1 A A R H U S U N I V E R S I T E T Faculty of Agricultural Sciences Analysis of fatty acid composition of milk Method improvement and validation Mette Krogh Larsen Dept. of Food Science ICH Validation Guidelines Specificity Linearity Range Accuracy Precision Detection limit Quantitation limit Robustness System suitability testing Current analysis Originally published 1959 (Folch) Fat extracted from sample 3 x Extraction solvent: Chloroform + methanol Suitable for most materials Methylating reagent: Na-methylate Solvent: Pentane Future needs Large amounts of samples require lots of time Permit to use chloroform may not last forever Need for a quantitative determination
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ICH Validation Guidelines Analysis of fatty acid ...
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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?
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