8th ISCNP, 17-20 May,2012, Lublin 1

Analysis of underivatized fatty acids by LC and charged aerosol detector

Grzegorz KiełbowiczCzesław Wawrzeńczyk

Department of Chemistry, Wrocław University of Environmental and Life Sciences,

O

O

O

O

O

R2

R1

P

O

O-

O CH2CH2N+

CH3

CH3

CH3

O

O

O

O

O

R2

R1

P

O

O-

O CH2CH2N+

H

H

H

O

O

O

O

O

R2

R1

O

R3

O

OH

RO

OH

R

O

OH

R

8th ISCNP, 17-20 May,2012, Lublin 2

Presentation plan

1. Introduction1.1. Detection of fatty acids1.2. Derivatization of fatty acids1.3. Charge aerosol detector (Corona™ CAD™)

2. Method development2.2. ECL (equivalent chain length)2.3. Organic solvent effect2.4. Temperature effect2.5. Programming method 2.6. Validation

3. Application to egg yolk lipids

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1.1. Detection of fatty acids

1. Gas chromatography (GC) Derivatization !!!• FID – the most commonly used detector

2. Liquid chromatography (LC)• UV Derivatization !!!

• Aerosol detectors (ELSD, CAD, CNLSD)• Mass spectrometry• Refractive index (RI)• Electrochemical detectors (ECD) …

e.g. methyl esters

chromophore

Derivatization

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Derivatization

Complication

Labor-intense

Time-consuming

Unwanted oxidation

Unwanted izomerization

Excess reagent orsolvents

1.2. Derivatization of fatty acids

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1.3. Charge aerosol detector (Corona™ CAD™)

http://coronacad.com

• Excellent Sensitivity and Reproducibility

• Consistent Response Response Independent of Chemical

Structure• Wide Dynamic Range• Broad Applicability Analyzes any Non-Volatile Molecule No need for a chromophore or

ionization• Intuitive Operation

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2.1. Equivalent chain length (ECL)

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13 14 15 16 17 18 19 20 210

0.5

1

1.5

2

2.5

Thermo Betasil C18

MeOH 90%/30°CLinear (MeOH 90%/30°C)ACN 90%/30°CLinear (ACN 90%/30°C)MeOH 80%/30°CLinear (MeOH 80%/30°C)ACN 80%/30°C

Carbon number

log

k

• logk of saturated fatty acids is linearly related to the solute carbon number • higher temperature , higher % of organic modifier lower retention

How to determine „real value” of unsaturated fatty acids ECL ?

ECL=(logk-intercept)/slope

1. Plots of logk vs carbon numbers of saturated fatty acids

2. Determination of ECL from the linear regression of the plots

8th ISCNP, 17-20 May,2012, Lublin 8

Fatty acidTheoretical ECL

ECL=N-2nEmpirical ECL

ECL=(logk-intercept)/slope

Docosahexaenoic 22:6 (DHA) 10 13.3

Eikozapentaenoic 20:5 (EPA) 10 12.8

Linolenic 18:3 12 13.2

Arachidonic 20:4 12 14.0

Mirystic 14:0 14 14.0

Palmitooleic 16:1 14 14.0

Linoleic18:2 14 14.5

Palmitic 16:0 16 16.0

Oleic 18:1 16 15.9

Stearic 18:0 18 18.0

Theoretical ECL vs Empirical ECL

Polyunsaturated fatty acids with three or more double bonds do not follow the ECL rule !!!

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75 80 85 900

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

14:0

16:0

16:118:1

18:2

18:3

20:4DHA

EPA

% MeOH

log

k

70 75 80 85 90 95-0.2

-1.66533453693773E-16

0.2

0.4

0.6

0.8

1

1.2

1.4

14:00

16:00

16:01

18:01

18:02

18:03

20:04

DHA

EPA

% ACN

log

k

2.2. Organic solvent effect (1)

1. The increase of the organic solvent composition decreased the polarity of the mobile phase and hence, the capacity factor was linearly decreased

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2. As the solvent strength increases, retention times for the less hydrophobic compounds will decrease faster

