Sample preparation of free sterols from vegetable oils by countercurrent chromatography in co-current mode.

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 (LPs) was pumped twice as fast as the mobile upper phase (UPm). This novel reversed ccCCC mode improved the performance but also required a higher demand of LPs compared to UPm. Therefore, the exact phase composition of UPm and LPs was determined by gas chromatography and Karl Fischer titration. This step enabled the direct preparation of LPs 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.

Gas chromatography with mass spectrometry analysis of 4,4-dimethylsterols and 4-methylsterols in edible oils after their enrichment by means of solid phase extraction.

4-Methylsterols (4-M-sterols) and 4,4-dimethylsterols (4,4-D-sterols) are a group of underexplored minor sterols that occur in almost all living organisms. Here, we developed a strategy for the determination of the biochemical precursors of the predominant 4-desmethylsterols in edible oils. Due to their low contribution to the sterol content in the samples, a solid phase extraction (SPE) method was developed for the enrichment of 4-M- and 4,4-D-sterols in the hexane extracts of saponified oils. In a two-fold SPE procedure, the bulk of 4,4-D-sterols was collected in one fraction. The residual sample was subjected to a second SPE step which targeted all 4-M-sterols and low shares of 4,4-D-sterols in one fraction and the predominant 4-desmethylsterols in another one. After silylation of the SPE fractions, gas chromatography with mass spectrometry (GC/MS) was used to analyze 4,4-D- and 4-M-sterols. The results were used to define eight subgroups whose characteristic structural features could be linked with the presence of specific m/z values. These m/z values were measured sensitively by GC/MS operated in selected ion monitoring (SIM) mode. Application of the GC/MS method to eighteen edible oils enabled the detection of 55 mostly very low abundant 4-M- and 4,4-D-sterols. Twenty-four of the 4-M- and 4,4-D-sterols could be assigned and the remaining 31 unknown sterols could be traced back to their basic structures.

Isolation of lanosterol and dihydrolanosterol from the unsaponifiable matter of lanolin by urea complexation and countercurrent chromatography in heart-cut recycling mode.

4,4-Dimethyl-substituted sterols are bioactive minor sterols of most animal fats and plant oils, but higher shares are present in lanolin (wool grease). Here, the isolation of the 4,4-dimethyl-substituted sterols dihydrolanosterol and lanosterol from lanolin by countercurrent chromatography (CCC) is described. An initial examination of the hexane extract of saponified lanolin showed the presence of relatively high portions of fatty alcohols which were known to co-elute with the target analytes in CCC. Hence, fatty alcohols were precipitated by urea complexation. Unexpectedly, 4,4-dimethyl-substituted sterols were also found in the crystalline fraction, while cholesterol and other desmethylsterols were detected in the liquid phase. Urea complexation represented a useful preparative method for the separation of desmethylsterols and 4,4-dimethyl-substituted sterols from lanolin. Shake flask experiments of 4,4-dimethyl-substituted sterols and fatty alcohols with 14 biphasic solvent systems indicated suitable partition coefficients (K values) with n-hexane/ethanol/water (12:8:1, v/v/v) and n-hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v). After initial tests with conventional CCC, the application of CCC in heart-cut recycling mode provided 4,4-dimethyl-substituted sterols with purities of 99 % (dihydrolanosterol) and 95 % (lanosterol).

Quantitation of 20 Phytosterols in 34 Different Accessions of Quinoa (Chenopodium quinoa).

Phytosterols were analyzed in 34 different quinoa accessions, which were obtained from the same field trial. Twenty different sterols were detected, and 17 could be structurally assigned by means of gas chromatography with mass spectrometry. Sterols were quantitated in selected ion monitoring mode (GC/MS-SIM) with the novel internal standard 3-O-tert-butyldimethylsilyl-cholestanol (cholestanyl-TBDMS). GC/MS-SIM response factors of minor sterols were determined after enrichment by countercurrent chromatography. The total sterol contents varied from 120 to 180 mg/100 g of seeds, which is higher than has been described in quinoa before. This was due to the fact that Δ7-sterols (e.g., Δ7-sitosterol, spinasterol, and Δ7-avenasterol) were quantitated for the first time in quinoa and contributed ∼64% to the total sterol content. Clustering allowed distributing of the 34 different quinoa accessions into four distinct groups on the basis of the different sterol patterns.

GC/EI-MS method for the determination of phytosterols in vegetable oils.

Sterols are a highly complex group of lipophilic compounds present in the unsaponifiable matter of virtually all living organisms. In this study, we developed a novel gas chromatography with mass spectrometry selected ion monitoring (GC/MS-SIM) method for the comprehensive analysis of sterols after saponification and silylation. A new referencing system was introduced by means of a series of saturated fatty acid pyrrolidides (FAPs) as internal standards. Linked with retention time locking (RTL), the resulting FAP retention indices (RIFAP) of the sterols could be determined with high precision. The GC/MS-SIM method was based on the parallel measurement of 17 SIM ions in four time windows. This set included eight molecular ions and seven diagnostic fragment ions of silylated sterols as well as two abundant ions of FAPs. Altogether, twenty molecular ions of C27- to C31-sterols with 0-3 double bonds were included in the final method. Screening of four common vegetable oils (sunflower oil, hemp oil, rapeseed oil, and corn oil) enabled the detection of 30 different sterols and triterpenes most of which could be identified.

Polyhalogenated Compounds (Halogenated Natural Products and POPs) in Sardine (Sardinops sagax) from the South Atlantic and Indian Oceans.

Halogenated natural products (HNPs) and persistent organic pollutants (POPs) were quantified in South African sardines (Sardinops sagax) from one site in the South Atlantic Ocean and one in the Indian Ocean. At both sites, HNPs [2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1), mixed halogenated compound 1 (MHC-1), 2,4,6-tribromoanisole (2,4,6-TBA), 2'-MeO-BDE 68 (BC-2), and 6-MeO-BDE 47 (BC-3)] were 1 order of magnitude higher concentrated than anthropogenic POPs [mainly polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT), ∼3 ng/g lipids]. MHC-1 and Q1 were the major HNPs in the samples from both sites, contributing with up to 49 and 52 ng/g lipids, respectively. The same 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDE)/PCB ratio suggested that the major POPs were evenly distributed at both sites. Different ratios of Q1/MHC-1 in the samples from the Indian (∼2:1) and South Atlantic (∼1:1) Oceans indicated that the occurrence of HNPs in seafood is difficult to predict and should be investigated more in detail. The PCB levels in sardines were found to pose no risk to human consumers, whereas HNPs could not be evaluated because of the lack of toxicological data.