (2014) 54–62

lipid compound log KOW

HEMWAT -7 KU/L

BTF KU/L

oleic acid 7.7 29 0.57

oleic acid methyl ester 7.5 41 2.4

sitosterol 9.7 16 2.2

squalane 14.6 550 62

cholesteryl stearate 15 1260 80

tripalmitin (PPP) 21 1490 60

excellent for FAMEs, sterols, tocopherols

BTF system in co-current* CCC mode

* introduced by Sutherland et al., theory by Berthod et al.

• both phases are moved

• accelerates elution of

analytes with high KU/L

• exponential increase

leads to co-elution of

analytes with high KU/L,

even if ∆KU/L is high

Ref.: S. Hammann, M. Englert, M. Müller, W. Vetter, Anal. Bioanal. Chem. 407 (2015) 9010-9027

0

5

10

15

20

25

30

0 20 40 60 80 100 120 140 160

3 m

L/m

in

part

ition

coe

ffici

ent

K U/L

CCC retention time [min]

curves: flow of “stationary” phase [mL/min]

mobile phase: 4 mL/min

Co-current CCC mode with the BTF system

0,0

0,2

0,4

0,6

0,8

1,0

1,2

10 20 30 40 50 60 70

campesterol

β-sitosterol

dicaprylin

tricaprylin

FAMEs free fatty acids

tripalmitin cholesteryl stearate

squalane

• elution of lipid compounds spreading from log KOW 3 – 30 within acceptable run time

• no separation of extremely nonpolar lipid compounds

elution range of triacylglycerols

standard mix


ELSD

sig

nal 0

0,2

0,4

0,6

0,8

0 10 20 30 40 50 60 70 80 90

ELSD

sig

nal


triacylglycerols

steryl esters

diacylglycerols

fatty acids phytosterols tocopherols sesamin

sesamolin

sesame oil

Co-current CCC mode with the BTF system

Ref.: S. Hammann, M. Englert, M. Müller, W. Vetter, Anal. Bioanal. Chem. 407 (2015) 9010-9027

10

15

20

25

30

35

40

45

50

10 15 20 25 30 35 40 45 50

CCC

rete

ntio

n tim

e [m

in]

GC retention time [min]

steryl esters

steryl ferulic acid esters

triacylglycerols

diacylglycerols

sterols

steradienes

tocotrienols

tocopherols

101520253035404550

8 18 28 38 48

CCC

rete

ntio

n tim

e [m

in]

GC retention time [min]

triacylglycerols

diacylglycerols

phytosterols

squalene

free fatty acids

bubble blot of the co-current CCC separation of 0.5 g rice bran oil Ref.: S. Hammann, A. Kröpfl, W. Vetter, J. Chromatogr. A 1476 (2016) 77-87

CCC isolation of vitamin E compounds

Ref.: M. Müller, S. Hammann, W. Vetter, Food Chem. 256 (2018) 327-332

Abso

rban

ce

0

20

60

100

140

[min] 25.0 50.0

𝛿𝛿-T3

𝛾𝛾-T3

𝛼𝛼-T3

𝛼𝛼-T1

𝛼𝛼-T

8.0 10.0 12.0 14.0 16.0 18.0 20.0 [min]

Irel

10.0 15.0 20.0 25.0 30.0 35.0 [min]

Irel

10.0 15.0 20.0 25.0 30.0 35.0 [min]

α-T3 Irel

10.0 15.0 20.0 25.0 30.0 35.0 [min]

Irel

γ-T3

δ-T3

α-tocomonoenol

O

HO

R1

R2

• dietary supplementary capsule made from palm oil

• each isolated at 10-65 mg/run (purity 99%)

Tocotrienol artefacts

17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 t [min]

Irel Fr 6 α-cholestane (ISTD

δ-T3 δ-T3

δ-T3

δ-T3

δ-T3

δ-T3+γ-T4

δ-T3

δ-T3

δ-T3+γ-T4

γ-T4 γ-T3

17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 t [min]

Irel Fr 16

γ-T3 γ-T3

γ-T3

γ-T3

γ-T3

γ-T3 γ-T3

γ-T3

γ-T3

γ-T3

γ-T3

γ-T3

γ-T3

γ-T3 γ-T3 γ-T3+α-T3 α-T3

17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 t [min]

Irel Fr 25

δ-T

γ-T1

α-T3

α-T3

α-T3

α-T3 α-T3

α-T3

α-T3

α-T3

α-T3

α-T3

α-T3

α-T3 α-T3 α-T3 α-T3 α-T3

α-cholestane (ISTD

α-cholestane (ISTD

Ref.: S. Hammann, A. Kröpfl, W. Vetter, J. Chromatogr. A 1476 (2016) 77-87

• 170 non-natural vitamin E compounds in a palm-oil based dietary supplementary oil

• ~80 tocotrienol isomers • tocotetra- & pentaenols • …

• formed during

inadequate sample processing

CCC-UV

Summary

• analysis of minor lipid compounds is a fascination and varied research field

• the actual relevance of minor fatty acids may currently be underrated

• unavailability of standards frequently hampers progress in the field (no standard = no research = no knowledge)

• lipid standards can be isolated by countercurrent chromatography

• our work is a mixture of basic research and applications

• and sometimes … … it´s like a road movie (the journey is the goal)

Thousand thanks!

• to my former and previous ph. d. students, master and bachelor students, especially those pictured in this presentation (people first)!

• to our research partners here, there and everywhere!

• to our funders (without money, no research)!

• to Analytical Division of AOCS for honoring our research with the Herbert J. Dutton Award

• to Dr. Perluigi Delmonte, US FDA, for inviting me to the AOCS meeting in Minneapolis

• to YOU for your attention

searching and finding unusual fatty acids and compounds of ... · anteiso-fatty acids acids •...

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