A Generic Method for Target (Group) Analysis in Edible Oils and Fats: Combined Normal-Phase Liquid Chromatography and Capillary Gas Chromatography

Sep 02, 2014

Edible oils and fats are complex mixtures of compounds. To understand the properties of an oil or fat, information on groups of molecules, as well as individual molecules, is needed. This article describes the role of normal-phase liquid chromatography (LC) as a generic sample pretreatment tool prior to detailed analysis by gas chromatography (GC). Moving from one group to another can be performed by simply adjusting the elution conditions and collection windows. Different applications of the new unified method will be discussed, including detailed analysis of partial acylglycerides, steradienes, and glycidylesters, as well as the use of the novel method for total-polarity mapping.

Edible oils and fats are an important part of the human diet. From an analytical perspective, these food ingredients are extremely complex mixtures, irrespective of whether they originate from animals or vegetables. Analytical measurements are therefore challenging. A wide range of analytical methods are available that allow a detailed mapping of the overall bulk composition, as well as an analysis of specific trace compounds. In all of these methods sample preparation is a crucial step. Historically, little standardization of the sample preparation and analysis methods was performed. As a result, edible oil and fat laboratories are now confronted with a huge diversity of almost identical, yet slightly different, methods. More generic approaches could help to contribute to the efficiency of these laboratories.

As in all analytical measurements, the key aspects in method development for edible oil and fat analysis are selectivity and sensitivity. The compounds of interest should be measured at the desired levels without interferences from other species. Sensitivity is a crucial factor in trace analysis, for example, in contaminant or vitamin analysis. Selectivity, that is, the ability to distinguish the compounds of interest from those that are not relevant, is a second important factor in trace analysis, but it is equally relevant in the assessment of the main compound classes in the oil. Selectivity in oil and fat analysis is basically related to isolating the compound or compound group of interest from the total sample by exploiting differences in the physicochemical properties (reactivity, size, or polarity) between the compounds of interest and the rest. Isolation is very often performed in the sample pretreatment step. Saponification followed by extraction is an important sample preparation step for removal of the bulk triacylglycerides (TAG) and isolation of the (non-hydrolyzable) nonpolar species. Saponification will not be considered here because it is not a universal approach. All compounds that can be hydrolyzed are lost in the procedure. This article will focus on methods for target-group isolation that isolate the molecules in their native, intact form. After the initial isolation of the compounds of interest, chromatographic techniques can be used to introduce additional selectivity and quantify the groups or individual compounds for which information is needed.

In the past a wide variety of methods have been developed for the pre-isolation of the target analytes from an oil or fat sample. Liquid–liquid partitioning, solid-phase extraction (SPE), thin-layer chromatography (TLC), size-exclusion chromatography (SEC), and complexation are the most common tools.

After the initial isolation both gas chromatography (GC) and liquid chromatography (LC), either in combination with or without mass spectrometry (MS), can be used for separation and quantification. It is clear that all the sample preparation methods, with the exception of SEC, exploit solubility and interactions as the mechanisms for separating the target compounds from the remaining sample material. It was from this that the idea to replace the wide variety of isolation methods by a single analytical isolation tool, normal-phase LC, arose. Much of the early work to replace complex sample work-up methods for oil and fat analysis by LC isolation of the fraction of interest was performed by Grob and coworkers (1). Normal-phase LC isolation of the compounds of interest provided an excellent selectivity. The drawback of LC isolation (the dilution of the fraction) was elegantly resolved by applying large volume sample transfer to the second dimension GC or LC method.

This article will describe previous work in the development of a single, unified analytical protocol that can replace most of the sample preparation procedures applied in the various edible oil analysis methods. Building on the pioneering work done by Grob, the use of combined normal-phase LC–GC for the analysis of compounds and compound classes that have been of interest for decades will be demonstrated. In addition, its use for the analysis of species that have only received interest recently will be illustrated. The latter group includes sterols, steroloxides, and glycidylesters.