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A A R H U S U N I V E R S I T E T

Faculty of Agricultural Sciences

Analysis of fatty acidcomposition of milk

Method improvementand validation

Mette Krogh LarsenDept. of Food Science

ICH Validation Guidelines

SpecificityLinearityRangeAccuracyPrecisionDetection limitQuantitation limitRobustnessSystem suitability testing

Current analysis

Originally published 1959 (Folch)Fat extracted from sample 3 xExtraction solvent: Chloroform + methanolSuitable for most materialsMethylating reagent: Na-methylateSolvent: Pentane

Future needs

Large amounts of samples require lots oftimePermit to use chloroform may not last foreverNeed for a quantitative determination

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Quantitative determination

Possibility of analysing a few interestingfatty acids

Statistical purposes (independent values)

Methylation procedure

Current analysis: 0.05 M Na-MethylateCommercially available: 25% - 5M

Badings & DeJong (1983):Low conc. result in much longer reactiontime (2M much better than 1M).Longer reaction time favors saponificationinstead of esterification

Methylation of triglyceridesLimited shelf life of Na-methylate?

0,000

0,200

0,400

0,600

0,800

1,000

1,200

1,400

1,600

0 200 400 600 800 1000 1200 1400

Minutter

Mg/

mg

C4 GlC16GlC17GlC4 NyC16 NyC17 NY

Quantitative determination

Quantitative results, sum: 700 mg FA/g fat Accuracy poor, precision fine!

Existing method is not suitable for quantitativedeterminations

(ingen) sammenhæng mellem afvejet mængde og mg FA/g fedt

y = 4,5945x + 734,16R2 = 0,1408

740

750

760

770

780

790

800

810

820

830

0 5 10 15

Serie1Lineær (Serie1)

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New methylation procedure10 mg fat in an Eppendorf tube (2 tubes per sample)Add 1 ml pentane in one tube, 1 ml of a solution ofInternal std-triglyceride in pentane in one tube.Add 10µl 2M Na-methylateWhirlmix 1 min, leave for 10 min at room temp.Centrifuge 13000 g 4C 5 minTransfer pentane to GC vials, run GC

Methylation of a model solutionby use of 5M Na-methylat

Kvantitativ beregning

0

0,5

1

1,5

2

2,5

3

50 100 150 200 250

µl prøve i arbejde

mg

C4 teoC4 fundetC16 teoC16 fundet

Centrifugation of milk fat

Cream separated from skim milk by centrifugation 3500g 4C 15 min

Centrifugation of cream 13000 g 20C 10 min

Heating of cream 60C 10 min

Centrifugation of cream 13000 g 40C 10 min

Yellow liquid fat used for analysis

Centrifugation used by

Murphy et al, 1990 (Teagasc, Cork) Neves et al, 2007 (Brasilia) McNamee et al, 2002 (Belfast)

Ryhänen et al, 2005 (Finland) Stockdale et al, 2003 (Australia)

Feng et al, 2004 (Nottingham)Luna et al, 2005 (Madrid)

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Precision

Milk sample analysed by extraction and methylated by use of dilute Na-methylate

Repeated on 5 days

ANOVA, relative results: Significantdifferences between days.%CV, relative results: 1-5% between days