Fatty acid Intercept (b)a Slope(a)aRatios of k at 80% and 90% MeOH

14:0 1.69 -0.3271 4.96

16:0 2.11 -0.3611 5.7116:1 1.77 -0.3411 5.14

18:0 2.54 -0.4100 7.7918:1 2.16 -0.3722 6.04

18:2 1.94 -0.3670 5.70

18:3 1.65 -0.3224 5.32

20:0 2.96 -0.4551 8.9920:4 1.86 -0.3584 5.99

20:5 1.61 -0.3181 5.29

22:6 1.83 -0.3517 5.70

Fatty acid Intercept (b)a Slope(a)aRatios of k at 80%

and 90% ACN

14:0 3.01 -0.0295 1.92

16:0 3.69 -0.0342 2.1616:1 3.22 -0.0319 2.06

18:0 4.60 -0.0415 2.4418:1 3.85 -0.0363 2.29

18:2 3.46 -0.0343 2.19

18:3 2.98 -0.0306 2.01

20:0 5.09 -0.0439 2.8220:4 3.47 -0.0356 2.28

20:5 2.99 -0.0316 2.09

22:6 3.34 -0.0350 2.22alog k = a(%ACN) + balog k = a(%MeOH) + b

2.2. Organic solvent effect (2)

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2.2. Organic solvent effect (3)

75 80 85 90 9513

14

15

16

17

16:0118:118:0218:0320:04DHAEPA

MeOH (%)

ECL

70 75 80 85 90 9511

12

13

14

15

16

17

16:01

18:01

18:02

18:03

20:04

DHA

EPA

ACN (%)

ECL

3. The decrease of the organic solvent composition inreased the overall resolution

4. The resolution was greater at low organic solvent composition

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2.3. The effect of temperature (Van’t Hoff equation)

1. The influence of temperature on solute capacity factor (k), is a function of the free energy changes in the interaction between the solute and thestationary phase

System enthalpy System entropy

Universal gas constantAbsolute temperature

System phase ratio

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0.00315 0.0032 0.00325 0.0033 0.00335 0.0034 0.00345 0.0035 0.00355 0.00360

0.5

1

1.5

2

2.5

3

1/T (K-1)

ln k

0.00310 0.00320 0.00330 0.00340 0.00350 0.003600

0.5

11.5

22.5

33.5

44.5

14:0016:0016:0118:0018:0118:0218:0320:0020:04EPADHA

1/T (K-1)

ln k

2.3. The effect of temperature (Van’t Hoff plots)

ACN:H2O 90:10MeOH:H2O 90:10

2. Logk should be linearly ralated to the inverse temperature if the system enthalpy and entropy are invariant

3. Linear behaviour at temperature 10-40°C (R2>0.99)

4. 25°C - „phase transition” of the C18 stationary phase from liquid-like structure to solid-like structure

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10 15 20 25 30 35 4013

14

15

16

17

Temperature (°C)

ECL

10 15 20 25 30 35 4011

12

13

14

15

16

16:0118:0118:0218:0320:04EPADHA

Temperature (°C)

ECL

2.3. The effect of temperture on ECL

5. The ECL was decreased as the temperature was decreased

6. 25°C - optimal temperature - column back pressure vs resolution

MeOH ACN

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2.5. Programming method

Detector: Corona™ CAD™Column: Thermo Betasil C18Mobile phase: ACN: Buffer (1%HCOOH, 0,1%TEA)Flow: 1mL/minTemperature: 25°CSegmented gradient elution

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Compound Range (µg) Power model(y=axb)

