Countercurrent chromatography (CCC) is a preparative instrumental method where both the mobile and stationary phases are liquids and which are predominantly used for the isolation of natural products. In this study, we widened the scope of CCC by using it as an instrumental method for the direct enrichment of the free sterol fraction from plant oils to which they contribute with ~ 1%. For the enrichment of sterols in a narrow band, we employed the so-called co-current CCC (ccCCC) mode in which both liquid phases of the solvent system (here: n-hexane/ethanol/methanol/water (34:11:12:2, v/v/v/v)) are moved at different flow rates in the same direction. Different from previous applications of ccCCC, the lower and predominant “stationary” phase (LP_s) was pumped twice as fast as the mobile upper phase (UP_m). This novel reversed ccCCC mode improved the performance but also required a higher demand of LP_s compared to UP_m. Therefore, the exact phase composition of UP_m and LP_s was determined by gas chromatography and Karl Fischer titration. This step enabled the direct preparation of LP_s which considerably reduced the waste of solvents. Internal standards (phenyl-substituted fatty acid alkyl esters) were synthesised and utilised to frame the free sterol fraction. This approach allowed a fractionation of free sterols based on the UV signal and compensated run-to-run variations. The reversed ccCCC method was then applied to the sample preparation of five vegetable oils. In addition to free sterols, free tocochromanols (tocopherols, vitamin E) were also eluted in the same fraction as free sterols. Graphical Abstract

Persistent organic pollutants (POPs) are ubiquitously present in food and environmental samples. Accompanied by similar compounds like halogenated natural products (HNPs), their regular monitoring in fish and their predators is an important task. While different procedures have been developed, the removal of the lipid matrix, mainly carried out by treatment with sulfuric acid or by gel permeation chromatography (GPC), is an indispensable step during sample preparation. Here, we present an alternative, non-destructive method using countercurrent chromatography operated in the co-current mode (ccCCC) for the removal of lipids from the fraction of polyhalogenated compounds in fish. The final method was developed using various POP and HNP standards as well as the most critical lipid compound, i.e. free docosahexaenoic acid (DHA). The recovery rates of polyhalogenated compounds ranged between 79 and 117 %. Applied to six herring (Clupea harengus) fillet samples from the North, the Baltic, and the Norwegian Seas, the results obtained with the new (using ccCCC for lipid removal) method agreed well with those determined with an established method using GPC. The quantitative results indicated that fish from contaminated areas is still a major source in the human uptake of POPs. HNP levels were on average only similar to two times lower than POPs which underscores that they should be monitored on a more regular basis.

Furan fatty acids (FuFAs) are lipophilic antioxidants that occur in small amounts (≤1% of the total fatty acids) in plants and animals. They are characterised by a furan substructure within an otherwise saturated acyl chain. Although FuFAs are valuable dietary components due to their beneficial effects, research is hampered by the unavailability of standard substances such as α'-propyl-substituted FuFAs, which are particularly common in fish oils. Here we succeeded in isolating 11-(3,4-dimethyl-5-propylfuran-2-yl)-undecanoic acid ethyl ester (11D3-EE) from gram-amounts of an eicosapentaenoic acid ethyl ester (EPA-EE) oil. After an initial enrichment by hydrogenation and urea complexation, the concentrated FuFA-EE fraction was separated by countercurrent chromatography (CCC) in two-dimensional heart-cut mode using the biphasic solvent system n-hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v). Comparative investigations showed that the head-to-tail mode was better suited than the tail-to-head mode. With the help of the ProMISE2 software, two time-shifted injections were carried out in one run with in total four heart-cuts. As a result, 1.2 mg of 11D3-EE with a purity of 95% (recovery: ∼2.5%), 11.3 mg 11D3-EE with a purity of 90% (recovery: ∼23.6%), and 1.2 mg of the trace compound 9D3-EE (95% purity; recovery not determinable) were isolated from the sample initially enriched by hydrogenation and urea complexation.