Summary
Many different analytical procedures for fatty acid analysis of infant formulae and human milk are described. The objective was to study possible pitfalls in the use of different acid-catalyzed procedures compared to a base-catalyzed procedure based on sodium-methoxide in methanol. The influence of the different methods on the relative fatty acid composition (wt% of total fatty acids) and the total fatty acid recovery rate (expressed as % of total lipids) was studied in two experimental LCP-containing formulae and a human milk sample. MeOH/HCl-procedures were found to result in an incomplete transesterification of triglycerides, if an additional unpolar solvent like toluene or hexane is not added and a water-free preparation is not guaranteed. In infant formulae the low transesterification of triglycerides (up to only 37 %) could result in an 100 %-overestimation of the relative amount of LCP, if these fatty acids primarily derive from phospholipids. This is the case in infant formulae containing egg lipids as raw materials. In formula containing fish oils and in human milk the efficacy of esterification results in incorrect absolute amounts of fatty acids, but has no remarkable effect on the relative fatty acid distribution. This is due to the fact that in these samples LCP are primarily bound to triglycerides. Furthermore, in formulae based on butterfat the derivatization procedure should be designed in such a way that losses of short-chain fatty acids due to evaporation steps can be avoided. The procedure based on sodium methoxide was found to result in a satisfactory (about 90 %) conversion of formula lipids and a reliable content of all individual fatty acids. Due to a possibly high amount of free fatty acids in human milk, which are not methylated by sodium-methoxide, caution is expressed about the use of this reagent for fatty acid analysis of mothers milk.
It is concluded that accurate fatty acid analysis of infant formulae and human milk requires a careful and quantitative derivatization of both polar and unpolar lipid classes. Sodium methoxide seems to be a reliable and time-saving method for routine fatty acid analysis of infant formulae, which should be validated by interlaboratory comparison. Anhydrous procedures based on methanolic hydrogen chloride including an additional unpolar solvent are also suitable for infant formulae but seem to be preferable for human milk samples.
Zusammenfassung
Zur gaschromatographischen Bestimmung des Fettsäuremusters von Säuglingsnahrungen und von Humanmilch werden zahlreiche unterschiedliche Derivatisierungsverfahren benutzt. Potentielle Fehlerquellen unterschiedlicher säurekatalysierter Verfahren sollten mit denen eines basischen Verfahrens auf der Grundlage von Natriummethylat in Methanol verglichen werden. Untersucht wurde die relative Fettsäurezusammensetzung (in Gew.% der Gesamtfettsäuren) von zwei experimentellen Säuglingsmilchnahrungen, die langkettighochungesättigte Fettsäuren (LCP) in Form von Eilipiden bzw. Fischölen enthielten und von einer Frauenmilchprobe. Zusätzlich wurde die Wiederfindungsrate der Gesamtfettsäuren bezogen auf die Lipideinwaage bestimmt. Es konnte gezeigt werden, daß die Derivatisierung mit methanolischer Salzsäure in einer unvollständigen Transesterifizierung der Triglyceride resultiert, wenn wasserfreie Reaktionsbedingungen und der Zusatz eines unpolaren Lösungsvermittlers (Toluol/Hexan) nicht gewährleistet waren. In einer Säuglingsmilchnahrung, die Eilipide als Rohstoff enthielt, resultierte die nur bis zu 37 %ige Umesterung der Triglyceride in bis zu 100 %-überhöhten relativen Anteilen der LCP-Fettsäuren, da diese sich in erster Linie aus den polaren Phospholipiden ableiten. In Frauenmilch und in der auf Fischöl basierenden Säuglingsmilchnahrung hatte die Effizienz der Transesterifizierung der Triglyceride nur einen geringfügigen Einfluß auf die relativen LCP-Gehalte, da die LCP hier überwiegend in Form der Triglyceride vorliegen. Bei der Fettsäureanalyse von Formelnahrungen die Butterfett enthalten, ist es weiterhin notwendig, das Derivatisierungsverfahren derart durchzuführen, daß der Verlust flüchtiger, kurzkettiger Fettsäuren vermieden wird.
Die schnelle und einfache Derivatisierung mit Natriummethylat führte zu zufriedenstellenden Umesterungsraten der Gesamtlipide und zu zuverlässigen Fettsäuremustern von Säuglingsmilchnahrungen. Es wird daher empfohlen, diese Methode in einem Ringversuch auf die Eignung als Routinemethode zu überprüfen. Da freie Fettsäuren mit der Natriummethylatmethode nicht derivatisiert werden, sind bei der Analyse von Frauenmilchproben säurekatalysierte Verfahren auf Basis methanolischer Salzsäure vorzuziehen. Derartige Verfahren sind auch für Säuglingsmilchnahrungen geeignet, wobei allerdings auf wasserfreie Reaktionsbedingungen und auf den Zusatz eines unpolaren Lösungsvermittlers zu achten ist.
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Abbreviations
- AA :
-
arachidonic acid (20:4 n-6)
- BF 3/MeOH:
-
boron trifluoride in methanol
- DHA :
-
docosahexaenoic acid (22:6 n-3)
- F :
-
infant formulae
- FAME :
-
fatty acid methyl ester
- HM :
-
human milk
- LCP :
-
long-chain polyunsaturated fatty acids (C20/C22)
- MeOH/HCl :
-
methanolic hydrogen chloride
- Na-methoxide/MeOH :
-
sodium methoxide in methanol
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Kohn, G., van der Ploeg, P., Möbius, M. et al. Influence of the derivatization procedure on the results of the gaschromatographic fatty acid analysis of human milk and infant formulae. Z Ernährungswiss 35, 226–234 (1996). https://doi.org/10.1007/BF01625685
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DOI: https://doi.org/10.1007/BF01625685