S (mV/µg)(Abxb-1)a

LOD (ng)(3.3 x σ/S)b

LOQ (ng)(10 x σ/S)b

Equation R2

1 Ricinoleic acid 0.018-4.48 5.4967(±0.01)x1.0526 (±0.01) 0.9985 4.7 11.0 33.0

2 Eicosapentaenoic acid (EPA) (20:5) 0.018-4.54 17.177(±0.06)x0.9354(±0.00) 0.9984 20.8 10.2 30.8

3 Linolenic acid (18:3) 0.017-4.18 17.140(±0.01)x0.9326(±0.01) 0.9974 21.0 1.5 4.6

4 Docosahexaenoic acid (DHA) (22:6) 0.019-4.93 20.600(±0.07)x0.9533(±0.00) 0.9985 23.6 9.7 29.3

5 Palmitoleic acid (16:1) 0.076-3.82 12.668(±0.07)x0.8096 (±0.00) 0.9941 16.7 13.6 41.1

6 Arachidonic acid(20:4) 0.018-4.57 22.615(±0.05)x0.9201 (±0.00) 0.9989 28.7 6.1 18.6

7 Linoleic acid(18:2) 0.017-4.21 19.868(±0.11)x0.9204 (±0.00) 0.9956 25.3 14.8 44.9

8 Palmitic acid(16:0) 0.015-3.85 15.246(±0.03)x1.0475 (±0.00) 0.9989 13.1 6.8 20.6

9 Oleic acid(18:1) 0.017-4.24 19.073(±0.01)x1.0794 (±0.00) 0.9978 14.9 1.85 5.6

10 Stearic acid(18:0) 0.017-4.27 30.532(±0.07)x1.0608 (±0.00) 0.9975 25.3 9.4 28.5

2.6. Validation (1) (Linearity, Sensitivity, LOD, LOQ)

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Compound Amount(µg)

Peak area(mV/min)

Retention time(min)

(µmol) Mean SD RSD% Mean SD RSD%

Ricinoleic acid 1.79 (0.006) 10.389 0.00 0.04 6.684 0.01 0.17

Eicosapentaenoic acid (EPA) (20:5) 1.81 (0.006) 30.094 0.14 0.48 11.897 0.01 0.15

Linolenic acid(18:3) 1.67 (0.006) 27.829 0.05 0.17 12.431 0.02 0.14

Docosahexaenoic acid (DHA)(22:6) 1.97 (0.006) 39.735 0.05 0.13 14.071 0.022 0.16

Palmitoleic acid (16:1) 1.53 (0.006) 18.021 0.09 0.48 14.451 0.018 0.13

Arachidonic acid(20:4) 1.83 (0.006) 39.591 0.08 0.20 15.082 0.020 0.13

Linoleic acid(18:2) 1.68 (0.006) 31.781 0.02 0.07 15.934 0.02 0.13

Palmitic acid(16:0) 1.54 (0.006) 25.309 0.07 0.29 19.141 0.00 0.03

Oleic acid(18:1) 1.69 (0.006) 36.856 0.17 0.48 19.633 0.00 0.03

Stearic acid(18:0) 1.71 (0.006) 57.350 0.19 0.33 21.395 0.00 0.03

2.6. Validation (2) (Repeatability of CAD response and retention times)

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3. Application to egg yolk lipids

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FA TAG (%)

SD %RSD PE (%)

SD %RSD PC (%)

SD %RSD

Myristic acid(14:0) 0.45 0.02 4.44 - - - - - -

Eicosapentaenoic acid (EPA) (20:5) - - - - - - - - -

Linolenic acid(18:3) 1.02 0.04 3.72 - - - - - -

Docosahexaenoic acid (DHA) (22:6) 0.1 0.01 5.97 6.96 0.17 2.44 2.24 0.14 6.37

Palmitoleic acid (16:1) 5.98 0.02 0.35 - - - 1.2 0.09 7.26

Arachidonic acid(20:4) 0.38 0.01 2.63 14.86 0.18 1.24 5.13 0.14 2.83

Linoleic acid(18:2) 17.43 0.20 1.16 14.2 0.19 1.34 15.4 0.04 0.23

Palmitic acid(16:0) 24.09 0.13 0.52 17.52 0.66 3.75 33.64 0.43 1.27

Oleic acid(18:1) 46.24 0.15 0.32 18.37 0.68 3.69 27.14 0.80 2.96

Stearic acid(18:0) 4.31 0.16 3.62 27.82 1.52 5.47 15.25 0.39 2.59

Egg yolk fatty acids profile

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Thank you for your attention !