ich validation guidelines analysis of fatty acid
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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