Comparison of methodsRelative amounts

4.42b0.249b0.248a0.268<0.0001C17:1

0.62b0.698b0.697a0.7050.0062C17

1.12a1.87b1.84a1.880.0012C16:1

0.87b26.02b26.19a26.470.0003C16

1.02b1.13b1.13a1.150.0104C15

5.850.7820.7580.8480.5273C14:1

0.93b10.26b10.25a10.42<0.0001C14

2.53a0.139b0.133a0.139<0.0001C13

1.36a2.67b2.62a2.690.006C12

3.35a0.251b0.239a0.255<0.0001C11

2.30a2.74b2.65a2.770.0144C10

2.67a1.32b1.28a1.350.019C8

1.142.352.32.340.6058C6

9.00a5.42a5.45b4.630.0078C4

CV%Conc. MethylatConc. MethylatDil. MethylatPr > FFatty Acid

CentrifugationEkstractionEkstractionF-test

Comparison of methodsRelative amounts

CV%Conc. MethylatConc. MethylatDil. MethylatPr > FFatty acid

CentrifugationEkstractionEkstractionF-test

4.02ab0.063b0.06a0.065<0.0001C23

37.08b0.139a0.181c0.082<0.0001C20-4

6.270.070.0790.0770.2513C20:3N6

3.33b0.09c0.087a0.093<0.0001C22

23.57a0.081a0.072b0.05<0.0001CLA 10-12

12.87c0.051a0.066b0.058<0.0001C21

0.461.251.251.240.4703CLA 9-11

0.861.171.151.160.2301C18:3N3

34.27c0.047b0.066a0.094<0.0001C20:1

1.91c0.157b0.161a0.1630.0005C20

0.661.511.521.530.2353C18:2N6CIS

0.78b23.79c23.61a23.980.0825C18:1CIS9

0.933.093.093.140.4793C18:1 Trans 11

1.19b0.416a0.426a0.422<0.0001C18:1Trans 9

1.65c12.22b12.45a12.630.0432C18

Quantitative results

6.8019.26%2.74%2.74%1.70%C17

15.933.38%1.71%2.49%1.80%C16:1

251.334.12%1.69%2.53%1.76%C16

11.103.43%1.92%3.24%2.33%C15

6.455.05%3.60%4.26%2.80%C14:1

91.093.14%1.76%3.98%2.88%C14

1.742.52%2.15%5.10%3.44%C13

27.702.65%2.20%5.69%3.68%C12

2.662.58%2.85%6.76%4.41%C11

28.532.49%2.43%7.53%4.46%C10

13.812.29%3.24%9.21%5.63%C8

26.423.26%6.23%12.35%7.08%C6

46.447.69%8.69%14.08%11.22%C4

avg mg/g fat10 milk samples analysed 2 days

10 milk samples, duplicatesMilk 2, 6 repMilk 1, 6 rep

ReproducabilityRepeatability

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Quantitative results

942.883.30%1.51%3.01%1.33%Sum

0.547.76%5.30%3.08%C20:5N3

1.1816.51%4.30%7.09%4.30%C20-4

0.634.48%2.54%2.44%C20:3N6

0.7022.99%10.86%4.85%5.61%C22

5.424.68%3.08%1.82%2.90%CLA 9-11

6.293.89%4.10%1.84%3.30%C18:3N3

0.997.08%5.99%7.86%6.51%C20:1

2.1410.86%5.54%5.86%7.18%C20

17.733.60%2.08%2.18%1.05%C18:2N6CIS

223.254.19%2.01%1.97%1.25%C18:1CIS 9

18.4015.13%2.59%1.94%1.26%C18:1 Trans 11

4.6113.98%10.31%4.44%2.41%C18:1Trans 9

131.975.79%2.28%2.05%1.42%C18

avg mg/g fat10 milk samples analysed 2 days

10 milk samples, duplicatesMilk 2, 6 repMilk 1, 6 rep

ReproducabilityRepeatability

Quantitative results

0.485.965.920.7903C17|Area

0.0017.417.40.9582C16:1|Area

1.16223.4227.10.4508C16|Area

0.328.988.940.7934C15|Area

0.106.886.870.957C14:1|Area

0.0884.484.30.9133C14|Area

8.551.141.010.2281C13|Area

2.2222.621.90.0889C12|Area

3.382.142.040.0043C11|Area

4.0123.622.30.0052C10|Area

5.7111.610.70.0007C8|Area

6.4020.8190.0021C6|Area

2.8249.251.20.48C4|Area

CV%Conc. MethylatConc. MethylatPr > FFatty acid

CentrifugationExtractionF-test

mg/g fat

Quantitative results

0.42863.1868.30.7747Sum

20.741.290.960.1633C20:5N3

29.541.422.170.1806C20-4

2.280.630.610.7588C20:3N6

10.880.70.60.2125C22

0.488.798.850.7666CLA 9-11

1.288.98.740.433C18:3N3

17.250.460.360.0964C20:1

1.601.311.340.3312C20

0.7912.5112.650.6456C18:2N6CIS

0.20215.9216.50.9162C18:1CIS 9

0.4622.922.750.8133C18:1 Trans 11

4.513.43.190.5306C18:1Trans 9

1.66105.2107.70.3394C18

CV%Conc. MethylatConc. MethylatPr > FFedtsyre

CentrifugationExtractionF-test

mg/g fat

Conclusions, Methylation

Analysis of relative amounts not affected

Quantitative determination possible

Much faster

Lower costs (worktime)

No handling of metallic sodium

Suitable for determination of fatty acidcomposition of triglycerides of isolated fat.

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Conclusions, Centrifugation

No use of chloroform or other solvents

Much less work

Results comparable to those obtained by extraction of fat

Only suitable for liquid milk

Shorter time of analysis may prevent degradationof sample during storage